Sample records for accelerates axon regeneration

  1. Brief post-surgical electrical stimulation accelerates axon regeneration and muscle reinnervation without affecting the functional measures in carpal tunnel syndrome patients.

    PubMed

    Gordon, Tessa; Amirjani, Nasim; Edwards, David C; Chan, K Ming

    2010-05-01

    Electrical stimulation (ES) of injured peripheral nerves accelerates axonal regeneration in laboratory animals. However, clinical applicability of this intervention has never been investigated in human subjects. The aim of this pilot study was to determine the effect of ES on axonal regeneration after surgery in patients with median nerve compression in the carpal tunnel causing marked motor axonal loss. A randomized control trial was conducted to provide proof of principle for ES-induced acceleration of axon regeneration in human patients. Carpel tunnel release surgery (CTRS) was performed and in the stimulation group of patients, stainless steel electrode wires placed alongside the median nerve proximal to the surgical decompression site for immediate 1 h 20 Hz bipolar ES. Subjects were followed for a year at regular intervals. Axonal regeneration was quantified using motor unit number estimation (MUNE) and sensory and motor nerve conduction studies. Purdue Pegboard Test, Semmes Weinstein Monofilaments, and Levine's Self-Assessment Questionnaire were used to assess functional recovery. The stimulation group had significant axonal regeneration 6-8 months after the CTRS when the MUNE increased to 290+/-140 (mean+/-SD) motor units (MU) from 150+/-62 MU at baseline (p<0.05). In comparison, MUNE did not significantly improve in the control group (p>0.2). Terminal motor latency significantly accelerated in the stimulation group but not the control group (p>0.1). Sensory nerve conduction values significantly improved in the stimulation group earlier than the controls. Other outcome measures showed a significant improvement in both patient groups. We conclude that brief low frequency ES accelerates axonal regeneration and target reinnervation in humans. Copyright 2009 Elsevier Inc. All rights reserved.

  2. Axon Regeneration in C. elegans

    PubMed Central

    Hammarlund, Marc; Jin, Yishi

    2014-01-01

    Single axon transection by laser surgery has made C. elegans a new model for axon regeneration. Multiple conserved molecular signaling modules have been discovered through powerful genetic screening. in vivo imaging with single cell and axon resolution has revealed unprecedented cellular dynamics in regenerating axons. Information from C. elegans has greatly expanded our knowledge of the molecular and cellular mechanisms of axon regeneration. PMID:24794753

  3. Inhibiting poly(ADP-ribosylation) improves axon regeneration.

    PubMed

    Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

    2016-10-04

    The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration.

  4. Inhibiting poly(ADP-ribosylation) improves axon regeneration

    PubMed Central

    Byrne, Alexandra B; McWhirter, Rebecca D; Sekine, Yuichi; Strittmatter, Stephen M; Miller, David M; Hammarlund, Marc

    2016-01-01

    The ability of a neuron to regenerate its axon after injury depends in part on its intrinsic regenerative potential. Here, we identify novel intrinsic regulators of axon regeneration: poly(ADP-ribose) glycohodrolases (PARGs) and poly(ADP-ribose) polymerases (PARPs). PARGs, which remove poly(ADP-ribose) from proteins, act in injured C. elegans GABA motor neurons to enhance axon regeneration. PARG expression is regulated by DLK signaling, and PARGs mediate DLK function in enhancing axon regeneration. Conversely, PARPs, which add poly(ADP-ribose) to proteins, inhibit axon regeneration of both C. elegans GABA neurons and mammalian cortical neurons. Furthermore, chemical PARP inhibitors improve axon regeneration when administered after injury. Our results indicate that regulation of poly(ADP-ribose) levels is a critical function of the DLK regeneration pathway, that poly-(ADP ribosylation) inhibits axon regeneration across species, and that chemical inhibition of PARPs can elicit axon regeneration. DOI: http://dx.doi.org/10.7554/eLife.12734.001 PMID:27697151

  5. Axonal regeneration in zebrafish spinal cord

    PubMed Central

    Hui, Subhra Prakash

    2018-01-01

    Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

  6. Axonal regeneration in zebrafish spinal cord.

    PubMed

    Ghosh, Sukla; Hui, Subhra Prakash

    2018-03-01

    In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

  7. Axon Regeneration in C. elegans: worming our way to mechanisms of axon regeneration

    PubMed Central

    Byrne, Alexandra B.; Hammarlund, Marc

    2016-01-01

    How axons repair themselves after injury is a fundamental question in neurobiology. With its conserved genome, relatively simple nervous system, and transparent body, C. elegans has recently emerged as a productive model to uncover the cellular mechanisms that regulate and execute axon regeneration. In this review, we discuss the strengths and weaknesses of the C. elegans model of regeneration. We explore the technical advances that enable the use of C. elegans for in vivo regeneration studies, review findings in C. elegans that have contributed to our understanding of the regeneration response across species, discuss the potential of C. elegans research to provide insight into mechanisms that function in the injured mammalian nervous system, and present potential future directions of axon regeneration research using C. elegans. PMID:27569538

  8. Mitochondria localize to injured axons to support regeneration

    PubMed Central

    Han, Sung Min; Baig, Huma S.; Hammarlund, Marc

    2016-01-01

    SUMMARY Axon regeneration is essential to restore the nervous system after axon injury. However, the neuronal cell biology that underlies axon regeneration is incompletely understood. Here we use in vivo single-neuron analysis to investigate the relationship between nerve injury, mitochondrial localization, and axon regeneration. Mitochondria translocate into injured axons, so that average mitochondria density increases after injury. Moreover, single-neuron analysis reveals that axons that fail to increase mitochondria have poor regeneration. Experimental alterations to axonal mitochondrial distribution or mitochondrial respiratory chain function result in corresponding changes to regeneration outcomes. Axonal mitochondria are specifically required for growth cone migration, identifying a key energy challenge for injured neurons. Finally, mitochondrial localization to the axon after injury is regulated in part by dual-leucine zipper kinase-1 (DLK-1), a conserved regulator of axon regeneration. These data identify regulation of axonal mitochondria as a new cell biological mechanism that helps determine the regenerative response of injured neurons. PMID:28009276

  9. Cell intrinsic control of axon regeneration

    PubMed Central

    Mar, Fernando M; Bonni, Azad; Sousa, Mónica M

    2014-01-01

    Although neurons execute a cell intrinsic program of axonal growth during development, following the establishment of connections, the developmental growth capacity declines. Besides environmental challenges, this switch largely accounts for the failure of adult central nervous system (CNS) axons to regenerate. Here, we discuss the cell intrinsic control of axon regeneration, including not only the regulation of transcriptional and epigenetic mechanisms, but also the modulation of local protein translation, retrograde and anterograde axonal transport, and microtubule dynamics. We further explore the causes underlying the failure of CNS neurons to mount a vigorous regenerative response, and the paradigms demonstrating the activation of cell intrinsic axon growth programs. Finally, we present potential mechanisms to support axon regeneration, as these may represent future therapeutic approaches to promote recovery following CNS injury and disease. PMID:24531721

  10. Learning to swim, again: Axon regeneration in fish.

    PubMed

    Rasmussen, Jeffrey P; Sagasti, Alvaro

    2017-01-01

    Damage to the central nervous system (CNS) of fish can often be repaired to restore function, but in mammals recovery from CNS injuries usually fails due to a lack of axon regeneration. The relatively growth-permissive environment of the fish CNS may reflect both the absence of axon inhibitors found in the mammalian CNS and the presence of pro-regenerative environmental factors. Despite their different capacities for axon regeneration, many of the physiological processes, intrinsic molecular pathways, and cellular behaviors that control an axon's ability to regrow are conserved between fish and mammals. Fish models have thus been useful both for identifying factors differing between mammals and fish that may account for differences in CNS regeneration and for characterizing conserved intrinsic pathways that regulate axon regeneration in all vertebrates. The majority of adult axon regeneration studies have focused on the optic nerve or spinal axons of the teleosts goldfish and zebrafish, which have been productive models for identifying genes associated with axon regeneration, cellular mechanisms of circuit reestablishment, and the basis of functional recovery. Lampreys, which are jawless fish lacking myelin, have provided an opportunity to study regeneration of well defined spinal cord circuits. Newer larval zebrafish models offer numerous genetic tools and the ability to monitor the dynamic behaviors of extrinsic cell types regulating axon regeneration in live animals. Recent advances in imaging and gene editing methods are making fish models yet more powerful for investigating the cellular and molecular underpinnings of axon regeneration. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

    PubMed

    Leibinger, Marco; Andreadaki, Anastasia; Golla, Renate; Levin, Evgeny; Hilla, Alexander M; Diekmann, Heike; Fischer, Dietmar

    2017-07-03

    Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3 S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3β reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3β KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2 T/A ). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2 T/A in GSK3 S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

  12. Dependence of regenerated sensory axons on continuous neurotrophin-3 delivery.

    PubMed

    Hou, Shaoping; Nicholson, LaShae; van Niekerk, Erna; Motsch, Melanie; Blesch, Armin

    2012-09-19

    Previous studies have shown that injured dorsal column sensory axons extend across a spinal cord lesion site if axons are guided by a gradient of neurotrophin-3 (NT-3) rostral to the lesion. Here we examined whether continuous NT-3 delivery is necessary to sustain regenerated axons in the injured spinal cord. Using tetracycline-regulated (tet-off) lentiviral gene delivery, NT-3 expression was tightly controlled by doxycycline administration. To examine axon growth responses to regulated NT-3 expression, adult rats underwent a C3 dorsal funiculus lesion. The lesion site was filled with bone marrow stromal cells, tet-off-NT-3 virus was injected rostral to the lesion site, and the intrinsic growth capacity of sensory neurons was activated by a conditioning lesion. When NT-3 gene expression was turned on, cholera toxin β-subunit-labeled sensory axons regenerated into and beyond the lesion/graft site. Surprisingly, the number of regenerated axons significantly declined when NT-3 expression was turned off, whereas continued NT-3 expression sustained regenerated axons. Quantification of axon numbers beyond the lesion demonstrated a significant decline of axon growth in animals with transient NT-3 expression, only some axons that had regenerated over longer distance were sustained. Regenerated axons were located in white matter and did not form axodendritic synapses but expressed presynaptic markers when closely associated with NG2-labeled cells. A decline in axon density was also observed within cellular grafts after NT-3 expression was turned off possibly via reduction in L1 and laminin expression in Schwann cells. Thus, multiple mechanisms underlie the inability of transient NT-3 expression to fully sustain regenerated sensory axons.

  13. Axon Regeneration Genes Identified by RNAi Screening in C. elegans

    PubMed Central

    Nix, Paola; Hammarlund, Marc; Hauth, Linda; Lachnit, Martina; Jorgensen, Erik M.

    2014-01-01

    Axons of the mammalian CNS lose the ability to regenerate soon after development due to both an inhibitory CNS environment and the loss of cell-intrinsic factors necessary for regeneration. The complex molecular events required for robust regeneration of mature neurons are not fully understood, particularly in vivo. To identify genes affecting axon regeneration in Caenorhabditis elegans, we performed both an RNAi-based screen for defective motor axon regeneration in unc-70/β-spectrin mutants and a candidate gene screen. From these screens, we identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions. Through our analysis of mutants, we shed new light on certain aspects of regeneration, including the role of β-spectrin and membrane dynamics, the antagonistic activity of MAP kinase signaling pathways, and the role of stress in promoting axon regeneration. Many gene candidates had not previously been associated with axon regeneration and implicate new pathways of interest for therapeutic intervention. PMID:24403161

  14. Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

    NASA Technical Reports Server (NTRS)

    Macias, M. Y.; Lehman, C. T.; Sanger, J. R.; Riley, D. A.

    1998-01-01

    Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median-ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies.

  15. The Genetics of Axon Guidance and Axon Regeneration in Caenorhabditis elegans

    PubMed Central

    Chisholm, Andrew D.; Hutter, Harald; Jin, Yishi; Wadsworth, William G.

    2016-01-01

    The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment. PMID:28114100

  16. Insulin/IGF1 Signaling Inhibits Age-Dependent Axon Regeneration

    PubMed Central

    Byrne, Alexandra B.; Walradt, Trent; Gardner, Kathryn E.; Hubbert, Austin; Reinke, Valerie; Hammarlund, Marc

    2014-01-01

    Summary The ability of injured axons to regenerate declines with age yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2’s function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron specific, and genetically regulated process. In addition, we found that daf-18/PTEN inhibits regeneration independently of age and FOXO signaling, via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and is required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons, and that this mechanism is independent of PTEN and TOR. PMID:24440228

  17. Syndecan promotes axon regeneration by stabilizing growth cone migration

    PubMed Central

    Edwards, Tyson J.; Hammarlund, Marc

    2014-01-01

    SUMMARY Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS) proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: 1) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains, and 2) a novel intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a novel regulator of a critical choke point in nervous system repair. PMID:25001284

  18. Syndecan promotes axon regeneration by stabilizing growth cone migration.

    PubMed

    Edwards, Tyson J; Hammarlund, Marc

    2014-07-10

    Growth cones facilitate the repair of nervous system damage by providing the driving force for axon regeneration. Using single-neuron laser axotomy and in vivo time-lapse imaging, we show that syndecan, a heparan sulfate (HS) proteoglycan, is required for growth cone function during axon regeneration in C. elegans. In the absence of syndecan, regenerating growth cones form but are unstable and collapse, decreasing the effective growth rate and impeding regrowth to target cells. We provide evidence that syndecan has two distinct functions during axon regeneration: (1) a canonical function in axon guidance that requires expression outside the nervous system and depends on HS chains and (2) an intrinsic function in growth cone stabilization that is mediated by the syndecan core protein, independently of HS. Thus, syndecan is a regulator of a critical choke point in nervous system repair. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration

    PubMed Central

    Rao, Kavitha; Stone, Michelle C.; Weiner, Alexis T.; Gheres, Kyle W.; Zhou, Chaoming; Deitcher, David L.; Levitan, Edwin S.; Rolls, Melissa M.

    2016-01-01

    Mutations in >50 genes, including spastin and atlastin, lead to hereditary spastic paraplegia (HSP). We previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin, and spichthyin were reduced. Impaired regeneration was dependent on genetic background and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting that HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. We examined other markers to identify additional changes associated with regeneration. Whereas mitochondria, endosomes, and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was observed only during axon regeneration and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. We propose that the HSP proteins spastin and atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip. PMID:27605706

  20. Can injured adult CNS axons regenerate by recapitulating development?

    PubMed

    Hilton, Brett J; Bradke, Frank

    2017-10-01

    In the adult mammalian central nervous system (CNS), neurons typically fail to regenerate their axons after injury. During development, by contrast, neurons extend axons effectively. A variety of intracellular mechanisms mediate this difference, including changes in gene expression, the ability to form a growth cone, differences in mitochondrial function/axonal transport and the efficacy of synaptic transmission. In turn, these intracellular processes are linked to extracellular differences between the developing and adult CNS. During development, the extracellular environment directs axon growth and circuit formation. In adulthood, by contrast, extracellular factors, such as myelin and the extracellular matrix, restrict axon growth. Here, we discuss whether the reactivation of developmental processes can elicit axon regeneration in the injured CNS. © 2017. Published by The Company of Biologists Ltd.

  1. Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury*

    PubMed Central

    van Niekerk, Erna A.; Tuszynski, Mark H.; Lu, Paul; Dulin, Jennifer N.

    2016-01-01

    Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. PMID:26695766

  2. Molecular Determinants Fundamental to Axon Regeneration after SCI

    DTIC Science & Technology

    2012-10-01

    functions. In the mammalian spinal cord, axon regeneration is frustrated by inhibitors such as chondroitin sulfate proteoglycans (CSPGs) expressed by...CG, Becker T (2002) Repellent guidance of regeneration optic axons by chondroitin sulfate glycosaminoglycans in zebrafish. J Neurosci 22(3): 842-853...Shen Y, Tenney AP, Busch SA, Horn KP, Cuascut FX, Liu K, He Z, Silver J, Flanagan JG (2009) PTPσ is a receptor for chondroitin sulfate

  3. Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration.

    PubMed

    Rao, Kavitha; Stone, Michelle C; Weiner, Alexis T; Gheres, Kyle W; Zhou, Chaoming; Deitcher, David L; Levitan, Edwin S; Rolls, Melissa M

    2016-11-01

    Mutations in >50 genes, including spastin and atlastin, lead to hereditary spastic paraplegia (HSP). We previously demonstrated that reduction of spastin leads to a deficit in axon regeneration in a Drosophila model. Axon regeneration was similarly impaired in neurons when HSP proteins atlastin, seipin, and spichthyin were reduced. Impaired regeneration was dependent on genetic background and was observed when partial reduction of HSP proteins was combined with expression of dominant-negative microtubule regulators, suggesting that HSP proteins work with microtubules to promote regeneration. Microtubule rearrangements triggered by axon injury were, however, normal in all genotypes. We examined other markers to identify additional changes associated with regeneration. Whereas mitochondria, endosomes, and ribosomes did not exhibit dramatic repatterning during regeneration, the endoplasmic reticulum (ER) was frequently concentrated near the tip of the growing axon. In atlastin RNAi and spastin mutant animals, ER accumulation near single growing axon tips was impaired. ER tip concentration was observed only during axon regeneration and not during dendrite regeneration. In addition, dendrite regeneration was unaffected by reduction of spastin or atlastin. We propose that the HSP proteins spastin and atlastin promote axon regeneration by coordinating concentration of the ER and microtubules at the growing axon tip. © 2016 Rao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. Different effects of astrocytes and Schwann cells on regenerating retinal axons.

    PubMed

    Campbell, Gregor; Kitching, Juliet; Anderson, Patrick N; Lieberman, A Robert

    2003-11-14

    Following a crush injury of the optic nerve in adult rats, the axons of retinal ganglion cells, stimulated to regenerate by a lens injury and growing within the optic nerve, are associated predominantly with astrocytes: they remain of small diameter (0.1-0.5 microm) and unmyelinated for > or = 2 months after the operation. In contrast, when the optic nerve is cut and a segment of a peripheral nerve is grafted to the ocular stump of the optic nerve, the regenerating retinal axons are associated predominantly with Schwann cells: they are of larger diameter than in the previous experiment and include unmyelinated axons (0.2-2.5 microm) and myelinated axons (mean diameter 2.3 microm). Thus, the grafted peripheral nerve, and presumably its Schwann cells, stimulate enlargement of the regenerating retinal axons leading to partial myelination, whereas the injured optic nerve itself, and presumably its astrocytes, does not. The result points to a marked difference of peripheral (Schwann cells) and central (astrocytes) glia in their effect on regenerating retinal axons.

  5. Molecular and Cellular Mechanisms of Axonal Regeneration After Spinal Cord Injury.

    PubMed

    van Niekerk, Erna A; Tuszynski, Mark H; Lu, Paul; Dulin, Jennifer N

    2016-02-01

    Following axotomy, a complex temporal and spatial coordination of molecular events enables regeneration of the peripheral nerve. In contrast, multiple intrinsic and extrinsic factors contribute to the general failure of axonal regeneration in the central nervous system. In this review, we examine the current understanding of differences in protein expression and post-translational modifications, activation of signaling networks, and environmental cues that may underlie the divergent regenerative capacity of central and peripheral axons. We also highlight key experimental strategies to enhance axonal regeneration via modulation of intraneuronal signaling networks and the extracellular milieu. Finally, we explore potential applications of proteomics to fill gaps in the current understanding of molecular mechanisms underlying regeneration, and to provide insight into the development of more effective approaches to promote axonal regeneration following injury to the nervous system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. The Mammalian-Specific Protein Armcx1 Regulates Mitochondrial Transport during Axon Regeneration.

    PubMed

    Cartoni, Romain; Norsworthy, Michael W; Bei, Fengfeng; Wang, Chen; Li, Siwei; Zhang, Yiling; Gabel, Christopher V; Schwarz, Thomas L; He, Zhigang

    2016-12-21

    Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Scar modulation in subacute and chronic CNS lesions: Effects on axonal regeneration.

    PubMed

    Stichel, Christine C.; Lausberg, Friederike; Hermanns, Susanne; Müller, Hans Werner

    1999-01-01

    After injury of the adult mammalian CNS axonal regeneration across or around the lesion scar is negligible. Previously, we have shown that the lesion-induced basal membrane (BM) within the lesion center participates in a growth barrier for axon regeneration and that its reduction by means of pharmacological or immunochemical treatment is a prerequisite and sufficient condition for regrowing axons to cross the lesion site. The present study was designed to further investigate this observation by analyzing the effect of a delayed treatment on the regeneration of both subacutely and chronically lesioned axons.Adult rats underwent unilateral transection of the postcommissural fornix. At one to five days after transection one group of animals received a local injection of 2, 2'-dipyridyl (DPY), an inhibitor of collagen triple helix formation and synthesis. Another group received a second transection within the former lesion site followed by an immediate DPY-injection at five days or 4 weeks after transection. Six weeks after the last surgery BM deposition and axonal regeneration were analysed using immunocytochemical methods.A local injection of DPY clearly reduced the lesion-induced BM deposition when applied within the first 3 days after transection. Under these conditions regrowing axons still crossed the former impermeable lesion site and regenerated within their normal pathway up to their former target, the mammillary body. However, in late subacute (5 d) and chronic stages (4 w) the double transection+injection paradigm failed to reduce BM deposition and, in consequence, also to induce axonal regeneration.These results demonstrate the potential of the collagen IV-reducing strategy to promote axonal regeneration across the lesion scar not only in acute but also in early subacute traumatic injuries.

  8. S6 Kinase Inhibits Intrinsic Axon Regeneration Capacity via AMP Kinase in Caenorhabditis elegans

    PubMed Central

    Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.

    2014-01-01

    The ability of axons to regrow after injury is determined by the complex interplay of intrinsic growth programs and external cues. In Caenorhabditis elegans mechanosensory neuron, axons exhibit robust regenerative regrowth following laser axotomy. By surveying conserved metabolic signaling pathways, we have identified the ribosomal S6 kinase RSKS-1 as a new cell-autonomous inhibitor of axon regeneration. RSKS-1 is not required for axonal development but inhibits axon regrowth after injury in multiple neuron types. Loss of function in rsks-1 results in more rapid growth cone formation after injury and accelerates subsequent axon extension. The enhanced regrowth of rsks-1 mutants is partly dependent on the DLK-1 MAPK cascade. An essential output of RSKS-1 in axon regrowth is the metabolic sensor AMP kinase, AAK-2. We further show that the antidiabetic drug phenformin, which activates AMP kinase, can promote axon regrowth. Our data reveal a new function for an S6 kinase acting through an AMP kinase in regenerative growth of injured axons. PMID:24431434

  9. Pharmacologically inhibiting kinesin-5 activity with monastrol promotes axonal regeneration following spinal cord injury

    PubMed Central

    Xu, Chen; Klaw, Michelle C.; Lemay, Michel A.; Baas, Peter W.; Tom, Veronica J.

    2014-01-01

    While it is well established that the axons of adult neurons have a lower capacity for regrowth, some regeneration of certain CNS populations after spinal cord injury (SCI) is possible if their axons are provided with a permissive substrate, such as an injured peripheral nerve. While some axons readily regenerate into a peripheral nerve graft (PNG), these axons almost always stall at the distal interface and fail to re-innervate spinal cord tissue. Treatment of the glial scar at the distal graft interface with chondroitinase ABC (ChABC) can improve regeneration, but most regenerated axons need further stimulation to extend beyond the interface. Previous studies demonstrate that pharmacologically inhibiting kinesin-5, a motor protein best known for its essential role in mitosis but also expressed in neurons, with the pharmacological agent monastrol increases axon growth on inhibitory substrates in vitro. We sought to determine if monastrol treatment after a SCI improves functional axon regeneration. Animals received complete thoracic level 7 (T7) transections and PNGs and were treated intrathecally with ChABC and either monastrol or DMSO vehicle. We found that combining ChABC with monastrol significantly enhanced axon regeneration. However, there were no further improvements in function or enhanced c-Fos induction upon stimulation of spinal cord rostral to the transection. This indicates that monastrol improves ChABC-mediated axon regeneration but that further treatments are needed to enhance the integration of these regrown axons. PMID:25447935

  10. Antisense Morpholino Oligonucleotides Reduce Neurofilament Synthesis and Inhibit Axon Regeneration in Lamprey Reticulospinal Neurons.

    PubMed

    Zhang, Guixin; Jin, Li-qing; Hu, Jianli; Rodemer, William; Selzer, Michael E

    2015-01-01

    The sea lamprey has been used as a model for the study of axonal regeneration after spinal cord injury. Previous studies have suggested that, unlike developing axons in mammal, the tips of regenerating axons in lamprey spinal cord are simple in shape, packed with neurofilaments (NFs), and contain very little F-actin. Thus it has been proposed that regeneration of axons in the central nervous system of mature vertebrates is not based on the canonical actin-dependent pulling mechanism of growth cones, but involves an internal protrusive force, perhaps generated by the transport or assembly of NFs in the distal axon. In order to assess this hypothesis, expression of NFs was manipulated by antisense morpholino oligonucleotides (MO). A standard, company-supplied MO was used as control. Axon retraction and regeneration were assessed at 2, 4 and 9 weeks after MOs were applied to a spinal cord transection (TX) site. Antisense MO inhibited NF180 expression compared to control MO. The effect of inhibiting NF expression on axon retraction and regeneration was studied by measuring the distance of axon tips from the TX site at 2 and 4 weeks post-TX, and counting the number of reticulospinal neurons (RNs) retrogradely labeled by fluorescently-tagged dextran injected caudal to the injury at 9 weeks post-TX. There was no statistically significant effect of MO on axon retraction at 2 weeks post-TX. However, at both 4 and 9 weeks post-TX, inhibition of NF expression inhibited axon regeneration.

  11. Molecular Determinants Fundamental to Axon Regeneration after SCI

    DTIC Science & Technology

    2014-09-01

    mammalian spinal cord, axon regeneration is frustrated by inhibitors such as chondroitin sulfate proteoglycans (CSPGs) expressed by reactive astrocytes... chondroitin sulfates . Publications, Abstracts and Presentations: Publications: 1. Katerina Vajn, Jeffery A Plunkett, Alexis Tapanes...Jeffery A. Plunkett. Axonal growth of primary zebrafish brainstem neurons across inhibitory chondroitin sulfate proteoglycans. Manuscript in

  12. Focal release of neurotrophic factors by biodegradable microspheres enhance motor and sensory axonal regeneration in vitro and in vivo.

    PubMed

    Santos, Daniel; Giudetti, Guido; Micera, Silvestro; Navarro, Xavier; Del Valle, Jaume

    2016-04-01

    Neurotrophic factors (NTFs) promote nerve regeneration and neuronal survival after peripheral nerve injury. However, drawbacks related with administration and bioactivity during long periods limit their therapeutic application. In this study, PLGA microspheres (MPs) were used to locally release different NTFs and evaluate whether they accelerate axonal regeneration in comparison with free NTFs or controls. ELISA, SEM, UV/visible light microscopy, organotypic cultures of DRG explants and spinal cord slices were used to characterize MP properties and the bioactivity of the released NTFs. Results of organotypic cultures showed that encapsulated NTFs maintain longer bioactivity and enhance neurite regeneration of both sensory and motor neurons compared with free NTFs. For in vivo assays, the rat sciatic nerve was transected and repaired with a silicone tube filled with collagen gel or collagen mixed with PBS encapsulated MPs (control groups) and with free or encapsulated NGF, BDNF, GDNF or FGF-2. After 20 days, a retrotracer was applied to the regenerated nerve to quantify motor and sensory axonal regeneration. NTF encapsulation in MPs improved regeneration of both motor and sensory axons, as evidenced by increased numbers of retrolabeled neurons. Hence, our results show that slow release of NTFs with PLGA MP enhance nerve regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Axon regeneration can facilitate or suppress hindlimb function after olfactory ensheathing glia transplantation.

    PubMed

    Takeoka, Aya; Jindrich, Devin L; Muñoz-Quiles, Cintia; Zhong, Hui; van den Brand, Rubia; Pham, Daniel L; Ziegler, Matthias D; Ramón-Cueto, Almudena; Roy, Roland R; Edgerton, V Reggie; Phelps, Patricia E

    2011-03-16

    Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.

  14. Rewiring of regenerated axons by combining treadmill training with semaphorin3A inhibition

    PubMed Central

    2014-01-01

    Background Rats exhibit extremely limited motor function recovery after total transection of the spinal cord (SCT). We previously reported that SM-216289, a semaphorin3A inhibitor, enhanced axon regeneration and motor function recovery in SCT adult rats. However, these effects were limited because most regenerated axons likely do not connect to the right targets. Thus, rebuilding the appropriate connections for regenerated axons may enhance recovery. In this study, we combined semaphorin3A inhibitor treatment with extensive treadmill training to determine whether combined treatment would further enhance the “rewiring” of regenerated axons. In this study, which aimed for clinical applicability, we administered a newly developed, potent semaphorin3A inhibitor, SM-345431 (Vinaxanthone), using a novel drug delivery system that enables continuous drug delivery over the period of the experiment. Results Treatment with SM-345431 using this delivery system enhanced axon regeneration and produced significant, but limited, hindlimb motor function recovery. Although extensive treadmill training combined with SM-345431 administration did not further improve axon regeneration, hindlimb motor performance was restored, as evidenced by the significant improvement in the execution of plantar steps on a treadmill. In contrast, control SCT rats could not execute plantar steps at any point during the experimental period. Further analyses suggested that this strategy reinforced the wiring of central pattern generators in lumbar spinal circuits, which, in turn, led to enhanced motor function recovery (especially in extensor muscles). Conclusions This study highlights the importance of combining treatments that promote axon regeneration with specific and appropriate rehabilitations that promote rewiring for the treatment of spinal cord injury. PMID:24618249

  15. MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3β expression.

    PubMed

    Jiang, J-J; Liu, C-M; Zhang, B-Y; Wang, X-W; Zhang, M; Saijilafu; Zhang, S-R; Hall, P; Hu, Y-W; Zhou, F-Q

    2015-08-27

    MicroRNAs are emerging to be important epigenetic factors that control axon regeneration. Here, we report that microRNA-26a (miR-26a) is a physiological regulator of mammalian axon regeneration in vivo. We demonstrated that endogenous miR-26a acted to target specifically glycogen synthase kinase 3β (GSK3β) in adult mouse sensory neurons in vitro and in vivo. Inhibition of endogenous miR-26a in sensory neurons impaired axon regeneration in vitro and in vivo. Moreover, the regulatory effect of miR-26a was mediated by increased expression of GSK3β because downregulation or pharmacological inhibition of GSK3β fully rescued axon regeneration. Our results also suggested that the miR-26a-GSK3β pathway regulated axon regeneration at the neuronal soma by controlling gene expression. We provided biochemical and functional evidences that the regeneration-associated transcription factor Smad1 acted downstream of miR-26a and GSK3β to control sensory axon regeneration. Our study reveals a novel miR-26a-GSK3β-Smad1 signaling pathway in the regulation of mammalian axon regeneration. Moreover, we provide the first evidence that, in addition to inhibition of GSK3β kinase activity, maintaining a lower protein level of GSK3β in neurons by the microRNA is necessary for efficient axon regeneration.

  16. The influence of predegenerated nerve grafts on axonal regeneration from prelesioned peripheral nerves.

    PubMed

    Hasan, N A; Neumann, M M; de Souky, M A; So, K F; Bedi, K S

    1996-10-01

    Recent in vitro work has indicated that predegenerated segments of peripheral nerve are more capable of supporting neurite growth from adult neurons than fresh segments of nerve, whereas previous in vivo studies which investigated whether predegenerated nerve segments used as grafts are capable of enhancing axonal regeneration produced conflicting results. We have reinvestigated this question by using predegenerated nerve grafts in combination with conditioning lesions of the host nerve to determine the optimal conditions for obtaining the maximal degree of regeneration of myelinated axons. The sciatic nerve of adult Dark Agouti rats were sectioned at midthigh level, and the distal portion was allowed to predegenerate for 0, 6 or 12 d in situ. 10-15 mm lengths of these distal nerve segments were then syngenically grafted onto the central stumps of sciatic nerves which had themselves received a conditioning lesion 0, 6, and 12 d previously, making a total of 9 different donor-host combinations. The grafts were assessed histologically 3 or 8 wk after grafting. Axonal regeneration in the 9 different donor-host combinations was determined by counting the numbers of myelinated axons in transverse sections through the grafts. All grafts examined contained regenerating myelinated axons. The rats given a 3 wk postgrafting survival period had an average of between 1400 and 5300 such axons. The rats given an 8 wk postgrafting survival period had between about 13,000 and 25,000 regenerating myelinated axons. Analysis of variance revealed significant main effects for both the Donor and Host conditions as well as Weeks (i.e. survival period after grafting). These results indicate that both a conditioning lesion of the host neurons and the degree of predegeneration of peripheral nerve segments to be used as grafts are of importance in influencing the degree of axonal regeneration. Of these 2 factors the conditioning lesion of the host appears to have the greater effect on the

  17. Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury.

    PubMed

    Gordon, Tessa; Chan, K Ming; Sulaiman, Olawale A R; Udina, Esther; Amirjani, Nasim; Brushart, Thomas M

    2009-10-01

    Injured peripheral nerves regenerate at very slow rates. Therefore, proximal injury sites such as the brachial plexus still present major challenges, and the outcomes of conventional treatments remain poor. This is in part attributable to a progressive decline in the Schwann cells' ability to provide a supportive milieu for the growth cone to extend and to find the appropriate target. These challenges are compounded by the often considerable delay of regeneration across the site of nerve laceration. Recently, low-frequency electrical stimulation (as brief as an hour) has shown promise, as it significantly accelerated regeneration in animal models through speeding of axon growth across the injury site. To test whether this might be a useful clinical tool, we carried out a randomized controlled trial in patients who had experienced substantial axonal loss in the median nerve owing to severe compression in the carpal tunnel. To further elucidate the potential mechanisms, we applied rolipram, a cyclic adenosine monophosphate agonist, to rats after axotomy of the femoral nerve. We demonstrated that effects similar to those observed in animal studies could also be attained in humans. The mechanisms of action of electrical stimulation likely operate through up-regulation of neurotrophic factors and cyclic adenosine monophosphate. Indeed, the application of rolipram significantly accelerated nerve regeneration. With new mechanistic insights into the influencing factors of peripheral nerve regeneration, the novel treatments described above could form part of an armament of synergistic therapies that could make a meaningful difference to patients with peripheral nerve injuries.

  18. Axon Regeneration Can Facilitate or Suppress Hindlimb Function after Olfactory Ensheathing Glia Transplantation

    PubMed Central

    Takeoka, Aya; Jindrich, Devin L.; Muñoz-Quiles, Cintia; Zhong, Hui; van den Brand, Rubia; Pham, Daniel L.; Ziegler, Matthias D.; Ramón-Cueto, Almudena; Roy, Roland R.; Edgerton, V. Reggie

    2011-01-01

    Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function. PMID:21411671

  19. Chondroitin sulfates do not impede axonal regeneration in goldfish spinal cord.

    PubMed

    Takeda, Akihito; Okada, Soichiro; Funakoshi, Kengo

    2017-10-15

    Chondroitin sulfate proteoglycans produced in glial scar tissue are a major inhibitory factor for axonal regeneration after central nervous system injury in mammals. The inhibition is largely due to chondroitin sulfates, whose effects differ according to the sulfation pattern. In contrast to mammals, fish nerves spontaneously regenerate beyond the scar tissue after spinal cord injury, although the mechanisms that allow for axons to pass through the scar are unclear. Here, we used immunohistochemistry to examine the expression of two chondroitin sulfates with different sulfation variants at the lesion site in goldfish spinal cord. The intact spinal cord was immunoreactive for both chondroitin sulfate-A (CS-A) and chondroitin sulfate-C (CS-C), and CS-A immunoreactivity overlapped extensively with glial processes positive for glial fibrillary acidic protein. At 1week after inducing the spinal lesion, CS-A immunoreactivity was observed in the cell bodies and extracellular matrix, as well as in glial processes surrounding the lesion center. At 2weeks after the spinal lesion, regenerating axons entering the lesion center overtook the CS-A abundant area. In contrast, at 1week after lesion induction, CS-C immunoreactivity was significantly decreased, and at 2weeks after lesion induction, CS-C immunoreactivity was observed along the regenerating axons entering the lesion center. The present findings suggest that after spinal cord injury in goldfish, chondroitin sulfate proteoglycans are deposited in the extracellular matrix at the lesion site but do not form an impenetrable barrier to the growth of regenerating axons. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Wnt3 and Gata4 regulate axon regeneration in adult mouse DRG neurons.

    PubMed

    Duan, Run-Shan; Liu, Pei-Pei; Xi, Feng; Wang, Wei-Hua; Tang, Gang-Bin; Wang, Rui-Ying; Saijilafu; Liu, Chang-Mei

    2018-05-05

    Neurons in the adult central nervous system (CNS) have a poor intrinsic axon growth potential after injury, but the underlying mechanisms are largely unknown. Wingless-related mouse mammary tumor virus integration site (WNT) family members regulate neural stem cell proliferation, axon tract and forebrain development in the nervous system. Here we report that Wnt3 is an important modulator of axon regeneration. Downregulation or overexpression of Wnt3 in adult dorsal root ganglion (DRG) neurons enhances or inhibits their axon regeneration ability respectively in vitro and in vivo. Especially, we show that Wnt3 modulates axon regeneration by repressing mRNA translation of the important transcription factor Gata4 via binding to the three prime untranslated region (3'UTR). Downregulation of Gata4 could restore the phenotype exhibited by Wnt3 downregulation in DRG neurons. Taken together, these data indicate that Wnt3 is a key intrinsic regulator of axon growth ability of the nervous system. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Lost in the jungle: new hurdles for optic nerve axon regeneration.

    PubMed

    Pernet, Vincent; Schwab, Martin E

    2014-07-01

    The poor regenerative capacity of injured central nervous system (CNS) axons leads to permanent neurological deficits after brain, spinal cord, or optic nerve lesions. In the optic nerve, recent studies showed that stimulation of the cytokine or mammalian target of rapamycin (mTOR) signaling pathways potently enhances sprouting and regeneration of injured retinal ganglion cell axons in adult mice, but does not allow the majority of axons to reach their main cerebral targets. New analyses have revealed axon navigation defects in the optic nerve and at the optic chiasm under conditions of strong growth stimulation. We propose that a balanced growth stimulatory treatment will have to be combined with guidance factors and suppression of local growth inhibitory factors to obtain the full regeneration of long CNS axonal tracts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Misdirection of Regenerating Axons and Functional Recovery Following Sciatic Nerve Injury in Rats

    PubMed Central

    Hamilton, Shirley K.; Hinkle, Marcus L.; Nicolini, Jennifer; Rambo, Lindsay N.; Rexwinkle, April M.; Rose, Sam J.; Sabatier, Manning J.; Backus, Deborah; English, Arthur W.

    2013-01-01

    Poor functional recovery found after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. We applied brief electrical stimulation (ES) to the common fibular (CF) but not the tibial (Tib) nerve just prior to transection and repair of the entire rat sciatic nerve, to attempt to influence the misdirection of its regenerating axons. The specificity with which regenerating axons reinnervated appropriate targets was evaluated physiologically using compound muscle action potentials (M responses) evoked from stimulation of the two nerve branches above the injury site. Functional recovery was assayed using the timing of electromyography (EMG) activity recorded from the tibialis anterior (TA) and soleus (Sol) muscles during treadmill locomotion and kinematic analysis of hindlimb locomotor movements. Selective ES of the CF nerve resulted in restored M-responses at earlier times than in unstimulated controls in both TA and Sol muscles. Stimulated CF axons reinnervated inappropriate targets to a greater extent than unstimulated Tib axons. During locomotion, functional antagonist muscles, TA and Sol, were coactivated both in stimulated rats and in unstimulated but injured rats. Hindlimb kinematics in stimulated rats were comparable to untreated rats, but significantly different from intact controls. Selective ES promotes enhanced axon regeneration but does so with decreased fidelity of muscle reinnervation. Functional recovery is neither improved nor degraded, suggesting that compensatory changes in the outputs of the spinal circuits driving locomotion may occur irrespective of the extent of misdirection of regenerating axons in the periphery. PMID:21120925

  3. Regenerated Sciatic Nerve Axons Stimulated through a Chronically Implanted Macro-Sieve Electrode.

    PubMed

    MacEwan, Matthew R; Zellmer, Erik R; Wheeler, Jesse J; Burton, Harold; Moran, Daniel W

    2016-01-01

    Sieve electrodes provide a chronic interface for stimulating peripheral nerve axons. Yet, successful utilization requires robust axonal regeneration through the implanted electrode. The present study determined the effect of large transit zones in enhancing axonal regeneration and revealed an intimate neural interface with an implanted sieve electrode. Fabrication of the polyimide sieve electrodes employed sacrificial photolithography. The manufactured macro-sieve electrode (MSE) contained nine large transit zones with areas of ~0.285 mm 2 surrounded by eight Pt-Ir metallized electrode sites. Prior to implantation, saline, or glial derived neurotropic factor (GDNF) was injected into nerve guidance silicone-conduits with or without a MSE. The MSE assembly or a nerve guidance conduit was implanted between transected ends of the sciatic nerve in adult male Lewis rats. At 3 months post-operation, fiber counts were similar through both implant types. Likewise, stimulation of nerves regenerated through a MSE or an open silicone conduit evoked comparable muscle forces. These results showed that nerve regeneration was comparable through MSE transit zones and an open conduit. GDNF had a minimal positive effect on the quality and morphology of fibers regenerating through the MSE; thus, the MSE may reduce reliance on GDNF to augment axonal regeneration. Selective stimulation of several individual muscles was achieved through monopolar stimulation of individual electrodes sites suggesting that the MSE might be an optimal platform for functional neuromuscular stimulation.

  4. Regenerated Sciatic Nerve Axons Stimulated through a Chronically Implanted Macro-Sieve Electrode

    PubMed Central

    MacEwan, Matthew R.; Zellmer, Erik R.; Wheeler, Jesse J.; Burton, Harold; Moran, Daniel W.

    2016-01-01

    Sieve electrodes provide a chronic interface for stimulating peripheral nerve axons. Yet, successful utilization requires robust axonal regeneration through the implanted electrode. The present study determined the effect of large transit zones in enhancing axonal regeneration and revealed an intimate neural interface with an implanted sieve electrode. Fabrication of the polyimide sieve electrodes employed sacrificial photolithography. The manufactured macro-sieve electrode (MSE) contained nine large transit zones with areas of ~0.285 mm2 surrounded by eight Pt-Ir metallized electrode sites. Prior to implantation, saline, or glial derived neurotropic factor (GDNF) was injected into nerve guidance silicone-conduits with or without a MSE. The MSE assembly or a nerve guidance conduit was implanted between transected ends of the sciatic nerve in adult male Lewis rats. At 3 months post-operation, fiber counts were similar through both implant types. Likewise, stimulation of nerves regenerated through a MSE or an open silicone conduit evoked comparable muscle forces. These results showed that nerve regeneration was comparable through MSE transit zones and an open conduit. GDNF had a minimal positive effect on the quality and morphology of fibers regenerating through the MSE; thus, the MSE may reduce reliance on GDNF to augment axonal regeneration. Selective stimulation of several individual muscles was achieved through monopolar stimulation of individual electrodes sites suggesting that the MSE might be an optimal platform for functional neuromuscular stimulation. PMID:28008303

  5. EGFR Activation Mediates Inhibition of Axon Regeneration by Myelin and Chondroitin Sulfate Proteoglycans

    NASA Astrophysics Data System (ADS)

    Koprivica, Vuk; Cho, Kin-Sang; Park, Jong Bae; Yiu, Glenn; Atwal, Jasvinder; Gore, Bryan; Kim, Jieun A.; Lin, Estelle; Tessier-Lavigne, Marc; Chen, Dong Feng; He, Zhigang

    2005-10-01

    Inhibitory molecules associated with myelin and the glial scar limit axon regeneration in the adult central nervous system (CNS), but the underlying signaling mechanisms of regeneration inhibition are not fully understood. Here, we show that suppressing the kinase function of the epidermal growth factor receptor (EGFR) blocks the activities of both myelin inhibitors and chondroitin sulfate proteoglycans in inhibiting neurite outgrowth. In addition, regeneration inhibitors trigger the phosphorylation of EGFR in a calcium-dependent manner. Local administration of EGFR inhibitors promotes significant regeneration of injured optic nerve fibers, pointing to a promising therapeutic avenue for enhancing axon regeneration after CNS injury.

  6. Cross-talk between KLF4 and STAT3 regulates axon regeneration

    NASA Astrophysics Data System (ADS)

    Qin, Song; Zou, Yuhua; Zhang, Chun-Li

    2013-10-01

    Cytokine-induced activation of signal transducer and activator of transcription 3 (STAT3) promotes the regrowth of damaged axons in the adult central nervous system (CNS). Here we show that KLF4 physically interacts with STAT3 upon cytokine-induced phosphorylation of tyrosine 705 (Y705) on STAT3. This interaction suppresses STAT3-dependent gene expression by blocking its DNA-binding activity. The deletion of KLF4 in vivo induces axon regeneration of adult retinal ganglion cells (RGCs) via Janus kinase (JAK)-STAT3 signalling. This regeneration can be greatly enhanced by exogenous cytokine treatment, or removal of an endogenous JAK-STAT3 pathway inhibitor called suppressor of cytokine signalling 3 (SOCS3). These findings reveal an unexpected cross-talk between KLF4 and activated STAT3 in the regulation of axon regeneration that might have therapeutic implications in promoting repair of injured adult CNS.

  7. Akt1-Inhibitor of DNA binding2 is essential for growth cone formation and axon growth and promotes central nervous system axon regeneration

    PubMed Central

    Ko, Hyo Rim; Kwon, Il-Sun; Hwang, Inwoo; Jin, Eun-Ju; Shin, Joo-Ho; Brennan-Minnella, Angela M; Swanson, Raymond; Cho, Sung-Woo; Lee, Kyung-Hoon; Ahn, Jee-Yin

    2016-01-01

    Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration. DOI: http://dx.doi.org/10.7554/eLife.20799.001 PMID:27938661

  8. Gene Manipulation Strategies to Identify Molecular Regulators of Axon Regeneration in the Central Nervous System

    PubMed Central

    Ribas, Vinicius T.; Costa, Marcos R.

    2017-01-01

    Limited axon regeneration in the injured adult mammalian central nervous system (CNS) usually results in irreversible functional deficits. Both the presence of extrinsic inhibitory molecules at the injury site and the intrinsically low capacity of adult neurons to grow axons are responsible for the diminished capacity of regeneration in the adult CNS. Conversely, in the embryonic CNS, neurons show a high regenerative capacity, mostly due to the expression of genes that positively control axon growth and downregulation of genes that inhibit axon growth. A better understanding of the role of these key genes controlling pro-regenerative mechanisms is pivotal to develop strategies to promote robust axon regeneration following adult CNS injury. Genetic manipulation techniques have been widely used to investigate the role of specific genes or a combination of different genes in axon regrowth. This review summarizes a myriad of studies that used genetic manipulations to promote axon growth in the injured CNS. We also review the roles of some of these genes during CNS development and suggest possible approaches to identify new candidate genes. Finally, we critically address the main advantages and pitfalls of gene-manipulation techniques, and discuss new strategies to promote robust axon regeneration in the mature CNS. PMID:28824380

  9. Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse

    PubMed Central

    Hossain-Ibrahim, Mohammed K; Rezajooi, Kia; Stallcup, William B; Lieberman, Alexander R; Anderson, Patrick N

    2007-01-01

    Background The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve. Results We studied axonal regeneration in the PNS and CNS of genetically engineered mice that do not express NG2, and in sex and age matched wild-type controls. In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve. Injury to these fibre tracts resulted in no difference between knockout and wild-type mice in the ability of CST axons or DC axons to enter or cross the lesion site. Similarly, after dorsal root injury (with conditioning lesion), most regenerating dorsal root axons failed to grow across the dorsal root entry zone in both transgenic and wild-type mice. Following sciatic nerve injuries, functional recovery was assessed by analysis of the toe-spreading reflex and cutaneous sensitivity to Von Frey hairs. Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation. We also examined functional and anatomical correlates of regeneration after injury of the facial nerve by assessing the time taken for whisker movements and corneal reflexes to recover and by retrograde labelling of regenerated axons with Fluorogold and DiAsp. None of the anatomical or functional analyses revealed significant

  10. Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam

    PubMed Central

    Song, Yuanquan; Ori-McKenney, Kassandra M.; Zheng, Yi; Han, Chun; Jan, Lily Yeh; Jan, Yuh Nung

    2012-01-01

    Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. Here we report that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. Our study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN–Akt pathway that is also important for axon regeneration. We thus established an important new model system—the fly da neuron regeneration model that resembles the mammalian injury model—with which to study and gain novel insights into the regeneration machinery. PMID:22759636

  11. GSK3β regulates AKT-induced central nervous system axon regeneration via an eIF2Bε-dependent, mTORC1-independent pathway.

    PubMed

    Guo, Xinzheng; Snider, William D; Chen, Bo

    2016-03-14

    Axons fail to regenerate after central nervous system (CNS) injury. Modulation of the PTEN/mTORC1 pathway in retinal ganglion cells (RGCs) promotes axon regeneration after optic nerve injury. Here, we report that AKT activation, downstream of Pten deletion, promotes axon regeneration and RGC survival. We further demonstrate that GSK3β plays an indispensable role in mediating AKT-induced axon regeneration. Deletion or inactivation of GSK3β promotes axon regeneration independently of the mTORC1 pathway, whereas constitutive activation of GSK3β reduces AKT-induced axon regeneration. Importantly, we have identified eIF2Bε as a novel downstream effector of GSK3β in regulating axon regeneration. Inactivation of eIF2Bε reduces both GSK3β and AKT-mediated effects on axon regeneration. Constitutive activation of eIF2Bε is sufficient to promote axon regeneration. Our results reveal a key role of the AKT-GSK3β-eIF2Bε signaling module in regulating axon regeneration in the adult mammalian CNS.

  12. Upslope treadmill exercise enhances motor axon regeneration but not functional recovery following peripheral nerve injury

    PubMed Central

    Cannoy, Jill; Crowley, Sam; Jarratt, Allen; Werts, Kelly LeFevere; Osborne, Krista; Park, Sohee

    2016-01-01

    Following peripheral nerve injury, moderate daily exercise conducted on a level treadmill results in enhanced axon regeneration and modest improvements in functional recovery. If the exercise is conducted on an upwardly inclined treadmill, even more motor axons regenerate successfully and reinnervate muscle targets. Whether this increased motor axon regeneration also results in greater improvement in functional recovery from sciatic nerve injury was studied. Axon regeneration and muscle reinnervation were studied in Lewis rats over an 11 wk postinjury period using stimulus evoked electromyographic (EMG) responses in the soleus muscle of awake animals. Motor axon regeneration and muscle reinnervation were enhanced in slope-trained rats. Direct muscle (M) responses reappeared faster in slope-trained animals than in other groups and ultimately were larger than untreated animals. The amplitude of monosynaptic H reflexes recorded from slope-trained rats remained significantly smaller than all other groups of animals for the duration of the study. The restoration of the amplitude and pattern of locomotor EMG activity in soleus and tibialis anterior and of hindblimb kinematics was studied during treadmill walking on different slopes. Slope-trained rats did not recover the ability to modulate the intensity of locomotor EMG activity with slope. Patterned EMG activity in flexor and extensor muscles was not noted in slope-trained rats. Neither hindblimb length nor limb orientation during level, upslope, or downslope walking was restored in slope-trained rats. Slope training enhanced motor axon regeneration but did not improve functional recovery following sciatic nerve transection and repair. PMID:27466130

  13. Expression of an Activated Integrin Promotes Long-Distance Sensory Axon Regeneration in the Spinal Cord

    PubMed Central

    Cheah, Menghon; Chew, Daniel J.; Moloney, Elizabeth B.; Verhaagen, Joost; Fässler, Reinhard

    2016-01-01

    After CNS injury, axon regeneration is blocked by an inhibitory environment consisting of the highly upregulated tenascin-C and chondroitin sulfate proteoglycans (CSPGs). Tenascin-C promotes growth of axons if they express a tenascin-binding integrin, particularly α9β1. Additionally, integrins can be inactivated by CSPGs, and this inhibition can be overcome by the presence of a β1-binding integrin activator, kindlin-1. We examined the synergistic effect of α9 integrin and kindlin-1 on sensory axon regeneration in adult rat spinal cord after dorsal root crush and adeno-associated virus transgene expression in dorsal root ganglia. After 12 weeks, axons from C6–C7 dorsal root ganglia regenerated through the tenascin-C-rich dorsal root entry zone into the dorsal column up to C1 level and above (>25 mm axon length) through a normal pathway. Animals also showed anatomical and electrophysiological evidence of reconnection to the dorsal horn and behavioral recovery in mechanical pressure, thermal pain, and ladder-walking tasks. Expression of α9 integrin or kindlin-1 alone promoted much less regeneration and recovery. SIGNIFICANCE STATEMENT The study demonstrates that long-distance sensory axon regeneration over a normal pathway and with sensory and sensory–motor recovery can be achieved. This was achieved by expressing an integrin that recognizes tenascin-C, one of the components of glial scar tissue, and an integrin activator. This enabled extensive long-distance (>25 mm) regeneration of both myelinated and unmyelinated sensory axons with topographically correct connections in the spinal cord. The extent of growth and recovery we have seen would probably be clinically significant. Restoration of sensation to hands, perineum, and genitalia would be a significant improvement for a spinal cord-injured patient. PMID:27383601

  14. The effect of hyperbaric oxygen treatment on early regeneration of sensory axons after nerve crush in the rat.

    PubMed

    Bajrović, Fajko F; Sketelj, Janez; Jug, Marko; Gril, Iztok; Mekjavić, Igor B

    2002-09-01

    Abstract The effect of hyperbaric oxygen treatment (HBO) on sensory axon regeneration was examined in the rat. The sciatic nerve was crushed in both legs. In addition, the distal stump of the sural nerve on one side was made acellular and its blood perfusion was compromised by freezing and thawing. Two experimental groups received hyperbaric exposures (2.5 ATA) to either compressed air (pO2 = 0.5 ATA) or 100% oxygen (pO2 = 2.5 ATA) 90 minutes per day for 6 days. Sensory axon regeneration in the sural nerve was thereafter assessed by the nerve pinch test and immunohistochemical reaction to neurofilament. HBO treatment increased the distances reached by the fastest regenerating sensory axons by about 15% in the distal nerve segments with preserved and with compromised blood perfusion. There was no significant difference between the rats treated with different oxygen tensions. The total number of regenerated axons in the distal sural nerve segments after a simple crush injury was not affected, whereas in the nerve segments with compromised blood perfusion treated by the higher pO2, the axon number was about 30% lower than that in the control group. It is concluded that the beneficial effect of HBO on sensory axon regeneration is not dose-dependent between 0.5 and 2.5 ATA pO2. Although the exposure to 2.5 ATA of pO2 moderately enhanced early regeneration of the fastest sensory axons, it decreased the number of regenerating axons in the injured nerves with compromised blood perfusion of the distal nerve stump.

  15. Spider Silk Constructs Enhance Axonal Regeneration and Remyelination in Long Nerve Defects in Sheep

    PubMed Central

    Radtke, Christine; Allmeling, Christina; Waldmann, Karl-Heinz; Reimers, Kerstin; Thies, Kerstin; Schenk, Henning C.; Hillmer, Anja; Guggenheim, Merlin; Brandes, Gudrun; Vogt, Peter M.

    2011-01-01

    Background Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep. Methodology/Principal Findings The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation. Conclusions/Significance This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery. PMID:21364921

  16. PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1

    NASA Astrophysics Data System (ADS)

    Saijilafu; Hur, Eun-Mi; Liu, Chang-Mei; Jiao, Zhongxian; Xu, Wen-Lin; Zhou, Feng-Quan

    2013-10-01

    In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.

  17. Blockade of Nogo Receptor Ligands Promotes Functional Regeneration of Sensory Axons After Dorsal Root Crush

    PubMed Central

    Harvey, Pamela A.; Lee, Daniel H.S.; Qian, Fang; Weinreb, Paul H.; Frank, Eric

    2010-01-01

    A major impediment for regeneration of axons within the central nervous system is the presence of multiple inhibitory factors associated with myelin. Three of these factors bind to the Nogo receptor, NgR, which is expressed on axons. Administration of exogenous blockers of NgR or NgR ligands promotes the regeneration of descending axonal projections after spinal cord hemisection. A more detailed analysis of CNS regeneration can be made by examining the growth of specific classes of sensory axons into the spinal cord after dorsal root crush injury . In this study, we assessed whether administration of a soluble peptide fragment of the NgR that binds to and blocks all three NgR ligands can promote regeneration after brachial dorsal root crush in adult rats. Intraventricular infusion of sNgR for one month results in extensive regrowth of myelinated sensory axons into the white and gray matter of the dorsal spinal cord, but unmyelinated sensory afferents do not regenerate. In concert with the anatomical growth of sensory axons into the cord, there is a gradual restoration of synaptic function in the denervated region, as revealed by extracellular microelectrode recordings from the spinal gray matter in response to stimulation of peripheral nerves. These positive synaptic responses are correlated with substantial improvements in use of the forelimb, as assessed by paw preference, paw withdrawal to tactile stimuli and the ability to grasp. These results suggest that sNgR may be a potential therapy for restoring sensory function following injuries to sensory roots. PMID:19439606

  18. Regulated viral BDNF delivery in combination with Schwann cells promotes axonal regeneration through capillary alginate hydrogels after spinal cord injury.

    PubMed

    Liu, Shengwen; Sandner, Beatrice; Schackel, Thomas; Nicholson, LaShae; Chtarto, Abdelwahed; Tenenbaum, Liliane; Puttagunta, Radhika; Müller, Rainer; Weidner, Norbert; Blesch, Armin

    2017-09-15

    Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord. Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within

  19. In Vitro Analysis of the Role of Schwann Cells on Axonal Degeneration and Regeneration Using Sensory Neurons from Dorsal Root Ganglia.

    PubMed

    López-Leal, Rodrigo; Diaz, Paula; Court, Felipe A

    2018-01-01

    Sensory neurons from dorsal root ganglion efficiently regenerate after peripheral nerve injuries. These neurons are widely used as a model system to study degenerative mechanisms of the soma and axons, as well as regenerative axonal growth in the peripheral nervous system. This chapter describes techniques associated to the study of axonal degeneration and regeneration using explant cultures of dorsal root ganglion sensory neurons in vitro in the presence or absence of Schwann cells. Schwann cells are extremely important due to their involvement in tissue clearance during axonal degeneration as well as their known pro-regenerative effect during regeneration in the peripheral nervous system. We describe methods to induce and study axonal degeneration triggered by axotomy (mechanical separation of the axon from its soma) and treatment with vinblastine (which blocks axonal transport), which constitute clinically relevant mechanical and toxic models of axonal degeneration. In addition, we describe three different methods to evaluate axonal regeneration using quantitative methods. These protocols constitute a valuable tool to analyze in vitro mechanisms associated to axonal degeneration and regeneration of sensory neurons and the role of Schwann cells in these processes.

  20. The Core Molecular Machinery Used for Engulfment of Apoptotic Cells Regulates the JNK Pathway Mediating Axon Regeneration in Caenorhabditis elegans.

    PubMed

    Pastuhov, Strahil Iv; Fujiki, Kota; Tsuge, Anna; Asai, Kazuma; Ishikawa, Sho; Hirose, Kazuya; Matsumoto, Kunihiro; Hisamoto, Naoki

    2016-09-14

    The mechanisms that govern the ability of specific neurons to regenerate their axons after injury are not well understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we identify two components functioning upstream of the JNK pathway: the Ste20-related protein kinase MAX-2 and the Rac-type GTPase CED-10. CED-10, when bound by GTP, interacts with MAX-2 and functions as its upstream regulator in axon regeneration. CED-10, in turn, is activated by axon injury via signals initiated from the integrin α-subunit INA-1 and the nonreceptor tyrosine kinase SRC-1 and transmitted via the signaling module CED-2/CrkII-CED-5/Dock180-CED-12/ELMO. This module is also known to regulate the engulfment of apoptotic cells during development. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. The molecular mechanisms of axon regeneration after injury remain poorly understood. In Caenorhabditis elegans, the initiation of axon regeneration is positively regulated by the JNK-MAPK pathway. In this study, we show that integrin, Rac-GTPase, and several other molecules, all of which are known to regulate engulfment of apoptotic cells during development, also regulate axon regeneration. This signaling module activates the JNK-MAPK cascade via MAX-2, a PAK-like protein kinase that binds Rac. Our findings thus reveal that the molecular machinery used for engulfment of apoptotic cells also promotes axon regeneration through activation of the JNK pathway. Copyright © 2016 the authors 0270-6474/16/369710-12$15.00/0.

  1. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

    PubMed

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A

    2016-02-24

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

  2. Studies in the development of a bridging device for guiding regenerating axons

    NASA Astrophysics Data System (ADS)

    Wen, Xuejun

    At present there is no clinically effective treatment for injuries or pathological processes that disrupt the continuity of axons in the mature central nervous system. However, a number of studies suggest that a tremendous potential exists for developing therapies. In particular biomaterials in the form of bridging substrates been shown to support at least some level of axonal regeneration across the lesion site, but display a limited capacity for directing axons toward their targets. To influence the directionality of the regeneration process filaments and tubes appear promising but the technology is far from optimized. As a step toward optimization, we investigated various components of a tissue-engineered bridging device consisting of numerous filaments surrounded by a semipermeable biodegradable hollow fiber membrane (HFM). In the first part of the thesis, we studied the influence of filament diameter and various extracellular matrix coatings on neuron regeneration suing a dorsal root ganglion explant model. We found that laminin surface treated filaments that approached the size of spinal axons support significantly longer regenerative outgrowth than similarly treated filaments of larger diameter, and exceed outgrowth distance on similarly sized filaments treated with fibronectin. Such substrates also consistently supported the attachment and alignment of glial cells and directed the outgrowth of regenerating axons along the long axis of the filaments. In the last part of the thesis, biodegradable hollow fiber membranes were fabricated and their physical, chemical and degradation properties were analyzed. We found that it is possible to use phase inversion methods to fabricate hollow fiber membranes of widely varying properties that degrade of the course of several months. We then evaluated the biocompatibility of the new materials after implantation in the CNS using an adult rat model. We found that the implants were well tolerated and elicited a reaction

  3. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury

    PubMed Central

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A.

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  4. Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System Following Neural Injury (Addendum)

    DTIC Science & Technology

    2016-03-01

    AD_________________ Award Number: W81XWH-12-1-0051 TITLE: Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the...Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System Following Neural Injury 5a. CONTRACT NUMBER...incapable of axon regeneration . There are currently two principal concepts that form the basis of our understanding of the inability of the mature

  5. Axonal degeneration and regeneration in sensory roots in a genital herpes model.

    PubMed

    Soffer, D; Martin, J R

    1989-01-01

    In a mouse model of genital herpes simplex virus type 2 (HSV-2) infection, roots of the lower spinal cord were examined 5 days to 6 months after inoculation. Using immunoperoxidase methods on paraffin sections, viral antigen was found in sensory ganglia, their proximal roots and distal nerves on days 5 and 6 after infection. In Epon sections, most mice had focal sensory root abnormalities in lower thoracic, lumbar or sacral levels. At days 7 and 10, lesions showed chiefly nerve fiber degeneration, particularly of large myelinated fibers. At 2 weeks, lesions contained relatively large bundles of small unmyelinated fibers with immature axon-Schwann cell relationships. From 3 to 6 weeks, lesions again contained many more small unmyelinated fibers than normal but, in increasing proportions, axons in bundles were isolated from their neighbors by Schwann cell cytoplasm, and Schwann cells having 1:1 relationships with axons showed mesaxon or thin myelin sheath formation. At later times, the proportion of small unmyelinated axons decreased in parallel with increased numbers of small myelinated axons. By 6 months, affected roots showed a relative reduction in large myelinated fibers, increased proportions of small myelinated fibers and Schwann cell nuclei. Numbers of unmyelinated fibers were reduced relative to 3- to 6-week lesions. Axonal degeneration and regeneration appears to be the chief pathological change in sensory roots in this model. If regenerated fibers arise from latently infected neurons, then establishment of latency is not a relatively silent event, but is associated with major long-lasting, morphologically detectable effects.

  6. Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.

    PubMed

    Nakamura, Yuka; Fujita, Yuki; Ueno, Masaki; Takai, Toshiyuki; Yamashita, Toshihide

    2011-01-21

    Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.

  7. Improved axonal regeneration of transected spinal cord mediated by multichannel collagen conduits functionalized with neurotrophin-3 gene.

    PubMed

    Yao, L; Daly, W; Newland, B; Yao, S; Wang, W; Chen, B K K; Madigan, N; Windebank, A; Pandit, A

    2013-12-01

    Functionalized biomaterial scaffolds targeted at improving axonal regeneration by enhancing guided axonal growth provide a promising approach for the repair of spinal cord injury. Collagen neural conduits provide structural guidance for neural tissue regeneration, and in this study it is shown that these conduits can also act as a reservoir for sustained gene delivery. Either a G-luciferase marker gene or a neurotrophin-3-encoding gene, complexed to a non-viral, cyclized, PEGylated transfection vector, was loaded within a multichannel collagen conduit. The complexed genes were then released in a controlled fashion using a dual release system both in vitro and in vivo. For evaluation of their biological performance, the loaded conduits were implanted into the completely transected rat thoracic spinal cord (T8-T10). Aligned axon regeneration through the channels of conduits was observed one month post-surgery. The conduits delivering neurotrophin-3 polyplexes resulted in significantly increased neurotrophin-3 levels in the surrounding tissue and a statistically higher number of regenerated axons versus the control conduits (P<0.05). This study suggests that collagen neural conduits delivering a highly effective non-viral therapeutic gene may hold promise for repair of the injured spinal cord.

  8. Preferential Enhancement of Sensory and Motor Axon Regeneration by Combining Extracellular Matrix Components with Neurotrophic Factors

    PubMed Central

    Santos, Daniel; González-Pérez, Francisco; Giudetti, Guido; Micera, Silvestro; Udina, Esther; Del Valle, Jaume; Navarro, Xavier

    2016-01-01

    After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation. PMID:28036084

  9. Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

    PubMed Central

    Berg, Alexander; Zelano, Johan; Pekna, Marcela; Wilhelmsson, Ulrika; Pekny, Milos; Cullheim, Staffan

    2013-01-01

    Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP–/–Vim–/– mice. After sciatic nerve crush in GFAP–/–Vim–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics. PMID:24223940

  10. Optically-Induced Neuronal Activity Is Sufficient to Promote Functional Motor Axon Regeneration In Vivo.

    PubMed

    Ward, Patricia J; Jones, Laura N; Mulligan, Amanda; Goolsby, William; Wilhelm, Jennifer C; English, Arthur W

    2016-01-01

    Peripheral nerve injuries are common, and functional recovery is very poor. Beyond surgical repair of the nerve, there are currently no treatment options for these patients. In experimental models of nerve injury, interventions (such as exercise and electrical stimulation) that increase neuronal activity of the injured neurons effectively enhance axon regeneration. Here, we utilized optogenetics to determine whether increased activity alone is sufficient to promote motor axon regeneration. In thy-1-ChR2/YFP transgenic mice in which a subset of motoneurons express the light-sensitive cation channel, channelrhodopsin (ChR2), we activated axons in the sciatic nerve using blue light immediately prior to transection and surgical repair of the sciatic nerve. At four weeks post-injury, direct muscle EMG responses evoked with both optical and electrical stimuli as well as the ratio of these optical/electrical evoked EMG responses were significantly greater in mice that received optical treatment. Thus, significantly more ChR2+ axons successfully re-innervated the gastrocnemius muscle in mice that received optical treatment. Sections of the gastrocnemius muscles were reacted with antibodies to Synaptic Vesicle Protein 2 (SV2) to quantify the number of re-occupied motor endplates. The number of SV2+ endplates was greater in mice that received optical treatment. The number of retrogradely-labeled motoneurons following intramuscular injection of cholera toxin subunit B (conjugated to Alexa Fluor 555) was greater in mice that received optical treatment. Thus, the acute (1 hour), one-time optical treatment resulted in robust, long-lasting effects compared to untreated animals as well as untreated axons (ChR2-). We conclude that neuronal activation is sufficient to promote motor axon regeneration, and this regenerative effect is specific to the activated neurons.

  11. Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

    PubMed Central

    Gey, Manuel; Wanner, Renate; Schilling, Corinna; Pedro, Maria T.; Sinske, Daniela

    2016-01-01

    Axon injury in the peripheral nervous system (PNS) induces a regeneration-associated gene (RAG) response. Atf3 (activating transcription factor 3) is such a RAG and ATF3's transcriptional activity might induce ‘effector’ RAGs (e.g. small proline rich protein 1a (Sprr1a), Galanin (Gal), growth-associated protein 43 (Gap43)) facilitating peripheral axon regeneration. We provide a first analysis of Atf3 mouse mutants in peripheral nerve regeneration. In Atf3 mutant mice, facial nerve regeneration and neurite outgrowth of adult ATF3-deficient primary dorsal root ganglia neurons was decreased. Using genome-wide transcriptomics, we identified a neuropeptide-encoding RAG cluster (vasoactive intestinal peptide (Vip), Ngf, Grp, Gal, Pacap) regulated by ATF3. Exogenous administration of neuropeptides enhanced neurite growth of Atf3 mutant mice suggesting that these molecules might be effector RAGs of ATF3's pro-regenerative function. In addition to the induction of growth-promoting molecules, we present data that ATF3 suppresses growth-inhibiting molecules such as chemokine (C-C motif) ligand 2. In summary, we show a pro-regenerative ATF3 function during PNS nerve regeneration involving transcriptional activation of a neuropeptide-encoding RAG cluster. ATF3 is a general injury-inducible factor, therefore ATF3-mediated mechanisms identified herein might apply to other cell and injury types. PMID:27581653

  12. NT-3 promotes proprioceptive axon regeneration when combined with activation of the mTor intrinsic growth pathway but not with reduction of myelin extrinsic inhibitors

    PubMed Central

    Liu, Yingpeng; Kelamangalath, Lakshmi; Kim, Hyukmin; Han, Seung Baek; Tang, Xiaoqing; Zhai, Jinbin; Hong, Jee W; Lin, Shen; Son, Young-Jin; Smith, George M.

    2016-01-01

    Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons. PMID:27264357

  13. Examination of Axonal Injury and Regeneration in Microfluidic Neuronal Culture Using Pulsed Laser Microbeam Dissection

    PubMed Central

    Hellman, Amy N.; Vahidi, Behrad; Kim, Hyung Joon; Mismar, Wael; Steward, Oswald; Jeon, Noo Li; Venugopalan, Vasan

    2010-01-01

    We describe the integrated use of pulsed laser microbeams and microfluidic cell culture to examine the dynamics of axonal injury and regeneration in vitro. Microfabrication methods are used to place high purity dissociated central nervous system neurons in specific regions that allow the axons to interact with permissive and inhibitory substrates. Acute injury to neuron bundles is produced via the delivery of single 180 ps duration, λ=532 nm laser pulses. Laser pulse energies of 400 nJ and 800 nJ produce partial and complete transection of the axons, respectively, resulting in elliptical lesions 25 μm and 50 μm in size. The dynamics of the resulting degeneration and regrowth of proximal and distal axonal segments are examined for up to 8 h using time-lapse microscopy. We find the proximal and distal dieback distances from the site of laser microbeam irradiation to be roughly equal for both partial and complete transection of the axons. In addition, distinct growth cones emerge from the proximal neurite segments within 1–2 h post-injury, followed by a uniform front of regenerating axons that originate from the proximal segment and traverse the injury site within 8 h. We also examine the use of EGTA to chelate the extracellular calcium and potentially reduce the severity of the axonal degeneration following injury. While we find the addition of EGTA to reduce the severity of the initial dieback, it also hampers neurite repair and interfere with the formation of neuronal growth cones to traverse the injury site. This integrated use of laser microbeam dissection within a microfluidic cell culture system to produce precise zones of neuronal injury shows potential for high-throughput screening of agents to promote neuronal regeneration. PMID:20532390

  14. Regulating Set-β's Subcellular Localization Toggles Its Function between Inhibiting and Promoting Axon Growth and Regeneration

    PubMed Central

    Wang, Yan; Morkin, Melina I.; Fernandez, Stephanie G.; Mlacker, Gregory M.; Shechter, Jesse M.; Liu, Xiongfei; Patel, Karan H.; Lapins, Allison; Yang, Steven; Dombrowski, Susan M.

    2014-01-01

    The failure of the CNS neurons to regenerate axons after injury or stroke is a major clinical problem. Transcriptional regulators like Set-β are well positioned to regulate intrinsic axon regeneration capacity, which declines developmentally in maturing CNS neurons. Set-β also functions at cellular membranes and its subcellular localization is disrupted in Alzheimer's disease, but many of its biological mechanisms have not been explored in neurons. We found that Set-β was upregulated postnatally in CNS neurons, and was primarily localized to the nucleus but was also detected in the cytoplasm and adjacent to the plasma membrane. Remarkably, nuclear Set-β suppressed, whereas Set-β localized to cytoplasmic membranes promoted neurite growth in rodent retinal ganglion cells and hippocampal neurons. Mimicking serine 9 phosphorylation, as found in Alzheimer's disease brains, delayed nuclear import and furthermore blocked the ability of nuclear Set-β to suppress neurite growth. We also present data on gene regulation and protein binding partner recruitment by Set-β in primary neurons, raising the hypothesis that nuclear Set-β may preferentially regulate gene expression whereas Set-β at cytoplasmic membranes may regulate unique cofactors, including PP2A, which we show also regulates axon growth in vitro. Finally, increasing recruitment of Set-β to cellular membranes promoted adult rat optic nerve axon regeneration after injury in vivo. Thus, Set-β differentially regulates axon growth and regeneration depending on subcellular localization and phosphorylation. PMID:24849368

  15. Variable laterality of corticospinal tract axons that regenerate after spinal cord injury as a result of PTEN deletion or knock-down

    PubMed Central

    Willenberg, Rafer; Zukor, Katherine; Liu, Kai; He, Zhigang; Steward, Oswald

    2016-01-01

    Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting PTEN in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the “wrong” side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical AAV-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral BDA injections in PTEN-deleted mice with spinal cord injury and in non-injured PTEN-floxed mice that had not received AAV-Cre. In non-injured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in grey matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side, in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using shRNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the non-injured CST, highlighting one aspect for how resultant circuitry from regenerating axons may differ from that of the uninjured CST. PMID:26878190

  16. Combining Constitutively Active Rheb Expression and Chondroitinase Promotes Functional Axonal Regeneration after Cervical Spinal Cord Injury.

    PubMed

    Wu, Di; Klaw, Michelle C; Connors, Theresa; Kholodilov, Nikolai; Burke, Robert E; Côté, Marie-Pascale; Tom, Veronica J

    2017-12-06

    After spinal cord injury (SCI), severed axons in the adult mammalian CNS are unable to mount a robust regenerative response. In addition, the glial scar at the lesion site further restricts the regenerative potential of axons. We hypothesized that a combinatorial approach coincidentally targeting these obstacles would promote axonal regeneration. We combined (1) transplantation of a growth-permissive peripheral nerve graft (PNG) into an incomplete, cervical lesion cavity; (2) transduction of neurons rostral to the SCI site to express constitutively active Rheb (caRheb; a Ras homolog enriched in brain), a GTPase that directly activates the growth-promoting pathway mammalian target of rapamycin (mTOR) via AAV-caRheb injection; and (3) digestion of growth-inhibitory chondroitin sulfate proteoglycans within the glial scar at the distal PNG interface using the bacterial enzyme chondroitinase ABC (ChABC). We found that expressing caRheb in neurons post-SCI results in modestly yet significantly more axons regenerating across a ChABC-treated distal graft interface into caudal spinal cord than either treatment alone. Excitingly, we found that caRheb+ChABC treatment significantly potentiates the formation of synapses in the host spinal cord and improves the animals' ability to use the affected forelimb. Thus, this combination strategy enhances functional axonal regeneration following a cervical SCI. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  17. Co-culture of oligodendrocytes and neurons can be used to assess drugs for axon regeneration in the central nervous system

    PubMed Central

    Gang, Lin; Yao, Yu-chen; Liu, Ying-fu; Li, Yi-peng; Yang, Kai; Lu, Lei; Cheng, Yuan-chi; Chen, Xu-yi; Tu, Yue

    2015-01-01

    We present a novel in vitro model in which to investigate the efficacy of experimental drugs for the promotion of axon regeneration in the central nervous system. We co-cultured rat hippocampal neurons and cerebral cortical oligodendrocytes, and tested the co-culture system using a Nogo-66 receptor antagonist peptide (NEP1–40), which promotes axonal growth. Primary cultured oligodendrocytes suppressed axonal growth in the rat hippocampus, but NEP1–40 stimulated axonal growth in the co-culture system. Our results confirm the validity of the neuron-oligodendrocyte co-culture system as an assay for the evaluation of drugs for axon regeneration in the central nervous system. PMID:26692858

  18. mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system.

    PubMed

    Miao, Linqing; Yang, Liu; Huang, Haoliang; Liang, Feisi; Ling, Chen; Hu, Yang

    2016-03-30

    Injured mature CNS axons do not regenerate in mammals. Deletion of PTEN, the negative regulator of PI3K, induces CNS axon regeneration through the activation of PI3K-mTOR signaling. We have conducted an extensive molecular dissection of the cross-regulating mechanisms in axon regeneration that involve the downstream effectors of PI3K, AKT and the two mTOR complexes (mTORC1 and mTORC2). We found that the predominant AKT isoform in CNS, AKT3, induces much more robust axon regeneration than AKT1 and that activation of mTORC1 and inhibition of GSK3β are two critical parallel pathways for AKT-induced axon regeneration. Surprisingly, phosphorylation of T308 and S473 of AKT play opposite roles in GSK3β phosphorylation and inhibition, by which mTORC2 and pAKT-S473 negatively regulate axon regeneration. Thus, our study revealed a complex neuron-intrinsic balancing mechanism involving AKT as the nodal point of PI3K, mTORC1/2 and GSK3β that coordinates both positive and negative cues to regulate adult CNS axon regeneration.

  19. Differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.

    PubMed

    Tong, Ling-Ling; Ding, You-Quan; Jing, Hong-Bo; Li, Xuan-Yang; Qi, Jian-Guo

    2015-05-06

    Peripheral nerve functional recovery after injuries relies on both axon regeneration and remyelination. Both axon regeneration and remyelination require intimate interactions between regenerating neurons and their accompanying Schwann cells. Previous studies have shown that motor and sensory neurons are intrinsically different in their regeneration potentials. Moreover, denervated Schwann cells accompanying myelinated motor and sensory axons have distinct gene expression profiles for regeneration-associated growth factors. However, it is unknown whether differential motor and sensory functional recovery exists. If so, the particular one among axon regeneration and remyelination responsible for this difference remains unclear. Here, we aimed to establish an adult rat sciatic nerve crush model with the nonserrated microneedle holders and measured rat motor and sensory functions during regeneration. Furthermore, axon regeneration and remyelination was evaluated by morphometric analysis of electron microscopic images on the basis of nerve fiber classification. Our results showed that Aα fiber-mediated motor function was successfully recovered in both male and female rats. Aδ fiber-mediated sensory function was partially restored in male rats, but completely recovered in female littermates. For both male and female rats, the numbers of regenerated motor and sensory axons were quite comparable. However, remyelination was diverse among myelinated motor and sensory nerve fibers. In detail, Aβ and Aδ fibers incompletely remyelinated in male, but not female rats, whereas Aα fibers fully remyelinated in both sexes. Our result indicated that differential motor and sensory functional recovery in male but not female adult rats is associated with remyelination rather than axon regeneration after sciatic nerve crush.

  20. Extrinsic and intrinsic regulation of axon regeneration at a crossroads

    PubMed Central

    Kaplan, Andrew; Ong Tone, Stephan; Fournier, Alyson E.

    2015-01-01

    Repair of the injured spinal cord is a major challenge in medicine. The limited intrinsic regenerative response mounted by adult central nervous system (CNS) neurons is further hampered by astrogliosis, myelin debris and scar tissue that characterize the damaged CNS. Improved axon regeneration and recovery can be elicited by targeting extrinsic factors as well as by boosting neuron-intrinsic growth regulators. Our knowledge of the molecular basis of intrinsic and extrinsic regulators of regeneration has expanded rapidly, resulting in promising new targets to promote repair. Intriguingly certain neuron-intrinsic growth regulators are emerging as promising targets to both stimulate growth and relieve extrinsic inhibition of regeneration. This crossroads between the intrinsic and extrinsic aspects of spinal cord injury is a promising target for effective therapies for this unmet need. PMID:26136657

  1. Extrinsic and intrinsic regulation of axon regeneration at a crossroads.

    PubMed

    Kaplan, Andrew; Ong Tone, Stephan; Fournier, Alyson E

    2015-01-01

    Repair of the injured spinal cord is a major challenge in medicine. The limited intrinsic regenerative response mounted by adult central nervous system (CNS) neurons is further hampered by astrogliosis, myelin debris and scar tissue that characterize the damaged CNS. Improved axon regeneration and recovery can be elicited by targeting extrinsic factors as well as by boosting neuron-intrinsic growth regulators. Our knowledge of the molecular basis of intrinsic and extrinsic regulators of regeneration has expanded rapidly, resulting in promising new targets to promote repair. Intriguingly certain neuron-intrinsic growth regulators are emerging as promising targets to both stimulate growth and relieve extrinsic inhibition of regeneration. This crossroads between the intrinsic and extrinsic aspects of spinal cord injury is a promising target for effective therapies for this unmet need.

  2. Human Periodontal Ligament-Derived Stem Cells Promote Retinal Ganglion Cell Survival and Axon Regeneration After Optic Nerve Injury.

    PubMed

    Cen, Ling-Ping; Ng, Tsz Kin; Liang, Jia-Jian; Zhuang, Xi; Yao, Xiaowu; Yam, Gary Hin-Fai; Chen, Haoyu; Cheung, Herman S; Zhang, Mingzhi; Pang, Chi Pui

    2018-06-01

    Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855. © AlphaMed Press 2018.

  3. Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site

    PubMed Central

    Tom, Veronica J.; Sandrow-Feinberg, Harra R.; Miller, Kassi; Domitrovich, Cheryl; Bouyer, Julien; Zhukareva, Victoria; Klaw, Michelle C.; Lemay, Michel A.; Houlé, John D.

    2016-01-01

    Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue. PMID:23022460

  4. Attenuation of TRPV1 by AMG-517 after nerve injury promotes peripheral axonal regeneration in rats.

    PubMed

    Bai, Juan; Liu, Fu; Wu, Li-Fei; Wang, Ya-Fang; Li, Xia-Qing

    2018-01-01

    Aims The main objective was to investigate the effects of the transient receptor potential cation channel subfamily V member 1 (TRPV1) on nerve regeneration following sciatic transection injury by functional blockage of TRPV1 using AMG-517, a specific blocker of TRPV1. Methods AMG-517 was injected into the area surrounding ipsilateral lumbar dorsal root ganglia 30 min after unilateral sciatic nerve transection. The number of sciatic axons and the expression of growth-associated protein-43 (GAP-43) and glial fibrillary acidic protein was examined using semithin sections, Western blot, and immunofluorescence analyses. Results Blockage of TRPV1 with AMG-517 markedly promoted axonal regeneration, especially at two weeks after sciatic injury; the number of axons was similar to the uninjured control group. After sciatic nerve transection, expression of glial fibrillary acidic protein was decreased and GAP-43 was increased at the proximal stump. However, the expression of both glial fibrillary acidic protein and GAP-43 increased significantly in AMG-517-treated groups. Conclusions TRPV1 may be an important therapeutic target to promote peripheral nerve regeneration after injury.

  5. Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

    PubMed

    Dash, P K; Tian, L M; Moore, A N

    1998-07-07

    Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

  6. Force recovery and axonal regeneration of the sternomastoid muscle reinnervated with the end-to-end nerve anastomosis

    PubMed Central

    Sobotka, Stanislaw; Mu, Liancai

    2012-01-01

    Background End-to-end nerve anastomosis (EEA) is a commonly used nerve repair technique. However, this method generally results in poor functional recovery. This study was designed to determine the correlation of functional recovery to the extent of axonal reinnervation after EEA procedure in a rat model. Materials and Methods Seven adult rats were subjected to the immediate reinnervation of an experimentally paralyzed sternomastoid (SM) muscle. The SM nerve was transected and immediately repaired with EEA. The SM muscle at the opposite side, without nerve transection, served as a control. Three months after EEA nerve repair, the muscle force of the SM muscle was measured and the regenerated axons in the muscle were detected using neurofilament immunohistochemistry. Results Three months after surgery, the reinnervated SM muscle produced limited anatomical and functional recovery (calculated as the percentage of the control). Specifically, the wet weight of the operated SM muscle (a measure of muscle mass recovery) was 78.0% of the control. The maximal tetanic force (a measure of muscle functional recovery) was 56.7% of the control. The area fraction of the neurofilament stained intramuscular axons (a measure of axonal regeneration and muscle reinnervation) was measured to be only 13.4% of the control. A positive correlation was revealed between the extent of muscle reinnervation and maximal muscle force. Conclusions The EEA reinnervated SM muscle in the rat yielded unsatisfactory muscle force recovery as a result of mild to moderate nerve regeneration. Further work is needed to improve the surgical procedure, enhance axonal regeneration, and/or develop novel treatment strategies for better functional recovery. PMID:23207170

  7. Force recovery and axonal regeneration of the sternomastoid muscle reinnervated with the end-to-end nerve anastomosis.

    PubMed

    Sobotka, Stanislaw; Mu, Liancai

    2013-06-15

    End-to-end nerve anastomosis (EEA) is a commonly used nerve repair technique. However, this method generally results in poor functional recovery. This study was designed to determine the correlation of functional recovery to the extent of axonal reinnervation after EEA procedure in a rat model. Seven adult rats were subjected to the immediate reinnervation of an experimentally paralyzed sternomastoid (SM) muscle. The SM nerve was transected and immediately repaired with EEA. The SM muscle at the opposite side, without nerve transection, served as a control. Three months after EEA nerve repair, the muscle force of the SM muscle was measured and the regenerated axons in the muscle were detected using neurofilament immunohistochemistry. Three months after surgery, the reinnervated SM muscle produced limited anatomical and functional recovery (calculated as the percentage of the control). Specifically, the wet weight of the operated SM muscle (a measure of muscle mass recovery) was 78.0% of the control. The maximal tetanic force (a measure of muscle functional recovery) was 56.7% of the control. The area fraction of the neurofilament stained intramuscular axons (a measure of axonal regeneration and muscle reinnervation) was measured to be only 13.4% of the control. A positive correlation was revealed between the extent of muscle reinnervation and maximal muscle force. The EEA reinnervated SM muscle in the rat yielded unsatisfactory muscle force recovery as a result of mild to moderate nerve regeneration. Further work is needed to improve the surgical procedure, enhance axonal regeneration, and/or develop novel treatment strategies for better functional recovery. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans.

    PubMed

    Knowlton, Wendy M; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D; Jin, Yishi

    2017-01-01

    The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo , we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1 . Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7 , and isp-1 , and the putative oxidoreductase rad-8 . In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1 . Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the

  9. A Select Subset of Electron Transport Chain Genes Associated with Optic Atrophy Link Mitochondria to Axon Regeneration in Caenorhabditis elegans

    PubMed Central

    Knowlton, Wendy M.; Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.; Jin, Yishi

    2017-01-01

    The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular

  10. Expressing Constitutively Active Rheb in Adult Neurons after a Complete Spinal Cord Injury Enhances Axonal Regeneration beyond a Chondroitinase-Treated Glial Scar

    PubMed Central

    Wu, Di; Klaw, Michelle C.; Connors, Theresa; Kholodilov, Nikolai; Burke, Robert E.

    2015-01-01

    After a spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to: (1) the presence of inhibitory molecules, e.g., chondroitin sulfate proteoglycans (CSPG), in the glial scar at the lesion; and (2) the diminished growth capacity of adult neurons. We sought to determine whether expressing a constitutively active form of the GTPase Rheb (caRheb) in adult neurons after a complete SCI in rats improves intrinsic growth potential to result in axon regeneration out of a growth-supportive peripheral nerve grafted (PNG) into the SCI cavity. We also hypothesized that treating the glial scar with chondroitinase ABC (ChABC), which digests CSPG, would further allow caRheb-transduced neurons to extend axons across the distal graft interface. We found that targeting this pathway at a clinically relevant post-SCI time point improves both sprouting and regeneration of axons. CaRheb increased the number of axons, but not the number of neurons, that projected into the PNG, indicative of augmented sprouting. We also saw that caRheb enhanced sprouting far rostral to the injury. CaRheb not only increased growth rostral and into the graft, it also resulted in significantly more regrowth of axons across a ChABC-treated scar into caudal spinal cord. CaRheb+ neurons had higher levels of growth-associated-43, suggestive of a newly identified mechanism for mTOR-mediated enhancement of regeneration. Thus, we demonstrate for the first time that simultaneously addressing intrinsic and scar-associated, extrinsic impediments to regeneration results in significant regrowth beyond an extremely challenging, complete SCI site. SIGNIFICANCE STATEMENT After spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to the diminished growth capacity of adult neurons and the presence of inhibitory molecules in the scar at the lesion. We sought to simultaneously counter both of these obstacles to achieve more robust

  11. Expressing Constitutively Active Rheb in Adult Neurons after a Complete Spinal Cord Injury Enhances Axonal Regeneration beyond a Chondroitinase-Treated Glial Scar.

    PubMed

    Wu, Di; Klaw, Michelle C; Connors, Theresa; Kholodilov, Nikolai; Burke, Robert E; Tom, Veronica J

    2015-08-05

    After a spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to: (1) the presence of inhibitory molecules, e.g., chondroitin sulfate proteoglycans (CSPG), in the glial scar at the lesion; and (2) the diminished growth capacity of adult neurons. We sought to determine whether expressing a constitutively active form of the GTPase Rheb (caRheb) in adult neurons after a complete SCI in rats improves intrinsic growth potential to result in axon regeneration out of a growth-supportive peripheral nerve grafted (PNG) into the SCI cavity. We also hypothesized that treating the glial scar with chondroitinase ABC (ChABC), which digests CSPG, would further allow caRheb-transduced neurons to extend axons across the distal graft interface. We found that targeting this pathway at a clinically relevant post-SCI time point improves both sprouting and regeneration of axons. CaRheb increased the number of axons, but not the number of neurons, that projected into the PNG, indicative of augmented sprouting. We also saw that caRheb enhanced sprouting far rostral to the injury. CaRheb not only increased growth rostral and into the graft, it also resulted in significantly more regrowth of axons across a ChABC-treated scar into caudal spinal cord. CaRheb(+) neurons had higher levels of growth-associated-43, suggestive of a newly identified mechanism for mTOR-mediated enhancement of regeneration. Thus, we demonstrate for the first time that simultaneously addressing intrinsic and scar-associated, extrinsic impediments to regeneration results in significant regrowth beyond an extremely challenging, complete SCI site. After spinal cord injury (SCI), CNS axons fail to regenerate, resulting in permanent deficits. This is due to the diminished growth capacity of adult neurons and the presence of inhibitory molecules in the scar at the lesion. We sought to simultaneously counter both of these obstacles to achieve more robust regeneration after

  12. Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

    PubMed Central

    Koch, Jan Christoph; Tönges, Lars; Michel, Uwe; Bähr, Mathias; Lingor, Paul

    2014-01-01

    The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush. PMID:25249936

  13. Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

    PubMed

    Iskandar, Bermans J; Rizk, Elias; Meier, Brenton; Hariharan, Nithya; Bottiglieri, Teodoro; Finnell, Richard H; Jarrard, David F; Banerjee, Ruma V; Skene, J H Pate; Nelson, Aaron; Patel, Nirav; Gherasim, Carmen; Simon, Kathleen; Cook, Thomas D; Hogan, Kirk J

    2010-05-01

    The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.

  14. Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation

    PubMed Central

    Iskandar, Bermans J.; Rizk, Elias; Meier, Brenton; Hariharan, Nithya; Bottiglieri, Teodoro; Finnell, Richard H.; Jarrard, David F.; Banerjee, Ruma V.; Skene, J.H. Pate; Nelson, Aaron; Patel, Nirav; Gherasim, Carmen; Simon, Kathleen; Cook, Thomas D.; Hogan, Kirk J.

    2010-01-01

    The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries. PMID:20424322

  15. Accelerated remyelination during inflammatory demyelination prevents axonal loss and improves functional recovery.

    PubMed

    Mei, Feng; Lehmann-Horn, Klaus; Shen, Yun-An A; Rankin, Kelsey A; Stebbins, Karin J; Lorrain, Daniel S; Pekarek, Kara; A Sagan, Sharon; Xiao, Lan; Teuscher, Cory; von Büdingen, H-Christian; Wess, Jürgen; Lawrence, J Josh; Green, Ari J; Fancy, Stephen Pj; Zamvil, Scott S; Chan, Jonah R

    2016-09-27

    Demyelination in MS disrupts nerve signals and contributes to axon degeneration. While remyelination promises to restore lost function, it remains unclear whether remyelination will prevent axonal loss. Inflammatory demyelination is accompanied by significant neuronal loss in the experimental autoimmune encephalomyelitis (EAE) mouse model and evidence for remyelination in this model is complicated by ongoing inflammation, degeneration and possible remyelination. Demonstrating the functional significance of remyelination necessitates selectively altering the timing of remyelination relative to inflammation and degeneration. We demonstrate accelerated remyelination after EAE induction by direct lineage analysis and hypothesize that newly formed myelin remains stable at the height of inflammation due in part to the absence of MOG expression in immature myelin. Oligodendroglial-specific genetic ablation of the M1 muscarinic receptor, a potent negative regulator of oligodendrocyte differentiation and myelination, results in accelerated remyelination, preventing axonal loss and improving functional recovery. Together our findings demonstrate that accelerated remyelination supports axonal integrity and neuronal function after inflammatory demyelination.

  16. Strategies to promote peripheral nerve regeneration: electrical stimulation and/or exercise

    PubMed Central

    Gordon, Tessa; English, Arthur W.

    2015-01-01

    Enhancing the regeneration of axons is often considered a therapeutic target for improving functional recovery after peripheral nerve injury. In this review, the evidence for the efficacy of electrical stimulation (ES), daily exercise, and their combination in promoting nerve regeneration after peripheral nerve injuries in both animal models and in human patients, is explored. The rationale, effectiveness, and molecular basis of ES and exercise in accelerating axon outgrowth are reviewed. In comparing the effects of ES and exercise in enhancing axon regeneration, increased neural activity, neurotrophins, and androgens are considered common requirements. Similar, gender-specific requirements are found for exercise to enhance axon regeneration in the periphery and for sustaining synaptic inputs onto injured motoneurons. ES promotes nerve regeneration after delayed nerve repair in humans and rats. The effectiveness of exercise is less clear. Although ES, but not exercise, results in a significant misdirection of regenerating motor axons to reinnervate different muscle targets, the loss of neuromuscular specificity encountered has only a very small impact on resulting functional recovery. Both ES and exercise are promising experimental treatments for peripheral nerve injury that seem ready to be translated to clinical use. PMID:26121368

  17. Comprehensive Corticospinal Labeling with mu-crystallin Transgene Reveals Axon Regeneration after Spinal Cord Trauma in ngr1−/− Mice

    PubMed Central

    Fink, Kathren L.

    2015-01-01

    Spinal cord injury interrupts descending motor tracts and creates persistent functional deficits due to the absence of spontaneous axon regeneration. Of descending pathways, the corticospinal tract (CST) is thought to be the most critical for voluntary function in primates. Even with multiple tracer injections and genetic tools, the CST is visualized to only a minor degree in experimental studies. Here, we identify and validate the mu-crystallin (crym) gene as a high-fidelity marker of the CST. In transgenic mice expressing green fluorescent protein (GFP) under crym regulatory elements (crym-GFP), comprehensive and near complete CST labeling is achieved throughout the spinal cord. Bilateral pyramidotomy eliminated the 17,000 GFP-positive CST axons that were reproducibly labeled in brainstem from the spinal cord. We show that CST tracing with crym-GFP is 10-fold more efficient than tracing with biotinylated dextran amine (BDA). Using crym-GFP, we reevaluated the CST in mice lacking nogo receptor 1 (NgR1), a protein implicated in limiting neural repair. The number and trajectory of CST axons in ngr1−/− mice without injury was indistinguishable from ngr1+/+ mice. After dorsal hemisection in the midthoracic cord, CST axons did not significantly regenerate in ngr1+/+ mice, but an average of 162 of the 6000 labeled thoracic CST axons (2.68%) regenerated >100 μm past the lesion site in crym-GFP ngr1−/− mice. Although traditional BDA tracing cannot reliably visualize regenerating ngr1−/− CST axons, their regenerative course is clear with crym-GFP. Therefore the crym-GFP transgenic mouse is a useful tool for studies of CST anatomy in experimental studies of motor pathways. SIGNIFICANCE STATEMENT Axon regeneration fails in the adult CNS, resulting in permanent functional deficits. Traditionally, inefficient extrinsic tracers such a biotinylated dextran amine (BDA) are used to label regenerating fibers after therapeutic intervention. We introduce crym

  18. Cell-type specific expression of constitutively-active Rheb promotes regeneration of bulbospinal respiratory axons following cervical SCI.

    PubMed

    Urban, Mark W; Ghosh, Biswarup; Strojny, Laura R; Block, Cole G; Blazejewski, Sara M; Wright, Megan C; Smith, George M; Lepore, Angelo C

    2018-05-01

    Damage to respiratory neural circuitry and consequent loss of diaphragm function is a major cause of morbidity and mortality in individuals suffering from traumatic cervical spinal cord injury (SCI). Repair of CNS axons after SCI remains a therapeutic challenge, despite current efforts. SCI disrupts inspiratory signals originating in the rostral ventral respiratory group (rVRG) of the medulla from their phrenic motor neuron (PhMN) targets, resulting in loss of diaphragm function. Using a rat model of cervical hemisection SCI, we aimed to restore rVRG-PhMN-diaphragm circuitry by stimulating regeneration of injured rVRG axons via targeted induction of Rheb (ras homolog enriched in brain), a signaling molecule that regulates neuronal-intrinsic axon growth potential. Following C2 hemisection, we performed intra-rVRG injection of an adeno-associated virus serotype-2 (AAV2) vector that drives expression of a constitutively-active form of Rheb (cRheb). rVRG neuron-specific cRheb expression robustly increased mTOR pathway activity within the transduced rVRG neuron population ipsilateral to the hemisection, as assessed by levels of phosphorylated ribosomal S6 kinase. By co-injecting our novel AAV2-mCherry/WGA anterograde/trans-synaptic axonal tracer into rVRG, we found that cRheb expression promoted regeneration of injured rVRG axons into the lesion site, while we observed no rVRG axon regrowth with AAV2-GFP control. AAV2-cRheb also significantly reduced rVRG axonal dieback within the intact spinal cord rostral to the lesion. However, cRheb expression did not promote any recovery of ipsilateral hemi-diaphragm function, as assessed by inspiratory electromyography (EMG) burst amplitudes. This lack of functional recovery was likely because regrowing rVRG fibers did not extend back into the caudal spinal cord to synaptically reinnervate PhMNs that we retrogradely-labeled with cholera toxin B from the ipsilateral hemi-diaphragm. Our findings demonstrate that enhancing neuronal

  19. Unraveling the mechanisms of synapse formation and axon regeneration: the awesome power of C. elegans genetics.

    PubMed

    Jin, YiShi

    2015-11-01

    Since Caenorhabditis elegans was chosen as a model organism by Sydney Brenner in 1960's, genetic studies in this organism have been instrumental in discovering the function of genes and in deciphering molecular signaling network. The small size of the organism and the simple nervous system enable the complete reconstruction of the first connectome. The stereotypic developmental program and the anatomical reproducibility of synaptic connections provide a blueprint to dissect the mechanisms underlying synapse formation. Recent technological innovation using laser surgery of single axons and in vivo imaging has also made C. elegans a new model for axon regeneration. Importantly, genes regulating synaptogenesis and axon regeneration are highly conserved in function across animal phyla. This mini-review will summarize the main approaches and the key findings in understanding the mechanisms underlying the development and maintenance of the nervous system. The impact of such findings underscores the awesome power of C. elegans genetics.

  20. Regeneration of neurosecretory axons into various types of intrahypothalamic graft is promoted by the absence of the blood-brain barrier: a neurophysin-immunohistochemical and horseradish peroxidase-histochemical study.

    PubMed

    Ouassat, M; Dellmann, H D

    1997-01-15

    In order to test the hypothesis that neurosecretory axon regeneration occurs only in the presence of specific vascular, perivascular, and glial microenvironments, isografts of neural lobe and optic nerve and autografts of sciatic nerve were transplanted into the hypothalamo-neurohypophysial tract at the lateral retrochiasmatic area of adult male rats. The integrity of the blood-brain barrier (BBB) to intravenously administered horseradish peroxidase (HRP), the regenerative process of neurosecretory axons, and functional recovery from lesion-induced diabetes insipidus were analyzed at 18 hr, 36 hr, 10 days, 30 days, and 80 days postsurgery. Neurophysin-positive axons invaded all grafts, as well as perivascular spaces of the adjacent hypothalamus. Wherever neurosecretory axon regeneration occurred, the BBB was breached. Reestablishment of the BBB was paralleled by a decrease in both density and staining intensity of regenerated neurophysin-positive axons. These observations illustrate that neurosecretory axon regeneration is tributary of the absence of BBB. It is speculated that blood-borne factors, provided when the BBB is breached, initiate and sustain neurosecretory axon regeneration. In addition, products of glial elements may enhance or complement the above stimulatory processes.

  1. ARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons.

    PubMed

    Eva, Richard; Crisp, Sarah; Marland, Jamie R K; Norman, Jim C; Kanamarlapudi, Venkateswarlu; ffrench-Constant, Charles; Fawcett, James W

    2012-07-25

    Integrins are involved in axon growth and regeneration. Manipulation of integrins is a route to promoting axon regeneration and understanding regeneration failure in the CNS. Expression of α9 integrin promotes axon regeneration, so we have investigated α9β1 trafficking and transport in axons and at the growth cone. We have previously found that α9 and β1 integrins traffic via Rab11-positive recycling endosomes in peripheral axons and growth cones. However, transport via Rab11 is slow, while rapid transport occurs in vesicles lacking Rab11. We have further studied α9 and β1 integrin transport and traffic in adult rat dorsal root ganglion axons and PC12 cells. Integrins are in ARF6 vesicles during rapid axonal transport and during trafficking in the growth cone. We report that rapid axonal transport of these integrins and their trafficking at the cell surface is regulated by ARF6. ARF6 inactivation by expression of ACAP1 leads to increased recycling of β1 integrins to the neuronal surface and to increased anterograde axonal transport. ARF6 activation by expression of the neuronal guanine nucleotide exchange factors, ARNO or EFA6, increases retrograde integrin transport in axons and increases integrin internalization. ARF6 inactivation increases integrin-mediated outgrowth, while activation decreases it. The coordinated changes in integrin transport and recycling resulting from ARF6 activation or inactivation are the probable mechanism behind this regulation of axon growth. Our data suggest a novel mechanism of integrin traffic and transport in peripheral axons, regulated by the activation state of ARF6, and suggest that ARF6 might be targeted to enhance integrin-dependent axon regeneration after injury.

  2. Complement Protein C1q Modulates Neurite Outgrowth In Vitro and Spinal Cord Axon Regeneration In Vivo

    PubMed Central

    Peterson, Sheri L.; Nguyen, Hal X.; Mendez, Oscar A.

    2015-01-01

    Traumatic injury to CNS fiber tracts is accompanied by failure of severed axons to regenerate and results in lifelong functional deficits. The inflammatory response to CNS trauma is mediated by a diverse set of cells and proteins with varied, overlapping, and opposing effects on histological and behavioral recovery. Importantly, the contribution of individual inflammatory complement proteins to spinal cord injury (SCI) pathology is not well understood. Although the presence of complement components increases after SCI in association with axons and myelin, it is unknown whether complement proteins affect axon growth or regeneration. We report a novel role for complement C1q in neurite outgrowth in vitro and axon regrowth after SCI. In culture, C1q increased neurite length on myelin. Protein and molecular assays revealed that C1q interacts directly with myelin associated glycoprotein (MAG) in myelin, resulting in reduced activation of growth inhibitory signaling in neurons. In agreement with a C1q-outgrowth-enhancing mechanism in which C1q binding to MAG reduces MAG signaling to neurons, complement C1q blocked both the growth inhibitory and repulsive turning effects of MAG in vitro. Furthermore, C1q KO mice demonstrated increased sensory axon turning within the spinal cord lesion after SCI with peripheral conditioning injury, consistent with C1q-mediated neutralization of MAG. Finally, we present data that extend the role for C1q in axon growth and guidance to include the sprouting patterns of descending corticospinal tract axons into spinal gray matter after dorsal column transection SCI. PMID:25762679

  3. [Experimental studies for the improvement of facial nerve regeneration].

    PubMed

    Guntinas-Lichius, O; Angelov, D N

    2008-02-01

    Using a combination of the following, it is possible to investigate procedures to improve the morphological and functional regeneration of the facial nerve in animal models: 1) retrograde fluorescence tracing to analyse collateral axonal sprouting and the selectivity of reinnervation of the mimic musculature, 2) immunohistochemistry to analyse both the terminal axonal sprouting in the muscles and the axon reaction within the nucleus of the facial nerve, the peripheral nerve, and its environment, and 3) digital motion analysis of the muscles. To obtain good functional facial nerve regeneration, a reduction of terminal sprouting in the mimic musculature seems to be more important than a reduction of collateral sprouting at the lesion site. Promising strategies include acceleration of nerve regeneration, forced induced use of the paralysed face, mechanical stimulation of the face, and transplantation of nerve-growth-promoting olfactory epithelium at the lesion site.

  4. MicroRNA-431 regulates axon regeneration in mature sensory neurons by targeting the Wnt antagonist Kremen1

    PubMed Central

    Wu, Di; Murashov, Alexander K.

    2013-01-01

    MicroRNAs (miRNAs) are small, non-coding RNAs that function as key post-transcriptional regulators in neural development, brain function, and neurological diseases. Growing evidence indicates that miRNAs are also important mediators of nerve regeneration, however, the affected signaling mechanisms are not clearly understood. In the present study, we show that nerve injury-induced miR-431 stimulates regenerative axon growth by silencing Kremen1, an antagonist of Wnt/beta-catenin signaling. Both the gain-of-function of miR-431 and knockdown of Kremen1 significantly enhance axon outgrowth in murine dorsal root ganglion neuronal cultures. Using cross-linking with AGO-2 immunoprecipitation, and 3′-untranslated region (UTR) luciferase reporter assay we demonstrate miR-431 direct interaction on the 3′-UTR of Kremen1 mRNA. Together, our results identify miR-431 as an important regulator of axonal regeneration and a promising therapeutic target. PMID:24167472

  5. Treadmill Training Enhances Axon Regeneration In Injured Mouse Peripheral Nerves Without Increased Loss of Topographic Specificity

    PubMed Central

    English, Arthur W.; Cucoranu, Delia; Mulligan, Amanda; Sabatier, Manning

    2009-01-01

    We investigated the extent of misdirection of regenerating axons when that regeneration was enhanced using treadmill training. Retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial and common fibular nerves two or four weeks after transection and surgical repair of the mouse sciatic nerve. The spatial locations of retrogradely labeled motoneurons were studied in untreated control mice and in mice receiving two weeks of treadmill training, either according to a continuous protocol (10 m/min, one hour/day, five day/week) or an interval protocol (20 m/min for two minutes, followed by a five minute rest, repeated 4 times, five days/week). More retrogradely labeled motoneurons were found in both treadmill trained groups. The magnitude of this increase was as great as or greater than that found after using other enhancement strategies. In both treadmill trained groups, the proportions of motoneurons labeled from tracer applied to the common fibular nerve that were found in spinal cord locations reserved for tibial motoneurons in intact mice was no greater than in untreated control mice and significantly less than found after electrical stimulation or chondroitinase treatment. Treadmill training in the first two weeks following peripheral nerve injury produces a marked enhancement of motor axon regeneration without increasing the propensity of those axons to choose pathways leading to functionally inappropriate targets. PMID:19731339

  6. Neural Regeneration in Caenorhabditis elegans

    PubMed Central

    El Bejjani, Rachid; Hammarlund, Marc

    2013-01-01

    Axon regeneration is a medically relevant process that can repair damaged neurons. This review describes current progress in understanding axon regeneration in the model organism Caenorhabditis elegans. Factors that regulate axon regeneration in C. elegans have broadly similar roles in vertebrate neurons. This means that using C. elegans as a tool to leverage discovery is a legitimate strategy for identifying conserved mechanisms of axon regeneration. PMID:22974301

  7. Combination of Engineered Schwann Cell Grafts to Secrete Neurotrophin and Chondroitinase Promotes Axonal Regeneration and Locomotion after Spinal Cord Injury

    PubMed Central

    Pressman, Yelena; Moody, Alison; Berg, Randall; Muir, Elizabeth M.; Rogers, John H.; Ozawa, Hiroshi; Itoi, Eiji; Pearse, Damien D.; Bunge, Mary Bartlett

    2014-01-01

    Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function. PMID:24478364

  8. Swimming Exercise in the Acute or Late Phase after Sciatic Nerve Crush Accelerates Nerve Regeneration

    PubMed Central

    Teodori, Rosana Macher; Betini, Joice; de Oliveira, Larissa Salgado; Sobral, Luciane Lobato; Takeda, Sibele Yoko Mattozo; Montebelo, Maria Imaculada de Lima

    2011-01-01

    There is no consensus about the best time to start exercise after peripheral nerve injury. We evaluated the morphological and functional characteristics of the sciatic nerves of rats that began to swim immediately after crush nerve injury (CS1), those that began to swim 14 days after injury (CS14), injured rats not submitted to swimming (C), and uninjured rats submitted to swimming (S). After 30 days the number of axons in CS1 and CS14 was lower than in C (P < 0.01). The diameter of axons and nerve fibers was larger in CS1 (P < 0.01) and CS14 (P < 0.05) than in C, and myelin sheath thickness was lower in all crushed groups (P < 0.05). There was no functional difference between CS1 and CS14 (P > 0.05). Swimming exercise applied during the acute or late phase of nerve injury accelerated nerve regeneration and synaptic elimination after axonotmesis, suggesting that exercise may be initiated immediately after injury. PMID:21876821

  9. Side-To-Side Nerve Bridges Support Donor Axon Regeneration Into Chronically Denervated Nerves and Are Associated With Characteristic Changes in Schwann Cell Phenotype.

    PubMed

    Hendry, J Michael; Alvarez-Veronesi, M Cecilia; Snyder-Warwick, Alison; Gordon, Tessa; Borschel, Gregory H

    2015-11-01

    Chronic denervation resulting from long nerve regeneration times and distances contributes greatly to suboptimal outcomes following nerve injuries. Recent studies showed that multiple nerve grafts inserted between an intact donor nerve and a denervated distal recipient nerve stump (termed "side-to-side nerve bridges") enhanced regeneration after delayed nerve repair. To examine the cellular aspects of axon growth across these bridges to explore the "protective" mechanism of donor axons on chronically denervated Schwann cells. In Sprague Dawley rats, 3 side-to-side nerve bridges were placed over a 10-mm distance between an intact donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) distal nerve stump. Green fluorescent protein-expressing TIB axons grew across the bridges and were counted in cross section after 4 weeks. Immunofluorescent axons and Schwann cells were imaged over a 4-month period. Denervated Schwann cells dedifferentiated to a proliferative, nonmyelinating phenotype within the bridges and the recipient denervated CP nerve stump. As donor TIB axons grew across the 3 side-to-side nerve bridges and into the denervated CP nerve, the Schwann cells redifferentiated to the myelinating phenotype. Bridge placement led to an increased mass of hind limb anterior compartment muscles after 4 months of denervation compared with muscles whose CP nerve was not "protected" by bridges. This study describes patterns of donor axon regeneration and myelination in the denervated recipient nerve stump and supports a mechanism where these donor axons sustain a proregenerative state to prevent deterioration in the face of chronic denervation.

  10. The Extracellular Environment of the CNS: Influence on Plasticity, Sprouting, and Axonal Regeneration after Spinal Cord Injury

    PubMed Central

    Forbes, Lindsey H.

    2018-01-01

    The extracellular environment of the central nervous system (CNS) becomes highly structured and organized as the nervous system matures. The extracellular space of the CNS along with its subdomains plays a crucial role in the function and stability of the CNS. In this review, we have focused on two components of the neuronal extracellular environment, which are important in regulating CNS plasticity including the extracellular matrix (ECM) and myelin. The ECM consists of chondroitin sulfate proteoglycans (CSPGs) and tenascins, which are organized into unique structures called perineuronal nets (PNNs). PNNs associate with the neuronal cell body and proximal dendrites of predominantly parvalbumin-positive interneurons, forming a robust lattice-like structure. These developmentally regulated structures are maintained in the adult CNS and enhance synaptic stability. After injury, however, CSPGs and tenascins contribute to the structure of the inhibitory glial scar, which actively prevents axonal regeneration. Myelin sheaths and mature adult oligodendrocytes, despite their important role in signal conduction in mature CNS axons, contribute to the inhibitory environment existing after injury. As such, unlike the peripheral nervous system, the CNS is unable to revert to a “developmental state” to aid neuronal repair. Modulation of these external factors, however, has been shown to promote growth, regeneration, and functional plasticity after injury. This review will highlight some of the factors that contribute to or prevent plasticity, sprouting, and axonal regeneration after spinal cord injury. PMID:29849554

  11. The mTOR Substrate S6 Kinase 1 (S6K1) Is a Negative Regulator of Axon Regeneration and a Potential Drug Target for Central Nervous System Injury

    PubMed Central

    Ding, Ying; Slepak, Tatiana; Sun, Yan; Martinez, Yania; Xu, Xiao-Ming

    2017-01-01

    The mammalian target of rapamycin (mTOR) positively regulates axon growth in the mammalian central nervous system (CNS). Although axon regeneration and functional recovery from CNS injuries are typically limited, knockdown or deletion of PTEN, a negative regulator of mTOR, increases mTOR activity and induces robust axon growth and regeneration. It has been suggested that inhibition of S6 kinase 1 (S6K1, gene symbol: RPS6KB1), a prominent mTOR target, would blunt mTOR's positive effect on axon growth. In contrast to this expectation, we demonstrate that inhibition of S6K1 in CNS neurons promotes neurite outgrowth in vitro by twofold to threefold. Biochemical analysis revealed that an mTOR-dependent induction of PI3K signaling is involved in mediating this effect of S6K1 inhibition. Importantly, treating female mice in vivo with PF-4708671, a selective S6K1 inhibitor, stimulated corticospinal tract regeneration across a dorsal spinal hemisection between the cervical 5 and 6 cord segments (C5/C6), increasing axon counts for at least 3 mm beyond the injury site at 8 weeks after injury. Concomitantly, treatment with PF-4708671 produced significant locomotor recovery. Pharmacological targeting of S6K1 may therefore constitute an attractive strategy for promoting axon regeneration following CNS injury, especially given that S6K1 inhibitors are being assessed in clinical trials for nononcological indications. SIGNIFICANCE STATEMENT Despite mTOR's well-established function in promoting axon regeneration, the role of its downstream target, S6 kinase 1 (S6K1), has been unclear. We used cellular assays with primary neurons to demonstrate that S6K1 is a negative regulator of neurite outgrowth, and a spinal cord injury model to show that it is a viable pharmacological target for inducing axon regeneration. We provide mechanistic evidence that S6K1's negative feedback to PI3K signaling is involved in axon growth inhibition, and show that phosphorylation of S6K1 is a more

  12. Rearrangement of potassium ions and Kv1.1/Kv1.2 potassium channels in regenerating axons following end-to-end neurorrhaphy: ionic images from TOF-SIMS.

    PubMed

    Liu, Chiung-Hui; Chang, Hung-Ming; Wu, Tsung-Huan; Chen, Li-You; Yang, Yin-Shuo; Tseng, To-Jung; Liao, Wen-Chieh

    2017-10-01

    The voltage-gated potassium channels Kv1.1 and Kv1.2 that cluster at juxtaparanodal (JXP) regions are essential in the regulation of nerve excitability and play a critical role in axonal conduction. When demyelination occurs, Kv1.1/Kv1.2 activity increases, suppressing the membrane potential nearly to the equilibrium potential of K + , which results in an axonal conduction blockade. The recovery of K + -dependent communication signals and proper clustering of Kv1.1/Kv1.2 channels at JXP regions may directly reflect nerve regeneration following peripheral nerve injury. However, little is known about potassium channel expression and its relationship with the dynamic potassium ion distribution at the node of Ranvier during the regenerative process of peripheral nerve injury (PNI). In the present study, end-to-end neurorrhaphy (EEN) was performed using an in vivo model of PNI. The distribution of K + at regenerating axons following EEN was detected by time-of-flight secondary-ion mass spectrometry. The specific localization and expression of Kv1.1/Kv1.2 channels were examined by confocal microscopy and western blotting. Our data showed that the re-establishment of K + distribution and intensity was correlated with the functional recovery of compound muscle action potential morphology in EEN rats. Furthermore, the re-clustering of Kv1.1/1.2 channels 1 and 3 months after EEN at the nodal region of the regenerating nerve corresponded to changes in the K + distribution. This study provided direct evidence of K + distribution in regenerating axons for the first time. We proposed that the Kv1.1/Kv1.2 channels re-clustered at the JXP regions of regenerating axons are essential for modulating the proper patterns of K + distribution in axons for maintaining membrane potential stability after EEN.

  13. Chronic recording of regenerating VIIIth nerve axons with a sieve electrode

    NASA Technical Reports Server (NTRS)

    Mensinger, A. F.; Anderson, D. J.; Buchko, C. J.; Johnson, M. A.; Martin, D. C.; Tresco, P. A.; Silver, R. B.; Highstein, S. M.

    2000-01-01

    A micromachined silicon substrate sieve electrode was implanted within transected toadfish (Opsanus tau) otolith nerves. High fidelity, single unit neural activity was recorded from seven alert and unrestrained fish 30 to 60 days after implantation. Fibrous coatings of genetically engineered bioactive protein polymers and nerve guide tubes increased the number of axons regenerating through the electrode pores when compared with controls. Sieve electrodes have potential as permanent interfaces to the nervous system and to bridge missing connections between severed or damaged nerves and muscles. Recorded impulses might also be amplified and used to control prosthetic devices.

  14. Functional Role of the Disulfide Isomerase ERp57 in Axonal Regeneration.

    PubMed

    Castillo, Valentina; Oñate, Maritza; Woehlbier, Ute; Rozas, Pablo; Andreu, Catherine; Medinas, Danilo; Valdés, Pamela; Osorio, Fabiola; Mercado, Gabriela; Vidal, René L; Kerr, Bredford; Court, Felipe A; Hetz, Claudio

    2015-01-01

    ERp57 (also known as grp58 and PDIA3) is a protein disulfide isomerase that catalyzes disulfide bonds formation of glycoproteins as part of the calnexin and calreticulin cycle. ERp57 is markedly upregulated in most common neurodegenerative diseases downstream of the endoplasmic reticulum (ER) stress response. Despite accumulating correlative evidence supporting a neuroprotective role of ERp57, the contribution of this foldase to the physiology of the nervous system remains unknown. Here we developed a transgenic mouse model that overexpresses ERp57 in the nervous system under the control of the prion promoter. We analyzed the susceptibility of ERp57 transgenic mice to undergo neurodegeneration. Unexpectedly, ERp57 overexpression did not affect dopaminergic neuron loss and striatal denervation after injection of a Parkinson's disease-inducing neurotoxin. In sharp contrast, ERp57 transgenic animals presented enhanced locomotor recovery after mechanical injury to the sciatic nerve. These protective effects were associated with enhanced myelin removal, macrophage infiltration and axonal regeneration. Our results suggest that ERp57 specifically contributes to peripheral nerve regeneration, whereas its activity is dispensable for the survival of a specific neuronal population of the central nervous system. These results demonstrate for the first time a functional role of a component of the ER proteostasis network in peripheral nerve regeneration.

  15. Selective rab11 transport and the intrinsic regenerative ability of CNS axons

    PubMed Central

    Koseki, Hiroaki; Donegá, Matteo; Lam, Brian YH; Petrova, Veselina; van Erp, Susan; Yeo, Giles SH; Kwok, Jessica CF; ffrench-Constant, Charles

    2017-01-01

    Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration. PMID:28829741

  16. Regeneration-associated genes on optic nerve regeneration in fish retina.

    PubMed

    Ogai, Kazuhiro; Nishitani, Maki; Kuwana, Ayaka; Mawatari, Kazuhiro; Koriyama, Yoshiki; Sugitani, Kayo; Nakashima, Hiroshi; Kato, Satoru

    2014-01-01

    It has been well documented that fish central nervous system, including retina and optic nerve, can regenerate and recover its function after nerve injury. Within a few decades, a number of regeneration-associated genes (RAGs) have been identified in fish retina following optic nerve injury (ONI). RAGs can be classified into two groups: cell survival- and axonal outgrowth-related genes. In fish retina after ONI, cell survival-related genes were upregulated in 1-6 days after ONI, which corresponds to the preparation stage for cell survival and axonal sprouting. Subsequently, axonal outgrowth-related genes were upregulated in 1-6 weeks after ONI, which corresponds to the axonal regrowth stage. Recently, we've found a novel type of RAGs, dedifferentiation-related genes, that are upregulated in overlapping time between cell survival and axonal regrowth (3-10 days after ONI). In this chapter we summarize these three types of RAGs that promote optic nerve regeneration in the fish retina after ONI.

  17. Integration and long distance axonal regeneration in the central nervous system from transplanted primitive neural stem cells.

    PubMed

    Zhao, Jiagang; Sun, Woong; Cho, Hyo Min; Ouyang, Hong; Li, Wenlin; Lin, Ying; Do, Jiun; Zhang, Liangfang; Ding, Sheng; Liu, Yizhi; Lu, Paul; Zhang, Kang

    2013-01-04

    Spinal cord injury (SCI) results in devastating motor and sensory deficits secondary to disrupted neuronal circuits and poor regenerative potential. Efforts to promote regeneration through cell extrinsic and intrinsic manipulations have met with limited success. Stem cells represent an as yet unrealized therapy in SCI. Recently, we identified novel culture methods to induce and maintain primitive neural stem cells (pNSCs) from human embryonic stem cells. We tested whether transplanted human pNSCs can integrate into the CNS of the developing chick neural tube and injured adult rat spinal cord. Following injection of pNSCs into the developing chick CNS, pNSCs integrated into the dorsal aspects of the neural tube, forming cell clusters that spontaneously differentiated into neurons. Furthermore, following transplantation of pNSCs into the lesioned rat spinal cord, grafted pNSCs survived, differentiated into neurons, and extended long distance axons through the scar tissue at the graft-host interface and into the host spinal cord to form terminal-like structures near host spinal neurons. Together, these findings suggest that pNSCs derived from human embryonic stem cells differentiate into neuronal cell types with the potential to extend axons that associate with circuits of the CNS and, more importantly, provide new insights into CNS integration and axonal regeneration, offering hope for repair in SCI.

  18. Neuroinflammation as Fuel for Axonal Regeneration in the Injured Vertebrate Central Nervous System

    PubMed Central

    Van houcke, Jessie

    2017-01-01

    Damage to the central nervous system (CNS) is one of the leading causes of morbidity and mortality in elderly, as repair after lesions or neurodegenerative disease usually fails because of the limited capacity of CNS regeneration. The causes underlying this limited regenerative potential are multifactorial, but one critical aspect is neuroinflammation. Although classically considered as harmful, it is now becoming increasingly clear that inflammation can also promote regeneration, if the appropriate context is provided. Here, we review the current knowledge on how acute inflammation is intertwined with axonal regeneration, an important component of CNS repair. After optic nerve or spinal cord injury, inflammatory stimulation and/or modification greatly improve the regenerative outcome in rodents. Moreover, the hypothesis of a beneficial role of inflammation is further supported by evidence from adult zebrafish, which possess the remarkable capability to repair CNS lesions and even restore functionality. Lastly, we shed light on the impact of aging processes on the regenerative capacity in the CNS of mammals and zebrafish. As aging not only affects the CNS, but also the immune system, the regeneration potential is expected to further decline in aged individuals, an element that should definitely be considered in the search for novel therapeutic strategies. PMID:28203046

  19. Roles of Chondroitin Sulfate and Dermatan Sulfate in the Formation of a Lesion Scar and Axonal Regeneration after Traumatic Injury of the Mouse Brain

    PubMed Central

    Li, Hong-Peng; Komuta, Yukari; Kimura-Kuroda, Junko; van Kuppevelt, Toin H.

    2013-01-01

    Abstract Dermatan sulfate (DS) is synthesized from chondroitin sulfate (CS) by epimerization of glucuronic acid of CS to yield iduronic acid. In the present study, the role of CS and DS was examined in mice that received transection of nigrostriatal dopaminergic pathway followed by injection of glycosaminoglycan degrading enzymes into the lesion site. Two weeks after injury, fibrotic and glial scars were formed around the lesion, and transected axons did not regenerate beyond the fibrotic scar. Injection of chondroitinase ABC (ChABC), which degrades both CS and DS, completely suppressed the fibrotic scar formation, reduced the glial scar, and promoted the regeneration of dopaminergic axons. Injection of the DS-degrading enzyme chondroitinase B (ChB) also yielded similar results. By contrast, injection of chondroitinase AC (ChAC), a CS-degrading enzyme, did not suppress the fibrotic and glial scar formation, but reduced CS immunoreactivity and promoted the axonal regeneration. Addition of transforming growth factor-β1 (TGF-β1) to a co-culture of meningeal fibroblasts and cerebral astrocytes induces a fibrotic scar-like cell cluster. The effect of TGF-β1 on cluster formation was suppressed by treatment with ChABC or ChB, but not by ChAC. TGF-β1-induced cell cluster repelled neurites of neonatal cerebellar neurons, but addition of ChABC or ChAC suppressed the inhibitory property of clusters on neurite outgrowth. The present study is the first to demonstrate that DS and CS play different functions after brain injury: DS is involved in the lesion scar formation, and CS inhibits axonal regeneration. PMID:23438307

  20. Outer Electrospun Polycaprolactone Shell Induces Massive Foreign Body Reaction and Impairs Axonal Regeneration through 3D Multichannel Chitosan Nerve Guides

    PubMed Central

    Behrens, Peter; Wienecke, Soenke; Chakradeo, Tanmay; Glasmacher, Birgit

    2014-01-01

    We report on the performance of composite nerve grafts with an inner 3D multichannel porous chitosan core and an outer electrospun polycaprolactone shell. The inner chitosan core provided multiple guidance channels for regrowing axons. To analyze the in vivo properties of the bare chitosan cores, we separately implanted them into an epineural sheath. The effects of both graft types on structural and functional regeneration across a 10 mm rat sciatic nerve gap were compared to autologous nerve transplantation (ANT). The mechanical biomaterial properties and the immunological impact of the grafts were assessed with histological techniques before and after transplantation in vivo. Furthermore during a 13-week examination period functional tests and electrophysiological recordings were performed and supplemented by nerve morphometry. The sheathing of the chitosan core with a polycaprolactone shell induced massive foreign body reaction and impairment of nerve regeneration. Although the isolated novel chitosan core did allow regeneration of axons in a similar size distribution as the ANT, the ANT was superior in terms of functional regeneration. We conclude that an outer polycaprolactone shell should not be used for the purpose of bioartificial nerve grafting, while 3D multichannel porous chitosan cores could be candidate scaffolds for structured nerve grafts. PMID:24818158

  1. Outer electrospun polycaprolactone shell induces massive foreign body reaction and impairs axonal regeneration through 3D multichannel chitosan nerve guides.

    PubMed

    Duda, Sven; Dreyer, Lutz; Behrens, Peter; Wienecke, Soenke; Chakradeo, Tanmay; Glasmacher, Birgit; Haastert-Talini, Kirsten

    2014-01-01

    We report on the performance of composite nerve grafts with an inner 3D multichannel porous chitosan core and an outer electrospun polycaprolactone shell. The inner chitosan core provided multiple guidance channels for regrowing axons. To analyze the in vivo properties of the bare chitosan cores, we separately implanted them into an epineural sheath. The effects of both graft types on structural and functional regeneration across a 10 mm rat sciatic nerve gap were compared to autologous nerve transplantation (ANT). The mechanical biomaterial properties and the immunological impact of the grafts were assessed with histological techniques before and after transplantation in vivo. Furthermore during a 13-week examination period functional tests and electrophysiological recordings were performed and supplemented by nerve morphometry. The sheathing of the chitosan core with a polycaprolactone shell induced massive foreign body reaction and impairment of nerve regeneration. Although the isolated novel chitosan core did allow regeneration of axons in a similar size distribution as the ANT, the ANT was superior in terms of functional regeneration. We conclude that an outer polycaprolactone shell should not be used for the purpose of bioartificial nerve grafting, while 3D multichannel porous chitosan cores could be candidate scaffolds for structured nerve grafts.

  2. Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System Following Neural Injury

    DTIC Science & Technology

    2017-08-01

    AWARD NUMBER: W81XWH-12-1-0051 TITLE: Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System ...Central Nervous System Following Neural Injury 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-12-1-0051 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Robert...induces re- growth of dopaminergic axons at 3 to 6 weeks after destruction by a neurotoxin. However, this approach cannot be used in humans because

  3. Maximizing functional axon repair in the injured central nervous system: Lessons from neuronal development.

    PubMed

    Kaplan, Andrew; Bueno, Mardja; Hua, Luyang; Fournier, Alyson E

    2018-01-01

    The failure of damaged axons to regrow underlies disability in central nervous system injury and disease. Therapies that stimulate axon repair will be critical to restore function. Extensive axon regeneration can be induced by manipulation of oncogenes and tumor suppressors; however, it has been difficult to translate this into functional recovery in models of spinal cord injury. The current challenge is to maximize the functional integration of regenerating axons to recover motor and sensory behaviors. Insights into axonal growth and wiring during nervous system development are helping guide new approaches to boost regeneration and functional connectivity after injury in the mature nervous system. Here we discuss our current understanding of axonal behavior after injury and prospects for the development of drugs to optimize axon regeneration and functional recovery after CNS injury. Developmental Dynamics 247:18-23, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  4. Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

    PubMed

    Gwee, Serene S L; Radford, Rowan A W; Chow, Sharron; Syal, Monisha D; Morsch, Marco; Formella, Isabel; Lee, Albert; Don, Emily K; Badrock, Andrew P; Cole, Nicholas J; West, Adrian K; Cheung, Steve N S; Chung, Roger S

    2018-02-21

    Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

  5. Intravitreally transplanted dental pulp stem cells promote neuroprotection and axon regeneration of retinal ganglion cells after optic nerve injury.

    PubMed

    Mead, Ben; Logan, Ann; Berry, Martin; Leadbeater, Wendy; Scheven, Ben A

    2013-11-15

    To investigate the potential therapeutic benefit of intravitreally implanted dental pulp stem cells (DPSCs) on axotomized adult rat retinal ganglion cells (RGCs) using in vitro and in vivo neural injury models. Conditioned media collected from cultured rat DPSCs and bone marrow-derived mesenchymal stem cells (BMSCs) were assayed for nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) secretion using ELISA. DPSCs or BMSCs were cocultured with retinal cells, with or without Fc-TrK inhibitors, in a Transwell system, and the number of surviving βIII-tubulin⁺ retinal cells and length/number of βIII-tubulin⁺ neurites were quantified. For the in vivo study, DPSCs or BMSCs were transplanted into the vitreous body of the eye after a surgically induced optic nerve crush injury. At 7, 14, and 21 days postlesion (dpl), optical coherence tomography (OCT) was used to measure the retinal nerve fiber layer thickness as a measure of axonal atrophy. At 21 dpl, numbers of Brn-3a⁺ RGCs in parasagittal retinal sections and growth-associated protein-43⁺ axons in longitudinal optic nerve sections were quantified as measures of RGC survival and axon regeneration, respectively. Both DPSCs and BMSCs secreted NGF, BDNF, and NT-3, with DPSCs secreting significantly higher titers of NGF and BDNF than BMSCs. DPSCs, and to a lesser extent BMSCs, promoted statistically significant survival and neuritogenesis/axogenesis of βIII-tubulin⁺ retinal cells in vitro and in vivo where the effects were abolished after TrK receptor blockade. Intravitreal transplants of DPSCs promoted significant neurotrophin-mediated RGC survival and axon regeneration after optic nerve injury.

  6. Axonal regeneration through acellular muscle grafts

    PubMed Central

    HALL, SUSAN

    1997-01-01

    The management of peripheral nerve injury remains a major clinical problem. Progress in this field will almost certainly depend upon manipulating the pathophysiological processes which are triggered by traumatic injuries. One of the most important determinants of functional outcome after the reconstruction of a transected peripheral nerve is the length of the gap between proximal and distal nerve stumps. Long defects (> 2 cm) must be bridged by a suitable conduit in order to support axonal regrowth. This review examines the cellular and acellular elements which facilitate axonal regrowth and the use of acellular muscle grafts in the repair of injuries in the peripheral nervous system. PMID:9034882

  7. NEUROTROPHIC FACTORS IN COMBINATORIAL APPROACHES FOR SPINAL CORD REGENERATION

    PubMed Central

    McCall, Julianne; Weidner, Norbert; Blesch, Armin

    2012-01-01

    Axonal regeneration is inhibited by a plethora of different mechanisms in the adult central nervous system (CNS). While neurotrophic factors have been shown to stimulate axonal growth in numerous animal models of nervous system injury, a lack of suitable growth substrates, an insufficient activation of neuron-intrinsic regenerative programs and extracellular inhibitors of regeneration limit the efficacy of neurotrophic factor delivery for anatomical and functional recovery after spinal cord injury. Thus, growth-stimulating factors will likely have to be combined with other treatment approaches to tap into the full potential of growth factor therapy for axonal regeneration. In addition, the temporal and spatial distribution of growth factors have to be tightly controlled to achieve biologically active concentrations, to allow for the chemotropic guidance of axons and to prevent adverse effects related to the widespread distribution of neurotrophic factors. Here, we will review the rationale for combinatorial treatments in axonal regeneration and summarize some recent progress in promoting axonal regeneration in the injured CNS using such approaches. PMID:22526621

  8. Sustained release of neurotrophin-3 via calcium phosphate-coated sutures promotes axonal regeneration after spinal cord injury.

    PubMed

    Hanna, Amgad; Thompson, Daniel L; Hellenbrand, Daniel J; Lee, Jae-Sung; Madura, Casey J; Wesley, Meredith G; Dillon, Natalie J; Sharma, Tapan; Enright, Connor J; Murphy, William L

    2016-07-01

    Because of the dynamics of spinal cord injury (SCI), the optimal treatment will almost certainly be a combination approach to control the environment and promote axonal growth. This study uses peripheral nerve grafts (PNGs) as scaffolds for axonal growth while delivering neurotrophin-3 (NT-3) via calcium phosphate (CaP) coatings on surgical sutures. CaP coating was grown on sutures, and NT-3 binding and release were characterized in vitro. Then, the NT-3-loaded sutures were tested in a complete SCI model. Rats were analyzed for functional improvement and axonal growth into the grafts. The CaP-coated sutures exhibited a burst release of NT-3, followed by a sustained release for at least 20 days. Functionally, the rats with PNGs + NT-3-loaded sutures and the rats treated with PNGs scored significantly higher than controls on day 56 postoperatively. However, functional scores in rats treated with PNGs + NT-3-loaded suture were not significantly different from those of rats treated with PNGs alone. Cholera toxin subunit B (CTB) labeling rostral to the graft was not observed in any controls, but CTB labeling rostral to the graft was observed in almost all rats that had had a PNG. Neurofilament labeling on transverse sections of the graft revealed that the rats treated with the NT-3-loaded sutures had significantly more axons per graft than rats treated with an NT-3 injection and rats without NT-3. These data demonstrate that PNGs serve as scaffolds for axonal growth after SCI and that CaP-coated sutures can efficiently release NT-3 to increase axonal regeneration. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  9. The Dyslexia-susceptibility Protein KIAA0319 Inhibits Axon Growth Through Smad2 Signaling

    PubMed Central

    Franquinho, Filipa; Nogueira-Rodrigues, Joana; Duarte, Joana M.; Esteves, Sofia S.; Carter-Su, Christin; Monaco, Anthony P.; Molnár, Zoltán; Velayos-Baeza, Antonio; Brites, Pedro; Sousa, Mónica M.

    2017-01-01

    Abstract KIAA0319 is a transmembrane protein associated with dyslexia with a presumed role in neuronal migration. Here we show that KIAA0319 expression is not restricted to the brain but also occurs in sensory and spinal cord neurons, increasing from early postnatal stages to adulthood and being downregulated by injury. This suggested that KIAA0319 participates in functions unrelated to neuronal migration. Supporting this hypothesis, overexpression of KIAA0319 repressed axon growth in hippocampal and dorsal root ganglia neurons; the intracellular domain of KIAA0319 was sufficient to elicit this effect. A similar inhibitory effect was observed in vivo as axon regeneration was impaired after transduction of sensory neurons with KIAA0319. Conversely, the deletion of Kiaa0319 in neurons increased neurite outgrowth in vitro and improved axon regeneration in vivo. At the mechanistic level, KIAA0319 engaged the JAK2-SH2B1 pathway to activate Smad2, which played a central role in KIAA0319-mediated repression of axon growth. In summary, we establish KIAA0319 as a novel player in axon growth and regeneration with the ability to repress the intrinsic growth potential of axons. This study describes a novel regulatory mechanism operating during peripheral nervous system and central nervous system axon growth, and offers novel targets for the development of effective therapies to promote axon regeneration. PMID:28334068

  10. Expressing Constitutively Active Rheb in Adult Dorsal Root Ganglion Neurons Enhances the Integration of Sensory Axons that Regenerate Across a Chondroitinase-Treated Dorsal Root Entry Zone Following Dorsal Root Crush

    PubMed Central

    Wu, Di; Klaw, Michelle C.; Kholodilov, Nikolai; Burke, Robert E.; Detloff, Megan R.; Côté, Marie-Pascale; Tom, Veronica J.

    2016-01-01

    While the peripheral branch of dorsal root ganglion neurons (DRG) can successfully regenerate after injury, lesioned central branch axons fail to regrow across the dorsal root entry zone (DREZ), the interface between the dorsal root and the spinal cord. This lack of regeneration is due to the limited regenerative capacity of adult sensory axons and the growth-inhibitory environment at the DREZ, which is similar to that found in the glial scar after a central nervous system (CNS) injury. We hypothesized that transduction of adult DRG neurons using adeno-associated virus (AAV) to express a constitutively-active form of the GTPase Rheb (caRheb) will increase their intrinsic growth potential after a dorsal root crush. Additionally, we posited that if we combined that approach with digestion of upregulated chondroitin sulfate proteoglycans (CSPG) at the DREZ with chondroitinase ABC (ChABC), we would promote regeneration of sensory axons across the DREZ into the spinal cord. We first assessed if this strategy promotes neuritic growth in an in vitro model of the glial scar containing CSPG. ChABC allowed for some regeneration across the once potently inhibitory substrate. Combining ChABC treatment with expression of caRheb in DRG significantly improved this growth. We then determined if this combination strategy also enhanced regeneration through the DREZ after dorsal root crush in adult rats in vivo. After unilaterally crushing C4-T1 dorsal roots, we injected AAV5-caRheb or AAV5-GFP into the ipsilateral C5-C8 DRGs. ChABC or PBS was injected into the ipsilateral dorsal horn at C5-C8 to digest CSPG, for a total of four animal groups (caRheb + ChABC, caRheb + PBS, GFP + ChABC, GFP + PBS). Regeneration was rarely observed in PBS-treated animals, whereas short-distance regrowth across the DREZ was observed in ChABC-treated animals. No difference in axon number or length between the ChABC groups was observed, which may be related to intraganglionic inflammation induced by the

  11. NEUROTROPHIN SELECTIVITY IN ORGANIZING TOPOGRAPHIC REGENERATION OF NOCICEPTIVE AFFERENTS

    PubMed Central

    Kelamangalath, Lakshmi; Tang, Xiaoqing; Bezik, Kathleen; Sterling, Noelle; Son, Young-Jin; Smith, George M.

    2015-01-01

    Neurotrophins represent some of the best candidates to enhance regeneration. In the current study, we investigated the effects of artemin, a member of the glial derived neurotrophic factor (GDNF) family, on sensory axon regeneration following a lumbar dorsal root injury and compared these effects with that observed after either NGF or GDNF expression in the rat spinal cord. Unlike previously published data, artemin failed to induce regeneration of large-diameter myelinated sensory afferents when expressed within either the spinal cord or DRG. However, artemin or NGF induced regeneration of calcitonin gene related peptide positive (CGRP+) axons only when expressed within the spinal cord. Accordingly, artemin or NGF enhanced recovery of only nociceptive behavior and showed a cFos distribution similar to the topography of regenerating axons. Artemin and GDNF signaling requires binding to different co-receptors (GFRα3 or GFRα1, respectively) prior to binding to the signaling receptor, cRet. Approximately 70% of DRG neurons express cRet, but only 35% express either co-receptor. To enhance artemin-induced regeneration, we co-expressed artemin with either GFRα3 or GDNF. Co-expression of artemin and GFRα3 only slightly enhanced regeneration of IB4+ non-peptidergic nociceptive axons, but not myelinated axons. Interestingly, this co-expression also disrupted the ability of artemin to produce topographic targeting and lead to significant increases in cFos immunoreactivity within the deep dorsal laminae. This study failed to demonstrate artemin-induced regeneration of myelinated axons, even with co-expression of GFR-α3, which only promoted mistargeted regeneration. PMID:26054884

  12. Neurotrophin selectivity in organizing topographic regeneration of nociceptive afferents.

    PubMed

    Kelamangalath, Lakshmi; Tang, Xiaoqing; Bezik, Kathleen; Sterling, Noelle; Son, Young-Jin; Smith, George M

    2015-09-01

    Neurotrophins represent some of the best candidates to enhance regeneration. In the current study, we investigated the effects of artemin, a member of the glial derived neurotrophic factor (GDNF) family, on sensory axon regeneration following a lumbar dorsal root injury and compared these effects with that observed after either NGF or GDNF expression in the rat spinal cord. Unlike previously published data, artemin failed to induce regeneration of large-diameter myelinated sensory afferents when expressed within either the spinal cord or DRG. However, artemin or NGF induced regeneration of calcitonin gene related peptide positive (CGRP(+)) axons only when expressed within the spinal cord. Accordingly, artemin or NGF enhanced recovery of only nociceptive behavior and showed a cFos distribution similar to the topography of regenerating axons. Artemin and GDNF signaling requires binding to different co-receptors (GFRα3 or GFRα1, respectively) prior to binding to the signaling receptor, cRet. Approximately 70% of DRG neurons express cRet, but only 35% express either co-receptor. To enhance artemin-induced regeneration, we co-expressed artemin with either GFRα3 or GDNF. Co-expression of artemin and GFRα3 only slightly enhanced regeneration of IB4(+) non-peptidergic nociceptive axons, but not myelinated axons. Interestingly, this co-expression also disrupted the ability of artemin to produce topographic targeting and lead to significant increases in cFos immunoreactivity within the deep dorsal laminae. This study failed to demonstrate artemin-induced regeneration of myelinated axons, even with co-expression of GFRα3, which only promoted mistargeted regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Highly Effective Photonic Cue for Repulsive Axonal Guidance

    PubMed Central

    Black, Bryan J.; Gu, Ling; Mohanty, Samarendra K.

    2014-01-01

    In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm. PMID:24717339

  14. Highly effective photonic cue for repulsive axonal guidance.

    PubMed

    Black, Bryan J; Gu, Ling; Mohanty, Samarendra K

    2014-01-01

    In vivo nerve repair requires not only the ability to regenerate damaged axons, but most importantly, the ability to guide developing or regenerating axons along paths that will result in functional connections. Furthermore, basic studies in neuroscience and neuro-electronic interface design require the ability to construct in vitro neural circuitry. Both these applications require the development of a noninvasive, highly effective tool for axonal growth-cone guidance. To date, a myriad of technologies have been introduced based on chemical, electrical, mechanical, and hybrid approaches (such as electro-chemical, optofluidic flow and photo-chemical methods). These methods are either lacking in desired spatial and temporal selectivity or require the introduction of invasive external factors. Within the last fifteen years however, several attractive guidance cues have been developed using purely light based cues to achieve axonal guidance. Here, we report a novel, purely optical repulsive guidance technique that uses low power, near infrared light, and demonstrates the guidance of primary goldfish retinal ganglion cell axons through turns of up to 120 degrees and over distances of ∼90 µm.

  15. TRPV1 Agonist, Capsaicin, Induces Axon Outgrowth after Injury via Ca2+/PKA Signaling.

    PubMed

    Frey, Erin; Karney-Grobe, Scott; Krolak, Trevor; Milbrandt, Jeff; DiAntonio, Aaron

    2018-01-01

    Preconditioning nerve injuries activate a pro-regenerative program that enhances axon regeneration for most classes of sensory neurons. However, nociceptive sensory neurons and central nervous system neurons regenerate poorly. In hopes of identifying novel mechanisms that promote regeneration, we screened for drugs that mimicked the preconditioning response and identified a nociceptive ligand that activates a preconditioning-like response to promote axon outgrowth. We show that activating the ion channel TRPV1 with capsaicin induces axon outgrowth of cultured dorsal root ganglion (DRG) sensory neurons, and that this effect is blocked in TRPV1 knockout neurons. Regeneration occurs only in NF200-negative nociceptive neurons, consistent with a cell-autonomous mechanism. Moreover, we identify a signaling pathway in which TRPV1 activation leads to calcium influx and protein kinase A (PKA) activation to induce a preconditioning-like response. Finally, capsaicin administration to the mouse sciatic nerve activates a similar preconditioning-like response and induces enhanced axonal outgrowth, indicating that this pathway can be induced in vivo . These findings highlight the use of local ligands to induce regeneration and suggest that it may be possible to target selective neuronal populations for repair, including cell types that often fail to regenerate.

  16. Neuronal intrinsic regenerative capacity: The impact of microtubule organization and axonal transport.

    PubMed

    Murillo, Blanca; Sousa, Mónica Mendes

    2018-05-08

    In the adult vertebrate central nervous system, axons generally fail to regenerate. In contrast, peripheral nervous system axons are able to form a growth cone and regenerate upon lesion. Among the multiple intrinsic mechanisms leading to the formation of a new growth cone and to successful axon regrowth, cytoskeleton organization and dynamics is central. Here we discuss how multiple pathways that define the regenerative capacity converge into the regulation of the axonal microtubule cytoskeleton and transport. We further explore the use of dorsal root ganglion neurons as a model to study the neuronal regenerative ability. Finally, we address some of the unanswered questions in the field, including the mechanisms by which axonal transport might be modulated by injury, and the relationship between microtubule organization, dynamics, and axonal transport. © 2018 Wiley Periodicals, Inc. Develop Neurobiol, 2018. © 2018 Wiley Periodicals, Inc.

  17. Neurosurgery: Functional regeneration after laser axotomy

    NASA Astrophysics Data System (ADS)

    Yanik, Mehmet Fatih; Cinar, Hulusi; Cinar, Hediye Nese; Chisholm, Andrew D.; Jin, Yishi; Ben-Yakar, Adela

    2004-12-01

    Understanding how nerves regenerate is an important step towards developing treatments for human neurological disease, but investigation has so far been limited to complex organisms (mouse and zebrafish) in the absence of precision techniques for severing axons (axotomy). Here we use femtosecond laser surgery for axotomy in the roundworm Caenorhabditis elegans and show that these axons functionally regenerate after the operation. Application of this precise surgical technique should enable nerve regeneration to be studied in vivo in its most evolutionarily simple form.

  18. Exclusion of Integrins from CNS Axons Is Regulated by Arf6 Activation and the AIS

    PubMed Central

    Franssen, Elske H. P.; Zhao, Rong-Rong; Koseki, Hiroaki; Kanamarlapudi, Venkateswarlu; Hoogenraad, Casper C.

    2015-01-01

    Integrins are adhesion and survival molecules involved in axon growth during CNS development, as well as axon regeneration after injury in the peripheral nervous system (PNS). Adult CNS axons do not regenerate after injury, partly due to a low intrinsic growth capacity. We have previously studied the role of integrins in axon growth in PNS axons; in the present study, we investigate whether integrin mechanisms involved in PNS regeneration may be altered or lacking from mature CNS axons by studying maturing CNS neurons in vitro. In rat cortical neurons, we find that integrins are present in axons during initial growth but later become restricted to the somato-dendritic domain. We investigated how this occurs and whether it can be altered to enhance axonal growth potential. We find a developmental change in integrin trafficking; transport becomes predominantly retrograde throughout axons, but not dendrites, as neurons mature. The directionality of transport is controlled through the activation state of ARF6, with developmental upregulation of the ARF6 GEF ARNO enhancing retrograde transport. Lowering ARF6 activity in mature neurons restores anterograde integrin flow, allows transport into axons, and increases axon growth. In addition, we found that the axon initial segment is partly responsible for exclusion of integrins and removal of this structure allows integrins into axons. Changing posttranslational modifications of tubulin with taxol also allows integrins into the proximal axon. The experiments suggest that the developmental loss of regenerative ability in CNS axons is due to exclusion of growth-related molecules due to changes in trafficking. PMID:26019348

  19. Limits to the capacity of transplants of olfactory glia to promote axonal regrowth in the CNS.

    PubMed

    Gudiño-Cabrera, G; Pastor, A M; de la Cruz, R R; Delgado-García, J M; Nieto-Sampedro, M

    2000-02-28

    Olfactory bulb ensheathing cell (OBEC) transplants promoted axonal regeneration in the spinal cord dorsal root entry zone and in the corticospinal tract. However, OBECs failed to promote abducens internuclear neuron axon regeneration when transplanted at the site of nerve fibre transection. In experiments performed in both cats and rats, OBECs survived for up to 2 months, lining themselves up along the portion of the regrowing axons proximal to the interneuron cell body. However, OBECs migrated preferentially towards abducens somata, in the direction opposite to the oculomotor nucleus target. OBECs seem to promote nerve fibre regeneration only where preferred direction of glial migration coincides with the direction of axonal growth towards its target.

  20. Optic nerve regeneration in the mouse is a complex trait modulated by genetic background

    PubMed Central

    Wang, Jiaxing; Li, Ying; King, Rebecca; Struebing, Felix L.

    2018-01-01

    Purpose The present study is designed to identify the influences of genetic background on optic nerve regeneration using the two parental strains (C57BL/6J and DBA/2J) and seven BXD recombinant inbred mouse strains. Methods To study regeneration in the optic nerve, Pten was knocked down in the retinal ganglion cells using adenoassociated virus (AAV) delivery of shRNA, and a mild inflammatory response was induced with an intravitreal injection of zymosan with CPT-cAMP. The axons of the retinal ganglion cells were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by cholera toxin B, and 2 days later, the regenerating axons within the optic nerve were examined. The number of axons at 0.5 mm and 1 mm from the crush site were counted. In addition, we measured the distance that five axons had grown down the nerve and the longest distance a single axon reached. Results The analysis revealed a considerable amount of differential axonal regeneration across the seven BXD strains and the parental strains. There was a statistically significant difference (p=0.014 Mann–Whitney U test) in the regenerative capacity in the number of axons reaching 0.5 mm from a low of 236.1±24.4 axons in the BXD102 mice to a high of 759.8±79.2 axons in the BXD29 mice. There were also statistically significant differences (p=0.014 Mann–Whitney U test) in the distance axons traveled. Looking at a minimum of five axons, the shortest distance was 787.2±46.5 µm in the BXD102 mice, and the maximum distance was 2025.5±223.3 µm in the BXD29 mice. Conclusions Differences in genetic background can have a profound effect on axonal regeneration causing a threefold increase in the number of regenerating axons at 0.5 mm from the crush site and a 2.5-fold increase in the distance traveled by at least five axons in the damaged optic nerve. PMID:29463955

  1. After facial nerve damage, regenerating axons become aberrant throughout the length of the nerve and not only at the site of the lesion: an experimental study.

    PubMed

    Choi, D; Raisman, G

    2004-02-01

    After facial nerve trauma, aberrant regeneration is associated with synkinesis. Animal models of mechanical nerve guides or reparative cell transplants at the site of a lesion have not been shown to improve disorganized regeneration. We examined whether this is because regenerating axons become disorganized throughout the length of the nerve and not only at the site of the lesion. In rats (n = 12), retrograde fluorescent tracer techniques were used to establish that most of the temporal branch fibres were carried in the superior half of the facial nerve trunk. In two further groups of rats (n = 24) a complete proximal facial nerve lesion was made, and the nerve immediately repaired by suture. After 4 weeks, at a second operation, the superior half of the facial nerve trunk was cut, either proximal or distal to the original lesion, and retrograde tracers were applied to distal branches of the nerve. It was possible to localize the points at which regenerating fibres became aberrant in their course by studying the number of labelled motoneurons in the facial nucleus after application of the tracer to the temporal branch of the nerve: this was similar in the distal and proximal hemisection groups, suggesting that aberrant axonal development occurred throughout the length of the nerve. Future strategies aimed at improving the organization of regeneration need to provide guidance cues not only at the site of the lesion as previously thought, but also throughout the length of the nerve.

  2. Role of Schwann cells in the regeneration of penile and peripheral nerves

    PubMed Central

    Wang, Lin; Sanford, Melissa T; Xin, Zhongcheng; Lin, Guiting; Lue, Tom F

    2015-01-01

    Schwann cells (SCs) are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED). Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1) the origin and development of SCs in the peripheral and penile nerve system; (2) Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3) how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4) and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED. PMID:25999359

  3. Regenerating reptile retinas: a comparative approach to restoring retinal ganglion cell function.

    PubMed

    Williams, D L

    2017-02-01

    Transection or damage to the mammalian optic nerve generally results in loss of retinal ganglion cells by apoptosis. This cell death is seen less in fish or amphibians where retinal ganglion cell survival and axon regeneration leads to recovery of sight. Reptiles lie somewhere in the middle of this spectrum of nerve regeneration, and different species have been reported to have a significant variation in their retinal ganglion cell regenerative capacity. The ornate dragon lizard Ctenophoris ornatus exhibits a profound capacity for regeneration, whereas the Tenerife wall lizard Gallotia galloti has a more variable response to optic nerve damage. Some individuals regain visual activity such as the pupillomotor responses, whereas in others axons fail to regenerate sufficiently. Even in Ctenophoris, although the retinal ganglion cell axons regenerate adequately enough to synapse in the tectum, they do not make long-term topographic connections allowing recovery of complex visually motivated behaviour. The question then centres on where these intraspecies differences originate. Is it variation in the innate ability of retinal ganglion cells from different species to regenerate with functional validity? Or is it variances between different species in the substrate within which the nerves regenerate, the extracellular environment of the damaged nerve or the supporting cells surrounding the regenerating axons? Investigations of retinal ganglion cell regeneration between different species of lower vertebrates in vivo may shed light on these questions. Or perhaps more interesting are in vitro studies comparing axon regeneration of retinal ganglion cells from various species placed on differing substrates.

  4. Atmospheric pressure plasma accelerates tail regeneration in tadpoles Xenopus laevis

    NASA Astrophysics Data System (ADS)

    Rivie, A.; Martus, K.; Menon, J.

    2017-08-01

    Atmospheric pressure plasma is a partially ionized gas composed of neutral and charged particles, including electrons and ions, as well as reactive oxygen species (ROS). Recently, it is utilized as possible therapy in oncology, sterilization, skin diseases, wound healing and tissue regeneration. In this study we focused on effect of plasma exposure on tail regeneration of tadpoles, Xenopus leavis with special emphasis on role of ROS, antioxidant defenses and morphological features of the regenerate. When amputated region of the tail was exposed to the helium plasma it resulted in a faster rate of growth, elevated ROS and increase in antioxidant enzymes in the regenerate compared to that of untreated control. An increase in nitric oxide (free radical) as well as activity of nitric oxide synthase(s) were observed once the cells of the regeneration blastema - a mass of proliferating cells are ready for differentiation. Microscopically the cells of the regenerate of plasma treated tadpoles show altered morphology and characteristics of cellular hypoxia and oxidative stress. We summarize that plasma exposure accelerates the dynamics of wound healing and tail regeneration through its effects on cell proliferation and differentiation as well as angiogenesis mediated through ROS signaling.

  5. Axonal sprouting and laminin appearance after destruction of glial sheaths.

    PubMed Central

    Masuda-Nakagawa, L M; Muller, K J; Nicholls, J G

    1993-01-01

    Laminin, a large extracellular matrix molecule, is associated with axonal outgrowth during development and regeneration of the nervous system in a variety of animals. In the leech central nervous system, laminin immunoreactivity appears after axon injury in advance of the regenerating axons. Although studies of vertebrate nervous system in culture have implicated glial and Schwann cells as possible sources, the cells that deposit laminin at sites crucial for regeneration in the living animal are not known. We have made a direct test to determine whether, in the central nervous system of the leech, cells other than ensheathing glial cells can produce laminin. Ensheathing glial cells of adult leeches were ablated selectively by intracellular injection of a protease. As a result, leech laminin accumulated within 10 days in regions of the central nervous system where it is not normally found, and undamaged, intact axons began to sprout extensively. In normal leeches laminin immunoreactivity is situated only in the basement membrane that surrounds the central nervous system, whereas after ablation of ensheathing glia it appeared in spaces through which neurons grew. Within days of ablation of the glial cell, small mobile phagocytes, or microglia, accumulated in the spaces formerly occupied by the glial cell. Microglia were concentrated at precisely the sites of new laminin appearance and axon sprouting. These results suggest that in the animal, as in culture, leech laminin promotes sprouting and that microglia may be responsible for its appearance. Images Fig. 1 Fig. 2 Fig. 3 PMID:8506343

  6. JNK1 induces hedgehog signaling from stellate cells to accelerate liver regeneration in mice.

    PubMed

    Langiewicz, Magda; Graf, Rolf; Humar, Bostjan; Clavien, Pierre A

    2018-04-28

    To improve outcomes of two-staged hepatectomies for large/multiple liver tumors, portal vein ligation (PVL) has been combined with parenchymal transection (associating liver partition and portal vein ligation for staged hepatectomy [coined ALPPS]) to greatly accelerate liver regeneration. In a novel ALPPS mouse model, we have reported paracrine Indian hedgehog (IHH) signaling from stellate cells as an early contributor to augmented regeneration. Here, we sought to identify upstream regulators of IHH. ALPPS in mice was compared against PVL and additional control surgeries. Potential IHH regulators were identified through in silico mining of transcriptomic data. c-Jun N-terminal kinase (JNK1 [Mapk8]) activity was reduced through SP600125 to evaluate its effects on IHH signaling. Recombinant IHH was injected after JNK1 diminution to substantiate their relationship during accelerated liver regeneration. Transcriptomic analysis linked Ihh to Mapk8. JNK1 upregulation after ALPPS was validated and preceded the IHH peak. On immunofluorescence, JNK1 and IHH co-localized in alpha-smooth muscle actin-positive non-parenchymal cells. Inhibition of JNK1 prior to ALPPS surgery reduced liver weight gain to PVL levels and was accompanied by downregulation of hepatocellular proliferation and the IHH-GLI1-CCND1 axis. In JNK1-inhibited mice, recombinant IHH restored ALPPS-like acceleration of regeneration and re-elevated JNK1 activity, suggesting the presence of a positive IHH-JNK1 feedback loop. JNK1-mediated induction of IHH paracrine signaling from hepatic stellate cells is essential for accelerated regeneration of parenchymal mass. The JNK1-IHH axis is a mechanism unique to ALPPS surgery and may point to therapeutic alternatives for patients with insufficient regenerative capacity. Associating liver partition and portal vein ligation for staged hepatectomy (so called ALPPS), is a new two-staged approach to hepatectomy, which induces an unprecedented acceleration of liver

  7. Axonal Regeneration in Mammals with Spinal Cord Injury

    DTIC Science & Technology

    1983-09-14

    Cajal, S. 1905. Notas preventivas sobre la degeneracion y regeneracion las vias nerviosos centrales . Trab. Lab. Invest. Biol. Univ. Madrid, 4: 295-301...S. 1914. Degeneracion y Regeneration del Sistema Nervioso , Vol. 1, 2. (Nicolas Moya, Madrid), Ramon y Cajal, S. 1928. Degeneration and Regeneration...field of central nervous system (CNS) regeneration research. These developments have revealed important aspects regarding the histology and

  8. Plasticity and regeneration in the injured spinal cord after cell transplantation therapy.

    PubMed

    Nori, Satoshi; Nakamura, Masaya; Okano, Hideyuki

    2017-01-01

    Spinal cord injury (SCI) typically damages the long axonal tracts of the spinal cord which results in permanent disability. However, regeneration of the injured spinal cord is approaching reality according to the advances in stem cell biology. Cell transplantation therapy holds potential to lead to recovery following SCI through some positive mechanisms. Grafted cells induce plasticity and regeneration in the injured spinal cord by promoting remyelination of damaged axons, reconstruction of neural circuits by synapse formation between host neurons and graft-derived neurons, and secreting neurotrophic factors to promote axonal elongation as well as reduce retrograde axonal degeneration. In this review, we will delineate (1) the microenvironment of the injured spinal cord that influence the plasticity and regeneration capacity after SCI, (2) a number of different kinds of cell transplantation therapies for SCI that has been extensively studied by researchers, and (3) potential mechanisms of grafted cell-induced regeneration and plasticity in the injured spinal cord. © 2017 Elsevier B.V. All rights reserved.

  9. Identifying motor and sensory myelinated axons in rabbit peripheral nerves by histochemical staining for carbonic anhydrase and cholinesterase activities

    NASA Technical Reports Server (NTRS)

    Riley, Danny A.; Sanger, James R.; Matloub, Hani S.; Yousif, N. John; Bain, James L. W.

    1988-01-01

    Carbonic anhydrase (CA) and cholinesterase (CE) histochemical staining of rabbit spinal nerve roots and dorsal root ganglia demonstrated that among the reactive myeliated axons, with minor exceptions, sensory axons were CA positive and CE negative whereas motor axons were CA negative and CE positive. The high specificity was achieved by adjusting reaction conditions to stain subpopulations of myelinated axons selectively while leaving 50 percent or so unstained. Fixation with glutaraldehyde appeared necessary for achieving selectivity. Following sciatic nerve transection, the reciprocal staining pattern persisted in damaged axons and their regenerating processes which formed neuromas within the proximal nerve stump. Within the neuromas, CA-stained sensory processes were elaborated earlier and in greater numbers than CE-stained regenerating motor processes. The present results indicate that histochemical axon typing can be exploited to reveal heterogeneous responses of motor and sensory axons to injury.

  10. Functional ionotropic glutamate receptors on peripheral axons and myelin.

    PubMed

    Christensen, Pia Crone; Welch, Nicole Cheryl; Brideau, Craig; Stys, Peter K

    2016-09-01

    Neurotransmitter-dependent signaling is traditionally restricted to axon terminals. However, receptors are present on myelinating glia, suggesting that chemical transmission may also occur along axons. Confocal microscopy and Ca(2+) -imaging using an axonally expressed FRET-based reporter was used to measure Ca(2+) changes and morphological alterations in myelin in response to stimulation of glutamate receptors. Activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors induced a Ca(2+) increase in axon cylinders. However, only the latter caused structural alterations in axons, despite similar Ca(2+) increases. Myelin morphology was significantly altered by NMDA receptor activation, but not by AMPA receptors. Cu(2+) ions influenced the NMDA receptor-dependent response, suggesting that this metal modulates axonal receptors. Glutamate increased ribosomal signal in Schwann cell cytoplasm. Axon cylinders and myelin of peripheral nervous system axons respond to glutamate, with a consequence being an increase in Schwann cell ribosomes. This may have implications for nerve pathology and regeneration. Muscle Nerve 54: 451-459, 2016. © 2016 Wiley Periodicals, Inc.

  11. Molecular mechanisms of optic axon guidance

    NASA Astrophysics Data System (ADS)

    Inatani, Masaru

    2005-12-01

    Axon guidance is one of the critical processes during vertebrate central nervous system (CNS) development. The optic nerve, which contains the axons of retinal ganglion cells, has been used as a powerful model to elucidate some of the mechanisms underlying axon guidance because it is easily manipulated experimentally, and its function is well understood. Recent molecular biology studies have revealed that numerous guidance molecules control the development of the visual pathway. This review introduces the molecular mechanisms involved in each critical step during optic axon guidance. Axonal projections to the optic disc are thought to depend on adhesion molecules and inhibitory extracellular matrices such as chondroitin sulfate. The formation of the head of the optic nerve and the optic chiasm require ligand-receptor interactions between netrin-1 and the deleted in colorectal cancer receptor, and Slit proteins and Robo receptors, respectively. The gradient distributions of ephrin ligands and Eph receptors are essential for correct ipsilateral projections at the optic chiasm and the topographic mapping of axons in the superior colliculus/optic tectum. The precise gradient is regulated by transcription factors determining the retinal dorso-ventral and nasal-temporal polarities. Moreover, the axon guidance activities by Slit and semaphorin 5A require the existence of heparan sulfate, which binds to numerous guidance molecules. Recent discoveries about the molecular mechanisms underlying optic nerve guidance will facilitate progress in CNS developmental biology and axon-regeneration therapy.

  12. LIMITED RECOVERY OF PINEAL FUNCTION AFTER REGENERATION OF PREGANGLIONIC SYMPATHETIC AXONS: EVIDENCE FOR LOSS OF GANGLIONIC SYNAPTIC SPECIFICITY

    PubMed Central

    Lingappa, Jaisri R.; Zigmond, Richard E.

    2013-01-01

    The cervical sympathetic trunks (CST) contain axons of preganglionic neurons that innervate the superior cervical ganglia (SCG). Since, regeneration of CST fibers can be extensive and can reestablish certain specific patterns of SCG connections, restoration of end organ function would be expected. This expectation was examined with respect to the pineal gland, an organ innervated by the two SCG. The activity of pineal serotonin N-acetyltransferase (NAT) exhibits a large circadian rhythm, with activity high at night, which is dependent on the gland’s sympathetic input. Thirty six hours after the CST were crushed bilaterally, nocturnal NAT was decreased by 99%. Three months later, enzyme activity had recovered only to 15% of control values, a recovery dependent on regeneration of CST fibers. Nevertheless, a small day-night rhythm was present in lesioned animals. Neither the density of the gland’s adrenergic innervation nor the ability of an adrenergic agonist to stimulate NAT activity was reduced in rats with regenerated CST. In addition, stimulation of the regenerated CST at a variety of frequencies was at least as effective in increasing NAT activity as seen with control nerves. These data suggest that the failure of pineal function to recover is not due to a quantitative deficit in the extent of reinnervation or in synaptic efficacy. Rather, we suggest that there is some loss of specificity in the synaptic connections made in the SCG during reinnervation, resulting in a loss of the central neuronal information necessary for directing a normal NAT rhythm and thus normal pineal function. PMID:23486957

  13. Role of Netrin-1 Signaling in Nerve Regeneration

    PubMed Central

    Dun, Xin-Peng; Parkinson, David B.

    2017-01-01

    Netrin-1 was the first axon guidance molecule to be discovered in vertebrates and has a strong chemotropic function for axonal guidance, cell migration, morphogenesis and angiogenesis. It is a secreted axon guidance cue that can trigger attraction by binding to its canonical receptors Deleted in Colorectal Cancer (DCC) and Neogenin or repulsion through binding the DCC/Uncoordinated (Unc5) A–D receptor complex. The crystal structures of Netrin-1/receptor complexes have recently been revealed. These studies have provided a structure based explanation of Netrin-1 bi-functionality. Netrin-1 and its receptor are continuously expressed in the adult nervous system and are differentially regulated after nerve injury. In the adult spinal cord and optic nerve, Netrin-1 has been considered as an inhibitor that contributes to axon regeneration failure after injury. In the peripheral nervous system, Netrin-1 receptors are expressed in Schwann cells, the cell bodies of sensory neurons and the axons of both motor and sensory neurons. Netrin-1 is expressed in Schwann cells and its expression is up-regulated after peripheral nerve transection injury. Recent studies indicated that Netrin-1 plays a positive role in promoting peripheral nerve regeneration, Schwann cell proliferation and migration. Targeting of the Netrin-1 signaling pathway could develop novel therapeutic strategies to promote peripheral nerve regeneration and functional recovery. PMID:28245592

  14. Stimulating effect of thyroid hormones in peripheral nerve regeneration: research history and future direction toward clinical therapy

    PubMed Central

    Barakat-Walter, I.; Kraftsik, R.

    2018-01-01

    Injury to peripheral nerves is often observed in the clinic and severe injuries may cause loss of motor and sensory functions. Despite extensive investigation, testing various surgical repair techniques and neurotrophic molecules, at present, a satisfactory method to ensuring successful recovery does not exist. For successful molecular therapy in nerve regeneration, it is essential to improve the intrinsic ability of neurons to survive and to increase the speed of axonal outgrowth. Also to induce Schwann cell phenotypical changes to prepare the local environment favorable for axonal regeneration and myelination. Therefore, any molecule that regulates gene expression of both neurons and Schwann cells could play a crucial role in peripheral nerve regeneration. Clinical and experimental studies have reported that thyroid hormones are essential for the normal development and function of the nervous system, so they could be candidates for nervous system regeneration. This review provides an overview of studies devoted to testing the effect of thyroid hormones on peripheral nerve regeneration. Also it emphasizes the importance of combining biodegradable tubes with local administration of triiodothyronine for future clinical therapy of human severe injured nerves. We highlight that the local and single administration of triiodothyronine within biodegradable nerve guide improves significantly the regeneration of severed peripheral nerves, and accelerates functional recovering. This technique provides a serious step towards future clinical application of triiodothyronine in human severe injured nerves. The possible regulatory mechanism by which triiodothyronine stimulates peripheral nerve regeneration is a rapid action on both axotomized neurons and Schwann cells. PMID:29722302

  15. Optofluidic control of axonal guidance

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Ordonez, Simon; Black, Bryan; Mohanty, Samarendra K.

    2013-03-01

    Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc) methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite particles were placed separately in one chamber and single particle was transported through microfluidic channel to another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.

  16. Sox11 Expression Promotes Regeneration of Some Retinal Ganglion Cell Types but Kills Others.

    PubMed

    Norsworthy, Michael W; Bei, Fengfeng; Kawaguchi, Riki; Wang, Qing; Tran, Nicholas M; Li, Yi; Brommer, Benedikt; Zhang, Yiming; Wang, Chen; Sanes, Joshua R; Coppola, Giovanni; He, Zhigang

    2017-06-21

    At least 30 types of retinal ganglion cells (RGCs) send distinct messages through the optic nerve to the brain. Available strategies of promoting axon regeneration act on only some of these types. Here we tested the hypothesis that overexpressing developmentally important transcription factors in adult RGCs could reprogram them to a "youthful" growth-competent state and promote regeneration of other types. From a screen of transcription factors, we identified Sox11 as one that could induce substantial axon regeneration. Transcriptome profiling indicated that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, α-RGCs, which preferentially regenerate following treatments such as Pten deletion, were killed by Sox11 overexpression. Thus, Sox11 promotes regeneration of non-α-RGCs, which are refractory to Pten deletion-induced regeneration. We conclude that Sox11 can reprogram adult RGCs to a growth-competent state, suggesting that different growth-promoting interventions promote regeneration in distinct neuronal types. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Review: peripheral nerve regeneration using non-tubular alginate gel crosslinked with covalent bonds.

    PubMed

    Hashimoto, Tadashi; Suzuki, Yoshihisa; Suzuki, Kyoko; Nakashima, Toshihide; Tanihara, Masao; Ide, Chizuka

    2005-06-01

    We have developed a nerve regeneration material consisting of alginate gel crosslinked with covalent bonds. in the first part of this study, we attempted to analyze nerve regeneration through alginate gel in the early stages within 2 weeks. in the second part, we tried to regenerate cat peripheral nerve by using alginate tubular or non-tubular nerve regeneration devices, and compared their efficacies. Four days after surgery, regenerating axons grew without Schwann cell investment through the partially degraded alginate gel, being in direct contact with the alginate without a basal lamina covering. One to 2 weeks after surgery, regenerating axons were surrounded by common Schwann cells, forming small bundles, with some axons at the periphery being partly in direct contact with alginate. At the distal stump, numerous Schwann cells had migrated into the alginate 8-14 days after surgery. Remarkable restorations of the 50-mm gap in cat sciatic nerve were obtained after a long term by using tubular or non-tubular nerve regeneration material consisting mainly of alginate gel. However, there was no significant difference between both groups at electrophysiological and morphological evaluation. Although, nowadays, nerve regeneration materials being marketed mostly have a tubular structure, our results suggest that the tubular structure is not indispensable for peripheral nerve regeneration.

  18. Nerve Cross-Bridging to Enhance Nerve Regeneration in a Rat Model of Delayed Nerve Repair

    PubMed Central

    2015-01-01

    There are currently no available options to promote nerve regeneration through chronically denervated distal nerve stumps. Here we used a rat model of delayed nerve repair asking of prior insertion of side-to-side cross-bridges between a donor tibial (TIB) nerve and a recipient denervated common peroneal (CP) nerve stump ameliorates poor nerve regeneration. First, numbers of retrogradely-labelled TIB neurons that grew axons into the nerve stump within three months, increased with the size of the perineurial windows opened in the TIB and CP nerves. Equal numbers of donor TIB axons regenerated into CP stumps either side of the cross-bridges, not being affected by target neurotrophic effects, or by removing the perineurium to insert 5-9 cross-bridges. Second, CP nerve stumps were coapted three months after inserting 0-9 cross-bridges and the number of 1) CP neurons that regenerated their axons within three months or 2) CP motor nerves that reinnervated the extensor digitorum longus (EDL) muscle within five months was determined by counting and motor unit number estimation (MUNE), respectively. We found that three but not more cross-bridges promoted the regeneration of axons and reinnervation of EDL muscle by all the CP motoneurons as compared to only 33% regenerating their axons when no cross-bridges were inserted. The same 3-fold increase in sensory nerve regeneration was found. In conclusion, side-to-side cross-bridges ameliorate poor regeneration after delayed nerve repair possibly by sustaining the growth-permissive state of denervated nerve stumps. Such autografts may be used in human repair surgery to improve outcomes after unavoidable delays. PMID:26016986

  19. Retention of retinal axon collateral is responsible for induced ipsilateral retinotectal projections in adult goldfish.

    PubMed

    Sharma, S C; Tsai, C

    1991-01-01

    In normal goldfish, optic axons innervate only the contralateral optic tectum. When one eye was enucleated and the optic nerve of the other eye crushed, the regenerating optic axons innervated both optic tecta. We studied the presence of bilaterally projecting retinal ganglion cells by double retrograde cell labeling methods using Nuclear Yellow and True Blue dyes. About 10% of the retinal ganglion cells were double labeled and these cells were found throughout the retina. In addition, HRP application to the ipsilateral tectum revealed retrogradely-labeled retinal ganglion cells of all morphological types. These results suggest that induced ipsilateral projections are formed by regenerating axon collaterals and that all cell types are involved in the generation of normal mirror image typography.

  20. The Impact of Motor Axon Misdirection and Attrition on Behavioral Deficit Following Experimental Nerve Injuries

    PubMed Central

    Alant, Jacob Daniel de Villiers; Senjaya, Ferry; Ivanovic, Aleksandra; Forden, Joanne; Shakhbazau, Antos; Midha, Rajiv

    2013-01-01

    Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r2=.67), motor neuron counts (attrition) (p<.0001, r2=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly

  1. Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat

    PubMed Central

    Jiang, Wenli; Wang, Yuexiang; Tang, Jie; Peng, Jiang; Wang, Yu; Guo, Quanyi; Guo, Zhiyuan; Li, Pan; Xiao, Bo; Zhang, Jinxing

    2016-01-01

    Low intensity pulsed ultrasound (LIPUS) has been widely used in clinic for the treatment of repairing pseudarthrosis, bone fractures and of healing in various soft tissues. Some reports indicated that LIPUS accelerated peripheral nerve regeneration including Schwann cells (SCs) and injured nerves. But little is known about its appropriate intensities on autograft nerves. This study was to investigate which intensity of LIPUS improved the regeneration of gold standard postsurgical nerves in experimental rat model. Sprague-Dawley rats were made into 10 mm right side sciatic nerve reversed autologous nerve transplantation and randomly treated with 250 mW/cm2, 500 mW/cm2 or 750 mW/cm2 LIPUS for 2–12 weeks after operation. Functional and pathological results showed that LIPUS of 250 mW/cm2 significantly induced faster rate of axonal regeneration. This suggested that autograft nerve regeneration was improved. PMID:27102358

  2. DRG axon elongation and growth cone collapse rate induced by Sema3A are differently dependent on NGF concentration.

    PubMed

    Kaselis, Andrius; Treinys, Rimantas; Vosyliūtė, Rūta; Šatkauskas, Saulius

    2014-03-01

    Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon's regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.

  3. Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury.

    PubMed

    Lin, Tao; Qiu, Shuai; Yan, Liwei; Zhu, Shuang; Zheng, Canbin; Zhu, Qingtang; Liu, Xiaolin

    2018-05-01

    Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Fifty Sprague-Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit-8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57: 821-828, 2018. © 2017 Wiley Periodicals, Inc.

  4. A cellular spinal cord scaffold seeded with rat adipose-derived stem cells facilitates functional recovery via enhancing axon regeneration in spinal cord injured rats

    PubMed Central

    Yin, Hong; Jiang, Tao; Deng, Xi; Yu, Miao; Xing, Hui; Ren, Xianjun

    2018-01-01

    Spinal cord injury (SCI), usually resulting in severe sensory and motor deficits, is a major public health concern. Adipose-derived stem cells (ADSCs), one type of adult stem cell, are free from ethical restriction, easily isolated and enriched. Therefore, ADSCs may provide a feasible cell source for cell-based therapies in treatment of SCI. The present study successfully isolated rat ADSCs (rADSCs) from Sprague-Dawley male rats and co-cultured them with acellular spinal cord scaffolds (ASCs). Then, a rat spinal cord hemisection model was built and rats were randomly divided into 3 groups: SCI only, ASC only, and ASC + ADSCs. Furthermore, behavioral tests were conducted to evaluate functional recovery. Hematoxylin & Eosin staining and immunofluorence were carried out to assess histopathological remodeling. In addition, biotinylated dextran amines anterograde tracing was employed to visualize axon regeneration. The data demonstrated that harvested cells, which were positive for cell surface antigen cluster of differentiation (CD) 29, CD44 and CD90 and negative for CD4, detected by flow cytometry analysis, held the potential to differentiate into osteocytes and adipocytes. Rats that received transplantation of ASCs seeded with rADSCs benefited greatly in functional recovery through facilitation of histopathological rehabilitation, axon regeneration and reduction of reactive gliosis. rADSCs co-cultured with ASCs may survive and integrate into the host spinal cord on day 14 post-SCI. PMID:29257299

  5. Electrical stimulation accelerates motor functional recovery in autograft-repaired 10 mm femoral nerve gap in rats.

    PubMed

    Huang, Jinghui; Hu, Xueyu; Lu, Lei; Ye, Zhengxu; Wang, Yuqing; Luo, Zhuojing

    2009-10-01

    Electrical stimulation has been shown to enhance peripheral nerve regeneration after nerve injury. However, the impact of electrical stimulation on motor functional recovery after nerve injuries, especially over long nerve gap lesions, has not been investigated in a comprehensive manner. In the present study, we aimed to determine whether electrical stimulation (1 h, 20 Hz) is beneficial for motor functional recovery after a 10 mm femoral nerve gap lesion in rats. The proximal nerve stump was electrically stimulated for 1 h at 20 Hz frequency prior to nerve repair with an autologous graft. The rate of motor functional recovery was evaluated by single frame motion analysis and electrophysiological studies, and the nerve regeneration was investigated by double labeling and histological analysis. We found that brief electrical stimulation significantly accelerated motor functional recovery and nerve regeneration. Although the final outcome, both in functional terms and morphological terms, was not improved by electrical stimulation, the observed acceleration of functional recovery and axon regeneration may be of therapeutic importance in clinical setting.

  6. The Role of Direct Current Electric Field-Guided Stem Cell Migration in Neural Regeneration.

    PubMed

    Yao, Li; Li, Yongchao

    2016-06-01

    Effective directional axonal growth and neural cell migration are crucial in the neural regeneration of the central nervous system (CNS). Endogenous currents have been detected in many developing nervous systems. Experiments have demonstrated that applied direct current (DC) electric fields (EFs) can guide axonal growth in vitro, and attempts have been made to enhance the regrowth of damaged spinal cord axons using DC EFs in in vivo experiments. Recent work has revealed that the migration of stem cells and stem cell-derived neural cells can be guided by DC EFs. These studies have raised the possibility that endogenous and applied DC EFs can be used to direct neural tissue regeneration. Although the mechanism of EF-directed axonal growth and cell migration has not been fully understood, studies have shown that the polarization of cell membrane proteins and the activation of intracellular signaling molecules are involved in the process. The application of EFs is a promising biotechnology for regeneration of the CNS.

  7. An ex vivo laser-induced spinal cord injury model to assess mechanisms of axonal degeneration in real-time.

    PubMed

    Okada, Starlyn L M; Stivers, Nicole S; Stys, Peter K; Stirling, David P

    2014-11-25

    Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular

  8. Axon-Sorting Multifunctional Nerve Guides: Accelerating Restoration of Nerve Function

    DTIC Science & Technology

    2014-10-01

    factor (singly & in selected combinations) in the organotypic model system for preferential sensory or motor axon extension. Use confocal microscopy to...track axon extension of labeled sensory or motor neurons from spinal cord slices (motor) or dorsal root ganglia ( DRG ) (sensory). 20 Thy1-YFP mice...RESEARCH ACCOMPLISHMENTS: • Established a system of color-coded mixed nerve tracking using GFP and RFP expressing motor and sensory neurons (Figure 1

  9. Multifunctional Silk Nerve Guides for Axon Outgrowth

    NASA Astrophysics Data System (ADS)

    Tupaj, Marie C.

    Peripheral nerve regeneration is a critical issue as 2.8% of trauma patients present with this type of injury, estimating a total of 200,000 nerve repair procedures yearly in the United States. While the peripheral nervous system exhibits slow regeneration, at a rate of 0.5 mm -- 9 mm/day following trauma, this regenerative ability is only possible under certain conditions. Clinical repairs have changed slightly in the last 30 years and standard methods of treatment include suturing damaged nerve ends, allografting, and autografting, with the autograft the gold standard of these approaches. Unfortunately, the use of autografts requires a second surgery and there is a shortage of nerves available for grafting. Allografts are a second option however allografts have lower success rates and are accompanied by the need of immunosuppressant drugs. Recently there has been a focus on developing nerve guides as an "off the shelf" approach. Although some natural and synthetic guidance channels have been approved by the FDA, these nerve guides are unfunctionalized and repair only short gaps, less than 3 cm in length. The goal of this project was to identify strategies for functionalizing peripheral nerve conduits for the outgrowth of neuron axons in vitro . To accomplish this, two strategies (bioelectrical and biophysical) were indentified for increasing axon outgrowth and promoting axon guidance. Bioelectrical strategies exploited electrical stimulation for increasing neurite outgrowth. Biophysical strategies tested a range of surface topographies for axon guidance. Novel methods were developed for integrating electrical and biophysical strategies into silk films in 2D. Finally, a functionalized nerve conduit system was developed that integrated all strategies for the purpose of attaching, elongating, and guiding nervous tissue in vitro. Future directions of this work include silk conduit translation into a rat sciatic nerve model in vivo for the purpose of repairing long

  10. BDNF gene delivery within and beyond templated agarose multi-channel guidance scaffolds enhances peripheral nerve regeneration

    NASA Astrophysics Data System (ADS)

    Gao, Mingyong; Lu, Paul; Lynam, Dan; Bednark, Bridget; Campana, W. Marie; Sakamoto, Jeff; Tuszynski, Mark

    2016-12-01

    Objective. We combined implantation of multi-channel templated agarose scaffolds with growth factor gene delivery to examine whether this combinatorial treatment can enhance peripheral axonal regeneration through long sciatic nerve gaps. Approach. 15 mm long scaffolds were templated into highly organized, strictly linear channels, mimicking the linear organization of natural nerves into fascicles of related function. Scaffolds were filled with syngeneic bone marrow stromal cells (MSCs) secreting the growth factor brain derived neurotrophic factor (BDNF), and lentiviral vectors expressing BDNF were injected into the sciatic nerve segment distal to the scaffold implantation site. Main results. Twelve weeks after injury, scaffolds supported highly linear regeneration of host axons across the 15 mm lesion gap. The incorporation of BDNF-secreting cells into scaffolds significantly increased axonal regeneration, and additional injection of viral vectors expressing BDNF into the distal segment of the transected nerve significantly enhanced axonal regeneration beyond the lesion. Significance. Combinatorial treatment with multichannel bioengineered scaffolds and distal growth factor delivery significantly improves peripheral nerve repair, rivaling the gold standard of autografts.

  11. Deficiency in Monocarboxylate Transporter 1 (MCT1) in Mice Delays Regeneration of Peripheral Nerves following Sciatic Nerve Crush

    PubMed Central

    Morrison, Brett M.; Tsingalia, Akivaga; Vidensky, Svetlana; Lee, Youngjin; Jin, Lin; Farah, Mohamed H.; Lengacher, Sylvain; Magistretti, Pierre J.; Pellerin, Luc; Rothstein, Jeffrey D.

    2014-01-01

    Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence and MCT1 tdTomato BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves in MCT1 heterozygous null mice are crushed and peripheral nerve regeneration quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21 days in wild-type mice to greater than 38 days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42 days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42 days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4 weeks and tibial mixed sensory and motor nerve at 3 weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly through failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush. PMID:25447940

  12. JUN regulates early transcriptional responses to axonal injury in retinal ganglion cells.

    PubMed

    Fernandes, Kimberly A; Harder, Jeffrey M; Kim, Jessica; Libby, Richard T

    2013-07-01

    The AP1 family transcription factor JUN is an important molecule in the neuronal response to injury. In retinal ganglion cells (RGCs), JUN is upregulated soon after axonal injury and disrupting JUN activity delays RGC death. JUN is known to participate in the control of many different injury response pathways in neurons, including pathways controlling cell death and axonal regeneration. The role of JUN in regulating genes involved in cell death, ER stress, and regeneration was tested to determine the overall importance of JUN in regulating RGC response to axonal injury. Genes from each of these pathways were transcriptionally controlled following axonal injury and Jun deficiency altered the expression of many of these genes. The differentially expressed genes included, Atf3, Ddit3, Ecel1, Gadd45α, Gal, Hrk, Pten, Socs3, and Sprr1a. Two of these genes, Hrk and Atf3, were tested for importance in RGC death using null alleles of each gene. Disruption of the prodeath Bcl2 family member Hrk did not affect the rate or amount of RGC death after axonal trauma. Deficiency in the ATF/CREB family transcription factor Atf3 did lessen the amount of RGC death after injury, though it did not provide long term protection to RGCs. Since JUN's dimerization partner determines its transcriptional targets, the expression of several candidate AP1 family members were examined. Multiple AP1 family members were induced by axonal injury and had a different expression profile in Jun deficient retinas compared to wildtype retinas (Fosl1, Fosl2 and Jund). Overall, JUN appears to play a multifaceted role in regulating RGC response to axonal injury. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Spatial temperature gradients guide axonal outgrowth

    PubMed Central

    Black, Bryan; Vishwakarma, Vivek; Dhakal, Kamal; Bhattarai, Samik; Pradhan, Prabhakar; Jain, Ankur; Kim, Young-tae; Mohanty, Samarendra

    2016-01-01

    Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects. PMID:27460512

  14. Spatial temperature gradients guide axonal outgrowth

    NASA Astrophysics Data System (ADS)

    Black, Bryan; Vishwakarma, Vivek; Dhakal, Kamal; Bhattarai, Samik; Pradhan, Prabhakar; Jain, Ankur; Kim, Young-Tae; Mohanty, Samarendra

    2016-07-01

    Formation of neural networks during development and regeneration after injury depends on accuracy of axonal pathfinding, which is primarily believed to be influenced by chemical cues. Recently, there is growing evidence that physical cues can play crucial role in axonal guidance. However, detailed mechanism involved in such guidance cues is lacking. By using weakly-focused near-infrared continuous wave (CW) laser microbeam in the path of an advancing axon, we discovered that the beam acts as a repulsive guidance cue. Here, we report that this highly-effective at-a-distance guidance is the result of a temperature field produced by the near-infrared laser light absorption. Since light absorption by extracellular medium increases when the laser wavelength was red shifted, the threshold laser power for reliable guidance was significantly lower in the near-infrared as compared to the visible spectrum. The spatial temperature gradient caused by the near-infrared laser beam at-a-distance was found to activate temperature-sensitive membrane receptors, resulting in an influx of calcium. The repulsive guidance effect was significantly reduced when extracellular calcium was depleted or in the presence of TRPV1-antagonist. Further, direct heating using micro-heater confirmed that the axonal guidance is caused by shallow temperature-gradient, eliminating the role of any non-photothermal effects.

  15. Spinal Injury: Regeneration, Recovery, and a Possible New Approach

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cohen, Avis

    Spinal injury is most frequent in young healthy men, desperate to walk. Most treatments have focused on regeneration of the injured axons, but no one has as yet achieved success with this approach. However, in the lamprey, a primitive fish with a spinal cord having all the critical features of the human spinal cored, spinal injury is followed by complete regeneration of injured axons. Additionally, the animal recovers the ability to swim, and in many, the swimming is normal. Unfortunately, in most others, it is highly abnormal. This talk will review evidence from the abnormal regeneration, why it bespeaks difficultiesmore » heretofore not considered, and suggest an alternate approach for the near future. In so doing, the speaker will introduce the normal function of the spinal cord, what happens in normal and abnormal regeneration, and the new techniques that employ methods from neuromorphic engineering, a synthesis of neuroscience and engineering to engineer smart devices.« less

  16. Spinal Injury: Regeneration, Recovery, and a Possible New Approach

    ScienceCinema

    Cohen, Avis [University of Maryland, College Park, Maryland, United States

    2017-12-09

    Spinal injury is most frequent in young healthy men, desperate to walk. Most treatments have focused on regeneration of the injured axons, but no one has as yet achieved success with this approach. However, in the lamprey, a primitive fish with a spinal cord having all the critical features of the human spinal cored, spinal injury is followed by complete regeneration of injured axons. Additionally, the animal recovers the ability to swim, and in many, the swimming is normal. Unfortunately, in most others, it is highly abnormal. This talk will review evidence from the abnormal regeneration, why it bespeaks difficulties heretofore not considered, and suggest an alternate approach for the near future. In so doing, the speaker will introduce the normal function of the spinal cord, what happens in normal and abnormal regeneration, and the new techniques that employ methods from neuromorphic engineering, a synthesis of neuroscience and engineering to engineer smart devices.

  17. Curcumin promotes nerve regeneration and functional recovery after sciatic nerve crush injury in diabetic rats.

    PubMed

    Ma, Junxiong; Yu, Hailong; Liu, Jun; Chen, Yu; Wang, Qi; Xiang, Liangbi

    2016-01-01

    Curcumin is capable of promoting peripheral nerve regeneration in normal condition. However, it is unclear whether its beneficial effect on nerve regeneration still exists under diabetic mellitus. The present study was designed to investigate such a possibility. Diabetes in rats was developed by a single dose of streptozotocin at 50 mg/kg. Immediately after nerve crush injury, the diabetic rats were intraperitoneally administrated daily for 4 weeks with curcumin (50 mg/kg, 100 mg/kg and 300 mg/kg), or normal saline, respectively. The axonal regeneration was investigated by morphometric analysis and retrograde labeling. The functional recovery was evaluated by electrophysiological studies and behavioral analysis. Axonal regeneration and functional recovery was significantly enhanced by curcumin, which were significantly better than those in vehicle saline group. In addition, high doses of curcumin (100 mg/kg and 300 mg/kg) achieved better axonal regeneration and functional recovery than low dose (50 mg/kg). In conclusion, curcumin is capable of promoting nerve regeneration after sciatic nerve crush injury in diabetes mellitus, highlighting its therapeutic values as a neuroprotective agent for peripheral nerve injury repair in diabetes mellitus. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Functional evaluation of peripheral nerve regeneration and target reinnervation in animal models: a critical overview.

    PubMed

    Navarro, Xavier

    2016-02-01

    Peripheral nerve injuries usually lead to severe loss of motor, sensory and autonomic functions in the patients. Due to the complex requirements for adequate axonal regeneration, functional recovery is often poorly achieved. Experimental models are useful to investigate the mechanisms related to axonal regeneration and tissue reinnervation, and to test new therapeutic strategies to improve functional recovery. Therefore, objective and reliable evaluation methods should be applied for the assessment of regeneration and function restitution after nerve injury in animal models. This review gives an overview of the most useful methods to assess nerve regeneration, target reinnervation and recovery of complex sensory and motor functions, their values and limitations. The selection of methods has to be adequate to the main objective of the research study, either enhancement of axonal regeneration, improving regeneration and reinnervation of target organs by different types of nerve fibres, or increasing recovery of complex sensory and motor functions. It is generally recommended to use more than one functional method for each purpose, and also to perform morphological studies of the injured nerve and the reinnervated targets. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  19. Nogo-A is involved in secondary axonal degeneration of thalamus in hypertensive rats with focal cortical infarction.

    PubMed

    Wang, Fang; Liang, Zhijian; Hou, Qinghua; Xing, Shihui; Ling, Li; He, Meixia; Pei, Zhong; Zeng, Jinsheng

    2007-05-07

    We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid betaA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.

  20. The "waiting period" of sensory and motor axons in early chick hindlimb: its role in axon pathfinding and neuronal maturation.

    PubMed

    Wang, G; Scott, S A

    2000-07-15

    During embryonic development motor axons in the chick hindlimb grow out slightly before sensory axons and wait in the plexus region at the base of the limb for approximately 24 hr before invading the limb itself (Tosney and Landmesser, 1985a). We have investigated the role of this waiting period by asking, Is the arrest of growth cones in the plexus region a general property of both sensory and motor axons? Why do axons wait? Does eliminating the waiting period affect the further development of motor and sensory neurons? Here we show that sensory axons, like motor axons, pause in the plexus region and that neither sensory nor motor axons require cues from the other population to wait in or exit from the plexus region. By transplanting older or younger donor limbs to host embryos, we show that host axons innervate donor limbs on a schedule consistent with the age of the grafted limbs. Thus, axons wait in the plexus region for maturational changes to occur in the limb rather than in the neurons themselves. Both sensory and motor axons innervate their appropriate peripheral targets when the waiting period is eliminated by grafting older donor limbs. Therefore, axons do not require a prolonged period in the plexus region to sort out and project appropriately. Eliminating the waiting period does, however, accelerate the onset of naturally occurring cell death, but it does not enhance the development of central projections or the biochemical maturation of sensory neurons.

  1. Chondroitin-4-sulfation negatively regulates axonal guidance and growth

    PubMed Central

    Wang, Hang; Katagiri, Yasuhiro; McCann, Thomas E.; Unsworth, Edward; Goldsmith, Paul; Yu, Zu-Xi; Tan, Fei; Santiago, Lizzie; Mills, Edward M.; Wang, Yu; Symes, Aviva J.; Geller, Herbert M.

    2008-01-01

    Summary Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition, and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate (CS) mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated CS GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings provide the evidence showing that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function. PMID:18768934

  2. Acceleration of Regeneration of Large Gap Peripheral Nerve Injuries Using Acellular Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS)

    DTIC Science & Technology

    2016-09-01

    AWARD NUMBER: W811XWH-13-1-0310 TITLE: Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Acellular Nerve Allografts...plus amniotic Fluid Derived Stem Cells (AFS). PRINCIPAL INVESTIGATOR: Zhongyu Li, MD, PhD RECIPIENT: Wake Forest University Health Sciences...REPORT DATE September 2016 2. REPORT TYPE Annual 3. DATES COVERED 1Sep2015 - 31Aug2016 4. TITLE AND SUBTITLE Acceleration of Regeneration of Large

  3. Recovery of function, peripheral sensitization and sensory neurone activation by novel pathways following axonal injury in Aplysia californica.

    PubMed

    Dulin, M F; Steffensen, I; Morris, C E; Walters, E T

    1995-10-01

    Recovery of behavioural and sensory function was examined following unilateral pedal nerve crush in Aplysia californica. Nerve crush that transected all axons connecting the tail to the central nervous system (CNS) eliminated the ipsilateral tail-evoked siphon reflex, whose sensory input travels in the crushed tail nerve (p9). The first reliable signs of recovery of this reflex were observed within 1 week, and most animals displayed tail-evoked siphon responses within 2 weeks. Wide-dynamic-range mechanosensory neurons with somata in the ventrocaudal (VC) cluster of the ipsilateral pleural ganglion exhibited a few receptive fields (RFs) on the tail 3 weeks after unilateral pedal nerve crush, indicating that the RFs had either regenerated or been reconnected to the central somata. These RFs were smaller and sensitized compared with corresponding RFs on the contralateral, uncrushed side. Centrally conducted axon responses of VC sensory neurones to electrical stimulation distal to the nerve crush site did not reappear until at least 10 days after the crush. Because the crush site was much closer to the CNS than to the tail, the failure of axon responses to be restored earlier than the behavioural responses indicates that early stages of reflex recovery are not due to regeneration of VC sensory neurone axons into the tail. Following nerve crush, VC sensory neurones often could be activated by stimulating central connectives or peripheral nerves that do not normally contain the sensory neurone's axons. These results suggest that recovery of behavioral function after nerve injury involves complex mechanisms, including regenerative growth of axotomized VC sensory neurones, sensitization of regenerating RFs and sprouting of VC sensory neurone fibres within the CNS. Furthermore, the rapidity of behavioural recovery indicates that its initial phases are mediated by additional mechanisms, perhaps centripetal regeneration of unidentified sensory neurones having peripheral

  4. Deficiency in monocarboxylate transporter 1 (MCT1) in mice delays regeneration of peripheral nerves following sciatic nerve crush.

    PubMed

    Morrison, Brett M; Tsingalia, Akivaga; Vidensky, Svetlana; Lee, Youngjin; Jin, Lin; Farah, Mohamed H; Lengacher, Sylvain; Magistretti, Pierre J; Pellerin, Luc; Rothstein, Jeffrey D

    2015-01-01

    Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence in wild-type mice and tdTomato fluorescence in MCT1 BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves of MCT1 heterozygous null mice are crushed and peripheral nerve regeneration was quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21 days in wild-type mice to greater than 38 days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42 days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42 days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4 weeks and tibial mixed sensory and motor nerve at 3 weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly due to failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Differential expression of GAP-43 and neurofilament during peripheral nerve regeneration through bio-artificial conduits.

    PubMed

    Carriel, Víctor; Garzón, Ingrid; Campos, Antonio; Cornelissen, Maria; Alaminos, Miguel

    2017-02-01

    Nerve conduits are promising alternatives for repairing nerve gaps; they provide a close microenvironment that supports nerve regeneration. In this sense, histological analysis of axonal growth is a determinant to achieve successful nerve regeneration. To evaluate this process, the most-used immunohistochemical markers are neurofilament (NF), β-III tubulin and, infrequently, GAP-43. However, GAP-43 expression in long-term nerve regeneration models is still poorly understood. In this study we analysed GAP-43 expression and its correlation with NF and S-100, using three tissue-engineering approaches with different regeneration profiles. A 10 mm gap was created in the sciatic nerve of 12 rats and repaired using collagen conduits or collagen conduits filled with fibrin-agarose hydrogels or with hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs). After 12 weeks the conduits were harvested for histological analysis. Our results confirm the long-term expression of GAP-43 in all groups. The expression of GAP-43 and NF was significantly higher in the group with ADMSCs. Interestingly, GAP-43 was observed in immature, newly formed axons and NF in thicker and mature axons. These proteins were not co-expressed, demonstrating their differential expression in newly formed nerve fascicles. Our descriptive and quantitative histological analysis of GAP-43 and NFL allowed us to determine, with high accuracy, the heterogenic population of axons at different stages of maturation in three tissue-engineering approaches. Finally, to perform a complete assessment of axonal regeneration, the quantitative immunohistochemical evaluation of both GAP-43 and NF could be a useful quality control in tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  6. In vivo assessment of peripheral nerve regeneration by diffusion tensor imaging.

    PubMed

    Morisaki, Shinsuke; Kawai, Yuko; Umeda, Masahiro; Nishi, Mayumi; Oda, Ryo; Fujiwara, Hiroyoshi; Yamada, Kei; Higuchi, Toshihiro; Tanaka, Chuzo; Kawata, Mitsuhiro; Kubo, Toshikazu

    2011-03-01

    To evaluate the sensitivity of diffusion tensor imaging (DTI) in assessing peripheral nerve regeneration in vivo. We assessed the changes in the DTI parameters and histological analyses after nerve injury to examine degeneration and regeneration in the rat sciatic nerves. For magnetic resonance imaging (MRI), 16 rats were randomly divided into two groups: group P (permanently crushed; n = 7) and group T (temporally crushed; n = 9). Serial MRI of the right leg was performed before the operation, and then performed at the timepoints of 1, 2, 3, and 4 weeks after the crush injury. The changes in fractional anisotropy (FA), axial diffusivity (λ(∥)), and radial diffusivity (λ(⟂)) were quantified. For histological analyses, the number of axons and the myelinated axon areas were quantified. Decreased FA and increased λ(⟂) were observed in the degenerative phase, and increased FA and decreased λ(⟂) were observed in the regenerative phase. The changes in FA and λ(⟂) were strongly correlated with histological changes, including axonal and myelin regeneration. DTI parameters, especially λ(⟂) , can be good indicators for peripheral nerve regeneration and can be applied as noninvasive diagnostic tools for a variety of neurological diseases. Copyright © 2011 Wiley-Liss, Inc.

  7. A regenerative microchannel neural interface for recording from and stimulating peripheral axons in vivo

    NASA Astrophysics Data System (ADS)

    FitzGerald, James J.; Lago, Natalia; Benmerah, Samia; Serra, Jordi; Watling, Christopher P.; Cameron, Ruth E.; Tarte, Edward; Lacour, Stéphanie P.; McMahon, Stephen B.; Fawcett, James W.

    2012-02-01

    Neural interfaces are implanted devices that couple the nervous system to electronic circuitry. They are intended for long term use to control assistive technologies such as muscle stimulators or prosthetics that compensate for loss of function due to injury. Here we present a novel design of interface for peripheral nerves. Recording from axons is complicated by the small size of extracellular potentials and the concentration of current flow at nodes of Ranvier. Confining axons to microchannels of ˜100 µm diameter produces amplified potentials that are independent of node position. After implantation of microchannel arrays into rat sciatic nerve, axons regenerated through the channels forming ‘mini-fascicles’, each typically containing ˜100 myelinated fibres and one or more blood vessels. Regenerated motor axons reconnected to distal muscles, as demonstrated by the recovery of an electromyogram and partial prevention of muscle atrophy. Efferent motor potentials and afferent signals evoked by muscle stretch or cutaneous stimulation were easily recorded from the mini-fascicles and were in the range of 35-170 µV. Individual motor units in distal musculature were activated from channels using stimulus currents in the microampere range. Microchannel interfaces are a potential solution for applications such as prosthetic limb control or enhancing recovery after nerve injury.

  8. Olfactory Ensheathing Cell Transplantation after a Complete Spinal Cord Transection Mediates Neuroprotective and Immunomodulatory Mechanisms to Facilitate Regeneration

    PubMed Central

    Khankan, Rana R.; Griffis, Khris G.; Haggerty-Skeans, James R.; Zhong, Hui; Roy, Roland R.; Edgerton, V. Reggie

    2016-01-01

    Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury. SIGNIFICANCE STATEMENT Spinal cord injury creates physical and chemical barriers to axon regeneration. We used a complete spinal cord transection model and olfactory ensheathing cell (OEC) or

  9. Aptamer antagonists of myelin-derived inhibitors promote axon growth.

    PubMed

    Wang, Yuxuan; Khaing, Zin Z; Li, Na; Hall, Brad; Schmidt, Christine E; Ellington, Andrew D

    2010-03-16

    Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.

  10. Aptamer Antagonists of Myelin-Derived Inhibitors Promote Axon Growth

    PubMed Central

    Wang, Yuxuan; Khaing, Zin Z.; Li, Na; Hall, Brad; Schmidt, Christine E.; Ellington, Andrew D.

    2010-01-01

    Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators. PMID:20300533

  11. The Molecular and Cellular Mechanisms of Axon Guidance in Mossy Fiber Sprouting

    PubMed Central

    Koyama, Ryuta; Ikegaya, Yuji

    2018-01-01

    The question of whether mossy fiber sprouting is epileptogenic has not been resolved; both sprouting-induced recurrent excitatory and inhibitory circuit hypotheses have been experimentally (but not fully) supported. Therefore, whether mossy fiber sprouting is a potential therapeutic target for epilepsy remains under debate. Moreover, the axon guidance mechanisms of mossy fiber sprouting have attracted the interest of neuroscientists. Sprouting of mossy fibers exhibits several uncommon axonal growth features in the basically non-plastic adult brain. For example, robust branching of axonal collaterals arises from pre-existing primary mossy fiber axons. Understanding the branching mechanisms in adulthood may contribute to axonal regeneration therapies in neuroregenerative medicine in which robust axonal re-growth is essential. Additionally, because granule cells are produced throughout life in the neurogenic dentate gyrus, it is interesting to examine whether the mossy fibers of newly generated granule cells follow the pre-existing trajectories of sprouted mossy fibers in the epileptic brain. Understanding these axon guidance mechanisms may contribute to neuron transplantation therapies, for which the incorporation of transplanted neurons into pre-existing neural circuits is essential. Thus, clarifying the axon guidance mechanisms of mossy fiber sprouting could lead to an understanding of central nervous system (CNS) network reorganization and plasticity. Here, we review the molecular and cellular mechanisms of axon guidance in mossy fiber sprouting by discussing mainly in vitro studies. PMID:29896153

  12. Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

    PubMed

    Quintá, Héctor R; Wilson, Carlos; Blidner, Ada G; González-Billault, Christian; Pasquini, Laura A; Rabinovich, Gabriel A; Pasquini, Juana M

    2016-09-01

    Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel

  13. Planarian GSK3s are involved in neural regeneration.

    PubMed

    Adell, Teresa; Marsal, Maria; Saló, Emili

    2008-02-01

    Glycogen synthase kinase-3 (GSK3) is a key element in several signaling cascades that is known to be involved in both patterning and neuronal organization. It is, therefore, a good candidate to play a role in neural regeneration in planarians. We report the characterization of three GSK3 genes in Schmidtea mediterranea. Phylogenetic analysis shows that Smed-GSK3.1 is highly conserved compared to GSK3 sequences from other species, whereas Smed-GSK3.2 and Smed-GSK3.3 are more divergent. Treatment of regenerating planarians with 1-azakenpaullone, a synthetic GSK3 inhibitor, suggests that planarian GSK3s are essential for normal differentiation and morphogenesis of the nervous system. Cephalic ganglia appear smaller and disconnected in 1-azakenpaullone-treated animals, whereas visual axons are ectopically projected, and the pharynx does not regenerate properly. This phenotype is consistent with a role for Smed-GSK3s in neuronal polarization and axonal growth.

  14. High plasticity of axonal pathology in Alzheimer's disease mouse models.

    PubMed

    Blazquez-Llorca, Lidia; Valero-Freitag, Susana; Rodrigues, Eva Ferreira; Merchán-Pérez, Ángel; Rodríguez, J Rodrigo; Dorostkar, Mario M; DeFelipe, Javier; Herms, Jochen

    2017-02-07

    Axonal dystrophies (AxDs) are swollen and tortuous neuronal processes that are associated with extracellular depositions of amyloid β (Aβ) and have been observed to contribute to synaptic alterations occurring in Alzheimer's disease. Understanding the temporal course of this axonal pathology is of high relevance to comprehend the progression of the disease over time. We performed a long-term in vivo study (up to 210 days of two-photon imaging) with two transgenic mouse models (dE9xGFP-M and APP-PS1xGFP-M). Interestingly, AxDs were formed only in a quarter of GFP-expressing axons near Aβ-plaques, which indicates a selective vulnerability. AxDs, especially those reaching larger sizes, had long lifetimes and appeared as highly plastic structures with large variations in size and shape and axonal sprouting over time. In the case of the APP-PS1 mouse only, the formation of new long axonal segments in dystrophic axons (re-growth phenomenon) was observed. Moreover, new AxDs could appear at the same point of the axon where a previous AxD had been located before disappearance (re-formation phenomenon). In addition, we observed that most AxDs were formed and developed during the imaging period, and numerous AxDs had already disappeared by the end of this time. This work is the first in vivo study analyzing quantitatively the high plasticity of the axonal pathology around Aβ plaques. We hypothesized that a therapeutically early prevention of Aβ plaque formation or their growth might halt disease progression and promote functional axon regeneration and the recovery of neural circuits.

  15. Schwann Cell Glycogen Selectively Supports Myelinated Axon Function

    PubMed Central

    Brown, Angus M; Evans, Richard D; Black, Joel; Ransom, Bruce R

    2012-01-01

    Objectives Interruption of energy supply to peripheral axons is a cause of axon loss. We determined if glycogen was present in mammalian peripheral nerve, and if it supported axon conduction during aglycemia. Methods We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. Results Glycogen was present in sciatic nerve, its concentration varying directly with ambient [glucose]. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time-course of glycogen loss. Latency to CAP failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. Interpretation Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. PMID:23034913

  16. Full spinal cord regeneration after total transection is not possible due to entropy change.

    PubMed

    Zielinski, P; Sokal, P

    2016-09-01

    Transected spinal cord regeneration is a main challenge of regenerative medicine. The mainstream of research is focused on the promotion of spinal axons growth, which is strongly inhibited in mammals. Assuming that the inhibition of the axonal growth may be ever overcome, the complexity of neural reconnections may be the second serious stand to overcome. Peripheral nerve axons regeneration seem to form a random pattern of their targets reconnections. The hypothesis is that due to the laws of entropy or irreversible information loss the full spinal cord restoration after the transection is not possible. The hypothesis is discussed based on several assumptions. Simplifying the dissertation spinal cord is represented by 2millions of pyramidal axons. After the transection each of these axons has to make a growth and reconnect with exactly matching targets below the transection, in the same number. Axons are guided by neurotrophic factors and afterwards reconnected with neuroplasticity mechanisms. Assuming random reconnections, there are 2,000,000! permutations [Formula: see text] , therefore the chance of ideally random but correct reconnection of pyramidal axons with adequate targets is 1/2,000,000!. Apart from pyramidal axons, there are other axons, like extrapyramidal, sensory and associative. Empirical data and analysis of neurotrophic factors and organogenesis mechanisms may seem to slightly contradict the hypothesis, but strictly adhering to the second law of thermodynamics and entropy laws the full restoration of the transected cord may never be possible. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Nonexpanded Adipose Stromal Vascular Fraction Local Therapy on Peripheral Nerve Regeneration Using Allografts.

    PubMed

    Mohammadi, Rahim; Mehrtash, Moein; Mehrtash, Moeid; Sajjadi, Seyedeh-Sepideh

    2016-06-01

    Adipose tissue possesses a population of multi-potent stem cells which can be differentiated to a Schwann cell phenotype and may be of benefit for treatment of peripheral nerve injuries. Effects of local therapy of nonexpanded adipose stromal vascular fraction (SVF) on peripheral nerve regeneration was studied using allografts in a rat sciatic nerve model. Thirty male white Wistar rats were divided into three experimental groups (n = 10), randomly: Sham-operated group (SHAM), allograft group (ALLO), SVF-treated group (ALLO/SVF). In SHAM group left sciatic nerve was exposed through a gluteal muscle incision and after homeostasis muscle was sutured. In the ALLO group the left sciatic nerve was exposed through a gluteal muscle incision and transected proximal to the tibio-peroneal bifurcation where a 10 mm segment was excised. The same procedure was performed in the ALLO/SVF group. The harvested nerves of the rats of ALLO group were served as allograft for ALLO/SVF group and vice versa. The SHAM and ALLO groups received 100 μL phosphate buffered saline and the ALLO/SVF group received 100 μL SVF (2.25 ± 0.45 × 10(7) cells) locally where the grafting was performed. Behavioral, functional, biomechanical, and gastrocnemius muscle mass showed earlier regeneration of axons in ALLO/SVF than in ALLO group (p < .05). Histomorphometic and immunohistochemical studies also showed earlier regeneration of axons in ALLO/SVF than in ALLO group (p < .05). Administration of nonexpanded SVF could accelerate functional recovery after nerve allografting in sciatic nerve. It may have clinical implications for the surgical management of patients after nerve transection.

  18. Schwann cell glycogen selectively supports myelinated axon function.

    PubMed

    Brown, Angus M; Evans, Richard D; Black, Joel; Ransom, Bruce R

    2012-09-01

    Interruption of energy supply to peripheral axons is a cause of axon loss. We determined whether glycogen was present in mammalian peripheral nerve, and whether it supported axon conduction during aglycemia. We used biochemical assay and electron microscopy to determine the presence of glycogen, and electrophysiology to monitor axon function. Glycogen was present in sciatic nerve, its concentration varying directly with ambient glucose. Electron microscopy detected glycogen granules primarily in myelinating Schwann cell cytoplasm, and these diminished after exposure to aglycemia. During aglycemia, conduction failure in large myelinated axons (A fibers) mirrored the time course of glycogen loss. Latency to compound action potential (CAP) failure was directly related to nerve glycogen content at aglycemia onset. Glycogen did not benefit the function of slow-conducting, small-diameter unmyelinated axons (C fibers) during aglycemia. Blocking glycogen breakdown pharmacologically accelerated CAP failure during aglycemia in A fibers, but not in C fibers. Lactate was as effective as glucose in supporting sciatic nerve function, and was continuously released into the extracellular space in the presence of glucose and fell rapidly during aglycemia. Our findings indicated that glycogen is present in peripheral nerve, primarily in myelinating Schwann cells, and exclusively supports large-diameter, myelinated axon conduction during aglycemia. Available evidence suggests that peripheral nerve glycogen breaks down during aglycemia and is passed, probably as lactate, to myelinated axons to support function. Unmyelinated axons are not protected by glycogen and are more vulnerable to dysfunction during periods of hypoglycemia. . Copyright © 2012 American Neurological Association.

  19. Astrocytes as gate-keepers in optic nerve regeneration--a mini-review.

    PubMed

    García, Dana M; Koke, Joseph R

    2009-02-01

    Animals that develop without extra-embryonic membranes (anamniotes--fish, amphibians) have impressive regenerative capacity, even to the extent of replacing entire limbs. In contrast, animals that develop within extra-embryonic membranes (amniotes--reptiles, birds, mammals) have limited capacity for regeneration as adults, particularly in the central nervous system (CNS). Much is known about the process of nerve development in fish and mammals and about regeneration after lesions in the CNS in fish and mammals. Because the retina of the eye and optic nerve are functionally part of the brain and are accessible in fish, frogs, and mice, optic nerve lesion and regeneration (ONR) has been extensively used as a model system for study of CNS nerve regeneration. When the optic nerve of a mouse is severed, the axons leading into the brain degenerate. Initially, the cut end of the axons on the proximal, eye-side of the injury sprout neurites which begin to grow into the lesion. Simultaneously, astrocytes of the optic nerve become activated to initiate wound repair as a first step in reestablishing the structural integrity of the optic nerve. This activation appears to initiate a cascade of molecular signals resulting in apoptotic cell death of the retinal ganglion cells axons of which make up the neural component of the optic nerve; regeneration fails and the injury is permanent. Evidence specifically implicating astrocytes comes from studies showing selective poisoning of astrocytes at the optic nerve lesion, along with activation of a gene whose product blocks apoptosis in retinal ganglion cells, creates conditions favorable to neurites sprouting from the cut proximal stump, growing through the lesion and into the distal portion of the injured nerve, eventually reaching appropriate targets in the brain. In anamniotes, astrocytes ostensibly present no such obstacle since optic nerve regeneration occurs without intervention; however, no systematic study of glial involvement

  20. Macroscopic in vivo imaging of facial nerve regeneration in Thy1-GFP rats.

    PubMed

    Placheta, Eva; Wood, Matthew D; Lafontaine, Christine; Frey, Manfred; Gordon, Tessa; Borschel, Gregory H

    2015-01-01

    Facial nerve injury leads to severe functional and aesthetic deficits. The transgenic Thy1-GFP rat is a new model for facial nerve injury and reconstruction research that will help improve clinical outcomes through translational facial nerve injury research. To determine whether serial in vivo imaging of nerve regeneration in the transgenic rat model is possible, facial nerve regeneration was imaged under the main paradigms of facial nerve injury and reconstruction. Fifteen male Thy1-GFP rats, which express green fluorescent protein (GFP) in their neural structures, were divided into 3 groups in the laboratory: crush-injury, direct repair, and cross-face nerve grafting (30-mm graft length). The distal nerve stump or nerve graft was predegenerated for 2 weeks. The facial nerve of the transgenic rats was serially imaged at the time of operation and after 2, 4, and 8 weeks of regeneration. The imaging was performed under a GFP-MDS-96/BN excitation stand (BLS Ltd). Facial nerve injury. Optical fluorescence of regenerating facial nerve axons. Serial in vivo imaging of the regeneration of GFP-positive axons in the Thy1-GFP rat model is possible. All animals survived the short imaging procedures well, and nerve regeneration was followed over clinically relevant distances. The predegeneration of the distal nerve stump or the cross-face nerve graft was, however, necessary to image the regeneration front at early time points. Crush injury was not suitable to sufficiently predegenerate the nerve (and to allow for degradation of the GFP through Wallerian degeneration). After direct repair, axons regenerated over the coaptation site in between 2 and 4 weeks. The GFP-positive nerve fibers reached the distal end of the 30-mm-long cross-face nervegrafts after 4 to 8 weeks of regeneration. The time course of facial nerve regeneration was studied by serial in vivo imaging in the transgenic rat model. Nerve regeneration was followed over clinically relevant distances in a small

  1. Intracellular calcium release through IP3R or RyR contributes to secondary axonal degeneration.

    PubMed

    Orem, Ben C; Pelisch, Nicolas; Williams, Joshua; Nally, Jacqueline M; Stirling, David P

    2017-10-01

    Severed CNS axons often retract or dieback away from the injury site and fail to regenerate. The precise mechanisms underlying acute axonal dieback and secondary axonal degeneration remain poorly understood. Here we investigate the role of Ca 2+ store mediated intra-axonal Ca 2+ release in acute axonal dieback and secondary axonal degeneration. To differentiate between primary (directly transected) and "bystander" axonal injury (axons spared by the initial injury but then succumb to secondary degeneration) in real-time we use our previously published highly focal laser-induced spinal cord injury (LiSCI) ex vivo model. Ascending spinal cord dorsal column axons that express YFP were severed using an 800 nm laser pulse while being imaged continuously using two-photon excitation microscopy. We inhibited two major intra-axonal Ca 2+ store channels, ryanodine receptors (RyR) and IP 3 R, with ryanodine or 2-APB, respectively, to individually determine their role in axonal dieback and secondary axonal degeneration. Each antagonist was dissolved in artificial CSF and applied 1h post-injury alone or in combination, and continuously perfused for the remainder of the imaging session. Initially following LiSCI, transected axons retracted equal distances both distal and proximal to the lesion. However, by 4h after injury, the distal axonal segments that are destined for Wallerian degeneration had significantly retracted further than their proximal counterparts. We also found that targeting either RyR or IP 3 R using pharmacological and genetic approaches significantly reduced proximal axonal dieback and "bystander" secondary degeneration of axons compared to vehicle controls at 6h post-injury. Combined treatment effects on secondary axonal degeneration were similar to either drug in isolation. Together, these results suggest that intra-axonal Ca 2+ store mediated Ca 2+ release through RyR or IP 3 R contributes to secondary axonal degeneration following SCI. Copyright © 2017

  2. Peripheral Nerve Regeneration by Secretomes of Stem Cells from Human Exfoliated Deciduous Teeth.

    PubMed

    Sugimura-Wakayama, Yukiko; Katagiri, Wataru; Osugi, Masashi; Kawai, Takamasa; Ogata, Kenichi; Sakaguchi, Kohei; Hibi, Hideharu

    2015-11-15

    Peripheral nerve regeneration across nerve gaps is often suboptimal, with poor functional recovery. Stem cell transplantation-based regenerative therapy is a promising approach for axon regeneration and functional recovery of peripheral nerve injury; however, the mechanisms remain controversial and unclear. Recent studies suggest that transplanted stem cells promote tissue regeneration through a paracrine mechanism. We investigated the effects of conditioned media derived from stem cells from human exfoliated deciduous teeth (SHED-CM) on peripheral nerve regeneration. In vitro, SHED-CM-treated Schwann cells exhibited significantly increased proliferation, migration, and the expression of neuron-, extracellular matrix (ECM)-, and angiogenesis-related genes. SHED-CM stimulated neuritogenesis of dorsal root ganglia and increased cell viability. Similarly, SHED-CM enhanced tube formation in an angiogenesis assay. In vivo, a 10-mm rat sciatic nerve gap model was bridged by silicon conduits containing SHED-CM or serum-free Dulbecco's modified Eagle's medium. Light and electron microscopy confirmed that the number of myelinated axons and axon-to-fiber ratio (G-ratio) were significantly higher in the SHED-CM group at 12 weeks after nerve transection surgery. The sciatic functional index (SFI) and gastrocnemius (target muscle) wet weight ratio demonstrated functional recovery. Increased compound muscle action potentials and increased SFI in the SHED-CM group suggested sciatic nerve reinnervation of the target muscle and improved functional recovery. We also observed reduced muscle atrophy in the SHED-CM group. Thus, SHEDs may secrete various trophic factors that enhance peripheral nerve regeneration through multiple mechanisms. SHED-CM may therefore provide a novel therapy that creates a more desirable extracellular microenvironment for peripheral nerve regeneration.

  3. A Novel Internal Fixator Device for Peripheral Nerve Regeneration

    PubMed Central

    Chuang, Ting-Hsien; Wilson, Robin E.; Love, James M.; Fisher, John P.

    2013-01-01

    Recovery from peripheral nerve damage, especially for a transected nerve, is rarely complete, resulting in impaired motor function, sensory loss, and chronic pain with inappropriate autonomic responses that seriously impair quality of life. In consequence, strategies for enhancing peripheral nerve repair are of high clinical importance. Tension is a key determinant of neuronal growth and function. In vitro and in vivo experiments have shown that moderate levels of imposed tension (strain) can encourage axonal outgrowth; however, few strategies of peripheral nerve repair emphasize the mechanical environment of the injured nerve. Toward the development of more effective nerve regeneration strategies, we demonstrate the design, fabrication, and implementation of a novel, modular nerve-lengthening device, which allows the imposition of moderate tensile loads in parallel with existing scaffold-based tissue engineering strategies for nerve repair. This concept would enable nerve regeneration in two superposed regimes of nerve extension—traditional extension through axonal outgrowth into a scaffold and extension in intact regions of the proximal nerve, such as that occurring during growth or limb-lengthening. Self-sizing silicone nerve cuffs were fabricated to grip nerve stumps without slippage, and nerves were deformed by actuating a telescoping internal fixator. Poly(lactic co-glycolic) acid (PLGA) constructs mounted on the telescoping rods were apposed to the nerve stumps to guide axonal outgrowth. Neuronal cells were exposed to PLGA using direct contact and extract methods, and they exhibited no signs of cytotoxic effects in terms of cell morphology and viability. We confirmed the feasibility of implanting and actuating our device within a sciatic nerve gap and observed axonal outgrowth following device implantation. The successful fabrication and implementation of our device provides a novel method for examining mechanical influences on nerve regeneration. PMID

  4. Uncultured undifferentiated adipose-derived nucleated cell fractions combined with inside-out artery graft accelerate sciatic nerve regeneration and functional recovery.

    PubMed

    Mohammadi, R; Asadollahi, A; Amini, K

    2014-09-01

    Effects of transplantation of adipose-derived nucleated cell fractions (ADNCs) on sciatic nerve regeneration were studied. A 10-mm sciatic nerve defect was bridged using artery graft filled with ADNCs. In control group, artery graft was filled with saline alone. Regenerated nerve fibres were studied for 12 weeks. In sham-operated group, sciatic nerve was only exposed and manipulated. Behavioural and functional studies confirmed faster recovery of regenerated axons in ADNCs transplanted animals than in control group (P<0.05). At the end of study period, animals in ADNCs transplanted group achieved a sciatic functional index (SFI) value of -31.6 ± -3.14, whereas in control group a value of -42.5 ± -3.7 was found. Gastrocnemius muscle mass in ADNCs transplanted animals was found to be significantly higher than that in control group (P=0.001). Morphometric indices of regenerated fibres showed the number and diameter of myelinated fibres to be significantly higher in ADNCs transplanted animals than in control group (P=0.001). On immunohistochemistry, there was more positive staining of S100 in the ADNCs transplanted animals than in control group. ADNCs transplantation into an artery graft could be considered a readily accessible technique that improves functional recovery of sciatic nerve. Copyright © 2014 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  5. Modelling the impact of altered axonal morphometry on the response of regenerative nervous tissue to electrical stimulation through macro-sieve electrodes.

    PubMed

    Zellmer, Erik R; MacEwan, Matthew R; Moran, Daniel W

    2018-04-01

    Regenerated peripheral nervous tissue possesses different morphometric properties compared to undisrupted nerve. It is poorly understood how these morphometric differences alter the response of the regenerated nerve to electrical stimulation. In this work, we use computational modeling to explore the electrophysiological response of regenerated and undisrupted nerve axons to electrical stimulation delivered by macro-sieve electrodes (MSEs). A 3D finite element model of a peripheral nerve segment populated with mammalian myelinated axons and implanted with a macro-sieve electrode has been developed. Fiber diameters and morphometric characteristics representative of undisrupted or regenerated peripheral nervous tissue were assigned to core conductor models to simulate the two tissue types. Simulations were carried out to quantify differences in thresholds and chronaxie between undisrupted and regenerated fiber populations. The model was also used to determine the influence of axonal caliber on recruitment thresholds for the two tissue types. Model accuracy was assessed through comparisons with in vivo recruitment data from chronically implanted MSEs. Recruitment thresholds of individual regenerated fibers with diameters  >2 µm were found to be lower compared to same caliber undisrupted fibers at electrode to fiber distances of less than about 90-140 µm but roughly equal or higher for larger distances. Caliber redistributions observed in regenerated nerve resulted in an overall increase in average recruitment thresholds and chronaxie during whole nerve stimulation. Modeling results also suggest that large diameter undisrupted fibers located close to a longitudinally restricted current source such as the MSE have higher average recruitment thresholds compared to small diameter fibers. In contrast, large diameter regenerated nerve fibers located in close proximity of MSE sites have, on average, lower recruitment thresholds compared to small fibers. Utilizing

  6. Modelling the impact of altered axonal morphometry on the response of regenerative nervous tissue to electrical stimulation through macro-sieve electrodes

    NASA Astrophysics Data System (ADS)

    Zellmer, Erik R.; MacEwan, Matthew R.; Moran, Daniel W.

    2018-04-01

    Objective. Regenerated peripheral nervous tissue possesses different morphometric properties compared to undisrupted nerve. It is poorly understood how these morphometric differences alter the response of the regenerated nerve to electrical stimulation. In this work, we use computational modeling to explore the electrophysiological response of regenerated and undisrupted nerve axons to electrical stimulation delivered by macro-sieve electrodes (MSEs). Approach. A 3D finite element model of a peripheral nerve segment populated with mammalian myelinated axons and implanted with a macro-sieve electrode has been developed. Fiber diameters and morphometric characteristics representative of undisrupted or regenerated peripheral nervous tissue were assigned to core conductor models to simulate the two tissue types. Simulations were carried out to quantify differences in thresholds and chronaxie between undisrupted and regenerated fiber populations. The model was also used to determine the influence of axonal caliber on recruitment thresholds for the two tissue types. Model accuracy was assessed through comparisons with in vivo recruitment data from chronically implanted MSEs. Main results. Recruitment thresholds of individual regenerated fibers with diameters  >2 µm were found to be lower compared to same caliber undisrupted fibers at electrode to fiber distances of less than about 90-140 µm but roughly equal or higher for larger distances. Caliber redistributions observed in regenerated nerve resulted in an overall increase in average recruitment thresholds and chronaxie during whole nerve stimulation. Modeling results also suggest that large diameter undisrupted fibers located close to a longitudinally restricted current source such as the MSE have higher average recruitment thresholds compared to small diameter fibers. In contrast, large diameter regenerated nerve fibers located in close proximity of MSE sites have, on average, lower recruitment thresholds

  7. Spontaneous regeneration of the corticospinal tract after transection in young rats: a key role of reactive astrocytes in making favorable and unfavorable conditions for regeneration.

    PubMed

    Iseda, T; Nishio, T; Kawaguchi, S; Yamanoto, M; Kawasaki, T; Wakisaka, S

    2004-01-01

    We demonstrated the occurrence of marked regeneration of the corticospinal tract (CST) after a single transection and failure of regeneration after a repeated transection in young rats. To provide convincing evidence for the complete transection and regeneration we used retrograde neuronal double labeling. Double-labeled neurons that took up the first tracer from the transection site and the second tracer from the injection site caudal to the transection site were observed in the sensorimotor cortex. The anterograde tracing method revealed various patterns of regeneration. In the most successful cases the vast majority of regenerated fibers descended in the normal tract and terminated normally whereas a trace amount of fibers coursed aberrantly. In the less successful cases fibers descended partly normally and partly aberrantly or totally aberrantly. To clarify the role of astrocytes in determining the success or failure of regeneration we compared expression of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) immunoreactivity (IR) in the lesion between single and repeated transections. In either transection, astrocytes disappeared from the CST near the lesion site as early as 3 h after lesioning. However, by 24 h after a single transection, immature astrocytes coexpressing GFAP- and vimentin-IR appeared in the former astrocyte-free area and NF-positive axons crossed the lesion. By contrast, after a repeated transection the astrocyte-free area spread and NF-positive axons never crossed the lesion. It appears likely that the major sign, and possibly cause of failure of regeneration is the prolonged disappearance of astrocytes in the lesioned tract area. Copyright 2004 IBRO

  8. Inhibition of KLF7-Targeting MicroRNA 146b Promotes Sciatic Nerve Regeneration.

    PubMed

    Li, Wen-Yuan; Zhang, Wei-Ting; Cheng, Yong-Xia; Liu, Yan-Cui; Zhai, Feng-Guo; Sun, Ping; Li, Hui-Ting; Deng, Ling-Xiao; Zhu, Xiao-Feng; Wang, Ying

    2018-06-01

    A previous study has indicated that Krüppel-like factor 7 (KLF7), a transcription factor that stimulates Schwann cell (SC) proliferation and axonal regeneration after peripheral nerve injury, is a promising therapeutic transcription factor in nerve injury. We aimed to identify whether inhibition of microRNA-146b (miR-146b) affected SC proliferation, migration, and myelinated axon regeneration following sciatic nerve injury by regulating its direct target KLF7. SCs were transfected with miRNA lentivirus, miRNA inhibitor lentivirus, or KLF7 siRNA lentivirus in vitro. The expression of miR146b and KLF7, as well as SC proliferation and migration, were subsequently evaluated. In vivo, an acellular nerve allograft (ANA) followed by injection of GFP control vector or a lentiviral vector encoding an miR-146b inhibitor was used to assess the repair potential in a model of sciatic nerve gap. miR-146b directly targeted KLF7 by binding to the 3'-UTR, suppressing KLF7. Up-regulation of miR-146b and KLF7 knockdown significantly reduced the proliferation and migration of SCs, whereas silencing miR-146b resulted in increased proliferation and migration. KLF7 protein was localized in SCs in which miR-146b was expressed in vivo. Similarly, 4 weeks after the ANA, anti-miR-146b increased KLF7 and its target gene nerve growth factor cascade, promoting axonal outgrowth. Closer analysis revealed improved nerve conduction and sciatic function index score, and enhanced expression of neurofilaments, P0 (anti-peripheral myelin), and myelinated axon regeneration. Our findings provide new insight into the regulation of KLF7 by miR-146b during peripheral nerve regeneration and suggest a potential therapeutic strategy for peripheral nerve injury.

  9. Pre-differentiation of mesenchymal stromal cells in combination with a microstructured nerve guide supports peripheral nerve regeneration in the rat sciatic nerve model.

    PubMed

    Boecker, Arne Hendrik; van Neerven, Sabien Geraldine Antonia; Scheffel, Juliane; Tank, Julian; Altinova, Haktan; Seidensticker, Katrin; Deumens, Ronald; Tolba, Rene; Weis, Joachim; Brook, Gary Anthony; Pallua, Norbert; Bozkurt, Ahmet

    2016-02-01

    Many bioartificial nerve guides have been investigated pre-clinically for their nerve regeneration-supporting function, often in comparison to autologous nerve transplantation, which is still regarded as the current clinical gold standard. Enrichment of these scaffolds with cells intended to support axonal regeneration has been explored as a strategy to boost axonal regeneration across these nerve guides Ansselin et al. (1998). In the present study, 20 mm rat sciatic nerve defects were implanted with a cell-seeded microstructured collagen nerve guide (Perimaix) or an autologous nerve graft. Under the influence of seeded, pre-differentiated mesenchymal stromal cells, axons regenerated well into the Perimaix nerve guide. Myelination-related parameters, like myelin sheath thickness, benefitted from an additional seeding with pre-differentiated mesenchymal stromal cells. Furthermore, both the number of retrogradely labelled sensory neurons and the axon density within the implant were elevated in the cell-seeded scaffold group with pre-differentiated mesenchymal stromal cells. However, a pre-differentiation had no influence on functional recovery. An additional cell seeding of the Perimaix nerve guide with mesenchymal stromal cells led to an extent of functional recovery, independent of the differentiation status, similar to autologous nerve transplantation. These findings encourage further investigations on pre-differentiated mesenchymal stromal cells as a cellular support for peripheral nerve regeneration. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Dose-Dependent Differential Effect of Neurotrophic Factors on In Vitro and In Vivo Regeneration of Motor and Sensory Neurons

    PubMed Central

    Santos, Daniel; Gonzalez-Perez, Francisco; Navarro, Xavier

    2016-01-01

    Although peripheral axons can regenerate after nerve transection and repair, functional recovery is usually poor due to inaccurate reinnervation. Neurotrophic factors promote directional guidance to regenerating axons and their selective application may help to improve functional recovery. Hence, we have characterized in organotypic cultures of spinal cord and dorsal root ganglia the effect of GDNF, FGF-2, NGF, NT-3, and BDNF at different concentrations on motor and sensory neurite outgrowth. In vitro results show that GDNF and FGF-2 enhanced both motor and sensory neurite outgrowth, NGF and NT-3 were the most selective to enhance sensory neurite outgrowth, and high doses of BDNF selectively enhanced motor neurite outgrowth. Then, NGF, NT-3, and BDNF (as the most selective factors) were delivered in a collagen matrix within a silicone tube to repair the severed sciatic nerve of rats. Quantification of Fluorogold retrolabeled neurons showed that NGF and NT-3 did not show preferential effect on sensory regeneration whereas BDNF preferentially promoted motor axons regeneration. Therefore, the selective effects of NGF and NT-3 shown in vitro are lost when they are applied in vivo, but a high dose of BDNF is able to selectively enhance motor neuron regeneration both in vitro and in vivo. PMID:27867665

  11. Diffuse axonal injury by assault.

    PubMed

    Imajo, T; Challener, R C; Roessmann, U

    1987-09-01

    A case of diffuse axonal injury (DAI) by assault is reported. The majority of DAI cases documented have been due to traffic accidents and some due to falls from height. DAI is caused by angular or rotational acceleration of the victim's head. The condition is common and is the second most important head injury after subdural hematoma with regard to death. Its clinical picture is characterized by immediate and prolonged coma or demented state. Because of the subtle nature of histological changes in DAI, awareness and intentional search for the lesion is essential. The triad of DAI is as follows: focal lesions (hemorrhages and/or lacerations) in the corpus callosum and brain stem, and microscopic demonstration of axonal damage--retraction balls. The concept of DAI will elucidate and enhance the understanding of many head trauma cases.

  12. Biodegradable and plasma-treated electrospun scaffolds coated with recombinant Olfactomedin-like 3 for accelerating wound healing and tissue regeneration.

    PubMed

    Dunn, Louise L; de Valence, Sarra; Tille, Jean-Christophe; Hammel, Philippe; Walpoth, Beat H; Stocker, Roland; Imhof, Beat A; Miljkovic-Licina, Marijana

    2016-11-01

    Three-dimensional biomimetic scaffolds resembling the native extracellular matrix (ECM) are widely used in tissue engineering, however they often lack optimal bioactive cues needed for acceleration of cell proliferation, neovascularization, and tissue regeneration. In this study, the use of the ECM-related protein Olfactomedin-like 3 (Olfml3) demonstrates the importance and feasibility of fabricating efficient bioactive scaffolds without in vitro cell seeding prior to in vivo implantation. First, in vivo proangiogenic properties of Olfml3 were shown in a murine wound healing model by accelerated wound closure and a 1.4-fold increase in wound vascularity. Second, subcutaneous implantation of tubular scaffolds coated with recombinant Olfml3 resulted in enhanced cell in-growth and neovascularization compared with control scaffolds. Together, our data indicates the potential of Olfml3 to accelerate neovascularization during tissue regeneration by promoting endothelial cell proliferation and migration. This study provides a promising concept for the reconstruction of damaged tissue using affordable and effective bioactive scaffolds. © 2016 by the Wound Healing Society.

  13. Platelet-Rich Plasma Promotes Axon Regeneration, Wound Healing, and Pain Reduction: Fact or Fiction.

    PubMed

    Kuffler, Damien P

    2015-10-01

    Platelet-rich plasma (PRP) has been tested in vitro, in animal models, and clinically for its efficacy in enhancing the rate of wound healing, reducing pain associated with injuries, and promoting axon regeneration. Although extensive data indicate that PRP-released factors induce these effects, the claims are often weakened because many studies were not rigorous or controlled, the data were limited, and other studies yielded contrary results. Critical to assessing whether PRP is effective are the large number of variables in these studies, including the method of PRP preparation, which influences the composition of PRP; type of application; type of wounds; target tissues; and diverse animal models and clinical studies. All these variables raise the question of whether one can anticipate consistent influences and raise the possibility that most of the results are correct under the circumstances where PRP was tested. This review examines evidence on the potential influences of PRP and whether PRP-released factors could induce the reported influences and concludes that the preponderance of evidence suggests that PRP has the capacity to induce all the claimed influences, although this position cannot be definitively argued. Well-defined and rigorously controlled studies of the potential influences of PRP are required in which PRP is isolated and applied using consistent techniques, protocols, and models. Finally, it is concluded that, because of the purported benefits of PRP administration and the lack of adverse events, further animal and clinical studies should be performed to explore the potential influences of PRP.

  14. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

    PubMed Central

    Li, Yiqing; Andereggen, Lukas; Yuki, Kenya; Omura, Kumiko; Yin, Yuqin; Gilbert, Hui-Ya; Erdogan, Burcu; Asdourian, Maria S.; Shrock, Christine; de Lima, Silmara; Apfel, Ulf-Peter; Zhuo, Yehong; Hershfinkel, Michal; Lippard, Stephen J.; Benowitz, Larry

    2017-01-01

    Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration. PMID:28049831

  15. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration.

    PubMed

    Li, Yiqing; Andereggen, Lukas; Yuki, Kenya; Omura, Kumiko; Yin, Yuqin; Gilbert, Hui-Ya; Erdogan, Burcu; Asdourian, Maria S; Shrock, Christine; de Lima, Silmara; Apfel, Ulf-Peter; Zhuo, Yehong; Hershfinkel, Michal; Lippard, Stephen J; Rosenberg, Paul A; Benowitz, Larry

    2017-01-10

    Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn 2+ ) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn 2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn 2+ accumulation in amacrine cell processes involves the Zn 2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn 2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn 2+ chelation extends for several days after nerve injury. These results show that retinal Zn 2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn 2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

  16. 3D axon growth by exogenous electrical stimulus and soluble factors.

    PubMed

    Tang-Schomer, Min D

    2018-01-01

    Axon growth and alignment are fundamental processes during nervous system development and neural regeneration after injury. The present study investigates the effects of exogenous stimulus of electrical signals and soluble factors on axon 3D growth, using a silk protein material-based 3D brain tissue model. Electrical stimulus was delivered via embedded gold wires positioned at the interface of the scaffold region and the center matrix gel-filled region, spanning the axon growth area. This setup delivered applied electrical field directly to growing axons, and the effects were compared to micro-needle assisted local delivery of soluble factors of extracellular (ECM) components and neurotrophins. Dissociated rat cortical neurons were exposed to an alternating field of 80 mV/mm at 0.5 Hz to 2 kHz or soluble factors for up to 4 days, and evaluated by of β III-tubulin immunostaining, confocal imaging and 3D neurite tracing. 0.5-20 Hz were found to promote axon growth, with 2 Hz producing the biggest effect of ∼30% axon length increase compared to control cultures. Delivery of ECM components of laminin and fibronectin resulted significantly greater axon initial length increases compared to neurotrophic factors, such as BDNF, GDNF, NGF and NT3 (all at 1 μM). Though axon lengths under 2 Hz stimulation and LN or FN exposure were statistically similar, significant AC-induced axon alignment was found under all frequencies tested. The effects included perpendicular orientation of axons trespassing an electrode, large populations of aligned axon tracts in parallel to the field direction with a few perpendicularly aligned along the middle point of the EF. These findings are consistent with the hypothesis that an electrode in AC field could act as an alternating cathode that attracts the growing tip of the axon. These results demonstrate the use of alternating electric field stimulation to direct axon 3D length growth and orientation. Our study provides basis

  17. Boosted Regeneration and Reduced Denervated Muscle Atrophy by NeuroHeal in a Pre-clinical Model of Lumbar Root Avulsion with Delayed Reimplantation.

    PubMed

    Romeo-Guitart, David; Forés, Joaquim; Navarro, Xavier; Casas, Caty

    2017-09-20

    The "gold standard" treatment of patients with spinal root injuries consists of delayed surgical reconnection of nerves. The sooner, the better, but problems such as injury-induced motor neuronal death and muscle atrophy due to long-term denervation mean that normal movement is not restored. Herein we describe a preclinical model of root avulsion with delayed reimplantation of lumbar roots that was used to establish a new adjuvant pharmacological treatment. Chronic treatment (up to 6 months) with NeuroHeal, a new combination drug therapy identified using a systems biology approach, exerted long-lasting neuroprotection, reduced gliosis and matrix proteoglycan content, accelerated nerve regeneration by activating the AKT pathway, promoted the formation of functional neuromuscular junctions, and reduced denervation-induced muscular atrophy. Thus, NeuroHeal is a promising treatment for spinal nerve root injuries and axonal regeneration after trauma.

  18. Characterization of Pax2 Expression in the Goldfish Optic Nerve Head during Retina Regeneration

    PubMed Central

    Parrilla, Marta; Lillo, Concepción; Herrero-Turrión, M. Javier; Arévalo, Rosario; Aijón, José; Lara, Juan M.; Velasco, Almudena

    2012-01-01

    The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2+ astrocytes located in the ONH and S100+ astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2+ and S100+ astrocytes have different responses. At 7 days after injury the number of Pax2+ cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100+ astrocytes in the retina surrounding the ONH and S100+ processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100+ astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2+ cells increase. At the same time, S100+/GFAP+/GS+ astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2+/PCNA+ astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2+ astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process. PMID:22384226

  19. Characterization of Pax2 expression in the goldfish optic nerve head during retina regeneration.

    PubMed

    Parrilla, Marta; Lillo, Concepción; Herrero-Turrión, M Javier; Arévalo, Rosario; Aijón, José; Lara, Juan M; Velasco, Almudena

    2012-01-01

    The Pax2 transcription factor plays a crucial role in axon-guidance and astrocyte differentiation in the optic nerve head (ONH) during vertebrate visual system development. However, little is known about its function during regeneration. The fish visual system is in continuous growth and can regenerate. Müller cells and astrocytes of the retina and ONH play an important role in these processes. We demonstrate that pax2a in goldfish is highly conserved and at least two pax2a transcripts are expressed in the optic nerve. Moreover, we show two different astrocyte populations in goldfish: Pax2(+) astrocytes located in the ONH and S100(+) astrocytes distributed throughout the retina and the ONH. After peripheral growth zone (PGZ) cryolesion, both Pax2(+) and S100(+) astrocytes have different responses. At 7 days after injury the number of Pax2(+) cells is reduced and coincides with the absence of young axons. In contrast, there is an increase of S100(+) astrocytes in the retina surrounding the ONH and S100(+) processes in the ONH. At 15 days post injury, the PGZ starts to regenerate and the number of S100(+) astrocytes increases in this region. Moreover, the regenerating axons reach the ONH and the pax2a gene expression levels and the number of Pax2(+) cells increase. At the same time, S100(+)/GFAP(+)/GS(+) astrocytes located in the posterior ONH react strongly. In the course of the regeneration, Müller cell vitreal processes surrounding the ONH are primarily disorganized and later increase in number. During the whole regenerative process we detect a source of Pax2(+)/PCNA(+) astrocytes surrounding the posterior ONH. We demonstrate that pax2a expression and the Pax2(+) astrocyte population in the ONH are modified during the PGZ regeneration, suggesting that they could play an important role in this process.

  20. Axon Targeting of the Alpha 7 Nicotinic Receptor in Developing Hippocampal Neurons by Gprin1 Regulates Growth

    PubMed Central

    Nordman, Jacob C.; Philips, Wiktor S.; Kodama, Nathan; Clark, Sarah G.; Negro, Christopher Del; Kabbani, Nadine

    2015-01-01

    Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone (GC). Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the GC. In particular, α7/Gprin1 interaction appears intimately linked to a Gαo, GAP-43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain. PMID:24350810

  1. Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

    PubMed Central

    Williams, Wes; Nix, Paola; Bastiani, Michael

    2011-01-01

    Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent

  2. Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina.

    PubMed

    White, David T; Sengupta, Sumitra; Saxena, Meera T; Xu, Qingguo; Hanes, Justin; Ding, Ding; Ji, Hongkai; Mumm, Jeff S

    2017-05-02

    Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of ( i ) rod cell clearance, ( ii ) MG/progenitor cell proliferation, and ( iii ) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions.

  3. Low Piconewton Towing of CNS Axons against Diffusing and Surface-Bound Repellents Requires the Inhibition of Motor Protein-Associated Pathways

    NASA Astrophysics Data System (ADS)

    Kilinc, Devrim; Blasiak, Agata; O'Mahony, James J.; Lee, Gil U.

    2014-11-01

    Growth cones, dynamic structures at axon tips, integrate chemical and physical stimuli and translate them into coordinated axon behaviour, e.g., elongation or turning. External force application to growth cones directs and enhances axon elongation in vitro; however, direct mechanical stimulation is rarely combined with chemotactic stimulation. We describe a microfluidic device that exposes isolated cortical axons to gradients of diffusing and substrate-bound molecules, and permits the simultaneous application of piconewton (pN) forces to multiple individual growth cones via magnetic tweezers. Axons treated with Y-27632, a RhoA kinase inhibitor, were successfully towed against Semaphorin 3A gradients, which repel untreated axons, with less than 12 pN acting on a small number of neural cell adhesion molecules. Treatment with Y-27632 or monastrol, a kinesin-5 inhibitor, promoted axon towing on substrates coated with chondroitin sulfate proteoglycans, potent axon repellents. Thus, modulating key molecular pathways that regulate contractile stress generation in axons counteracts the effects of repellent molecules and promotes tension-induced growth. The demonstration of parallel towing of axons towards inhibitory environments with minute forces suggests that mechanochemical stimulation may be a promising therapeutic approach for the repair of the damaged central nervous system, where regenerating axons face repellent factors over-expressed in the glial scar.

  4. BDNF is required for taste axon regeneration following unilateral chorda tympani nerve section.

    PubMed

    Meng, Lingbin; Huang, Tao; Sun, Chengsan; Hill, David L; Krimm, Robin

    2017-07-01

    Taste nerves readily regenerate to reinnervate denervated taste buds; however, factors required for regeneration have not yet been identified. When the chorda tympani nerve is sectioned, expression of brain-derived neurotrophic factor (BDNF) remains high in the geniculate ganglion and lingual epithelium, despite the loss of taste buds. These observations suggest that BDNF is present in the taste system after nerve section and may support taste nerve regeneration. To test this hypothesis, we inducibly deleted Bdnf during adulthood in mice. Shortly after Bdnf gene recombination, the chorda tympani nerve was unilaterally sectioned causing a loss of both taste buds and neurons, irrespective of BDNF levels. Eight weeks after nerve section, however, regeneration was differentially affected by Bdnf deletion. In control mice, there was regeneration of the chorda tympani nerve and taste buds reappeared with innervation. In contrast, few taste buds were reinnervated in mice lacking normal Bdnf expression such that taste bud number remained low. In all genotypes, taste buds that were reinnervated were normal-sized, but non-innervated taste buds remained small and atrophic. On the side of the tongue contralateral to the nerve section, taste buds for some genotypes became larger and all taste buds remained innervated. Our findings suggest that BDNF is required for nerve regeneration following gustatory nerve section. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Use of a Y-tube conduit after facial nerve injury reduces collateral axonal branching at the lesion site but neither reduces polyinnervation of motor endplates nor improves functional recovery.

    PubMed

    Hizay, Arzu; Ozsoy, Umut; Demirel, Bahadir Murat; Ozsoy, Ozlem; Angelova, Srebrina K; Ankerne, Janina; Sarikcioglu, Sureyya Bilmen; Dunlop, Sarah A; Angelov, Doychin N; Sarikcioglu, Levent

    2012-06-01

    Despite increased understanding of peripheral nerve regeneration, functional recovery after surgical repair remains disappointing. A major contributing factor is the extensive collateral branching at the lesion site, which leads to inaccurate axonal navigation and aberrant reinnervation of targets. To determine whether the Y tube reconstruction improved axonal regrowth and whether this was associated with improved function. We used a Y-tube conduit with the aim of improving navigation of regenerating axons after facial nerve transection in rats. Retrograde labeling from the zygomatic and buccal branches showed a halving in the number of double-labeled facial motor neurons (15% vs 8%; P < .05) after Y tube reconstruction compared with facial-facial anastomosis coaptation. However, in both surgical groups, the proportion of polyinnervated motor endplates was similar (≈ 30%; P > .05), and video-based motion analysis of whisking revealed similarly poor function. Although Y-tube reconstruction decreases axonal branching at the lesion site and improves axonal navigation compared with facial-facial anastomosis coaptation, it fails to promote monoinnervation of motor endplates and confers no functional benefit.

  6. Resveratrol Promotes Nerve Regeneration via Activation of p300 Acetyltransferase-Mediated VEGF Signaling in a Rat Model of Sciatic Nerve Crush Injury.

    PubMed

    Ding, Zhuofeng; Cao, Jiawei; Shen, Yu; Zou, Yu; Yang, Xin; Zhou, Wen; Guo, Qulian; Huang, Changsheng

    2018-01-01

    Peripheral nerve injuries are generally associated with incomplete restoration of motor function. The slow rate of nerve regeneration after injury may account for this. Although many benefits of resveratrol have been shown in the nervous system, it is not clear whether resveratrol could promote fast nerve regeneration and motor repair after peripheral nerve injury. This study showed that the motor deficits caused by sciatic nerve crush injury were alleviated by daily systematic resveratrol treatment within 10 days. Resveratrol increased the number of axons in the distal part of the injured nerve, indicating enhanced nerve regeneration. In the affected ventral spinal cord, resveratrol enhanced the expression of several vascular endothelial growth factor family proteins (VEGFs) and increased the phosphorylation of p300 through Akt signaling, indicating activation of p300 acetyltransferase. Inactivation of p300 acetyltransferase reversed the resveratrol-induced expression of VEGFs and motor repair in rats that had undergone sciatic nerve crush injury. The above results indicated that daily systematic resveratrol treatment promoted nerve regeneration and led to rapid motor repair. Resveratrol activated p300 acetyltransferase-mediated VEGF signaling in the affected ventral spinal cord, which may have thus contributed to the acceleration of nerve regeneration and motor repair.

  7. Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert.

    PubMed

    Lee, Geoffrey; Espirito Santo, Ana Isabel; Zwingenberger, Stefan; Cai, Lawrence; Vogl, Thomas; Feldmann, Marc; Horwood, Nicole J; Chan, James K; Nanchahal, Jagdeep

    2018-05-08

    A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1 -/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G 0 to G Alert HMGB1 also transitions human stem and progenitor cells to G Alert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery. Copyright © 2018 the Author(s). Published by PNAS.

  8. Hyperinnervation improves Xenopus laevis limb regeneration.

    PubMed

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Akira

    2018-01-15

    Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Nerve stepping stone has minimal impact in aiding regeneration across long acellular nerve allografts.

    PubMed

    Yan, Ying; Hunter, Daniel A; Schellhardt, Lauren; Ee, Xueping; Snyder-Warwick, Alison K; Moore, Amy M; Mackinnon, Susan E; Wood, Matthew D

    2018-02-01

    Acellular nerve allografts (ANAs) yield less consistent favorable outcomes compared with autografts for long gap reconstructions. We evaluated whether a hybrid ANA can improve 6-cm gap reconstruction. Rat sciatic nerve was transected and repaired with either 6-cm hybrid or control ANAs. Hybrid ANAs were generated using a 1-cm cellular isograft between 2.5-cm ANAs, whereas control ANAs had no isograft. Outcomes were assessed by graft gene and marker expression (n = 4; at 4 weeks) and motor recovery and nerve histology (n = 10; at 20 weeks). Hybrid ANAs modified graft gene and marker expression and promoted modest axon regeneration across the 6-cm defect compared with control ANA (P < 0.05), but yielded no muscle recovery. Control ANAs had no appreciable axon regeneration across the 6-cm defect. A hybrid ANA confers minimal motor recovery benefits for regeneration across long gaps. Clinically, the authors will continue to reconstruct long nerve gaps with autografts. Muscle Nerve 57: 260-267, 2018. © 2017 Wiley Periodicals, Inc.

  10. Effect of low-level laser therapy (LLLT) on peripheral nerve regeneration using fibrin glue derived from snake venom.

    PubMed

    Buchaim, Rogerio Leone; Andreo, Jesus Carlos; Barraviera, Benedito; Ferreira Junior, Rui Seabra; Buchaim, Daniela Vieira; Rosa Junior, Geraldo Marco; de Oliveira, Alexandre Leite Rodrigues; de Castro Rodrigues, Antonio

    2015-04-01

    The purpose of this study was to assess whether the adhesive permits the collateral repair of axons originating from a vagus nerve to the interior of a sural nerve graft, and whether low-level laser therapy (LLLT) assists in the regeneration process. Study sample consisted of 32 rats randomly separated into three groups: Control Group (CG; n=8), from which the intact sural nerve was collected; Experimental Group (EG; n=12), in which one of the ends of the sural nerve graft was coapted to the vagus nerve using the fibrin glue; and Experimental Group Laser (EGL; n=12), in which the animals underwent the same procedures as those in EG with the addition of LLLT. Ten weeks after surgery, the animals were euthanized. Morphological analysis by means of optical and electron microscopy, and morphometry of the regenerated fibers were employed to evaluate the results. Collateral regeneration of axons was observed from the vagus nerve to the interior of the autologous graft in EG and EGL, and in CG all dimensions measured were greater and presented a significant difference in relation to EG and EGL, except for the area and thickness of the myelin sheath, that showed significant difference only in relation to the EG. The present study demonstrated that the fibrin glue makes axonal regeneration feasible and is an efficient method to recover injured peripheral nerves, and the use of low-level laser therapy enhances nerve regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. A novel and efficient gene transfer strategy reduces glial reactivity and improves neuronal survival and axonal growth in vitro.

    PubMed

    Desclaux, Mathieu; Teigell, Marisa; Amar, Lahouari; Vogel, Roland; Gimenez Y Ribotta, Minerva; Privat, Alain; Mallet, Jacques

    2009-07-14

    The lack of axonal regeneration in the central nervous system is attributed among other factors to the formation of a glial scar. This cellular structure is mainly composed of reactive astrocytes that overexpress two intermediate filament proteins, the glial fibrillary acidic protein (GFAP) and vimentin. Indeed, in vitro, astrocytes lacking GFAP or both GFAP and vimentin were shown to be the substrate for increased neuronal plasticity. Moreover, double knockout mice lacking both GFAP and vimentin presented lower levels of glial reactivity in vivo, significant axonal regrowth and improved functional recovery in comparison with wild-type mice after spinal cord hemisection. From these results, our objective was to develop a novel therapeutic strategy for axonal regeneration, based on the targeted suppression of astroglial reactivity and scarring by lentiviral-mediated RNA-interference (RNAi). In this study, we constructed two lentiviral vectors, Lv-shGFAP and Lv-shVIM, which allow efficient and stable RNAi-mediated silencing of endogenous GFAP or vimentin in vitro. In cultured cortical and spinal reactive astrocytes, the use of these vectors resulted in a specific, stable and highly significant decrease in the corresponding protein levels. In a second model -- scratched primary cultured astrocytes -- Lv-shGFAP, alone or associated with Lv-shVIM, decreased astrocytic reactivity and glial scarring. Finally, in a heterotopic coculture model, cortical neurons displayed higher survival rates and increased neurite growth when cultured with astrocytes in which GFAP and vimentin had been invalidated by lentiviral-mediated RNAi. Lentiviral-mediated knockdown of GFAP and vimentin in astrocytes show that GFAP is a key target for modulating reactive gliosis and monitoring neuron/glia interactions. Thus, manipulation of reactive astrocytes with the Lv-shGFAP vector constitutes a promising therapeutic strategy for increasing glial permissiveness and permitting axonal regeneration

  12. Myosin-Va-Dependent Cell-To-Cell Transfer of RNA from Schwann Cells to Axons

    PubMed Central

    Sotelo, José R.; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R.; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A.

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration. PMID:23626749

  13. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    PubMed

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  14. A morphological study of diffuse axonal injury in a rat model by lateral head rotation trauma.

    PubMed

    Xiaoshengi, He; Guitao, Yang; Xiang, Zhang; Zhou, Fei

    2010-03-01

    Morphology in diffuse axonal injury (DAI) by lateral head rotation was investigated. SD rats were divided into injury (n=9) and sham (n=3) groups. A device was used to produce lateral rotational acceleration of the rats' heads. At different survival times three rats were killed for light and electron microscopic examination of the brain tissue. Sagittal sections were made from medulla oblongata and immunolabelled for NF68. At post-traumatic 30 min, NF68 immunolabelling showed a small number ofswollen and irregular axons. Ultrastructurally slightly-separated myelin lamellae and disorderly arranged neurofilaments occurred. At 2 and 24 h axonal damage became more severe. Increases in immunolabelled axonal swellings, disconnected axons and axonal retraction bulbs appeared. EM provided evidence of myelin separation, peri-axonal spaces, blank areas in axoplasm, loss of microtubules, peripheral accumulation of mitochondria and clumped neurofilaments for DAI. A tendency was noted for greater labelling with NF68 as axonal damage increased. The disorderly arrangement of NFs occurred at early stage of post-traumatic axonal changes.

  15. ENHANCING ADULT NERVE REGENERATION THROUGH THE KNOCKDOWN OF RETINOBLASTOMA PROTEIN

    PubMed Central

    Christie, Kimberly J.; Krishnan, Anand; Martinez, Jose A.; Purdy, Kaylynn; Singh, Bhagat; Eaton, Shane; Zochodne, Douglas

    2016-01-01

    Tumour suppressor pathways may offer novel targets capable of altering the plasticity of post-mitotic adult neurons. Here we describe a role for retinoblastoma (Rb) protein, widely expressed in adult sensory neurons and their axons, during regeneration. In adult sensory neurons, Rb siRNA knockdown or Rb1 deletion in vitro enhances neurite outgrowth and branching. Plasticity is achieved in part through upregulation of neuronal PPARγ; its antagonism inhibits Rb siRNA plasticity whereas a PPARγ agonist increases growth. In an in vivo regenerative paradigm following complete peripheral nerve trunk transection, direct delivery of Rb siRNA prompts increased outgrowth of axons from proximal stumps and entrains Schwann cells to accompany them for greater distances. Similarly Rb siRNA delivery following a nerve crush improves behavioural indices of motor and sensory recovery in mice. The overall findings indicate that inhibition of tumour suppressor molecules has a role to play in promoting adult neuron regeneration. PMID:24752312

  16. Chondroitinase C Selectively Degrades Chondroitin Sulfate Glycosaminoglycans that Inhibit Axonal Growth within the Endoneurium of Peripheral Nerve.

    PubMed

    Graham, James B; Muir, David

    2016-01-01

    The success of peripheral nerve regeneration is highly dependent on the regrowth of axons within the endoneurial basal lamina tubes that promote target-oriented pathfinding and appropriate reinnervation. Restoration of nerve continuity at this structural level after nerve transection injury by direct repair and nerve grafting remains a major surgical challenge. Recently, biological approaches that alter the balance of growth inhibitors and promoters in nerve have shown promise to improve appropriate axonal regeneration and recovery of peripheral nerve function. Chondroitin sulfate proteoglycans (CSPGs) are known inhibitors of axonal growth. This growth inhibition is mainly associated with a CSPG's glycosaminoglycan chains. Enzymatic degradation of these chains with chondroitinase eliminates this inhibitory activity and, when applied in vivo, can improve the outcome of nerve repair. To date, these encouraging findings were obtained with chondroitinase ABC (a pan-specific chondroitinase). The aim of this study was to examine the distribution of CSPG subtypes in rodent, rabbit, and human peripheral nerve and to test more selective biological enzymatic approaches to improve appropriate axonal growth within the endoneurium and minimize aberrant growth. Here we provide evidence that the endoneurium, but not the surrounding epineurium, is rich in CSPGs that have glycosaminoglycan chains readily degraded by chondroitinase C. Biochemical studies indicate that chondroitinase C has degradation specificity for 6-sulfated glycosaminoglycans found in peripheral nerve. We found that chondroitinase C degrades and inactivates inhibitory CSPGs within the endoneurium but not so much in the surrounding nerve compartments. Cryoculture bioassays (neurons grown on tissue sections) show that chondroitinase C selectively and significantly enhanced neuritic growth associated with the endoneurial basal laminae without changing growth-inhibiting properties of the surrounding epineurium

  17. Dynein mediates retrograde neurofilament transport within axons and anterograde delivery of NFs from perikarya into axons: regulation by multiple phosphorylation events.

    PubMed

    Motil, Jennifer; Chan, Walter K-H; Dubey, Maya; Chaudhury, Pulkit; Pimenta, Aurea; Chylinski, Teresa M; Ortiz, Daniela T; Shea, Thomas B

    2006-05-01

    We examined the respective roles of dynein and kinesin in axonal transport of neurofilaments (NFs). Differentiated NB2a/d1 cells were transfected with green fluorescent protein-NF-M (GFP-M) and dynein function was inhibited by co-transfection with a construct expressing myc-tagged dynamitin, or by intracellular delivery of purified dynamitin and two antibodies against dynein's cargo domain. Monitoring of the bulk distribution of GFP signal within axonal neurites, recovery of GFP signal within photobleached regions, and real-time monitoring of individual NFs/punctate structures each revealed that pertubation of dynein function inhibited retrograde transport and accelerated anterograde, confirming that dynein mediated retrograde axonal transport, while intracellular delivery of two anti-kinesin antibodies selectively inhibited NF anterograde transport. In addition, dynamitin overexpression inhibited the initial translocation of newly-expressed NFs out of perikarya and into neurites, indicating that dynein participated in the initial anterograde delivery of NFs into neurites. Delivery of NFs to the axon hillock inner plasma membrane surface, and their subsequent translocation into neurites, was also prevented by vinblastine-mediated inhibition of microtubule assembly. These data collectively suggest that some NFs enter axons as cargo of microtubues that are themselves undergoing transport into axons via dynein-mediated interactions with the actin cortex and/or larger microtubules. C-terminal NF phosphorylation regulates motor association, since anti-dynein selectively coprecipitated extensively phosphorylated NFs, while anti-kinesin selectively coprecipitated less phosphorylated NFs. In addition, however, the MAP kinase inhibitor PD98059 also inhibited transport of a constitutively-phosphorylated NF construct, indicating that one or more additional, non-NF phosphorylation events also regulated NF association with dynein or kinesin. Copyright 2006 Wiley-Liss, Inc.

  18. Activation of mTor Signaling by Gene Transduction to Induce Axon Regeneration in the Central Nervous System Following Neural Injury

    DTIC Science & Technology

    2014-03-01

    bundle (MFB); quantification by confocal optical dissection of either GFP-positive axons in the MFB in transgenic TH- GFP mice or of Tomato -positive...axons following transduction with anterograde tracer Tomato -Tau. As anticipated, based on anatomical evidence showing an inability of AAV eIF4E to re...which the axon-targeted fusion protein Tomato -Tau is delivered to SN neurons by AAV and expression is driven by the robust chicken-beta actin promoter

  19. Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

    PubMed Central

    White, David T.; Sengupta, Sumitra; Saxena, Meera T.; Xu, Qingguo; Hanes, Justin; Ding, Ding; Ji, Hongkai

    2017-01-01

    Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration—i.e., selective cell-loss paradigms akin to degenerative disease—are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions. PMID:28416692

  20. Axon tension regulates fasciculation/defasciculation through the control of axon shaft zippering

    PubMed Central

    Šmít, Daniel; Fouquet, Coralie; Pincet, Frédéric; Zapotocky, Martin; Trembleau, Alain

    2017-01-01

    While axon fasciculation plays a key role in the development of neural networks, very little is known about its dynamics and the underlying biophysical mechanisms. In a model system composed of neurons grown ex vivo from explants of embryonic mouse olfactory epithelia, we observed that axons dynamically interact with each other through their shafts, leading to zippering and unzippering behavior that regulates their fasciculation. Taking advantage of this new preparation suitable for studying such interactions, we carried out a detailed biophysical analysis of zippering, occurring either spontaneously or induced by micromanipulations and pharmacological treatments. We show that zippering arises from the competition of axon-axon adhesion and mechanical tension in the axons, and provide the first quantification of the force of axon-axon adhesion. Furthermore, we introduce a biophysical model of the zippering dynamics, and we quantitatively relate the individual zipper properties to global characteristics of the developing axon network. Our study uncovers a new role of mechanical tension in neural development: the regulation of axon fasciculation. DOI: http://dx.doi.org/10.7554/eLife.19907.001 PMID:28422009

  1. Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy

    PubMed Central

    Bombeiro, André L.; Santini, Júlio C.; Thomé, Rodolfo; Ferreira, Elisângela R. L.; Nunes, Sérgio L. O.; Moreira, Bárbara M.; Bonet, Ivan J. M.; Sartori, Cesar R.; Verinaud, Liana; Oliveira, Alexandre L. R.

    2016-01-01

    Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest

  2. Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

    PubMed

    Spitzbarth, Ingo; Lempp, Charlotte; Kegler, Kristel; Ulrich, Reiner; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Seehusen, Frauke

    2016-07-01

    CDV-DL (Canine distemper virus-induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination. Immunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed. Increased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up-regulated in the chronic stage compared to dogs with subacute CDV-DL. However, immunohistochemically, hints for axonal regeneration were restricted to up-regulated axonal positivity of hypoxia-inducible factor 1 alpha, while growth-associated protein 43, erythropoietin and its receptor were not or even down-regulated. Periaxin-positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions. The present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV-DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

  3. Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ.

    PubMed

    Zarei, Kasra; Scheetz, Todd E; Christopher, Mark; Miller, Kathy; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G; Fingert, John H; Abràmoff, Michael David

    2016-05-26

    We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ's performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice.

  4. Automated Axon Counting in Rodent Optic Nerve Sections with AxonJ

    NASA Astrophysics Data System (ADS)

    Zarei, Kasra; Scheetz, Todd E.; Christopher, Mark; Miller, Kathy; Hedberg-Buenz, Adam; Tandon, Anamika; Anderson, Michael G.; Fingert, John H.; Abràmoff, Michael David

    2016-05-01

    We have developed a publicly available tool, AxonJ, which quantifies the axons in optic nerve sections of rodents stained with paraphenylenediamine (PPD). In this study, we compare AxonJ’s performance to human experts on 100x and 40x images of optic nerve sections obtained from multiple strains of mice, including mice with defects relevant to glaucoma. AxonJ produced reliable axon counts with high sensitivity of 0.959 and high precision of 0.907, high repeatability of 0.95 when compared to a gold-standard of manual assessments and high correlation of 0.882 to the glaucoma damage staging of a previously published dataset. AxonJ allows analyses that are quantitative, consistent, fully-automated, parameter-free, and rapid on whole optic nerve sections at 40x. As a freely available ImageJ plugin that requires no highly specialized equipment to utilize, AxonJ represents a powerful new community resource augmenting studies of the optic nerve using mice.

  5. HIP/PAP accelerates liver regeneration and protects against acetaminophen injury in mice.

    PubMed

    Lieu, Hanh-Tu; Batteux, Frédéric; Simon, Marie-Thérèse; Cortes, Alexandre; Nicco, Carole; Zavala, Flora; Pauloin, Alain; Tralhao, José Guilherme; Soubrane, Olivier; Weill, Bernard; Bréchot, Christian; Christa, Laurence

    2005-09-01

    Human hepatocarcinoma-intestine-pancreas/pancreatic-associated protein HIP/PAP is a secreted C-type lectin belonging to group VII, according to Drickamer's classification. HIP/PAP is overexpressed in liver carcinoma; however, its functional role remains unclear. In this study, we demonstrate that HIP/PAP is a paracrine hepatic growth factor promoting both proliferation and viability of liver cells in vivo. First, a low number of implanted hepatocytes deriving from HIP/PAP-transgenic mice (<1:1,000) was sufficient to stimulate overall recipient severe combined immunodeficiency liver regeneration after partial hepatectomy. After a single injection of HIP/PAP protein, the percentages of bromodeoxyuridine-positive nuclei and mitosis were statistically higher than after saline injection, indicating that HIP/PAP acts as a paracrine mitogenic growth factor for the liver. Comparison of the early events posthepatectomy in control and transgenic mice indicated that HIP/PAP accelerates the accumulation/degradation of nuclear phospho-signal transducer activator transcription factor 3 and tumor necrosis factor alpha level, thus reflecting that HIP/PAP accelerates liver regeneration. Second, we showed that 80% of the HIP/PAP-transgenic mice versus 25% of the control mice were protected against lethal acetaminophen-induced fulminate hepatitis. A single injection of recombinant HIP/PAP induced a similar cytoprotective effect, demonstrating the antiapoptotic effect of HIP/PAP. Comparison of Cu/Zn superoxide dismutase activity and glutathione reductase-like effects in control and transgenic liver mice indicated that HIP/PAP exerts an antioxidant activity and prevents reactive oxygen species-induced mitochondrial damage by acetaminophen overdose. In conclusion, the present data offer new insights into the biological functions of C-type lectins. In addition, HIP/PAP is a promising candidate for the prevention and treatment of liver failure.

  6. Dendrosomatic Sonic Hedgehog Signaling in Hippocampal Neurons Regulates Axon Elongation

    PubMed Central

    Petralia, Ronald S.; Ott, Carolyn; Wang, Ya-Xian; Lippincott-Schwartz, Jennifer; Mattson, Mark P.

    2015-01-01

    The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits. PMID:26658865

  7. Spectraplakins promote microtubule-mediated axonal growth by functioning as structural MAPs and EB1-dependent +TIPs

    PubMed Central

    Alves-Silva, J.; Sánchez-Soriano, N.; Beaven, R.; Klein, M.; Parkin, J.; Millard, T.H.; Bellen, H. J; Venken, K. J.T.; Ballestrem, C.; Kammerer, R.A.; Prokop, A.

    2013-01-01

    The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homologue Short stop/Shot similarly cause severe axon shortening and microtubule disorganisation. How spectraplakins perform these functions is not known. Here we show that axonal growth promoting roles of Shot require interaction with EB1 (End binding protein) at polymerising plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot’s carboxyterminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organisation. In addition, we report that Shot localises along microtubule shafts and stabilises them against pharmacologically induced depolymerisation. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (plus end regulators) and structural MAPs (microtubule associated proteins). From our data we deduce a model that relates the different features of the spectraplakin carboxy-terminus to the two functions of Shot during axonal growth. PMID:22764224

  8. cAMP-responsive Element-binding Protein (CREB) and cAMP Co-regulate Activator Protein 1 (AP1)-dependent Regeneration-associated Gene Expression and Neurite Growth*

    PubMed Central

    Ma, Thong C.; Barco, Angel; Ratan, Rajiv R.; Willis, Dianna E.

    2014-01-01

    To regenerate damaged axons, neurons must express a cassette of regeneration-associated genes (RAGs) that increases intrinsic growth capacity and confers resistance to extrinsic inhibitory cues. Here we show that dibutyrl-cAMP or forskolin combined with constitutive-active CREB are superior to either agent alone in driving neurite growth on permissive and inhibitory substrates. Of the RAGs examined, only arginase 1 (Arg1) expression correlated with the increased neurite growth induced by the cAMP/CREB combination, both of which were AP1-dependent. This suggests that cAMP-induced AP1 activity is necessary and interacts with CREB to drive expression of RAGs relevant for regeneration and demonstrates that combining a small molecule (cAMP) with an activated transcription factor (CREB) stimulates the gene expression necessary to enhance axonal regeneration. PMID:25296755

  9. Axonal GABAA receptors.

    PubMed

    Trigo, Federico F; Marty, Alain; Stell, Brandon M

    2008-09-01

    Type A GABA receptors (GABA(A)Rs) are well established as the main inhibitory receptors in the mature mammalian forebrain. In recent years, evidence has accumulated showing that GABA(A)Rs are prevalent not only in the somatodendritic compartment of CNS neurons, but also in their axonal compartment. Evidence for axonal GABA(A)Rs includes new immunohistochemical and immunogold data: direct recording from single axonal terminals; and effects of local applications of GABA(A)R modulators on action potential generation, on axonal calcium signalling, and on neurotransmitter release. Strikingly, whereas presynaptic GABA(A)Rs have long been considered inhibitory, the new studies in the mammalian brain mostly indicate an excitatory action. Depending on the neuron that is under study, axonal GABA(A)Rs can be activated by ambient GABA, by GABA spillover, or by an autocrine action, to increase either action potential firing and/or transmitter release. In certain neurons, the excitatory effects of axonal GABA(A)Rs persist into adulthood. Altogether, axonal GABA(A)Rs appear as potent neuronal modulators of the mammalian CNS.

  10. Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Acellular Nerve Allografts plus amniotic Fluid Derived Stem Cells (AFS)

    DTIC Science & Technology

    2016-09-01

    AWARD NUMBER: W81XWH-13-1-0309 TITLE: Acceleration of Regeneration of Large-Gap Peripheral Nerve Injuries Using Acellular Nerve Allografts...plus amniotic Fluid Derived Stem Cells (AFS). PRINCIPAL INVESTIGATOR: Thomas L. Smith, PhD RECIPIENT: Wake Forest University Health Sciences

  11. Pathfinding in a large vertebrate axon tract: isotypic interactions guide retinotectal axons at multiple choice points

    PubMed Central

    Pittman, Andrew J.; Law, Mei-Yee; Chien, Chi-Bin

    2008-01-01

    Summary Navigating axons respond to environmental guidance signals, but can also follow axons that have gone before—pioneer axons. Pioneers have been studied extensively in simple systems, but the role of axon-axon interactions remains largely unexplored in large vertebrate axon tracts, where cohorts of identical axons could potentially use isotypic interactions to guide each other through multiple choice points. Furthermore, the relative importance of axon-axon interactions compared to axon-autonomous receptor function has not been assessed. Here we test the role of axon-axon interactions in retinotectal development, by devising a technique to selectively remove or replace early-born retinal ganglion cells (RGCs). We find that early RGCs are both necessary and sufficient for later axons to exit the eye. Furthermore, introducing misrouted axons by transplantation reveals that guidance from eye to tectum relies heavily on interactions between axons, including both pioneer-follower and community effects. We conclude that axon-axon interactions and ligand-receptor signaling have coequal roles, cooperating to ensure the fidelity of axon guidance in developing vertebrate tracts. PMID:18653554

  12. Mitofusin2 mutations disrupt axonal mitochondrial positioning and promote axon degeneration

    PubMed Central

    Misko, Albert; Sasaki, Yo; Tuck, Elizabeth; Milbrandt, Jeffrey; Baloh, Robert H.

    2012-01-01

    Summary Alterations in mitochondrial dynamics (fission, fusion and movement) are implicated in many neurodegenerative diseases, from rare genetic disorders such as Charcot-Marie-Tooth disease, to common conditions including Alzheimer’s disease. However, the relationship between altered mitochondrial dynamics and neurodegeneration is incompletely understood. Here we show that disease associated MFN2 proteins suppressed both mitochondrial fusion and transport, and produced classic features of segmental axonal degeneration without cell body death, including neurofilament filled swellings, loss of calcium homeostasis, and accumulation of reactive oxygen species. By contrast, depletion of Opa1 suppressed mitochondrial fusion while sparing transport, and did not induce axonal degeneration. Axon degeneration induced by mutant MFN2 proteins correlated with the disruption of the proper mitochondrial positioning within axons, rather than loss of overall mitochondrial movement, or global mitochondrial dysfunction. We also found that augmenting expression of MFN1 rescued the axonal degeneration caused by MFN2 mutants, suggesting a possible therapeutic strategy for Charcot-Marie-Tooth disease. These experiments provide evidence that the ability of mitochondria to sense energy requirements and localize properly within axons is key to maintaining axonal integrity, and may be a common pathway by which disruptions in axonal transport contribute to neurodegeneration. PMID:22442078

  13. A Fully Automated Microfluidic Femtosecond Laser Axotomy Platform for Nerve Regeneration Studies in C. elegans

    PubMed Central

    Gokce, Sertan Kutal; Guo, Samuel X.; Ghorashian, Navid; Everett, W. Neil; Jarrell, Travis; Kottek, Aubri; Bovik, Alan C.; Ben-Yakar, Adela

    2014-01-01

    Femtosecond laser nanosurgery has been widely accepted as an axonal injury model, enabling nerve regeneration studies in the small model organism, Caenorhabditis elegans. To overcome the time limitations of manual worm handling techniques, automation and new immobilization technologies must be adopted to improve throughput in these studies. While new microfluidic immobilization techniques have been developed that promise to reduce the time required for axotomies, there is a need for automated procedures to minimize the required amount of human intervention and accelerate the axotomy processes crucial for high-throughput. Here, we report a fully automated microfluidic platform for performing laser axotomies of fluorescently tagged neurons in living Caenorhabditis elegans. The presented automation process reduces the time required to perform axotomies within individual worms to ∼17 s/worm, at least one order of magnitude faster than manual approaches. The full automation is achieved with a unique chip design and an operation sequence that is fully computer controlled and synchronized with efficient and accurate image processing algorithms. The microfluidic device includes a T-shaped architecture and three-dimensional microfluidic interconnects to serially transport, position, and immobilize worms. The image processing algorithms can identify and precisely position axons targeted for ablation. There were no statistically significant differences observed in reconnection probabilities between axotomies carried out with the automated system and those performed manually with anesthetics. The overall success rate of automated axotomies was 67.4±3.2% of the cases (236/350) at an average processing rate of 17.0±2.4 s. This fully automated platform establishes a promising methodology for prospective genome-wide screening of nerve regeneration in C. elegans in a truly high-throughput manner. PMID:25470130

  14. Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail.

    PubMed

    Tokuyama, Minami A; Xu, Cindy; Fisher, Rebecca E; Wilson-Rawls, Jeanne; Kusumi, Kenro; Newbern, Jason M

    2018-01-15

    Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Partial Denervation of Subbasal Axons Persists Following Debridement Wounds to the Mouse Cornea

    PubMed Central

    Pajoohesh-Ganji, Ahdeah; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Kyne, Briana M.; Saban, Daniel R.; Stepp, Mary Ann

    2015-01-01

    Although sensory reinnervation occurs after injury in the PNS, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify subbasal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of subbasal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7d after superficial trephination, subbasal axon density returns to control levels; by 28d the vortex reforms. Although axon density is similar to control 14d after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14d, axons retract from the center leaving the subbasal axon density reduced by 37.2% and 36.8% at 28d after dulled blade and rotating burr wounding, respectively, compared to control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration associated genes (RAGs) involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7d after injury and by 14d and 28d after wounding, many of these basal cells undergo apoptosis and die. While subbasal axons are restored to their normal density and morphology after superficial trephination, subbasal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14d

  16. Structural parameters of collagen nerve grafts influence peripheral nerve regeneration.

    PubMed

    Stang, Felix; Fansa, Hisham; Wolf, Gerald; Reppin, Michael; Keilhoff, Gerburg

    2005-06-01

    Large nerve defects require nerve grafts to allow regeneration. To avoid donor nerve problems the concept of tissue engineering was introduced into nerve surgery. However, non-neuronal grafts support axonal regeneration only to a certain extent. They lack viable Schwann cells which provide neurotrophic and neurotopic factors and guide the sprouting nerve. This experimental study used the rat sciatic nerve to bridge 2 cm nerve gaps with collagen (type I/III) tubes. The tubes were different in their physical structure (hollow versus inner collagen skeleton, different inner diameters). To improve regeneration Schwann cells were implanted. After 8 weeks the regeneration process was monitored clinically, histologically and morphometrically. Autologous nerve grafts and collagen tubes without Schwann cells served as control. In all parameters autologous nerve grafts showed best regeneration. Nerve regeneration in a noteworthy quality was also seen with hollow collagen tubes and tubes with reduced lumen, both filled with Schwann cells. The inner skeleton, however, impaired nerve regeneration independent of whether Schwann cells were added or not. This indicates that not only viable Schwann cells are an imperative prerequisite but also structural parameters determine peripheral nerve regeneration.

  17. Functional role of NT-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro

    PubMed Central

    Wang, Qiong; Green, Steven H.

    2011-01-01

    Spiral ganglion neurons (SGNs) are postsynaptic to hair cells and project to the brainstem. The inner hair cell (IHC) to SGN synapse is susceptible to glutamate excitotoxicity and to acoustic trauma, with potentially adverse consequences to long-term SGN survival. We used a cochlear explant culture from P6 rat pups consisting of a portion of organ of Corti maintained intact with the corresponding portion of spiral ganglion to investigate excitotoxic damage to IHC-SGN synapses in vitro. The normal innervation pattern is preserved in vitro. Brief treatment with NMDA and kainate results in loss of IHC–SGN synapses and degeneration of the distal type 1 SGN peripheral axons, mimicking damage to SGN peripheral axons caused by excitotoxicity or noise in vivo. The number of IHC presynaptic ribbons is not significantly altered. Reinnervation of IHCs occurs and regenerating axons remain restricted to the IHC row. However, the number of postsynaptic densities (PSDs) does not fully recover and not all axons regrow to the IHCs. Addition of either NT-3 or BDNF increases axon growth and synaptogenesis. Selective blockade of endogenous NT-3 signaling with TrkC-IgG reduced regeneration of axons and PSDs, but TrkB-IgG, which blocks BDNF, has no such effect, indicating that endogenous NT-3 is necessary for SGN axon growth and synaptogenesis. Remarkably, TrkC-IgG reduced axon growth and synaptogenesis even in the presence of BDNF, indicating that endogenous NT-3 has a distinctive role, not mimicked by BDNF, in promoting SGN axon growth in the organ of Corti and synaptogenesis on IHCs. PMID:21613508

  18. [Review of the regeneration mechanism of complete spinal cord injury].

    PubMed

    Li, Jiayin; Li, Xing; Xiao, Zhifeng; Dai, Jianwu

    2018-06-01

    Spinal cord injury (SCI), especially the complete SCI, usually results in complete paralysis below the level of the injury and seriously affects the patient's quality of life. SCI repair is still a worldwide medical problem. In the last twenty years, Professor DAI Jianwu and his team pioneered complete SCI model by removing spinal tissue with varied lengths in rodents, canine, and non-human primates to verify therapeutic effect of different repair strategies. Moreover, they also started the first clinical study of functional collagen scaffold on patients with acute complete SCI on January 16th, 2015. This review mainly focusses on the possible mechanisms responsible for complete SCI. In common, recovery of some sensory and motor functions post complete SCI include the following three contributing reasons. ① Regeneration of long ascending and descending axons throughout the lesion site to re-connect the original targets; ② New neural circuits formed in the lesion site by newly generated neurons post injury, which effectively re-connect the transected stumps; ③ The combined effect of ① and ②. The numerous studies have confirmed that neural circuits rebuilt across the injury site by newborn neurons might be the main mechanisms for functional recovery of animals from rodents to dogs. In many SCI model, especially the complete spinal cord transection model, many studies have convincingly demonstrated that the quantity and length of regenerated long descending axons, particularly like CST fibers, are too few to across the lesion site that is millimeters in length to realize motor functional recovery. Hence, it is more feasible in guiding neuronal relays formation by bio-scaffolds implantation than directing long motor axons regeneration in improving motor function of animals with complete spinal cord transection. However, some other issues such as promoting more neuronal relays formation, debugging wrong connections, and maintaining adequate neural circuits for

  19. Drosophila growth cones: a genetically tractable platform for the analysis of axonal growth dynamics.

    PubMed

    Sánchez-Soriano, Natalia; Gonçalves-Pimentel, Catarina; Beaven, Robin; Haessler, Ulrike; Ofner-Ziegenfuss, Lisa; Ballestrem, Christoph; Prokop, Andreas

    2010-01-01

    The formation of neuronal networks, during development and regeneration, requires outgrowth of axons along reproducible paths toward their appropriate postsynaptic target cells. Axonal extension occurs at growth cones (GCs) at the tips of axons. GC advance and navigation requires the activity of their cytoskeletal networks, comprising filamentous actin (F-actin) in lamellipodia and filopodia as well as dynamic microtubules (MTs) emanating from bundles of the axonal core. The molecular mechanisms governing these two cytoskeletal networks, their cross-talk, and their response to extracellular signaling cues are only partially understood, hindering our conceptual understanding of how regulated changes in GC behavior are controlled. Here, we introduce Drosophila GCs as a suitable model to address these mechanisms. Morphological and cytoskeletal readouts of Drosophila GCs are similar to those of other models, including mammals, as demonstrated here for MT and F-actin dynamics, axonal growth rates, filopodial structure and motility, organizational principles of MT networks, and subcellular marker localization. Therefore, we expect fundamental insights gained in Drosophila to be translatable into vertebrate biology. The advantage of the Drosophila model over others is its enormous amenability to combinatorial genetics as a powerful strategy to address the complexity of regulatory networks governing axonal growth. Thus, using pharmacological and genetic manipulations, we demonstrate a role of the actin cytoskeleton in a specific form of MT organization (loop formation), known to regulate GC pausing behavior. We demonstrate these events to be mediated by the actin-MT linking factor Short stop, thus identifying an essential molecular player in this context.

  20. Development of a central nervous system axonal myelination assay for high throughput screening.

    PubMed

    Lariosa-Willingham, Karen D; Rosler, Elen S; Tung, Jay S; Dugas, Jason C; Collins, Tassie L; Leonoudakis, Dmitri

    2016-04-22

    Regeneration of new myelin is impaired in persistent multiple sclerosis (MS) lesions, leaving neurons unable to function properly and subject to further degeneration. Current MS therapies attempt to ameliorate autoimmune-mediated demyelination, but none directly promote the regeneration of lost and damaged myelin of the central nervous system (CNS). Development of new drugs that stimulate remyelination has been hampered by the inability to evaluate axonal myelination in a rapid CNS culture system. We established a high throughput cell-based assay to identify compounds that promote myelination. Culture methods were developed for initiating myelination in vitro using primary embryonic rat cortical cells. We developed an immunofluorescent phenotypic image analysis method to quantify the morphological alignment of myelin characteristic of the initiation of myelination. Using γ-secretase inhibitors as promoters of myelination, the optimal growth, time course and compound treatment conditions were established in a 96 well plate format. We have characterized the cortical myelination assay by evaluating the cellular composition of the cultures and expression of markers of differentiation over the time course of the assay. We have validated the assay scalability and consistency by screening the NIH clinical collection library of 727 compounds and identified ten compounds that promote myelination. Half maximal effective concentration (EC50) values for these compounds were determined to rank them according to potency. We have designed the first high capacity in vitro assay that assesses myelination of live axons. This assay will be ideal for screening large compound libraries to identify new drugs that stimulate myelination. Identification of agents capable of promoting the myelination of axons will likely lead to the development of new therapeutics for MS patients.

  1. Histochemical discrimination of fibers in regenerating rat infraorbital nerve

    NASA Technical Reports Server (NTRS)

    Wilke, R. A.; Riley, D. A.; Sanger, J. R.

    1992-01-01

    In rat dorsal root ganglia, histochemical staining of carbonic anhydrase (CA) and cholinesterase (CE) yields a reciprocal pattern of activity: Sensory processes are CA positive and CE negative, whereas motor processes are CA negative and CE positive. In rat infraorbital nerve (a sensory peripheral nerve), we saw extensive CA staining of nearly 100% of the myelinated axons. Although CE reactivity in myelinated axons was extremely rare, we did observe CE staining of unmyelinated autonomic fibers. Four weeks after transection of infraorbital nerves, CA-stained longitudinal sections of the proximal stump demonstrated 3 distinct morphological zones. A fraction of the viable axons retained CA activity to within 2 mm of the distal extent of the stump, and the stain is capable of resolving growth sprouts being regenerated from these fibers. Staining of unmyelinated autonomic fibers in serial sections shows that CE activity was not retained as far distally as is the CA sensory staining.

  2. Functional Multichannel Poly(Propylene Fumarate)-Collagen Scaffold with Collagen-Binding Neurotrophic Factor 3 Promotes Neural Regeneration After Transected Spinal Cord Injury.

    PubMed

    Chen, Xi; Zhao, Yannan; Li, Xing; Xiao, Zhifeng; Yao, Yuanjiang; Chu, Yun; Farkas, Balázs; Romano, Ilaria; Brandi, Fernando; Dai, Jianwu

    2018-06-19

    Many factors contribute to the poor axonal regrowth and ineffective functional recovery after spinal cord injury (SCI). Biomaterials have been used for SCI repair by promoting bridge formation and reconnecting the neural tissue at the lesion site. The mechanical properties of biomaterials are critical for successful design to ensure the stable support as soon as possible when compressed by the surrounding spine and musculature. Poly(propylene fumarate) (PPF) scaffolds with high mechanical strength have been shown to provide firm spatial maintenance and to promote repair of tissue defects. A multichannel PPF scaffold is combined with collagen biomaterial to build a novel biocompatible delivery system coated with neurotrophin-3 containing an engineered collagen-binding domain (CBD-NT3). The parallel-aligned multichannel structure of PPF scaffolds guide the direction of neural tissue regeneration across the lesion site and promote reestablishment of bridge connectivity. The combinatorial treatment consisting of PPF and collagen loaded with CBD-NT3 improves the inhibitory microenvironment, facilitates axonal and neuronal regeneration, survival of various types of functional neurons and remyelination and synapse formation of regenerated axons following SCI. This novel treatment strategy for SCI repair effectively promotes neural tissue regeneration after transected spinal injury by providing a regrowth-supportive microenvironment and eventually induces functional improvement. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Synergistic effects of micropatterned biodegradable conduits and Schwann cells on sciatic nerve regeneration

    NASA Astrophysics Data System (ADS)

    Rutkowski, Gregory E.; Miller, Cheryl A.; Jeftinija, Srdija; Mallapragada, Surya K.

    2004-09-01

    This paper describes a novel biodegradable conduit that provides a combination of physical, chemical and biological cues at the cellular level to facilitate peripheral nerve regeneration. The conduit consists of a porous poly(D,L-lactic acid) (PDLLA) tubular support structure with a micropatterned inner lumen. Schwann cells were pre-seeded into the lumen to provide additional trophic support. Conduits with micropatterned inner lumens pre-seeded with Schwann cells (MS) were fabricated and compared with three types of conduits used as controls: M (conduits with micropatterned inner lumens without pre-seeded Schwann cells), NS (conduits without micropatterned inner lumens pre-seeded with Schwann cells) and N (conduits without micropatterned inner lumens, without pre-seeded Schwann cells). The conduits were implanted in rats with 1 cm sciatic nerve transections and the regeneration and functional recovery were compared in the four different cases. The number or size of regenerated axons did not vary significantly among the different conduits. The time of recovery, and the sciatic function index, however, were significantly enhanced using the MS conduits, based on qualitative observations as well as quantitative measurements using walking track analysis. This demonstrates that biodegradable micropatterned conduits pre-seeded with Schwann cells that provide a combination of physical, chemical and biological guidance cues for regenerating axons at the cellular level offer a better alternative for repairing sciatic nerve transactions than conventional biodegradable conduits.

  4. The beneficial effect of genetically engineered Schwann cells with enhanced motility in peripheral nerve regeneration: review.

    PubMed

    Gravvanis, A I; Lavdas, A A; Papalois, A; Tsoutsos, D A; Matsas, R

    2007-01-01

    The importance of Schwann cells in promoting nerve regeneration across a conduit has been extensively reported in the literature, and Schwann cell motility has been acknowledged as a prerequisite for myelination of the peripheral nervous system during regeneration after injury. Review of recent literature and retrospective analysis of our studies with genetically modified Schwann Cells with increased motility in order to identify the underlying mechanism of action and outline the future trends in peripheral nerve repair. Schwann cell transduction with the pREV-retrovirus, for expression of Sialyl-Transferase-X, resulting in conferring Polysialyl-residues (PSA) on NCAM, increases their motility in-vitro and ensures nerve regeneration through silicone tubes after end-to-side neurorraphy in the rat sciatic nerve model, thus significantly promoting fiber maturation and functional outcome. An artificial nerve graft consisting of a type I collagen tube lined with the genetically modified Schwann cells with increased motility, used to bridge a defect in end-to-end fashion in the rat sciatic nerve model, was shown to promote nerve regeneration to a level equal to that of a nerve autograft. The use of genetically engineered Schwann cells with enhanced motility for grafting endoneural tubes promotes axonal regeneration, by virtue of the interaction of the transplanted cells with regenerating axonal growth cones as well as via the recruitment of endogenous Schwann cells. It is envisaged that mixed populations of Schwann cells, expressing PSA and one or more trophic factors, might further enhance the regenerating and remyelinating potential of the lesioned nerves.

  5. Action Potential Dynamics in Fine Axons Probed with an Axonally Targeted Optical Voltage Sensor.

    PubMed

    Ma, Yihe; Bayguinov, Peter O; Jackson, Meyer B

    2017-01-01

    The complex and malleable conduction properties of axons determine how action potentials propagate through extensive axonal arbors to reach synaptic terminals. The excitability of axonal membranes plays a major role in neural circuit function, but because most axons are too thin for conventional electrical recording, their properties remain largely unexplored. To overcome this obstacle, we used a genetically encoded hybrid voltage sensor (hVOS) harboring an axonal targeting motif. Expressing this probe in transgenic mice enabled us to monitor voltage changes optically in two populations of axons in hippocampal slices, the large axons of dentate granule cells (mossy fibers) in the stratum lucidum of the CA3 region and the much finer axons of hilar mossy cells in the inner molecular layer of the dentate gyrus. Action potentials propagated with distinct velocities in each type of axon. Repetitive firing broadened action potentials in both populations, but at an intermediate frequency the degree of broadening differed. Repetitive firing also attenuated action potential amplitudes in both mossy cell and granule cell axons. These results indicate that the features of use-dependent action potential broadening, and possible failure, observed previously in large nerve terminals also appear in much finer unmyelinated axons. Subtle differences in the frequency dependences could influence the propagation of activity through different pathways to excite different populations of neurons. The axonally targeted hVOS probe used here opens up the diverse repertoire of neuronal processes to detailed biophysical study.

  6. Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function.

    PubMed

    Cioni, Jean-Michel; Wong, Hovy Ho-Wai; Bressan, Dario; Kodama, Lay; Harris, William A; Holt, Christine E

    2018-03-07

    The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2's function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Permanent central synaptic disconnection of proprioceptors after nerve injury and regeneration. I. Loss of VGLUT1/IA synapses on motoneurons

    PubMed Central

    Titus-Mitchell, Haley E.; Bullinger, Katie L.; Kraszpulski, Michal; Nardelli, Paul; Cope, Timothy C.

    2011-01-01

    Motor and sensory proprioceptive axons reinnervate muscles after peripheral nerve transections followed by microsurgical reattachment; nevertheless, motor coordination remains abnormal and stretch reflexes absent. We analyzed the possibility that permanent losses of central IA afferent synapses, as a consequence of peripheral nerve injury, are responsible for this deficit. VGLUT1 was used as a marker of proprioceptive synapses on rat motoneurons. After nerve injuries synapses are stripped from motoneurons, but while other excitatory and inhibitory inputs eventually recover, VGLUT1 synapses are permanently lost on the cell body (75–95% synaptic losses) and on the proximal 100 μm of dendrite (50% loss). Lost VGLUT1 synapses did not recover, even many months after muscle reinnervation. Interestingly, VGLUT1 density in more distal dendrites did not change. To investigate whether losses are due to VGLUT1 downregulation in injured IA afferents or to complete synaptic disassembly and regression of IA ventral projections, we studied the central trajectories and synaptic varicosities of axon collaterals from control and regenerated afferents with IA-like responses to stretch that were intracellularly filled with neurobiotin. VGLUT1 was present in all synaptic varicosities, identified with the synaptic marker SV2, of control and regenerated afferents. However, regenerated afferents lacked axon collaterals and synapses in lamina IX. In conjunction with the companion electrophysiological study [Bullinger KL, Nardelli P, Pinter MJ, Alvarez FJ, Cope TC. J Neurophysiol (August 10, 2011). doi:10.1152/jn.01097.2010], we conclude that peripheral nerve injuries cause a permanent retraction of IA afferent synaptic varicosities from lamina IX and disconnection with motoneurons that is not recovered after peripheral regeneration and reinnervation of muscle by sensory and motor axons. PMID:21832035

  8. A polymer foam conduit seeded with Schwann cells promotes guided peripheral nerve regeneration.

    PubMed

    Hadlock, T; Sundback, C; Hunter, D; Cheney, M; Vacanti, J P

    2000-04-01

    axonal regeneration compared with autografts (n = 6). At 6 weeks, axonal regeneration was observed in the midconduit region of all five channels in each experimental animal. The cross-sectional area comprising axons relative to the open conduit cross sectional area (mean 26.3%, SD 10. 1%) compared favorably with autografts (mean 23.8%, SD 3.6%). Our methodology can be used to create polymer foam conduits containing longitudinally aligned channels, to introduce Schwann cells into them, and to implant them into surgically created neural defects. These conduits provide an environment permissive to axonal regeneration. Furthermore, this polymer foam-processing method and unique channeled architecture allows the introduction of neurotrophic factors into the conduit in a controlled fashion. Deposition of different factors into distinct regions within the conduit may be possible to promote more precisely guided neural regeneration.

  9. Partial denervation of sub-basal axons persists following debridement wounds to the mouse cornea.

    PubMed

    Pajoohesh-Ganji, Ahdeah; Pal-Ghosh, Sonali; Tadvalkar, Gauri; Kyne, Briana M; Saban, Daniel R; Stepp, Mary Ann

    2015-11-01

    Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal

  10. Combination of small RNAs for skeletal muscle regeneration.

    PubMed

    Kim, NaJung; Yoo, James J; Atala, Anthony; Lee, Sang Jin

    2016-03-01

    Selectively controlling the expression of the target genes through RNA interference (RNAi) has significant therapeutic potential for injuries or diseases of tissues. We used this strategy to accelerate and enhance skeletal muscle regeneration for the treatment of muscular atrophy. In this study, we used myostatin small interfering (si)RNA (siGDF-8), a major inhibitory factor in the development and postnatal regeneration of skeletal muscle and muscle-specific microRNAs (miR-1 and -206) to further accelerate muscle regeneration. This combination of 3 small RNAs significantly improved the gene expression of myogenic regulatory factors in vitro, suggesting myogenic activation. Moreover, cell proliferation and myotube formation improved without compromising each other, which indicates the myogenic potential of this combination of small RNAs. The recovery of chemically injured tibialis anterior muscles in rats was significantly accelerated, both functionally and structurally. This novel combination of siRNA and miRNAs has promising therapeutic potential to improve in situ skeletal muscle regeneration. © FASEB.

  11. Physical Biology of Axonal Damage.

    PubMed

    de Rooij, Rijk; Kuhl, Ellen

    2018-01-01

    Excessive physical impacts to the head have direct implications on the structural integrity at the axonal level. Increasing evidence suggests that tau, an intrinsically disordered protein that stabilizes axonal microtubules, plays a critical role in the physical biology of axonal injury. However, the precise mechanisms of axonal damage remain incompletely understood. Here we propose a biophysical model of the axon to correlate the dynamic behavior of individual tau proteins under external physical forces to the evolution of axonal damage. To propagate damage across the scales, we adopt a consistent three-step strategy: First, we characterize the axonal response to external stretches and stretch rates for varying tau crosslink bond strengths using a discrete axonal damage model. Then, for each combination of stretch rates and bond strengths, we average the axonal force-stretch response of n = 10 discrete simulations, from which we derive and calibrate a homogenized constitutive model. Finally, we embed this homogenized model into a continuum axonal damage model of [1-d]-type in which d is a scalar damage parameter that is driven by the axonal stretch and stretch rate. We demonstrate that axonal damage emerges naturally from the interplay of physical forces and biological crosslinking. Our study reveals an emergent feature of the crosslink dynamics: With increasing loading rate, the axonal failure stretch increases, but axonal damage evolves earlier in time. For a wide range of physical stretch rates, from 0.1 to 10 /s, and biological bond strengths, from 1 to 100 pN, our model predicts a relatively narrow window of critical damage stretch thresholds, from 1.01 to 1.30, which agrees well with experimental observations. Our biophysical damage model can help explain the development and progression of axonal damage across the scales and will provide useful guidelines to identify critical damage level thresholds in response to excessive physical forces.

  12. Tissue sparing, behavioral recovery, supraspinal axonal sparing/regeneration following sub-acute glial transplantation in a model of spinal cord contusion.

    PubMed

    Barbour, Helen R; Plant, Christine D; Harvey, Alan R; Plant, Giles W

    2013-09-27

    It has been shown that olfactory ensheathing glia (OEG) and Schwann cell (SCs) transplantation are beneficial as cellular treatments for spinal cord injury (SCI), especially acute and sub-acute time points. In this study, we transplanted DsRED transduced adult OEG and SCs sub-acutely (14 days) following a T10 moderate spinal cord contusion injury in the rat. Behaviour was measured by open field (BBB) and horizontal ladder walking tests to ascertain improvements in locomotor function. Fluorogold staining was injected into the distal spinal cord to determine the extent of supraspinal and propriospinal axonal sparing/regeneration at 4 months post injection time point. The purpose of this study was to investigate if OEG and SCs cells injected sub acutely (14 days after injury) could: (i) improve behavioral outcomes, (ii) induce sparing/regeneration of propriospinal and supraspinal projections, and (iii) reduce tissue loss. OEG and SCs transplanted rats showed significant increased locomotion when compared to control injury only in the open field tests (BBB). However, the ladder walk test did not show statistically significant differences between treatment and control groups. Fluorogold retrograde tracing showed a statistically significant increase in the number of supraspinal nuclei projecting into the distal spinal cord in both OEG and SCs transplanted rats. These included the raphe, reticular and vestibular systems. Further pairwise multiple comparison tests also showed a statistically significant increase in raphe projecting neurons in OEG transplanted rats when compared to SCs transplanted animals. Immunohistochemistry of spinal cord sections short term (2 weeks) and long term (4 months) showed differences in host glial activity, migration and proteoglycan deposits between the two cell types. Histochemical staining revealed that the volume of tissue remaining at the lesion site had increased in all OEG and SCs treated groups. Significant tissue sparing was

  13. In vivo imaging reveals mitophagy independence in the maintenance of axonal mitochondria during normal aging.

    PubMed

    Cao, Xu; Wang, Haiqiong; Wang, Zhao; Wang, Qingyao; Zhang, Shuang; Deng, Yuanping; Fang, Yanshan

    2017-10-01

    Mitophagy is thought to be a critical mitochondrial quality control mechanism in neurons and has been extensively studied in neurological disorders such as Parkinson's disease. However, little is known about how mitochondria are maintained in the lengthy neuronal axons in the context of physiological aging. Here, we utilized the unique Drosophila wing nerve model and in vivo imaging to rigorously profile changes in axonal mitochondria during aging. We revealed that mitochondria became fragmented and accumulated in aged axons. However, lack of Pink1 or Parkin did not lead to the accumulation of axonal mitochondria or axonal degeneration. Further, unlike in in vitro cultured neurons, we found that mitophagy rarely occurred in intact axons in vivo, even in aged animals. Furthermore, blocking overall mitophagy by knockdown of the core autophagy genes Atg12 or Atg17 had little effect on the turnover of axonal mitochondria or axonal integrity, suggesting that mitophagy is not required for axonal maintenance; this is regardless of whether the mitophagy is PINK1-Parkin dependent or independent. In contrast, downregulation of mitochondrial fission-fusion genes caused age-dependent axonal degeneration. Moreover, Opa1 expression in the fly head was significantly decreased with age, which may underlie the accumulation of fragmented mitochondria in aged axons. Finally, we showed that adult-onset, neuronal downregulation of the fission-fusion, but not mitophagy genes, dramatically accelerated features of aging. We propose that axonal mitochondria are maintained independently of mitophagy and that mitophagy-independent mechanisms such as fission-fusion may be central to the maintenance of axonal mitochondria and neural integrity during normal aging. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  14. Current Opportunities for Clinical Monitoring of Axonal Pathology in Traumatic Brain Injury

    PubMed Central

    Tsitsopoulos, Parmenion P.; Abu Hamdeh, Sami; Marklund, Niklas

    2017-01-01

    Traumatic brain injury (TBI) is a multidimensional and highly complex disease commonly resulting in widespread injury to axons, due to rapid inertial acceleration/deceleration forces transmitted to the brain during impact. Axonal injury leads to brain network dysfunction, significantly contributing to cognitive and functional impairments frequently observed in TBI survivors. Diffuse axonal injury (DAI) is a clinical entity suggested by impaired level of consciousness and coma on clinical examination and characterized by widespread injury to the hemispheric white matter tracts, the corpus callosum and the brain stem. The clinical course of DAI is commonly unpredictable and it remains a challenging entity with limited therapeutic options, to date. Although axonal integrity may be disrupted at impact, the majority of axonal pathology evolves over time, resulting from delayed activation of complex intracellular biochemical cascades. Activation of these secondary biochemical pathways may lead to axonal transection, named secondary axotomy, and be responsible for the clinical decline of DAI patients. Advances in the neurocritical care of TBI patients have been achieved by refinements in multimodality monitoring for prevention and early detection of secondary injury factors, which can be applied also to DAI. There is an emerging role for biomarkers in blood, cerebrospinal fluid, and interstitial fluid using microdialysis in the evaluation of axonal injury in TBI. These biomarker studies have assessed various axonal and neuroglial markers as well as inflammatory mediators, such as cytokines and chemokines. Moreover, modern neuroimaging can detect subtle or overt DAI/white matter changes in diffuse TBI patients across all injury severities using magnetic resonance spectroscopy, diffusion tensor imaging, and positron emission tomography. Importantly, serial neuroimaging studies provide evidence for evolving axonal injury. Since axonal injury may be a key risk factor for

  15. Glia to axon RNA transfer.

    PubMed

    Sotelo, José Roberto; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José Roberto; Calliari, Aldo; Cal, Karina; Bresque, Mariana; Dipaolo, Andrés; Farias, Joaquina; Mercer, John A

    2014-03-01

    The existence of RNA in axons has been a matter of dispute for decades. Evidence for RNA and ribosomes has now accumulated to a point at which it is difficult to question, much of the disputes turned to the origin of these axonal RNAs. In this review, we focus on studies addressing the origin of axonal RNAs and ribosomes. The neuronal soma as the source of most axonal RNAs has been demonstrated and is indisputable. However, the surrounding glial cells may be a supplemental source of axonal RNAs, a matter scarcely investigated in the literature. Here, we review the few papers that have demonstrated that glial-to-axon RNA transfer is not only feasible, but likely. We describe this process in both invertebrate axons and vertebrate axons. Schwann cell to axon ribosomes transfer was conclusively demonstrated (Court et al. [2008]: J. Neurosci 28:11024-11029; Court et al. [2011]: Glia 59:1529-1539). However, mRNA transfer still remains to be demonstrated in a conclusive way. The intercellular transport of mRNA has interesting implications, particularly with respect to the integration of glial and axonal function. This evolving field is likely to impact our understanding of the cell biology of the axon in both normal and pathological conditions. Most importantly, if the synthesis of proteins in the axon can be controlled by interacting glia, the possibilities for clinical interventions in injury and neurodegeneration are greatly increased. Copyright © 2013 Wiley Periodicals, Inc.

  16. Spinal cord regeneration: lessons for mammals from non-mammalian vertebrates.

    PubMed

    Lee-Liu, Dasfne; Edwards-Faret, Gabriela; Tapia, Víctor S; Larraín, Juan

    2013-08-01

    Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative-permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so. Copyright © 2013 Wiley Periodicals, Inc.

  17. Nerve Regeneration in the Peripheral Nervous System versus the Central Nervous System and the Relevance to Speech and Hearing after Nerve Injuries

    ERIC Educational Resources Information Center

    Gordon, Tessa; Gordon, Karen

    2010-01-01

    Schwann cells normally form myelin sheaths around axons in the peripheral nervous system (PNS) and support nerve regeneration after nerve injury. In contrast, nerve regeneration in the central nervous system (CNS) is not supported by the myelinating cells known as oligodendrocytes. We have found that: 1) low frequency electrical stimulation can be…

  18. The discovery of the growth cone and its influence on the study of axon guidance

    PubMed Central

    Tamariz, Elisa; Varela-Echavarría, Alfredo

    2015-01-01

    For over a century, there has been a great deal of interest in understanding how neural connectivity is established during development and regeneration. Interest in the latter arises from the possibility that knowledge of this process can be used to re-establish lost connections after lesion or neurodegeneration. At the end of the XIX century, Santiago Ramón y Cajal discovered that the distal tip of growing axons contained a structure that he called the growth cone. He proposed that this structure enabled the axon’s oriented growth in response to attractants, now known as chemotropic molecules. He further proposed that the physical properties of the surrounding tissues could influence the growth cone and the direction of growth. This seminal discovery afforded a plausible explanation for directed axonal growth and has led to the discovery of axon guidance mechanisms that include diffusible attractants and repellants and guidance cues anchored to cell membranes or extracellular matrix. In this review the major events in the development of this field are discussed. PMID:26029056

  19. KINETICS OF ION MOVEMENT IN THE SQUID GIANT AXON

    PubMed Central

    Shanes, Abraham M.; Berman, Morris D.

    1955-01-01

    The loss of Na22, K42, and Cl36 from single giant axons of the squid, Loligo pealii, following exposure to an artificial sea water containing these radioisotopes, occurs in two stages, an initial rapid one followed by an exponential decline. The time constants of the latter stage for the 3 ion species are, respectively, 290, 200, and 175 minutes. The outflux of sodium is depressed while that of potassium is accelerated in the absence of oxygen; the emergence of potassium is slowed by cocaine, while that of sodium is unaffected. One cm. ends of the axons take up about twice as much radiosodium as the central segment; this difference in activity is largely preserved during exposure to inactive solution. Such marked differences are not observed with radiopotassium. From the experimental data estimates are given of the influxes and outfluxes of the individual ions. The kinetics of outflux suggests a cortical layer of measureable thickness which contains the ions in different proportions from those in the medium and which governs the rate of emergence of these ions from the axon as though it contained very few but large (relative to ion dimensions) pores. PMID:13271727

  20. Nerve Degeneration and Regeneration Associated with NF1 Tumors

    DTIC Science & Technology

    2014-09-01

    burden for this collection of information is estimated to average 1 hour per response , including the time for reviewing instructions, searching existing...structure with an impenetrable fibrotic mass. Even though afflicted neurons remain viable they fail to regrow axons into the fibrotic mass (even when...devoid of tumor cells). The main conclusion of our findings is that tumor eradication does not induce or support nerve regeneration. Instead, a fibrotic

  1. Interleukin 6-dependent genomic instability heralds accelerated carcinogenesis following liver regeneration on a background of chronic hepatitis.

    PubMed

    Lanton, Tali; Shriki, Anat; Nechemia-Arbely, Yael; Abramovitch, Rinat; Levkovitch, Orr; Adar, Revital; Rosenberg, Nofar; Paldor, Mor; Goldenberg, Daniel; Sonnenblick, Amir; Peled, Amnon; Rose-John, Stefan; Galun, Eithan; Axelrod, Jonathan H

    2017-05-01

    Liver cancer, which typically develops on a background of chronic liver inflammation, is now the second leading cause of cancer mortality worldwide. For patients with liver cancer, surgical resection is a principal treatment modality that offers a chance of prolonged survival. However, tumor recurrence after resection, the mechanisms of which remain obscure, markedly limits the long-term survival of these patients. We have shown that partial hepatectomy in multidrug resistance 2 knockout (Mdr2 -/- ) mice, a model of chronic inflammation-associated liver cancer, significantly accelerates hepatocarcinogenesis. Here, we explore the postsurgical mechanisms that drive accelerated hepatocarcinogenesis in Mdr2 -/- mice by perioperative pharmacological inhibition of interleukin-6 (IL6), which is a crucial liver regeneration priming cytokine. We demonstrate that inhibition of IL6 signaling dramatically impedes tumorigenesis following partial hepatectomy without compromising survival or liver mass recovery. IL6 blockade significantly inhibited hepatocyte cell cycle progression while promoting a hypertrophic regenerative response, without increasing apoptosis. Mdr2 -/- mice contain hepatocytes with a notable persistent DNA damage response (γH2AX, 53BP1) due to chronic inflammation. We show that liver regeneration in this microenvironment leads to a striking increase in hepatocytes bearing micronuclei, a marker of genomic instability, which is suppressed by IL6 blockade. Our findings indicate that genomic instability derived during the IL6-mediated liver regenerative response within a milieu of chronic inflammation links partial hepatectomy to accelerated hepatocarcinogenesis; this suggests a new therapeutic approach through the usage of an anti-IL6 treatment to extend the tumor-free survival of patients undergoing surgical resection. (Hepatology 2017;65:1600-1611). © 2016 by the American Association for the Study of Liver Diseases.

  2. L1CAM/Neuroglian controls the axon-axon interactions establishing layered and lobular mushroom body architecture.

    PubMed

    Siegenthaler, Dominique; Enneking, Eva-Maria; Moreno, Eliza; Pielage, Jan

    2015-03-30

    The establishment of neuronal circuits depends on the guidance of axons both along and in between axonal populations of different identity; however, the molecular principles controlling axon-axon interactions in vivo remain largely elusive. We demonstrate that the Drosophila melanogaster L1CAM homologue Neuroglian mediates adhesion between functionally distinct mushroom body axon populations to enforce and control appropriate projections into distinct axonal layers and lobes essential for olfactory learning and memory. We addressed the regulatory mechanisms controlling homophilic Neuroglian-mediated cell adhesion by analyzing targeted mutations of extra- and intracellular Neuroglian domains in combination with cell type-specific rescue assays in vivo. We demonstrate independent and cooperative domain requirements: intercalating growth depends on homophilic adhesion mediated by extracellular Ig domains. For functional cluster formation, intracellular Ankyrin2 association is sufficient on one side of the trans-axonal complex whereas Moesin association is likely required simultaneously in both interacting axonal populations. Together, our results provide novel mechanistic insights into cell adhesion molecule-mediated axon-axon interactions that enable precise assembly of complex neuronal circuits. © 2015 Siegenthaler et al.

  3. Neutron source, linear-accelerator fuel enricher and regenerator and associated methods

    DOEpatents

    Steinberg, Meyer; Powell, James R.; Takahashi, Hiroshi; Grand, Pierre; Kouts, Herbert

    1982-01-01

    A device for producing fissile material inside of fabricated nuclear elements so that they can be used to produce power in nuclear power reactors. Fuel elements, for example, of a LWR are placed in pressure tubes in a vessel surrounding a liquid lead-bismuth flowing columnar target. A linear-accelerator proton beam enters the side of the vessel and impinges on the dispersed liquid lead-bismuth columns and produces neutrons which radiate through the surrounding pressure tube assembly or blanket containing the nuclear fuel elements. These neutrons are absorbed by the natural fertile uranium-238 elements and are transformed to fissile plutonium-239. The fertile fuel is thus enriched in fissile material to a concentration whereby they can be used in power reactors. After use in the power reactors, dispensed depleted fuel elements can be reinserted into the pressure tubes surrounding the target and the nuclear fuel regenerated for further burning in the power reactor.

  4. Nerve regeneration in nerve grafts conditioned by vibration exposure.

    PubMed

    Bergman, S; Widerberg, A; Danielsen, N; Lundborg, G; Dahlin, L B

    1995-01-01

    Regeneration distances were studied in nerves from vibration-exposed limbs. One hind limb of anaesthetized rats was attached to a vibration exciter and exposed to vibration (80 Hz/32 m/s2) for 5 h/day for 2 or 5 days. Seven days after the latest vibration period a 10-mm long nerve graft was taken from the vibrated sciatic nerve and sutured into a corresponding defect in the con-tralateral sciatic nerve and vice versa, thereby creating two different models within the same animal: (i) regeneration from a freshly transected unvibrated nerve into a vibrated graft and (ii) regeneration from a vibrated nerve into a fresh nerve graft (vibrated recipient side). Four, 6 or 8 days postoperatively (p.o.) the distances achieved by the regenerating axons were determined using the pinch reflex test. Two days of vibration did not influence the regeneration, but 5 days of vibration reduced the initial delay period and a slight reduction of regeneration rate was observed. After 5 days of vibration an increased regeneration distance was observed in both models at day 4 p.o. and at day 6 p.o. in vibrated grafts. This study demonstrates that vibration can condition peripheral nerves and this may be caused by local changes in the peripheral nerve trunk and in the neuron itself.

  5. A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3′ untranslated region AU-rich regulatory element

    PubMed Central

    Yoo, Soonmoon; Kim, Hak Hee; Kim, Paul; Donnelly, Christopher J.; Kalinski, Ashley L.; Vuppalanchi, Deepika; Park, Michael; Lee, Seung Joon; Merianda, Tanuja T.; Perrone-Bizzozero, Nora I.; Twiss, Jeffery L.

    2013-01-01

    Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3′UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich element (ARE) in GAP-43’s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 coimmunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3′UTR competing with endogenous GAP-43 mRNA. Conversely, overexpressing GAP-43 coding sequence with it’s 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43’s 3′UTR. PMID:23586486

  6. Paeoniae alba Radix Promotes Peripheral Nerve Regeneration

    PubMed Central

    Huang, Kun-Shan; Lin, Jaung-Geng; Lee, Han-Chung; Tsai, Fuu-Jen; Bau, Da-Tian; Huang, Chih-Yang; Yao, Chun-Hsu; Chen, Yueh-Sheng

    2011-01-01

    The present study provides in vitro and in vivo evaluation of Paeoniae alba Radix (PR) on peripheral nerve regeneration. In the in vitro study, we found the PR caused a marked enhancement of the nerve growth factor-mediated neurite outgrowth from PC12 cells as well as their expression of growth associated protein 43 and synapsin I. In the in vivo study, silicone rubber chambers filled with the PR water extract were used to bridge a 10-mm sciatic nerve defect in rats. At the conclusion of 8 weeks, regenerated nerves in the PR groups, especially at 1.25 mg ml−1 had a higher rate of successful regeneration across the wide gap, relatively larger mean values of total nerve area, myelinated axon count and blood vessel number, and a significantly larger nerve conductive velocity compared to the control group (P  <  .05). These results suggest that the PR extract can be a potential nerve growth-promoting factor, being salutary in aiding the growth of injured peripheral nerve. PMID:19687191

  7. STAT3 Controls the Long-Term Survival and Phenotype of Repair Schwann Cells during Nerve Regeneration.

    PubMed

    Benito, Cristina; Davis, Catherine M; Gomez-Sanchez, Jose A; Turmaine, Mark; Meijer, Dies; Poli, Valeria; Mirsky, Rhona; Jessen, Kristjan R

    2017-04-19

    After nerve injury, Schwann cells convert to a phenotype specialized to promote repair. But during the slow process of axonal regrowth, these repair Schwann cells gradually lose their regeneration-supportive features and eventually die. Although this is a key reason for the frequent regeneration failures in humans, the transcriptional mechanisms that control long-term survival and phenotype of repair cells have not been studied, and the molecular signaling underlying their decline is obscure. We show, in mice, that Schwann cell STAT3 has a dual role. It supports the long-term survival of repair Schwann cells and is required for the maintenance of repair Schwann cell properties. In contrast, STAT3 is less important for the initial generation of repair Schwann cells after injury. In repair Schwann cells, we find that Schwann cell STAT3 activation by Tyr705 phosphorylation is sustained during long-term denervation. STAT3 is required for maintaining autocrine Schwann cell survival signaling, and inactivation of Schwann cell STAT3 results in a striking loss of repair cells from chronically denervated distal stumps. STAT3 inactivation also results in abnormal morphology of repair cells and regeneration tracks, and failure to sustain expression of repair cell markers, including Shh, GDNF, and BDNF. Because Schwann cell development proceeds normally without STAT3, the function of this factor appears restricted to Schwann cells after injury. This identification of transcriptional mechanisms that support long-term survival and differentiation of repair cells will help identify, and eventually correct, the failures that lead to the deterioration of this important cell population. SIGNIFICANCE STATEMENT Although injured peripheral nerves contain repair Schwann cells that provide signals and spatial clues for promoting regeneration, the clinical outcome after nerve damage is frequently poor. A key reason for this is that, during the slow growth of axons through the proximal

  8. Curcumin mitigates axonal injury and neuronal cell apoptosis through the PERK/Nrf2 signaling pathway following diffuse axonal injury.

    PubMed

    Huang, Tingqin; Zhao, Junjie; Guo, Dan; Pang, Honggang; Zhao, Yonglin; Song, Jinning

    2018-05-23

    Diffuse axonal injury (DAI) accounts for more than 50% of all traumatic brain injury. In response to the mechanical damage associated with DAI, the abnormal proteins produced in the neurons and axons, namely, β-APP and p-tau, induce endoplasmic reticulum (ER) stress. Curcumin, a major component extracted from the rhizome of Curcuma longa, has shown potent anti-inflammatory, antioxidant, anti-infection, and antitumor activity in previous studies. Moreover, curcumin is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2) and promotes its nuclear translocation. In this study, we evaluated the therapeutic potential of curcumin for the treatment of DAI and investigated the mechanisms underlying the protective effects of curcumin against neural cell death and axonal injury after DAI. Rats subjected to a model of DAI by head rotational acceleration were treated with vehicle or curcumin to evaluate the effect of curcumin on neuronal and axonal injury. We observed that curcumin (20 mg/kg intraperitoneal) administered 1 h after DAI induction alleviated the aggregation of p-tau and β-APP in neurons, reduced ER-stress-related cell apoptosis, and ameliorated neurological deficits. Further investigation showed that the protective effect of curcumin in DAI was mediated by the PERK/Nrf2 pathway. Curcumin promoted PERK phosphorylation, and then Nrf2 dissociated from Keap1 and was translocated to the nucleus, which activated ATF4, an important bZIP transcription factor that maintains intracellular homeostasis, but inhibited the CHOP, a hallmark of ER stress and ER-associated programmed cell death. In summary, we demonstrate for the first time that curcumin confers protection against abnormal proteins and neuronal apoptosis after DAI, that the process is mediated by strengthening of the unfolded protein response to overcome ER stress, and that the protective effect of curcumin against DAI is dependent on the activation of Nrf2.

  9. Nogo-66 Receptor Antagonist Peptide (NEP1-40) Administration Promotes Functional Recovery and Axonal Growth After Lateral Funiculus Injury in the Adult Rat

    PubMed Central

    Cao, Y.; Shumsky, J. S.; Sabol, M. A.; Kushner, R. A.; Strittmatter, S.; Hamers, F. P. T.; Lee, D. H. S.; Rabacchi, S. A.; Murray, M.

    2010-01-01

    Objective The myelin protein Nogo inhibits axon regeneration by binding to its receptor (NgR) on axons. Intrathecal delivery of an NgR antagonist (NEP1-40) promotes growth of injured corticospinal axons and recovery of motor function following a dorsal hemisection. The authors used a similar design to examine recovery and repair after a lesion that interrupts the rubrospinal tract (RST). Methods Rats received a lateral funiculotomy at C4 and NEP1-40 or vehicle was delivered to the cervical spinal cord for 4 weeks. Outcome measures included motor and sensory tests and immunohistochemistry. Results Gait analysis showed recovery in the NEP1-40-treated group compared to operated controls, and a test of forelimb usage also showed a beneficial effect. The density of labeled RST axons increased ipsilaterally in the NEP1-40 group in the lateral funiculus rostral to the lesion and contralaterally in both gray and white matter. Thus, rubrospinal axons exhibited diminished dieback and/or growth up to the lesion site. This was accompanied by greater density of 5 HT and calcitonin gene-related peptide axons adjacent to and into the lesion/matrix site in the NEP1-40 group. Conclusions NgR blockade after RST injury is associated with axonal growth and/or diminished dieback of severed RST axons up to but not into or beyond the lesion/matrix site, and growth of serotonergic and dorsal root axons adjacent to and into the lesion/matrix site. NgR blockade also supported partial recovery of function. The authors’ results indicate that severed rubrospinal axons respond to NEP1-40 treatment but less robustly than corticospinal, raphe-spinal, or dorsal root axons. PMID:18056009

  10. Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

    PubMed Central

    Sato, Ayako; Kajiya, Hiroshi; Mori, Nana; Sato, Hironobu; Fukushima, Tadao; Kido, Hirofumi

    2017-01-01

    The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration. PMID:28060874

  11. Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

    PubMed

    Alves-Silva, Juliana; Sánchez-Soriano, Natalia; Beaven, Robin; Klein, Melanie; Parkin, Jill; Millard, Thomas H; Bellen, Hugo J; Venken, Koen J T; Ballestrem, Christoph; Kammerer, Richard A; Prokop, Andreas

    2012-07-04

    The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

  12. Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor to bone marrow stromal cells promotes axonal regeneration after transplantation in completely transected adult rat spinal cord

    PubMed Central

    Kamada, Takahito; Hashimoto, Masayuki; Murakami, Masazumi; Shirasawa, Hiroshi; Sakao, Seiichiro; Ino, Hidetoshi; Yoshinaga, Katsunori; Koshizuka, Shuhei; Moriya, Hideshige; Yamazaki, Masashi

    2007-01-01

    The aim of this study was to evaluate the efficacy in adult rat completely transected spinal cord of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to bone marrow stromal cells (BMSC). BMSC were infected with adenovirus vectors carrying β-galactosidase (AxCALacZ) or BDNF (AxCABDNF) genes. The T8 segment of spinal cord was removed and replaced by graft containing Matrigel alone (MG group) or Matrigel and BMSC infected by AxCALacZ (BMSC-LacZ group) or AxCABDNF (BMSC-BDNF group). Axons in the graft were evaluated by immunohistochemistry and functional recovery was assessed with BBB locomotor scale. In the BMSC-BDNF group, the number of fibers positive for growth associated protein-43, tyrosine hydroxylase, and calcitonin gene-related peptide was significantly larger than numbers found for the MG and BMSC-LacZ groups. Rats from BMSC-BDNF and BMSC-LacZ groups showed significant recovery of hind limb function compared with MG rats; however, there was no significant difference between groups in degree of functional recovery. These findings demonstrate that adenovirus vector-mediated ex vivo gene transfer of BDNF enhances the capacity of BMSC to promote axonal regeneration in this completely transected spinal cord model; however, BDNF failed to enhance hind limb functional recovery. Further investigation is needed to establish an optimal combination of cell therapy and neurotrophin gene transfer for cases of spinal cord injury. PMID:17885772

  13. Calpain-Mediated Proteolysis of Talin and FAK Regulates Adhesion Dynamics Necessary for Axon Guidance

    PubMed Central

    Kerstein, Patrick C.; Patel, Kevin M.

    2017-01-01

    Guidance of axons to their proper synaptic target sites requires spatially and temporally precise modulation of biochemical signals within growth cones. Ionic calcium (Ca2+) is an essential signal for axon guidance that mediates opposing effects on growth cone motility. The diverse effects of Ca2+ arise from the precise localization of Ca2+ signals into microdomains containing specific Ca2+ effectors. For example, differences in the mechanical and chemical composition of the underlying substrata elicit local Ca2+ signals within growth cone filopodia that regulate axon guidance through activation of the protease calpain. However, how calpain regulates growth cone motility remains unclear. Here, we identify the adhesion proteins talin and focal adhesion kinase (FAK) as proteolytic targets of calpain in Xenopus laevis spinal cord neurons both in vivo and in vitro. Inhibition of calpain increases the localization of endogenous adhesion signaling to growth cone filopodia. Using live cell microscopy and specific calpain-resistant point-mutants of talin (L432G) and FAK (V744G), we find that calpain inhibits paxillin-based adhesion assembly through cleavage of talin and FAK, and adhesion disassembly through cleavage of FAK. Blocking calpain cleavage of talin and FAK inhibits repulsive turning from focal uncaging of Ca2+ within filopodia. In addition, blocking calpain cleavage of talin and FAK in vivo promotes Rohon–Beard peripheral axon extension into the skin. These data demonstrate that filopodial Ca2+ signals regulate axon outgrowth and guidance through calpain regulation of adhesion dynamics through specific cleavage of talin and FAK. SIGNIFICANCE STATEMENT The proper formation of neuronal networks requires accurate guidance of axons and dendrites during development by motile structures known as growth cones. Understanding the intracellular signaling mechanisms that govern growth cone motility will clarify how the nervous system develops and regenerates, and may

  14. NT3-chitosan enables de novo regeneration and functional recovery in monkeys after spinal cord injury.

    PubMed

    Rao, Jia-Sheng; Zhao, Can; Zhang, Aifeng; Duan, Hongmei; Hao, Peng; Wei, Rui-Han; Shang, Junkui; Zhao, Wen; Liu, Zuxiang; Yu, Juehua; Fan, Kevin S; Tian, Zhaolong; He, Qihua; Song, Wei; Yang, Zhaoyang; Sun, Yi Eve; Li, Xiaoguang

    2018-06-12

    Spinal cord injury (SCI) often leads to permanent loss of motor, sensory, and autonomic functions. We have previously shown that neurotrophin3 (NT3)-loaded chitosan biodegradable material allowed for prolonged slow release of NT3 for 14 weeks under physiological conditions. Here we report that NT3-loaded chitosan, when inserted into a 1-cm gap of hemisectioned and excised adult rhesus monkey thoracic spinal cord, elicited robust axonal regeneration. Labeling of cortical motor neurons indicated motor axons in the corticospinal tract not only entered the injury site within the biomaterial but also grew across the 1-cm-long lesion area and into the distal spinal cord. Through a combination of magnetic resonance diffusion tensor imaging, functional MRI, electrophysiology, and kinematics-based quantitative walking behavioral analyses, we demonstrated that NT3-chitosan enabled robust neural regeneration accompanied by motor and sensory functional recovery. Given that monkeys and humans share similar genetics and physiology, our method is likely translatable to human SCI repair.

  15. A HuD-ZBP1 ribonucleoprotein complex localizes GAP-43 mRNA into axons through its 3' untranslated region AU-rich regulatory element.

    PubMed

    Yoo, Soonmoon; Kim, Hak H; Kim, Paul; Donnelly, Christopher J; Kalinski, Ashley L; Vuppalanchi, Deepika; Park, Michael; Lee, Seung J; Merianda, Tanuja T; Perrone-Bizzozero, Nora I; Twiss, Jeffery L

    2013-09-01

    Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β-actin and GAP-43 mRNAs. β-actin 3'UTR has a defined element for interaction with ZBP1, but GAP-43 mRNA shows no homology to this RNA sequence. Here, we show that an AU-rich regulatory element (ARE) in GAP-43's 3'UTR is necessary and sufficient for its axonal localization. Axonal GAP-43 mRNA levels increase after in vivo injury, and GAP-43 mRNA shows an increased half-life in regenerating axons. GAP-43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co-immunoprecipitate in an RNA-dependent fashion. Reporter mRNA with the GAP-43 ARE competes with endogenous β-actin mRNA for axonal localization and decreases axon length and branching similar to the β-actin 3'UTR competing with endogenous GAP-43 mRNA. Conversely, over-expressing GAP-43 coding sequence with its 3'UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP-43's 3'UTR. We have recently found that over-expression of GAP-43 using an axonally targeted construct with the 3'UTRs of GAP-43 promoted elongating growth of axons, while restricting the mRNA to the cell body with the 3'UTR of γ-actin had minimal effect on axon length. In this study, we show that the ARE in GAP-43's 3'UTR is responsible for localization of GAP-43 mRNA into axons and is sufficient for GAP-43 protein's role in elongating axonal growth. © 2013 International Society for Neurochemistry.

  16. Axonal abnormalities in vanishing white matter.

    PubMed

    Klok, Melanie D; Bugiani, Marianna; de Vries, Sharon I; Gerritsen, Wouter; Breur, Marjolein; van der Sluis, Sophie; Heine, Vivi M; Kole, Maarten H P; Baron, Wia; van der Knaap, Marjo S

    2018-04-01

    We aimed to study the occurrence and development of axonal pathology and the influence of astrocytes in vanishing white matter. Axons and myelin were analyzed using electron microscopy and immunohistochemistry on Eif2b4 and Eif2b5 single- and double-mutant mice and patient brain tissue. In addition, astrocyte-forebrain co-culture studies were performed. In the corpus callosum of Eif2b5- mutant mice, myelin sheath thickness, axonal diameter, and G-ratio developed normally up to 4 months. At 7 months, however, axons had become thinner, while in control mice axonal diameters had increased further. Myelin sheath thickness remained close to normal, resulting in an abnormally low G-ratio in Eif2b5- mutant mice. In more severely affected Eif2b4-Eif2b5 double-mutants, similar abnormalities were already present at 4 months, while in milder affected Eif2b4 mutants, few abnormalities were observed at 7 months. Additionally, from 2 months onward an increased percentage of thin, unmyelinated axons and increased axonal density were present in Eif2b5 -mutant mice. Co-cultures showed that Eif2b5 mutant astrocytes induced increased axonal density, also in control forebrain tissue, and that control astrocytes induced normal axonal density, also in mutant forebrain tissue. In vanishing white matter patient brains, axons and myelin sheaths were thinner than normal in moderately and severely affected white matter. In mutant mice and patients, signs of axonal transport defects and cytoskeletal abnormalities were minimal. In vanishing white matter, axons are initially normal and atrophy later. Astrocytes are central in this process. If therapy becomes available, axonal pathology may be prevented with early intervention.

  17. The Parkinsonian mimetic, 6-OHDA, impairs axonal transport in dopaminergic axons

    PubMed Central

    2014-01-01

    6-hydroxydopamine (6-OHDA) is one of the most commonly used toxins for modeling degeneration of dopaminergic (DA) neurons in Parkinson's disease. 6-OHDA also causes axonal degeneration, a process that appears to precede the death of DA neurons. To understand the processes involved in 6-OHDA-mediated axonal degeneration, a microdevice designed to isolate axons fluidically from cell bodies was used in conjunction with green fluorescent protein (GFP)-labeled DA neurons. Results showed that 6-OHDA quickly induced mitochondrial transport dysfunction in both DA and non-DA axons. This appeared to be a general effect on transport function since 6-OHDA also disrupted transport of synaptophysin-tagged vesicles. The effects of 6-OHDA on mitochondrial transport were blocked by the addition of the SOD1-mimetic, Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), as well as the anti-oxidant N-acetyl-cysteine (NAC) suggesting that free radical species played a role in this process. Temporally, microtubule disruption and autophagy occurred after transport dysfunction yet before DA cell death following 6-OHDA treatment. The results from the study suggest that ROS-mediated transport dysfunction occurs early and plays a significant role in inducing axonal degeneration in response to 6-OHDA treatment. PMID:24885281

  18. PERSPECTIVE: Electrical activity enhances neuronal survival and regeneration

    NASA Astrophysics Data System (ADS)

    Corredor, Raul G.; Goldberg, Jeffrey L.

    2009-10-01

    The failure of regeneration in the central nervous system (CNS) remains an enormous scientific and clinical challenge. After injury or in degenerative diseases, neurons in the adult mammalian CNS fail to regrow their axons and reconnect with their normal targets, and furthermore the neurons frequently die and are not normally replaced. While significant progress has been made in understanding the molecular basis for this lack of regenerative ability, a second approach has gained momentum: replacing lost neurons or lost connections with artificial electrical circuits that interface with the nervous system. In the visual system, gene therapy-based 'optogenetics' prostheses represent a competing technology. Now, the two approaches are converging, as recent data suggest that electrical activity itself, via the molecular signaling pathways such activity stimulates, is sufficient to induce neuronal survival and regeneration, particularly in retinal ganglion cells. Here, we review these data, discuss the effects of electrical activity on neurons' molecular signaling pathways and propose specific mechanisms by which exogenous electrical activity may be acting to enhance survival and regeneration.

  19. Axonal interferon responses and alphaherpesvirus neuroinvasion

    NASA Astrophysics Data System (ADS)

    Song, Ren

    Infection by alphaherpesviruses, including herpes simplex virus (HSV) and pseudorabies virus (PRV), typically begins at a peripheral epithelial surface and continues into the peripheral nervous system (PNS) that innervates this tissue. Inflammatory responses are induced at the infected peripheral site prior to viral invasion of the PNS. PNS neurons are highly polarized cells with long axonal processes that connect to distant targets. When the peripheral tissue is first infected, only the innervating axons are exposed to this inflammatory milieu, which include type I interferon (e.g. IFNbeta) and type II interferon (i.e. IFNgamma). IFNbeta can be produced by all types of cells, while IFNgamma is secreted by some specific types of immune cells. And both types of IFN induce antiviral responses in surrounding cells that express the IFN receptors. The fundamental question is how do PNS neurons respond to the inflammatory milieu experienced only by their axons. Axons must act as potential front-line barriers to prevent PNS infection and damage. Using compartmented cultures that physically separate neuron axons from cell bodies, I found that pretreating isolated axons with IFNbeta or IFNgamma significantly diminished the number of HSV-1 and PRV particles moving from axons to the cell bodies in an IFN receptor-dependent manner. Furthermore, I found the responses in axons are activated differentially by the two types of IFNs. The response to IFNbeta is a rapid, axon-only response, while the response to IFNgamma involves long distance signaling to the PNS cell body. For example, exposing axons to IFNbeta induced STAT1 phosphorylation (p-STAT1) only in axons, while exposure of axons to IFNgamma induced p-STAT1 accumulation in distant cell body nuclei. Blocking transcription in cell bodies eliminated IFNgamma-, but not IFNbeta-mediated antiviral effects. Proteomic analysis of IFNbeta- or IFNgamma-treated axons identified several differentially regulated proteins. Therefore

  20. Signal propagation along the axon.

    PubMed

    Rama, Sylvain; Zbili, Mickaël; Debanne, Dominique

    2018-03-08

    Axons link distant brain regions and are usually considered as simple transmission cables in which reliable propagation occurs once an action potential has been generated. Safe propagation of action potentials relies on specific ion channel expression at strategic points of the axon such as nodes of Ranvier or axonal branch points. However, while action potentials are generally considered as the quantum of neuronal information, their signaling is not entirely digital. In fact, both their shape and their conduction speed have been shown to be modulated by activity, leading to regulations of synaptic latency and synaptic strength. We report here newly identified mechanisms of (1) safe spike propagation along the axon, (2) compartmentalization of action potential shape in the axon, (3) analog modulation of spike-evoked synaptic transmission and (4) alteration in conduction time after persistent regulation of axon morphology in central neurons. We discuss the contribution of these regulations in information processing. Copyright © 2018 Elsevier Ltd. All rights reserved.

  1. Shock wave treatment improves nerve regeneration in the rat.

    PubMed

    Mense, Siegfried; Hoheisel, Ulrich

    2013-05-01

    The aims of the experiments were to: (1) determine whether low-energy shock wave treatment accelerates the recovery of muscle sensitivity and functionality after a nerve lesion; and (2) assess the effect of shock waves on the regeneration of injured nerve fibers. After compression of a muscle nerve in rats the effects of shock wave treatment on the sequelae of the lesion were tested. In non-anesthetized animals, pressure pain thresholds and exploratory activity were determined. The influence of the treatment on the distance of nerve regeneration was studied in immunohistochemical experiments. Both behavioral and immunohistochemical data show that shock wave treatment accelerates the recovery of muscle sensitivity and functionality and promotes regeneration of injured nerve fibers. Treatment with focused shock waves induces an improvement of nerve regeneration in a rodent model of nerve compression. Copyright © 2012 Wiley Periodicals, Inc.

  2. Rabies Virus Hijacks and Accelerates the p75NTR Retrograde Axonal Transport Machinery

    PubMed Central

    Gluska, Shani; Zahavi, Eitan Erez; Chein, Michael; Gradus, Tal; Bauer, Anja; Finke, Stefan; Perlson, Eran

    2014-01-01

    Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS. PMID:25165859

  3. Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration.

    PubMed

    Jung, Namhee; Park, Saeyoung; Choi, Yoonyoung; Park, Joo-Won; Hong, Young Bin; Park, Hyun Ho Choi; Yu, Yeonsil; Kwak, Geon; Kim, Han Su; Ryu, Kyung-Ha; Kim, Jae Kwang; Jo, Inho; Choi, Byung-Ok; Jung, Sung-Chul

    2016-11-09

    Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19 , GFAP , MBP , NGFR , S100B , and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR , S100B , and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

  4. Floor plate chemoattracts crossed axons and chemorepels uncrossed axons in the vertebrate brain.

    PubMed

    Tamada, A; Shirasaki, R; Murakami, F

    1995-05-01

    In the bilaterally symmetrical vertebrate CNS, all developing axons must choose between remaining on the same side of the midline or growing across it. The mechanism underlying this axonal pathfinding is, however, poorly understood. Here we demonstrate that the ventral midline floor plate (FP) chemorepels two types of ipsilaterally projecting axons, one from the alar plate and another from the basal plate in the mesencephalon. We further demonstrate that the FP chemoattracts contralaterally projecting myelencephalic as well as metencephalic axons. The FP at all axial levels displayed both chemoattractive and chemorepellent activities, suggesting that FP chemoattraction and chemorepulsion may be at work throughout the neuraxis. Chemotropic guidance by the FP may therefore play a key role in the establishment of neuronal projection laterality.

  5. Neuronal Dynamics and Axonal Flow, V. The Semisolid State of the Moving Axonal Column

    PubMed Central

    Weiss, Paul A.

    1972-01-01

    Evidence assembled since the first comprehensive description of “axonal flow”, by deformation analysis, electron microscopy, cinemicrography, and microrheology, has confirmed that the axon of the mature neuron is (a) a semisolid column; (b) in cellulifugal motion at about 1 μm/min (1 mm per day); (c) continuously reproduced at its perikaryal base; (d) propelled by a microperistaltic pulse wave in its surface; and (e) undergoing internal dissolution at the nerve ending. The axon thus “flows” as a structural entity (“axonal flow”), in contradistinction to fast “intraaxonal transport” of molecules and molecular assemblies along internal routes and by mechanisms that are still unknown. Images PMID:4111049

  6. Regeneration and maintenance of the planarian midline is regulated by a slit orthologue.

    PubMed

    Cebrià, Francesc; Guo, Tingxia; Jopek, Jessica; Newmark, Phillip A

    2007-07-15

    Several families of evolutionarily conserved axon guidance cues orchestrate the precise wiring of the nervous system during embryonic development. The remarkable plasticity of freshwater planarians provides the opportunity to study these molecules in the context of neural regeneration and maintenance. Here we characterize a homologue of the Slit family of guidance cues from the planarian Schmidtea mediterranea. Smed-slit is expressed along the planarian midline, in both dorsal and ventral domains. RNA interference (RNAi) targeting Smed-slit results in the collapse of many newly regenerated tissues at the midline; these include the cephalic ganglia, ventral nerve cords, photoreceptors, and the posterior digestive system. Surprisingly, Smed-slit RNAi knockdown animals also develop morphologically distinguishable, ectopic neural structures near the midline in uninjured regions of intact and regenerating planarians. These results suggest that Smed-slit acts not only as a repulsive cue required for proper midline formation during regeneration but that it may also act to regulate the behavior of neural precursors at the midline in intact planarians.

  7. Axons take a dive

    PubMed Central

    Tong, Cheuk Ka; Cebrián-Silla, Arantxa; Paredes, Mercedes F; Huang, Eric J; García-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2015-01-01

    In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular–subventricular zone (V–SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells. PMID:26413556

  8. Oligodendroglia: metabolic supporters of axons.

    PubMed

    Morrison, Brett M; Lee, Youngjin; Rothstein, Jeffrey D

    2013-12-01

    Axons are specialized extensions of neurons that are critical for the organization of the nervous system. To maintain function in axons that often extend some distance from the cell body, specialized mechanisms of energy delivery are likely to be necessary. Over the past decade, greater understanding of human demyelinating diseases and the development of animal models have suggested that oligodendroglia are critical for maintaining the function of axons. In this review, we discuss evidence for the vulnerability of neurons to energy deprivation, the importance of oligodendrocytes for axon function and survival, and recent data suggesting that transfer of energy metabolites from oligodendroglia to axons through monocarboxylate transporter 1 (MCT1) may be critical for the survival of axons. This pathway has important implications both for the basic biology of the nervous system and for human neurological disease. New insights into the role of oligodendroglial biology provide an exciting opportunity for revisions in nervous system biology, understanding myelin-based disorders, and therapeutics development. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Meninges-derived cues control axon guidance.

    PubMed

    Suter, Tracey A C S; DeLoughery, Zachary J; Jaworski, Alexander

    2017-10-01

    The axons of developing neurons travel long distances along stereotyped pathways under the direction of extracellular cues sensed by the axonal growth cone. Guidance cues are either secreted proteins that diffuse freely or bind the extracellular matrix, or membrane-anchored proteins. Different populations of axons express distinct sets of receptors for guidance cues, which results in differential responses to specific ligands. The full repertoire of axon guidance cues and receptors and the identity of the tissues producing these cues remain to be elucidated. The meninges are connective tissue layers enveloping the vertebrate brain and spinal cord that serve to protect the central nervous system (CNS). The meninges also instruct nervous system development by regulating the generation and migration of neural progenitors, but it has not been determined whether they help guide axons to their targets. Here, we investigate a possible role for the meninges in neuronal wiring. Using mouse neural tissue explants, we show that developing spinal cord meninges produce secreted attractive and repulsive cues that can guide multiple types of axons in vitro. We find that motor and sensory neurons, which project axons across the CNS-peripheral nervous system (PNS) boundary, are attracted by meninges. Conversely, axons of both ipsi- and contralaterally projecting dorsal spinal cord interneurons are repelled by meninges. The responses of these axonal populations to the meninges are consistent with their trajectories relative to meninges in vivo, suggesting that meningeal guidance factors contribute to nervous system wiring and control which axons are able to traverse the CNS-PNS boundary. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. A compound scaffold with uniform longitudinally oriented guidance cues and a porous sheath promotes peripheral nerve regeneration in vivo.

    PubMed

    Huang, Liangliang; Zhu, Lei; Shi, Xiaowei; Xia, Bing; Liu, Zhongyang; Zhu, Shu; Yang, Yafeng; Ma, Teng; Cheng, Pengzhen; Luo, Kai; Huang, Jinghui; Luo, Zhuojing

    2018-03-01

    Scaffolds with inner fillers that convey directional guidance cues represent promising candidates for nerve repair. However, incorrect positioning or non-uniform distribution of intraluminal fillers might result in regeneration failure. In addition, proper porosity (to enhance nutrient and oxygen exchange but prevent fibroblast infiltration) and mechanical properties (to ensure fixation and to protect regenerating axons from compression) of the outer sheath are also highly important for constructing advanced nerve scaffolds. In this study, we constructed a compound scaffold using a stage-wise strategy, including directionally freezing orientated collagen-chitosan (O-CCH) filler, electrospinning poly(ε-caprolactone) (PCL) sheaths and assembling O-CCH/PCL scaffolds. Based on scanning electron microscopy (SEM) and mechanical tests, a blend of collagen/chitosan (1:1) was selected for filler fabrication, and a wall thickness of 400 μm was selected for PCL sheath production. SEM and three-dimensional (3D) reconstruction further revealed that the O-CCH filler exhibited a uniform, longitudinally oriented microstructure (over 85% of pores were 20-50 μm in diameter). The electrospun PCL porous sheath with pore sizes of 6.5 ± 3.3 μm prevented fibroblast invasion. The PCL sheath exhibited comparable mechanical properties to commercially available nerve conduits, and the O-CCH filler showed a physiologically relevant substrate stiffness of 2.0 ± 0.4 kPa. The differential degradation time of the filler and sheath allows the O-CCH/PCL scaffold to protect regenerating axons from compression stress while providing enough space for regenerating nerves. In vitro and in vivo studies indicated that the O-CCH/PCL scaffolds could promote axonal regeneration and Schwann cell migration. More importantly, functional results indicated that the CCH/PCL compound scaffold induced comparable functional recovery to that of the autograft group at the end of the study. Our

  11. Short-Term Depression of Axonal Spikes at the Mouse Hippocampal Mossy Fibers and Sodium Channel-Dependent Modulation

    PubMed Central

    Ohura, Shunsuke

    2018-01-01

    Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the functional significance, possible activity-dependent modulation of axonal spikes has not been studied extensively, partly due to inaccessibility of the small structures of axons for electrophysiological recordings. In this study, we tested the possibility of use-dependent changes in axonal spikes at the hippocampal mossy fibers, where direct recordings from the axon terminals are readily feasible. Hippocampal slices were made from mice of either sex, and loose-patch clamp recordings were obtained from the visually identified giant mossy fiber boutons located in the stratum lucidum of the CA3 region. Stimulation of the granule cell layer of the dentate gyrus elicited axonal spikes at the single bouton which occurred in all or none fashion. Unexpected from the digital nature of spike signaling, the peak amplitude of the second spikes in response to paired stimuli at a 50-ms interval was slightly but reproducibly smaller than the first spikes. Repetitive stimuli at 20 or 100 Hz also caused progressive use-dependent depression during the train. Notably, veratridine, an inhibitor of inactivation of sodium channels, significantly accelerated the depression with minimal effect on the initial spikes. These results suggest that sodium channels contribute to use-dependent depression of axonal spikes at the hippocampal mossy fibers, possibly by shaping the afterdepolarization (ADP) following axonal spikes. Prolonged depolarization during ADP may inactivate a fraction of sodium channels and thereby suppresses the subsequent spikes at the hippocampal mossy fibers. PMID:29468192

  12. Development of microarray device for functional evaluation of PC12D cell axonal extension ability

    NASA Astrophysics Data System (ADS)

    Nakamachi, Eiji; Yanagimoto, Junpei; Murakami, Shinya; Morita, Yusuke

    2014-04-01

    In this study, we developed a microarray bio-MEMS device that could trap PC12D (rat pheochromocytoma cells) cells to examine the intercellular interaction effect on the cell activation and the axonal extension ability. This is needed to assign particular patterns of PC12D cells to establish a cell functional evaluation technique. This experimental observation-based technique can be used for design of the cell sheet and scaffold for peripheral and central nerve regeneration. We have fabricated a micropillar-array bio-MEMS device, whose diameter was approximately 10 μm, by using thick photoresist SU-8 on the glass slide substrate. A maximum trapped PC12D cell ratio, 48.5%, was achieved. Through experimental observation of patterned PC12D "bi-cells" activation, we obtained the following results. Most of the PC12D "bi-cells" which had distances between 40 and 100 μm were connected after 24 h with a high probability. On the other hand, "bi-cells" which had distances between 110 and 200 μm were not connected. In addition, we measured axonal extension velocities in cases where the intercellular distance was between 40 and 100 μm. A maximum axonal extension velocity, 86.4 μm/h, was obtained at the intercellular distance of 40 μm.

  13. A Fat-Facets-Dscam1-JNK Pathway Enhances Axonal Growth in Development and after Injury

    PubMed Central

    Koch, Marta; Nicolas, Maya; Zschaetzsch, Marlen; de Geest, Natalie; Claeys, Annelies; Yan, Jiekun; Morgan, Matthew J.; Erfurth, Maria-Luise; Holt, Matthew; Schmucker, Dietmar; Hassan, Bassem A.

    2018-01-01

    Injury to the adult central nervous systems (CNS) can result in severe long-term disability because damaged CNS connections fail to regenerate after trauma. Identification of regulators that enhance the intrinsic growth capacity of severed axons is a first step to restore function. Here, we conducted a gain-of-function genetic screen in Drosophila to identify strong inducers of axonal growth after injury. We focus on a novel axis the Down Syndrome Cell Adhesion Molecule (Dscam1), the de-ubiquitinating enzyme Fat Facets (Faf)/Usp9x and the Jun N-Terminal Kinase (JNK) pathway transcription factor Kayak (Kay)/Fos. Genetic and biochemical analyses link these genes in a common signaling pathway whereby Faf stabilizes Dscam1 protein levels, by acting on the 3′-UTR of its mRNA, and Dscam1 acts upstream of the growth-promoting JNK signal. The mammalian homolog of Faf, Usp9x/FAM, shares both the regenerative and Dscam1 stabilizing activities, suggesting a conserved mechanism. PMID:29472843

  14. Axonal Transport: How High Microtubule Density Can Compensate for Boundary Effects in Small-Caliber Axons

    PubMed Central

    Wortman, Juliana C.; Shrestha, Uttam M.; Barry, Devin M.; Garcia, Michael L.; Gross, Steven P.; Yu, Clare C.

    2014-01-01

    Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall. We find that having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus minimizes the effects of any opposition to transport. Even if microtubules are randomly placed in axons, we find that the higher density of microtubules found in small-caliber axons increases the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. The boundary effect is not a factor in transport in large-caliber axons where the microtubule density is lower. PMID:24559984

  15. Evidence That Descending Cortical Axons Are Essential for Thalamocortical Axons to Cross the Pallial-Subpallial Boundary in the Embryonic Forebrain

    PubMed Central

    Chen, Yijing; Magnani, Dario; Theil, Thomas; Pratt, Thomas; Price, David J.

    2012-01-01

    Developing thalamocortical axons traverse the subpallium to reach the cortex located in the pallium. We tested the hypothesis that descending corticofugal axons are important for guiding thalamocortical axons across the pallial-subpallial boundary, using conditional mutagenesis to assess the effects of blocking corticofugal axonal development without disrupting thalamus, subpallium or the pallial-subpallial boundary. We found that thalamic axons still traversed the subpallium in topographic order but did not cross the pallial-subpallial boundary. Co-culture experiments indicated that the inability of thalamic axons to cross the boundary was not explained by mutant cortex developing a long-range chemorepulsive action on thalamic axons. On the contrary, cortex from conditional mutants retained its thalamic axonal growth-promoting activity and continued to express Nrg-1, which is responsible for this stimulatory effect. When mutant cortex was replaced with control cortex, corticofugal efferents were restored and thalamic axons from conditional mutants associated with them and crossed the pallial-subpallial boundary. Our study provides the most compelling evidence to date that cortical efferents are required to guide thalamocortical axons across the pallial-subpallial boundary, which is otherwise hostile to thalamic axons. These results support the hypothesis that thalamic axons grow from subpallium to cortex guided by cortical efferents, with stimulation from diffusible cortical growth-promoting factors. PMID:22412988

  16. Neuregulin Facilitates Nerve Regeneration by Speeding Schwann Cell Migration via ErbB2/3-Dependent FAK Pathway

    PubMed Central

    Chang, Hung-Ming; Shyu, Ming-Kwang; Tseng, Guo-Fang; Liu, Chiung-Hui; Chang, Hung-Shuo; Lan, Chyn-Tair; Hsu, Wen-Ming; Liao, Wen-Chieh

    2013-01-01

    Background Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches. Methodology and Principal Findings Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles. Conclusions As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI. PMID:23301073

  17. Key changes in denervated muscles and their impact on regeneration and reinnervation

    PubMed Central

    Wu, Peng; Chawla, Aditya; Spinner, Robert J.; Yu, Cong; Yaszemski, Michael J.; Windebank, Anthony J.; Wang, Huan

    2014-01-01

    The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle's residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair. PMID:25422641

  18. Overexpression of IGF-1 attenuates skeletal muscle damage and accelerates muscle regeneration and functional recovery after disuse

    PubMed Central

    Ye, Fan; Mathur, Sunita; Liu, Min; Borst, Stephen E.; Walter, Glenn A.; Sweeney, H. Lee; Vandenborne, Krista

    2014-01-01

    Skeletal muscle is a highly dynamic tissue that responds to endogenous and external stimuli, including alterations in mechanical loading and growth factors. In particular, the antigravity soleus muscle experiences significant muscle atrophy during disuse and extensive muscle damage upon reloading. Since insulin-like growth factor-1 (IGF-1) has been implicated as a central regulator of muscle repair and modulation of muscle size, we examined the effect of viral mediated overexpression of IGF-1 on the soleus muscle following hindlimb cast immobilization and upon reloading. Recombinant IGF-1 cDNA virus was injected into one of the posterior hindlimbs of the mice, while the contralateral limb was injected with saline (control). At 20 weeks of age, both hindlimbs were immobilized for two weeks to induce muscle atrophy in the soleus and ankle plantar flexor muscle group. Subsequently, the mice were allowed to reambulate and muscle damage and recovery was monitored over a period of 2 to 21 days. The primary finding of this study was that IGF-1 overexpression attenuated reloading-induced muscle damage in the soleus muscle, and accelerated muscle regeneration and force recovery. Muscle T2 assessed by MRI, a nonspecific marker of muscle damage, was significantly lower in IGF-1 injected, compared to contralateral soleus muscles at 2 and 5 days reambulation (P<0.05). The reduced prevalence of muscle damage in IGF-1 injected soleus muscles was confirmed on histology, with a lower fraction area of abnormal muscle tissue in IGF-I injected muscles at 2 days reambulation (33.2±3.3%vs 54.1±3.6%, P<0.05). Evidence of the effect of IGF-1 on muscle regeneration included timely increases in the number of central nuclei (21% at 5 days reambulation), paired-box transcription factor 7 (36% at 5 days), embryonic myosin (37% at 10 days), and elevated MyoD mRNA (7-fold at 2 days) in IGF-1 injected limbs (P<0.05). These findings demonstrate a potential role of IGF-1 in protecting unloaded

  19. Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cheng, Lei; Liu, Yi; Zhao, Hua

    Highlights: •CDNF was successfully transfected by a lentiviral vector into the distal sciatic nerve. •CDNF improved S-100, NF200 expression and nerve regeneration after sciatic injury. •CDNF improved the remyelination and thickness of the regenerated sciatic nerve. •CDNF improved gastrocnemius muscle weight and sciatic functional recovery. -- Abstract: Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediatedmore » transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which

  20. Mice with hepatocyte-specific deficiency of type 3 deiodinase have intact liver regeneration and accelerated recovery from nonthyroidal illness after toxin-induced hepatonecrosis.

    PubMed

    Castroneves, Luciana A; Jugo, Rebecca H; Maynard, Michelle A; Lee, Jennifer S; Wassner, Ari J; Dorfman, David; Bronson, Roderick T; Ukomadu, Chinweike; Agoston, Agoston T; Ding, Lai; Luongo, Cristina; Guo, Cuicui; Song, Huaidong; Demchev, Valeriy; Lee, Nicholas Y; Feldman, Henry A; Vella, Kristen R; Peake, Roy W; Hartigan, Christina; Kellogg, Mark D; Desai, Anal; Salvatore, Domenico; Dentice, Monica; Huang, Stephen A

    2014-10-01

    Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome.

  1. Mice With Hepatocyte-Specific Deficiency of Type 3 Deiodinase Have Intact Liver Regeneration and Accelerated Recovery From Nonthyroidal Illness After Toxin-Induced Hepatonecrosis

    PubMed Central

    Castroneves, Luciana A.; Jugo, Rebecca H.; Maynard, Michelle A.; Lee, Jennifer S.; Wassner, Ari J.; Dorfman, David; Bronson, Roderick T.; Ukomadu, Chinweike; Agoston, Agoston T.; Ding, Lai; Luongo, Cristina; Guo, Cuicui; Song, Huaidong; Demchev, Valeriy; Lee, Nicholas Y.; Feldman, Henry A.; Vella, Kristen R.; Peake, Roy W.; Hartigan, Christina; Kellogg, Mark D.; Desai, Anal; Salvatore, Domenico; Dentice, Monica

    2014-01-01

    Type 3 deiodinase (D3), the physiologic inactivator of thyroid hormones, is induced during tissue injury and regeneration. This has led to the hypotheses that D3 impacts injury tolerance by reducing local T3 signaling and contributes to the fall in serum triiodothyronine (T3) observed in up to 75% of sick patients (termed the low T3 syndrome). Here we show that a novel mutant mouse with hepatocyte-specific D3 deficiency has normal local responses to toxin-induced hepatonecrosis, including normal degrees of tissue necrosis and intact regeneration, but accelerated systemic recovery from illness-induced hypothyroxinemia and hypotriiodothyroninemia, demonstrating that peripheral D3 expression is a key modulator of the low T3 syndrome. PMID:25004090

  2. MAPK signaling promotes axonal degeneration by speeding the turnover of the axonal maintenance factor NMNAT2

    PubMed Central

    Walker, Lauren J; Summers, Daniel W; Sasaki, Yo; Brace, EJ; Milbrandt, Jeffrey; DiAntonio, Aaron

    2017-01-01

    Injury-induced (Wallerian) axonal degeneration is regulated via the opposing actions of pro-degenerative factors such as SARM1 and a MAPK signal and pro-survival factors, the most important of which is the NAD+ biosynthetic enzyme NMNAT2 that inhibits activation of the SARM1 pathway. Here we investigate the mechanism by which MAPK signaling facilitates axonal degeneration. We show that MAPK signaling promotes the turnover of the axonal survival factor NMNAT2 in cultured mammalian neurons as well as the Drosophila ortholog dNMNAT in motoneurons. The increased levels of NMNAT2 are required for the axonal protection caused by loss of MAPK signaling. Regulation of NMNAT2 by MAPK signaling does not require SARM1, and so cannot be downstream of SARM1. Hence, pro-degenerative MAPK signaling functions upstream of SARM1 by limiting the levels of the essential axonal survival factor NMNAT2 to promote injury-dependent SARM1 activation. These findings are consistent with a linear molecular pathway for the axonal degeneration program. DOI: http://dx.doi.org/10.7554/eLife.22540.001 PMID:28095293

  3. Acute nutritional axonal neuropathy.

    PubMed

    Hamel, Johanna; Logigian, Eric L

    2018-01-01

    This study describes clinical, laboratory, and electrodiagnostic features of a severe acute axonal polyneuropathy common to patients with acute nutritional deficiency in the setting of alcoholism, bariatric surgery (BS), or anorexia. Retrospective analysis of clinical, electrodiagnostic, and laboratory data of patients with acute axonal neuropathy. Thirteen patients were identified with a severe, painful, sensory or sensorimotor axonal polyneuropathy that developed over 2-12 weeks with sensory ataxia, areflexia, variable muscle weakness, poor nutritional status, and weight loss, often with prolonged vomiting and normal cerebrospinal fluid protein. Vitamin B6 was low in half and thiamine was low in all patients when obtained before supplementation. Patients improved with weight gain and vitamin supplementation, with motor greater than sensory recovery. We suggest that acute or subacute axonal neuropathy in patients with weight loss or vomiting associated with alcohol abuse, BS, or dietary deficiency is one syndrome, caused by micronutrient deficiencies. Muscle Nerve 57: 33-39, 2018. © 2017 Wiley Periodicals, Inc.

  4. Corneal nerve regeneration. Correlation between morphology and restoration of sensitivity.

    PubMed

    de Leeuw, A M; Chan, K Y

    1989-09-01

    Corneal nerve regeneration was determined in albino rabbits after deepithelialization of the cornea using heptanol. Regeneration was monitored up to 10 weeks by measuring corneal tactile sensitivity using an esthesiometer and by examining stromal and intraepithelial nerve patterns following gold chloride impregnation and acetylcholinesterase staining. Tactile sensitivity was much reduced, possibly absent, up to 2 weeks after wounding. From 2.5-4 weeks, sensitivity recovered rapidly to 60% of prewounding levels and remained unchanged thereafter. In control corneas, a distinct orientation of the basoepithelial leashes towards the nasal-most limbus was observed in the central two-thirds of the cornea. Three days after wounding, neurites that were oriented radially towards the wound center extended into the periphery of the wound area from just beyond the wound margin. At 1 week, regenerating axons were present as single neurites and in the form of modified leashes, mainly at the periphery of the wound area but also more towards the center. At 3 weeks, neurites, regenerated leashes and networks of terminals with terminal endings were found throughout the regenerated epithelium. Regional changes in the orientation of the regenerated leashes were observed also. No further change in the intraepithelial nerve pattern was detectable thereafter up to 10 weeks after wounding. It was concluded that partial restoration of tactile sensitivity following deepithelialization of the cornea is a function of the establishment of a near-normal nerve pattern in the regenerated epithelium and is correlated with the subnormal neural density observed in a previous study.

  5. Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons.

    PubMed

    Aschrafi, Armaz; Gioio, Anthony E; Dong, Lijin; Kaplan, Barry B

    2017-01-01

    Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3'untranslated region (3'UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis- acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal.

  6. Extrinsic Repair of Injured Dendrites as a Paradigm for Regeneration by Fusion in Caenorhabditis elegans

    PubMed Central

    Oren-Suissa, Meital; Gattegno, Tamar; Kravtsov, Veronika; Podbilewicz, Benjamin

    2017-01-01

    Injury triggers regeneration of axons and dendrites. Research has identified factors required for axonal regeneration outside the CNS, but little is known about regeneration triggered by dendrotomy. Here, we study neuronal plasticity triggered by dendrotomy and determine the fate of complex PVD arbors following laser surgery of dendrites. We find that severed primary dendrites grow toward each other and reconnect via branch fusion. Simultaneously, terminal branches lose self-avoidance and grow toward each other, meeting and fusing at the tips via an AFF-1-mediated process. Ectopic branch growth is identified as a step in the regeneration process required for bypassing the lesion site. Failure of reconnection to the severed dendrites results in degeneration of the distal end of the neuron. We discover pruning of excess branches via EFF-1 that acts to recover the original wild-type arborization pattern in a late stage of the process. In contrast, AFF-1 activity during dendritic auto-fusion is derived from the lateral seam cells and not autonomously from the PVD neuron. We propose a model in which AFF-1-vesicles derived from the epidermal seam cells fuse neuronal dendrites. Thus, EFF-1 and AFF-1 fusion proteins emerge as new players in neuronal arborization and maintenance of arbor connectivity following injury in Caenorhabditis elegans. Our results demonstrate that there is a genetically determined multi-step pathway to repair broken dendrites in which EFF-1 and AFF-1 act on different steps of the pathway. EFF-1 is essential for dendritic pruning after injury and extrinsic AFF-1 mediates dendrite fusion to bypass injuries. PMID:28283540

  7. Extrinsic Repair of Injured Dendrites as a Paradigm for Regeneration by Fusion in Caenorhabditis elegans.

    PubMed

    Oren-Suissa, Meital; Gattegno, Tamar; Kravtsov, Veronika; Podbilewicz, Benjamin

    2017-05-01

    Injury triggers regeneration of axons and dendrites. Research has identified factors required for axonal regeneration outside the CNS, but little is known about regeneration triggered by dendrotomy. Here, we study neuronal plasticity triggered by dendrotomy and determine the fate of complex PVD arbors following laser surgery of dendrites. We find that severed primary dendrites grow toward each other and reconnect via branch fusion. Simultaneously, terminal branches lose self-avoidance and grow toward each other, meeting and fusing at the tips via an AFF-1-mediated process. Ectopic branch growth is identified as a step in the regeneration process required for bypassing the lesion site. Failure of reconnection to the severed dendrites results in degeneration of the distal end of the neuron. We discover pruning of excess branches via EFF-1 that acts to recover the original wild-type arborization pattern in a late stage of the process. In contrast, AFF-1 activity during dendritic auto-fusion is derived from the lateral seam cells and not autonomously from the PVD neuron. We propose a model in which AFF-1-vesicles derived from the epidermal seam cells fuse neuronal dendrites. Thus, EFF-1 and AFF-1 fusion proteins emerge as new players in neuronal arborization and maintenance of arbor connectivity following injury in Caenorhabditis elegans Our results demonstrate that there is a genetically determined multi-step pathway to repair broken dendrites in which EFF-1 and AFF-1 act on different steps of the pathway. EFF-1 is essential for dendritic pruning after injury and extrinsic AFF-1 mediates dendrite fusion to bypass injuries. Copyright © 2017 by the Genetics Society of America.

  8. Npn-1 Contributes to Axon-Axon Interactions That Differentially Control Sensory and Motor Innervation of the Limb

    PubMed Central

    Bianchi, Elisa; Novitch, Bennett G.; Huber, Andrea B.

    2011-01-01

    The initiation, execution, and completion of complex locomotor behaviors are depending on precisely integrated neural circuitries consisting of motor pathways that activate muscles in the extremities and sensory afferents that deliver feedback to motoneurons. These projections form in tight temporal and spatial vicinities during development, yet the molecular mechanisms and cues coordinating these processes are not well understood. Using cell-type specific ablation of the axon guidance receptor Neuropilin-1 (Npn-1) in spinal motoneurons or in sensory neurons in the dorsal root ganglia (DRG), we have explored the contribution of this signaling pathway to correct innervation of the limb. We show that Npn-1 controls the fasciculation of both projections and mediates inter-axonal communication. Removal of Npn-1 from sensory neurons results in defasciculation of sensory axons and, surprisingly, also of motor axons. In addition, the tight coupling between these two heterotypic axonal populations is lifted with sensory fibers now leading the spinal nerve projection. These findings are corroborated by partial genetic elimination of sensory neurons, which causes defasciculation of motor projections to the limb. Deletion of Npn-1 from motoneurons leads to severe defasciculation of motor axons in the distal limb and dorsal-ventral pathfinding errors, while outgrowth and fasciculation of sensory trajectories into the limb remain unaffected. Genetic elimination of motoneurons, however, revealed that sensory axons need only minimal scaffolding by motor axons to establish their projections in the distal limb. Thus, motor and sensory axons are mutually dependent on each other for the generation of their trajectories and interact in part through Npn-1-mediated fasciculation before and within the plexus region of the limbs. PMID:21364975

  9. Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation.

    PubMed

    Mathur, Chhavi; Johnson, Kory R; Tong, Brian A; Miranda, Pablo; Srikumar, Deepa; Basilio, Daniel; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

    2018-02-02

    Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker K V channel into excised squid giant axons. We found that total K + currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

  10. Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

    PubMed

    Nichol, Robert H; Hagen, Kate M; Lumbard, Derek C; Dent, Erik W; Gómez, Timothy M

    2016-02-17

    Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and

  11. Curcumin accelerates the repair of sciatic nerve injury in rats through reducing Schwann cells apoptosis and promoting myelinization.

    PubMed

    Zhao, Zhiwei; Li, Xiaoling; Li, Qing

    2017-08-01

    Schwann cells (SCs) play an indispensable role in the repair and regeneration of injured peripheral nerve. Curcumin can reduce SCs apoptosis, and promote the regeneration and functional recovery of injured peripheral nerves. However, the corresponding mechanisms are not clear. The article was aimed to explore the effect and corresponding mechanisms of curcumin on the repair of sciatic nerve injury in rats. After surgery induced sciatic nerve injury, the model rats were divided into three groups and treated with curcumin, curcumin+PD98059 and curcumin+IGF-1 respectively for 4days. The phosphorylation of Erk1/2 and Akt, and the expression of LC3-II, Beclin 1 and p62 were measured using western blotting. After treatment for 60days, myelination of the injured sciatic nerve was evaluated by MBP immunohistochemical staining and the expression of PMP22, Fibrin and S100 were determined using qRT-PCR and western blotting. In vitro, RSC96 cells were starved for 12h to induce autophagy, and received DMSO, curcumin, PD98059+curcumin, IGF-1+curcumin and BFA1 respectively. The phosphorylation of Erk1/2、Akt and the expression of LC3-II, Beclin 1, p62, PMP22, Fibrin and S100 were measured using western blotting, and the cell apoptosis was detected by flow cytometry. Curcumin could promote injury-induced cell autophagy, remyelination and axon regeneration in sciatic nerve of rats. In vitro, curcumin could accelerate cell autophagy through regulating autophagy related Erk1/2 and Akt pathway, prevent cell apoptosis and promote expression of PMP22 and S100, and reduced deposition of Fibrin in cultured RSC96 SCs. Curcumin could accelerate injured sciatic nerve repair in rats through reducing SCs apoptosis and promoting myelinization. Copyright © 2017. Published by Elsevier Masson SAS.

  12. Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons

    PubMed Central

    Gioio, Anthony E.

    2017-01-01

    Abstract Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters. In a previous communication, evidence was provided that TH mRNA is trafficked to the axon, where it is locally translated. In addition, a 50-bp sequence element in the 3′untranslated region (3’UTR) of TH mRNA was identified that directs TH mRNA to distal axons (i.e., zip-code). In the present study, the hypothesis was tested that local translation of TH plays an important role in the biosynthesis of the catecholamine neurotransmitters in the axon and/or presynaptic nerve terminal. Toward this end, a targeted deletion of the axonal transport sequence element was developed, using the lentiviral delivery of the CRISPR/Cas9 system, and two guide RNA (gRNA) sequences flanking the 50-bp cis-acting regulatory element in rat superior cervical ganglion (SCG) neurons. Deletion of the axonal transport element reduced TH mRNA levels in the distal axons and reduced the axonal protein levels of TH and TH activity as measured by phosphorylation of SER40 in SCG neurons. Moreover, deletion of the zip-code diminished the axonal levels of dopamine (DA) and norepinephrine (NE). Conversely, the local translation of exogenous TH mRNA in the distal axon enhanced TH levels and activity, and elevated axonal NE levels. Taken together, these results provide direct evidence to support the hypothesis that TH mRNA trafficking and local synthesis of TH play an important role in the synthesis of catecholamines in the axon and presynaptic terminal. PMID:28630892

  13. Axon diameter and intra-axonal volume fraction of the corticospinal tract in idiopathic normal pressure hydrocephalus measured by q-space imaging.

    PubMed

    Kamiya, Kouhei; Hori, Masaaki; Miyajima, Masakazu; Nakajima, Madoka; Suzuki, Yuriko; Kamagata, Koji; Suzuki, Michimasa; Arai, Hajime; Ohtomo, Kuni; Aoki, Shigeki

    2014-01-01

    Previous studies suggest that compression and stretching of the corticospinal tract (CST) potentially cause treatable gait disturbance in patients with idiopathic normal pressure hydrocephalus (iNPH). Measurement of axon diameter with diffusion MRI has recently been used to investigate microstructural alterations in neurological diseases. In this study, we investigated alterations in the axon diameter and intra-axonal fraction of the CST in iNPH by q-space imaging (QSI) analysis. Nineteen patients with iNPH and 10 age-matched controls were recruited. QSI data were obtained with a 3-T system by using a single-shot echo planar imaging sequence with the diffusion gradient applied parallel to the antero-posterior axis. By using a two-component low-q fit model, the root mean square displacements of intra-axonal space ( =  axon diameter) and intra-axonal volume fraction of the CST were calculated at the levels of the internal capsule and body of the lateral ventricle, respectively. Wilcoxon's rank-sum test revealed a significant increase in CST intra-axonal volume fraction at the paraventricular level in patients (p<0.001), whereas no significant difference was observed in the axon diameter. At the level of the internal capsule, neither axon diameter nor intra-axonal volume fraction differed significantly between the two groups. Our results suggest that in patients with iNPH, the CST does not undergo irreversible axonal damage but is rather compressed and/or stretched owing to pressure from the enlarged ventricle. These analyses of axon diameter and intra-axonal fraction yield insights into microstructural alterations of the CST in iNPH.

  14. Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy.

    PubMed

    Liau, Ee Shan; Yen, Ya-Ping; Chen, Jun-An

    2018-05-11

    Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

  15. Axonal Membranes and Their Domains: Assembly and Function of the Axon Initial Segment and Node of Ranvier

    PubMed Central

    Nelson, Andrew D.; Jenkins, Paul M.

    2017-01-01

    Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS) and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of Ranvier, and how

  16. [11C]choline uptake in regenerating liver after partial hepatectomy or CCl4-administration.

    PubMed

    Sasaki, Toru

    2004-02-01

    To characterize [methyl-(11)C]choline ([(11)C]choline) as an oncologic PET radiopharmaceutical, [(11)C]choline uptake in regenerating livers after partial hepatectomy as a model of typical proliferating tissue and after CCl(4) insult as that of proliferating tissue with inflammation, was studied in rats. [(11)C]Choline, [(18)F]2-fluoro-2-deoxy-D-glucose ([(18)F]FDG) and [2-(14)C]thymidine ([(14)C]TdR) uptake was studied in regenerating rat liver after 70% partial hepatectomy or CCl(4)-administration. [(11)C]Choline uptake in regenerating liver after partial hepatectomy was significantly increased with [(14)C]TdR uptake as a marker of DNA synthesis at 18 hours after surgery. On the other hand, the uptake was not accelerated by CCl(4)-administration, though it significantly increased [(14)C]TdR uptake. There were no differences of [(11)C]choline uptake acceleration following partial hepatectomy among the three parts of the regenerating liver. [(18)F]FDG uptake was accelerated in the regenerating liver on either partial hepatectomy or CCl(4)-administration. The magnitude of the increase in [(18)F]FDG uptake in the regenerating liver induced by partial hepatectomy was greater than that for [(11)C]choline. [(11)C]Choline uptake in the liver was accelerated by partial hepatectomy, but not by CCl(4)-administration. This might be expected given that the differentiation between proliferating tissues such as tumor and inflammatory tissue was possible by [(11)C]choline-PET.

  17. Compensatory axon sprouting for very slow axonal die-back in a transgenic model of spinal muscular atrophy type III.

    PubMed

    Udina, Esther; Putman, Charles T; Harris, Luke R; Tyreman, Neil; Cook, Victoria E; Gordon, Tessa

    2017-03-01

    Smn +/- transgenic mouse is a model of the mildest form of spinal muscular atrophy. Although there is a loss of spinal motoneurons in 11-month-old animals, muscular force is maintained. This maintained muscular force is mediated by reinnervation of the denervated fibres by surviving motoneurons. The spinal motoneurons in these animals do not show an increased susceptibility to death after nerve injury and they retain their regenerative capacity. We conclude that the hypothesized immaturity of the neuromuscular system in this model cannot explain the loss of motoneurons by systematic die-back. Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and is the leading genetic cause of infantile death. Patients lack the SMN1 gene with the severity of the disease depending on the number of copies of the highly homologous SMN2 gene. Although motoneuron death in the Smn +/- transgenic mouse model of the mildest form of SMA, SMA type III, has been reported, we have used retrograde tracing of sciatic and femoral motoneurons in the hindlimb with recording of muscle and motor unit isometric forces to count the number of motoneurons with intact neuromuscular connections. Thereby, we investigated whether incomplete maturation of the neuromuscular system induced by survival motoneuron protein (SMN) defects is responsible for die-back of axons relative to survival of motoneurons. First, a reduction of ∼30% of backlabelled motoneurons began relatively late, at 11 months of age, with a significant loss of 19% at 7 months. Motor axon die-back was affirmed by motor unit number estimation. Loss of functional motor units was fully compensated by axonal sprouting to retain normal contractile force in four hindlimb muscles (three fast-twitch and one slow-twitch) innervated by branches of the sciatic nerve. Second, our evaluation of whether axotomy of motoneurons in the adult Smn +/- transgenic mouse increases their susceptibility to cell death demonstrated

  18. Action Potentials Initiate in the Axon Initial Segment and Propagate Through Axon Collaterals Reliably in Cerebellar Purkinje Neurons

    PubMed Central

    Foust, Amanda; Popovic, Marko; Zecevic, Dejan; McCormick, David A.

    2010-01-01

    Purkinje neurons are the output cells of the cerebellar cortex and generate spikes in two distinct modes, known as simple and complex spikes. Revealing the point of origin of these action potentials, and how they conduct into local axon collaterals, is important for understanding local and distal neuronal processing and communication. By utilizing a recent improvement in voltage sensitive dye imaging technique that provided exceptional spatial and temporal resolution, we were able to resolve the region of spike initiation as well as follow spike propagation into axon collaterals for each action potential initiated on single trials. All fast action potentials, for both simple and complex spikes, whether occurring spontaneously or in response to a somatic current pulse or synaptic input, initiated in the axon initial segment. At discharge frequencies of less than approximately 250 Hz, spikes propagated faithfully through the axon and axon collaterals, in a saltatory manner. Propagation failures were only observed for very high frequencies or for the spikelets associated with complex spikes. These results demonstrate that the axon initial segment is a critical decision point in Purkinje cell processing and that the properties of axon branch points are adjusted to maintain faithful transmission. PMID:20484631

  19. Identification of adequate vehicles to carry nerve regeneration inducers using tubulisation.

    PubMed

    do Nascimento-Elias, Adriana Helena; Fresnesdas, Bruno César; Schiavoni, Maria Cristina Lopes; de Almeida, Natália Fernanda Gaspar; Santos, Ana Paula; de Oliveira Ramos, Jean; Junior, Wilson Marques; Barreira, Amilton Antunes

    2012-08-14

    Axonal regeneration depends on many factors, such as the type of injury and repair, age, distance from the cell body and distance of the denervated muscle, loss of surrounding tissue and the type of injured nerve. Experimental models use tubulisation with a silicone tube to research regenerative factors and substances to induce regeneration. Agarose, collagen and DMEM (Dulbecco's modified Eagle's medium) can be used as vehicles. In this study, we compared the ability of these vehicles to induce rat sciatic nerve regeneration with the intent of finding the least active or inert substance. The experiment used 47 female Wistar rats, which were divided into four experimental groups (agarose 4%, agarose 0.4%, collagen, DMEM) and one normal control group. The right sciatic nerve was exposed, and an incision was made that created a 10 mm gap between the distal and proximal stumps. A silicone tube was grafted onto each stump, and the tubes were filled with the respective media. After 70 days, the sciatic nerve was removed. We evaluated the formation of a regeneration cable, nerve fibre growth, and the functional viability of the regenerated fibres. Comparison among the three vehicles showed that 0.4% agarose gels had almost no effect on provoking the regeneration of peripheral nerves and that 4% agarose gels completely prevented fibre growth. The others substances were associated with profuse nerve fibre growth. In the appropriate concentration, agarose gel may be an important vehicle for testing factors that induce regeneration without interfering with nerve growth.

  20. Enhanced peripheral nerve regeneration by the combination of a polycaprolactone tubular prosthesis and a scaffold of collagen with supramolecular organization

    PubMed Central

    Maturana, Luiz G; Pierucci, Amauri; Simões, Gustavo F; Vidigal, Mateus; Duek, Eliana A R; Vidal, Benedicto C; Oliveira, Alexandre L R

    2013-01-01

    The purpose of this study was to investigate the influence of implanting collagen with a supramolecular organization on peripheral nerve regeneration, using the sciatic nerve tubulization technique. For this purpose, adult female Sprague Dawley rats were divided into five groups: (1) TP – sciatic nerve repaired with empty polyethylene tubular prothesis (n = 10), (2) TPCL – nerve repair with empty polycaprolactone (PCL) tubing (n = 8), (3) TPCLF – repair with PCL tubing filled with an implant of collagen with a supramolecular organization (n = 10), (4) AG – animals that received a peripheral nerve autograft (n = 8), and (5) Normal nerves (n = 8). The results were assessed by quantification of the regenerated fibers, nerve morphometry, and transmission electron microscopy, 60 days after surgery. Immunohistochemistry and polarization microscopy were also used to analyze the regenerated nerve structure and cellular elements. The results showed that the AG group presented a larger number of regenerated axons. However, the TPCL and TPCLF groups presented more compact regenerated fibers with a morphometric profile closer to normal, both at the tube midpoint and 2 mm distal to the prosthesis. These findings were reinforced by polarization microscopy, which indicated a better collagen/axons suprastructural organization in the TPCLF derived samples. In addition, the immunohistochemical results obtained using the antibody anti-p75NTR as a Schwann cell reactivity marker demonstrated that the Schwann cells were more reactive during the regenerative process in the TPCLF group as compared to the TPCL group and the normal sciatic nerve. Altogether, the results of this study indicated that the implant of collagen with a supramolecular organization positively influenced and stimulated the regeneration process through the nerve gap, resulting in the formation of a better morphologically arranged tissue. PMID:24381812

  1. Nerve growth factor loaded heparin/chitosan scaffolds for accelerating peripheral nerve regeneration.

    PubMed

    Li, Guicai; Xiao, Qinzhi; Zhang, Luzhong; Zhao, Yahong; Yang, Yumin

    2017-09-01

    Artificial chitosan scaffolds have been widely investigated for peripheral nerve regeneration. However, the effect was not as good as that of autologous grafts and therefore could not meet the clinical requirement. In the present study, the nerve growth factor (NGF) loaded heparin/chitosan scaffolds were fabricated via electrostatic interaction for further improving nerve regeneration. The physicochemical properties including morphology, wettability and composition were measured. The heparin immobilization, NGF loading and release were quantitatively and qualitatively characterized, respectively. The effect of NGF loaded heparin/chitosan scaffolds on nerve regeneration was evaluated by Schwann cells culture for different periods. The results showed that the heparin immobilization and NGF loading did not cause the change of bulk properties of chitosan scaffolds except for morphology and wettability. The pre-immobilization of heparin in chitosan scaffolds could enhance the stability of subsequently loaded NGF. The NGF loaded heparin/chitosan scaffolds could obviously improve the attachment and proliferation of Schwann cells in vitro. More importantly, the NGF loaded heparin/chitosan scaffolds could effectively promote the morphology development of Schwann cells. The study may provide a useful experimental basis to design and develop artificial implants for peripheral nerve regeneration and other tissue regeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. AxonPacking: An Open-Source Software to Simulate Arrangements of Axons in White Matter

    PubMed Central

    Mingasson, Tom; Duval, Tanguy; Stikov, Nikola; Cohen-Adad, Julien

    2017-01-01

    HIGHLIGHTS AxonPacking: Open-source software for simulating white matter microstructure.Validation on a theoretical disk packing problem.Reproducible and stable for various densities and diameter distributions.Can be used to study interplay between myelin/fiber density and restricted fraction. Quantitative Magnetic Resonance Imaging (MRI) can provide parameters that describe white matter microstructure, such as the fiber volume fraction (FVF), the myelin volume fraction (MVF) or the axon volume fraction (AVF) via the fraction of restricted water (fr). While already being used for clinical application, the complex interplay between these parameters requires thorough validation via simulations. These simulations required a realistic, controlled and adaptable model of the white matter axons with the surrounding myelin sheath. While there already exist useful algorithms to perform this task, none of them combine optimisation of axon packing, presence of myelin sheath and availability as free and open source software. Here, we introduce a novel disk packing algorithm that addresses these issues. The performance of the algorithm is tested in term of reproducibility over 50 runs, resulting density, and stability over iterations. This tool was then used to derive multiple values of FVF and to study the impact of this parameter on fr and MVF in light of the known microstructure based on histology sample. The standard deviation of the axon density over runs was lower than 10−3 and the expected hexagonal packing for monodisperse disks was obtained with a density close to the optimal density (obtained: 0.892, theoretical: 0.907). Using an FVF ranging within [0.58, 0.82] and a mean inter-axon gap ranging within [0.1, 1.1] μm, MVF ranged within [0.32, 0.44] and fr ranged within [0.39, 0.71], which is consistent with the histology. The proposed algorithm is implemented in the open-source software AxonPacking (https://github.com/neuropoly/axonpacking) and can be useful for

  3. SDF-1 overexpression by mesenchymal stem cells enhances GAP-43-positive axonal growth following spinal cord injury.

    PubMed

    Stewart, Andrew Nathaniel; Matyas, Jessica Jane; Welchko, Ryan Matthew; Goldsmith, Alison Delanie; Zeiler, Sarah Elizabeth; Hochgeschwender, Ute; Lu, Ming; Nan, Zhenhong; Rossignol, Julien; Dunbar, Gary Leo

    2017-01-01

    Utilizing genetic overexpression of trophic molecules in cell populations has been a promising strategy to develop cell replacement therapies for spinal cord injury (SCI). Over-expressing the chemokine, stromal derived factor-1 (SDF-1α), which has chemotactic effects on many cells of the nervous system, offers a promising strategy to promote axonal regrowth following SCI. The purpose of this study was to explore the effects of human SDF-1α, when overexpressed by mesenchymal stem cells (MSCs), on axonal growth and motor behavior in a contusive rat model of SCI. Using a transwell migration assay, the paracrine effects of MSCs, which were engineered to secrete human SDF-1α (SDF-1-MSCs), were assessed on cultured neural stem cells (NSCs). For in vivo analyses, the SDF-1-MSCs, unaltered MSCs, or Hanks Buffered Saline Solution (vehicle) were injected into the lesion epicenter of rats at 9-days post-SCI. Behavior was analyzed for 7-weeks post-injury, using the Basso, Beattie, and Bresnahan (BBB) scale of locomotor functions. Immunohistochemistry was performed to evaluate major histopathological outcomes, including gliosis, inflammation, white matter sparing, and cavitation. New axonal outgrowth was characterized using immunohistochemistry against the neuron specific growth-associated protein-43 (GAP-43). The results of these experiments demonstrate that the overexpression of SDF-1α by MSCs can enhance the migration of NSCs in vitro. Although only modest functional improvements were observed following transplantation of SDF-1-MSCs, a significant reduction in cavitation surrounding the lesion, and an increased density of GAP-43-positive axons inside the SCI lesion/graft site were found. The results from these experiments support the potential role for utilizing SDF-1α as a treatment for enhancing growth and regeneration of axons after traumatic SCI.

  4. Epoxyeicosanoids promote organ and tissue regeneration.

    PubMed

    Panigrahy, Dipak; Kalish, Brian T; Huang, Sui; Bielenberg, Diane R; Le, Hau D; Yang, Jun; Edin, Matthew L; Lee, Craig R; Benny, Ofra; Mudge, Dayna K; Butterfield, Catherine E; Mammoto, Akiko; Mammoto, Tadanori; Inceoglu, Bora; Jenkins, Roger L; Simpson, Mary A; Akino, Tomoshige; Lih, Fred B; Tomer, Kenneth B; Ingber, Donald E; Hammock, Bruce D; Falck, John R; Manthati, Vijaya L; Kaipainen, Arja; D'Amore, Patricia A; Puder, Mark; Zeldin, Darryl C; Kieran, Mark W

    2013-08-13

    Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

  5. G-CSF prevents caspase 3 activation in Schwann cells after sciatic nerve transection, but does not improve nerve regeneration.

    PubMed

    Frost, Hanna K; Kodama, Akira; Ekström, Per; Dahlin, Lars B

    2016-10-15

    Exogenous granulocyte-colony stimulating factor (G-CSF) has emerged as a drug candidate for improving the outcome after peripheral nerve injuries. We raised the question if exogenous G-CSF can improve nerve regeneration following a clinically relevant model - nerve transection and repair - in healthy and diabetic rats. In short-term experiments, distance of axonal regeneration and extent of injury-induced Schwann cell death was quantified by staining for neurofilaments and cleaved caspase 3, respectively, seven days after repair. There was no difference in axonal outgrowth between G-CSF-treated and non-treated rats, regardless if healthy Wistar or diabetic Goto-Kakizaki (GK) rats were examined. However, G-CSF treatment caused a significant 13% decrease of cleaved caspase 3-positive Schwann cells at the lesion site in healthy rats, but only a trend in diabetic rats. In the distal nerve segments of healthy rats a similar trend was observed. In long-term experiments of healthy rats, regeneration outcome was evaluated at 90days after repair by presence of neurofilaments, wet weight of gastrocnemius muscle, and perception of touch (von Frey monofilament testing weekly). The presence of neurofilaments distal to the suture line was similar in G-CSF-treated and non-treated rats. The weight ratio of ipsi-over contralateral gastrocnemius muscles, and perception of touch at any time point, were likewise not affected by G-CSF treatment. In addition, the inflammatory response in short- and long-term experiments was studied by analyzing ED1 stainable macrophages in healthy rats, but in neither case was any attenuation seen at the injury site or distal to it. G-CSF can prevent caspase 3 activation in Schwann cells in the short-term, but does not detectably affect the inflammatory response, nor improve early or late axonal outgrowth or functional recovery. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Autologous Bone Marrow Concentrates and Concentrated Growth Factors Accelerate Bone Regeneration After Enucleation of Mandibular Pathologic Lesions.

    PubMed

    Talaat, Wael M; Ghoneim, Mohamed M; Salah, Omar; Adly, Osama A

    2018-02-23

    Stem cell therapy is a revolutionary new way to stimulate mesenchymal tissue regeneration. The platelets concentrate products started with platelet-rich plasma (PRP), followed by platelet-rich fibrin (PRF), whereas concentrated growth factors (CGF) are the latest generation of the platelets concentrate products which were found in 2011. The aim of the present study was to evaluate the potential of combining autologous bone marrow concentrates and CGF for treatment of bone defects resulting from enucleation of mandibular pathologic lesions. Twenty patients (13 males and 7 females) with mandibular benign unilateral lesions were included, and divided into 2 groups. Group I consisted of 10 patients who underwent enucleation of the lesions followed by grafting of the bony defects with autologous bone marrow concentrates and CGF. Group II consisted of 10 patients who underwent enucleation of the lesions without grafting (control). Radiographic examinations were done immediately postoperative, then at 1, 3, 6, and 12 months, to evaluate the reduction in size and changes in bone density at the bony defects. Results indicated a significant increase in bone density with respect to the baseline levels in both groups (P < 0.05). The increase in bone density was significantly higher in group I compared with group II at the 6- and 12-month follow-up examinations (P < 0.05). The percent of reduction in the defects' size was significantly higher in group I compared with group II after 12 months (P = 0.00001). In conclusion, the clinical application of autologous bone marrow concentrates with CGF is a cost effective and safe biotechnology, which accelerates bone regeneration and improves the density of regenerated bone.

  7. Degeneration and regeneration of motor and sensory nerves: a stereological study of crush lesions in rat facial and mental nerves.

    PubMed

    Barghash, Z; Larsen, J O; Al-Bishri, A; Kahnberg, K-E

    2013-12-01

    The aim of this study was to evaluate the degeneration and regeneration of a sensory nerve and a motor nerve at the histological level after a crush injury. Twenty-five female Wistar rats had their mental nerve and the buccal branch of their facial nerve compressed unilaterally against a glass rod for 30s. Specimens of the compressed nerves and the corresponding control nerves were dissected at 3, 7, and 19 days after surgery. Nerve cross-sections were stained with osmium tetroxide and toluidine blue and analysed using two-dimensional stereology. We found differences between the two nerves both in the normal anatomy and in the regenerative pattern. The mental nerve had a larger cross-sectional area including all tissue components. The mental nerve had a larger volume fraction of myelinated axons and a correspondingly smaller volume fraction of endoneurium. No differences were observed in the degenerative pattern; however, at day 19 the buccal branch had regenerated to the normal number of axons, whereas the mental nerve had only regained 50% of the normal number of axons. We conclude that the regenerative process is faster and/or more complete in the facial nerve (motor function) than it is in the mental nerve (somatosensory function). Copyright © 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  8. Axonal neurofilaments are nonessential elements of toxicant-induced reductions in fast axonal transport: video-enhanced differential interference microscopy in peripheral nervous system axons.

    PubMed

    Stone, J D; Peterson, A P; Eyer, J; Oblak, T G; Sickles, D W

    1999-11-15

    Neurofilament modification and accumulation, occurring in toxicant-induced neuropathies, has been proposed to compromise fast axonal transport and contribute to neurological symptoms or pathology. The current study compares the effects of the neurotoxicants acrylamide (ACR) and 2,5-hexanedione (2,5-HD) on the quantity of fast, bidirectional vesicular traffic within isolated mouse sciatic nerve axons from transgenic mice lacking axonal neurofilaments (Eyer and Peterson, Neuron 12, 1-20, 1994) and nontransgenic littermates possessing neurofilaments. Fast anterograde and retrograde membrane bound organelle (MBO) traffic was quantitated within axons, before and after toxicant exposure, using video-enhanced differential interference contrast (AVEC-DIC) microscopy. Addition of 0.7 mM ACR to the buffer bathing the nerve produced a time-dependent reduction in bidirectional transport with a similar time to onset and magnitude in both transgenic and nontransgenic mice. 2,5-HD (4 mM) exposure reduced bidirectional vesicle traffic by a similar amount in both transgenic and nontransgenic animals. The time to onset of the transport reduction was less and the magnitude of the reduction was greater with 2,5-HD compared to ACR. A single 10-min exposure to ACR or 2,5-HD produced a similar reduction in transport to that produced by prolonged (1 h) exposure. Nonneurotoxic propionamide or 3,4-hexanedione (3,4-HD) produced no changes in bidirectional transport in either transgenic or nontransgenic animals. We conclude that ACR or 2,5-HD produces a rapid, saturable, nonreversible, neurotoxicant-specific reduction in fast bidirectional transport within isolated peripheral nerve axons. These actions are mediated through direct modification of axonal component(s), which are independent of toxicant-induced modifications of, or accumulations of, neurofilaments. Copyright 1999 Academic Press.

  9. Differential effects of myostatin deficiency on motor and sensory axons.

    PubMed

    Jones, Maria R; Villalón, Eric; Northcutt, Adam J; Calcutt, Nigel A; Garcia, Michael L

    2017-12-01

    Deletion of myostatin in mice (MSTN -/- ) alters structural properties of peripheral axons. However, properties like axon diameter and myelin thickness were analyzed in mixed nerves, so it is unclear whether loss of myostatin affects motor, sensory, or both types of axons. Using the MSTN -/- mouse model, we analyzed the effects of increasing the number of muscle fibers on axon diameter, myelin thickness, and internode length in motor and sensory axons. Axon diameter and myelin thickness were increased in motor axons of MSTN -/- mice without affecting internode length or axon number. The number of sensory axons was increased without affecting their structural properties. These results suggest that motor and sensory axons establish structural properties by independent mechanisms. Moreover, in motor axons, instructive cues from the neuromuscular junction may play a role in co-regulating axon diameter and myelin thickness, whereas internode length is established independently. Muscle Nerve 56: E100-E107, 2017. © 2017 Wiley Periodicals, Inc.

  10. let-7 miRNA controls CED-7 homotypic adhesion and EFF-1–mediated axonal self-fusion to restore touch sensation following injury

    PubMed Central

    Basu, Atrayee; Dey, Shirshendu; Puri, Dharmendra; Das Saha, Nilanjana; Sabharwal, Vidur; Thyagarajan, Pankajam; Srivastava, Prerna; Koushika, Sandhya Padmanabhan

    2017-01-01

    Neuronal injury often leads to devastating consequences such as loss of senses or locomotion. Restoration of function after injury relies on whether the injured axons can find their target cells. Although fusion between injured proximal axon and distal fragment has been observed in many organisms, its functional significance is not clear. Here, using Caenorhabditis elegans mechanosensory neurons, we address this question. Using two femtosecond lasers simultaneously, we could scan and sever posterior lateral microtubule neurons [posterior lateral microtubules (PLMs)] on both sides of the worm. We showed that axotomy of both PLMs leads to a dramatic loss of posterior touch sensation. During the regenerative phase, only axons that fuse to their distal counterparts contribute to functional recovery. Loss of let-7 miRNA promotes functional restoration in both larval and adult stages. In the L4 stage, loss of let-7 increases fusion events by increasing the mRNA level of one of the cell-recognition molecules, CED-7. The ability to establish cytoplasmic continuity between the proximal and distal ends declines with age. Loss of let-7 overcomes this barrier by promoting axonal transport and enrichment of the EFF-1 fusogen at the growing tip of cut processes. Our data reveal the functional property of a regenerating neuron. PMID:29109254

  11. The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

    PubMed

    Godzik, Katharina; Coleman, Michael P

    2015-04-01

    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

  12. Motoneuron regeneration accuracy and recovery of gait after femoral nerve injuries in rats.

    PubMed

    Kruspe, M; Thieme, H; Guntinas-Lichius, O; Irintchev, A

    2014-11-07

    The rat femoral nerve is a valuable model allowing studies on specificity of motor axon regeneration. Despite common use of this model, the functional consequences of femoral nerve lesions and their relationship to precision of axonal regeneration have not been evaluated. Here we assessed gait recovery after femoral nerve injuries of varying severity in adult female Wistar rats using a video-based approach, single-frame motion analysis (SFMA). After nerve crush, recovery was complete at 4 weeks after injury (99% of maximum 100% as estimated by a recovery index). Functional restoration after nerve section/suture was much slower and incomplete (84%) even 20 weeks post-surgery. A 5-mm gap between the distal and proximal nerve stumps additionally delayed recovery and worsened the outcome (68% recovery). As assessed by retrograde labeling in the same rats at 20 weeks after injury, the anatomical outcome was also dependent on lesion severity. After nerve crush, 97% of the femoral motoneurons (MNs) had axons correctly projecting only into the distal quadriceps branch of the femoral nerve. The percentage of correctly projecting MNs was only 55% and 15% after nerve suture and gap repair, respectively. As indicated by regression analyses, better functional recovery was associated with higher numbers of correctly projecting MNs and, unexpectedly, lower numbers of MNs projecting to both muscle and skin. The data show that type of nerve injury and repair profoundly influence selectivity of motor reinnervation and, in parallel, functional outcome. The results also suggest that MNs' projection patterns may influence their contribution to muscle performance. In addition to the experiments described above, we performed repeated measurements and statistical analyses to validate the SFMA. The results revealed high accuracy and reproducibility of the SFMA measurements. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis

    PubMed Central

    Zambonin, Jessica L.; Zhao, Chao; Ohno, Nobuhiko; Campbell, Graham R.; Engeham, Sarah; Ziabreva, Iryna; Schwarz, Nadine; Lee, Sok Ee; Frischer, Josa M.; Turnbull, Doug M.; Trapp, Bruce D.; Lassmann, Hans; Franklin, Robin J. M.

    2011-01-01

    Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in

  14. Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis.

    PubMed

    Zambonin, Jessica L; Zhao, Chao; Ohno, Nobuhiko; Campbell, Graham R; Engeham, Sarah; Ziabreva, Iryna; Schwarz, Nadine; Lee, Sok Ee; Frischer, Josa M; Turnbull, Doug M; Trapp, Bruce D; Lassmann, Hans; Franklin, Robin J M; Mahad, Don J

    2011-07-01

    Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in

  15. Creatine pretreatment protects cortical axons from energy depletion in vitro

    PubMed Central

    Shen, Hua; Goldberg, Mark P.

    2012-01-01

    Creatine is a natural nitrogenous guanidino compound involved in bioenergy metabolism. Although creatine has been shown to protect neurons of the central nervous system (CNS) from experimental hypoxia/ischemia, it remains unclear if creatine may also protect CNS axons, and if the potential axonal protection depends on glial cells. To evaluate the direct impact of creatine on CNS axons, cortical axons were cultured in a separate compartment from their somas and proximal neurites using a modified two-compartment culture device. Axons in the axon compartment were subjected to acute energy depletion, an in vitro model of white matter ischemia, by exposure to 6 mM sodium azide for 30 min in the absence of glucose and pyruvate. Energy depletion reduced axonal ATP by 65%, depolarized axonal resting potential, and damaged 75% of axons. Application of creatine (10 mM) to both compartments of the culture at 24 h prior to energy depletion significantly reduced axonal damage by 50%. In line with the role of creatine in the bioenergy metabolism, this application also alleviated the axonal ATP loss and depolarization. Inhibition of axonal depolarization by blocking sodium influx with tetrodotoxin also effectively reduced the axonal damage caused by energy depletion. Further study revealed that the creatine effect was independent of glial cells, as axonal protection was sustained even when creatine was applied only to the axon compartment (free from somas and glial cells) for as little as 2 h. In contrast, application of creatine after energy depletion did not protect axons. The data provide the first evidence that creatine pretreatment may directly protect CNS axons from energy deficiency. PMID:22521466

  16. Maintenance of long-term adaptation of synaptic transmission requires axonal transport following induction in an identified crayfish motoneuron.

    PubMed

    Nguyen, P V; Atwood, H L

    1992-03-01

    Motoneurons can adapt to altered levels of electrical activity by effecting semi-permanent changes in their neuromuscular synaptic physiology. In the present study, we tested the hypothesis that maintenance of activity-dependent long-term adaptation of synaptic transmission in a crayfish abdominal extensor motoneuron (phasic axon 3) required axonal transport following induction. Intact crayfish were chronically wired for periodic in vivo stimulation of axon 3. Periodic unilateral stimulation for 3-5 consecutive days (2 h/day) induced long-term adaptation (LTA) of neuromuscular synaptic transmission in axon 3. Initial EPSP amplitudes (measured at 0.1 Hz) were significantly reduced to approximately 40% of contralateral control amplitudes over a 7-day poststimulation period. Additionally, synaptic depression during 5 Hz test stimulation of axon 3 was significantly less in chronically stimulated neurons: excitatory postsynaptic potential (EPSP) amplitudes measured after 20 min of 5 Hz test stimulation (final EPSPs) were significantly larger in conditioned neurons than in unstimulated controls. The depression of initial EPSP amplitudes persisted for 7 days postinduction, while the increased synaptic stamina persisted for 4 days but was absent at 7 days postinduction. Axotomy of axon 3 following induction of LTA had no effect on long-term maintenance of the activity-induced reduction in initial EPSP amplitudes. Initial EPSP amplitudes in conditioned, axotomized neurons were still reduced to 42% of control amplitudes over the 7-day postinduction period. In contrast, postinduction axotomy of axon 3 elicited an accelerated decay of the enhanced synaptic stamina. Following axotomy, final EPSP amplitudes were significantly larger in conditioned neurons for only 1 day poststimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Axon growth regulation by a bistable molecular switch.

    PubMed

    Padmanabhan, Pranesh; Goodhill, Geoffrey J

    2018-04-25

    For the brain to function properly, its neurons must make the right connections during neural development. A key aspect of this process is the tight regulation of axon growth as axons navigate towards their targets. Neuronal growth cones at the tips of developing axons switch between growth and paused states during axonal pathfinding, and this switching behaviour determines the heterogeneous axon growth rates observed during brain development. The mechanisms controlling this switching behaviour, however, remain largely unknown. Here, using mathematical modelling, we predict that the molecular interaction network involved in axon growth can exhibit bistability, with one state representing a fast-growing growth cone state and the other a paused growth cone state. Owing to stochastic effects, even in an unchanging environment, model growth cones reversibly switch between growth and paused states. Our model further predicts that environmental signals could regulate axon growth rate by controlling the rates of switching between the two states. Our study presents a new conceptual understanding of growth cone switching behaviour, and suggests that axon guidance may be controlled by both cell-extrinsic factors and cell-intrinsic growth regulatory mechanisms. © 2018 The Author(s).

  18. Axonal transport: cargo-specific mechanisms of motility and regulation.

    PubMed

    Maday, Sandra; Twelvetrees, Alison E; Moughamian, Armen J; Holzbaur, Erika L F

    2014-10-22

    Axonal transport is essential for neuronal function, and many neurodevelopmental and neurodegenerative diseases result from mutations in the axonal transport machinery. Anterograde transport supplies distal axons with newly synthesized proteins and lipids, including synaptic components required to maintain presynaptic activity. Retrograde transport is required to maintain homeostasis by removing aging proteins and organelles from the distal axon for degradation and recycling of components. Retrograde axonal transport also plays a major role in neurotrophic and injury response signaling. This review provides an overview of axonal transport pathways and discusses their role in neuronal function.

  19. FGF and BMP derived from dorsal root ganglia regulate blastema induction in limb regeneration in Ambystoma mexicanum.

    PubMed

    Satoh, Akira; Makanae, Aki; Nishimoto, Yurie; Mitogawa, Kazumasa

    2016-09-01

    Urodele amphibians have a remarkable organ regeneration ability that is regulated by neural inputs. The identification of these neural inputs has been a challenge. Recently, Fibroblast growth factor (Fgf) and Bone morphogenic protein (Bmp) were shown to substitute for nerve functions in limb and tail regeneration in urodele amphibians. However, direct evidence of Fgf and Bmp being secreted from nerve endings and regulating regeneration has not yet been shown. Thus, it remained uncertain whether they were the nerve factors responsible for successful limb regeneration. To gather experimental evidence, the technical difficulties involved in the usage of axolotls had to be overcome. We achieved this by modifying the electroporation method. When Fgf8-AcGFP or Bmp7-AcGFP was electroporated into the axolotl dorsal root ganglia (DRG), GFP signals were detectable in the regenerating limb region. This suggested that Fgf8 and Bmp7 synthesized in neural cells in the DRG were delivered to the limbs through the long axons. Further knockdown experiments with double-stranded RNA interference resulted in impaired limb regeneration ability. These results strongly suggest that Fgf and Bmp are the major neural inputs that control the organ regeneration ability. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Evolution of the Mauthner axon cap.

    PubMed

    Bierman, Hilary S; Zottoli, Steven J; Hale, Melina E

    2009-01-01

    Studies of vertebrate brain evolution have focused primarily on patterns of gene expression or changes in size and organization of major brain regions. The Mauthner cell, an important reticulospinal neuron that functions in the startle response of many species, provides an opportunity for evolutionary comparisons at the cellular level. Despite broad interspecific similarities in Mauthner cell morphology, the motor patterns and startle behaviors it initiates vary markedly. Response diversity has been hypothesized to result, in part, from differences in the structure and function of the Mauthner cell-associated axon cap. We used light microscopy techniques to compare axon cap morphology across a wide range of species, including all four extant basal actinopterygian orders, representatives of a variety of teleost lineages and lungfishes, and we combined our data with published descriptions of axon cap structure. The 'composite' axon cap, observed in teleosts, is an organized conglomeration of glia and fibers of inhibitory and excitatory interneurons. Lungfish, amphibian tadpoles and several basal actinopterygian fishes have 'simple' axon caps that appear to lack glia and include few fibers. Several other basal actinopterygian fishes have 'simple-dense' caps that include greater numbers of fibers than simple caps, but lack the additional elements and organization of composite caps. Phylogenetic mapping shows that through evolution there are discrete transitions in axon cap morphology occurring at the base of gnathostomes, within basal actinopterygians, and at the base of the teleost radiation. Comparing axon cap evolution to the evolution of startle behavior and motor pattern provides insight into the relationship between Mauthner cell-associated structures and their functions in behavior. Copyright 2009 S. Karger AG, Basel.

  1. Commissural axons of the mouse cochlear nucleus.

    PubMed

    Brown, M Christian; Drottar, Marie; Benson, Thane E; Darrow, Keith

    2013-05-01

    The axons of commissural neurons that project from one cochlear nucleus to the other were studied after labeling with anterograde tracer. Injections were made into the dorsal subdivision of the cochlear nucleus in order to restrict labeling only to the group of commissural neurons that gave off collaterals to, or were located in, this subdivision. The number of labeled commissural axons in each injection was correlated with the number of labeled radiate multipolar neurons, suggesting radiate neurons as the predominant origin of the axons. The radiate commissural axons are thick and myelinated, and they exit the dorsal acoustic stria of the injected cochlear nucleus to cross the brainstem in the dorsal half, near the crossing position of the olivocochlear bundle. They enter the opposite cochlear nucleus via the dorsal and ventral acoustic stria and at its medial border. Reconstructions of single axons demonstrate that terminations are mostly in the core and typically within a single subdivision of the cochlear nucleus. Extents of termination range from narrow to broad along both the dorsoventral (i.e., tonotopic) and the rostrocaudal dimensions. In the electron microscope, labeled swellings form synapses that are symmetric (in that there is little postsynaptic density), a characteristic of inhibitory synapses. Our labeled axons do not appear to include excitatory commissural axons that end in edge regions of the nucleus. Radiate commissural axons could mediate the broadband inhibition observed in responses to contralateral sound, and they may balance input from the two ears with a quick time course. Copyright © 2012 Wiley Periodicals, Inc.

  2. Commissural Axons of the Mouse Cochlear Nucleus

    PubMed Central

    Brown, M. Christian; Drottar, Marie; Benson, Thane E.; Darrow, Keith

    2012-01-01

    The axons of commissural neurons that project from one cochlear nucleus to the other were studied after labeling with anterograde tracer. Injections were made into the dorsal subdivision of the cochlear nucleus in order to restrict labeling only to the group of commissural neurons that gave off collaterals to, or were located in, this subdivision. The number of labeled commissural axons in each injection was correlated with the number of labeled radiate multipolar neurons, suggesting radiate neurons as the predominant origin of the axons. The radiate commissural axons are thick and myelinated, and they exit the dorsal acoustic stria of the injected cochlear nucleus to cross the brainstem in the dorsal half, near the crossing position of the olivocochlear bundle. They enter the opposite cochlear nucleus via the dorsal and ventral acoustic stria and at its medial border. Reconstructions of single axons demonstrate that terminations are mostly in the core and typically within a single subdivision of the cochlear nucleus. Extents of termination range from narrow to broad along both the dorso-ventral (i.e. tonotopic) and rostro-caudal dimensions. In the electron microscope, labeled swellings form synapses that are symmetric (in that there is little postsynaptic density), a characteristic of inhibitory synapses. Our labeled axons do not appear to include excitatory commissural axons that end in edge regions of the nucleus. Radiate commissural axons could mediate the broad-band inhibition observed in responses to contralateral sound, and they may balance input from the two ears on a quick time course. PMID:23124982

  3. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins.

    PubMed

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-07-25

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function.

  4. Regulation of Conduction Time along Axons

    PubMed Central

    Seidl, Armin H.

    2013-01-01

    Timely delivery of information is essential for proper function of the nervous system. Precise regulation of nerve conduction velocity is needed for correct exertion of motor skills, sensory integration and cognitive functions. In vertebrates, the rapid transmission of signals along nerve fibers is made possible by the myelination of axons and the resulting saltatory conduction in between nodes of Ranvier. Myelin is a specialization of glia cells and is provided by oligodendrocytes in the central nervous system. Myelination not only maximizes conduction velocity, but also provides a means to systematically regulate conduction times in the nervous system. Systematic regulation of conduction velocity along axons, and thus systematic regulation of conduction time in between neural areas, is a common occurrence in the nervous system. To date, little is understood about the mechanism that underlies systematic conduction velocity regulation and conduction time synchrony. Node assembly, internode distance (node spacing) and axon diameter - all parameters determining the speed of signal propagation along axons - are controlled by myelinating glia. Therefore, an interaction between glial cells and neurons has been suggested. This review summarizes examples of neural systems in which conduction velocity is regulated by anatomical variations along axons. While functional implications in these systems are not always clear, recent studies in the auditory system of birds and mammals present examples of conduction velocity regulation in systems with high temporal precision and a defined biological function. Together these findings suggest an active process that shapes the interaction between axons and myelinating glia to control conduction velocity along axons. Future studies involving these systems may provide further insight into how specific conduction times in the brain are established and maintained in development. Throughout the text, conduction velocity is used for the

  5. Novel DLK-independent neuronal regeneration in Caenorhabditis elegans shares links with activity-dependent ectopic outgrowth

    PubMed Central

    Awal, Mehraj R.; Shay, James; McLoed, Melissa M.; Mazur, Eric; Gabel, Christopher V.

    2016-01-01

    During development, a neuron transitions from a state of rapid growth to a stable morphology, and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury. Here, we identify a novel form of neuronal regeneration, which is remarkably independent of DLK-1/DLK, KGB-1/JNK, and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans, Drosophila, and mammals. This DLK-independent regeneration in C. elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type. Both neuron outgrowth types are triggered by physical lesion of the sensory dendrite or mutations disrupting sensory activity, calcium signaling, or genes that restrict outgrowth during neuronal maturation, such as SAX-1/NDR kinase or UNC-43/CaMKII. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration. Moreover, we note numerous similarities between C. elegans DLK-independent regeneration and lesion conditioning, a phenomenon producing robust regeneration in the mammalian CNS. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP. Taken as a whole, our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system’s intrinsic regenerative capacity. PMID:27078101

  6. Remodeling of motor units after nerve regeneration studied by quantitative electromyography.

    PubMed

    Krarup, Christian; Boeckstyns, Michel; Ibsen, Allan; Moldovan, Mihai; Archibald, Simon

    2016-02-01

    Peripheral nerve has the capacity to regenerate after nerve lesions; during reinnervation of muscle motor units are gradually reestablished. The aim of this study was to follow the time course of reestablishing and remodeling of motor units in relation to recovery of force after different types of nerve repair. Reinnervation of muscle was compared clinically and electrophysiologically in complete median or ulnar nerve lesions with short gap lengths in the distal forearm repaired with a collagen nerve conduit (11 nerves) or nerve suture (10 nerves). Reestablishment of motor units was studied by quantitative EMG and recording of evoked compound muscle action potential (CMAP) during a 24-month observation period after nerve repair. Force recovered partially to about 80% of normal. Denervation activity gradually decreased during reinnervation though it was still increased at 24 months. Nascent motor unit potentials (MUPs) at early reinnervation were prolonged and polyphasic. During longitudinal studies, MUPs remained prolonged and their amplitudes gradually increased markedly. Firing of MUPs was unstable throughout the study. CMAPs gradually increased and the number of motor units recovered to approximately 20% of normal. There was weak evidence of CMAP amplitude recovery after suture ahead of conduit repair but without treatment related differences at 2 years. Surgical repair of nerve lesions with a nerve conduit or suture supported recovery of force and of motor unit reinnervation to the same extent. Changes occurred at a higher rate during early regeneration and slower after 12 months but should be followed for at least 2 years to assess outcome. EMG changes reflected extensive remodeling of motor units from early nascent units to a mature state with greatly enlarged units due to axonal regeneration and collateral sprouting and maturation of regenerated nerve and reinnervated muscle fibers after both types of repair. Remodeling of motor units after peripheral nerve

  7. Modeling of axonal endoplasmic reticulum network by spastic paraplegia proteins

    PubMed Central

    Yalçın, Belgin; Zhao, Lu; Stofanko, Martin; O'Sullivan, Niamh C; Kang, Zi Han; Roost, Annika; Thomas, Matthew R; Zaessinger, Sophie; Blard, Olivier; Patto, Alex L; Sohail, Anood; Baena, Valentina; Terasaki, Mark; O'Kane, Cahir J

    2017-01-01

    Axons contain a smooth tubular endoplasmic reticulum (ER) network that is thought to be continuous with ER throughout the neuron; the mechanisms that form this axonal network are unknown. Mutations affecting reticulon or REEP proteins, with intramembrane hairpin domains that model ER membranes, cause an axon degenerative disease, hereditary spastic paraplegia (HSP). We show that Drosophila axons have a dynamic axonal ER network, which these proteins help to model. Loss of HSP hairpin proteins causes ER sheet expansion, partial loss of ER from distal motor axons, and occasional discontinuities in axonal ER. Ultrastructural analysis reveals an extensive ER network in axons, which shows larger and fewer tubules in larvae that lack reticulon and REEP proteins, consistent with loss of membrane curvature. Therefore HSP hairpin-containing proteins are required for shaping and continuity of axonal ER, thus suggesting roles for ER modeling in axon maintenance and function. DOI: http://dx.doi.org/10.7554/eLife.23882.001 PMID:28742022

  8. Effects of ozone therapy on facial nerve regeneration.

    PubMed

    Ozbay, Isa; Ital, Ilker; Kucur, Cuneyt; Akcılar, Raziye; Deger, Aysenur; Aktas, Savas; Oghan, Fatih

    Ozone may promote moderate oxidative stress, which increases antioxidant endogenous systems. There are a number of antioxidants that have been investigated therapeutically for improving peripheral nerve regeneration. However, no previous studies have reported the effect of ozone therapy on facial nerve regeneration. We aimed to evaluate the effect of ozone therapy on facial nerve regeneration. Fourteen Wistar albino rats were randomly divided into two groups with experimental nerve crush injuries: a control group, which received saline treatment post-crush, and an experimental group, which received ozone treatment. All animals underwent surgery in which the left facial nerve was exposed and crushed. Treatment with saline or ozone began on the day of the nerve crush. Left facial nerve stimulation thresholds were measured before crush, immediately after crush, and after 30 days. After measuring nerve stimulation thresholds at 30 days post-injury, the crushed facial nerve was excised. All specimens were studied using light and electron microscopy. Post-crushing, the ozone-treated group had lower stimulation thresholds than the saline group. Although this did not achieve statistical significance, it is indicative of greater functional improvement in the ozone group. Significant differences were found in vascular congestion, macrovacuolization, and myelin thickness between the ozone and control groups. Significant differences were also found in axonal degeneration and myelin ultrastructure between the two groups. We found that ozone therapy exerted beneficial effect on the regeneration of crushed facial nerves in rats. Copyright © 2016 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.

  9. Potential Roles of Dental Pulp Stem Cells in Neural Regeneration and Repair

    PubMed Central

    Luo, Lihua; Wang, Xiaoyan; Key, Brian; Lee, Bae Hoon

    2018-01-01

    This review summarizes current advances in dental pulp stem cells (DPSCs) and their potential applications in the nervous diseases. Injured adult mammalian nervous system has a limited regenerative capacity due to an insufficient pool of precursor cells in both central and peripheral nervous systems. Nerve growth is also constrained by inhibitory factors (associated with central myelin) and barrier tissues (glial scarring). Stem cells, possessing the capacity of self-renewal and multicellular differentiation, promise new therapeutic strategies for overcoming these impediments to neural regeneration. Dental pulp stem cells (DPSCs) derive from a cranial neural crest lineage, retain a remarkable potential for neuronal differentiation, and additionally express multiple factors that are suitable for neuronal and axonal regeneration. DPSCs can also express immunomodulatory factors that stimulate formation of blood vessels and enhance regeneration and repair of injured nerve. These unique properties together with their ready accessibility make DPSCs an attractive cell source for tissue engineering in injured and diseased nervous systems. In this review, we interrogate the neuronal differentiation potential as well as the neuroprotective, neurotrophic, angiogenic, and immunomodulatory properties of DPSCs and its application in the injured nervous system. Taken together, DPSCs are an ideal stem cell resource for therapeutic approaches to neural repair and regeneration in nerve diseases. PMID:29853908

  10. Axonal synapse sorting in medial entorhinal cortex

    NASA Astrophysics Data System (ADS)

    Schmidt, Helene; Gour, Anjali; Straehle, Jakob; Boergens, Kevin M.; Brecht, Michael; Helmstaedter, Moritz

    2017-09-01

    Research on neuronal connectivity in the cerebral cortex has focused on the existence and strength of synapses between neurons, and their location on the cell bodies and dendrites of postsynaptic neurons. The synaptic architecture of individual presynaptic axonal trees, however, remains largely unknown. Here we used dense reconstructions from three-dimensional electron microscopy in rats to study the synaptic organization of local presynaptic axons in layer 2 of the medial entorhinal cortex, the site of grid-like spatial representations. We observe path-length-dependent axonal synapse sorting, such that axons of excitatory neurons sequentially target inhibitory neurons followed by excitatory neurons. Connectivity analysis revealed a cellular feedforward inhibition circuit involving wide, myelinated inhibitory axons and dendritic synapse clustering. Simulations show that this high-precision circuit can control the propagation of synchronized activity in the medial entorhinal cortex, which is known for temporally precise discharges.

  11. Death Receptor 6 Promotes Wallerian Degeneration in Peripheral Axons.

    PubMed

    Gamage, Kanchana K; Cheng, Irene; Park, Rachel E; Karim, Mardeen S; Edamura, Kazusa; Hughes, Christopher; Spano, Anthony J; Erisir, Alev; Deppmann, Christopher D

    2017-03-20

    Axon degeneration during development is required to sculpt a functional nervous system and is also a hallmark of pathological insult, such as injury [1, 2]. Despite similar morphological characteristics, very little overlap in molecular mechanisms has been reported between pathological and developmental degeneration [3-5]. In the peripheral nervous system (PNS), developmental axon pruning relies on receptor-mediated extrinsic degeneration mechanisms to determine which axons are maintained or degenerated [5-7]. Receptors have not been implicated in Wallerian axon degeneration; instead, axon autonomous, intrinsic mechanisms are thought to be the primary driver for this type of axon disintegration [8-10]. Here we survey the role of neuronally expressed, paralogous tumor necrosis factor receptor super family (TNFRSF) members in Wallerian degeneration. We find that an orphan receptor, death receptor 6 (DR6), is required to drive axon degeneration after axotomy in sympathetic and sensory neurons cultured in microfluidic devices. We sought to validate these in vitro findings in vivo using a transected sciatic nerve model. Consistent with the in vitro findings, DR6 -/- animals displayed preserved axons up to 4 weeks after injury. In contrast to phenotypes observed in Wld s and Sarm1 -/- mice, preserved axons in DR6 -/- animals display profound myelin remodeling. This indicates that deterioration of axons and myelin after axotomy are mechanistically distinct processes. Finally, we find that JNK signaling after injury requires DR6, suggesting a link between this novel extrinsic pathway and the axon autonomous, intrinsic pathways that have become established for Wallerian degeneration. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Natural healing-inspired collagen-targeting surgical protein glue for accelerated scarless skin regeneration.

    PubMed

    Jeon, Eun Young; Choi, Bong-Hyuk; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon

    2017-07-01

    Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Anatomical evidence for the influence of degenerating pathways on regenerating optic fibers following surgical manipulations in the visual system of the goldfish.

    PubMed

    Lo, R Y; Levine, R L

    1981-04-06

    We have used [3H]proline radioautography to trace regenerating optic fibers in the goldfish following: (1) the removal of the right tectal lobe and the right eye, and (2) the removal of both tectal lobes. Our results indicate that following the removal of the right tectal lobe and the right eye, both the denervated tectal efferent pathways, and the denervated visual pathways and terminal zones of the enucleated eye were penetrated by the regenerating optic fibers. In addition, following bilateral lobectomy, the denervated tectal efferent pathways were bilaterally penetrated by the regenerating fibers. Since, in both types of operations, these denervated pathways and terminal zones should undergo degeneration, our results support the suggestion that the presence of degenerating axonal debris and proliferating glia may play an important role in guiding regenerating optic fibers in the visual system of the goldfish.

  14. Neuronal growth cones respond to laser-induced axonal damage

    PubMed Central

    Wu, Tao; Mohanty, Samarendra; Gomez-Godinez, Veronica; Shi, Linda Z.; Liaw, Lih-Huei; Miotke, Jill; Meyer, Ronald L.; Berns, Michael W.

    2012-01-01

    Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness (‘thinning’) within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model. PMID:21831892

  15. Electrophysiology of Axonal Constrictions

    NASA Astrophysics Data System (ADS)

    Johnson, Christopher; Jung, Peter; Brown, Anthony

    2013-03-01

    Axons of myelinated neurons are constricted at the nodes of Ranvier, where they are directly exposed to the extracellular space and where the vast majority of the ion channels are located. These constrictions are generated by local regulation of the kinetics of neurofilaments the most important cytoskeletal elements of the axon. In this paper we discuss how this shape affects the electrophysiological function of the neuron. Specifically, although the nodes are short (about 1 μm) in comparison to the distance between nodes (hundreds of μm) they have a substantial influence on the conduction velocity of neurons. We show through computational modeling that nodal constrictions (all other features such as numbers of ion channels left constant) reduce the required fiber diameter for a given target conduction velocity by up to 50% in comparison to an unconstricted axon. We further show that the predicted optimal fiber morphologies closely match reported fiber morphologies. Supported by The National Science Foundation (IOS 1146789)

  16. Oligodendrocytes: Myelination and Axonal Support

    PubMed Central

    Simons, Mikael; Nave, Klaus-Armin

    2016-01-01

    Myelinated nerve fibers have evolved to enable fast and efficient transduction of electrical signals in the nervous system. To act as an electric insulator, the myelin sheath is formed as a multilamellar membrane structure by the spiral wrapping and subsequent compaction of the oligodendroglial plasma membrane around central nervous system (CNS) axons. Current evidence indicates that the myelin sheath is more than an inert insulating membrane structure. Oligodendrocytes are metabolically active and functionally connected to the subjacent axon via cytoplasmic-rich myelinic channels for movement of macromolecules to and from the internodal periaxonal space under the myelin sheath. This review summarizes our current understanding of how myelin is generated and also the role of oligodendrocytes in supporting the long-term integrity of myelinated axons. PMID:26101081

  17. Contribution of cytoskeletal elements to the axonal mechanical properties

    PubMed Central

    2013-01-01

    Background Microtubules, microfilaments, and neurofilaments are cytoskeletal elements that affect cell morphology, cellular processes, and mechanical structures in neural cells. The objective of the current study was to investigate the contribution of each type of cytoskeletal element to the mechanical properties of axons of dorsal root and sympathetic ganglia cells in chick embryos. Results Microtubules, microfilaments, and neurofilaments in axons were disrupted by nocodazole, cytochalasin D, and acrylamide, respectively, or a combination of the three. An atomic force microscope (AFM) was then used to compress the treated axons, and the resulting corresponding force-deformation information was analyzed to estimate the mechanical properties of axons that were partially or fully disrupted. Conclusion We have found that the mechanical stiffness was most reduced in microtubules-disrupted-axons, followed by neurofilaments-disrupted- and microfilaments-disrupted-axons. This suggests that microtubules contribute the most of the mechanical stiffness to axons. PMID:24007256

  18. Overexpression of insulin-like growth factor-1 attenuates skeletal muscle damage and accelerates muscle regeneration and functional recovery after disuse.

    PubMed

    Ye, Fan; Mathur, Sunita; Liu, Min; Borst, Stephen E; Walter, Glenn A; Sweeney, H Lee; Vandenborne, Krista

    2013-05-01

    Skeletal muscle is a highly dynamic tissue that responds to endogenous and external stimuli, including alterations in mechanical loading and growth factors. In particular, the antigravity soleus muscle experiences significant muscle atrophy during disuse and extensive muscle damage upon reloading. Given that insulin-like growth factor-1 (IGF-1) has been implicated as a central regulator of muscle repair and modulation of muscle size, we examined the effect of virally mediated overexpression of IGF-1 on the soleus muscle following hindlimb cast immobilization and upon reloading. Recombinant IGF-1 cDNA virus was injected into one of the posterior hindlimbs of the mice, while the contralateral limb was injected with saline (control). At 20 weeks of age, both hindlimbs were immobilized for 2 weeks to induce muscle atrophy in the soleus and ankle plantarflexor muscle group. Subsequently, the mice were allowed to reambulate, and muscle damage and recovery were monitored over a period of 2-21 days. The primary finding of this study was that IGF-1 overexpression attenuated reloading-induced muscle damage in the soleus muscle, and accelerated muscle regeneration and force recovery. Muscle T2 assessed by magnetic resonance imaging, a non-specific marker of muscle damage, was significantly lower in IGF-1-injected compared with contralateral soleus muscles at 2 and 5 days reambulation (P<0.05). The reduced prevalence of muscle damage in IGF-1-injected soleus muscles was confirmed on histology, with a lower fractional area of abnormal muscle tissue in IGF-1-injected muscles at 2 days reambulation (33.2±3.3 versus 54.1±3.6%, P<0.05). Evidence of the effect of IGF-1 on muscle regeneration included timely increases in the number of central nuclei (21% at 5 days reambulation), paired-box transcription factor 7 (36% at 5 days), embryonic myosin (37% at 10 days) and elevated MyoD mRNA (7-fold at 2 days) in IGF-1-injected limbs (P<0.05). These findings demonstrate a potential role

  19. Peptide drugs accelerate BMP‐2‐induced calvarial bone regeneration and stimulate osteoblast differentiation through mTORC1 signaling

    PubMed Central

    Sugamori, Yasutaka; Mise‐Omata, Setsuko; Maeda, Chizuko; Aoki, Shigeki; Tabata, Yasuhiko; Murali, Ramachandran; Yasuda, Hisataka; Udagawa, Nobuyuki; Suzuki, Hiroshi; Honma, Masashi

    2016-01-01

    Both W9 and OP3‐4 were known to bind the receptor activator of NF‐κB ligand (RANKL), inhibiting osteoclastogenesis. Recently, both peptides were shown to stimulate osteoblast differentiation; however, the mechanism underlying the activity of these peptides remains to be clarified. A primary osteoblast culture showed that rapamycin, an mTORC1 inhibitor, which was recently demonstrated to be an important serine/threonine kinase for bone formation, inhibited the peptide‐induced alkaline phosphatase activity. Furthermore, both peptides promoted the phosphorylation of Akt and S6K1, an upstream molecule of mTORC1 and the effector molecule of mTORC1, respectively. In the in vivo calvarial defect model, W9 and OP3‐4 accelerated BMP‐2‐induced bone formation to a similar extent, which was confirmed by histomorphometric analyses using fluorescence images of undecalcified sections. Our data suggest that these RANKL‐binding peptides could stimulate the mTORC1 activity, which might play a role in the acceleration of BMP‐2‐induced bone regeneration by the RANKL‐binding peptides. PMID:27345003

  20. The axonal guidance cue semaphorin 3C contributes to alveolar growth and repair.

    PubMed

    Vadivel, Arul; Alphonse, Rajesh S; Collins, Jennifer J P; van Haaften, Tim; O'Reilly, Megan; Eaton, Farah; Thébaud, Bernard

    2013-01-01

    Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.

  1. Modeling molecular mechanisms in the axon

    NASA Astrophysics Data System (ADS)

    de Rooij, R.; Miller, K. E.; Kuhl, E.

    2017-03-01

    Axons are living systems that display highly dynamic changes in stiffness, viscosity, and internal stress. However, the mechanistic origin of these phenomenological properties remains elusive. Here we establish a computational mechanics model that interprets cellular-level characteristics as emergent properties from molecular-level events. We create an axon model of discrete microtubules, which are connected to neighboring microtubules via discrete crosslinking mechanisms that obey a set of simple rules. We explore two types of mechanisms: passive and active crosslinking. Our passive and active simulations suggest that the stiffness and viscosity of the axon increase linearly with the crosslink density, and that both are highly sensitive to the crosslink detachment and reattachment times. Our model explains how active crosslinking with dynein motors generates internal stresses and actively drives axon elongation. We anticipate that our model will allow us to probe a wide variety of molecular phenomena—both in isolation and in interaction—to explore emergent cellular-level features under physiological and pathological conditions.

  2. Regeneration of anion exchange resins by catalyzed electrochemical reduction

    DOEpatents

    Gu, Baohua; Brown, Gilbert M.

    2002-01-01

    Anion exchange resins sorbed with perchlorate may be regenerated by a combination of chemical reduction of perchlorate to chloride using a reducing agent and an electrochemical reduction of the oxidized reducing agent. Transitional metals including Ti, Re, and V are preferred chemical reagents for the reduction of perchlorate to chloride. Complexing agents such as oxalate are used to prevent the precipitation of the oxidized Ti(IV) species, and ethyl alcohol may be added to accelerate the reduction kinetics of perchlorate. The regeneration may be performed by continuously recycling the regenerating solution through the resin bed and an electrochemical cell so that the secondary waste generation is minimized.

  3. Impaired peripheral nerve regeneration in type-2 diabetic mouse model.

    PubMed

    Pham, Vuong M; Tu, Nguyen Huu; Katano, Tayo; Matsumura, Shinji; Saito, Akira; Yamada, Akihiro; Furue, Hidemasa; Ito, Seiji

    2018-01-01

    Peripheral neuropathy is one of the most common and serious complications of type-2 diabetes. Diabetic neuropathy is characterized by a distal symmetrical sensorimotor polyneuropathy, and its incidence increases in patients 40 years of age or older. In spite of extensive research over decades, there are few effective treatments for diabetic neuropathy besides glucose control and improved lifestyle. The earliest changes in diabetic neuropathy occur in sensory nerve fibers, with initial degeneration and regeneration resulting in pain. To seek its effective treatment, here we prepared a type-2 diabetic mouse model by giving mice 2 injections of streptozotocin and nicotinamide and examining the ability for nerve regeneration by using a sciatic nerve transection-regeneration model previously established by us. Seventeen weeks after the last injection, the mice exhibited symptoms of type-2 diabetes, that is, impaired glucose tolerance, decreased insulin level, mechanical hyperalgesia, and impaired sensory nerve fibers in the plantar skin. These mice showed delayed functional recovery and nerve regeneration by 2 weeks compared with young healthy mice and by 1 week compared with age-matched non-diabetic mice after axotomy. Furthermore, type-2 diabetic mice displayed increased expression of PTEN in their DRG neurons. Administration of a PTEN inhibitor at the cutting site of the nerve for 4 weeks promoted the axonal transport and functional recovery remarkably. This study demonstrates that peripheral nerve regeneration was impaired in type-2 diabetic model and that its combination with sciatic nerve transection is suitable for the study of the pathogenesis and treatment of early diabetic neuropathy. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  4. Calpains mediate axonal cytoskeleton disintegration during Wallerian degeneration

    PubMed Central

    Ma, Marek; Ferguson, Toby A.; Schoch, Kathleen M.; Li, Jian; Qian, Yaping; Shofer, Frances S.; Saatman, Kathryn E.; Neumar, Robert W.

    2013-01-01

    In both the central nervous system (CNS) and peripheral nervous system (PNS), transected axons undergo Wallerian degeneration. Even though Augustus Waller first described this process after transection of axons in 1850, the molecular mechanisms may be shared, at least in part, by many human diseases. Early pathology includes failure of synaptic transmission, target denervation, and granular disintegration of the axonal cytoskeleton (GDC). The Ca2+-dependent proteases calpains have been implicated in GDC but causality has not been established. To test the hypothesis that calpains play a causal role in axonal and synaptic degeneration in vivo, we studied transgenic mice that express human calpastatin (hCAST), the endogenous calpain inhibitor, in optic and sciatic nerve axons. Five days after optic nerve transection and 48 hours after sciatic nerve transection, robust neurofilament proteolysis observed in wild-type controls was reduced in hCAST transgenic mice. Protection of the axonal cytoskeleton in sciatic nerves of hCAST mice was nearly complete 48 hours post-transection. In addition, hCAST expression preserved the morphological integrity of neuromuscular junctions. However, compound muscle action potential amplitudes after nerve transection were similar in wild-type and hCAST mice. These results, in total, provide direct evidence that calpains are responsible for the morphological degeneration of the axon and synapse during Wallerian degeneration. PMID:23542511

  5. Polymer Scaffolds with Preferential Parallel Grooves Enhance Nerve Regeneration

    PubMed Central

    Mobasseri, Atefeh; Faroni, Alessandro; Minogue, Ben M.; Downes, Sandra; Reid, Adam J.

    2015-01-01

    We have modified the surface topography of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) blended films to improve cell proliferation and to guide the regeneration of peripheral nerves. Films with differing shaped grooves were made using patterned silicon templates, sloped walls (SL), V-shaped (V), and square-shaped (SQ), and compared with nongrooved surfaces with micropits. The solvent cast films were tested in vitro using adult adipose-derived stem cells differentiated to Schwann cell-like cells. Cell attachment, proliferation, and cell orientation were all improved on the grooved surfaces, with SL grooves giving the best results. We present in vivo data on Sprague-Dawley rat sciatic nerve injury with a 10-mm gap, evaluating nerve regeneration at 3 weeks across a polymer nerve conduit modified with intraluminal grooves (SL, V, and SQ) and differing wall thicknesses (70, 100, 120, and 210 μm). The SL-grooved nerve conduit showed a significant improvement over the other topographical-shaped grooves, while increasing the conduit wall thickness saw no positive effect on the biological response of the regenerating nerve. Furthermore, the preferred SL-grooved conduit (C) with 70 μm wall thickness was compared with the current clinical gold standard of autologous nerve graft (Ag) in the rat 10-mm sciatic nerve gap model. At 3 weeks postsurgery, all nerve gaps across both groups were bridged with regenerated nerve fibers. At 16 weeks, features of regenerated axons were comparable between the autograft (Ag) and conduit (C) groups. End organ assessments of muscle weight, electromyography, and skin reinnervation were also similar between the groups. The comparable experimental outcome between conduit and autograft, suggests that the PCL/PLA conduit with inner lumen microstructured grooves could be used as a potential alternative treatment for peripheral nerve repair. PMID:25435096

  6. Guidance of retinal axons in mammals.

    PubMed

    Herrera, Eloísa; Erskine, Lynda; Morenilla-Palao, Cruz

    2017-11-26

    In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face 'choices' in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Sustained Delivery of Activated Rho GTPases and BDNF Promotes Axon Growth in CSPG-Rich Regions Following Spinal Cord Injury

    PubMed Central

    Jain, Anjana; McKeon, Robert J.; Brady-Kalnay, Susann M.; Bellamkonda, Ravi V.

    2011-01-01

    Background Spinal cord injury (SCI) often results in permanent functional loss. This physical trauma leads to secondary events, such as the deposition of inhibitory chondroitin sulfate proteoglycan (CSPG) within astroglial scar tissue at the lesion. Methodology/Principal Findings We examined whether local delivery of constitutively active (CA) Rho GTPases, Cdc42 and Rac1 to the lesion site alleviated CSPG-mediated inhibition of regenerating axons. A dorsal over-hemisection lesion was created in the rat spinal cord and the resulting cavity was conformally filled with an in situ gelling hydrogel combined with lipid microtubes that slowly released constitutively active (CA) Cdc42, Rac1, or Brain-derived neurotrophic factor (BDNF). Treatment with BDNF, CA-Cdc42, or CA-Rac1 reduced the number of GFAP-positive astrocytes, as well as CSPG deposition, at the interface of the implanted hydrogel and host tissue. Neurofilament 160kDa positively stained axons traversed the glial scar extensively, entering the hydrogel-filled cavity in the treatments with BDNF and CA-Rho GTPases. The treated animals had a higher percentage of axons from the corticospinal tract that traversed the CSPG-rich regions located proximal to the lesion site. Conclusion Local delivery of CA-Cdc42, CA-Rac1, and BDNF may have a significant therapeutic role in overcoming CSPG-mediated regenerative failure after SCI. PMID:21283639

  8. Tissue-engineered spiral nerve guidance conduit for peripheral nerve regeneration.

    PubMed

    Chang, Wei; Shah, Munish B; Lee, Paul; Yu, Xiaojun

    2018-06-01

    Recently in peripheral nerve regeneration, preclinical studies have shown that the use of nerve guidance conduits (NGCs) with multiple longitudinally channels and intra-luminal topography enhance the functional outcomes when bridging a nerve gap caused by traumatic injury. These features not only provide guidance cues for regenerating nerve, but also become the essential approaches for developing a novel NGC. In this study, a novel spiral NGC with aligned nanofibers and wrapped with an outer nanofibrous tube was first developed and investigated. Using the common rat sciatic 10-mm nerve defect model, the in vivo study showed that a novel spiral NGC (with and without inner nanofibers) increased the successful rate of nerve regeneration after 6 weeks recovery. Substantial improvements in nerve regeneration were achieved by combining the spiral NGC with inner nanofibers and outer nanofibrous tube, based on the results of walking track analysis, electrophysiology, nerve histological assessment, and gastrocnemius muscle measurement. This demonstrated that the novel spiral NGC with inner aligned nanofibers and wrapped with an outer nanofibrous tube provided a better environment for peripheral nerve regeneration than standard tubular NGCs. Results from this study will benefit for future NGC design to optimize tissue-engineering strategies for peripheral nerve regeneration. We developed a novel spiral nerve guidance conduit (NGC) with coated aligned nanofibers. The spiral structure increases surface area by 4.5 fold relative to a tubular NGC. Furthermore, the aligned nanofibers was coated on the spiral walls, providing cues for guiding neurite extension. Finally, the outside of spiral NGC was wrapped with randomly nanofibers to enhance mechanical strength that can stabilize the spiral NGC. Our nerve histological data have shown that the spiral NGC had 50% more myelinated axons than a tubular structure for nerve regeneration across a 10 mm gap in a rat sciatic nerve

  9. Molecular, Cellular and Functional Events in Axonal Sprouting after Stroke

    PubMed Central

    Kathirvelu, Balachander; Schweppe, Catherine A; Nie, Esther H

    2016-01-01

    Stroke is the leading cause of adult disability. Yet there is a limited degree of recovery in this disease. One of the mechanisms of recovery is the formation of new connections in the brain and spinal cord after stroke: post-stroke axonal sprouting. Studies indicate that post-stroke axonal sprouting occurs in mice, rats, primates and humans. Inducing post-stroke axonal sprouting in specific connections enhances recovery; blocking axonal sprouting impairs recovery. Behavioral activity patterns after stroke modify the axonal sprouting response. A unique regenerative molecular program mediates this aspect of tissue repair in the CNS. The types of connections that are formed after stroke indicate three patterns of axonal sprouting after stroke: Reactive, Reparative and Unbounded Axonal Sprouting. These differ in mechanism, location, relationship to behavioral recovery and, importantly, in their prospect for therapeutic manipulation to enhance tissue repair. PMID:26874223

  10. Planarian homologs of netrin and netrin receptor are required for proper regeneration of the central nervous system and the maintenance of nervous system architecture.

    PubMed

    Cebrià, Francesc; Newmark, Phillip A

    2005-08-01

    Conserved axon guidance mechanisms are essential for proper wiring of the nervous system during embryogenesis; however, the functions of these cues in adults and during regeneration remain poorly understood. Because freshwater planarians can regenerate a functional central nervous system (CNS) from almost any portion of their body, they are useful models in which to study the roles of guidance cues during neural regeneration. Here, we characterize two netrin homologs and one netrin receptor family member from Schmidtea mediterranea. RNAi analyses indicate that Smed-netR (netrin receptor) and Smed-netrin2 are required for proper CNS regeneration and that Smed-netR may mediate the response to Smed-netrin2. Remarkably, Smed-netR and Smed-netrin2 are also required in intact planarians to maintain the proper patterning of the CNS. These results suggest a crucial role for guidance cues, not only in CNS regeneration but also in maintenance of neural architecture.

  11. Axonal Conduction Delays, Brain State, and Corticogeniculate Communication

    PubMed Central

    2017-01-01

    Thalamocortical conduction times are short, but layer 6 corticothalamic axons display an enormous range of conduction times, some exceeding 40–50 ms. Here, we investigate (1) how axonal conduction times of corticogeniculate (CG) neurons are related to the visual information conveyed to the thalamus, and (2) how alert versus nonalert awake brain states affect visual processing across the spectrum of CG conduction times. In awake female Dutch-Belted rabbits, we found 58% of CG neurons to be visually responsive, and 42% to be unresponsive. All responsive CG neurons had simple, orientation-selective receptive fields, and generated sustained responses to stationary stimuli. CG axonal conduction times were strongly related to modulated firing rates (F1 values) generated by drifting grating stimuli, and their associated interspike interval distributions, suggesting a continuum of visual responsiveness spanning the spectrum of axonal conduction times. CG conduction times were also significantly related to visual response latency, contrast sensitivity (C-50 values), directional selectivity, and optimal stimulus velocity. Increasing alertness did not cause visually unresponsive CG neurons to become responsive and did not change the response linearity (F1/F0 ratios) of visually responsive CG neurons. However, for visually responsive CG neurons, increased alertness nearly doubled the modulated response amplitude to optimal visual stimulation (F1 values), significantly shortened response latency, and dramatically increased response reliability. These effects of alertness were uniform across the broad spectrum of CG axonal conduction times. SIGNIFICANCE STATEMENT Corticothalamic neurons of layer 6 send a dense feedback projection to thalamic nuclei that provide input to sensory neocortex. While sensory information reaches the cortex after brief thalamocortical axonal delays, corticothalamic axons can exhibit conduction delays of <2 ms to 40–50 ms. Here, in the

  12. Axonal Conduction Delays, Brain State, and Corticogeniculate Communication.

    PubMed

    Stoelzel, Carl R; Bereshpolova, Yulia; Alonso, Jose-Manuel; Swadlow, Harvey A

    2017-06-28

    Thalamocortical conduction times are short, but layer 6 corticothalamic axons display an enormous range of conduction times, some exceeding 40-50 ms. Here, we investigate (1) how axonal conduction times of corticogeniculate (CG) neurons are related to the visual information conveyed to the thalamus, and (2) how alert versus nonalert awake brain states affect visual processing across the spectrum of CG conduction times. In awake female Dutch-Belted rabbits, we found 58% of CG neurons to be visually responsive, and 42% to be unresponsive. All responsive CG neurons had simple, orientation-selective receptive fields, and generated sustained responses to stationary stimuli. CG axonal conduction times were strongly related to modulated firing rates (F1 values) generated by drifting grating stimuli, and their associated interspike interval distributions, suggesting a continuum of visual responsiveness spanning the spectrum of axonal conduction times. CG conduction times were also significantly related to visual response latency, contrast sensitivity (C-50 values), directional selectivity, and optimal stimulus velocity. Increasing alertness did not cause visually unresponsive CG neurons to become responsive and did not change the response linearity (F1/F0 ratios) of visually responsive CG neurons. However, for visually responsive CG neurons, increased alertness nearly doubled the modulated response amplitude to optimal visual stimulation (F1 values), significantly shortened response latency, and dramatically increased response reliability. These effects of alertness were uniform across the broad spectrum of CG axonal conduction times. SIGNIFICANCE STATEMENT Corticothalamic neurons of layer 6 send a dense feedback projection to thalamic nuclei that provide input to sensory neocortex. While sensory information reaches the cortex after brief thalamocortical axonal delays, corticothalamic axons can exhibit conduction delays of <2 ms to 40-50 ms. Here, in the corticogeniculate

  13. Effect of Surface Pore Structure of Nerve Guide Conduit on Peripheral Nerve Regeneration

    PubMed Central

    Oh, Se Heang; Kim, Jin Rae; Kwon, Gu Birm; Namgung, Uk; Song, Kyu Sang

    2013-01-01

    Polycaprolactone (PCL)/Pluronic F127 nerve guide conduits (NGCs) with different surface pore structures (nano-porous inner surface vs. micro-porous inner surface) but similar physical and chemical properties were fabricated by rolling the opposite side of asymmetrically porous PCL/F127 membranes. The effect of the pore structure on peripheral nerve regeneration through the NGCs was investigated using a sciatic nerve defect model of rats. The nerve fibers and tissues were shown to have regenerated along the longitudinal direction through the NGC with a nano-porous inner surface (Nanopore NGC), while they grew toward the porous wall of the NGC with a micro-porous inner surface (Micropore NGC) and, thus, their growth was restricted when compared with the Nanopore NGC, as investigated by immunohistochemical evaluations (by fluorescence microscopy with anti-neurofilament staining and Hoechst staining for growth pattern of nerve fibers), histological evaluations (by light microscopy with Meyer's modified trichrome staining and Toluidine blue staining and transmission electron microscopy for the regeneration of axon and myelin sheath), and FluoroGold retrograde tracing (for reconnection between proximal and distal stumps). The effect of nerve growth factor (NGF) immobilized on the pore surfaces of the NGCs on nerve regeneration was not so significant when compared with NGCs not containing immobilized NGF. The NGC system with different surface pore structures but the same chemical/physical properties seems to be a good tool that is used for elucidating the surface pore effect of NGCs on nerve regeneration. PMID:22871377

  14. Axonal Degeneration Is Mediated by the Mitochondrial Permeability Transition Pore

    PubMed Central

    Barrientos, Sebastian A.; Martinez, Nicolas W.; Yoo, Soonmoon; Jara, Juan S.; Zamorano, Sebastian; Hetz, Claudio; Twiss, Jeffery L.; Alvarez, Jaime; Court, Felipe A.

    2011-01-01

    Axonal degeneration is an active process that has been associated with neurodegenerative conditions triggered by mechanical, metabolic, infectious, toxic, hereditary and inflammatory stimuli. This degenerative process can cause permanent loss of function, so it represents a focus for neuroprotective strategies. Several signaling pathways are implicated in axonal degeneration, but identification of an integrative mechanism for this self-destructive process has remained elusive. Here, we show that rapid axonal degeneration triggered by distinct mechanical and toxic insults is dependent on the activation of the mitochondrial permeability transition pore (mPTP). Both pharmacological and genetic targeting of cyclophilin D, a functional component of the mPTP, protects severed axons and vincristine-treated neurons from axonal degeneration in ex vivo and in vitro mouse and rat model systems. These effects were observed in axons from both the peripheral and central nervous system. Our results suggest that the mPTP is a key effector of axonal degeneration, upon which several independent signaling pathways converge. Since axonal and synapse degeneration are increasingly considered early pathological events in neurodegeneration, our work identifies a potential target for therapeutic intervention in a wide variety of conditions that lead to loss of axons and subsequent functional impairment. PMID:21248121

  15. Microfluidic device for unidirectional axon growth

    NASA Astrophysics Data System (ADS)

    Malishev, E.; Pimashkin, A.; Gladkov, A.; Pigareva, Y.; Bukatin, A.; Kazantsev, V.; Mukhina, I.; Dubina, M.

    2015-11-01

    In order to better understand the communication and connectivity development of neuron networks, we designed microfluidic devices with several chambers for growing dissociated neuronal cultures from mice fetal hippocampus (E18). The chambers were connected with microchannels providing unidirectional axonal growth between “Source” and “Target” neural sub-networks. Experiments were performed in a hippocampal cultures plated in a poly-dimethylsiloxane (PDMS) microfluidic chip, aligned with a 60 microelectrode array (MEA). Axonal growth through microchannels was observed with brightfield, phase-contrast and fluorescence microscopy, and after 7 days in vitro electrical activity was recorded. Visual inspection and spike propagation analysis showed the predominant axonal growth in microchannels in a direction from “Source” to “Target”.

  16. Hindsight regulates photoreceptor axon targeting through transcriptional control of jitterbug/Filamin and multiple genes involved in axon guidance in Drosophila.

    PubMed

    Oliva, Carlos; Molina-Fernandez, Claudia; Maureira, Miguel; Candia, Noemi; López, Estefanía; Hassan, Bassem; Aerts, Stein; Cánovas, José; Olguín, Patricio; Sierralta, Jimena

    2015-09-01

    During axon targeting, a stereotyped pattern of connectivity is achieved by the integration of intrinsic genetic programs and the response to extrinsic long and short-range directional cues. How this coordination occurs is the subject of intense study. Transcription factors play a central role due to their ability to regulate the expression of multiple genes required to sense and respond to these cues during development. Here we show that the transcription factor HNT regulates layer-specific photoreceptor axon targeting in Drosophila through transcriptional control of jbug/Filamin and multiple genes involved in axon guidance and cytoskeleton organization.Using a microarray analysis we identified 235 genes whose expression levels were changed by HNT overexpression in the eye primordia. We analyzed nine candidate genes involved in cytoskeleton regulation and axon guidance, six of which displayed significantly altered gene expression levels in hnt mutant retinas. Functional analysis confirmed the role of OTK/PTK7 in photoreceptor axon targeting and uncovered Tiggrin, an integrin ligand, and Jbug/Filamin, a conserved actin- binding protein, as new factors that participate of photoreceptor axon targeting. Moreover, we provided in silico and molecular evidence that supports jbug/Filamin as a direct transcriptional target of HNT and that HNT acts partially through Jbug/Filamin in vivo to regulate axon guidance. Our work broadens the understanding of how HNT regulates the coordinated expression of a group of genes to achieve the correct connectivity pattern in the Drosophila visual system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1018-1032, 2015. © 2015 Wiley Periodicals, Inc.

  17. Dock and Pak regulate olfactory axon pathfinding in Drosophila.

    PubMed

    Ang, Lay-Hong; Kim, Jenny; Stepensky, Vitaly; Hing, Huey

    2003-04-01

    The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. We report here that the SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, we observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). Our findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli.

  18. Neurotrophin Signaling via Long-Distance Axonal Transport

    NASA Astrophysics Data System (ADS)

    Chowdary, Praveen D.; Che, Dung L.; Cui, Bianxiao

    2012-05-01

    Neurotrophins are a family of target-derived growth factors that support survival, development, and maintenance of innervating neurons. Owing to the unique architecture of neurons, neurotrophins that act locally on the axonal terminals must convey their signals across the entire axon for subsequent regulation of gene transcription in the cell nucleus. This long-distance retrograde signaling, a motor-driven process that can take hours or days, has been a subject of intense interest. In the last decade, live-cell imaging with high sensitivity has significantly increased our capability to track the transport of neurotrophins, their receptors, and subsequent signals in real time. This review summarizes recent research progress in understanding neurotrophin-receptor interactions at the axonal terminal and their transport dynamics along the axon. We emphasize high-resolution studies at the single-molecule level and also discuss recent technical advances in the field.

  19. Axonal inclusions in the crab Hemigrapsus nudus.

    PubMed

    Smith, R S

    1978-10-01

    Light microscopic examination of living giant axons from the walking legs of Hemigrapsus nudus revealed intra-axonal inclusions which were usually several tens of micrometers long and about 5 micron wide. The inclusions were filled with small light-scattering particles. The inclusions were shown, by thin section electron microscopy, to be composed largely 68% by volume) of mitochondria. Each inclusion was surrounded by membrane bounded spaces which are presumed to represent a part of the smooth endoplasmic reticulum. Similar inclusions were not found in the leg axons of a variety of other decapod crustaceans.

  20. Con-nectin axons and dendrites.

    PubMed

    Beaudoin, Gerard M J

    2006-07-03

    Unlike adherens junctions, synapses are asymmetric connections, usually between axons and dendrites, that rely on various cell adhesion molecules for structural stability and function. Two cell types of adhesion molecules found at adherens junctions, cadherins and nectins, are thought to mediate homophilic interaction between neighboring cells. In this issue, Togashi et al. (see p. 141) demonstrate that the differential localization of two heterophilic interacting nectins mediates the selective attraction of axons and dendrites in cooperation with cadherins.

  1. Oligodendroglia metabolically support axons and contribute to neurodegeneration

    PubMed Central

    Lee, Youngjin; Morrison, Brett M.; Li, Yun; Lengacher, Sylvain; Farah, Mohamed H.; Hoffman, Paul N.; Liu, Yiting; Tsingalia, Akivaga; Jin, Lin; Zhang, Ping-Wu; Pellerin, Luc; Magistretti, Pierre J.; Rothstein, Jeffrey D.

    2012-01-01

    Summary Oligodendroglia support axon survival and function through mechanisms independent of myelination and their dysfunction leads to axon degeneration in several diseases. The cause of this degeneration has not been determined, but lack of energy metabolites such as glucose or lactate has been hypothesized. Lactate is transported exclusively by monocarboxylate transporters, and changes to these transporters alter lactate production and utilization. We show the most abundant lactate transporter in the CNS, monocarboxylate transporter 1 (MCT1), is highly enriched within oligodendroglia and that disruption of this transporter produces axon damage and neuron loss in animal and cell culture models. In addition, this same transporter is reduced in patients with, and mouse models of, amyotrophic lateral sclerosis (ALS), suggesting a role for oligodendroglial MCT1 in pathogenesis. The role of oligodendroglia in axon function and neuron survival has been elusive; this study defines a new fundamental mechanism by which oligodendroglia support neurons and axons. PMID:22801498

  2. Tracking individual action potentials throughout mammalian axonal arbors.

    PubMed

    Radivojevic, Milos; Franke, Felix; Altermatt, Michael; Müller, Jan; Hierlemann, Andreas; Bakkum, Douglas J

    2017-10-09

    Axons are neuronal processes specialized for conduction of action potentials (APs). The timing and temporal precision of APs when they reach each of the synapses are fundamentally important for information processing in the brain. Due to small diameters of axons, direct recording of single AP transmission is challenging. Consequently, most knowledge about axonal conductance derives from modeling studies or indirect measurements. We demonstrate a method to noninvasively and directly record individual APs propagating along millimeter-length axonal arbors in cortical cultures with hundreds of microelectrodes at microsecond temporal resolution. We find that cortical axons conduct single APs with high temporal precision (~100 µs arrival time jitter per mm length) and reliability: in more than 8,000,000 recorded APs, we did not observe any conduction or branch-point failures. Upon high-frequency stimulation at 100 Hz, successive became slower, and their arrival time precision decreased by 20% and 12% for the 100th AP, respectively.

  3. Mechanisms of Distal Axonal Degeneration in Peripheral Neuropathies

    PubMed Central

    Cashman, Christopher R.; Höke, Ahmet

    2015-01-01

    Peripheral neuropathy is a common complication of a variety of diseases and treatments, including diabetes, cancer chemotherapy, and infectious causes (HIV, hepatitis C, and Campylobacter jejuni). Despite the fundamental difference between these insults, peripheral neuropathy develops as a combination of just six primary mechanisms: altered metabolism, covalent modification, altered organelle function and reactive oxygen species formation, altered intracellular and inflammatory signaling, slowed axonal transport, and altered ion channel dynamics and expression. All of these pathways converge to lead to axon dysfunction and symptoms of neuropathy. The detailed mechanisms of axon degeneration itself have begun to be elucidated with studies of animal models with altered degeneration kinetics, including the slowed Wallerian degeneration (Wlds) and Sarmknockout animal models. These studies have shown axonal degeneration to occur througha programmed pathway of injury signaling and cytoskeletal degradation. Insights into the common disease insults that converge on the axonal degeneration pathway promise to facilitate the development of therapeutics that may be effective against other mechanisms of neurodegeneration. PMID:25617478

  4. Mechanosensing is critical for axon growth in the developing brain

    PubMed Central

    Pillai, Eva K.; Sheridan, Graham K.; Svoboda, Hanno; Viana, Matheus; da F. Costa, Luciano; Guck, Jochen; Holt, Christine E.; Franze, Kristian

    2016-01-01

    During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signalling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell (RGC) axons. In vivo atomic force microscopy revealed striking stiffness gradient patterns in the embryonic brain. RGC axons grew towards the tissue’s softer side, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically, and knocked down the mechanosensitive ion channel Piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness–read out by mechanosensitive ion channels–is critically involved in instructing neuronal growth in vivo. PMID:27643431

  5. Axonal Transport and Morphology: How Myelination gets Nerves into Shape

    NASA Astrophysics Data System (ADS)

    Jung, Peter; Zhao, Peng; Monsma, Paula; Brown, Tony

    2011-03-01

    The local caliber of mature axons is largely determined by neurofilament (NF) content. The axoskeleton, mainly consisting of NFs, however, is dynamic. NFs are assembled in the cell body and are transported by molecular motors on microtubule tracks along the axon at a slow rate of fractions of mm per day. We combine live cell fluorescent imaging techniques to access NF transport in myelinated and non-myelinated segments of axons with computational modeling of the active NF flow to show that a), myelination locally slows NF transport rates by regulating duty ratios and b), that the predicted increase in axon caliber agrees well with experiments. This study, for the first time, links NF kinetics directly to axonal morphology, providing a novel conceptual framework for the physical understanding of processes leading to the formation of axonal structures such as the ``Nodes of Ranvier'' as well as abnormal axonal swellings associated with neurodegenerative diseases like Amyotrophic lateral sclerosis (ALS). NSF grants # IOS-0818412(PJ) and IOS-0818653 (AB).

  6. Axonal localization and mitochondrial association of precursor microRNA 338

    PubMed Central

    Vargas, Jose Norberto S.; Kar, Amar N.; Kowalak, Jeffrey A.; Gale, Jenna R.; Aschrafi, Armaz; Chen, Cai-Yun; Gioio, Anthony E.; Kaplan, Barry B.

    2016-01-01

    microRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can be locally processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatio-temporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs. PMID:27229124

  7. Fibrin matrices with affinity-based delivery systems and neurotrophic factors promote functional nerve regeneration.

    PubMed

    Wood, Matthew D; MacEwan, Matthew R; French, Alexander R; Moore, Amy M; Hunter, Daniel A; Mackinnon, Susan E; Moran, Daniel W; Borschel, Gregory H; Sakiyama-Elbert, Shelly E

    2010-08-15

    Glial-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity-based delivery of GDNF or NGF from fibrin-filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity-based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury.

  8. Retrograde and Wallerian Axonal Degeneration Occur Synchronously after Retinal Ganglion Cell Axotomy

    PubMed Central

    Kanamori, Akiyasu; Catrinescu, Maria-Magdalena; Belisle, Jonathan M.; Costantino, Santiago; Levin, Leonard A.

    2013-01-01

    Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration. PMID:22642911

  9. Axonal loss in the multiple sclerosis spinal cord revisited.

    PubMed

    Petrova, Natalia; Carassiti, Daniele; Altmann, Daniel R; Baker, David; Schmierer, Klaus

    2018-05-01

    Preventing chronic disease deterioration is an unmet need in people with multiple sclerosis, where axonal loss is considered a key substrate of disability. Clinically, chronic multiple sclerosis often presents as progressive myelopathy. Spinal cord cross-sectional area (CSA) assessed using MRI predicts increasing disability and has, by inference, been proposed as an indirect index of axonal degeneration. However, the association between CSA and axonal loss, and their correlation with demyelination, have never been systematically investigated using human post mortem tissue. We extensively sampled spinal cords of seven women and six men with multiple sclerosis (mean disease duration= 29 years) and five healthy controls to quantify axonal density and its association with demyelination and CSA. 396 tissue blocks were embedded in paraffin and immuno-stained for myelin basic protein and phosphorylated neurofilaments. Measurements included total CSA, areas of (i) lateral cortico-spinal tracts, (ii) gray matter, (iii) white matter, (iv) demyelination, and the number of axons within the lateral cortico-spinal tracts. Linear mixed models were used to analyze relationships. In multiple sclerosis CSA reduction at cervical, thoracic and lumbar levels ranged between 19 and 24% with white (19-24%) and gray (17-21%) matter atrophy contributing equally across levels. Axonal density in multiple sclerosis was lower by 57-62% across all levels and affected all fibers regardless of diameter. Demyelination affected 24-48% of the gray matter, most extensively at the thoracic level, and 11-13% of the white matter, with no significant differences across levels. Disease duration was associated with reduced axonal density, however not with any area index. Significant association was detected between focal demyelination and decreased axonal density. In conclusion, over nearly 30 years multiple sclerosis reduces axonal density by 60% throughout the spinal cord. Spinal cord cross sectional area

  10. Low-level laser therapy (LLLT) accelerates the sternomastoid muscle regeneration process after myonecrosis due to bupivacaine.

    PubMed

    Alessi Pissulin, Cristiane Neves; Henrique Fernandes, Ana Angélica; Sanchez Orellana, Alejandro Manuel; Rossi E Silva, Renata Calciolari; Michelin Matheus, Selma Maria

    2017-03-01

    Because of its long-lasting analgesic action, bupivacaine is an anesthetic used for peripheral nerve block and relief of postoperative pain. Muscle degeneration and neurotoxicity are its main limitations. There is strong evidence that low-level laser therapy (LLLT) assists in muscle and nerve repair. The authors evaluated the effects of a Gallium Arsenide laser (GaAs), on the regeneration of muscle fibers of the sternomastoid muscle and accessory nerve after injection of bupivacaine. In total, 30 Wistar adult rats were divided into 2 groups: control group (C: n=15) and laser group (L: n=15). The groups were subdivided by antimere, with 0.5% bupivacaine injected on the right and 0.9% sodium chloride on the left. LLLT (GaAs 904nm, 0,05W, 2.8J per point) was administered for 5 consecutive days, starting 24h after injection of the solutions. Seven days after the trial period, blood samples were collected for determination of creatine kinase (CK). The sternomastoid nerve was removed for morphological and morphometric analyses; the surface portion of the sternomastoid muscle was used for histopathological and ultrastructural analyses. Muscle CK and TNFα protein levels were measured. The anesthetic promoted myonecrosis and increased muscle CK without neurotoxic effects. The LLLT reduced myonecrosis, characterized by a decrease in muscle CK levels, inflammation, necrosis, and atrophy, as well as the number of central nuclei in the muscle fibers and the percentage of collagen. TNFα values remained constant. LLLT, at the dose used, reduced fibrosis and myonecrosis in the sternomastoid muscle triggered by bupivacaine, accelerating the muscle regeneration process. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Low-frequency pulsed electromagnetic field pretreated bone marrow-derived mesenchymal stem cells promote the regeneration of crush-injured rat mental nerve.

    PubMed

    Seo, NaRi; Lee, Sung-Ho; Ju, Kyung Won; Woo, JaeMan; Kim, BongJu; Kim, SoungMin; Jahng, Jeong Won; Lee, Jong-Ho

    2018-01-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) have been shown to promote the regeneration of injured peripheral nerves. Pulsed electromagnetic field (PEMF) reportedly promotes the proliferation and neuronal differentiation of BMSCs. Low-frequency PEMF can induce the neuronal differentiation of BMSCs in the absence of nerve growth factors. This study was designed to investigate the effects of low-frequency PEMF pretreatment on the proliferation and function of BMSCs and the effects of low-frequency PEMF pre-treated BMSCs on the regeneration of injured peripheral nerve using in vitro and in vivo experiments. In in vitro experiments, quantitative DNA analysis was performed to determine the proliferation of BMSCs, and reverse transcription-polymerase chain reaction was performed to detect S100 (Schwann cell marker), glial fibrillary acidic protein (astrocyte marker), and brain-derived neurotrophic factor and nerve growth factor (neurotrophic factors) mRNA expression. In the in vivo experiments, rat models of crush-injured mental nerve established using clamp method were randomly injected with low-frequency PEMF pretreated BMSCs, unpretreated BMSCs or PBS at the injury site (1 × 10 6 cells). DiI-labeled BMSCs injected at the injury site were counted under the fluorescence microscope to determine cell survival. One or two weeks after cell injection, functional recovery of the injured nerve was assessed using the sensory test with von Frey filaments. Two weeks after cell injection, axonal regeneration was evaluated using histomorphometric analysis and retrograde labeling of trigeminal ganglion neurons. In vitro experiment results revealed that low-frequency PEMF pretreated BMSCs proliferated faster and had greater mRNA expression of growth factors than unpretreated BMSCs. In vivo experiment results revealed that compared with injection of unpretreated BMSCs, injection of low-frequency PEMF pretreated BMSCs led to higher myelinated axon count and axon density and

  12. Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration

    PubMed Central

    Gerardy-Schahn, Rita; Hildebrandt, Herbert

    2014-01-01

    Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity

  13. Facilitation of facial nerve regeneration using chitosan-β-glycerophosphate-nerve growth factor hydrogel.

    PubMed

    Chao, Xiuhua; Xu, Lei; Li, Jianfeng; Han, Yuechen; Li, Xiaofei; Mao, YanYan; Shang, Haiqiong; Fan, Zhaomin; Wang, Haibo

    2016-06-01

    Conclusion C/GP hydrogel was demonstrated to be an ideal drug delivery vehicle and scaffold in the vein conduit. Combined use autologous vein and NGF continuously delivered by C/GP-NGF hydrogel can improve the recovery of facial nerve defects. Objective This study investigated the effects of chitosan-β-glycerophosphate-nerve growth factor (C/GP-NGF) hydrogel combined with autologous vein conduit on the recovery of damaged facial nerve in a rat model. Methods A 5 mm gap in the buccal branch of a rat facial nerve was reconstructed with an autologous vein. Next, C/GP-NGF hydrogel was injected into the vein conduit. In negative control groups, NGF solution or phosphate-buffered saline (PBS) was injected into the vein conduits, respectively. Autologous implantation was used as a positive control group. Vibrissae movement, electrophysiological assessment, and morphological analysis of regenerated nerves were performed to assess nerve regeneration. Results NGF continuously released from C/GP-NGF hydrogel in vitro. The recovery rate of vibrissae movement and the compound muscle action potentials of regenerated facial nerve in the C/GP-NGF group were similar to those in the Auto group, and significantly better than those in the NGF group. Furthermore, larger regenerated axons and thicker myelin sheaths were obtained in the C/GP-NGF group than those in the NGF group.

  14. Neuron-to-neuron transmission of α-synuclein fibrils through axonal transport

    PubMed Central

    Freundt, Eric C.; Maynard, Nate; Clancy, Eileen K.; Roy, Shyamali; Bousset, Luc; Sourigues, Yannick; Covert, Markus; Melki, Ronald; Kirkegaard, Karla; Brahic, Michel

    2012-01-01

    Objective The lesions of Parkinson's disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. Methods We grew primary cortical mouse neurons in microfluidic devices to separate soma from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. Results Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. Interpretation These results support the hypothesis that the progression of Parkinson's disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be transsynaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extra-cellular phase of their journey. PMID:23109146

  15. [The comparative characteristics of crystalline lens and limb regeneration in newts operated on before and after the completion of an orbital space flight].

    PubMed

    Tuchkova, S Ia; Brushlinskaia, N V; Grigorian, E N; Mitashov, V I

    1994-01-01

    It has been already established that a tendency towards synchronization and acceleration of the forelimb and lens regeneration is observed in Pleurodeles waltlii under the effect of space flight factors. Here we present the results obtained after 16-day space flight of two groups of newts. In animals of group I forelimbs were amputated and lenses were removed 14 and 7 days before the space flight, respectively. Intact animals of group II were operated on the day of the sputnik landing. Regenerates of the flight and corresponding control animals were fixed at the same time after the operation. For evaluation of the regeneration rate morphological criteria were used: morphological stages of regeneration were compared in the experiment and the control. For quantitative assay of the regeneration rate we determined the index of nuclei labelled with 3H-thymidine in the blastema and lens rudiment cells and used morphometry of the lens regenerates. Acceleration of forelimb and lens regeneration was observed in both groups of animals. In group II more than two-fold increase of the index of labelled nuclei was found in the blastema cells at the comparable stages of development. The size of lens regenerates in flight groups I and II exceeded reliably those in the control animals. The results obtained suggest a prolonged effect of the space flight factors on forelimb and lens regeneration. Under the conditions of space flight the lens regenerates reached more advanced stages of regeneration, as compared with the control animals operated after the space flight. These results also suggest acceleration of regeneration in lower vertebrates.

  16. Epithelial–Mesenchymal Interactions as a Working Concept for Oral Mucosa Regeneration

    PubMed Central

    Liu, Jiarong

    2011-01-01

    Oral mucosa consists of two tissue layers, the superficial epithelium and the underlying lamina propria. Together, oral mucosa functions as a barrier against exogenous substances and pathogens. In development, interactions of stem/progenitor cells of the epithelium and mesenchyme are crucial to the morphogenesis of oral mucosa. Previous work in oral mucosa regeneration has yielded important clues for several meritorious proof-of-concept approaches. Tissue engineering offers a broad array of novel tools for oral mucosa regeneration with reduced donor site trauma and accelerated clinical translation. However, the developmental concept of epithelial–mesenchymal interactions (EMIs) is rarely considered in oral mucosa regeneration. EMIs in postnatal oral mucosa regeneration likely will not be a simple recapitulation of prenatal oral mucosa development. Biomaterial scaffolds play an indispensible role for oral mucosa regeneration and should provide a conducive environment for pivotal EMIs. Autocrine and paracrine factors, either exogenously delivered or innately produced, have rarely been and should be harnessed to promote oral mucosa regeneration. This review focuses on a working concept of epithelial and mesenchymal interactions in oral mucosa regeneration. PMID:21062224

  17. Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish

    PubMed Central

    Mokalled, Mayssa H.; Patra, Chinmoy; Dickson, Amy L.; Endo, Toyokazu; Stainier, Didier Y. R.; Poss, Kenneth D.

    2016-01-01

    Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration, a relatively unexplored process. Here, we performed a genome-wide profiling screen for secreted factors that are upregulated during zebrafish spinal cord regeneration. We find that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupt spinal cord repair, while transgenic ctgfa overexpression and local human CTGF recombinant protein delivery accelerate bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration. PMID:27811277

  18. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation.

    PubMed

    Diez, Héctor; Benitez, Ma José; Fernandez, Silvia; Torres-Aleman, Ignacio; Garrido, Juan José; Wandosell, Francisco

    2016-11-01

    PI3K proteins family have multiple and essential functions in most cellular events. This family is composed of class I, class II and class III PI3Ks, which upstream and downstream elements are not completely elucidated. Previous studies using the broad PI3K inhibitor, LY294002 allowed to propose that PI3 kinase>Akt pathway is a key element in the determination of axonal polarity in hippocampal neurons. Recently, new inhibitors with a higher selectivity for class I PI3K have been characterized. In the present study we have examined this widely accepted theory using a new class I PI3K inhibitor (GDC-0941), as well as Akt inhibitors, and PTEN phosphatase constructs to reduce PIP3 levels. Our present data show that both, class I PI3K inhibitor and Akt inhibitor did not alter axon specification in hippocampal neurons, but greatly reduced axon length. However, in the same experiments LY294002 effectively impeded axonal polarization, as previously reported. Our biochemical data show that both, class I PI3K and Akt inhibitors, effectively block downstream elements from Akt to S6K1 activity. Both inhibitors are stable in culture medium along the time period analysed, maintaining the inhibition better than LY294002. Besides, we found evidence that LY294002 directly inhibits mTORC1. However, further analysis using an mTORC1 inhibitor showed no change in neuron polarity. Same result was obtained using a general class III PI3K inhibitor. Interestingly, we found that either, wild-type PTEN, or a phosphatase-dead form of PTEN, disrupted axonal polarization, strongly suggesting that the role of PTEN in axonal polarity can be independent of PIP3. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Enhancing nerve regeneration in the peripheral nervous system using polymeric scaffolds, stem cell engineering and nanoparticle delivery system

    NASA Astrophysics Data System (ADS)

    Sharma, Anup Dutt

    Peripheral nerve regeneration is a complex biological process responsible for regrowth of neural tissue following a nerve injury. The main objective of this project was to enhance peripheral nerve regeneration using interdisciplinary approaches involving polymeric scaffolds, stem cell therapy, drug delivery and high content screening. Biocompatible and biodegradable polymeric materials such as poly (lactic acid) were used for engineering conduits with micropatterns capable of providing mechanical support and orientation to the regenerating axons and polyanhydrides for fabricating nano/microparticles for localized delivery of neurotrophic growth factors and cytokines at the site of injury. Transdifferentiated bone marrow stromal cells or mesenchymal stem cells (MSCs) were used as cellular replacements for lost native Schwann cells (SCs) at the injured nerve tissue. MSCs that have been transdifferentiated into an SC-like phenotype were tested as a substitute for the myelinating SCs. Also, genetically modified MSCs were engineered to hypersecrete brain- derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) to secrete therapeutic factors which Schwann cell secrete. To further enhance the regeneration, nerve growth factor (NGF) and interleukin-4 (IL4) releasing polyanhydrides nano/microparticles were fabricated and characterized in vitro for their efficacy. Synergistic use of these proposed techniques was used for fabricating a multifunctional nerve regeneration conduit which can be used as an efficient tool for enhancing peripheral nerve regeneration.

  20. Defining a mechanistic link between pigment epithelium-derived factor, docosahexaenoic acid, and corneal nerve regeneration.

    PubMed

    Pham, Thang Luong; He, Jiucheng; Kakazu, Azucena H; Jun, Bokkyoo; Bazan, Nicolas G; Bazan, Haydee E P

    2017-11-10

    The cornea is densely innervated to sustain the integrity of the ocular surface. Corneal nerve damage produced by aging, diabetes, refractive surgeries, and viral or bacterial infections impairs tear production, the blinking reflex, and epithelial wound healing, resulting in loss of transparency and vision. A combination of the known neuroprotective molecule, pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA), has been shown to stimulate corneal nerve regeneration, but the mechanisms involved are unclear. Here, we sought to define the molecular events of this effect in an in vivo mouse injury model. We first confirmed that PEDF + DHA increased nerve regeneration in the mouse cornea. Treatment with PEDF activates the phospholipase A 2 activity of the PEDF-receptor (PEDF-R) leading to the release of DHA; this free DHA led to enhanced docosanoid synthesis and induction of bdnf, ngf , and the axon growth promoter semaphorin 7a ( sema7a ), and as a consequence, their products appeared in the mouse tears. Surprisingly, corneal injury and treatment with PEDF + DHA induced transcription of neuropeptide y ( npy ), small proline-rich protein 1a ( sprr1a ), and vasoactive intestinal peptide ( vip ) in the trigeminal ganglia (TG). The PEDF-R inhibitor, atglistatin, blocked all of these changes in the cornea and TG. In conclusion, we uncovered here an active cornea-TG axis, driven by PEDF-R activation, that fosters axon outgrowth in the cornea. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Identification of a Peripheral Nerve Neurite Growth-Promoting Activity by Development and Use of an in vitro Bioassay

    NASA Astrophysics Data System (ADS)

    Sandrock, Alfred W.; Matthew, William D.

    1987-10-01

    The effective regeneration of severed neuronal axons in the peripheral nerves of adult mammals may be explained by the presence of molecules in situ that promote the effective elongation of neurites. The absence of such molecules in the central nervous system of these animals may underlie the relative inability of axons to regenerate in this tissue after injury. In an effort to identify neurite growth-promoting molecules in tissues that support effective axonal regeneration, we have developed an in vitro bioassay that is sensitive to substrate-bound factors of peripheral nerve that influence the growth of neurites. In this assay, neonatal rat superior cervical ganglion explants are placed on longitudinal cryostat sections of fresh-frozen sciatic nerve, and the regrowing axons are visualized by catecholamine histofluorescence. Axons are found to regenerate effectively over sciatic nerve tissue sections. When ganglia are similarly explanted onto cryostat sections of adult rat central nervous system tissue, however, axonal regeneration is virtually absent. We have begun to identify the molecules in peripheral nerve that promote effective axonal regeneration by examining the effect of antibodies that interfere with the activity of previously described neurite growth-promoting factors. Axonal elongation over sciatic nerve tissue was found to be sensitive to the inhibitory effects of INO (for inhibitor of neurite outgrowth), a monoclonal antibody that recognizes and inhibits a neurite growth-promoting activity from PC-12 cell-conditioned medium. The INO antigen appears to be a molecular complex of laminin and heparan sulfate proteoglycan. In contrast, a rabbit antiserum that recognizes laminin purified from mouse Engelbreth-Holm-Swarm (EHS) sarcoma, stains the Schwann cell basal lamina of peripheral nerve, and inhibits neurite growth over purified laminin substrata has no detectable effect on the rate of axonal regeneration in our assay.

  2. Delivery of adipose-derived stem cells in poloxamer hydrogel improves peripheral nerve regeneration.

    PubMed

    Allbright, Kassandra O; Bliley, Jacqueline M; Havis, Emmanuelle; Kim, Deok-Yeol; Dibernardo, Gabriella A; Grybowski, Damian; Waldner, Matthias; James, Isaac B; Sivak, Wesley N; Rubin, J Peter; Marra, Kacey G

    2018-02-06

    Peripheral nerve damage is associated with high long-term morbidity. Because of beneficial secretome, immunomodulatory effects, and ease of clinical translation, transplantation with adipose-derived stem cells (ASC) represents a promising therapeutic modality. Effect of ASC delivery in poloxamer hydrogel was assessed in a rat sciatic nerve model of critical-sized (1.5 cm) peripheral nerve injury. Nerve/muscle unit regeneration was assessed via immunostaining explanted nerve, quantitative polymerase chain reaction (qPCR), and histological analysis of reinnervating gastrocnemius muscle. On the basis of viability data, 10% poloxamer hydrogel was selected for in vivo study. Six weeks after transection and repair, the group treated with poloxamer delivered ASCs demonstrated longest axonal regrowth. The qPCR results indicated that the inclusion of ASCs appeared to result in expression of factors that aid in reinnervating muscle tissue. Delivery of ASCs in poloxamer addresses multiple facets of the complexity of nerve/muscle unit regeneration, representing a promising avenue for further study. Muscle Nerve, 2018. © 2018 Wiley Periodicals, Inc.

  3. Neural stem cells promote nerve regeneration through IL12-induced Schwann cell differentiation.

    PubMed

    Lee, Don-Ching; Chen, Jong-Hang; Hsu, Tai-Yu; Chang, Li-Hsun; Chang, Hsu; Chi, Ya-Hui; Chiu, Ing-Ming

    2017-03-01

    Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6-fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5-fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. The effect of aging on efferent nerve fibers regeneration in mice.

    PubMed

    Verdú, E; Butí, M; Navarro, X

    1995-10-23

    This study evaluates the influence of aging on nerve regeneration and reinnervation of target organs in mice aged 2, 6, 9, 12, 18 and 24 months. In animals of each age group the sciatic nerve was subjected to crush, section or section and suture. Reinnervation of plantar muscles and sweat glands (SG) was evaluated over three months after operation by functional methods. Reappearance of SG secretion and motor responses occurred slightly earlier in young than older mice. The degree of motor and sudomotor reinnervation, with respect to preoperative control values, was also significantly higher in young than old animals. The differences were more pronounced after 12 months of age. The degree of recovery progressively decreased with the severity of the lesion, differences being more marked in older mice. Neurorraphy improved recovery, comparatively more in older than in young mice. These results indicate that, after injuries of peripheral nerves, axonal regeneration and reinnervation are maintained throughout life, but tend to be more delayed and slightly less effective with aging.

  5. Clinical progression in Parkinson disease and the neurobiology of axons.

    PubMed

    Cheng, Hsiao-Chun; Ulane, Christina M; Burke, Robert E

    2010-06-01

    Despite tremendous growth in recent years in our knowledge of the molecular basis of Parkinson disease (PD) and the molecular pathways of cell injury and death, we remain without therapies that forestall disease progression. Although there are many possible explanations for this lack of success, one is that experimental therapeutics to date have not adequately focused on an important component of the disease process, that of axon degeneration. It remains unknown what neuronal compartment, either the soma or the axon, is involved at disease onset, although some have proposed that it is the axons and their terminals that take the initial brunt of injury. Nevertheless, this concept has not been formally incorporated into many of the current theories of disease pathogenesis, and it has not achieved a wide consensus. More importantly, in view of growing evidence that the molecular mechanisms of axon degeneration are separate and distinct from the canonical pathways of programmed cell death that mediate soma destruction, the possibility of early involvement of axons in PD has not been adequately emphasized as a rationale to explore the neurobiology of axons for novel therapeutic targets. We propose that ongoing degeneration of axons, not cell bodies, is the primary determinant of clinically apparent progression of disease, and that future experimental therapeutics intended to forestall disease progression will benefit from a new focus on the distinct mechanisms of axon degeneration.

  6. Glypican Is a Modulator of Netrin-Mediated Axon Guidance

    PubMed Central

    Blanchette, Cassandra R.; Perrat, Paola N.; Thackeray, Andrea; Bénard, Claire Y.

    2015-01-01

    Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration. PMID:26148345

  7. At the interface: convergence of neural regeneration and neural prostheses for restoration of function.

    PubMed

    Grill, W M; McDonald, J W; Peckham, P H; Heetderks, W; Kocsis, J; Weinrich, M

    2001-01-01

    The rapid pace of recent advances in development and application of electrical stimulation of the nervous system and in neural regeneration has created opportunities to combine these two approaches to restoration of function. This paper relates the discussion on this topic from a workshop at the International Functional Electrical Stimulation Society. The goals of this workshop were to discuss the current state of interaction between the fields of neural regeneration and neural prostheses and to identify potential areas of future research that would have the greatest impact on achieving the common goal of restoring function after neurological damage. Identified areas include enhancement of axonal regeneration with applied electric fields, development of hybrid neural interfaces combining synthetic silicon and biologically derived elements, and investigation of the role of patterned neural activity in regulating various neuronal processes and neurorehabilitation. Increased communication and cooperation between the two communities and recognition by each field that the other has something to contribute to their efforts are needed to take advantage of these opportunities. In addition, creative grants combining the two approaches and more flexible funding mechanisms to support the convergence of their perspectives are necessary to achieve common objectives.

  8. Time course of ongoing activity during neuritis and following axonal transport disruption.

    PubMed

    Satkeviciute, Ieva; Goodwin, George; Bove, Geoffrey M; Dilley, Andrew

    2018-05-01

    Local nerve inflammation (neuritis) leads to ongoing activity and axonal mechanical sensitivity (AMS) along intact nociceptor axons and disrupts axonal transport. This phenomenon forms the most feasible cause of radiating pain, such as sciatica. We have previously shown that axonal transport disruption without inflammation or degeneration also leads to AMS but does not cause ongoing activity at the time point when AMS occurs, despite causing cutaneous hypersensitivity. However, there have been no systematic studies of ongoing activity during neuritis or noninflammatory axonal transport disruption. In this study, we present the time course of ongoing activity from primary sensory neurons following neuritis and vinblastine-induced axonal transport disruption. Whereas 24% of C/slow Aδ-fiber neurons had ongoing activity during neuritis, few (<10%) A- and C-fiber neurons showed ongoing activity 1-15 days following vinblastine treatment. In contrast, AMS increased transiently at the vinblastine treatment site, peaking on days 4-5 (28% of C/slow Aδ-fiber neurons) and resolved by day 15. Conduction velocities were slowed in all groups. In summary, the disruption of axonal transport without inflammation does not lead to ongoing activity in sensory neurons, including nociceptors, but does cause a rapid and transient development of AMS. Because it is proposed that AMS underlies mechanically induced radiating pain, and a transient disruption of axonal transport (as previously reported) leads to transient AMS, it follows that processes that disrupt axonal transport, such as neuritis, must persist to maintain AMS and the associated symptoms. NEW & NOTEWORTHY Many patients with radiating pain lack signs of nerve injury on clinical examination but may have neuritis, which disrupts axonal transport. We have shown that axonal transport disruption does not induce ongoing activity in primary sensory neurons but does cause transient axonal mechanical sensitivity. The present data

  9. Netrin-1 attracts axons through FAK-dependent mechanotransduction.

    PubMed

    Moore, Simon W; Zhang, Xian; Lynch, Christopher D; Sheetz, Michael P

    2012-08-22

    The mechanism by which extracellular cues influence intracellular biochemical cascades that guide axons is important, yet poorly understood. Because of the mechanical nature of axon extension, we explored whether the physical interactions of growth cones with their guidance cues might be involved. In the context of mouse spinal commissural neuron axon attraction to netrin-1, we found that mechanical attachment of netrin-1 to the substrate was required for axon outgrowth, growth cone expansion, axon attraction and phosphorylation of focal adhesion kinase (FAK) and Crk-associated substrate (CAS). Myosin II activity was necessary for traction forces >30 pN on netrin-1. Interestingly, while these myosin II-dependent forces on netrin-1 substrates or beads were needed to increase the kinase activity and phosphorylation of FAK, they were not necessary for netrin-1 to increase CAS phosphorylation. When FAK kinase activity was inhibited, the growth cone's ability to recruit additional adhesions and to generate forces >60 pN on netrin-1 was disrupted. Together, these findings demonstrate an important role for mechanotransduction during chemoattraction to netrin-1 and that mechanical activation of FAK reinforces interactions with netrin-1 allowing greater forces to be exerted.

  10. Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology

    PubMed Central

    Gowrishankar, Swetha; Wu, Yumei

    2017-01-01

    Lysosomes robustly accumulate within axonal swellings at Alzheimer’s disease (AD) amyloid plaques. However, the underlying mechanisms and disease relevance of such lysosome accumulations are not well understood. Motivated by these problems, we identified JNK-interacting protein 3 (JIP3) as an important regulator of axonal lysosome transport and maturation. JIP3 knockout mouse neuron primary cultures accumulate lysosomes within focal axonal swellings that resemble the dystrophic axons at amyloid plaques. These swellings contain high levels of amyloid precursor protein processing enzymes (BACE1 and presenilin 2) and are accompanied by elevated Aβ peptide levels. The in vivo importance of the JIP3-dependent regulation of axonal lysosomes was revealed by the worsening of the amyloid plaque pathology arising from JIP3 haploinsufficiency in a mouse model of AD. These results establish the critical role of JIP3-dependent axonal lysosome transport in regulating amyloidogenic amyloid precursor protein processing and support a model wherein Aβ production is amplified by plaque-induced axonal lysosome transport defects. PMID:28784610

  11. TRANSVERSE ELECTRIC IMPEDANCE OF THE SQUID GIANT AXON

    PubMed Central

    Curtis, Howard J.; Cole, Kenneth S.

    1938-01-01

    The impedance of the excised giant axon from hindmost stellar nerve of Loligo pealii has been measured over the frequency range from 1 to 2500 kilocycles per second. The measurements have been made with the current flow perpendicular to the axis of the axon to permit a relatively simple analysis of the data. It has been found that the axon membrane has a polarization impedance with an average phase angle of 76° and an average capacity of 1.1µf./cm2 at 1 kilocycle. The direct current resistance of the membrane could not be measured, but was greater than 3 ohm cm.2 and the average internal specific resistance was four times that of sea water. There was no detectable change in the membrane impedance when the axon lost excitability, but some time later it decreased to zero. PMID:19873081

  12. Axonal degeneration in Alzheimer’s disease: When signaling abnormalities meet the axonal transport system

    PubMed Central

    Kanaan, Nicholas M.; Pigino, Gustavo F.; Brady, Scott T.; Lazarov, Orly; Binder, Lester I.; Morfini, Gerardo A.

    2012-01-01

    Alzheimer’s disease (AD) is characterized by progressive, age-dependent degeneration of neurons in the central nervous system. A large body of evidence indicates that neurons affected in AD follow a dying-back pattern of degeneration, where abnormalities in synaptic function and axonal connectivity long precede somatic cell death. Mechanisms underlying dying-back degeneration of neurons in AD remain elusive but several have been proposed, including deficits in fast axonal transport (FAT). Accordingly, genetic evidence linked alterations in FAT to dying-back degeneration of neurons, and FAT defects have been widely documented in various AD models. In light of these findings, we discuss experimental evidence linking several AD-related pathogenic polypeptides to aberrant activation of signaling pathways involved in the phosphoregulation of microtubule-based motor proteins. While each pathway appears to affect FAT in a unique manner, in the context of AD, many of these pathways might work synergistically to compromise the delivery of molecular components critical for the maintenance and function of synapses and axons. Therapeutic approaches aimed at preventing FAT deficits by normalizing the activity of specific protein kinases may help prevent degeneration of vulnerable neurons in AD. PMID:22721767

  13. End-to-side neurorrhaphy repairs peripheral nerve injury: sensory nerve induces motor nerve regeneration.

    PubMed

    Yu, Qing; Zhang, She-Hong; Wang, Tao; Peng, Feng; Han, Dong; Gu, Yu-Dong

    2017-10-01

    End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve. It involves suturing the distal stump of the disconnected nerve (recipient nerve) to the side of the intimate adjacent nerve (donor nerve). However, the motor-sensory specificity after end-to-side neurorrhaphy remains unclear. This study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves after end-to-side neurorrhaphy. Thirty rats were randomized into three groups: (1) end-to-side neurorrhaphy using the ulnar nerve (mixed sensory and motor) as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve; (2) the sham group: ulnar nerve and cutaneous antebrachii medialis nerve were just exposed; and (3) the transected nerve group: cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied. At 5 months, acetylcholinesterase staining results showed that 34% ± 16% of the myelinated axons were stained in the end-to-side group, and none of the myelinated axons were stained in either the sham or transected nerve groups. Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%. In contrast, no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment. These results confirmed that motor neuron regeneration occurred after cutaneous nerve end-to-side neurorrhaphy.

  14. End-to-side neurorrhaphy repairs peripheral nerve injury: sensory nerve induces motor nerve regeneration

    PubMed Central

    Yu, Qing; Zhang, She-hong; Wang, Tao; Peng, Feng; Han, Dong; Gu, Yu-dong

    2017-01-01

    End-to-side neurorrhaphy is an option in the treatment of the long segment defects of a nerve. It involves suturing the distal stump of the disconnected nerve (recipient nerve) to the side of the intimate adjacent nerve (donor nerve). However, the motor-sensory specificity after end-to-side neurorrhaphy remains unclear. This study sought to evaluate whether cutaneous sensory nerve regeneration induces motor nerves after end-to-side neurorrhaphy. Thirty rats were randomized into three groups: (1) end-to-side neurorrhaphy using the ulnar nerve (mixed sensory and motor) as the donor nerve and the cutaneous antebrachii medialis nerve as the recipient nerve; (2) the sham group: ulnar nerve and cutaneous antebrachii medialis nerve were just exposed; and (3) the transected nerve group: cutaneous antebrachii medialis nerve was transected and the stumps were turned over and tied. At 5 months, acetylcholinesterase staining results showed that 34% ± 16% of the myelinated axons were stained in the end-to-side group, and none of the myelinated axons were stained in either the sham or transected nerve groups. Retrograde fluorescent tracing of spinal motor neurons and dorsal root ganglion showed the proportion of motor neurons from the cutaneous antebrachii medialis nerve of the end-to-side group was 21% ± 5%. In contrast, no motor neurons from the cutaneous antebrachii medialis nerve of the sham group and transected nerve group were found in the spinal cord segment. These results confirmed that motor neuron regeneration occurred after cutaneous nerve end-to-side neurorrhaphy. PMID:29171436

  15. Axon Transport and Neuropathy

    PubMed Central

    Tourtellotte, Warren G.

    2017-01-01

    Peripheral neuropathies are highly prevalent and are most often associated with chronic disease, side effects from chemotherapy, or toxic-metabolic abnormalities. Neuropathies are less commonly caused by genetic mutations, but studies of the normal function of mutated proteins have identified particular vulnerabilities that often implicate mitochondrial dynamics and axon transport mechanisms. Hereditary sensory and autonomic neuropathies are a group of phenotypically related diseases caused by monogenic mutations that primarily affect sympathetic and sensory neurons. Here, I review evidence to indicate that many genetic neuropathies are caused by abnormalities in axon transport. Moreover, in hereditary sensory and autonomic neuropathies. There may be specific convergence on gene mutations that disrupt nerve growth factor signaling, upon which sympathetic and sensory neurons critically depend. PMID:26724390

  16. Localization of mRNA in vertebrate axonal compartments by in situ hybridization.

    PubMed

    Sotelo-Silveira, José Roberto; Calliari, Aldo; Kun, Alejandra; Elizondo, Victoria; Canclini, Lucía; Sotelo, José Roberto

    2011-01-01

    The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

  17. An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

    PubMed

    Huang, Claire Yu-Mei; Zhang, Chuansheng; Zollinger, Daniel R; Leterrier, Christophe; Rasband, Matthew N

    2017-11-22

    Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with βIV and βII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1 f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K + channels. We show that the density of nodal βIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1 f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier. SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in

  18. Removal of sodium inactivation and block of sodium channels by chloramine-T in crayfish and squid giant axons.

    PubMed Central

    Huang, J. M.; Tanguy, J.; Yeh, J. Z.

    1987-01-01

    Modification of sodium channels by chloramine-T was examined in voltage clamped internally perfused crayfish and squid giant axons using the double sucrose gap and axial wire technique, respectively. Freshly prepared chloramine-T solution exerted two major actions on sodium channels: (a) an irreversible removal of the fast Na inactivation, and (b) a reversible block of the Na current. Both effects were observed when chloramine-T was applied internally or externally (5-10 mM) to axons. The first effect was studied in crayfish axons. We found that the removal of the fast Na inactivation did not depend on the states of the channel since the channel could be modified by chloramine-T at holding potential (from -80 to -100 mV) or at depolarized potential of -30 mV. After removal of fast Na inactivation, the slow inactivation mechanism was still present, and more channels could undergo slow inactivation. This result indicates that in crayfish axons the transition through the fast inactivated state is not a prerequisite for the slow inactivation to occur. During chloramine-T treatment, a distinct blocking phase occurred, which recovered upon washing out the drug. This second effect of chloramine-T was studied in detail in squid axons. After 24 h, chloramine-T solution lost its ability to remove fast inactivation but retained its blocking action. After removal of the fast Na inactivation, both fresh and aged chloramine-T solutions blocked the Na currents with a similar potency and in a voltage-dependent manner, being more pronounced at lower depolarizing potentials. A similar voltage-dependent block was observed with aged chloramine-T solution in an axon with intact inactivation. In contrast to the action of the fresh solution, the aged chloramine-T solution was found to accelerate the decay of Na currents.These results suggest that chloramine-T solution contains at least two active molecular forms that act at different sites in the Na channel. PMID:2444276

  19. Brain injury tolerance limit based on computation of axonal strain.

    PubMed

    Sahoo, Debasis; Deck, Caroline; Willinger, Rémy

    2016-07-01

    Traumatic brain injury (TBI) is the leading cause of death and permanent impairment over the last decades. In both the severe and mild TBIs, diffuse axonal injury (DAI) is the most common pathology and leads to axonal degeneration. Computation of axonal strain by using finite element head model in numerical simulation can enlighten the DAI mechanism and help to establish advanced head injury criteria. The main objective of this study is to develop a brain injury criterion based on computation of axonal strain. To achieve the objective a state-of-the-art finite element head model with enhanced brain and skull material laws, was used for numerical computation of real world head trauma. The implementation of new medical imaging data such as, fractional anisotropy and axonal fiber orientation from Diffusion Tensor Imaging (DTI) of 12 healthy patients into the finite element brain model was performed to improve the brain constitutive material law with more efficient heterogeneous anisotropic visco hyper-elastic material law. The brain behavior has been validated in terms of brain deformation against Hardy et al. (2001), Hardy et al. (2007), and in terms of brain pressure against Nahum et al. (1977) and Trosseille et al. (1992) experiments. Verification of model stability has been conducted as well. Further, 109 well-documented TBI cases were simulated and axonal strain computed to derive brain injury tolerance curve. Based on an in-depth statistical analysis of different intra-cerebral parameters (brain axonal strain rate, axonal strain, first principal strain, Von Mises strain, first principal stress, Von Mises stress, CSDM (0.10), CSDM (0.15) and CSDM (0.25)), it was shown that axonal strain was the most appropriate candidate parameter to predict DAI. The proposed brain injury tolerance limit for a 50% risk of DAI has been established at 14.65% of axonal strain. This study provides a key step for a realistic novel injury metric for DAI. Copyright © 2016 Elsevier Ltd

  20. Chlorpyrifos-Oxon Disrupts Zebrafish Axonal Growth and Motor Behavior

    PubMed Central

    Yang, Dongren; Lauridsen, Holly; Buels, Kalmia; Chi, Lai-Har; La Du, Jane; Bruun, Donald A.; Olson, James R.; Tanguay, Robert L.; Lein, Pamela J.

    2011-01-01

    Axonal morphology is a critical determinant of neuronal connectivity, and perturbation of the rate or extent of axonal growth during development has been linked to neurobehavioral deficits in animal models and humans. We previously demonstrated that the organophosphorus pesticide (OP) chlorpyrifos (CPF) inhibits axonal growth in cultured neurons. In this study, we used a zebrafish model to determine whether CPF, its oxon metabolite (CPFO), or the excreted metabolite trichloro-2-pyridinol (TCPy) alter spatiotemporal patterns of axonal growth in vivo. Static waterborne exposure to CPFO, but not CPF or TCPy, at concentrations ≥ 0.03μM from 24- to 72-h post fertilization significantly inhibited acetylcholinesterase, and high-performance liquid chromatography detected significantly more TCPy in zebrafish exposed to 0.1μM CPFO versus 1.0μM CPF. These data suggest that zebrafish lack the metabolic enzymes to activate CPF during these early developmental stages. Consistent with this, CPFO, but not CPF, significantly inhibited axonal growth of sensory neurons, primary motoneurons, and secondary motoneurons at concentrations ≥ 0.1μM. Secondary motoneurons were the most sensitive to axonal growth inhibition by CPFO, which was observed at concentrations that did not cause mortality, gross developmental defects, or aberrant somatic muscle differentiation. CPFO effects on axonal growth correlated with adverse effects on touch-induced swimming behavior, suggesting the functional relevance of these structural changes. These data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of CPF and demonstrate the relevance of zebrafish as a model for studying OP developmental neurotoxicity. PMID:21346248

  1. Rheological and Performance Research on a Regenerable Polyvinyl Alcohol Fracturing Fluid

    PubMed Central

    Shang, Xiaosen; Ding, Yunhong; Wang, Yonghui; Yang, Lifeng

    2015-01-01

    A regenerable polyvinyl alcohol/organic boron fracturing fluid system with 1.6 wt% polyvinyl alcohol (PVOH) and 1.2 wt% organic boron (OBT) was studied, and its main regeneration mechanism is the reversible cross-linking reaction between B(OH)4 - and hydroxyl groups of PVOH as the change of pH. Results of rheology evaluations show that both the apparent viscosity and the thermal stability of the fracturing fluid decreased with the regeneration number of times increasing. In addition, the apparent viscosity of the fluid which was without regeneration was more sensitive to the shear action compared with that of the fluid with regeneration once or twice. When the fracturing fluid was without regeneration, the elasticity was dominating due to the three-dimensional network structure of the formed gel; the viscosity gradually occupied the advantage when the fracturing fluid was regenerated once or twice. The settling velocity of proppant was accelerated by both the regeneration process and the increasing temperature, but it was decelerated when the proppant ratio increased. Results of core damage tests indicate that less permeability damage was caused by the PVOH/OBT fracturing fluid compared with that caused by the guar gum fracturing fluid after gel breaking. PMID:26641857

  2. Rheological and Performance Research on a Regenerable Polyvinyl Alcohol Fracturing Fluid.

    PubMed

    Shang, Xiaosen; Ding, Yunhong; Wang, Yonghui; Yang, Lifeng

    2015-01-01

    A regenerable polyvinyl alcohol/organic boron fracturing fluid system with 1.6 wt% polyvinyl alcohol (PVOH) and 1.2 wt% organic boron (OBT) was studied, and its main regeneration mechanism is the reversible cross-linking reaction between B(OH)4- and hydroxyl groups of PVOH as the change of pH. Results of rheology evaluations show that both the apparent viscosity and the thermal stability of the fracturing fluid decreased with the regeneration number of times increasing. In addition, the apparent viscosity of the fluid which was without regeneration was more sensitive to the shear action compared with that of the fluid with regeneration once or twice. When the fracturing fluid was without regeneration, the elasticity was dominating due to the three-dimensional network structure of the formed gel; the viscosity gradually occupied the advantage when the fracturing fluid was regenerated once or twice. The settling velocity of proppant was accelerated by both the regeneration process and the increasing temperature, but it was decelerated when the proppant ratio increased. Results of core damage tests indicate that less permeability damage was caused by the PVOH/OBT fracturing fluid compared with that caused by the guar gum fracturing fluid after gel breaking.

  3. Mechanosensitivity in axon growth and guidance

    NASA Astrophysics Data System (ADS)

    Urbach, Jeff

    2013-03-01

    In the developing nervous system, axons respond to a diverse array of cues to generate the intricate connection network required for proper function. The growth cone, a highly motile structure at the tip of a growing axon, integrates information about the local environment and modulates outgrowth and guidance, but little is known about effects of external mechanical cues and internal mechanical forces on growth cone behavior. We have investigated axon outgrowth and force generation on soft elastic substrates for dorsal root ganglion (DRG) neurons (from the peripheral nervous system) and hippocampal neurons (from the central) to see how the mechanics of the microenvironment affect different populations. We find that force generation and stiffness-dependent outgrowth are strongly dependent on cell type. We also observe very different internal dynamics and substrate coupling in the two populations, suggesting that the difference in force generation is due to stronger adhesions and therefore stronger substrate engagement in the peripheral nervous system neurons. We will discuss the biological origins of these differences, and recent analyses of the dynamic aspects of growth cone force generation and the implications for the role of mechanosensitivity in axon guidance. In collaboration with D. Koch, W. Rosoff, and H. M. Geller. Supported by NINDS grant 1R01NS064250-01 (J.S.U.) and the NHLBI Intramural Research Program (H.M.G.).

  4. A Communication Theoretical Modeling of Axonal Propagation in Hippocampal Pyramidal Neurons.

    PubMed

    Ramezani, Hamideh; Akan, Ozgur B

    2017-06-01

    Understanding the fundamentals of communication among neurons, known as neuro-spike communication, leads to reach bio-inspired nanoscale communication paradigms. In this paper, we focus on a part of neuro-spike communication, known as axonal transmission, and propose a realistic model for it. The shape of the spike during axonal transmission varies according to previously applied stimulations to the neuron, and these variations affect the amount of information communicated between neurons. Hence, to reach an accurate model for neuro-spike communication, the memory of axon and its effect on the axonal transmission should be considered, which are not studied in the existing literature. In this paper, we extract the important factors on the memory of axon and define memory states based on these factors. We also describe the transition among these states and the properties of axonal transmission in each of them. Finally, we demonstrate that the proposed model can follow changes in the axonal functionality properly by simulating the proposed model and reporting the root mean square error between simulation results and experimental data.

  5. In vivo testing of a 3D bifurcating microchannel scaffold inducing separation of regenerating axon bundles in peripheral nerves

    NASA Astrophysics Data System (ADS)

    Stoyanova, Irina I.; van Wezel, Richard J. A.; Rutten, Wim L. C.

    2013-12-01

    Artificial nerve guidance channels enhance the regenerative effectiveness in an injured peripheral nerve but the existing design so far has been limited to basic straight tubes simply guiding the growth to bridge the gap. Hence, one of the goals in development of more effective neuroprostheses is to create bidirectional highly selective neuro-electronic interface between a prosthetic device and the severed nerve. A step towards improving selectivity for both recording and stimulation have been made with some recent in vitro studies which showed that three-dimensional (3D) bifurcating microchannels can separate neurites growing on a planar surface and bring them into contact with individual electrodes. Since the growing axons in vivo have the innate tendency to group in bundles surrounded by connective tissue, one of the big challenges in neuro-prosthetic interface design is how to overcome it. Therefore, we performed experiments with 3D bifurcating guidance scaffolds implanted in the sciatic nerve of rats to test if this new channel architecture could trigger separation pattern of ingrowth also in vivo. Our results showed that this new method enabled the re-growth of neurites into channels with gradually diminished width (80, 40 and 20 µm) and facilitated the separation of the axonal bundles with 91% success. It seems that the 3D bifurcating scaffold might contribute towards conveying detailed neural control and sensory feedback to users of prosthetic devices, and thus could improve the quality of their daily life.

  6. Perspectives on stem cell therapy for cardiac regeneration. Advances and challenges.

    PubMed

    Choi, Sung Hyun; Jung, Seok Yun; Kwon, Sang-Mo; Baek, Sang Hong

    2012-01-01

    Ischemic heart disease (IHD) accelerates cardiomyocyte loss, but the developing stem cell research could be useful for regenerating a variety of tissue cells, including cardiomyocytes. Diverse sources of stem cells for IHD have been reported, including embryonic stem cells, induced pluripotent stem cells, skeletal myoblasts, bone marrow-derived stem cells, mesenchymal stem cells, and cardiac stem cells. However, stem cells have unique advantages and disadvantages for cardiac tissue regeneration, which are important considerations in determining the specific cells for improving cell survival and long-term engraftment after transplantation. Additionally, the dosage and administration method of stem cells need to be standardized to increase stability and efficacy for clinical applications. Accordingly, this review presents a summary of the stem cell therapies that have been studied for cardiac regeneration thus far, and discusses the direction of future cardiac regeneration research for stem cells.

  7. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  8. Regeneration of axotomized olfactory neurons in young and adult locusts quantified by fasciclin I immunofluorescence.

    PubMed

    Wasser, Hannah; Biller, Alexandra; Antonopoulos, Georgios; Meyer, Heiko; Bicker, Gerd; Stern, Michael

    2017-04-01

    The olfactory pathway of the locust Locusta migratoria is characterized by a multiglomerular innervation of the antennal lobe (AL) by olfactory receptor neurons (ORNs). After crushing the antenna and thereby severing ORN axons, changes in the AL were monitored. First, volume changes were measured at different times post-crush with scanning laser optical tomography in 5th instar nymphs. AL volume decreased significantly to a minimum volume at 4 days post-crush, followed by an increase. Second, anterograde labeling was used to visualize details in the AL and antennal nerve (AN) during de- and regeneration. Within 24 h post-crush (hpc) the ORN fragments distal to the lesion degenerated. After 48 hpc, regenerating fibers grew through the crush site. In the AL, labeled ORN projections disappeared completely and reappeared after a few days. A weak topographic match between ORN origin on the antenna and the position of innervated glomeruli that was present in untreated controls did not reappear after regeneration. Third, the cell surface marker fasciclin I that is expressed in ORNs was used for quantifying purposes. Immunofluorescence was measured in the AL during de- and regeneration in adults and 5th instar nymphs: after a rapid but transient, decrease, it reappeared. Both processes happen faster in 5th instar nymphs than in adults.

  9. A model of axonal transport drug delivery

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Andrey V.

    2012-04-01

    In this paper a model of targeted drug delivery by means of active (motor-driven) axonal transport is developed. The model is motivated by recent experimental research by Filler et al. (A.G. Filler, G.T. Whiteside, M. Bacon, M. Frederickson, F.A. Howe, M.D. Rabinowitz, A.J. Sokoloff, T.W. Deacon, C. Abell, R. Munglani, J.R. Griffiths, B.A. Bell, A.M.L. Lever, Tri-partite complex for axonal transport drug delivery achieves pharmacological effect, Bmc Neuroscience 11 (2010) 8) that reported synthesis and pharmacological efficiency tests of a tri-partite complex designed for axonal transport drug delivery. The developed model accounts for two populations of pharmaceutical agent complexes (PACs): PACs that are transported retrogradely by dynein motors and PACs that are accumulated in the axon at the Nodes of Ranvier. The transitions between these two populations of PACs are described by first-order reactions. An analytical solution of the coupled system of transient equations describing conservations of these two populations of PACs is obtained by using Laplace transform. Numerical results for various combinations of parameter values are presented and their physical significance is discussed.

  10. Dynamics of terminal arbor formation and target approach of retinotectal axons in living zebrafish embryos: a time-lapse study of single axons.

    PubMed

    Kaethner, R J; Stuermer, C A

    1992-08-01

    In a variety of species, developing retinal axons branch initially more widely in their visual target centers and only gradually restrict their terminal arbors to smaller and defined territories. Retinotectal axons in fish, however, appeared to grow in a directed manner and to arborize only at their retinotopic target sites. To visualize the dynamics of retinal axon growth and arbor formation in fish, time-lapse recordings were made of individual retinal ganglion cell axons in the tectum in live zebrafish embryos. Axons were labeled with the fluorescent carbocyanine dyes Dil or DiO inserted as crystals into defined regions of the retina, viewed with 40x and 100x objectives with an SIT camera, and recorded, with exposure times of 200 msec at 30 or 60 sec intervals, over time periods of up to 13 hr. (1) Growth cones advanced rapidly, but the advance was punctuated by periods of rest. During the rest periods, the growth cones broadened and developed filopodia, but during extension they were more streamlined. (2) Growth cones traveled unerringly into the direction of their retinotopic targets without branching en route. At their target and only there, the axons began to form terminal arborizations, a process that involved the emission and retraction of numerous short side branches. The area that was permanently occupied or touched by transient branches of the terminal arbor--"the exploration field"--was small and almost circular and covered not more than 5.3% of the entire tectal surface area, but represented up to six times the size of the arbor at any one time. These findings are consistent with the idea that retinal axons are guided to their retinotopic target sites by sets of positional markers, with a graded distribution over the axes of the tectum.

  11. Pharmacogenetic stimulation of neuronal activity increases myelination in an axon-specific manner.

    PubMed

    Mitew, Stanislaw; Gobius, Ilan; Fenlon, Laura R; McDougall, Stuart J; Hawkes, David; Xing, Yao Lulu; Bujalka, Helena; Gundlach, Andrew L; Richards, Linda J; Kilpatrick, Trevor J; Merson, Tobias D; Emery, Ben

    2018-01-22

    Mounting evidence suggests that neuronal activity influences myelination, potentially allowing for experience-driven modulation of neural circuitry. The degree to which neuronal activity is capable of regulating myelination at the individual axon level is unclear. Here we demonstrate that stimulation of somatosensory axons in the mouse brain increases proliferation and differentiation of oligodendrocyte progenitor cells (OPCs) within the underlying white matter. Stimulated axons display an increased probability of being myelinated compared to neighboring non-stimulated axons, in addition to being ensheathed with thicker myelin. Conversely, attenuating neuronal firing reduces axonal myelination in a selective activity-dependent manner. Our findings reveal that the process of selecting axons for myelination is strongly influenced by the relative activity of individual axons within a population. These observed cellular changes are consistent with the emerging concept that adaptive myelination is a key mechanism for the fine-tuning of neuronal circuitry in the mammalian CNS.

  12. Age related optic nerve axonal loss in adult Brown Norway rats.

    PubMed

    Cepurna, William O; Kayton, Robert J; Johnson, Elaine C; Morrison, John C

    2005-06-01

    The effect of age on the number and morphology of optic nerve axons in adult Brown Norway rats (5-31 months old) (n=29) was examined using transmission electron microscopy (TEM). By manually counting every axon in areas representing 60% of the optic nerve cross-section, we found a significant negative correlation between age and axon count (R(2)=0.18, P<0.05). However, when the oldest animals were omitted, the relationship was no longer statistically significant. Simultaneously, the proportion of spontaneously degenerating axons increased at an exponential rate (R(2)=0.79, P<0.05), with significantly more degeneration in the 31-month group than in 5-month-old animals (ANOVA, P<0.05). This study demonstrates, using quantitative TEM methods, that optic nerve axonal numbers are relatively constant throughout the majority of the adult life of the Brown Norway rat, an increasingly popular strain for glaucoma research. Total axonal loss with aging is substantially less than that reported for other strains. The reduction in axonal numbers and the rate of axonal degeneration do not appear significantly altered until the last few months of life, failing to support some studies that have concluded that optic nerve axon loss in adult rats is linear. However, they do agree with other studies in the rat, and a similar study performed in non-human primate eyes, that concluded that aging changes in the optic nerve and retina follow a complex pattern. Therefore, the impact of animal age must be considered when modeling the course and pathophysiology of experimental glaucomatous optic nerve damage in rats.

  13. Axon Response to Guidance Cues Is Stimulated by Acetylcholine in Caenorhabditis elegans

    PubMed Central

    Xu, Yan; Ren, Xing-Cong; Quinn, Christopher C.; Wadsworth, William G.

    2011-01-01

    Gradients of acetylcholine can stimulate growth cone turning when applied to neurons grown in culture, and it has been suggested that acetylcholine could act as a guidance cue. However, the role acetylcholine plays in directing axon migrations in vivo is not clear. Here, we show that acetylcholine positively regulates signaling pathways that mediate axon responses to guidance cues in Caenorhabditis elegans. Mutations that disrupt acetylcholine synthesis, transportation, and secretion affect circumferential axon guidance of the AVM neuron and in these mutants exogenously supplied acetylcholine improves AVM circumferential axon guidance. These effects are not observed for the circumferential guidance of the DD and VD motor neuron axons, which are neighbors of the AVM axon. Circumferential guidance is directed by the UNC-6 (netrin) and SLT-1 (slit) extracellular cues, and exogenously supplied acetylcholine can improve AVM axon guidance in mutants when either UNC-6– or SLT-1–induced signaling is disrupted, but not when both signaling pathways are perturbed. Not in any of the mutants does exogenously supplied acetylcholine improve DD and VD axon guidance. The ability of acetylcholine to enhance AVM axon guidance only in the presence of either UNC-6 or SLT-1 indicates that acetylcholine potentiates UNC-6 and SLT-1 guidance activity, rather than acting itself as a guidance cue. Together, our results show that for specific neurons acetylcholine plays an important role in vivo as a modulator of axon responses to guidance cues. PMID:21868605

  14. The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea

    PubMed Central

    Sasidharan, Vidyanand; Marepally, Srujan; Elliott, Sarah A.; Baid, Srishti; Lakshmanan, Vairavan; Nayyar, Nishtha; Bansal, Dhiru; Sánchez Alvarado, Alejandro; Vemula, Praveen Kumar

    2017-01-01

    Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system. PMID:28807895

  15. Relationship of acute axonal damage, Wallerian degeneration, and clinical disability in multiple sclerosis.

    PubMed

    Singh, Shailender; Dallenga, Tobias; Winkler, Anne; Roemer, Shanu; Maruschak, Brigitte; Siebert, Heike; Brück, Wolfgang; Stadelmann, Christine

    2017-03-17

    Axonal damage and loss substantially contribute to the incremental accumulation of clinical disability in progressive multiple sclerosis. Here, we assessed the amount of Wallerian degeneration in brain tissue of multiple sclerosis patients in relation to demyelinating lesion activity and asked whether a transient blockade of Wallerian degeneration decreases axonal loss and clinical disability in a mouse model of inflammatory demyelination. Wallerian degeneration and acute axonal damage were determined immunohistochemically in the periplaque white matter of multiple sclerosis patients with early actively demyelinating lesions, chronic active lesions, and inactive lesions. Furthermore, we studied the effects of Wallerian degeneration blockage on clinical severity, inflammatory pathology, acute axonal damage, and long-term axonal loss in experimental autoimmune encephalomyelitis using Wallerian degeneration slow (Wld S ) mutant mice. The highest numbers of axons undergoing Wallerian degeneration were found in the perilesional white matter of multiple sclerosis patients early in the disease course and with actively demyelinating lesions. Furthermore, Wallerian degeneration was more abundant in patients harboring chronic active as compared to chronic inactive lesions. No co-localization of neuropeptide Y-Y1 receptor, a bona fide immunohistochemical marker of Wallerian degeneration, with amyloid precursor protein, frequently used as an indicator of acute axonal transport disturbance, was observed in human and mouse tissue, indicating distinct axon-degenerative processes. Experimentally, a delay of Wallerian degeneration, as observed in Wld S mice, did not result in a reduction of clinical disability or acute axonal damage in experimental autoimmune encephalomyelitis, further supporting that acute axonal damage as reflected by axonal transport disturbances does not share common molecular mechanisms with Wallerian degeneration. Furthermore, delaying Wallerian degeneration

  16. The Pseudopod System for Axon-Glia Interactions: Stimulation and Isolation of Schwann Cell Protrusions that Form in Response to Axonal Membranes.

    PubMed

    Poitelon, Yannick; Feltri, M Laura

    2018-01-01

    In the peripheral nervous system, axons dictate the differentiation state of Schwann cells. Most of this axonal influence on Schwann cells is due to juxtacrine interactions between axonal transmembrane molecules (e.g., the neuregulin growth factor) and receptors on the Schwann cell (e.g., the ErbB2/ErbB3 receptor). The fleeting nature of this interaction together with the lack of synchronicity in the development of the Schwann cell population limits our capability to study this phenomenon in vivo. Here we present a simple Boyden Chamber-based method to study this important cell-cell interaction event. We isolate the early protrusions of Schwann cells that are generated in response to juxtacrine stimulation by sensory neuronal membranes. This method is compatible with a large array of current biochemical analyses and provides an effective approach to study biomolecules that are differentially localized in Schwann cell protrusions and cell bodies in response to axonal signals. A similar approach can be extended to different kinds of cell-cell interactions.

  17. Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons

    NASA Technical Reports Server (NTRS)

    Kirkpatrick, L. L.; Witt, A. S.; Payne, H. R.; Shine, H. D.; Brady, S. T.

    2001-01-01

    Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

  18. Characterization of axon formation in the embryonic stem cell-derived motoneuron.

    PubMed

    Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

    2011-01-01

    The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

  19. Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

    PubMed Central

    Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

    2012-01-01

    Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

  20. Imaging axonal transport in the rat visual pathway.

    PubMed

    Abbott, Carla J; Choe, Tiffany E; Lusardi, Theresa A; Burgoyne, Claude F; Wang, Lin; Fortune, Brad

    2013-02-01

    A technique was developed for assaying axonal transport in retinal ganglion cells using 2 µl injections of 1% cholera toxin b-subunit conjugated to AlexaFluor488 (CTB). In vivo retinal and post-mortem brain imaging by confocal scanning laser ophthalmoscopy and post-mortem microscopy were performed. The transport of CTB was sensitive to colchicine, which disrupts axonal microtubules. The bulk rates of transport were determined to be approximately 80-90 mm/day (anterograde) and 160 mm/day (retrograde). Results demonstrate that axonal transport of CTB can be monitored in vivo in the rodent anterior visual pathway, is dependent on intact microtubules, and occurs by active transport mechanisms.

  1. Acutely damaged axons are remyelinated in multiple sclerosis and experimental models of demyelination.

    PubMed

    Schultz, Verena; van der Meer, Franziska; Wrzos, Claudia; Scheidt, Uta; Bahn, Erik; Stadelmann, Christine; Brück, Wolfgang; Junker, Andreas

    2017-08-01

    Remyelination is in the center of new therapies for the treatment of multiple sclerosis to resolve and improve disease symptoms and protect axons from further damage. Although remyelination is considered beneficial in the long term, it is not known, whether this is also the case early in lesion formation. Additionally, the precise timing of acute axonal damage and remyelination has not been assessed so far. To shed light onto the interrelation between axons and the myelin sheath during de- and remyelination, we employed cuprizone- and focal lysolecithin-induced demyelination and performed time course experiments assessing the evolution of early and late stage remyelination and axonal damage. We observed damaged axons with signs of remyelination after cuprizone diet cessation and lysolecithin injection. Similar observations were made in early multiple sclerosis lesions. To assess the correlation of remyelination and axonal damage in multiple sclerosis lesions, we took advantage of a cohort of patients with early and late stage remyelinated lesions and assessed the number of APP- and SMI32- positive damaged axons and the density of SMI31-positive and silver impregnated preserved axons. Early de- and remyelinating lesions did not differ with respect to axonal density and axonal damage, but we observed a lower axonal density in late stage demyelinated multiple sclerosis lesions than in remyelinated multiple sclerosis lesions. Our findings suggest that remyelination may not only be protective over a long period of time, but may play an important role in the immediate axonal recuperation after a demyelinating insult. © 2017 The Authors GLIA Published by Wiley Periodicals, Inc.

  2. Spinal cord injury: overview of experimental approaches used to restore locomotor activity.

    PubMed

    Fakhoury, Marc

    2015-01-01

    Spinal cord injury affects more than 2.5 million people worldwide and can lead to paraplegia and quadriplegia. Anatomical discontinuity in the spinal cord results in disruption of the impulse conduction that causes temporary or permanent changes in the cord's normal functions. Although axonal regeneration is limited, damage to the spinal cord is often accompanied by spontaneous plasticity and axon regeneration that help improve sensory and motor skills. The recovery process depends mainly on synaptic plasticity in the preexisting circuits and on the formation of new pathways through collateral sprouting into neighboring denervated territories. However, spontaneous recovery after spinal cord injury can go on for several years, and the degree of recovery is very limited. Therefore, the development of new approaches that could accelerate the gain of motor function is of high priority to patients with damaged spinal cord. Although there are no fully restorative treatments for spinal injury, various rehabilitative approaches have been tested in animal models and have reached clinical trials. In this paper, a closer look will be given at the potential therapies that could facilitate axonal regeneration and improve locomotor recovery after injury to the spinal cord. This article highlights the application of several interventions including locomotor training, molecular and cellular treatments, and spinal cord stimulation in the field of rehabilitation research. Studies investigating therapeutic approaches in both animal models and individuals with injured spinal cords will be presented.

  3. Stress Increases Peripheral Axon Growth and Regeneration through Glucocorticoid Receptor-Dependent Transcriptional Programs

    PubMed Central

    Alexander, Jessica K.; Madalena, Kathryn M.; Motti, Dario; Quach, Tam; Zha, Alicia; Webster Marketon, Jeanette

    2017-01-01

    Abstract Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice (Mus musculus) basal GC receptor (Nr3c1, also known as GR) expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity. PMID:28828403

  4. Squid Giant Axon Contains Neurofilament Protein mRNA but does not Synthesize Neurofilament Proteins.

    PubMed

    Gainer, Harold; House, Shirley; Kim, Dong Sun; Chin, Hemin; Pant, Harish C

    2017-04-01

    When isolated squid giant axons are incubated in radioactive amino acids, abundant newly synthesized proteins are found in the axoplasm. These proteins are translated in the adaxonal Schwann cells and subsequently transferred into the giant axon. The question as to whether any de novo protein synthesis occurs in the giant axon itself is difficult to resolve because the small contribution of the proteins possibly synthesized intra-axonally is not easily distinguished from the large amounts of the proteins being supplied from the Schwann cells. In this paper, we reexamine this issue by studying the synthesis of endogenous neurofilament (NF) proteins in the axon. Our laboratory previously showed that NF mRNA and protein are present in the squid giant axon, but not in the surrounding adaxonal glia. Therefore, if the isolated squid axon could be shown to contain newly synthesized NF protein de novo, it could not arise from the adaxonal glia. The results of experiments in this paper show that abundant 3H-labeled NF protein is synthesized in the squid giant fiber lobe containing the giant axon's neuronal cell bodies, but despite the presence of NF mRNA in the giant axon no labeled NF protein is detected in the giant axon. This lends support to the glia-axon protein transfer hypothesis which posits that the squid giant axon obtains newly synthesized protein by Schwann cell transfer and not through intra-axonal protein synthesis, and further suggests that the NF mRNA in the axon is in a translationally repressed state.

  5. Axodendritic sorting and pathological missorting of Tau are isoform-specific and determined by axon initial segment architecture.

    PubMed

    Zempel, Hans; Dennissen, Frank J A; Kumar, Yatender; Luedtke, Julia; Biernat, Jacek; Mandelkow, Eva-Maria; Mandelkow, Eckhard

    2017-07-21

    Subcellular mislocalization of the microtubule-associated protein Tau is a hallmark of Alzheimer disease (AD) and other tauopathies. Six Tau isoforms, differentiated by the presence or absence of a second repeat or of N-terminal inserts, exist in the human CNS, but their physiological and pathological differences have long remained elusive. Here, we investigated the properties and distributions of human and rodent Tau isoforms in primary forebrain rodent neurons. We found that the Tau diffusion barrier (TDB), located within the axon initial segment (AIS), controls retrograde (axon-to-soma) and anterograde (soma-to-axon) traffic of Tau. Tau isoforms without the N-terminal inserts were sorted efficiently into the axon. However, the longest isoform (2N4R-Tau) was partially retained in cell bodies and dendrites, where it accelerated spine and dendrite growth. The TDB (located within the AIS) was impaired when AIS components (ankyrin G, EB1) were knocked down or when glycogen synthase kinase-3β (GSK3β; an AD-associated kinase tethered to the AIS) was overexpressed. Using superresolution nanoscopy and live-cell imaging, we observed that microtubules within the AIS appeared highly dynamic, a feature essential for the TDB. Pathomechanistically, amyloid-β insult caused cofilin activation and F-actin remodeling and decreased microtubule dynamics in the AIS. Concomitantly with these amyloid-β-induced disruptions, the AIS/TDB sorting function failed, causing AD-like Tau missorting. In summary, we provide evidence that the human and rodent Tau isoforms differ in axodendritic sorting and amyloid-β-induced missorting and that the axodendritic distribution of Tau depends on AIS integrity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Regeneration of the Exocrine Pancreas Is Delayed in Telomere-Dysfunctional Mice

    PubMed Central

    von Figura, Guido; Wagner, Martin; Nalapareddy, Kodandaramireddy; Hartmann, Daniel; Kleger, Alexander; Guachalla, Luis Miguel; Rolyan, Harshvardhan; Adler, Guido; Rudolph, Karl Lenhard

    2011-01-01

    Introduction Telomere shortening is a cell-intrinsic mechanism that limits cell proliferation by induction of DNA damage responses resulting either in apoptosis or cellular senescence. Shortening of telomeres has been shown to occur during human aging and in chronic diseases that accelerate cell turnover, such as chronic hepatitis. Telomere shortening can limit organ homeostasis and regeneration in response to injury. Whether the same holds true for pancreas regeneration in response to injury is not known. Methods In the present study, pancreatic regeneration after acute cerulein-induced pancreatitis was studied in late generation telomerase knockout mice with short telomeres compared to telomerase wild-type mice with long telomeres. Results Late generation telomerase knockout mice exhibited impaired exocrine pancreatic regeneration after acute pancreatitis as seen by persistence of metaplastic acinar cells and markedly reduced proliferation. The expression levels of p53 and p21 were not significantly increased in regenerating pancreas of late generation telomerase knockout mice compared to wild-type mice. Conclusion Our results indicate that pancreatic regeneration is limited in the context of telomere dysfunction without evidence for p53 checkpoint activation. PMID:21364961

  7. Nano-biomimetics for nano/micro tissue regeneration.

    PubMed

    Singh, Dolly; Singh, Deepti; Zo, Sunmi; Han, Sung Soo

    2014-10-01

    Nanostructured biomimetics have recently shown great promise in the field of tissue engineering. They can be used as nanoscaffolds and tailored at the molecular level. The scaffold topography closely resembles the native extracellular matrix in terms of framing, porosity and bio-functionality. This review covers the approaches used for biomimetic fabrication, including soft lithography, the plasmonic nanohybrid matrix method and multilayer self-assembly scaffolds for tissue regeneration. It brings together knowledge from different arenas about the synthesis, characterization and functionalization of matrices to accelerate the tissue regeneration process. Every tissue in the body presents different challenges and requires a specific fabrication process designed to identify and mirror the particular organ. For example, microfluidics systems aim to mimic the extracellular matrix of vascular and cartilage tissue, and these systems have different parts with completely different mechanical strength, cellular adhesion and interplay between matrix and cells. A fully functional nanomatrix designed by a self-assembling methodology for use as a vascular tissue engineering scaffold needs to have intrinsic microvessels that facilitate the transportation of metabolites and nutrients. Similarly, in the case of peripheral nerve regeneration, a scaffold needs to have sufficient mechanical strength to protect the regenerating tissue, yet be biodegradable enough to avoid a possible second surgery. To enhance the functionality of scaffolds, increasing focus has been placed on in vitro and in vivo research to achieve optimal scaffold design. Nanobiomimetics unarguably offer the most suitable physicochemical scaffold properties for tissue regeneration.

  8. Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans.

    PubMed

    Meng, Lingfeng; Zhang, Albert; Jin, Yishi; Yan, Dong

    2016-10-21

    Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4 . Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.

  9. LONGITUDINAL IMPEDANCE OF THE SQUID GIANT AXON

    PubMed Central

    Cole, Kenneth S.; Baker, Richard F.

    1941-01-01

    Longitudinal alternating current impedance measurements have been made on the squid giant axon over the frequency range from 30 cycles per second to 200 kc. per second. Large sea water electrodes were used and the inter-electrode length was immersed in oil. The impedance at high frequency was approximately as predicted theoretically on the basis of the poorly conducting dielectric characteristics of the membrane previously determined. For the large majority of the axons, the impedance reached a maximum at a low frequency and the reactance then vanished at a frequency between 150 and 300 cycles per second. Below this frequency, the reactance was inductive, reaching a maximum and then approaching zero as the frequency was decreased. The inductive reactance is a property of the axon and requires that it contain an inductive structure. The variation of the impedance with interpolar distance indicates that the inductance is in the membrane. The impedance characteristics of the membrane as calculated from the measured longitudinal impedance of the axon may be expressed by an equivalent membrane circuit containing inductance, capacity, and resistance. For a square centimeter of membrane the capacity of 1 µf with dielectric loss is shunted by the series combination of a resistance of 400 ohms and an inductance of one-fifth henry. PMID:19873252

  10. Cloning and characteristics of fish glial fibrillary acidic protein: implications for optic nerve regeneration.

    PubMed

    Cohen, I; Shani, Y; Schwartz, M

    1993-08-15

    Mammalian central nervous system neurons do not regenerate after axonal injury, unlike their counterparts in fish and amphibians. After axonal injury, glial cells in mammals do not support regrowth of axons, while in fish they support the regeneration process. Controversy exists as to whether or not the intact fish optic nerve expresses glial fibrillary acidic protein, a well-known marker for mature astrocytes, and thus whether its astrocytes differ in this respect from those of the brain and spinal cord, as well as from optic nerve astrocytes of other species. In an attempt to resolve this question we cloned fish glial fibrillary acidic protein. Two different complementary DNA clones were isolated from a carp brain complementary DNA library, each encoding a different form of glial fibrillary acidic protein apparently originating from different genes. Monospecific polyclonal antibodies were raised against a peptide synthesized according to the predicted amino acid sequence, and used to identify and localize the fish glial fibrillary acidic protein. Two glial fibrillary acidic proteins (of 49 kDa and 51 kDa) were identified by the antibodies in all tested fish central nervous system tissues. The antibodies were then used to examine glial fibrillary acidic protein immunoreactivity in sections taken from uninjured and injured optic nerves of goldfish. Injury was followed by an elevation in glial fibrillary acidic protein immunoreactivity along the whole length of the nerve, except at the site of the injury, where--as in the case of vimentin--no immunoreactivity was detectable. However, in contrast to vimentin-positive glial cells, which repopulate the site of the injury soon after the optic nerve is injured, glial fibrillary acidic protein-positive glial cells remained outside the injury site for as long as 6 weeks after the injury. Despite the injury-induced changes in glial fibrillary acidic protein immunoreactivity, no change was observed in the level of transcript

  11. The nano-architecture of the axonal cytoskeleton.

    PubMed

    Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit

    2017-12-01

    The corporeal beauty of the neuronal cytoskeleton has captured the imagination of generations of scientists. One of the easiest cellular structures to visualize by light microscopy, its existence has been known for well over 100 years, yet we have only recently begun to fully appreciate its intricacy and diversity. Recent studies combining new probes with super-resolution microscopy and live imaging have revealed surprising details about the axonal cytoskeleton and, in particular, have discovered previously unknown actin-based structures. Along with traditional electron microscopy, these newer techniques offer a nanoscale view of the axonal cytoskeleton, which is important for our understanding of neuronal form and function, and lay the foundation for future studies. In this Review, we summarize existing concepts in the field and highlight contemporary discoveries that have fundamentally altered our perception of the axonal cytoskeleton.

  12. GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

    PubMed Central

    Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

    2011-01-01

    Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

  13. The use of shock waves in peripheral nerve regeneration: new perspectives?

    PubMed

    Hausner, Thomas; Nógrádi, Antal

    2013-01-01

    Low-energy extracorporeal shock wave treatment (ESWT) is a relatively new therapeutic tool that is widely used for the treatment of epicondylitis and plantar fasciitis and to foster bone and wound healing. Shock waves, sonic pulses with high energy impact, are thought to induce biochemical changes within the targeted tissues through mechanotransduction. The biological effects of ESWT are manifested in improved vascularization, the local release of growth factors, and local anti-inflammatory effects, but the target cells too are influenced. ESWT appears to have differential effects on peripheral nerves and has been proved to promote axonal regeneration after axotomy. This review discusses the effects of ESWT on intact and injured peripheral nerves and suggests a multiple mechanism of action. © 2013 Elsevier Inc. All rights reserved.

  14. Shank3 is localized in axons and presynaptic specializations of developing hippocampal neurons and involved in the modulation of NMDA receptor levels at axon terminals.

    PubMed

    Halbedl, Sonja; Schoen, Michael; Feiler, Marisa S; Boeckers, Tobias M; Schmeisser, Michael J

    2016-04-01

    Autism-related Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses. A few studies, however, have already indicated that within a neuron, the presence of Shank family members is not limited to the postsynaptic density. By separating axons from dendrites of developing hippocampal neurons in microfluidic chambers, we show that RNA of all three Shank family members is present within axons. Immunostaining confirms these findings as all three Shanks are indeed found within separated axons and further co-localize with well-known proteins of the presynaptic specialization in axon terminals. Therefore, Shank proteins might not only serve as postsynaptic scaffold proteins, but also play a crucial role during axonal outgrowth and presynaptic development and function. This is supported by our findings that shRNA-mediated knockdown of Shank3 results in up-regulation of the NMDA receptor subunit GluN1 in axon terminals. Taken together, our findings will have major implications for the future analysis of neuronal Shank biology in both health and disease. Shank1, Shank2, and Shank3 are major postsynaptic scaffold proteins of excitatory glutamatergic synapses strongly related to several neuropsychiatric disorders. However, a few studies have already implicated a functional role of the Shanks beyond the postsynaptic density (PSD). We here show that all three Shanks are localized in both axons and pre-synaptic specializiations of developing hippocampal neurons in culture. We further provide evidence that Shank3 is involved in the modulation of NMDA receptor levels at axon terminals. Taken together, our study will open up novel avenues for the future analysis of neuronal Shank biology in both health and disease. © 2016 International Society for Neurochemistry.

  15. Golgi bypass for local delivery of axonal proteins, fact or fiction?

    PubMed

    González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

    2018-04-06

    Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

  16. Protein and peptide-based therapeutics in periodontal regeneration.

    PubMed

    Reynolds, Mark A; Aichelmann-Reidy, Mary E

    2012-09-01

    Protein and peptide-based therapeutics provide a unique strategy for controlling highly specific and complex biologic actions that cannot be accomplished by simple devices or chemical compounds. This article reviews some of the key characteristics and summarizes the clinical effectiveness of protein and peptide-based therapeutics targeting periodontal regeneration. A literature search was conducted of randomized clinical trials and systematic reviews evaluating protein and peptide-based therapeutics for the regeneration of periodontal tissues of at least 6 months duration. Data sources included PubMed and Embase electronic databases, hand-searched journals, and the ClinicalTrials.gov registry. Commercially marketed protein and peptide-based therapeutics for periodontal regeneration provide gains in clinical attachment level and bone formation that are comparable or superior to other regenerative approaches. Results from several clinical trials indicate that protein and peptide-based therapies can accelerate repair and regeneration when compared with other treatments and that improvements in clinical parameters continue beyond 12 months. Protein and peptide-based therapies also exhibit the capacity to increase the predictability of treatment outcomes. Clinical and histologic studies support the effectiveness of protein- and peptide-based therapeutics for periodontal regeneration. Emerging evidence suggests that the delivery devices/scaffolds play a critical role in determining the effectiveness of this class of therapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Modeling Axonal Defects in Hereditary Spastic Paraplegia with Human Pluripotent Stem Cells

    PubMed Central

    Denton, Kyle R.; Xu, Chongchong; Shah, Harsh; Li, Xue-Jun

    2016-01-01

    BACKGROUND Cortical motor neurons, also known as upper motor neurons, are large projection neurons whose axons convey signals to lower motor neurons to control the muscle movements. Degeneration of cortical motor neuron axons is implicated in several debilitating disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis (ALS). Since the discovery of the first HSP gene, SPAST that encodes spastin, over 70 distinct genetic loci associated with HSP have been identified. How the mutations of these functionally diverse genes result in axonal degeneration and why certain axons are affected in HSP remains largely unknown. The development of induced pluripotent stem cell (iPSC) technology has provided researchers an excellent resource to generate patient-specific human neurons to model human neuropathologic processes including axonal defects. METHODS In this article, we will frst review the pathology and pathways affected in the common forms of HSP subtypes by searching the PubMed database. We will then summurize the findings and insights gained from studies using iPSC-based models, and discuss the challenges and future directions. RESULTS HSPs, a heterogeneous group of genetic neurodegenerative disorders, are characterized by lower extremity weakness and spasticity that result from retrograde axonal degeneration of cortical motor neurons. Recently, iPSCs have been generated from several common forms of HSP including SPG4, SPG3A, and SPG11 patients. Neurons derived from HSP iPSCs exhibit disease-relevant axonal defects, such as impaired neurite outgrowth, increased axonal swellings, and reduced axonal transport. CONCLUSION These patient-derived neurons offer unique tools to study the pathogenic mechanisms and explore the treatments for rescuing axonal defects in HSP, as well as other diseases involving axonopathy. PMID:27956894

  18. Hair follicle stem cell proliferation, Akt and Wnt signaling activation in TPA-induced hair regeneration.

    PubMed

    Qiu, Weiming; Lei, Mingxing; Zhou, Ling; Bai, Xiufeng; Lai, Xiangdong; Yu, Yu; Yang, Tian; Lian, Xiaohua

    2017-06-01

    Regeneration of hair follicles relies on activation of hair follicle stem cells during telogen to anagen transition process in hair cycle. This process is rigorously controlled by intrinsic and environmental factors. 12-o-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, accelerates reentry of hair follicles into anagen phase. However, it is unclear that how TPA promotes the hair regeneration. In the present study, we topically applied TPA onto the dorsal skin of 2-month-old C57BL/6 female mice to examine the activity of hair follicle stem cells and alteration of signaling pathways during hair regeneration. We found that refractory telogen hair follicles entered anagen prematurely after TPA treatment, with the enhanced proliferation of CD34-positive hair follicle stem cells. Meanwhile, we observed Akt signaling was activated in epidermis, hair infundibulum, bulge and hair bulb, and Wnt signaling was also activated after hair follicle stem cells proliferation. Importantly, after overexpression of DKK1, a specific Wnt signaling inhibitor, the accelerated reentry of hair follicles into anagen induced by TPA was abolished. Our data indicated that TPA-induced hair follicle regeneration is associated with activation of Akt and Wnt/β-catenin signaling.

  19. Carboxylated graphene oxide promoted axonal guidance growth by activating Netrin-1/deleted in colorectal cancer signaling in rat primary cultured cortical neurons.

    PubMed

    Liu, Meili; Jia, Zhengtai; Xiao, Xiongfu; Zhang, Zhifa; Li, Ping; Zhou, Gang; Fan, Yubo

    2018-06-01

    Nanomaterials of graphene and its derivatives have been widely applied in recent years, but whose impacts on the neuronal guidance growth are still not reported. In the present study, graphene oxide (GO) and carboxylated graphene oxide (GO-COOH) were used to investigate the potential effects on axonal guidance growth in the primary cultured cortical neurons. In addition, we characterized the structure and chemical composition of synthesized GO and GO-COOH using Fourier transform infrared spectrophotometer and scanning electron microscope assays and Raman analysis. GO is not neurotoxic and not conductive in a soluble form. However, GO-COOH has higher solubility and conductivity. Cell viability was assessed using CCK-8 assays and fluorescein diacetate after GO and GO-COOH treatment (0, 1, 2, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 50, and 100 µg/mL). There are significant increases of cell viability and axonal growth after GO (2 and 4 μg/mL) and GO-COOH treatment (2 and 4 μg/mL). We further investigated the molecular mechanism of axonal guidance growth after GO and GO-COOH (2 and 4 μg/mL) application. Additionally, GO and GO-COOH up-regulated expression of Netrin-1 and its receptor, deleted in colorectal cancer by immunofluorescence assays and western blots assay. Our study demonstrated that GO-COOH activated Cdc42 and Rac1 and dramatically decreased RhoA. Thus, GO-COOH (2 µg/mL) is much better to be nanocarriers than GO for axonal guidance and growth in this study. GO-COOH may be used to facilitate guidance for regenerating neurons in the future. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1500-1510, 2018. © 2018 Wiley Periodicals, Inc.

  20. Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

    PubMed Central

    Rao, Mala V.; Garcia, Michael L.; Miyazaki, Yukio; Gotow, Takahiro; Yuan, Aidong; Mattina, Salvatore; Ward, Chris M.; Calcutt, Nigel A.; Uchiyama, Yasuo; Nixon, Ralph A.; Cleveland, Don W.

    2002-01-01

    The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine–serine–proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits. PMID:12186852

  1. Functional recovery of odor representations in regenerated sensory inputs to the olfactory bulb

    PubMed Central

    Cheung, Man C.; Jang, Woochan; Schwob, James E.; Wachowiak, Matt

    2014-01-01

    The olfactory system has a unique capacity for recovery from peripheral damage. After injury to the olfactory epithelium (OE), olfactory sensory neurons (OSNs) regenerate and re-converge on target glomeruli of the olfactory bulb (OB). Thus far, this process has been described anatomically for only a few defined populations of OSNs. Here we characterize this regeneration at a functional level by assessing how odor representations carried by OSN inputs to the OB recover after massive loss and regeneration of the sensory neuron population. We used chronic imaging of mice expressing synaptopHluorin in OSNs to monitor odor representations in the dorsal OB before lesion by the olfactotoxin methyl bromide and after a 12 week recovery period. Methyl bromide eliminated functional inputs to the OB, and these inputs recovered to near-normal levels of response magnitude within 12 weeks. We also found that the functional topography of odor representations recovered after lesion, with odorants evoking OSN input to glomerular foci within the same functional domains as before lesion. At a finer spatial scale, however, we found evidence for mistargeting of regenerated OSN axons onto OB targets, with odorants evoking synaptopHluorin signals in small foci that did not conform to a typical glomerular structure but whose distribution was nonetheless odorant-specific. These results indicate that OSNs have a robust ability to reestablish functional inputs to the OB and that the mechanisms underlying the topography of bulbar reinnervation during development persist in the adult and allow primary sensory representations to be largely restored after massive sensory neuron loss. PMID:24431990

  2. Ionized calcium concentrations in squid axons

    PubMed Central

    1976-01-01

    Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca. PMID:818340

  3. Molecular determinants of Cytochrome C oxidase IV mRNA axonal trafficking

    PubMed Central

    Kar, Amar N.; Vargas, Jose Norberto S.; Chen, Cai-Yun; Kowalak, Jeffrey A; Gioio, Anthony E.; Kaplan, Barry B.

    2017-01-01

    In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA. PMID:28161363

  4. Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones.

    PubMed

    Ahnert-Hilger, G; Höltje, M; Grosse, G; Pickert, G; Mucke, C; Nixdorf-Bergweiler, B; Boquet, P; Hofmann, F; Just, I

    2004-07-01

    Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton. While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development. Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth. Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted. The opposite phenotype was observed when a constitutively active RhoA variant was expressed. Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3(lim) or Staphylococcus aureus, C3(stau2)), diminished axonal branching. By contrast, extracellularly applied nanomolar concentrations of C3 from C. botulinum (C3(bot)) or enzymatically dead C3(bot) significantly increased axon growth and axon branching. Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation. However, extracellular application of enzymatically active or dead C3(bot) exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.

  5. Cortical Interneuron Subtypes Vary in Their Axonal Action Potential Properties

    PubMed Central

    Casale, Amanda E.; Foust, Amanda J.; Bal, Thierry

    2015-01-01

    The role of interneurons in cortical microcircuits is strongly influenced by their passive and active electrical properties. Although different types of interneurons exhibit unique electrophysiological properties recorded at the soma, it is not yet clear whether these differences are also manifested in other neuronal compartments. To address this question, we have used voltage-sensitive dye to image the propagation of action potentials into the fine collaterals of axons and dendrites in two of the largest cortical interneuron subtypes in the mouse: fast-spiking interneurons, which are typically basket or chandelier neurons; and somatostatin containing interneurons, which are typically regular spiking Martinotti cells. We found that fast-spiking and somatostatin-expressing interneurons differed in their electrophysiological characteristics along their entire dendrosomatoaxonal extent. The action potentials generated in the somata and axons, including axon collaterals, of somatostatin-expressing interneurons are significantly broader than those generated in the same compartments of fast-spiking inhibitory interneurons. In addition, action potentials back-propagated into the dendrites of somatostatin-expressing interneurons much more readily than fast-spiking interneurons. Pharmacological investigations suggested that axonal action potential repolarization in both cell types depends critically upon Kv1 channels, whereas the axonal and somatic action potentials of somatostatin-expressing interneurons also depend on BK Ca2+-activated K+ channels. These results indicate that the two broad classes of interneurons studied here have expressly different subcellular physiological properties, allowing them to perform unique computational roles in cortical circuit operations. SIGNIFICANCE STATEMENT Neurons in the cerebral cortex are of two major types: excitatory and inhibitory. The proper balance of excitation and inhibition in the brain is critical for its operation. Neurons

  6. Cortical Interneuron Subtypes Vary in Their Axonal Action Potential Properties.

    PubMed

    Casale, Amanda E; Foust, Amanda J; Bal, Thierry; McCormick, David A

    2015-11-25

    The role of interneurons in cortical microcircuits is strongly influenced by their passive and active electrical properties. Although different types of interneurons exhibit unique electrophysiological properties recorded at the soma, it is not yet clear whether these differences are also manifested in other neuronal compartments. To address this question, we have used voltage-sensitive dye to image the propagation of action potentials into the fine collaterals of axons and dendrites in two of the largest cortical interneuron subtypes in the mouse: fast-spiking interneurons, which are typically basket or chandelier neurons; and somatostatin containing interneurons, which are typically regular spiking Martinotti cells. We found that fast-spiking and somatostatin-expressing interneurons differed in their electrophysiological characteristics along their entire dendrosomatoaxonal extent. The action potentials generated in the somata and axons, including axon collaterals, of somatostatin-expressing interneurons are significantly broader than those generated in the same compartments of fast-spiking inhibitory interneurons. In addition, action potentials back-propagated into the dendrites of somatostatin-expressing interneurons much more readily than fast-spiking interneurons. Pharmacological investigations suggested that axonal action potential repolarization in both cell types depends critically upon Kv1 channels, whereas the axonal and somatic action potentials of somatostatin-expressing interneurons also depend on BK Ca(2+)-activated K(+) channels. These results indicate that the two broad classes of interneurons studied here have expressly different subcellular physiological properties, allowing them to perform unique computational roles in cortical circuit operations. Neurons in the cerebral cortex are of two major types: excitatory and inhibitory. The proper balance of excitation and inhibition in the brain is critical for its operation. Neurons contain three main

  7. Assembly and turnover of neurofilaments in growing axonal neurites.

    PubMed

    Boumil, Edward F; Vohnoutka, Rishel; Lee, Sangmook; Pant, Harish; Shea, Thomas B

    2018-01-26

    Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites ('bundled NFs'). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered. © 2018. Published by The Company of Biologists Ltd.

  8. Assembly and turnover of neurofilaments in growing axonal neurites

    PubMed Central

    Boumil, Edward F.; Vohnoutka, Rishel; Lee, Sangmook; Pant, Harish

    2018-01-01

    ABSTRACT Neurofilaments (NFs) are thought to provide stability to the axon. We examined NF dynamics within axonal neurites of NB2a/d1 neuroblastoma by transient transfection with green fluorescent protein-tagged NF-heavy (GFP-H) under the control of a tetracycline-inducible promoter. Immunofluorescent and biochemical analyses demonstrated that GFP-H expressed early during neurite outgrowth associated with a population of centrally-situated, highly-phosphorylated crosslinked NFs along the length of axonal neurites (‘bundled NFs’). By contrast, GFP-H expressed after considerable neurite outgrowth displayed markedly reduced association with bundled NFs and was instead more evenly distributed throughout the axon. This differential localization was maintained for up to 2 weeks in culture. Once considerable neurite outgrowth had progressed, GFP that had previously associated with the NF bundle during early expression was irreversibly depleted by photobleaching. Cessation of expression allowed monitoring of NF turnover. GFP-H associated bundled NFs underwent slower decay than GFP-H associated with surrounding, less-phosphorylated NFs. Notably, GFP associated with bundled NFs underwent similar decay rates within the core and edges of this bundle. These results are consistent with previous demonstration of a resident NF population within axonal neurites, but suggest that this population is more dynamic than previously considered. PMID:29158321

  9. X-linked microtubule-associated protein, Mid1, regulates axon development

    PubMed Central

    Lu, Tingjia; Chen, Renchao; Cox, Timothy C.; Moldrich, Randal X.; Kurniawan, Nyoman; Tan, Guohe; Perry, Jo K.; Ashworth, Alan; Bartlett, Perry F.; Xu, Li; Zhang, Jing; Lu, Bin; Wu, Mingyue; Shen, Qi; Liu, Yuanyuan; Richards, Linda J.; Xiong, Zhiqi

    2013-01-01

    Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS. PMID:24194544

  10. X-linked microtubule-associated protein, Mid1, regulates axon development.

    PubMed

    Lu, Tingjia; Chen, Renchao; Cox, Timothy C; Moldrich, Randal X; Kurniawan, Nyoman; Tan, Guohe; Perry, Jo K; Ashworth, Alan; Bartlett, Perry F; Xu, Li; Zhang, Jing; Lu, Bin; Wu, Mingyue; Shen, Qi; Liu, Yuanyuan; Richards, Linda J; Xiong, Zhiqi

    2013-11-19

    Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS.

  11. Recurrent laryngeal nerve regeneration using an oriented collagen scaffold containing Schwann cells.

    PubMed

    Chitose, Shun-Ichi; Sato, Kiminori; Fukahori, Mioko; Sueyoshi, Shintaro; Kurita, Takashi; Umeno, Hirohito

    2017-07-01

    Regeneration of the recurrent laryngeal nerve (RLN), which innervates the intrinsic laryngeal muscles such that they can perform complex functions, is particularly difficult to achieve. Synkinesis after RLN neogenesis leads to uncoordinated movement of laryngeal muscles. Recently, some basic research studies have used cultured Schwann cells (SCs) to repair peripheral nerve injuries. This study aimed to regenerate the RLN using an oriented collagen scaffold containing cultured SCs. Preliminary animal experiment. A 10-mm-long autologous canine cervical ansa was harvested. The nerve tissue was scattered and subcultured on oriented collagen sheets in reduced serum medium. After verifying that the smaller cultivated cells with high nucleus-cytoplasm ratios were SCs, collagen sheets with longitudinally oriented cells were rolled and inserted into a 20-mm collagen conduit. The fabricated scaffolds containing SCs were autotransplanted to a 20-mm deficient RLN, and vocal fold movements and histological characteristics were observed. Scaffolds containing cultured SCs were successfully fabricated. Immunocytochemical examination revealed that these isolated and cultured cells, identified as SCs, expressed S-100 protein and GFAP but not vimentin. The orientation of SCs matched that of the oriented collagen sheet. Two months after successful transplantation, laryngeal endoscopy revealed coordinated movement of the bilateral vocal folds by external stimulation under light general anesthesia. Hematoxylin and eosin staining showed that the regenerated RLN lacked epineurium surrounding the nerve fibers and was interspersed with collagen fibers. Myelin protein zero was expressed around many axons. Partial regeneration of RLN was achieved through the use of oriented collagen scaffolding. NA Laryngoscope, 127:1622-1627, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  12. Silk fibroin enhances peripheral nerve regeneration by improving vascularization within nerve conduits.

    PubMed

    Wang, Chunyang; Jia, Yachao; Yang, Weichao; Zhang, Cheng; Zhang, Kuihua; Chai, Yimin

    2018-07-01

    Silk fibroin (SF)-based nerve conduits have been widely used to bridge peripheral nerve defects. Our previous study showed that nerve regeneration in a SF-blended poly (l-lactide-co-ɛ-caprolactone) [P(LLA-CL)] nerve conduit is better than that in a P(LLA-CL) conduit. However, the involved mechanisms remain unclarified. Because angiogenesis within a nerve conduit plays an important role in nerve regeneration, vascularization of SF/P(LLA-CL) and P(LLA-CL) conduits was compared both in vitro and in vivo. In the present study, we observed that SF/P(LLA-CL) nanofibers significantly promoted fibroblast proliferation, and vascular endothelial growth factor secreted by fibroblasts seeded in SF/P(LLA-CL) nanofibers was more than seven-fold higher than that in P(LLA-CL) nanofibers. Conditioned medium of fibroblasts in the SF/P(LLA-CL) group stimulated more human umbilical vein endothelial cells (HUVEC) to form capillary-like networks and promoted faster HUVEC migration. The two kinds of nerve conduits were used to bridge 10-mm-length nerve defects in rats. At 3 weeks of reparation, the blood vessel area in the SF/P(LLA-CL) group was significantly larger than that in the P(LLA-CL) group. More regenerated axons and Schwann cells were also observed in the SF/P(LLA-CL) group, which was consistent with the results of blood vessels. Collectively, our data revealed that the SF/P(LLA-CL) nerve conduit enhances peripheral nerve regeneration by improving angiogenesis within the conduit. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2070-2077, 2018. © 2018 Wiley Periodicals, Inc.

  13. CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*

    PubMed Central

    Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira

    2013-01-01

    CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360

  14. Jab1 regulates Schwann cell proliferation and axonal sorting through p27

    PubMed Central

    Porrello, Emanuela; Rivellini, Cristina; Dina, Giorgia; Triolo, Daniela; Del Carro, Ubaldo; Ungaro, Daniela; Panattoni, Martina; Feltri, Maria Laura; Wrabetz, Lawrence; Pardi, Ruggero; Quattrini, Angelo

    2014-01-01

    Axonal sorting is a crucial event in nerve formation and requires proper Schwann cell proliferation, differentiation, and contact with axons. Any defect in axonal sorting results in dysmyelinating peripheral neuropathies. Evidence from mouse models shows that axonal sorting is regulated by laminin211– and, possibly, neuregulin 1 (Nrg1)–derived signals. However, how these signals are integrated in Schwann cells is largely unknown. We now report that the nuclear Jun activation domain–binding protein 1 (Jab1) may transduce laminin211 signals to regulate Schwann cell number and differentiation during axonal sorting. Mice with inactivation of Jab1 in Schwann cells develop a dysmyelinating neuropathy with axonal sorting defects. Loss of Jab1 increases p27 levels in Schwann cells, which causes defective cell cycle progression and aberrant differentiation. Genetic down-regulation of p27 levels in Jab1-null mice restores Schwann cell number, differentiation, and axonal sorting and rescues the dysmyelinating neuropathy. Thus, Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation. Finally, Jab1 may constitute a key molecule in the pathogenesis of dysmyelinating neuropathies. PMID:24344238

  15. GDF10 Is a Signal for Axonal Sprouting and Functional Recovery after Stroke

    PubMed Central

    Li, S; Nie, EH; Yin, Y; Benowitz, LI; Tung, S; Vinters, HV; Bahjat, FR; Stenzel-Poore, MP; Kawaguchi, R; Coppola, G; Carmichael, ST

    2016-01-01

    Stroke produces a limited process of neural repair. Axonal sprouting in cortex adjacent to the infarct is part of this recovery process, but the signal that initiates axonal sprouting is not known. Growth and Differentiation Factor 10 (GDF10) is induced in peri-infarct neurons in mouse, non-human primate and human. GDF10 promotes axonal outgrowth in vitro in mouse, rat and human neurons through TGFβRI/II signaling. Using pharmacogenetic gain and loss of function studies, GDF10 produces axonal sprouting and enhanced functional recovery after stroke; knocking down GDF10 blocks axonal sprouting and reduces recovery. RNA-seq from peri-infarct cortical neurons indicates that GDF10 downregulates PTEN and upregulates PI3 kinase signaling and induces specific axonal guidance molecules. Unsupervised genome-wide association analysis of the GDF10 transcriptome shows that it is not related to neurodevelopment but may partially overlap with other CNS injury patterns. GDF10 is a stroke-induced signal for axonal sprouting and functional recovery. PMID:26502261

  16. Target-Derived Neurotrophins Coordinate Transcription and Transport of Bclw to Prevent Axonal Degeneration

    PubMed Central

    Cosker, Katharina E.; Pazyra-Murphy, Maria F.; Fenstermacher, Sara J.

    2013-01-01

    Establishment of neuronal circuitry depends on both formation and refinement of neural connections. During this process, target-derived neurotrophins regulate both transcription and translation to enable selective axon survival or elimination. However, it is not known whether retrograde signaling pathways that control transcription are coordinated with neurotrophin-regulated actions that transpire in the axon. Here we report that target-derived neurotrophins coordinate transcription of the antiapoptotic gene bclw with transport of bclw mRNA to the axon, and thereby prevent axonal degeneration in rat and mouse sensory neurons. We show that neurotrophin stimulation of nerve terminals elicits new bclw transcripts that are immediately transported to the axons and translated into protein. Bclw interacts with Bax and suppresses the caspase6 apoptotic cascade that fosters axonal degeneration. The scope of bclw regulation at the levels of transcription, transport, and translation provides a mechanism whereby sustained neurotrophin stimulation can be integrated over time, so that axonal survival is restricted to neurons connected within a stable circuit. PMID:23516285

  17. Premyelinated central axons express neurotoxic NMDA receptors: relevance to early developing white-matter injury

    PubMed Central

    Huria, Tahani; Beeraka, Narasimha Murthy; Al-Ghamdi, Badrah; Fern, Robert

    2015-01-01

    Ischemic-type injury to developing white matter is associated with the significant clinical condition cerebral palsy and with the cognitive deficits associated with premature birth. Premyelinated axons are the major cellular component of fetal white matter and loss of axon function underlies the disability, but the cellular mechanisms producing ischemic injury to premyelinated axons have not previously been described. Injury was found to require longer periods of modelled ischemia than at latter developmental points. Ischemia produced initial hyperexcitability in axons followed by loss of function after Na+ and Ca2+ influx. N-methyl-D-aspartate- (NMDA) type glutamate receptor (GluR) agonists potentiated axon injury while antagonists were protective. The NMDA GluR obligatory Nr1 subunit colocalized with markers of small premyelinated axons and expression was found at focal regions of axon injury. Ischemic injury of glial cells present in early developing white matter was NMDA GluR independent. Axons in human postconception week 18 to 23 white matter had a uniform prediameter expansion phenotype and postembedded immuno-gold labelling showed Nr1 subunit expression on the membrane of these axons, demonstrating a shared key neuropathologic feature with the rodent model. Premyelinated central axons therefore express high levels of functional NMDA GluRs that confer sensitivity to ischemic injury. PMID:25515212

  18. SRF phosphorylation by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons.

    PubMed

    Li, Cong L; Sathyamurthy, Aruna; Oldenborg, Anna; Tank, Dharmesh; Ramanan, Narendrakumar

    2014-03-12

    The growth of axons is an intricately regulated process involving intracellular signaling cascades and gene transcription. We had previously shown that the stimulus-dependent transcription factor, serum response factor (SRF), plays a critical role in regulating axon growth in the mammalian brain. However, the molecular mechanisms underlying SRF-dependent axon growth remains unknown. Here we report that SRF is phosphorylated and activated by GSK-3 to promote axon outgrowth in mouse hippocampal neurons. GSK-3 binds to and directly phosphorylates SRF on a highly conserved serine residue. This serine phosphorylation is necessary for SRF activity and for its interaction with MKL-family cofactors, MKL1 and MKL2, but not with TCF-family cofactor, ELK-1. Axonal growth deficits caused by GSK-3 inhibition could be rescued by expression of a constitutively active SRF. The SRF target gene and actin-binding protein, vinculin, is sufficient to overcome the axonal growth deficits of SRF-deficient and GSK-3-inhibited neurons. Furthermore, short hairpin RNA-mediated knockdown of vinculin also attenuated axonal growth. Thus, our findings reveal a novel phosphorylation and activation of SRF by GSK-3 that is critical for SRF-dependent axon growth in mammalian central neurons.

  19. Efficacy of Laser Photobiomodulation on Morphological and Functional Repair of the Facial Nerve.

    PubMed

    Buchaim, Daniela Vieira; Andreo, Jesus Carlos; Ferreira Junior, Rui Seabra; Barraviera, Benedito; Rodrigues, Antonio de Castro; Macedo, Mariana de Cássia; Rosa Junior, Geraldo Marco; Shinohara, Andre Luis; Santos German, Iris Jasmin; Pomini, Karina Torres; Buchaim, Rogerio Leone

    2017-08-01

    Evaluate the efficacy of low-level laser therapy (LLLT) on qualitative, quantitative, and functional aspects in the facial nerve regeneration process. Forty-two male Wistar rats were used, randomly divided into a control group (CG; n = 10), in which the facial nerve without lesion was collected, and four experimental groups: (1) suture experimental group (SEG) and (2) fibrin experimental group (FEG), consisting of 16 animals in which the buccal branch of the facial nerve was sectioned on both sides of the face; an end-to-end epineural suture was performed on the right side, and a fibrin sealant was used on the left side for coaptation of the stumps; and (3) laser suture experimental group (LSEG) and (4) laser fibrin experimental group (LFEG), consisting of 16 animals that underwent the same surgical procedures as SEG and FEG with the addition of laser application at three different points along the surgical site (pulsed laser of 830 nm wavelength, optical output power of 30 mW, power density of 0.2586 W/cm 2 , energy density of 6.2 J/cm 2 , beam area of 0.116 cm 2 , exposure time of 24 sec per point, total energy per session of 2.16 J, and cumulative dose of 34.56 J). The animals were submitted to functional analysis (subjective observation of whisker movement) and the data obtained were compared using Fisher's exact test. Euthanasia was performed at 5 and 10 weeks postoperative. The total number and density of regenerated axons were analyzed using the unpaired t-test (p < 0.05). Laser therapy resulted in a significant increase in the number and density of regenerated axons. The LSEG and LFEG presented better scores in functional analysis in comparison with the SEG and FEG. LLLT enhanced axonal regeneration and accelerated functional recovery of the whiskers, and both repair techniques allowed the growth of axons.

  20. Netrin-4 regulates thalamocortical axon branching in an activity-dependent fashion.

    PubMed

    Hayano, Yasufumi; Sasaki, Kensuke; Ohmura, Nami; Takemoto, Makoto; Maeda, Yurie; Yamashita, Toshihide; Hata, Yoshio; Kitada, Kazuhiro; Yamamoto, Nobuhiko

    2014-10-21

    Axon branching is remodeled by sensory-evoked and spontaneous neuronal activity. However, the underlying molecular mechanism is largely unknown. Here, we demonstrate that the netrin family member netrin-4 (NTN4) contributes to activity-dependent thalamocortical (TC) axon branching. In the postnatal developmental stages of rodents, ntn4 expression was abundant in and around the TC recipient layers of sensory cortices. Neuronal activity dramatically altered the ntn4 expression level in the cortex in vitro and in vivo. TC axon branching was promoted by exogenous NTN4 and suppressed by depletion of the endogenous protein. Moreover, unc-5 homolog B (Unc5B), which strongly bound to NTN4, was expressed in the sensory thalamus, and knockdown of Unc5B in thalamic cells markedly reduced TC axon branching. These results suggest that NTN4 acts as a positive regulator for TC axon branching through activity-dependent expression.