Autofluorescence of bovine ligamentum nuchae, cartilage, heart valve and lung measured by microscopy and fibre optics.

PubMed

Swatland, H J

1988-09-01

The fluorescence of bovine tissues was measured post mortem by microscopy of frozen sections and by using optical fibres to excite fluorescence and to measure fluorescence emission spectra. Mechanical disruption of the tissue (by comminution or sectioning) did not appreciably change tissue fluorescence spectra. Ligamentum nuchae had the strongest fluorescence and lung tissue had the weakest. In samples measured with a minimum prior exposure to ultraviolet light, the peak fluorescence emission was at 410 or 420 nm (with excitation at 365 nm). Exposure to ultraviolet light for about 1 minute shifted the fluorescence peak to 450 to 470 nm. Further exposure (about 30 minutes) caused a loss of the 450 to 470 nm fluorescence peak, while emissions above 530 nm were maintained or strengthened. Microscopy showed that the fluorescence that was measured by fibre optics from intact connective tissues originated mostly from collagen and elastin fibres.

Improved method of producing satisfactory sections of whole eyeball by routine histology.

PubMed

Arko-Boham, Benjamin; Ahenkorah, John; Hottor, Bismarck Afedo; Dennis, Esther; Addai, Frederick Kwaku

2014-02-01

To overcome the loss of structural integrity when eyeball sections are prepared by wax embedding, we experimentally modified the routine histological procedure and report satisfactorily well-preserved antero-posterior sections of whole eyeballs for teaching/learning purposes. Presently histological sections of whole eyeballs are not readily available because substantial structural distortions attributable to variable consistency of tissue components (and their undesired differential shrinkage) result from routine processing. Notably, at the dehydration stage of processing, the soft, gel-like vitreous humor considerably shrinks relative to the tough fibrous sclera causing collapse of the ocular globe. Additionally, the combined effects of fixation, dehydration, and embedding at 60°C renders the eye lens too hard for microtome slicing at thicknesses suitable for light microscopy. We satisfactorily preserved intact antero-posterior sections of eyeballs via routine paraffin wax processing procedure entailing two main modifications; (i) careful needle aspiration of vitreous humor and replacement with molten wax prior to wax infiltration; (ii) softening of lens in trimmed wax block by placing a drop of concentrated liquid phenol on it for 3 h during microtomy. These variations of the routine histological method produced intact whole eyeball sections with retinal detachment as the only structural distortion. Intact sections of the eyeball obtained compares well with the laborious, expensive, and 8-week long celloidin method. Our method has wider potential usability than costly freeze drying method which requires special skills and equipment (cryotome) and does not produce whole eyeball sections. Copyright © 2013 Wiley Periodicals, Inc.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

Collagen fibre characterisation in arterial tissue under load using SALS.

PubMed

Gaul, R T; Nolan, D R; Lally, C

2017-11-01

The collagen fibre architecture of arterial tissue is known to play a key role in its resultant mechanical behaviour, while maladaptive remodelling of this architecture may be linked to disease. Many of the techniques currently used to analyse collagen fibre architecture require time consuming tissue preparation procedures and are destructive in nature. The aim of this study is to fully explore Small Angle Light Scattering (SALS) as a means to non-destructively assess collagen fibre architecture in arterial tissue and subsequently gain insights into load induced reorientation. The optimised configuration of the SALS system for arterial tissue was determined using quantitative comparisons to histological analyses of porcine carotid artery as its basis. Once established, layer specific fibre orientation and the influence of tissue loading was determined for thin sections of carotid artery using SALS. This process was subsequently repeated for intact carotid artery layers. A single family of circumferentially orientated collagen fibres were found in the intima (- 0.1 ± 1.4° (5.5°)) and media (- 1.7 ± 1.9° (4.7°)) while two perpendicular families of fibres were identified in the adventitia (- 6.4 ± 0.7° (37.7°)) and (118.3 ± 2.7 (39.9°)). An increase in fibre alignment in response to a 20% circumferential strain was also identified using SALS, characterised by an increase in scattered light eccentricity. determined using SALS agreed with those found using traditional destructive techniques, however SALS has the important benefits of allowing vessel layers to remain intact, and has a fast processing time. SALS unique ability to identify load induced reorganisation in intact arterial layers offers an efficient means to gain crucial insights into arterial disease and its development over time. Copyright © 2017 Elsevier Ltd. All rights reserved.

49 CFR 173.137 - Class 8-Assignment of packing group.

Code of Federal Regulations, 2010 CFR

2010-10-01

... subchapter) as follows: (a) Packing Group I. Materials that cause full thickness destruction of intact skin... full thickness destruction of intact skin tissue within an observation period of up to 14 days starting... destruction of intact skin tissue within an observation period of up to 14 days starting after the exposure...

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PubMed

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

PubMed Central

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

The Structure Of Intact Side Tissue Loss Based On FTIR Spectroscopic Measurements

NASA Astrophysics Data System (ADS)

Hussain, N.; Al-Hadithi, K. O.; Jaafar, M. S.

2009-09-01

Laser applications in dentistry were strongly evolved during the last three decades. Among those applications are laser ablation of dental hard tissue, caries inhibition treatments by localized surface heating, and surface conditioning for bonding. In addition, infra-red lasers are ideally suited for the selective and precise removal of carious dental hard tissue while minimizing the healthy tissue loss. In the present study we applied laser spectroscopy technique FTIR for the study of the structure of intact side tissue of teeth. The aim of the recent work is to study the effect of race and sex (genealogy) on the structure of intact side tissue loss. Our sample consists of twenty Malay females' teeth where the FTIR has been applied. The data show a decrease in the amounts of main substances (like Hydroxyapatite crystals ([Ca5(PO4)3(OH)4], CaF2) than those in healthy teeth. The measured spectra represent the enamel with the characteristic peaks due to the phosphate group in carbonated, hydroxyapatite at 1000 cm-1 and two small peaks near 1500 cm-1 due to the carbonate group. The data explains the effect of the several factors on the intact side tissue loss.

Quantifying the ultrastructure of carotid arteries using high-resolution micro-diffusion tensor imaging—comparison of intact versus open cut tissue

NASA Astrophysics Data System (ADS)

Salman Shahid, Syed; Gaul, Robert T.; Kerskens, Christian; Flamini, Vittoria; Lally, Caitríona

2017-12-01

Diffusion magnetic resonance imaging (dMRI) can provide insights into the microstructure of intact arterial tissue. The current study employed high magnetic field MRI to obtain ultra-high resolution dMRI at an isotropic voxel resolution of 117 µm3 in less than 2 h of scan time. A parameter selective single shell (128 directions) diffusion-encoding scheme based on Stejskel-Tanner sequence with echo-planar imaging (EPI) readout was used. EPI segmentation was used to reduce the echo time (TE) and to minimise the susceptibility-induced artefacts. The study utilised the dMRI analysis with diffusion tensor imaging (DTI) framework to investigate structural heterogeneity in intact arterial tissue and to quantify variations in tissue composition when the tissue is cut open and flattened. For intact arterial samples, the region of interest base comparison showed significant differences in fractional anisotropy and mean diffusivity across the media layer (p  <  0.05). For open cut flat samples, DTI based directionally invariant indices did not show significant differences across the media layer. For intact samples, fibre tractography based indices such as calculated helical angle and fibre dispersion showed near circumferential alignment and a high degree of fibre dispersion, respectively. This study demonstrates the feasibility of fast dMRI acquisition with ultra-high spatial and angular resolution at 7 T. Using the optimised sequence parameters, this study shows that DTI based markers are sensitive to local structural changes in intact arterial tissue samples and these markers may have clinical relevance in the diagnosis of atherosclerosis and aneurysm.

A Novel Ex Vivo Method for Visualizing Live-Cell Calcium Response Behavior in Intact Human Tumors.

PubMed

Koh, James; Hogue, Joyce A; Sosa, Julie A

2016-01-01

The functional impact of intratumoral heterogeneity has been difficult to assess in the absence of a means to interrogate dynamic, live-cell biochemical events in the native tissue context of a human tumor. Conventional histological methods can reveal morphology and static biomarker expression patterns but do not provide a means to probe and evaluate tumor functional behavior and live-cell responsiveness to experimentally controlled stimuli. Here, we describe an approach that couples vibratome-mediated viable tissue sectioning with live-cell confocal microscopy imaging to visualize human parathyroid adenoma tumor cell responsiveness to extracellular calcium challenge. Tumor sections prepared as 300 micron-thick tissue slices retain viability throughout a >24 hour observation period and retain the native architecture of the parental tumor. Live-cell observation of biochemical signaling in response to extracellular calcium challenge in the intact tissue slices reveals discrete, heterogeneous kinetic waveform categories of calcium agonist reactivity within each tumor. Plotting the proportion of maximally responsive tumor cells as a function of calcium concentration yields a sigmoid dose-response curve with a calculated calcium EC50 value significantly elevated above published reference values for wild-type calcium-sensing receptor (CASR) sensitivity. Subsequent fixation and immunofluorescence analysis of the functionally evaluated tissue specimens allows alignment and mapping of the physical characteristics of individual cells within the tumor to specific calcium response behaviors. Evaluation of the relative abundance of intracellular PTH in tissue slices challenged with variable calcium concentrations demonstrates that production of the hormone can be dynamically manipulated ex vivo. The capability of visualizing live human tumor tissue behavior in response to experimentally controlled conditions opens a wide range of possibilities for personalized ex vivo therapeutic testing. This highly adaptable system provides a unique platform for live-cell ex vivo provocative testing of human tumor responsiveness to a range of physiological agonists or candidate therapeutic compounds.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.

PubMed

Treweek, Jennifer B; Chan, Ken Y; Flytzanis, Nicholas C; Yang, Bin; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2015-11-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.

Label-free volumetric optical imaging of intact murine brains

NASA Astrophysics Data System (ADS)

Ren, Jian; Choi, Heejin; Chung, Kwanghun; Bouma, Brett E.

2017-04-01

A central effort of today’s neuroscience is to study the brain’s ’wiring diagram’. The nervous system is believed to be a network of neurons interacting with each other through synaptic connection between axons and dendrites, therefore the neuronal connectivity map not only depicts the underlying anatomy, but also has important behavioral implications. Different approaches have been utilized to decipher neuronal circuits, including electron microscopy (EM) and light microscopy (LM). However, these approaches typically demand extensive sectioning and reconstruction for a brain sample. Recently, tissue clearing methods have enabled the investigation of a fully assembled biological system with greatly improved light penetration. Yet, most of these implementations, still require either genetic or exogenous contrast labeling for light microscopy. Here we demonstrate a high-speed approach, termed as Clearing Assisted Scattering Tomography (CAST), where intact brains can be imaged at optical resolution without labeling by leveraging tissue clearing and the scattering contrast of optical frequency domain imaging (OFDI).

Variability of ribosomal RNA genes in Rauwolfia species: parallelism between tissue culture-induced rearrangements and interspecies polymorphism.

PubMed

Andreev, I O; Spiridonova, K V; Solovyan, V T; Kunakh, V A

2005-01-01

An analysis of 18S-25S and 5S rRNA genes in intact plants and cultured tissues of some Rauwolfia species was performed to compare these sequences variability occurred as a result of the species evolution in nature and that induced by tissue culture. The restriction fragment length polymorphism of 18S-25S and 5S rDNA was found both in intact plants of various Rauwolfia species and in long-term Rauwolfia serpentina tissue cultures. In addition, changes in the amount of 18S-25S rRNA genes were observed in long-term R. serpentina tissue cultures. The results demonstrate that rDNA variability observed in intact plants as well as in long-term cultures is attributed to differences in the same regions of ribosomal RNA genes.

Collagenolytic protease expression in cranial cruciate ligament and stifle synovial fluid in dogs with cranial cruciate ligament rupture.

PubMed

Muir, Peter; Danova, Nichole A; Argyle, David J; Manley, Paul A; Hao, Zhengling

2005-01-01

To determine expression of collagenolytic genes and collagen degradation in stifle tissues of dogs with ruptured cranial cruciate ligament (CCL). Six dogs with CCL rupture and 11 dogs with intact CCL. Gene expression in CCL tissue and synovial fluid cells was studied using reverse transcriptase-polymerase chain reaction (RT-PCR). Collagen degradation was studied using CCL explant cultures and a synovial fluid bioassay. Expression of matrix metalloproteases (MMP) was not found in young Beagles with intact CCL; however, increased expression of MMP-3 was found in CCL tissue from older hounds with intact CCL, when compared with young Beagles. In dogs with ruptured CCL, expression of MMP-2 and -9 was increased in stifle tissues, when compared with dogs with intact CCL. Similar to MMP-9, expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin S was only found in stifle tissues from dogs with ruptured CCL; in contrast, expression of cathepsin K was found in all ruptured and intact CCL. Collagen degradation was increased in ruptured CCL, when compared with intact CCL. Rupture of the CCL is associated with up-regulation of expression of MMP-2 and -9 (gelatinase A and B), TRAP, and cathepsin S, and increased degradation of collagen. These findings suggest that MMP-2, -9, cathepsin S, and TRAP may be important mediators of progressive joint destruction in dogs with CCL rupture. These genes are markers for macrophages and dendritic cells. MMP and cathepsin S pathways may offer novel targets for anti-inflammatory medical therapy aimed at ameliorating joint degradation associated with inflammatory arthritis.

Giant kidney worm (Dioctophyma renale) infection mimicking retroperitoneal neoplasm.

PubMed

Sun, T; Turnbull, A; Lieberman, P H; Sternberg, S S

1986-07-01

A 50-year-old Chinese man was found by ultrasound and computed tomography to have a retroperitoneal mass in the right upper quadrant of the abdomen. At operation, a hemorrhagic cyst was detected at the upper pole of the right kidney adjacent to the adrenal gland. Microscopic examination revealed that the cyst wall was composed of granulomatous tissue loaded with eggs and cross-sections of parasites, identified as Dioctophyma renale. The eggs were characterized by a birefringent striated double wall. The presence of cross sections of adult worms of D. renale in human tissue has not been previously described. Another unique feature of this case was that the right kidney was intact, as examined grossly at laparotomy and by intravenous pyelography. Eggs were not detected in the urine.

Internalization of Escherichia coli O157:H7 gfp+ in rocket and Swiss chard baby leaves as affected by abiotic and biotic damage.

PubMed

Hartmann, R; Fricke, A; Stützel, H; Mansourian, S; Dekker, T; Wohanka, W; Alsanius, B

2017-07-01

Internalization of human pathogens in edible parts of vegetables eaten raw is a major concern, since once internalized they are protected from sanitizing treatments. In this study, we examined the invasion of gfp-labelled Escherichia coli O157:H7 into intact and biotically (infection with Xanthomonas campestris/Pseudomonas syringae) and abiotically (grating with silicon carbide) damaged leaves of wild rocket (Diplotaxis tenuifolia) and Swiss chard (Beta vulgaris subsp. cicla) using laser scanning confocal microscopy. Bacterial cells were found in internal locations of the tissue, irrespective of tissue health status. Contaminated leaf sections of biotically and abiotically damaged wild rocket leaves showed higher susceptibility to microbial invasion, while the pathogen was internalized in greater numbers into intact Swiss chard leaf sections when abiotically, but not biotically, damaged. The greatest differences were observed between the plant species; after surface sanitization, E. coli O157:H7 was still detected in wild rocket leaves, but not in Swiss chard leaves. Contamination of leafy vegetables with Escherichia coli O157:H7 is a growing problem, as reported outbreaks are increasing. However, establishment of this human pathogen in the phyllosphere is not completely understood. Using laser scanning confocal microscopy, we demonstrated that E. coli O157:H7gfp+ can invade plant tissue of Swiss chard and wild rocket leaves and that the bacterium is more sensitive to surface sanitization of Swiss chard leaves. Damage to leaf tissue promoted leaf invasion, but the nature of the damage (abiotic or biotic) and plant species had an impact. © 2017 The Society for Applied Microbiology.

Three-Dimensional Optical Mapping of Nanoparticle Distribution in Intact Tissues.

PubMed

Sindhwani, Shrey; Syed, Abdullah Muhammad; Wilhelm, Stefan; Glancy, Dylan R; Chen, Yih Yang; Dobosz, Michael; Chan, Warren C W

2016-05-24

The role of tissue architecture in mediating nanoparticle transport, targeting, and biological effects is unknown due to the lack of tools for imaging nanomaterials in whole organs. Here, we developed a rapid optical mapping technique to image nanomaterials in intact organs ex vivo and in three-dimensions (3D). We engineered a high-throughput electrophoretic flow device to simultaneously transform up to 48 tissues into optically transparent structures, allowing subcellular imaging of nanomaterials more than 1 mm deep into tissues which is 25-fold greater than current techniques. A key finding is that nanomaterials can be retained in the processed tissue by chemical cross-linking of surface adsorbed serum proteins to the tissue matrix, which enables nanomaterials to be imaged with respect to cells, blood vessels, and other structures. We developed a computational algorithm to analyze and quantitatively map nanomaterial distribution. This method can be universally applied to visualize the distribution and interactions of materials in whole tissues and animals including such applications as the imaging of nanomaterials, tissue engineered constructs, and biosensors within their intact biological environment.

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

PubMed Central

Treweek, Jennifer B; Deverman, Benjamin E; Greenbaum, Alon; Lignell, Antti; Xiao, Cheng; Cai, Long; Ladinsky, Mark S; Bjorkman, Pamela J; Fowlkes, Charless C; Gradinaru, Viviana

2016-01-01

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks. PMID:26492141

Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

PubMed

Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

2014-07-23

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

The sensitivity in the IR spectrum of the intact and pathological tissues by laser biophotometry.

PubMed

Ravariu, Cristian; Bondarciuc, Ala

2014-03-01

In this paper, we use the laser biophotometry for in vivo investigations, searching the most sensitive interactions of the near-infrared spectrum with different tissues. The experimental methods are based on the average reflection coefficient (ARC) measurements. For healthy persons, ARC is the average of five values provided by the biophotometer. The probe is applied on dry skin with minimum pilosity, in five regions: left-right shank, left-right forearm, and epigastrium. For the pathological tissues, the emitting terminal is moved over the suspected area, controlling the reflection coefficient level, till a minimum value occurs, as ARC-Pathological. Then, the probe is moved on the symmetrical healthy region of the body to read the complementary coefficient from intact tissue, ARC-Intact, from the same patient. The experimental results show an ARC range between 67 and 59 mW for intact tissues and a lower range, up to 58-42 mW, for pathological tissues. The method is efficient only in those pathological processes accompanied by variable skin depigmentation, water retention, inflammation, thrombosis, or swelling. Frequently, the ARC ranges are overlapping for some diseases. This induces uncertain diagnosis. Therefore, a statistical algorithm is adopted for a differential diagnosis. The laser biophotometry provides a quantitative biometric parameter, ARC, suitable for fast diagnosis in the internal and emergency medicine. These laser biophotometry measurements are representatives for the Romanian clinical trials.

Evaluation of mast cells, eosinophils, blood capillaries in oral lichen planus and oral lichenoid mucositis.

PubMed

Reddy, D Santhosh; Sivapathasundharam, B; Saraswathi, T R; SriRam, G

2012-01-01

Mast cells are granule containing secretory cells present in oral mucosal and connective tissue environment. Oral lichen planus and oral lichenoid lesions are commonly occurring oral diseases and have some similarity clinically and histologically. Both are characterized by an extensive sub epithelial infiltrate of T cells, together with mast cells, eosinophils and blood capillaries. In this study mast cell and eosinophil densities along with number of blood capillaries were studied to find out if they could aid in histopathological distinction between oral lichen planus and lichenoid mucositis. To enumerate mast cells and compare the status of Mast Cells (Intact or Degranulated) in Lichen planus, Lichenoid mucositis and normal buccal mucosa in tissue sections stained with Toluidine Blue, and also to enumerate Eosinophils and blood capillaries in tissue sections stained with H and E. The study group included 30 cases each of oral lichen planus and oral lichenoid mucositis. 10 cases of clinically normal oral buccal mucosa formed the control group. All the sections were stained with Toluidine blue and H and E separately. Histopathological analysis was done using binocular light microscope equipped with square ocular grid to standardize the field of evaluation. The result of the study showed. · Significant increase in number of mast cells in oral lichen planus and oral lichenoid mucositis compared to normal buccal mucosa. · Significant increase of intact mast cells suepithelially within the inflammatory cell infiltrate in oral lichen planus compared to oral lichenoid mucositis. · Significant increase of degranulated mast cells in oral lichenoid mucositis to oral lichen planus, and increase in number of eosinophil densities in oral lichenoid mucositis compared to oral lichen planus. · Significant increase in number of capillaries in oral lichenoid mucositis compared to oral lichen planus. The findings of increased number of intact mast cells sub epithelially in oral lichen planus to oral lichenoid mucositis and increase in number of degranulated mast cells as well as capillaries subepithelially in oral lichenoid mucositis to oral lichen planus can be used as reliable criteria for histologic distinction between these two lesions. The increase of eosinophils in oral lichenoid mucositis to oral lichen planus could be used as adjunct histologic criterion in the diagnosis of oral lichenoid mucositis.

Visualisation of Collagen in fixed skeletal muscle tissue using fluorescently tagged Collagen binding protein CNA35.

PubMed

Mohammadkhah, Melika; Simms, Ciaran K; Murphy, Paula

2017-02-01

Detection and visualisation of Collagen structure are important to understand the relationship between mechanical behaviour and microstructure in skeletal muscle since Collagen is the main structural protein in animal connective tissues, and is primarily responsible for their passive load-bearing properties. In the current study, the direct detection and visualization of Collagen using fluorescently tagged CNA35 binding protein (fused to EGFP or tdTomato) is reported for the first time on fixed skeletal muscle tissue. This Technical Note also establishes a working protocol by examining tissue preparation, dilution factor, exposure time etc. for sensitivity and specificity. Penetration of the binding protein into intact mature skeletal muscle was found to be very limited, but detection works well on tissue sections with higher sensitivity on wax embedded sections compared to frozen sections. CNA35 fused to tdTomato has a higher sensitivity than CNA35 fused to EGFP but both show specific detection. Best results were obtained with 15μm wax embedded sections, with blocking of non-specific binding in 1% BSA and antigen retrieval in Sodium Citrate. There was a play-off between dilution of the binding protein and time of incubation but both CNA35-tdTomato and CNA35-EGFP worked well with approximately 100μg/ml of purified protein with overnight incubation, while CNA35-tdTomato could be utilized at 5 fold less concentration. This approach can be applied to study the relationship between skeletal muscle micro-structure and to observe mechanical response to applied deformation. It can be used more broadly to detect Collagen in a variety of fixed tissues, useful for structure-functions studies, constitutive modelling, tissue engineering and assessment of muscle tissue pathologies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Osteonal effects on elastic modulus and fatigue life in equine bone.

PubMed

Gibson, V A; Stover, S M; Gibeling, J C; Hazelwood, S J; Martin, R B

2006-01-01

We hypothesized that recently formed, incompletely mineralized, and thus, relatively deformable osteons in the equine third metacarpus enhance in vitro load-controlled fatigue life in two ways. Macroscopically, there is a compliance effect, because reduced tissue elastic modulus diminishes the stress required to reach a given strain. Microscopically, there is a cement line effect, in which new osteons and their cement lines more effectively serve as barriers to crack propagation. We studied 18 4 x 10 x 100 mm beams from the medial, lateral, and dorsal cortices of metacarpal bones from 6 thoroughbred racehorses. Following load-controlled fatigue testing to fracture in 4 point bending, a transverse, 100 microm thick, basic fuchsin-stained cross-section was taken from the load-bearing region. The number and diameter of all intact (and thus recently formed/compliant) secondary osteons in a 3.8 x 3.8 mm region in the center of the section were determined. The associated area fraction and cement line length of intact osteons were calculated, and the relationships between these variables, elastic modulus (E), and the logarithm of fatigue life (logN(F)) were analyzed. As expected, logN(F) was negatively correlated with E, which was in turn negatively correlated with intact osteon area fraction and density. (LogN(F))/E increased in proportion to intact osteon density and nonlinearly with cement line density (mm/mm(2)). These results support the hypothesis that remodeling extends load-controlled fatigue life both through the creation of osteonal barriers to microdamage propagation and modulus reduction.

The importance of the biomimetic composites components for recreating the optical properties and molecular composition of intact dental tissues.

NASA Astrophysics Data System (ADS)

Seredin, P. V.; Goloshchapov, D. L.; Gushchin, M. S.; Ippolitov, Y. A.; Prutskij, T.

2017-11-01

The objective of this paper was to investigate whether it is possible to obtain biomimetic materials recreating the luminescent properties and molecular composition of intact dental tissues. Biomimetic materials were produced and their properties compared with native dental tissues. In addition, the overall contribution of the organic and non-organic components in the photoluminescence band was investigated. The results showed that it is possible to develop biomimetic materials with similar molecular composition and optical properties to native dental tissues for the early identification of dental caries.

Miniaturized fiber-coupled confocal fluorescence microscope with an electrowetting variable focus lens using no moving parts

PubMed Central

Ozbay, Baris N.; Losacco, Justin T.; Cormack, Robert; Weir, Richard; Bright, Victor M.; Gopinath, Juliet T.; Restrepo, Diego; Gibson, Emily A.

2015-01-01

We report a miniature, lightweight fiber-coupled confocal fluorescence microscope that incorporates an electrowetting variable focus lens to provide axial scanning for full three-dimensional (3D) imaging. Lateral scanning is accomplished by coupling our device to a laser-scanning confocal microscope through a coherent imaging fiber-bundle. The optical components of the device are combined in a custom 3D-printed adapter with an assembled weight of <2 g that can be mounted onto the head of a mouse. Confocal sectioning provides an axial resolution of ~12 µm and an axial scan range of ~80 µm. The lateral field-of-view is 300 µm, and the lateral resolution is 1.8 µm. We determined these parameters by imaging fixed sections of mouse neuronal tissue labeled with green fluorescent protein (GFP) and fluorescent bead samples in agarose gel. To demonstrate viability for imaging intact tissue, we resolved multiple optical sections of ex vivo mouse olfactory nerve fibers expressing yellow fluorescent protein (YFP). PMID:26030555

Novel Micropatterned Cardiac Cell Cultures with Realistic Ventricular Microstructure

PubMed Central

Badie, Nima; Bursac, Nenad

2009-01-01

Systematic studies of cardiac structure-function relationships to date have been hindered by the intrinsic complexity and variability of in vivo and ex vivo model systems. Thus, we set out to develop a reproducible cell culture system that can accurately replicate the realistic microstructure of native cardiac tissues. Using cell micropatterning techniques, we aligned cultured cardiomyocytes at micro- and macroscopic spatial scales to follow local directions of cardiac fibers in murine ventricular cross sections, as measured by high-resolution diffusion tensor magnetic resonance imaging. To elucidate the roles of ventricular tissue microstructure in macroscopic impulse conduction, we optically mapped membrane potentials in micropatterned cardiac cultures with realistic tissue boundaries and natural cell orientation, cardiac cultures with realistic tissue boundaries but random cell orientation, and standard isotropic monolayers. At 2 Hz pacing, both microscopic changes in cell orientation and ventricular tissue boundaries independently and synergistically increased the spatial dispersion of conduction velocity, but not the action potential duration. The realistic variations in intramural microstructure created unique spatial signatures in micro- and macroscopic impulse propagation within ventricular cross-section cultures. This novel in vitro model system is expected to help bridge the existing gap between experimental structure-function studies in standard cardiac monolayers and intact heart tissues. PMID:19413993

Lateral ligament repair and reconstruction restore neither contact mechanics of the ankle joint nor motion patterns of the hindfoot.

PubMed

Prisk, Victor R; Imhauser, Carl W; O'Loughlin, Padhraig F; Kennedy, John G

2010-10-20

Ankle sprains may damage both the lateral ligaments of the hindfoot and the osteochondral tissue of the ankle joint. When nonoperative treatment fails, operative approaches are indicated to restore both native motion patterns at the hindfoot and ankle joint contact mechanics. The goal of the present study was to determine the effect of lateral ligament injury, repair, and reconstruction on ankle joint contact mechanics and hindfoot motion patterns. Eight cadaveric specimens were tested with use of robotic technology to apply combined compressive (200-N) and inversion (4.5-Nm) loads to the hindfoot at 0° and 20° of plantar flexion. Contact mechanics at the ankle joint were simultaneously measured. A repeated-measures experiment was designed with use of the intact condition as control, with the other conditions including sectioned anterior talofibular and calcaneofibular ligaments, the Broström and Broström-Gould repairs, and graft reconstruction. Ligament sectioning decreased contact area and caused a medial and anterior shift in the center of pressure with inversion loads relative to those with the intact condition. There were no significant differences in inversion or coupled axial rotation with inversion between the Broström repair and the intact condition; however, medial translation of the center of pressure remained elevated after the Broström repair relative to the intact condition. The Gould modification of the Broström procedure provided additional support to the hindfoot relative to the Broström repair, reducing inversion and axial rotation with inversion beyond that of intact ligaments. There were no significant differences in center-of-pressure excursion patterns between the Broström-Gould repair and the intact ligament condition, but this repair increased contact area beyond that with the ligaments intact. Graft reconstruction more closely restored inversion motion than did the Broström-Gould repair at 20° of plantar flexion but limited coupled axial rotation. Graft reconstruction also increased contact areas beyond the lateral ligament-deficient conditions but altered center-of-pressure excursion patterns relative to the intact condition. No lateral ankle ligament reconstruction completely restored native contact mechanics of the ankle joint and hindfoot motion patterns.

Comparison of the Pharmacological Effects of a Novel Selective Androgen Receptor Modulator, the 5α-Reductase Inhibitor Finasteride, and the Antiandrogen Hydroxyflutamide in Intact Rats: New Approach for Benign Prostate Hyperplasia

PubMed Central

Gao, Wenqing; Kearbey, Jeffrey D.; Nair, Vipin A.; Chung, Kiwon; Parlow, A. F.; Miller, Duane D.; Dalton, James T.

2007-01-01

Tissue-selective androgen receptor modulators (SARMs) demonstrate tissue selectivity in both castrated and intact male rats, behaving as partial agonists in androgenic tissues (i.e. prostate and seminal vesicle), but full agonists in anabolic tissues (i.e. levator ani muscle). The partial agonist activity of SARMs (compounds S-1 and S-4) in the prostate of intact rats suggested that SARM could be used for androgen suppression in the treatment of benign prostate hyperplasia (BPH). This study was designed to explore the mechanisms of action of SARM and to characterize the tissue selectivity of S-1 in intact male rats compared with that of hydroxyflutamide (antiandrogen) and finasteride (5α-reductase inhibitor), two major drugs used for androgen suppression treatment of BPH. In intact male rats, S-1 (5, 10, and 25 mg/kg) selectively decreased the prostate weight with similar efficacy to finasteride (5 mg/kg), without affecting the levator ani muscle or increasing the plasma levels of testosterone, LH, and FSH. Hydroxyflutamide (0.5, 1, 5, 10, and 25 mg/kg), however, decreased both the prostate and levator ani muscle weights without any selectivity and increased plasma hormone levels in a dose-dependent manner. Furthermore, S-1 and S-4 showed very weak inhibitory effects toward transiently expressed type I and II human 5α-reductase (Ki, >20 µM) during in vitro assays. Therefore, although S-1 and finasteride showed very similar suppressive effects in the prostate of intact male rats, they decreased prostate size via different mechanisms of action. S-1 simply worked as androgen receptor partial agonist, whereas finasteride inhibited prostatic 5α-reductase. These studies indicate that SARMs may demonstrate clinical utility as single agent or combination therapy for BPH. PMID:15308613

Multiaxial mechanical response and constitutive modeling of esophageal tissues: Impact on esophageal tissue engineering.

PubMed

Sommer, Gerhard; Schriefl, Andreas; Zeindlinger, Georg; Katzensteiner, Andreas; Ainödhofer, Herwig; Saxena, Amulya; Holzapfel, Gerhard A

2013-12-01

Congenital defects of the esophagus are relatively frequent, with 1 out of 2500 babies suffering from such a defect. A new method of treatment by implanting tissue engineered esophagi into newborns is currently being developed and tested using ovine esophagi. For the reconstruction of the biological function of native tissues with engineered esophagi, their cellular structure as well as their mechanical properties must be considered. Since very limited mechanical and structural data for the esophagus are available, the aim of this study was to investigate the multiaxial mechanical behavior of the ovine esophagus and the underlying microstructure. Therefore, uniaxial tensile, biaxial tensile and extension-inflation tests on esophagi were performed. The underlying microstructure was examined in stained histological sections through standard optical microscopy techniques. Moreover, the uniaxial ultimate tensile strength and residual deformations of the tissue were determined. Both the mucosa-submucosa and the muscle layers showed nonlinear and anisotropic mechanical behavior during uniaxial, biaxial and inflation testing. Cyclical inflation of the intact esophageal tube caused marked softening of the passive esophagi in the circumferential direction. The rupture strength of the mucosa-submucosa layer was much higher than that of the muscle layer. Overall, the ovine esophagus showed a heterogeneous and anisotropic behavior with different mechanical properties for the individual layers. The intact and layer-specific multiaxial properties were characterized using a well-known three-dimensional microstructurally based strain-energy function. This novel and complete set of data serves the basis for a better understanding of tissue remodeling in diseased esophagi and can be used to perform computer simulations of surgical interventions or medical-device applications. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Assessment of eosinophil peroxidase as a potential diagnostic and prognostic marker in dogs with inflammatory bowel disease.

PubMed

Bastan, Idil; Robinson, Nicholas A; Ge, Xiao Na; Rendahl, Aaron K; Rao, Savita P; Washabau, Robert J; Sriramarao, P

2017-01-01

OBJECTIVE To evaluate a method for identifying intact and degranulated eosinophils in the small intestine of dogs with inflammatory bowel disease (IBD) by use of a monoclonal antibody (mAb) against eosinophil peroxidase (EPX). ANIMALS 11 untreated dogs with IBD, 5 dogs with IBD treated with prednisolone, and 8 control dogs with no clinical evidence of gastrointestinal tract disease and no immunosuppressive treatment. PROCEDURES 4-μm-thick sections of paraffin-embedded tissues from necropsy specimens were immunostained with EPX mAb. Stained intact and degranulated eosinophils in consecutive microscopic fields (400X magnification) of the upper (villus tips) and lower (between the muscularis mucosae and crypts) regions of the lamina propria of the jejunum were manually counted. RESULTS Compared with control and treated IBD dogs, untreated IBD dogs had a significantly higher number of degranulated eosinophils in the lower region of the lamina propria. However, no significant differences were detected in the number of intact eosinophils in this region among groups. In the upper region of the lamina propria, untreated IBD dogs had a significantly higher number of degranulated and intact eosinophils, compared with control and treated IBD dogs. Number of degranulated and intact eosinophils did not differ significantly between control and treated IBD dogs. CONCLUSIONS AND CLINICAL RELEVANCE Immunohistologic analysis with EPX mAb yielded prominent granule staining that allowed reliable morphological identification of degranulated and intact eosinophils, which may provide a strategy for quantitative and selective evaluation of eosinophils in gastrointestinal biopsy specimens and a potential method to diagnose IBD and evaluate treatment outcome.

In situ characterization of the brain-microdevice interface using Device Capture Histology

PubMed Central

Woolley, Andrew J.; Desai, Himanshi A.; Steckbeck, Mitchell A.; Patel, Neil K.; Otto, Kevin J.

2011-01-01

Accurate assessment of brain-implantable microdevice bio-integration remains a formidable challenge. Prevailing histological methods require device extraction prior to tissue processing, often disrupting and removing the tissue of interest which had been surrounding the device. The Device-Capture Histology method, presented here, overcomes many limitations of the conventional Device-Explant Histology method, by collecting the device and surrounding tissue intact for subsequent labeling. With the implant remaining in situ, accurate and precise imaging of the morphologically preserved tissue at the brain/microdevice interface can then be collected and quantified. First, this article presents the Device-Capture Histology method for obtaining and processing the intact, undisturbed microdevice-tissue interface, and images using fluorescent labeling and confocal microscopy. Second, this article gives examples of how to quantify features found in the captured peridevice tissue. We also share histological data capturing 1) the impact of microdevice implantation on tissue, 2) the effects of an experimental anti-inflammatory coating, 3) a dense grouping of cell nuclei encapsulating a long-term implant, and 4) atypical oligodendrocyte organization neighboring a longterm implant. Data sets collected using the Device-Capture Histology method are presented to demonstrate the significant advantages of processing the intact microdevice-tissue interface, and to underscore the utility of the method in understanding the effects of the brain-implantable microdevices on nearby tissue. PMID:21802446

In vitro comparison of human fibroblasts from intact and ruptured ACL for use in tissue engineering.

PubMed

Brune, T; Borel, A; Gilbert, T W; Franceschi, J P; Badylak, S F; Sommer, P

2007-12-17

The present study compares fibroblasts extracted from intact and ruptured human anterior cruciate ligaments (ACL) for creation of a tissue engineered ACL-construct, made of porcine small intestinal submucosal extracellular matrix (SIS-ECM) seeded with these ACL cells. The comparison is based on histological, immunohistochemical and RT-PCR analyses. Differences were observed between cells in a ruptured ACL (rACL) and cells in an intact ACL (iACL), particularly with regard to the expression of integrin subunits and smooth muscle actin (SMA). Despite these differences in the cell source, both cell populations behaved similarly when seeded on an SIS-ECM scaffold, with similar cell morphology, connective tissue organization and composition, SMA and integrin expression. This study shows the usefulness of naturally occurring scaffolds such as SIS-ECM for the study of cell behaviour in vitro, and illustrates the possibility to use autologous cells extracted from ruptured ACL biopsies as a source for tissue engineered ACL constructs.

An immunohistochemical identification key for cell types in adult mouse prostatic and urethral tissue sections

PubMed Central

Turco, Anne E.; Gottschalk, Adam; Halberg, Richard B.; Guo, Jinjin; McMahon, Jill A.; McMahon, Andrew P.

2017-01-01

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available. PMID:29145476

A novel, modernized Golgi-Cox stain optimized for CLARITY cleared tissue.

PubMed

Kassem, Mustafa S; Fok, Sandra Y Y; Smith, Kristie L; Kuligowski, Michael; Balleine, Bernard W

2018-01-15

High resolution neuronal information is extraordinarily useful in understanding the brain's functionality. The development of the Golgi-Cox stain allowed observation of the neuron in its entirety with unrivalled detail. Tissue clearing techniques, e.g., CLARITY and CUBIC, provide the potential to observe entire neuronal circuits intact within tissue and without previous restrictions with regard to section thickness. Here we describe an improved Golgi-Cox stain method, optimised for use with CLARITY and CUBIC that can be used in both fresh and fixed tissue. Using this method, we were able to observe neurons in their entirety within a fraction of the time traditionally taken to clear tissue (48h). We were also able to show for the first-time that Golgi stained tissue is fluorescent when visualized using a multi-photon microscope, allowing us to image synaptic spines with a detail previously unachievable. These novel methods provide cheap and easy to use techniques to investigate the morphology of cellular processes in the brain at a new-found depth, speed, utility and detail, without previous restrictions of time, tissue type and section thickness. This is the first application of a Golgi-Cox stain to cleared brain tissue, it is investigated and discussed in detail, describing different methodologies that may be used, a comparison between the different clearing techniques and lastly the novel interaction of these techniques with this ultra-rapid stain. Copyright © 2017 Elsevier B.V. All rights reserved.

Histological assessment of testicular residues in lambs and calves after Burdizzo castration.

PubMed

Stoffel, M H; von Rotz, A; Kocher, M; Merkli, M; Boesch, D; Steiner, A

2009-04-25

To assess the reliability of the Burdizzo procedure for castrating calves and lambs, testicular tissue from 63 bull calves (15 intact and 48 castrated) and 69 male lambs (35 intact and 34 castrated) was collected at slaughter and assessed histologically. The bull calves were castrated at either one, four to five or 12 to 16 weeks of age and the lambs at either one or 10 weeks. There was clear evidence of spermatogenesis in testicular tissue from all the intact animals. In the samples from the calves that had been castrated at 12 to 16 weeks functional testicular tissue was completely lacking. However, there was evidence of spermatogenesis and steroidogenesis in the calves that had been castrated at one week or four to five weeks, respectively. Failure to achieve complete involution of the testicular parenchyma was observed in the majority of lambs, irrespective of the age at which they had been castrated.

Proteasome activity and their subunit composition in endometrial cancer tissue: correlations with clinical morphological parameters.

PubMed

Spirina, L V; Kondakova, I V; Koval', V D; Kolomiets, L A; Chernyshova, A L; Choinzonov, E L; Sharova, N P

2012-08-01

The development of endometrial cancer is related to the status of the intracellular proteasome system. Total proteasome activity and pools 26S and 20S activities are higher in tumor tissue than in intact endometrium, and their composition is different. The expression of α1α2α3α5α6α7 is lower in endometrial cancer tissue in comparison with intact endometrium and the content of immune subunits LMP7, LMP2, and PA28β is increased. Total proteasome activity depends on the disease stage.

Cytological and ultrastructural preservation in Eocene Metasequoia leaves from the Canadian High Arctic.

PubMed

Schoenhut, Karimah; Vann, David R; Lepage, Ben A

2004-06-01

The ultrastructural examination by transmission electron microscopy of 45-million-year-old mummified leaves of Metasequoia extracted from the Upper Coal member of the Buchanan Lake Formation in Napartulik on Axel Heiberg Island revealed the preservation of intact chloroplasts and chloroplast components. Abundant tanniferous cell inclusions may indicate that the 3-mo period of constant daylight during the Artic summer induced high concentrations of tannins in the leaf tissues, which may have arrested microbial degradation of the litter. Quantified differences in the extent of chloroplast preservation through a vertical section of the lignite suggest that short-term shifts in the depositional environment took place, perhaps influencing the exposure of the leaf tissues to conditions that would either promote or inhibit decomposition.

Rapid wall relaxation in elongating tissues.

PubMed

Matyssek, R; Maruyama, S; Boyer, J S

1988-04-01

Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max [L.] Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, we investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. We found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached to the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. Previous reports of wall relaxation in pea included slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold should be constant during so short a time, thus reflecting the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species. The presence of slowly growing or mature tissue delays wall relaxation and should be avoided during such measurements. However, this delay can be used to advantage when turgor of intact growing tissues is being measured using excised tissues because turgor does not change for a considerable time after excision.

Rapid Wall Relaxation in Elongating Tissues 1

PubMed Central

Matyssek, Rainer; Maruyama, Sachio; Boyer, John S.

1988-01-01

Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max [L.] Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, we investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. We found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached to the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. Previous reports of wall relaxation in pea included slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold should be constant during so short a time, thus reflecting the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species. The presence of slowly growing or mature tissue delays wall relaxation and should be avoided during such measurements. However, this delay can be used to advantage when turgor of intact growing tissues is being measured using excised tissues because turgor does not change for a considerable time after excision. PMID:16666048

50 CFR 622.186 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

50 CFR 622.186 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.186 Section 622...-Grouper Fishery of the South Atlantic Region § 622.186 Landing fish intact. (a) South Atlantic snapper... specified in paragraph (b) of this section. Such fish may be eviscerated, gilled, and scaled, but must...

In vivo imaging of coral tissue and skeleton with optical coherence tomography

PubMed Central

Wentzel, Camilla; Jacques, Steven L.; Wagner, Michael

2017-01-01

Application of optical coherence tomography (OCT) for in vivo imaging of tissue and skeleton structure of intact living corals enabled the non-invasive visualization of coral tissue layers (endoderm versus ectoderm), skeletal cavities and special structures such as mesenterial filaments and mucus release from intact living corals. Coral host chromatophores containing green fluorescent protein-like pigment granules appeared hyper-reflective to near-infrared radiation allowing for excellent optical contrast in OCT and a rapid characterization of chromatophore size, distribution and abundance. In vivo tissue plasticity could be quantified by the linear contraction velocity of coral tissues upon illumination resulting in dynamic changes in the live coral tissue surface area, which varied by a factor of 2 between the contracted and expanded state of a coral. Our study provides a novel view on the in vivo organization of coral tissue and skeleton and highlights the importance of microstructural dynamics for coral ecophysiology. PMID:28250104

Rapid quantification of inflammation in tissue samples using perfluorocarbon emulsion and fluorine-19 nuclear magnetic resonance

PubMed Central

Ahrens, Eric T.; Young, Won-Bin; Xu, Hongyan; Pusateri, Lisa K.

2016-01-01

Quantification of inflammation in tissue samples can be a time-intensive bottleneck in therapeutic discovery and preclinical endeavors. We describe a versatile and rapid approach to quantitatively assay macrophage burden in intact tissue samples. Perfluorocarbon (PFC) emulsion is injected intravenously, and the emulsion droplets are effectively taken up by monocytes and macrophages. These ‘in situ’ labeled cells participate in inflammatory events in vivo resulting in PFC accumulation at inflammatory loci. Necropsied tissues or intact organs are subjected to conventional fluorine-19 (19F) NMR spectroscopy to quantify the total fluorine content per sample, proportional to the macrophage burden. We applied these methods to a rat model of experimental allergic encephalomyelitis (EAE) exhibiting extensive inflammation and demyelination in the central nervous system (CNS), particularly in the spinal cord. In a cohort of EAE rats, we used 19F NMR to derive an inflammation index (IFI) in intact CNS tissues. Immunohistochemistry was used to confirm intracellular colocalization of the PFC droplets within CNS CD68+ cells having macrophage morphology. The IFI linearly correlated to mRNA levels of CD68 via real-time PCR analysis. This 19F NMR approach can accelerate tissue analysis by at least an order of magnitude compared with histological approaches. PMID:21548906

Lateral Ligament Repair and Reconstruction Restore Neither Contact Mechanics of the Ankle Joint nor Motion Patterns of the Hindfoot

PubMed Central

Prisk, Victor R.; Imhauser, Carl W.; O'Loughlin, Padhraig F.; Kennedy, John G.

2010-01-01

Background: Ankle sprains may damage both the lateral ligaments of the hindfoot and the osteochondral tissue of the ankle joint. When nonoperative treatment fails, operative approaches are indicated to restore both native motion patterns at the hindfoot and ankle joint contact mechanics. The goal of the present study was to determine the effect of lateral ligament injury, repair, and reconstruction on ankle joint contact mechanics and hindfoot motion patterns. Methods: Eight cadaveric specimens were tested with use of robotic technology to apply combined compressive (200-N) and inversion (4.5-Nm) loads to the hindfoot at 0° and 20° of plantar flexion. Contact mechanics at the ankle joint were simultaneously measured. A repeated-measures experiment was designed with use of the intact condition as control, with the other conditions including sectioned anterior talofibular and calcaneofibular ligaments, the Broström and Broström-Gould repairs, and graft reconstruction. Results: Ligament sectioning decreased contact area and caused a medial and anterior shift in the center of pressure with inversion loads relative to those with the intact condition. There were no significant differences in inversion or coupled axial rotation with inversion between the Broström repair and the intact condition; however, medial translation of the center of pressure remained elevated after the Broström repair relative to the intact condition. The Gould modification of the Broström procedure provided additional support to the hindfoot relative to the Broström repair, reducing inversion and axial rotation with inversion beyond that of intact ligaments. There were no significant differences in center-of-pressure excursion patterns between the Broström-Gould repair and the intact ligament condition, but this repair increased contact area beyond that with the ligaments intact. Graft reconstruction more closely restored inversion motion than did the Broström-Gould repair at 20° of plantar flexion but limited coupled axial rotation. Graft reconstruction also increased contact areas beyond the lateral ligament-deficient conditions but altered center-of-pressure excursion patterns relative to the intact condition. Conclusions: No lateral ankle ligament reconstruction completely restored native contact mechanics of the ankle joint and hindfoot motion patterns. Clinical Relevance: Our results provide a rationale for conducting long-term, prospective, comparative, in vivo studies to assess the impact of altered mechanics following lateral ligament injury, and its nonoperative and operative treatment, on the development of ankle osteoarthritis. PMID:20962188

POWASSAN VIRUS: MORPHOLOGY AND CYTOPATHOLOGY.

PubMed

ABDELWAHAB, K S; ALMEIDA, J D; DOANE, F W; MCLEAN, D M

1964-05-02

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22 degrees C., first appeared in tissue culture supernatant fluids four days after inoculation.Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed.

Macroscopic multiphoton biomedical imaging using semiconductor saturable Bragg reflector mode-locked lasers

NASA Astrophysics Data System (ADS)

Girkin, John M.; Burns, David; Dawson, Martin D.

1999-06-01

We report on the development of practical and user friendly lasers for multiphoton imaging of biological material. The laser developed for the work is a laser diode pumped Cr:LiSAF source modelocked using a saturable Bragg reflector as the passive modelocking element. For this system we routinely obtain 100 fs pulses at a repetition rate 200 MHz with an average output power of 20 mW. The laser has a single operator control and is particularly suitable for use by non-laser specialists. We have used the source developed to image a range of biologically significant samples. The initial work has centered on the imaging of intact human dental tissue. The first two-photon images of dental tissue are reported showing the development of early dental disease from depths up to 500 micrometers into the tooth. These results demonstrate the detection of carious lesions before the more conventional techniques currently used by dental practitioners. Work on other living intact biological tissue is also reported, in particular plants containing a genetically bred fluorescent marker to enable the examination of complete and intact living plant tissue.

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Landing fish intact. 622.38 Section 622.38... Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish... specified in paragraphs (c) and (d) of this section. Such fish may be eviscerated, gilled, and scaled, but...

Gender discrimination in the influence of hyperglycemia and hyperosmolarity on rat aortic tissue responses to insulin.

PubMed

Wong, Nikki L; Achike, Francis I

2010-08-09

Hyperglycaemia initiates endothelial dysfunction causing diabetic macro- and micro-vasculopathy, the main causes of morbidity and mortality in diabetes mellitus. The vasculopathy exhibits gender peculiarities. We therefore explored gender differences in comparing the effects of hyperglycaemia (50 mM) per se with its hyperosmolar (50 mM) effects on vascular tissue responses to insulin. Endothelium-intact or denuded thoracic aortic rings from age-matched male and female Sprague-Dawley rats were incubated for 10 min or 6 h (acute versus chronic exposure) in normal, hyperglycaemic or hyperosmolar Krebs solution. Relaxant responses to insulin (6.9x10(-7)-6.9x10(-5) M) of the phenylephrine-contracted tissues were recorded. Endothelium denudation in both genders inhibited relaxation to insulin in all conditions, more significantly in female than in male tissues, suggesting the female response to insulin is more endothelium-dependent than the male. Acutely and chronically exposed normoglycemic endothelium-intact or -denuded tissues responded similarly to insulin. Chronic hyperglycemic or hyperosmolar exposure did not alter the endothelium-denuded tissue responses to insulin, whereas the responses of the endothelium-intact male and female hyperosmolar, and male hyperglycemic tissues were enhanced. The results show that insulin exerts an endothelium-dependent and independent relaxation with the female tissue responses more endothelium-dependent than the male. The data also suggest that hyperosmolarity per se enhances aortic tissue relaxant responses to insulin whereas hyperglycemia per se inhibits the same and more so in female than male tissues. These effects are endothelium-dependent. Copyright (c) 2010 Elsevier B.V. All rights reserved.

The response of MRI contrast parameters in in vitro tissues and tissue mimicking phantoms to fractionation by histotripsy

NASA Astrophysics Data System (ADS)

Allen, Steven P.; Vlaisavljevich, Eli; Shi, Jiaqi; Hernandez-Garcia, Luis; Cain, Charles A.; Xu, Zhen; Hall, Timothy L.

2017-09-01

Histotripsy is a non-invasive, focused ultrasound lesioning technique that can ablate precise volumes of soft tissue using a novel mechanical fractionation mechanism. Previous research suggests that magnetic resonance imaging (MRI) may be a sensitive image-based feedback mechanism for histotripsy. However, there are insufficient data to form some unified understanding of the response of the MR contrast mechanisms in tissues to histotripsy. In this paper, we investigate the response of the MR contrast parameters R1, R2, and the apparent diffusion coefficient (ADC) to various treatment levels of histotripsy in in vitro porcine liver, kidney, muscle, and blood clot as well in formulations of bovine red blood cells suspended in agar gel. We also make a histological analysis of histotripsy lesions in porcine liver. We find that R2 and the ADC are both sensitive to ablation in all materials tested here, and the degree of response varies with tissue type. Correspondingly, under histologic analysis, the porcine liver exhibited various levels of mechanical disruption and necrotic debris that are characteristic of histotripsy. While the area of intact red blood cells and nuclei found within these lesions both decreased with increasing amounts of treatment, the area of red blood cells decreased much more rapidly than the area of intact nuclei. Additionally, the decrease in area of intact red blood cells saturated at the same treatment levels at which the response of the R2 saturated while the area of intact nuclei appeared to vary linearly with the response of the ADC.

Contact lens-induced peripheral ulcers with extended wear of disposable hydrogel lenses: histopathologic observations on the nature and type of corneal infiltrate.

PubMed

Holden, B A; Reddy, M K; Sankaridurg, P R; Buddi, R; Sharma, S; Willcox, M D; Sweeney, D F; Rao, G N

1999-09-01

Contact lens-induced peripheral ulcer (CLPU), a sudden-onset adverse event observed with extended wear of hydrogel lenses, is characterized by a single, small, circular, focal anterior stromal infiltrate in the corneal periphery or midperiphery. The condition is always associated with a significant overlying epithelial loss and resolves in a scar. The aim was to determine, by using histopathologic techniques, the nature and type of the corneal infiltrate of these events. Three CLPUs observed in three patients using disposable hydrogel lenses on an extended-wear schedule were examined. The eye was topically anesthetized, and a corneal section including all of the infiltrate was taken. A small triangular piece of conjunctiva immediately adjacent to the infiltrate was sectioned. The tissue was immediately fixed, processed, stained using hematoxylin and eosin and periodic acid-Schiff stains, and examined by using light microscopy. The diameter of these three corneal infiltrates varied from 0.3 to 0.6 mm. Histopathology of the corneal sections revealed a focal epithelial loss corresponding to the infiltrated stroma in all three patients. The adjacent epithelium was thinned. Bowman's layer was intact in two patients and had a localized area of loss in the remaining patient. The anterior stroma was densely infiltrated with polymorphonuclear leukocytes and had focal areas of necrosis. The infiltration was most dense in the region immediately underlying Bowman's layer. No other infiltrative cell type was seen in any of the sections. Histopathology of the conjunctiva revealed features consistent with normal conjunctival tissue. On histopathology of CLPU, distinctive features (i.e., focal corneal epithelial loss, an intact Bowman's membrane, and a localized infiltration of the anterior stroma with polymorphonuclear leukocytes) were seen. These features suggest that the event is an acute inflammatory process and probably noninfective in nature.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Preparation of wholemount mouse intestine for high-resolution three-dimensional imaging using two-photon microscopy.

PubMed

Appleton, P L; Quyn, A J; Swift, S; Näthke, I

2009-05-01

Visualizing overall tissue architecture in three dimensions is fundamental for validating and integrating biochemical, cell biological and visual data from less complex systems such as cultured cells. Here, we describe a method to generate high-resolution three-dimensional image data of intact mouse gut tissue. Regions of highest interest lie between 50 and 200 mum within this tissue. The quality and usefulness of three-dimensional image data of tissue with such depth is limited owing to problems associated with scattered light, photobleaching and spherical aberration. Furthermore, the highest-quality oil-immersion lenses are designed to work at a maximum distance of

Fluorophilia: Fluorophore-containing compounds adhere non-specifically to injured neurons

PubMed Central

Hawkins, Bridget E.; Frederickson, Christopher J.; DeWitt, Douglas S.; Prough, Donald S.

2012-01-01

Ionic (free) zinc (Zn2+) is implicated in apoptotic neuronal degeneration and death. In our attempt to examine the effects of Zn2+ in neurodegeneration following brain injury, we serendipitously discovered that injured neurons bind fluorescein moieties, either alone or as part of an indicator dye, in histologic sections. This phenomenon, that we have termed “fluorophilia”, is analogous to the ability of degenerating neuronal somata and axons to bind silver ions (argyrophilia — the basis of silver degeneration stains). To provide evidence that fluorophilia occurs in sections of brain tissue, we used a wide variety of indicators such as Fluoro-Jade (FJ), a slightly modified fluorescein sold as a marker for degenerating neurons; Newport Green, a fluorescein-containing Zn2+ probe; Rhod-5N, a rhodamine-containing Ca2+ probe; and plain fluorescein. All yielded remarkably similar staining of degenerating neurons in the traumatic brain-injured tissue with the absence of staining in our sham-injured brains. Staining of presumptive injured neurons by these agents was not modified when Zn2+ in the brain section was removed by prior chelation with EDTA or TPEN, whereas staining by a non-fluorescein containing Zn2+ probe, N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ), was suppressed by prior chelation. Thus, certain fluorophore-containing compounds nonspecifically stain degenerating neuronal tissue in histologic sections and may not reflect the presence of Zn2+. This may be of concern to researchers using indicator dyes to detect metals in brain tissue sections. Further experiments may be advised to clarify whether Zn2+-binding dyes bind more specifically in intact neurons in culture or organotypic slices. PMID:22137653

Localization of ergot alkaloids in sclerotia of Claviceps purpurea by matrix-assisted laser desorption/ionization mass spectrometry imaging.

PubMed

Dopstadt, Julian; Vens-Cappell, Simeon; Neubauer, Lisa; Tudzynski, Paul; Cramer, Benedikt; Dreisewerd, Klaus; Humpf, Hans-Ulrich

2017-02-01

The fungus Claviceps purpurea produces highly toxic ergot alkaloids and accumulates these in the hardened bodies of fungal mycelium. These so-called sclerotia, or ergot bodies, replace the crop seed of infected plants, which can include numerous important food- and feedstuff such as rye and wheat. While several studies have explored details of the infection process and development of ergot bodies, little information is available on the spatial distribution of the mycotoxins in the sclerotia. Here we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at a lateral resolution of 35 μm to visualize the distribution of two representative alkaloids, ergocristine and ergometrine, produced by Ecc93 and Gal 310 variants of C. purpurea, respectively, after infection of rye. To improve cryosectioning of this fragile biological material tissue with complex texture, we developed a practical embedding protocol based on cellulose polymers. The MALDI-MS images recorded from the so produced intact tissues sections revealed that ergometrine exhibited a relatively homogeneous distribution throughout the ergot body, whereas ergocristine was found to be enriched in the proximal region. This finding can be correlated to the morphological development of sclerotia as ergot alkaloids are only produced in the sphacelial stage. The ability to localize toxins and other secondary metabolites in intact sections of crop-infecting fungi with high lateral resolution renders MALDI-MSI a powerful tool for investigating biosynthetic pathways and for obtaining a deeper understanding of the parasite-host interaction. Graphical abstract Workflow for identification and spatial localization of ergot alkaloids in infected rye grains.

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI)

PubMed Central

Hainsworth, A. H.; Lee, S.; Patel, A.; Poon, W. W.; Knight, A. E.

2018-01-01

Aims The spatial resolution of light microscopy is limited by the wavelength of visible light (the ‘diffraction limit’, approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Methods Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8–32 nm) and for SOFI (effective pixel size 80 nm). Results In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Conclusions Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. PMID:28696566

Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

PubMed

Hainsworth, A H; Lee, S; Foot, P; Patel, A; Poon, W W; Knight, A E

2018-06-01

The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach. © 2017 British Neuropathological Society.

Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology.

PubMed

Zijlmans, H J; Bonnet, J; Burton, J; Kardos, K; Vail, T; Niedbala, R S; Tanke, H J

1999-02-01

A novel luminescent reporter for the sensitive detection of antigens in tissue sections or on cell membranes is described. It consists of submicron-size phosphor crystals (0.2-0.4 microm), which are surface labeled with avidin or antibodies and capable of binding specifically to antigens on intact cells or in tissue sections. These phosphor reporters exhibit two-photon, anti-Stokes luminescence by up-converting infrared to visible light and are named Up-converting Phosphor Technology (UPT). They typically consist of yttriumoxysulfides doped with two different lanthanides exhibiting photostable, strong emission in the visible (blue, green, and red) upon excitation in the infrared. This report describes the conjugation of phosphor particles to NeutrAvidin with the subsequent use of this conjugate in a model system consisting of prostate-specific antigen in tissue sections and the CD4 membrane antigen on human lymphocytes. An epi-illumination fluorescence microscope was adapted to provide near-IR excitation using a xenon lamp for visualization of the visible emission. Advantages of UPT are (i) permanent, strong, anti-Stokes emission of discrete wavelengths; (ii) unmatched contrast in biological specimens due to the absence of autofluorescence upon excitation with IR light; (iii) simultaneous detection of multiple target analytes; and (iv) low-cost microscope modifications. The new methodology has not only high potential value in diagnostic pathology as described here, but may offer advantages for the detection of proteins or nucleic acids when applied to molecular biology, genomic research, virology, and microbiology. Copyright 1999 Academic Press.

In vivo imaging of coral tissue and skeleton with optical coherence tomography.

PubMed

Wangpraseurt, Daniel; Wentzel, Camilla; Jacques, Steven L; Wagner, Michael; Kühl, Michael

2017-03-01

Application of optical coherence tomography (OCT) for in vivo imaging of tissue and skeleton structure of intact living corals enabled the non-invasive visualization of coral tissue layers (endoderm versus ectoderm), skeletal cavities and special structures such as mesenterial filaments and mucus release from intact living corals. Coral host chromatophores containing green fluorescent protein-like pigment granules appeared hyper-reflective to near-infrared radiation allowing for excellent optical contrast in OCT and a rapid characterization of chromatophore size, distribution and abundance. In vivo tissue plasticity could be quantified by the linear contraction velocity of coral tissues upon illumination resulting in dynamic changes in the live coral tissue surface area, which varied by a factor of 2 between the contracted and expanded state of a coral. Our study provides a novel view on the in vivo organization of coral tissue and skeleton and highlights the importance of microstructural dynamics for coral ecophysiology. © 2017 The Author(s).

Extracellular matrix fragmentation in young, healthy cartilaginous tissues.

PubMed

Craddock, R J; Hodson, N W; Ozols, M; Shearer, T; Hoyland, J A; Sherratt, M J

2018-02-09

Although the composition and structure of cartilaginous tissues is complex, collagen II fibrils and aggrecan are the most abundant assemblies in both articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact aggrecan ( containing three globular domains) is well characterised, the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months), bovine AC and NP tissues, it was shown that, whilst the ultrastructure of intact aggrecan was tissue-dependent, most molecules (AC: 95 %; NP: 99.5 %) were fragmented (lacking one or more globular domains). Fragments were significantly smaller and more structurally heterogeneous in the NP compared with the AC (molecular area; AC: 8543 nm2; NP: 4625 nm2; p < 0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue-invariant. Molecular fragmentation is considered indicative of a pathology; however, these young, skeletally mature tissues were histologically and mechanically (reduced modulus: AC: ≈ 500 kPa; NP: ≈ 80 kPa) comparable to healthy tissues and devoid of notable gelatinase activity (compared with rat dermis). As aggrecan fragmentation was prevalent in neonatal bovine AC (99.5 % fragmented, molecular area: 5137 nm2) as compared with mature AC (95.0 % fragmented, molecular area: 8667 nm2), it was hypothesised that targeted proteolysis might be an adaptive process that modified aggrecan packing (as simulated computationally) and, hence, tissue charge density, mechanical properties and porosity. These observations provided a baseline against which pathological and/or age-related fragmentation of aggrecan could be assessed and suggested that new strategies might be required to engineer constructs that mimic the mechanical properties of native cartilaginous tissues.

50 CFR 622.381 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise be...

50 CFR 622.381 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.381 Section 622... Migratory Pelagic Resources (Gulf of Mexico and South Atlantic) § 622.381 Landing fish intact. (a) Cobia... head and fins intact. Such fish may be eviscerated, gilled, and scaled, but must otherwise be...

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

NASA Astrophysics Data System (ADS)

Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

2013-03-01

Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun

PubMed Central

O'Brien, John A; Lummis, Sarah CR

2009-01-01

Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h. PMID:17406443

50 CFR 622.276 - Landing fish intact.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated, gilled...

50 CFR 622.276 - Landing fish intact.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact. 622.276 Section 622... Wahoo Fishery Off the Atlantic States § 622.276 Landing fish intact. (a) Dolphin and wahoo in or from the Atlantic EEZ must be maintained with head and fins intact. Such fish may be eviscerated, gilled...

Orthotopic Implantation of Intact Tumor Tissue Leads to Metastasis of OCUM-2MD3 Human Gastric Cancer in Nude Mice Visualized in Real Time by Intravital Fluorescence Imaging.

PubMed

Murakami, Takashi; Zhang, Yong; Wang, Xiaoen; Hiroshima, Yukihiko; Kasashima, Hiroaki; Yashiro, Masakazu; Hirakawa, Kosei; Miwa, Atsushi; Kiyuna, Tasuku; Matsuyama, Ryusei; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Hoffman, Robert M

2016-05-01

Orthotopic (literally "correct place") implantation of cancer in nude mice has long been known to be superior to subcutaneous transplantation because the orthotopic tumor can metastasize. We reported previously on surgical orthotopic implantation (SOI) of gastric cancer tissue in nude mice resulting in the formation of metastases in 100% of the mice with extensive primary growth to the regional lymph nodes, liver, and lung. In contrast, when cell suspensions were used to inject gastric cancer cells orthotopically, metastases occurred in only 6.7% of the mice with local tumor formation, emphasizing the importance of orthotopically implanting intact tissue to allow full expression of metastatic potential. However, the different behavior of tumors implanted orthotopically by the two methods has not been visualized in real time. OCUM-2MD3 human gastric cancer cells labeled with the fluorescent protein Azami-Green were implanted orthotopically as cells or tissue in nude mice. Orthotopic implantation of cells resulted in local spread on the stomach. In contrast, SOI of tumor tissue of OCUM-2MD3 resulted in vessel spread of the Azami-Green-expressing cancer cells. Metastasis was also observed in the left lobe of the liver after SOI. These results demonstrate the physiological importance of intact cancer tissue for orthotopic implantation in order for tumors to properly grow and express their metastatic potential. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Time-Dependent Changes in T1 during Fracture Healing in Juvenile Rats: A Quantitative MR Approach

PubMed Central

Baron, Katharina; Neumayer, Bernhard; Amerstorfer, Eva; Scheurer, Eva; Diwoky, Clemens; Stollberger, Rudolf; Sprenger, Hanna; Weinberg, Annelie M.

2016-01-01

Quantitative magnetic resonance imaging (qMRI) offers several advantages in imaging and determination of soft tissue alterations when compared to qualitative imaging techniques. Although applications in brain and muscle tissues are well studied, its suitability to quantify relaxation times of intact and injured bone tissue, especially in children, is widely unknown. The objective observation of a fracture including its age determination can become of legal interest in cases of child abuse or maltreatment. Therefore, the aim of this study is the determination of time dependent changes in intact and corresponding injured bones in immature rats via qMRI, to provide the basis for an objective and radiation-free approach for fracture dating. Thirty-five MR scans of 7 Sprague-Dawley rats (male, 4 weeks old, 100 ± 5 g) were acquired on a 3T MRI scanner (TimTrio, Siemens AG, Erlangen, Germany) after the surgical infliction of an epiphyseal fracture in the tibia. The images were taken at days 1, 3, 7, 14, 28, 42 and 82 post-surgery. A proton density-weighted and a T1-weighted 3D FLASH sequence were acquired to calculate the longitudinal relaxation time T1 of the fractured region and the surrounding tissues. The calculation of T1 in intact and injured bone resulted in a quantitative observation of bone development in intact juvenile tibiae as well as the bone healing process in the injured tibiae. In both areas, T1 decreased over time. To evaluate the differences in T1 behaviour between the intact and injured bone, the relative T1 values (bone-fracture) were calculated, showing clear detectable alterations of T1 after fracture occurrence. These results indicate that qMRI has a high potential not only for clinically relevant applications to detect growth defects or developmental alterations in juvenile bones, but also for forensically relevant applications such as the dating of fractures in cases of child abuse or maltreatment. PMID:27832068

Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue.

PubMed

Delgado-Goñi, Teresa; Campo, Sonia; Martín-Sitjar, Juana; Cabañas, Miquel E; San Segundo, Blanca; Arús, Carles

2013-08-01

In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4-2.5 g of glucose; and 0.73-2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.

Powassan Virus: Morphology and Cytopathology

PubMed Central

Abdelwahab, K. S. E.; Almeida, J. D.; Doane, F. W.; McLean, D. M.

1964-01-01

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22° C., first appeared in tissue culture supernatant fluids four days after inoculation. Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5 PMID:14146854

The coming paradigm shift: A transition from manual to automated microscopy.

PubMed

Farahani, Navid; Monteith, Corey E

2016-01-01

The field of pathology has used light microscopy (LM) extensively since the mid-19(th) century for examination of histological tissue preparations. This technology has remained the foremost tool in use by pathologists even as other fields have undergone a great change in recent years through new technologies. However, as new microscopy techniques are perfected and made available, this reliance on the standard LM will likely begin to change. Advanced imaging involving both diffraction-limited and subdiffraction techniques are bringing nondestructive, high-resolution, molecular-level imaging to pathology. Some of these technologies can produce three-dimensional (3D) datasets from sampled tissues. In addition, block-face/tissue-sectioning techniques are already providing automated, large-scale 3D datasets of whole specimens. These datasets allow pathologists to see an entire sample with all of its spatial information intact, and furthermore allow image analysis such as detection, segmentation, and classification, which are impossible in standard LM. It is likely that these technologies herald a major paradigm shift in the field of pathology.

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy

PubMed Central

Zhang, Yuying; Akins, Meredith L.; Murari, Kartikeya; Xi, Jiefeng; Li, Ming-Jun; Luby-Phelps, Katherine; Mahendroo, Mala; Li, Xingde

2012-01-01

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth. PMID:22826263

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 50 Wildlife and Fisheries 12 2012-10-01 2012-10-01 false Landing fish intact. 622.38 Section 622... § 622.38 Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish on that vessel in the EEZ are maintained intact and, if taken from the EEZ, are...

50 CFR 622.38 - Landing fish intact.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Landing fish intact. 622.38 Section 622... § 622.38 Landing fish intact. The operator of a vessel that fishes in the EEZ is responsible for ensuring that fish on that vessel in the EEZ are maintained intact and, if taken from the EEZ, are...

Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

PubMed

Hayashi, Kei; Bhandal, Jitender; Kim, Sun Young; Rodriguez, Carlos O; Entwistle, Rachel; Naydan, Diane; Kapatkin, Amy; Stover, Susan M

2011-02-01

To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL. In vitro study. Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions. Intact CCL were collected from fresh canine cadavers free from stifle pathology. CCL tissue was processed for immunohistochemistry and stained for factor VIII and laminin. Vascular density was determined by histomorphometric analysis. Specific vascular staining was sparsely identified throughout the CCL; however, the proximal portion of the CCL appears to have a greater number of vessels than the middle or distal portion of the ligament. The CCL is a hypovascular tissue and its vascular distribution is not homogeneous. © Copyright 2010 by The American College of Veterinary Surgeons.

Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy

PubMed Central

German, Christopher L.; Gudheti, Manasa V.; Fleckenstein, Annette E.; Jorgensen, Erik M.

2018-01-01

Localization microscopy techniques – such as photoactivation localization microscopy (PALM), fluorescent PALM (FPALM), ground state depletion (GSD), and stochastic optical reconstruction microscopy (STORM) – provide the highest precision for single molecule localization currently available. However, localization microscopy has been largely limited to cell cultures due to the difficulties that arise in imaging thicker tissue sections. Sample fixation and antibody staining, background fluorescence, fluorophore photoinstability, light scattering in thick sections, and sample movement create significant challenges for imaging intact tissue. We have developed a sample preparation and image acquisition protocol to address these challenges in rat brain slices. The sample preparation combined multiple fixation steps, saponin permeabilization, and tissue clarification. Together, these preserve intracellular structures, promote antibody penetration, reduce background fluorescence and light scattering, and allow acquisition of images deep in a 30 μm thick slice. Image acquisition challenges were resolved by overlaying samples with a permeable agarose pad and custom-built stainless steel imaging adapter, and sealing the imaging chamber. This approach kept slices flat, immobile, bathed in imaging buffer, and prevented buffer oxidation during imaging. Using this protocol, we consistently obtained single molecule localizations of synaptic vesicle and active zone proteins in three-dimensions within individual synaptic terminals of the striatum in rat brain slices. These techniques may be easily adapted to the preparation and imaging of other tissues, substantially broadening the application of super-resolution imaging. PMID:28924666

3D confocal reconstruction of gene expression in mouse.

PubMed

Hecksher-Sørensen, J; Sharpe, J

2001-01-01

Three-dimensional computer reconstructions of gene expression data will become a valuable tool in biomedical research in the near future. However, at present the process of converting in situ expression data into 3D models is a highly specialized and time-consuming procedure. Here we present a method which allows rapid reconstruction of whole-mount in situ data from mouse embryos. Mid-gestation embryos were stained with the alkaline phosphotase substrate Fast Red, which can be detected using confocal laser scanning microscopy (CLSM), and cut into 70 microm sections. Each section was then scanned and digitally reconstructed. Using this method it took two days to section, digitize and reconstruct the full expression pattern of Shh in an E9.5 embryo (a 3D model of this embryo can be seen at genex.hgu.mrc.ac.uk). Additionally we demonstrate that this technique allows gene expression to be studied at the single cell level in intact tissue.

Metabolic changes in psoriatic skin under topical corticosteroid treatment.

PubMed

Sitter, Beathe; Johnsson, Margareta Karin; Halgunset, Jostein; Bathen, Tone Frost

2013-08-14

MR spectroscopy of intact biopsies can provide a metabolic snapshot of the investigated tissue. The aim of the present study was to explore the metabolic pattern of uninvolved skin, psoriatic skin and corticosteroid treated psoriatic skin. The three types of skin biopsy samples were excised from patients with psoriasis (N = 10). Lesions were evaluated clinically, and tissue biopsies were excised and analyzed by one-dimensional 1H MR spectroscopy. Relative levels were calculated for nine tissue metabolites. Subsequently, relative amounts of epidermis, dermis and subcutaneous tissue were scored by histopathological evaluation of HES stained sections. Seven out of 10 patients experienced at least 40% reduction in clinical score after corticosteroid treatment. Tissue biopsies from psoriatic skin contained lower levels of the metabolites myo-inositol and glucose, and higher levels of choline and taurine compared to uninvolved skin. In corticosteroid treated psoriatic skin, tissue levels of glucose, myo-inositol, GPC and glycine were increased, whereas choline was reduced, in patients with good therapeutic effect. These tissue levels are becoming more similar to metabolite levels in uninvolved skin. This MR method demonstrates that metabolism in psoriatic skin becomes similar to that of uninvolved skin after effective corticosteroid treatment. MR profiling of skin lesions reflect metabolic alterations related to pathogenesis and treatment effects.

Determination of emamectin residues in the tissues of Atlantic salmon (Salmo salar L.) using HPLC with fluorescence detection.

PubMed

Kim-Kang, H; Crouch, L S; Bova, A; Robinson, R A; Wu, J

2001-11-01

An accurate, reliable, and reproducible assay for the determination of residual concentrations of emamectin B(1a) in muscle, skin, and intact muscle/skin in natural proportions from Atlantic salmon treated with SCH 58854 (emamectin benzoate) is described. The determinative method was developed and validated using fortified control tissues at five levels over a range of 50-800 ng/g as well as tissues containing incurred levels in the same range. Incurred tissues were obtained from a metabolism study of [(3)H]emamectin benzoate in Atlantic salmon. The assay employs processing of a tissue ethyl acetate extract on a propylsulfonic acid solid phase extraction cartridge, followed by derivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. Following separation using reversed phase HPLC, the amount of derivatized emamectin B(1a) is determined by fluorescence detection. The theoretical limits of detection were determined from the analysis of control tissue matrices to be 2.6, 3.3, and 3.8 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. Likewise, the theoretical limits of quantitation (LOQ) were determined to be 6.9, 8.1, and 9.5 ng/g as emamectin B(1a) for muscle, skin, and intact muscle/skin, respectively. The lowest fortification level used for method validation was 50 ng/g, which served as the effective LOQ for the method. The overall percent recoveries (+/-% CV) were 94.4 +/- 6.89% (n = 25) for muscle, 88.4 +/- 5.35% (n = 25) for skin, and 88.0 +/- 3.73% for intact muscle/skin (n = 25). Accuracy, precision, linearity, selectivity, and ruggedness were demonstrated. The structure of the final fluorescent derivative of emamectin B(1a) free base was identified by ESI(+)/LC-MS. The frozen storage stability of [(3)H]emamectin B(1a) in tissues with incurred residues was demonstrated for approximately 15 months by radiometric analysis and for an additional approximately 13 months by fluorometric analysis for a total of approximately 28 months.

Express diagnostics of intact and pathological dental hard tissues by optical PNC method

NASA Astrophysics Data System (ADS)

Masychev, Victor I.; Alexandrov, Michail T.

2000-03-01

The results of hard tooth tissues research by the optical PNC- method in experimental and clinical conditions are presented. In the experiment under 90 test-sample of tooth slices with thickness about 1 mm (enamel, dentine and cement) were researched. The results of the experiment were processed by the method of correlation analyze. Clinical researches were executed on teeth of 210 patients. The regions of tooth tissue diseases with initial, moderate and deep caries were investigated. Spectral characteristics of intact and pathologically changed tooth tissues are presented and their peculiar features are discussed. The results the optical PNC- method application while processing tooth carious cavities are presented in order to estimate efficiency of the mechanical and antiseptic processing of teeth. It is revealed that the PNC-method can be used as for differential diagnostics of a degree dental carious stage, as for estimating of carefulness of tooth cavity processing before filling.

Biodistribution of iodine-125 and indium-111 labeled OV-TL 3 intact antibodies and F(ab')2 fragments in tumor-bearing athymic mice.

PubMed

Massuger, L F; Boerman, O C; Corstens, F H; Verheijen, R H; Claessens, R A; Poels, L G; van den Broek, W J; Kenemans, P

1991-01-01

The monoclonal antibody OV-TL 3, directed against an ovarian carcinoma-associated antigenic determinant, was tested as a vehicle for radioimmunolocalization of ovarian carcinomas in athymic mice bearing NIH:OVCAR-3 xenografts. The biodistribution of intact. OV-TL 3 was compared with the distribution of OC 125. Tumor uptake with OV-TL 3 was significantly higher than with OC 125, and almost 7 times higher than with a non-specific control antibody (OV-TL 19). Administration of a mixture of intact OV-TL 3 and OC 125 did not improve tumor uptake in comparison with OV-TL 3 alone. Subsequently, intact OV-TL 3 and its F(ab')2 fragments were labeled with either 111In or 125I. The highest tumor uptake was obtained with 111In-labeled intact OV-TL 3 (14.7% ID/g, 48 hr p.i.). For both antibody forms uptake of 111In in liver, spleen and kidneys was very high. Furthermore, 111In cleared more slowly from most tissues than 125I. As a result, tumor/tissue ratios with 111In-labeled OV-TL 3 were lower than with 125I-labeled OV-TL 3. The highest tumor/tissue ratios (6.9 to 53) were obtained with 125I-labeled OV-TL 3 F(ab')2 fragments, 48 hr post injection. 111In-labeled OV-TL 3 F(ab')2 has already been shown to be a clinically useful label for the detection of ovarian cancer. The results of our comparative animal study suggest that these clinical results may even be improved by using 123I-labeled OV-TL 3 F(ab')2.

Relaxant effect of ghrelin on guinea pig isolated tracheal smooth muscle: role of epithelial NO and PGE2.

PubMed

Al-Ayed, Mohammed Saeed Zayed

2018-06-01

This study aimed at investigating the potential ghrelin relaxing effect on guinea pig isolated tracheal smooth muscle (TSM). Using an in vitro experimental approach, the physiological role of the airway epithelium on smooth muscle relaxation has been investigated by analyzing the dose-response curves for carbachol- or histamine-induced contractions on epithelium intact versus denuded tracheal tissue. The relaxant effect of ghrelin (5-200 μmol/L) then investigated on carbachol-contracted, non-sensitized, and ovalbumin (OVA)-sensitized guinea pig TSM with an intact or denuded epithelium. The isolated TSMs from identical guinea pigs were incubated in Krebs solution aerated with 95% O 2 and 5% CO 2 through an automated tissue organ bath system (n = 6 for each group). The ghrelin relaxation mechanism was assessed by adding L-NAME, indomethacin, and YIL-781 for GHS-R1 into the tissue chamber. The spasmogens carbachol and histamine have shown a significantly higher contracting effect on epithelium-denuded than in epithelium-intact TSM confirmed by the significantly higher mean pEC50 of both agonists on the epithelium-denuded trachea (p < 0.05). Ghrelin has shown a concentration-dependent relaxing effect on carbachol-contracted TSM (r = 0.96, p = 0.00). The effect was more evident in the intact non-sensitized than in epithelium-denuded or OVA-sensitized groups (p < 0.05). Preincubation with nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) inhibitors has significantly reduced the ghrelin-induced relaxation on epithelium-intact TSM suggesting an epithelium-dependant mechanism. However, GHS-R1a antagonist has also succeeded to reduce ghrelin relaxant effect, which needs further clarification. Ghrelin proved to have a potential TSM relaxant effect possibly through epithelium-dependant mechanisms involving NO and PGE 2 .

Ultrastructural preservation of tissues and their reaction to the infection with trichinella in the El Plomo mummy: Muscle fiber ultrastructure and trichinosis/mummy of the Cerro El Plomo.

PubMed

Héctor, Rodríguez; Omar, Espinoza-Navarro; González, Mercedes; Mario, Castro

2017-08-01

The El Plomo mummy was a pre-Columbian Incan child who was found mummified in the Andes Mountains above an altitude of 17,700 feet. In the environment, natural mummification occurred due to low temperatures and strong winds. Dating measurements (relative dating) by experts from the National Museum of Natural History of Chile established that the mummified body corresponds the Inca period (1,450 to 1,500 AD). In 2003, the body was transferred to the University of Chile Medical School for exhaustive medical examination. Tissue samples from the right quadriceps muscle were extracted and fixed in glutaraldehyde and postfixed in osmium tetroxide to obtain ultrathin sections to be observed by transmission electron microscope. Images were recorded on photographic paper, digitalized and analyzed by experts on morphology. Results showed a preservation of cell boundaries in striated muscle cells, but specific subcellular organelles or contractile sarcomeric units (actin and myosin) were unable to be recognized. However, the classical ultrastructural morphology of the polypeptide collagen type I was preserved intact both in primary and secondary organization. Therefore, we concluded that the process of natural mummification by freezing and strong winds is capable of damaging the ultrastructure of muscle cells and preserving collagen type I intact. © 2017 Wiley Periodicals, Inc.

A novel slice preparation to study medullary oromotor and autonomic circuits in vitro

PubMed Central

Nasse, Jason S.

2014-01-01

Background The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. New method This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. Results: Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. Comparison with existing methods This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. Conclusions Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section. PMID:25196216

A novel slice preparation to study medullary oromotor and autonomic circuits in vitro.

PubMed

Nasse, Jason S

2014-11-30

The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. Immunohistochemical staining against choline-acetyl-transferase and dopamine-β-hydroxylase demonstrated that within a 450 μm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section. Published by Elsevier B.V.

Fast widefield techniques for fluorescence and phase endomicroscopy

NASA Astrophysics Data System (ADS)

Ford, Tim N.

Endomicroscopy is a recent development in biomedical optics which gives researchers and physicians microscope-resolution views of intact tissue to complement macroscopic visualization during endoscopy screening. This thesis presents HiLo endomicroscopy and oblique back-illumination endomicroscopy, fast wide-field imaging techniques with fluorescence and phase contrast, respectively. Fluorescence imaging in thick tissue is often hampered by strong out-of-focus background signal. Laser scanning confocal endomicroscopy has been developed for optically-sectioned imaging free from background, but reliance on mechanical scanning fundamentally limits the frame rate and represents significant complexity and expense. HiLo is a fast, simple, widefield fluorescence imaging technique which rejects out-of-focus background signal without the need for scanning. It works by acquiring two images of the sample under uniform and structured illumination and synthesizing an optically sectioned result with real-time image processing. Oblique back-illumination microscopy (OBM) is a label-free technique which allows, for the first time, phase gradient imaging of sub-surface morphology in thick scattering tissue with a reflection geometry. OBM works by back-illuminating the sample with the oblique diffuse reflectance from light delivered via off-axis optical fibers. The use of two diametrically opposed illumination fibers allows simultaneous and independent measurement of phase gradients and absorption contrast. Video-rate single-exposure operation using wavelength multiplexing is demonstrated.

Non-destructive optical clearing technique enhances optical coherence tomography (OCT) for real-time, 3D histomorphometry of brain tissue (Conference Presentation)

NASA Astrophysics Data System (ADS)

Paul, Akshay; Chang, Theodore H.; Chou, Li-Dek; Ramalingam, Tirunelveli S.

2016-03-01

Evaluation of neurodegenerative disease often requires examination of brain morphology. Volumetric analysis of brain regions and structures can be used to track developmental changes, progression of disease, and the presence of transgenic phenotypes. Current standards for microscopic investigation of brain morphology are limited to detection of superficial structures at a maximum depth of 300μm. While histological techniques can provide detailed cross-sections of brain structures, they require complicated tissue preparation and the ultimate destruction of the sample. A non-invasive, label-free imaging modality known as Optical Coherence Tomography (OCT) can produce 3-dimensional reconstructions through high-speed, cross-sectional scans of biological tissue. Although OCT allows for the preservation of intact samples, the highly scattering and absorbing properties of biological tissue limit imaging depth to 1-2mm. Optical clearing agents have been utilized to increase imaging depth by index matching and lipid digestion, however, these contemporary techniques are expensive and harsh on tissues, often irreversibly denaturing proteins. Here we present an ideal optical clearing agent that offers ease-of-use and reversibility. Similar to how SeeDB has been effective for microscopy, our fructose-based, reversible optical clearing technique provides improved OCT imaging and functional immunohistochemical mapping of disease. Fructose is a natural, non-toxic sugar with excellent water solubility, capable of increasing tissue transparency and reducing light scattering. We will demonstrate the improved depth-resolving performance of OCT for enhanced whole-brain imaging of normal and diseased murine brains following a fructose clearing treatment. This technique potentially enables rapid, 3-dimensional evaluation of biological tissues at axial and lateral resolutions comparable to histopathology.

A pliable electroporation patch (ep-Patch) for efficient delivery of nucleic acid molecules into animal tissues with irregular surface shapes.

PubMed

Wei, Zewen; Huang, Yuanyu; Zhao, Deyao; Hu, Zhiyuan; Li, Zhihong; Liang, Zicai

2015-01-05

Delivery of nucleic acids into animal tissues by electroporation is an appealing approach for various types of gene therapy, but efficiency of existing methodsis not satisfactory. Here we present the validation of novel electroporation patch (ep-Patch) for efficient delivery of DNA and siRNA into mouse tissues. Using micromachining technology, closely spaced gold electrodes were made on the pliable parylene substrate to form a patch-like electroporation metrics. It enabled large coverage of the target tissues and close surface contact between the tissues and electrodes, thus providing a uniform electric field to deliver nucleic acids into tissues, even beneath intact skin. Using this ep-Patch for efficiently delivery of both DNA and siRNA, non-invasive electroporation of healthy mouse muscle tissue was successfully achieved. Delivery of these nucleic acids was performed to intact tumors with satisfactory results. Silencing of tumor genes using the ep-Patch was also demonstrated on mice. This pliable electroporation patch method constitutes a novel way of in vivo delivery of siRNA and DNA to certain tissues or organs to circumvent the disadvantages of existing methodologies for in vivo delivery of nucleic acid molecules.

Skin autofluorescence associates with vascular calcification in chronic kidney disease.

PubMed

Wang, Angela Yee-Moon; Wong, Chun-Kwok; Yau, Yat-Yin; Wong, Sharon; Chan, Iris Hiu-Shuen; Lam, Christopher Wai-Kei

2014-08-01

This study aims to evaluate the relationship between tissue advanced glycation end products, as reflected by skin autofluorescence, and vascular calcification in chronic kidney disease. Three hundred patients with stage 3 to 5 chronic kidney disease underwent multislice computed tomography to estimate total coronary artery calcium score (CACS) and had tissue advanced glycation end product assessed using a skin autofluorescence reader. Intact parathyroid hormone (P<0.001) displaced estimated glomerular filtration rate as third most significant factor associated with skin autofluorescence after age (P<0.001) and diabetes mellitus (P<0.001) in multiple regression analysis. On univariate multinomial logistic regression analysis, every 1-U increase in skin autofluorescence was associated with a 7.43-fold (95% confidence intervals, 3.59-15.37; P<0.001) increased odds of having CACS ≥400 compared with those with zero CACS. Skin autofluorescence retained significance in predicting CACS ≥400 (odds ratio, 3.63; 95% confidence intervals, 1.44-9.18; P=0.006) when adjusting for age, sex, serum calcium, phosphate, albumin, C-reactive protein, lipids, blood pressure, estimated glomerular filtration rate, and intact parathyroid hormone but marginally lost significance when additionally adjusting for diabetes mellitus (odds ratio, 2.23; 95% confidence intervals, 0.81-6.14; P=0.1). Combination of diabetes mellitus and higher intact parathyroid hormone was associated with greater skin autofluorescence and CACS versus those without diabetes mellitus and having lower intact parathyroid hormone. Tissue advanced glycation end product, as reflected by skin autofluorescence, showed a significant novel association with vascular calcification in chronic kidney disease. These data suggest that increased tissue advanced glycation end product may contribute to vascular calcification in chronic kidney disease and diabetes mellitus and warrant further experimental investigation. © 2014 American Heart Association, Inc.

A physiological basis for variation in the contractile properties of isolated rat heart.

PubMed Central

Lin, L E; McClellan, G; Weisberg, A; Winegrad, S

1991-01-01

1. The maximum Ca(2+)-activated force, maximum velocity of unloaded shortening and both Ca(2+)- and actin-activated ATPase activities of myosin have been measured in detergent-skinned preparations of isolated bundles of rat right ventricle after exposure of the intact tissue to different conditions of superfusion, mechanical activity and temperature. 2. Maximum Ca(2+)-activated force per unit cross-sectional area decreases with increasing cross-sectional area, and, in the absence of electrical stimulation, with the duration of superfusion. Maximum velocity of unloaded shortening is not influenced by these differences. 3. Actin-activated ATPase activity of myosin decreases as cross-sectional area increases and duration of superfusion increases, but the extent of the decrease in enzymatic activity is less than that of developed force. Ca(2+)-activated ATPase activity is independent of these differences. 4. Actin-activated ATPase activity in cryostatic sections of quickly frozen tissue is not uniform across the transverse section. In thin bundles, it is highest in the centre and lowest at the edge of the section, which correspond, respectively, to the centre and the surface of the tissue bundle. Exposure of the tissue section to 1 microM-cyclic AMP increases the actin-activated ATPase activity of myosin with the largest increase in activity occurring at or near the surface of the bundle. 5. Ca(2+)-activated ATPase activity of myosin is uniform across the transverse section and is not changed by cyclic AMP. 6. Electrical stimulation, elevated Ca2+ concentration in the superfusion medium, or isoprenaline partially or completely reverse the decline in maximum Ca(2+)-activated force produced by prolonged superfusion of the bundle before its skinning. 7. These observations are similar in many ways to those made on frog skeletal muscles by Elzinga, Howarth, Rull, Wilson & Woledge (1989a). An explanation based on the existence of a physiological mechanism for regulating the properties of force generators is proposed. Regulation of the attachment of the cross-bridge to an actin filament may be the basis for the regulatory mechanism. Images Fig. 4 Fig. 7 PMID:1667804

Transport of sup 14 C-IAA and sup 14 C-ACC within floral organs of Ipomoea nil

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

Kiss, H.G.; Maurice, H.R.; Koning, R.E.

1989-04-01

The transport of {sup 14}C-IAA {sup 14}C-ACC from agarose donor blocks applied to I. nil filaments their recovery as {sup 14}C-accumulation into floral organs was examined. The accumulation of the isotopes in the corolla tissue was greater when {sup 14}C-ACC was applied than {sup 14}C-IAA in intact isolated flower buds. Greater levels of the isotopes accumulated in the pistil, with minimal levels in receptacle and calyx tissues from isolated buds. With intact buds, greater levels of the isotopes were recovered in pistil, calyx receptacle tissues. This study provides further evidence for the role of the filaments as transport vectors formore » IAA ACC for the production of ethylene.« less

Rapid wall relaxation in elongating tissues. [Glycine max (L. ); Pisum sativum L

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

Matyssek, R.; Maruyama, S.; Boyer, J.S.

1988-01-01

Reported differences in the relaxation of cell walls in enlarging stem tissues of soybean (Glycine max (L.) Merr.) and pea (Pisum sativum L.) cause measurements of the yield threshold turgor, an important growth parameter, to be in doubt. Using the pressure probe and guillotine psychrometer, the authors investigated wall relaxation in these species by excising the elongating tissue in air to remove the water supply. The authors found that the rapid kinetics usually exhibited by soybean could be delayed and made similar to the slow kinetics previously reported for pea if slowly growing or mature tissue was left attached tomore » the rapidly growing tissue when relaxation was initiated. The greater the amount of attached tissue, the slower the relaxation, suggesting that slowly growing tissue acted as a water source. Consistent with this concept was a lower water potential in the rapidly elongating tissue than in the slowly growing tissue. If this tissue was removed from pea, relaxation became as rapid as usually exhibited by soybean. It is concluded that the true relaxation of cell walls to the yield threshold requires only a few minutes and that the yield threshold in the intact plant before excision. Under these conditions, the yield threshold was close to the turgor in the intact plant regardless of the species.« less

Assays for the activities of polyamine biosynthetic enzymes using intact tissues

Treesearch

Rakesh Minocha; Stephanie Long; Hisae Maki; Subhash C. Minocha

1999-01-01

Traditionally, most enzyme assays utilize homogenized cell extracts with or without dialysis. Homogenization and centrifugation of large numbers of samples for screening of mutants and transgenic cell lines is quite cumbersome and generally requires sufficiently large amounts (hundreds of milligrams) of tissue. However, in situations where the tissue is available in...

Novel Passive Clearing Methods for the Rapid Production of Optical Transparency in Whole CNS Tissue.

PubMed

Woo, Jiwon; Lee, Eunice Yoojin; Park, Hyo-Suk; Park, Jeong Yoon; Cho, Yong Eun

2018-05-08

Since the development of CLARITY, a bioelectrochemical clearing technique that allows for three-dimensional phenotype mapping within transparent tissues, a multitude of novel clearing methodologies including CUBIC (clear, unobstructed brain imaging cocktails and computational analysis), SWITCH (system-wide control of interaction time and kinetics of chemicals), MAP (magnified analysis of the proteome), and PACT (passive clarity technique), have been established to further expand the existing toolkit for the microscopic analysis of biological tissues. The present study aims to improve upon and optimize the original PACT procedure for an array of intact rodent tissues, including the whole central nervous system (CNS), kidneys, spleen, and whole mouse embryos. Termed psPACT (process-separate PACT) and mPACT (modified PACT), these novel techniques provide highly efficacious means of mapping cell circuitry and visualizing subcellular structures in intact normal and pathological tissues. In the following protocol, we provide a detailed, step-by-step outline on how to achieve maximal tissue clearance with minimal invasion of their structural integrity via psPACT and mPACT.

Mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development.

PubMed

Walton, Katherine D; Kolterud, Asa

2014-09-04

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine(1). Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought(1). The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth(2). Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Systematic Quantification of Stabilizing Effects of Subtalar Joint Soft-Tissue Constraints in a Novel Cadaveric Model.

PubMed

Pellegrini, Manuel J; Glisson, Richard R; Wurm, Markus; Ousema, Paul H; Romash, Michael M; Nunley, James A; Easley, Mark E

2016-05-18

Distinguishing between ankle instability and subtalar joint instability is challenging because the contributions of the subtalar joint's soft-tissue constraints are poorly understood. This study quantified the effects on joint stability of systematic sectioning of these constraints followed by application of torsional and drawer loads simulating a manual clinical examination. Subtalar joint motion in response to carefully controlled inversion, eversion, internal rotation, and external rotation moments and multidirectional drawer forces was quantified in fresh-frozen cadaver limbs. Sequential measurements were obtained under axial load approximating a non-weight-bearing clinical setting with the foot in neutral, 10° of dorsiflexion, and 10° and 20° of plantar flexion. The contributions of the components of the inferior extensor retinaculum were documented after incremental sectioning. The calcaneofibular, cervical, and interosseous talocalcaneal ligaments were then sectioned sequentially, in two different orders, to produce five different ligament-insufficiency scenarios. Incremental detachment of the components of the inferior extensor retinaculum had no effect on subtalar motion independent of foot position. Regardless of the subsequent ligament-sectioning order, significant motion increases relative to the intact condition occurred only after transection of the calcaneofibular ligament. Sectioning of this ligament produced increased inversion and external rotation, which was most evident with the foot dorsiflexed. Calcaneofibular ligament disruption results in increases in subtalar inversion and external rotation that might be detectable during a manual examination. Insufficiency of other subtalar joint constraints may result in motion increases that are too subtle to be perceptible. If calcaneofibular ligament insufficiency is established, its reconstruction or repair should receive priority over that of other ankle or subtalar periarticular soft-tissue structures. Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.

Low proton conductance of plant cuticles and its relevance to the acid-growth theory

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

Dreyer, S.A.; Seymour, V.; Cleland, R.E.

1981-09-01

Evidence obtained on the relation between the pH of the medium and the growth of intact stem sections is compatible with the acid-growth theory only if the proton conductance of the cuticle is an effective barrier to the entry or exit of protons from the tissue. By measuring the rate at which protons cross frozen-thawed epidermal strips of sunflower (Helianthus annus L.) and soybean hypocotyls (Glycine max Morr.) and enzymically isolated cuticles of Berberis aquifolium Persh. and tomato (Lycopersicum esculentum Mill.) fruit, we have now demonstrated the low proton conductance of the cuticular layer. Unless the conductance is enhanced bymore » abrasion of the cuticle or by removal of the cuticular waxes, proton movement into and out of a tissue across the cuticle will be significant only over long time periods.« less

Comparison of Ripening Processes in Intact Tomato Fruit and Excised Pericarp Discs 1

PubMed Central

Campbell, Alan D.; Huysamer, Marius; Stotz, Henrik U.; Greve, L. Carl; Labavitch, John M.

1990-01-01

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L*a*b* color space, changing from green (a* < −5) to red (a* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO2 production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a* = −5), appeared as discs turned yellow-red (a* = 5), and increased another eight-fold as discs became full red (a* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a* = 0) to red. PMID:16667893

Distribution of internal pressure around bony prominences: implications to deep tissue injury and effectiveness of intermittent electrical stimulation.

PubMed

Solis, Leandro R; Liggins, Adrian; Uwiera, Richard R E; Poppe, Niek; Pehowich, Enid; Seres, Peter; Thompson, Richard B; Mushahwar, Vivian K

2012-08-01

The overall goal of this project is to develop interventions for the prevention of deep tissue injury (DTI), a form of pressure ulcers that originates in deep tissue around bony prominences. The present study focused on: (1) obtaining detailed measures of the distribution of pressure experienced by tissue around the ischial tuberosities, and (2) investigating the effectiveness of intermittent electrical stimulation (IES), a novel strategy for the prevention of DTI, in alleviating pressure in regions at risk of breakdown due to sustained loading. The experiments were conducted in adult pigs. Five animals had intact spinal cords and healthy muscles and one had a spinal cord injury that led to substantial muscle atrophy at the time of the experiment. A force-controlled servomotor was used to load the region of the buttocks to levels corresponding to 25%, 50% or 75% of each animal's body weight. A pressure transducer embedded in a catheter was advanced into the tissue to measure pressure along a three dimensional grid around the ischial tuberosity of one hind leg. For all levels of external loading in intact animals, average peak internal pressure was 2.01 ± 0.08 times larger than the maximal interfacial pressure measured at the level of the skin. In the animal with spinal cord injury, similar absolute values of internal pressure as that in intact animals were recorded, but the substantial muscle atrophy produced larger maximal interfacial pressures. Average peak internal pressure in this animal was 1.43 ± 0.055 times larger than the maximal interfacial pressure. Peak internal pressure was localized within a ±2 cm region medio-laterally and dorso-ventrally from the bone in intact animals and ±1 cm in the animal with spinal cord injury. IES significantly redistributed internal pressure, shifting the peak values away from the bone in spinally intact and injured animals. These findings provide critical information regarding the relationship between internal and interfacial pressure around the ischial tuberosities during loading levels equivalent to those experienced while sitting. The information could guide future computer models investigating the etiology of DTI, as well as inform the design and prescription of seating cushions for people with reduced mobility. The findings also suggest that IES may be an effective strategy for the prevention of DTI.

46 CFR 178.325 - Intact stability requirements-monohull sailing vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-monohull sailing vessels....325 Intact stability requirements—monohull sailing vessels. (a) As permitted by § 178.310(c) of this part, a monohull sailing vessel may demonstrate compliance with paragraphs (b) or (c) of this section...

46 CFR 178.320 - Intact stability requirements-non-sailing vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability requirements-non-sailing vessels. 178.320 Section 178.320 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER... Intact stability requirements—non-sailing vessels. (a) As permitted by § 178.310(c) of this part, the...

50 CFR 622.10 - Landing fish intact--general.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing fish intact--general. 622.10... ATLANTIC General Provisions § 622.10 Landing fish intact—-general. This section contains requirements for landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified...

50 CFR 622.10 - Landing fish intact--general.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing fish intact--general. 622.10... ATLANTIC General Provisions § 622.10 Landing fish intact—-general. This section contains requirements for landing fish intact that are broadly applicable to finfish in the Gulf EEZ and Caribbean EEZ, as specified...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

46 CFR 28.570 - Intact righting energy.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Intact righting energy. 28.570 Section 28.570 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY UNINSPECTED VESSELS REQUIREMENTS FOR COMMERCIAL FISHING INDUSTRY VESSELS Stability § 28.570 Intact righting energy. (a) Except as provided in paragraph (c) of this...

The Effect of Antitumor Glycosides on Glioma Cells and Tissues as Studied by Proton HR-MAS NMR Spectroscopy

PubMed Central

García-Álvarez, Isabel; Garrido, Leoncio; Romero-Ramírez, Lorenzo; Nieto-Sampedro, Manuel; Fernández-Mayoralas, Alfonso; Campos-Olivas, Ramón

2013-01-01

The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning (1H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the 1H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death. PMID:24194925

Optimization of immunolabeling and clearing techniques for indelibly-labeled memory traces.

PubMed

Pavlova, Ina P; Shipley, Shannon C; Lanio, Marcos; Hen, René; Denny, Christine A

2018-04-16

Recent genetic tools have allowed researchers to visualize and manipulate memory traces (i.e. engrams) in small brain regions. However, the ultimate goal is to visualize memory traces across the entire brain in order to better understand how memories are stored in neural networks and how multiple memories may coexist. Intact tissue clearing and imaging is a new and rapidly growing area of focus that could accomplish this task. Here, we utilized the leading protocols for whole-brain clearing and applied them to the ArcCreER T2 mice, a murine line that allows for the indelible labeling of memory traces. We found that CLARITY and PACT greatly distorted the tissue, and iDISCO quenched enhanced yellow fluorescent protein (EYFP) fluorescence and hindered immunolabeling. Alternative clearing solutions, such as tert-Butanol, circumvented these harmful effects, but still did not permit whole-brain immunolabeling. CUBIC and CUBIC with Reagent 1A produced improved antibody penetration and preserved EYFP fluorescence, but also did not allow for whole-brain memory trace visualization. Modification of CUBIC with Reagent-1A resulted in EYFP fluorescence preservation and immunolabeling of the immediate early gene (IEG) Arc in deep brain areas; however, optimized memory trace labeling still required tissue slicing into mm-thick tissue sections. In summary, our data show that CUBIC with Reagent-1A* is the ideal method for reproducible clearing and immunolabeling for the visualization of memory traces in mm-thick tissue sections from ArcCreER T2 mice. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

From static to animated: Measuring mechanical forces in tissues

PubMed Central

2017-01-01

Cells are physical objects that exert mechanical forces on their surroundings as they migrate and take their places within tissues. New techniques are now poised to enable the measurement of cell-generated mechanical forces in intact tissues in vivo, which will illuminate the secret dynamic lives of cells and change our current perception of cell biology. PMID:28003332

Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces.

PubMed

Lao, Martin; Marino, Victor; Bartold, P Mark

2006-10-01

Periodontal disease is marked by inflammation and damage to tooth-supporting tissues. In particular, damage occurs to factors present in cementum that are thought to have the ability to influence the regeneration of surrounding tissues. Bone sialoprotein and osteopontin are major non-collagenous proteins in mineralized connective tissues associated with precementoblast chemo-attraction, adhesion to the root surface, and cell differentiation. The purpose of this investigation was to determine whether the expression and distribution of bone sialoprotein and osteopontin on root surfaces affected by periodontitis are altered compared to healthy, non-diseased root surfaces. Thirty healthy and 30 periodontitis-affected teeth were collected. Following fixation and demineralization, specimens were embedded in paraffin, sectioned, and exposed to antibodies against bone sialoprotein and osteopontin. Stained sections were assessed using light microscopy. Bone sialoprotein was not detected in the exposed cementum (absence of overlying periodontal ligament) of diseased teeth. In most areas where the periodontal ligament was intact, bone sialoprotein was detected for healthy and diseased teeth. For teeth reactive for bone sialoprotein, the matrix of the cementum just below the periodontal ligament was moderately stained. A similar immunoreactivity pattern for osteopontin was observed. The absence of bone sialoprotein and osteopontin staining along exposed cementum surfaces may be due to structural and compositional changes in matrix components associated with periodontal disease. This may influence the ability for regeneration and new connective tissue attachment onto previously diseased root surfaces.

Top-Down and Bottom-Up Identification of Proteins by Liquid Extraction Surface Analysis Mass Spectrometry of Healthy and Diseased Human Liver Tissue

NASA Astrophysics Data System (ADS)

Sarsby, Joscelyn; Martin, Nicholas J.; Lalor, Patricia F.; Bunch, Josephine; Cooper, Helen J.

2014-09-01

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15-25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue.

PubMed

Sarsby, Joscelyn; Martin, Nicholas J; Lalor, Patricia F; Bunch, Josephine; Cooper, Helen J

2014-11-01

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15-25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies.

Feasibility study of hidden flow imaging based on laser speckle technique using multiperspectives contrast images

NASA Astrophysics Data System (ADS)

Abookasis, David; Moshe, Tomer

2014-11-01

This paper demonstrates the insertion of lens array in the front of a CCD camera in a laser speckle imaging (LSI) like-technique to acquire multiple speckle reflectance projections for imaging blood flow in an intact biological tissue. In some of LSI applications, flow imaging is obtained by thinning or removing of the upper tissue layers to access blood vessels. In contrast, with the proposed approach flow imaging can be achieved while the tissue is intact. In the system, each lens from an hexagonal lens array observed the sample from slightly different perspectives and captured with a CCD camera. In the computer, these multiview raw images are converted to speckled contrast maps. Then, a self-deconvolution shift-and-add algorithm is employed for processing yields high contrast flow information. The method is experimentally validated first with a plastic tube filled with scattering liquid running at different controlled flow rates hidden in a biological tissue and then extensively tested for imaging of cerebral blood flow in an intact rodent head experience different conditions. A total of fifteen mice were used in the experiments divided randomly into three groups as follows: Group 1 (n=5) consisted of injured mice experience hypoxic ischemic brain injury monitored for ~40 min. Group 2 (n=5) injured mice experience anoxic brain injury monitored up to 20 min. Group 3 (n=5) experience functional activation monitored up to ~35 min. To increase tissue transparency and the penetration depth of photons through head tissue layers, an optical clearing method was employed. To our knowledge, this work presents for the first time the use of lens array in LSI scheme.

A study of the intrarenal recycling of urea in the rat with chronic experimental pyelonephritis.

PubMed Central

Gilbert, R M; Weber, H; Turchin, L; Fine, L G; Bourgoignie, J J; Bricker, N S

1976-01-01

The concentrating ability of the kidney was studied by clearance and micropuncture techniques and tissue slice analyses in normal rats with two intact kidneys (intact controls), normal rats with a solitary kidney (uninephrectomized controls), and uremic rats with a single pyelonephritic kidney. Urinary osmolality after water deprivation for 24 h and administration of antidiuretic hormone was 2,501+/-217 and 2,874+/-392 mosmol/kg H2O in intact and uninephrectomized control rats, respectively, and 929+/-130 mosmol/kg H2O in pyelonephritic rats (P less than 0.001 compared to each control group). Fractional water reabsorption and concentrating ability were significantly decreased in the pyelonephritic group, and, to achieve an equivalent fractional excretion of urea, a greater fractional excretion of water was required in the pyelonephritic rats than in the control rats. Whole animal glomerular filtration rate was 1.57+/-0.19 ml/min and 1.39+/-0.18 ml/min in intact and in uninephrectomized controls, respectively, and 0.30+/-0.07 ml/min in pyelonephritic rats (P less than 0.001 compared to each control group). Single nephron glomerular filtration rate was 35.6+/-3.8 nl/min in intact control rats and was significantly increased (P less than 0.05) in both uninephrectomized (88.0+/-10.8 nl/min) and pyelonephritic rats (71.5+/-14.4 nl/min). In all groups fractional water delivery and fractional sodium delivery were closely comparable at the end of the proximal convoluted tubule and at the beginning of the distal convoluted tubule. In contrast, fractional urea delivery out of the proximal tubule was greater in the intact control group (73+/-8%) than in either the uninephrectomized (52+/-2%) or the pyelonephritic group (53+/-3%) (P less than 0.005). Fractional urea delivery at the early part of the distal tubule increased significantly to 137+/-11% and 93+/-6% of the filtered load in intact control and uninephrectomized control rats, respectively (P less than 0.001 compared to the late proximal values of each group), but failed to increase significantly in pyelonephritic rats (65+/-13%), indicating interruption of the normal recycling of urea in the latter group. Analysis of tissue slices demonstrated a rising corticopapillary gradient for total tissue water solute concentration as well as for tissue water urea concentration in both groups of control rats. In contrast, the pyelonephritic animals exhibited no similar gradients from cortex to papilla. These data indicate that the pyelonephritic kidney fails to recycle urea and accumulate interstitial solute. The latter must inevitably lead to a concentrating defect. Images PMID:993348

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

Gibeault, J.D.; Cravens, R.B. Jr.; Chvapil, M.

A lathyrogen, ({sup 14}C)aminopropionitrile (beta APN), was administered to 34 rats either in K-Y jelly or saline vehicles onto intact shaven skin or onto a healed splinted deep excision wound. The dynamics of beta APN transport and content in the skin or repair tissue was observed after 2, 5, 8, and 24 hr of topical administration. The repair tissue quickly absorbed the lathyrogen and reached maximum at the 2-hr sampling. The content of beta APN in the repair tissue was twice as high as that in K-Y jelly vehicle and remained high and stable for at least 24 hr onlymore » when beta APN was administered through a saline vehicle. The transport of beta APN through intact skin, irrespective of the vehicle tested, was slow and continuously increased. The study showed that almost 20% of the beta APN administered onto the wounded skin area was transported into the repair tissue within 2 hr. We suggest that, due to the absence of epidermal stratum corneum from the repair tissue, drugs, such as beta APN, penetrate quickly into the wound.« less

Ultrasound elastography assessment of bone/soft tissue interface

NASA Astrophysics Data System (ADS)

Parmar, Biren J.; Yang, Xu; Chaudhry, Anuj; Shafeeq Shajudeen, Peer; Nair, Sanjay P.; Weiner, Bradley K.; Tasciotti, Ennio; Krouskop, Thomas A.; Righetti, Raffaella

2016-01-01

We report on the use of elastographic imaging techniques to assess the bone/soft tissue interface, a region that has not been previously investigated but may provide important information about fracture and bone healing. The performance of axial strain elastograms and axial shear strain elastograms at the bone/soft tissue interface was studied ex vivo on intact and fractured canine and ovine tibias. Selected ex vivo results were corroborated on intact sheep tibias in vivo. The elastography results were statistically analyzed using elastographic image quality tools. The results of this study demonstrate distinct patterns in the distribution of the normalized local axial strains and axial shear strains at the bone/soft tissue interface with respect to the background soft tissue. They also show that the relative strength and distribution of the elastographic parameters change in the presence of a fracture and depend on the degree of misalignment between the fracture fragments. Thus, elastographic imaging modalities might be used in the future to obtain information regarding the integrity of bones and to assess the severity of fractures, alignment of bone fragments as well as to follow bone healing.

Expression of cyclooxygenase-2 in the canine lower urinary tract with regard to the effects of gonadal status and gender.

PubMed

Ponglowhapan, S; Church, D B; Khalid, M

2009-05-01

As pituitary gonadotrophins can induce prostaglandin (PG) synthesis and receptors for LH and FSH are present in the canine lower urinary tract (LUT), the objectives of this study were to (i) investigate the expression of COX-2, a key rate-limiting enzyme in PG production, in the canine LUT and (ii) determine if COX-2 expression differs between gender, gonadal status (intact and gonadectomised) and LUT regions. Four regions (body and neck of the bladder as well as proximal and distal urethra) of the LUT were obtained from 20 clinically healthy dogs (5 intact males, 5 intact anoestrous females, 4 castrated males, 6 spayed females). In situ hybridization and immunohistochemistry were performed to determine the presence of COX-2 mRNA and protein, respectively. The mRNA and protein expression was semi-quantitatively assessed. The scoring system combined both the distribution and intensity of positive staining and was carried out separately on the three tissue layers (epithelium, sub-epithelial stroma and muscle) for each of four regions of the LUT. In comparison to intact dogs, lower expression (P<0.001) of COX-2 and its mRNA in gonadectomised males and females was observed in all tissue layers of each region of the LUT except in the distal urethra where there was no difference in mRNA expression between gonadal statuses. Regardless of region and tissue layer, intact females expressed more (P<0.05) COX-2 and its mRNA than intact males. However, in gonadectomised dogs, mRNA expression of COX-2 did not differ between genders; males had higher (P<0.001) protein level of COX-2 compared to females. In conclusion, both COX-2 and its mRNA were expressed in the canine LUT and COX-2-regulated PG synthesis in the canine LUT may differ between gonadal statuses and genders. The lower expression of COX-2 in gonadectomised dogs may impair normal function of the LUT and probably implicated in the development of neutering-induced urinary incontinence in the dog.

Microstructural Characterization of Vocal Folds toward a Strain-Energy Model of Collagen Remodeling

PubMed Central

Miri, Amir K.; Heris, Hossein K.; Tripathy, Umakanta; Wiseman, Paul W.; Mongeau, Luc

2013-01-01

Collagen fibrils are believed to control the immediate deformation of soft tissues under biomechanical load. Most extracellular matrix proteins remain intact during frozen sectioning, which allows them to be scanned using atomic force microscopy (AFM). Collagen fibrils are distinguishable because of their helical shape. In the present study, the shape and organization of collagen fibrils in dissected porcine vocal folds were quantified using nonlinear laser scanning microscopy data at the micrometer scale and AFM data at the nanometer scale. Rope-shape collagen fibrils were observed. Geometric characteristics for the fibrils were fed to a hyperelastic model to predict the biomechanical response of the tissue. The model simulates the micrometer-scale unlocking behavior of collagen bundles when extended from their unloaded configuration. Force spectroscopy using AFM was used to estimate the stiffness of collagen fibrils (1 ± 0.5 MPa). The presence of rope-shape fibrils is postulated to change the slope of the force-deflection response near the onset of nonlinearity. The proposed model could ultimately be used to evaluate changes in elasticity of soft tissues that result from the collagen remodeling. PMID:23643604

Factors Affecting the Extraction of Intact Ribonucleic Acid from Plant Tissues Containing Interfering Phenolic Compounds

PubMed Central

Newbury, H. John; Possingham, John V.

1977-01-01

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions. PMID:16660134

Coherent X-Ray Imaging of Collagen Fibril Distributions within Intact Tendons

PubMed Central

Berenguer, Felisa; Bean, Richard J.; Bozec, Laurent; Vila-Comamala, Joan; Zhang, Fucai; Kewish, Cameron M.; Bunk, Oliver; Rodenburg, John M.; Robinson, Ian K.

2014-01-01

The characterization of the structure of highly hierarchical biosamples such as collagen-based tissues at the scale of tens of nanometers is essential to correlate the tissue structure with its growth processes. Coherent x-ray Bragg ptychography is an innovative imaging technique that gives high resolution images of the ordered parts of such samples. Herein, we report how we used this method to image the collagen fibrillar ultrastructure of intact rat tail tendons. The images show ordered fibrils extending over 10–20 μm in length, with a quantifiable D-banding spacing variation of 0.2%. Occasional defects in the fibrils distribution have also been observed, likely indicating fibrillar fusion events. PMID:24461021

Novel in vivo techniques to visualize kidney anatomy and function.

PubMed

Peti-Peterdi, János; Kidokoro, Kengo; Riquier-Brison, Anne

2015-07-01

Intravital imaging using multiphoton microscopy (MPM) has become an increasingly popular and widely used experimental technique in kidney research over the past few years. MPM allows deep optical sectioning of the intact, living kidney tissue with submicron resolution, which is unparalleled among intravital imaging approaches. MPM has solved a long-standing critical technical barrier in renal research to study several complex and inaccessible cell types and anatomical structures in vivo in their native environment. Comprehensive and quantitative kidney structure and function MPM studies helped our better understanding of the cellular and molecular mechanisms of the healthy and diseased kidney. This review summarizes recent in vivo MPM studies with a focus on the glomerulus and the filtration barrier, although select, glomerulus-related renal vascular and tubular functions are also mentioned. The latest applications of serial MPM of the same glomerulus in vivo, in the intact kidney over several days, during the progression of glomerular disease are discussed. This visual approach, in combination with genetically encoded fluorescent markers of cell lineage, has helped track the fate and function (e.g., cell calcium changes) of single podocytes during the development of glomerular pathologies, and provided visual proof for the highly dynamic, rather than static, nature of the glomerular environment. Future intravital imaging applications have the promise to further push the limits of optical microscopy, and to advance our understanding of the mechanisms of kidney injury. Also, MPM will help to study new mechanisms of tissue repair and regeneration, a cutting-edge area of kidney research.

40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 26 2014-07-01 2014-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 25 2010-07-01 2010-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 27 2012-07-01 2012-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 27 2013-07-01 2013-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

40 CFR 261.40 - Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 26 2011-07-01 2011-07-01 false Conditional Exclusion for Used, Intact Cathode Ray Tubes (CRTs) Exported for Recycling. 261.40 Section 261.40 Protection of Environment...) Exported for Recycling. Used, intact CRTs exported for recycling are not solid wastes if they meet the...

Standard versus physiologic bone preparation in total knee arthroplasty and the effect on joint space opening.

PubMed

Burkhart, Timothy A; Herman, Benjamin V; Perry, Kevin; Vandekerckhove, Pieter-Jan; Howard, James; Lanting, Brent

2017-11-01

Total knee arthroplasty is an effective treatment for osteoarthritis. Restoration of physiologic varus alignment may restore the native soft tissue tension and improve outcomes. Six paired fresh-frozen knee specimens were used to perform total knee arthroplastys. The left and right sides of were randomly assigned to have either a physiologic alignment cut or a standard of care neutral alignment bony cut prior to the implantation. Loads of 100 and 200N were applied at 0, 30, 60, and 90° of flexion and the magnitude of the medial and lateral compartment distraction was measured. The loads were applied with the knee specimen intact and post arthroplasty. The physiologic alignment had no difference between medial and lateral gaps at either load. With 100N of load the physiologic alignment had a greater gap at 90° than at full extension while the standard alignment had significantly more gap at 60° of flexion than full extension. The physiologic alignment had a significantly greater gap with the implant compared to the intact condition at both loads. The standard alignment had no significant difference in overall gap between the implant and intact condition with any load. Although performing a physiologic aligned TKA resulted in medial-lateral soft tissue balance, the flexion gap was found to have greater magnitude than the intact knee. Notably, a neutral aligned TKA was found to be balanced, but also was found to recreate the intact knee flexion gaps. These results suggest that coronal plane stability can be achieved with physiologic alignment objectives, but the clinician needs to be aware of the potential to have greater laxity than the intact and neutral alignment surgical objectives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones.

PubMed

Mark, Christina; Zór, Kinga; Heiskanen, Arto; Dufva, Martin; Emnéus, Jenny; Finnie, Christine

2016-12-15

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts. Copyright © 2016 Elsevier Inc. All rights reserved.

Hydrolyzable tannins of tamaricaceous plants. IV: Micropropagation and ellagitannin production in shoot cultures of Tamarix tetrandra.

PubMed

Orabi, Mohamed A A; Taniguchi, Shoko; Terabayashi, Susumu; Hatano, Tsutomu

2011-11-01

Shoot cultures of Tamarix tetrandra on Linsmaier-Skoog (LS) agar medium with 30 g l(-1) sucrose, 2.13 mg l(-1) indoleacetic acid and 2.25 mg l(-1) benzyl adenine produced ellagitannins found in intact plants of the Tamaricaceae. This was demonstrated by the isolation of 14 monomeric-tetrameric ellagitannins from the aq. Me2CO extract of the cultured tissues. This is the first report on the production of ellagitannin tetramers by plant tissue culture. The effects of light and certain medium constituents on tissue growth and ellagitannin production were examined. The contents of representative tannins of different types [i.e., tellimagrandin II (monomer), hirtellin A (linear GOG-type dimer), hirtellin B (hellinoyl-type dimer), hirtellin C (macrocyclic-type dimer), and hirtellin T1 (linear GOG-type trimer)] in the resultant tissues in response to these factors were estimated by HPLC, and the optimal condition for production of these tannins were established. Shoots cultured on LS hormone-free medium promoted root development, and regenerated plants could adapt to ordinary soil and climate. Acclimatized and intact T. tetrandra plants that were collected in November and May, respectively, demonstrated seasonal differences in individual ellagitannin contents. HPLC comparison of individual ellagitannin contents in different plant materials (i.e., leaves, stems, and roots) of intact T. tetrandra plants is also reported. The results are discussed with respect to cellular deposition and biosynthetic relationship of tannins. Copyright © 2011 Elsevier Ltd. All rights reserved.

N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

PubMed Central

Madathil, Bernadette K.; Anil Kumar, Pallickaveedu RajanAsari; Kumary, Thrikkovil Variyath

2014-01-01

Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty. PMID:25003113

Femtosecond pump-probe microscopy generates virtual cross-sections in historic artwork

PubMed Central

Villafana, Tana Elizabeth; Brown, William P.; Delaney, John K.; Palmer, Michael; Warren, Warren S.; Fischer, Martin C.

2014-01-01

The layering structure of a painting contains a wealth of information about the artist's choice of materials and working methods, but currently, no 3D noninvasive method exists to replace the taking of small paint samples in the study of the stratigraphy. Here, we adapt femtosecond pump-probe imaging, previously shown in tissue, to the case of the color palette in paintings, where chromophores have much greater variety. We show that combining the contrasts of multispectral and multidelay pump-probe spectroscopy permits nondestructive 3D imaging of paintings with molecular and structural contrast, even for pigments with linear absorption spectra that are broad and relatively featureless. We show virtual cross-sectioning capabilities in mockup paintings, with pigment separation and nondestructive imaging on an intact 14th century painting (The Crucifixion by Puccio Capanna). Our approach makes it possible to extract microscopic information for a broad range of applications to cultural heritage. PMID:24449855

Bradykinin regulates human colonic ion transport in vitro

PubMed Central

Baird, A W; Skelly, M M; O'Donoghue, D P; Barrett, K E; Keely, S J

2008-01-01

Background and purpose: Kinins are acknowledged as important regulators of intestinal function during inflammation; however, their effects on human intestinal ion transport have not been reported. Here, we used muscle-stripped human colonic tissue and cultured T84-cell monolayers to study bradykinin (BK) actions on human intestinal ion transport. Experimental approach: Ion transport was measured as changes in short-circuit current (Isc) across colonic epithelia mounted in Ussing chambers. Key results: In intact tissue, there was a distinct polarity to BK-elicited Isc responses. Whereas basolateral BK stimulated sustained responses (EC50=0.5±0.1 μM), those to apical BK were more rapid and transient (EC50=4.1±1.2 nM). In T84 cells, responses to both apical and basolateral BK were similar to those seen upon apical addition to intact tissues. Cross-desensitization between apical and basolateral domains was not observed. BK-induced responses were largely due to Cl− secretion as shown by their sensitivity to bumetanide and removal of Cl− from the bathing solution. Studies using selective agonists and antagonists indicate responses to BK are mediated by B2 receptors. Finally, responses to basolateral BK in intact tissues were inhibited by tetrodotoxin (1 μM), atropine (1 μM), capsaicin (100 μM) and piroxicam (10 μM). BK-stimulated prostaglandin (PG)E2 release from colonic tissue. Conclusions: BK stimulates human colonic Cl− secretion by activation of apical and basolateral B2 receptors. Responses to apical BK reflect a direct action on epithelial cells, whereas those to basolateral BK are amplified by stimulation of enteric nerves and PG synthesis. PMID:18604228

Debrided Skin as a Source of Autologous Stem Cells for Wound Repair

DTIC Science & Technology

2011-08-01

dermal tissue shows the presence of hyalinized collagen (bold arrows) with loss of individual collagen bundles and cellular necrosis . The hypodermal...region consisted of intact adipo- cytes separated by intact interlobular septae and thermally collapsed areas with complete necrosis of both fat cells...and no dsASCs showed predom- inantly acellular multifocal amorphous matrix (Supporting In- formation Fig. S3A, S3B) and was avascular (Supporting Infor

Understanding Collagen Organization in Breast Tumors to Predict and Prevent Metastasis

DTIC Science & Technology

2014-09-01

Harmonic Generation to Image the Extracellular Matrix During Tumor Progression. Invited Perspective Intravital Manuscript Submitted. Sullivan K...harmonic generation (the SHG “F/B ratio”) in thick intact tissue, with a single image scan. This will be necessary for us to pursue our goal of...quantifying matrix changes dynamically, in intact tumor models. The first method determines F/B by generating a series of backscattered images using a series

Tumor margin assessment in Mohs surgery using reflectance, fluorescence and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Nguyen, Hieu T. M.; Moy, Austin J.; Zhang, Yao; Feng, Xu; Reichenberg, Jason S.; Fox, Matthew; Tunnell, James W.

2017-02-01

Mohs surgery is the current gold standard to treat large, aggressive or high-risk non-melanoma skin cancer (NMSC) cases. While Mohs surgery is an effective treatment, the procedure is time-consuming and expensive for physicians as well as burdensome for patients as they wait for frozen section histology. Our group has recently demonstrated high diagnostic accuracy using a noninvasive "spectral biopsy" (combination of diffuse reflectance (DRS), fluorescence (FS) and Raman spectroscopy (RS)) to classify NMSC vs. normal lesion in a screening setting of intact tissue. Here, we examine the sensitivity of spectral biopsy to pathology in excised Mohs sections. The system is designed with three modalities integrated into one fiber probe, which is utilized to measure DRS, FS, and RS of freshly excised skin from patients with various NMSC pathologies including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), where each measurement location is correlated to histopathology. The spectral biopsy provides complimentary physiological information including the reduced scattering coefficient, hemoglobin content and oxygen saturation from DRS, NADH and collagen contribution from FS and information regarding multiple proteins and lipids from RS. We then apply logistic regression model to the extracted physiological parameters to classify NMSC vs. normal tissue. The results on the excised tissue are generally consistent with in vivo measurements showing decreased scattering within the tumor and reduced fluorescence. Due to the high sensitivity of RS to lipids, subcutaneous fat often dominates the RS signal. This pilot study demonstrates the potential for a spectral biopsy to classify NMSC vs. normal tissue, indicating the opportunity to guide Mohs excisions.

Vital-dye-enhanced multimodal imaging of neoplastic progression in a mouse model of oral carcinogenesis

NASA Astrophysics Data System (ADS)

Hellebust, Anne; Rosbach, Kelsey; Wu, Jessica Keren; Nguyen, Jennifer; Gillenwater, Ann; Vigneswaran, Nadarajah; Richards-Kortum, Rebecca

2013-12-01

In this longitudinal study, a mouse model of 4-nitroquinoline 1-oxide chemically induced tongue carcinogenesis was used to assess the ability of optical imaging with exogenous and endogenous contrast to detect neoplastic lesions in a heterogeneous mucosal surface. Widefield autofluorescence and fluorescence images of intact 2-NBDG-stained and proflavine-stained tissues were acquired at multiple time points in the carcinogenesis process. Confocal fluorescence images of transverse fresh tissue slices from the same specimens were acquired to investigate how changes in tissue microarchitecture affect widefield fluorescence images of intact tissue. Widefield images were analyzed to develop and evaluate an algorithm to delineate areas of dysplasia and cancer. A classification algorithm for the presence of neoplasia based on the mean fluorescence intensity of 2-NBDG staining and the standard deviation of the fluorescence intensity of proflavine staining was found to separate moderate dysplasia, severe dysplasia, and cancer from non-neoplastic regions of interest with 91% sensitivity and specificity. Results suggest this combination of noninvasive optical imaging modalities can be used in vivo to discriminate non-neoplastic from neoplastic tissue in this model with the potential to translate this technology to the clinic.

Association of CagPAI integrity with severeness of Helicobacter pylori infection in patients with gastritis.

PubMed

Ahmadzadeh, A; Ghalehnoei, H; Farzi, N; Yadegar, A; Alebouyeh, M; Aghdaei, H A; Molaei, M; Zali, M R; Pour Hossein Gholi, M A

2015-12-01

The Helicobacter pylori cag pathogenicity island (cagPAI) is involved in delivery of CagA effector protein and peptidoglycan into host cells and also in IL-8 induction in the human gastric tissue. Diversity of cagPAI may affect disease status and clinical outcome of the infected patients. Our study was aimed to investigate diversity of this island and its intactness in Iranian patients to investigate possible associations between cagPAI integrity and pathological changes of the infected tissue. Out of the 75 patients, H. pylori strains were obtained from 30 patients with severe active gastritis (SAG) (n=11), moderate chronic gastritis (CG) (n=14) and intestinal metaplasia/dysplasia (IM) (n=5). Intactness of the cagPAI was determined using 12 sets of primer pairs specific for functionally important loci of cagPAI by polymerase chain reaction (PCR). The cagPAI positive strains were significantly observed in patients with SAG (52.4%) in comparison to those presenting CG (33.3%) and IM (14.3%). In addition, the presence of intact cagPAI was 87.5% in H. pylori strains isolated from patients with SAG, which was higher than those obtained from patients with CG (12.5%) or IM (0%). A significant increase in the frequency of cagα-cagY and cagW-cagT segments, as exterior proteins of the CagPAI, was illustrated in strains from SAG patients compared with those from patients with CG. Overall, these results strongly proposed an association between the severity of histopathological changes and intactness of cagPAI in the gastric tissue of patients infected with H. pylori. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Porcine endogenous retroviral nucleic acid in peripheral tissues is associated with migration of porcine cells post islet transplant.

PubMed

Binette, Tanya M; Seeberger, Karen L; Lyon, James G; Rajotte, Ray V; Korbutt, Gregory S

2004-07-01

Porcine islets represent an alternative source of insulin-producing tissue, however, porcine endogenous retrovirus (PERV) remains a concern. In this study, SCID mice were transplanted with nonencapsulated (non-EC), microencapsulated (EC) or macroencapsulated (in a TheraCyte trade mark device) neonatal porcine islets (NPIs), and peripheral tissues were screened for presence of viral DNA and mRNA. To understand the role of an intact immune system in PERV incidence, mice with established NPI grafts were reconstituted with splenocytes. Peripheral tissues were screened for PERV and porcine DNA using PCR. Tissues with positive DNA were analyzed for PERV mRNA using RT-PCR. No significant difference was observed between non-EC and EC transplants regarding presence of PERV or porcine-specific DNA or mRNA. In reconstituted animals, little PERV or porcine DNA, and no PERV mRNA was detected. No PERV or porcine-specific DNA was observed in animals implanted with a TheraCyte trade mark device. In conclusion, an intact immune system significantly lowered the presence of PERV. Microencapsulation of islets did not alter PERV presence, however, macroencapsulation in the TheraCyte device did. Lower PERV incidence coincided with lower porcine DNA in peripheral tissues, linking the presence of PERV to migration of porcine cells.

Simplified methods for screening cowpea cultivars for manganese leaf-tissue tolerance. [Vigna unguiculata

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

Wissemeier, A.H.; Horst, W.J.

In cowpea (Vigna unguiculata (L.) Walp.) dark brown speckles on old leaves are typical symptoms of Mn toxicity and indicate Mn sensitivity of leaf tissue. Induction and subsequent quantification of brown Mn speckles in leaf tissues were used to screen cowpea cultivars for Mn leaf-tissue tolerance using three different techniques: (i) leaf cuttings cultured for 22 days in solution culture with 20 {mu}M MnSO{sub 4}, (ii) leaf rings mounted on leaves of intact plants and filled with 500 {mu}M MnSO{sub 4} for 5 days, and (iii) leaf disks floated for 3 days on 500 {mu}M MnSO{sub 4}. Density of brownmore » speckles differed considerably among the six cultivars tested, and was not related to the Mn concentrations of the leaf tissues. There were close relationships between genotypic Mn-toxicity symptom expression and depression of dry matter production of the cultivars at high Mn supply in a long-term sand culture experiment. The floating leaf-disk method is particularly suited for screening large numbers of cowpea cultivars for Mn leaf-tissue tolerance because it requires only 3 days. The ranking of the cultivars for Mn tolerance was highly correlated to Mn tolerance of intact plants.« less

Astrocyte activation and wound healing in intact-skull mouse after focal brain injury.

PubMed

Suzuki, Takayuki; Sakata, Honami; Kato, Chiaki; Connor, John A; Morita, Mitsuhiro

2012-12-01

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery. © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Phytohormone ecology : Herbivory byThrips tabaci induces greater ethylene production in intact onions than mechanical damage alone.

PubMed

Kendall, D M; Bjostad, L B

1990-03-01

Herbivory byThrips tabaci affected production of the phytohormone ethylene from living onion foliage. Ethylene analysis was performed by gas chromatography on intact onion tissue. Thrips feeding damage and a crushed thrips extract stimulated significantly greater production of eihylene than could be explained by either one-time or semicontinuous mechanical damage alone, suggesting that ethylene-inducing cues may be transferred to the plant during feeding. This is the first demonstration of increased ethylene production from insect-infested intact plants. This study suggests that herbivores affect both the phytohormone physiology and secondary chemistry of living plants because ethylene has been shown to enhance production of defensive phytochemicals.

The optical diagnostics of parameters of biological tissues of human intact skin in near-infrared range

NASA Astrophysics Data System (ADS)

Petruk, Vasyl; Kvaternyuk, Sergii; Bolyuh, Boris; Bolyuh, Dmitry; Dronenko, Vladimir; Harasim, Damian; Annabayev, Azamat

2016-09-01

Melanoma skin is difficult to diagnose in the early stages of development despite its location outside. Melanoma is difficult to visually differentiate from benign melanocytic nevi. In the work we investigated parameters of human intact skin in near-infrared range for different racial and gender groups. This allows to analyze statistical differences in the coefficient of diffuse reflection and use them in the differential diagnosis of cancer by optical methods subject.

Near infrared laser-tissue welding using nanoshells as an exogenous absorber.

PubMed

Gobin, Andre M; O'Neal, D Patrick; Watkins, Daniel M; Halas, Naomi J; Drezek, Rebekah A; West, Jennifer L

2005-08-01

Gold nanoshells are a new class of nanoparticles that can be designed to strongly absorb light in the near infrared (NIR). These particles provide much larger absorption cross-sections and efficiency than can be achieved with currently used chemical chromophores without photobleaching. In these studies, we have investigated the use of gold nanoshells as exogenous NIR absorbers to facilitate NIR laser-tissue welding. Gold nanoshells with peak extinction matching the NIR wavelength of the laser being used were manufactured and suspended in an albumin solder. Optimization work was performed on ex vivo muscle samples and then translated into testing in an in vivo rat skin wound-healing model. Mechanical testing of the muscle samples was immediately performed and compared to intact tissue mechanical properties. In the in vivo study, full thickness incisions in the dorsal skin of rats were welded, and samples of skin were excised at 0, 5, 10, 21, and 32 days for analysis of strength and wound healing response. Mechanical testing of nanoshell-solder welds in muscle revealed successful fusion of tissues with tensile strengths of the weld site equal to the uncut tissue. No welding was accomplished with this light source when using solder formulations without nanoshells. Mechanical testing of the skin wounds showed sufficient strength for closure and strength increased over time. Histological examination showed good wound-healing response in the soldered skin. The use of nanoshells as an exogenous absorber allows the usage of light sources that are minimally absorbed by tissue components, thereby, minimizing damage to surrounding tissue and allowing welding of thicker tissues. (c) 2005 Wiley-Liss, Inc.

Differentiation and Cell-Cell Interactions of Neural Progenitor Cells Transplanted into Intact Adult Brain.

PubMed

Sukhinich, K K; Kosykh, A V; Aleksandrova, M A

2015-11-01

We studied the behavior and cell-cell interactions of embryonic brain cell from GFP-reporter mice after their transplantation into the intact adult brain. Fragments or cell suspensions of fetal neocortical cells at different stages of development were transplanted into the neocortex and striatum of adult recipients. Even in intact brain, the processes of transplanted neurons formed extensive networks in the striatum and neocortical layers I and V-VI. Processes of transplanted cells at different stages of development attained the rostral areas of the frontal cortex and some of them reached the internal capsule. However, the cells transplanted in suspension had lower process growth potency than cells from tissue fragments. Tyrosine hydroxylase fibers penetrated from the recipient brain into grafts at both early and late stages of development. Our experiments demonstrated the formation of extensive reciprocal networks between the transplanted fetal neural cells and recipient brain neurons even in intact brain.

Measurement of the Mechanical Properties of Intact Collagen Fibrils

NASA Astrophysics Data System (ADS)

Mercedes, H.; Heim, A.; Matthews, W. G.; Koob, T.

2006-03-01

Motivated by the genetic disorder Ehlers-Danlos syndrome (EDS), in which proper collagen synthesis is interrupted, we are investigating the structural and mechanical properties of collagen fibrils. The fibrous glycoprotein collagen is the most abundant protein found in the human body and plays a key role in the extracellular matrix of the connective tissue, the properties of which are altered in EDS. We have selected as our model system the collagen fibrils of the sea cucumber dermis, a naturally mutable tissue. This system allows us to work with native fibrils which have their proteoglycan complement intact, something that is not possible with reconstituted mammalian collagen fibrils. Using atomic force microscopy, we measure, as a function of the concentration of divalent cations, the fibril diameter, its response to force loading, and the changes in its rigidity. Through these experiments, we will shed light on the mechanisms which control the properties of the sea cucumber dermis and hope to help explain the altered connective tissue extracellular matrix properties associated with EDS.

Automated cell-type classification in intact tissues by single-cell molecular profiling

PubMed Central

2018-01-01

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research. PMID:29319504

Linewidth narrowing for 31Phosphorus MRI of cell membranes

NASA Astrophysics Data System (ADS)

Barrett, Sean; Frey, Merideth; Madri, Joseph; Michaud, Michael

2011-03-01

Most 31 P Magnetic Resonance Spectroscopy studies of tissues try to avoid contamination by a relatively large, but broad, spectral feature attributed to cell membrane phospholipids. MRI using this broad 31 P membrane spectrum is not even attempted, since the spatial resolution and signal-to-noise would be poor, relative to conventional MRI using the narrow 1 H water spectrum. This long-standing barrier has been overcome by a novel pulse sequence, recently discovered in fundamental quantum computation research, which narrows the broad 31 P spectrum by ~ 1000 × . Applying time-dependent gradients in synch with a repeating pulse block enables a new route to high spatial resolution, 3D 31 P MRI of the soft solid components of cells and tissues. So far, intact and sectioned samples of ex vivo fixed mouse organs have been imaged, with (sub-mm)3 voxels. Extending the reach of MRI to broad spectra in natural and artificial tissues opens a new window into cells, enabling progress in biomedical research. W.J. Thoma et al., J. MR 61, 141 (1985); E.J. Murphy et al., MR Med 12, 282 (1989); R. McNamara et al., NMR Biomed 7, 237 (1994).

SEM evaluation of pulp reaction to different pulp capping materials in dog’s teeth

PubMed Central

Asgary, Saeed; Parirokh, Masoud; Eghbal, Mohammad Jafar; Ghoddusi, Jamileh

2006-01-01

Introduction: This investigation evaluates the effects of mineral trioxide aggregate (MTA), calcium hydroxide (CH) and calcium enriched mixture (CEM) as pulp capping materials on dental pulp tissues. Materials and Methods: The experimental procedures were performed on eighteen intact dog canine teeth. The pulps were exposed. Cavities were randomly filled with CEM, MTA, or CH followed by glass ionomer filling. After 2 months, animals were sacrificed, each tooth was sectioned into halves, and the interface between each capping material and pulp tissue was evaluated by scanning electron microscope (SEM) in profile view of the specimens. Results: Dentinal bridge formation as the most characteristic reaction was resulted from SEM observation in all examined groups. Odontoblast-like cells were formed and create dens collagen network, which was calcified gradually by deposition of calcosphirit structures to form newly dentinal bridge. Conclusion: Based on the results of this in vivo study, it was concluded that these test materials are able to produce calcified tissue in underlying pulp in the case of being used as a pulp capping agent. Additionally, it appears that CEM has the potential to be used as a direct pulp capping material during vital pulp therapy. PMID:24379876

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.

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

Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress

PubMed Central

Sepehr, Reyhaneh; Staniszewski, Kevin; Maleki, Sepideh; Jacobs, Elizabeth R.; Audi, Said

2012-01-01

Abstract. Ventilation with enhanced fractions of O2 (hyperoxia) is a common and necessary treatment for hypoxemia in patients with lung failure, but prolonged exposure to hyperoxia causes lung injury. Ischemia-reperfusion (IR) injury of lung tissue is common in lung transplant or crush injury to the chest. These conditions are associated with apoptosis and decreased survival of lung tissue. The objective of this work is to use cryoimaging to evaluate the effect of exposure to hyperoxia and IR injury on lung tissue mitochondrial redox state in rats. The autofluorescent mitochondrial metabolic coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are electron carriers in ATP generation. These intrinsic fluorophores were imaged for rat lungs using low-temperature fluorescence imaging (cryoimaging). Perfused lungs from four groups of rats were studied: normoxia (control), control perfused with an mitochondrial complex IV inhibitor (potassium cyanide, KCN), rats exposed to hyperoxia (85% O2) for seven days, and from rats subjected to lung IR in vivo 24 hours prior to study. Each lung was sectioned sequentially in the transverse direction, and the images were used to reconstruct a three-dimensional (3-D) rendering. In KCN perfused lungs the respiratory chain was more reduced, whereas hyperoxic and IR lung tissue have a more oxidized respiratory chain than control lung tissue, consistent with previously measured mitochondrial dysfunction in both hyperoxic and IR lungs. PMID:22559688

Low-Magnitude, High-Frequency Vibration Fails to Accelerate Ligament Healing but Stimulates Collagen Synthesis in the Achilles Tendon.

PubMed

Thompson, William R; Keller, Benjamin V; Davis, Matthew L; Dahners, Laurence E; Weinhold, Paul S

2015-05-01

Low-magnitude, high-frequency vibration accelerates fracture and wound healing and prevents disuse atrophy in musculoskeletal tissues. To investigate the role of low-magnitude, high-frequency vibration as a treatment to accelerate healing of an acute ligament injury and to examine gene expression in the intact Achilles tendon of the injured limb after low-magnitude, high-frequency vibration. Controlled laboratory study. Complete surgical transection of the medial collateral ligament (MCL) was performed in 32 Sprague-Dawley rats, divided into control and low-magnitude, high-frequency vibration groups. Low-magnitude, high-frequency vibration started on postoperative day 2, and rats received vibration for 30 minutes a day for 12 days. All rats were sacrificed 2 weeks after the operation, and their intact and injured MCLs were biomechanically tested or used for histological analysis. Intact Achilles tendons from the injured limb were evaluated for differences in gene expression. Mechanical testing revealed no differences in the ultimate tensile load or the structural stiffness between the control and vibration groups for either the injured or intact MCL. Vibration exposure increased gene expression of collagen 1 alpha (3-fold), interleukin 6 (7-fold), cyclooxygenase 2 (5-fold), and bone morphogenetic protein 12 (4-fold) in the intact Achilles tendon when compared with control tendons ( P < .05). While no differences were observed in the mechanical or histological properties of the fully transected MCL after low-magnitude, high-frequency vibration treatment, significant enhancements in gene expression were observed in the intact Achilles tendon. These included collagen, several inflammatory cytokines, and growth factors critical for tendons. As low-magnitude, high-frequency vibration had no negative effects on ligament healing, vibration therapy may be a useful tool to accelerate healing of other tissues (bone) in multitrauma injuries without inhibiting ligament healing. Additionally, the enhanced gene expression in response to low-magnitude, high-frequency vibration in the intact Achilles tendon suggests the need to further study its potential to accelerate tendon healing in partial injury or repair models.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria.

PubMed

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50-100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed 'Brownian motion'-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as 'Cytobacts' and 'Peribacts', respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host.

Live cell imaging reveals extensive intracellular cytoplasmic colonization of banana by normally non-cultivable endophytic bacteria

PubMed Central

Thomas, Pious; Sekhar, Aparna Chandra

2014-01-01

It is generally believed that endophytic microorganisms are intercellular inhabitants present in either cultivable or non-cultivable form primarily as root colonizers. The objective of this study was to determine whether the actively mobile micro-particles observed in the intracellular matrix of fresh tissue sections of banana included endophytic bacteria. Tissue sections (50–100 µm) from apical leaf sheaths of surface-disinfected suckers (cv. Grand Naine) displayed ‘Brownian motion’-reminiscent abundant motile micro-particles under bright-field and phase-contrast (×1000), which appeared similar in size and motility to the pure cultures of endophytes previously isolated from banana. Observations on callus, embryonic cells and protoplasts with intact cell wall/plasma membrane confirmed their cytoplasmic nature. The motility of these entities reduced or ceased upon tissue fixation or staining with safranin/crystal violet (0.5 % w/v), but continued uninterrupted following treatment with actin-disrupting drugs, ruling out the possibility of micro-organelles like peroxisomes. Staining with 2,3,5-triphenyl tetrazolium chloride (TTC) confirmed them to be live bacteria with similar observations after dilute safranin (0.005 %) treatment. Tissue staining with SYTO-9 coupled with epi-fluorescence or confocal laser scanning microscopy showed bacterial colonization along the peri-space between cell wall and plasma membrane initially. SYTO-9 counterstaining on TTC- or safranin-treated tissue and those subjected to enzymatic permeabilization revealed the cytoplasmic bacteria. These included organisms moving freely in the cytoplasm and those adhering to the nuclear envelope or vacuoles and the intravacuolar colonizers. The observations appeared ubiquitous to different genomes and genotypes of banana. Plating the tissue homogenate on nutrient media seldom yielded colony growth. This study, supported largely by live cell video-imaging, demonstrates enormous intracellular colonization in bananas by normally non-cultivable endophytic bacteria in two niches, namely cytoplasmic and periplasmic, designated as ‘Cytobacts’ and ‘Peribacts’, respectively. The integral intracellular association with their clonal perpetuation suggests a mutualistic relationship between endophytes and the host. PMID:24790123

Secondary osteons scale allometrically in mammalian humerus and femur

PubMed Central

Phillips, C.; Cornish, H.; Cooke, M.; Hutchinson, J. R.; Doube, M.

2017-01-01

Intra-cortical bone remodelling is a cell-driven process that replaces existing bone tissue with new bone tissue in the bone cortex, leaving behind histological features called secondary osteons. While the scaling of bone dimensions on a macroscopic scale is well known, less is known about how the spatial dimensions of secondary osteons vary in relation to the adult body size of the species. We measured the cross-sectional area of individual intact secondary osteons and their central Haversian canals in transverse sections from 40 stylopodal bones of 39 mammalian species (body mass 0.3–21 000 kg). Scaling analysis of our data shows that mean osteonal resorption area (negative allometry, exponent 0.23,R2 0.54,p<0.005) and Haversian canal area (negative allometry, exponent 0.31,R2 0.45,p<0.005) are significantly related to body mass, independent of phylogeny. This study is the most comprehensive of its kind to date, and allows us to describe overall trends in the scaling behaviour of secondary osteon dimensions, supporting the inference that the osteonal resorption area may be limited by the need to avoid fracture in smaller mammalian species, but the need to maintain osteocyte viability in larger mammalian species. PMID:29291052

Release of Esterase Following Germination of Lettuce Seed (Lactuca sativa L.)

PubMed Central

Chandra, G. Ram; Toole, Vivian K.

1977-01-01

Light-insensitive lettuce seeds, Lactuca sativa L. cv. Great Lakes, release esterases for a period following radicle protrusion. Very little or no enzymes are released prior to 24 hours or after 48 hours of germination. As compared to intact seeds, half-seeds readily release esterases and the release is not affected by far red irradiation. Bulk of the released esterases are derived from the endosperm tissue and presumably exists in the intact seed as a component of the extraembryonic fluid. PMID:16659992

A two-layered mechanical model of the rat esophagus. Experiment and theory

PubMed Central

Fan, Yanhua; Gregersen, Hans; Kassab, Ghassan S

2004-01-01

Background The function of esophagus is to move food by peristaltic motion which is the result of the interaction of the tissue forces in the esophageal wall and the hydrodynamic forces in the food bolus. The structure of the esophagus is layered. In this paper, the esophagus is treated as a two-layered structure consisting of an inner collagen-rich submucosa layer and an outer muscle layer. We developed a model and experimental setup for determination of elastic moduli in the two layers in circumferential direction and related the measured elastic modulus of the intact esophagus to the elastic modulus computed from the elastic moduli of the two layers. Methods Inflation experiments were done at in vivo length and pressure-diameters relations were recorded for the rat esophagus. Furthermore, the zero-stress state was taken into consideration. Results The radius and the strain increased as function of pressure in the intact as well as in the individual layers of the esophagus. At pressures higher than 1.5 cmH2O the muscle layer had a larger radius and strain than the mucosa-submucosa layer. The strain for the intact esophagus and for the muscle layer was negative at low pressures indicating the presence of residual strains in the tissue. The stress-strain curve for the submucosa-mucosa layer was shifted to the left of the curves for the muscle layer and for the intact esophagus at strains higher than 0.3. The tangent modulus was highest in the submucosa-mucosa layer, indicating that the submucosa-mucosa has the highest stiffness. A good agreement was found between the measured elastic modulus of the intact esophagus and the elastic modulus computed from the elastic moduli of the two separated layers. PMID:15518591

Acute radiation impacts contractility of guinea-pig bladder strips affecting mucosal-detrusor interactions.

PubMed

McDonnell, Bronagh M; Buchanan, Paul J; Prise, Kevin M; McCloskey, Karen D

2018-01-01

Radiation-induced bladder toxicity is associated with radiation therapy for pelvic malignancies, arising from unavoidable irradiation of neighbouring normal bladder tissue. This study aimed to investigate the acute impact of ionizing radiation on the contractility of bladder strips and identify the radiation-sensitivity of the mucosa vs the detrusor. Guinea-pig bladder strips (intact or mucosa-free) received ex vivo sham or 20Gy irradiation and were studied with in vitro myography, electrical field stimulation and Ca2+-fluorescence imaging. Frequency-dependent, neurogenic contractions in intact strips were reduced by irradiation across the force-frequency graph. The radiation-difference persisted in atropine (1μM); subsequent addition of PPADs (100μM) blocked the radiation effect at higher stimulation frequencies and decreased the force-frequency plot. Conversely, neurogenic contractions in mucosa-free strips were radiation-insensitive. Radiation did not affect agonist-evoked contractions (1μM carbachol, 5mM ATP) in intact or mucosa-free strips. Interestingly, agonist-evoked contractions were larger in irradiated mucosa-free strips vs irradiated intact strips suggesting that radiation may have unmasked an inhibitory mucosal element. Spontaneous activity was larger in control intact vs mucosa-free preparations; this difference was absent in irradiated strips. Spontaneous Ca2+-transients in smooth muscle cells within tissue preparations were reduced by radiation. Radiation affected neurogenic and agonist-evoked bladder contractions and also reduced Ca2+-signalling events in smooth muscle cells when the mucosal layer was present. Radiation eliminated a positive modulatory effect on spontaneous activity by the mucosa layer. Overall, the findings suggest that radiation impairs contractility via mucosal regulatory mechanisms independent of the development of radiation cystitis.

Jueming prescription and its ingredients, semen cassiae and Rhizoma Curcumae Longae, stimulate lipolysis and enhance the phosphorylation of hormone‑sensitive lipase in cultured rat white adipose tissue.

PubMed

Zhang, Yue; Li, Jiaojiao; Wen, Xiuying

2017-11-01

The present study aimed to investigate the effect of jueming prescription (JMP) and its ingredients, semen cassiae (SC) and Rhizoma Curcumae Longae (RCL), on lipolysis, and to examine their effect on the phosphorylation of hormone‑sensitive lipase (HSL) in cultured rat white adipose tissue (WAT). Retroperitoneal WAT was aseptically excised from adult male Sprague‑Dawley rats, minced into uniform sections and subjected to ex vivo culture for 24 h. The tissue sections were then distributed into a 24‑well culture plate and treated with normal saline (vehicle), isoproterenol (ISO), JMP, SC and RCL. Non‑esterified fatty acid (NEFA) and glycerol release from the intact WAT explants were determined as a measurement of lipolysis, which were measured using NEFA and glycerol assay kits. The phosphorylation of HSL at Ser563 (P‑HSL S563) and 660 residues (P‑HSL S660) were determined using western blot analysis. The size of the adipocytes was visualized using hematoxylin and eosin (H&E) staining. It was found that JMP‑, SC‑ and RCL‑stimulated lipolysis was responsible for increasing the release of NEFAs and glycerol from the intact WAT in vitro. In addition, JMP, SC and RCL increased the levels of P‑HSL Ser563: JMP water (JW) extract, 3.52‑fold; JMP ethanol (JE) extract, 3.38‑fold; SC water (SW) extract, 4.60‑fold; SC ethanol (SE) extract, 4.20‑fold; RCL water (RW) extract, 6.98‑fold; RCL ethanol (RE) extract, 6.60‑fold. JMP, SC and RCL also increased the levels of P‑HSL Ser660: JW extract, 3.16‑fold; JE extract, 2.92‑fold; SW extract, 4.57‑fold; SE extract, 4.13‑fold; RW extract, 5.41‑fold; RE 4.96‑fold) in the WAT. The RW extract had the most marked effect. The HE staining revealed that JMP, SC and RCL reduced the size of adipocytes in the WAT. In conclusion, JMP and its ingredients, SC and RC, stimulated lipolysis and reduced the size of adipocytes, possibly via the phosphorylation of HSL in cultured rat WAT.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator.

PubMed

Rossano, Adam J; Romero, Michael F

2017-08-11

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H + ) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Slack length reduces the contractile phenotype of the Swine carotid artery.

PubMed

Rembold, Christopher M; Garvey, Sean M; Tejani, Ankit D

2013-01-01

Contraction is the primary function of adult arterial smooth muscle. However, in response to vessel injury or inflammation, arterial smooth muscle is able to phenotypically modulate from the contractile state to several 'synthetic' states characterized by proliferation, migration and/or increased cytokine secretion. We examined the effect of tissue length (L) on the phenotype of intact, isometrically held, initially contractile swine carotid artery tissues. Tissues were studied (1) without prolonged incubation at the optimal length for force generation (1.0 Lo, control), (2) with prolonged incubation for 17 h at 1.0 Lo, or (3) with prolonged incubation at slack length (0.6 Lo) for 16 h and then restoration to 1.0 Lo for 1 h. Prolonged incubation at 1.0 Lo minimally reduced the contractile force without substantially altering the mediators of contraction (crossbridge phosphorylation, shortening velocity or stimulated actin polymerization). Prolonged incubation of tissues at slack length (0.6 Lo), despite return of length to 1.0 Lo, substantially reduced contractile force, reduced crossbridge phosphorylation, nearly abolished crossbridge cycling (shortening velocity) and abolished stimulated actin polymerization. These data suggest that (1) slack length treatment significantly alters the contractile phenotype of arterial tissue, and (2) slack length treatment is a model to study acute phenotypic modulation of intact arterial smooth muscle. Copyright © 2013 S. Karger AG, Basel.

Construction of biocompatible porous tissue scaffold from the decellularized umbilical artery.

PubMed

Xin, Yi; Wu, Guanghui; Wu, Man; Zhang, Xiaoxia; Velot, Emilie; Decot, Véronique; Cui, Wei; Huang, Yimin; Stoltz, Jean-Francois; Du, Jie; Li, Na

2015-01-01

The scaffolds prepared from the tissue decellularization conserve the porous 3-D structure and provide an optimal matrix for the tissue regeneration. Since decade, the enzymatic digestion, chemical reagent treatment and mechanical actions such as eversion and abrasion have been used to remove the cells from the intact matrix. In this study, we optimized an enzymatic method to decellularize the umbilical artery to construct a 3-D porous scaffold which is suitable for the culture of mesenchymal stem cells (MSCs). The scaffold maintained the interconnected porous structure. It remained the similar high water content 95.3 ± 1% compared to 94.9 ± 0.6% in the intact umbilical artery (p>0.05). The decellularization process decreased the stress from 0.24 ± 0.05 mPa to 0.15 ± 0.06 mPa (p<0.05). However the decellularization did not change the strain of the artery (45 ± 15% vs. 53 ± 10%, p>0.05). When the scaffold was transplanted to the subcutaneous tissue in the wild type mice, there were less T cells appeared in the surrounding tissue which meant the decreased the immunogenicity by decellularization. This scaffold also supported the adhesion and proliferation of the MSCs. In this study, we constructed a biological compatible porous scaffold from the decellularized umbilical artery which may provide a suitable scaffold for cell-matrix interaction studies and for tissue engineering.

Transconjunctival penetration of mitomycin C.

PubMed

Velpandian, T; Sihota, Ramanjit; Sinha, Ankur; Gupta, Viney

2008-01-01

The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon's tissue following application over the intact conjunctiva before routine trabeculectomy. Institution-based case series. In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon's capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC). Statistical analysis was performed using STATA 8.0 version software (STATA Corporation, Houston, TX, USA). In this study, P -values less than 0.05 were considered as statistically significant. The average weight of the sample of Tenon's tissue excised was 5.51+/-4.42 mg (range: 0.9-17.1) and the average estimated MMC concentration found to be present in Tenon's tissue using HPLC was 18.67+/-32.36 x 10(-6) moles/kg of the tissue (range: 0.38-197.05 x 10(-6)). In 36 of the 41 patients (87.80%), the MMC concentration reached above 2 x 10(-6) moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1

PubMed Central

McCord, Lauren A.; Li, Feixue; Rosewell, Katherine L.; Brännström, Mats; Curry, Thomas E.

2011-01-01

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization. PMID:22116802

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat.

PubMed

McCord, Lauren A; Li, Feixue; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2012-03-01

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

Identification and characterization of long non-coding RNAs in subcutaneous adipose tissue from castrated and intact full-sib pair Huainan male pigs

USDA-ARS?s Scientific Manuscript database

Testosterone deficiency is associated with obesity in humans. It has been proven that long non-coding RNAs (lncRNAs) regulate adipose tissue metabolism; therefore, we first study the role of lncRNAs on testosterone deficiency-induced fat deposition using castrated male pigs as the model animal. The ...

Handling, storage, and preparation of human tissues.

PubMed

Dressler, L G; Visscher, D

2001-05-01

Human tissue for flow cytometry must be prepared as an adequate single-cell suspension. The appropriate methods for tissue collection, transport, storage, and dissociation depend on the cell parameters being measured and the localization of the markers. This unit includes a general method for collecting and transporting human tissue and preparing a tissue imprint. Protocols are supplied for tissue disaggregation by either mechanical or enzymatic means and for preparation of single-cell suspensions of whole cells from fine-needle aspirates, pleural effusions, abdominal fluids, or other body fluids. Other protocols detail preparation of intact nuclei from fresh, frozen, or paraffin-embedded tissue. Support protocols cover fixation, cryospin preparation, cryopreservation, and removal of debris.

A Biological Tissue Adhesive and Dissolvent System for Intraocular Tumor Plaque Brachytherapy.

PubMed

Zloto, Ofira; Vishnevskia-Dai, Vicktoria; Moisseiev, Joseph; Belkin, Michael; Fabian, Ido Didi

2016-02-01

To examine a novel technique for simplified placement and removal of plaque brachytherapy by fibrin glue and urokinase (medac Gmbh, Hamburg, Germany). In six enucleated porcine eyes, plaques were placed on the episclera and fibrin glue was applied to cover it. Urokinase was used to dissolve the glue in three eyes and saline was used in three eyes. Adhesion strength was measured further on 15 plaques affixed to porcine eyes (glued in five with intact conjunctiva, glued in five with removed conjunctiva, and sutured in five). Saline had no effect on the glue-plaque-eye complex, whereas the urokinase (0.38 mL ± 0.08 mL) easily dissolved the adhesion between the glue layer and surrounding tissues. The weight required to detach the plaques was 0.349 kg ± 0.173 kg for glued eyes with intact conjunctiva, 0.405 kg ± 0.083 kg for sutured eyes (P = .59), and 0.032 kg ± 0.004 kg for glued eyes without intact conjunctiva (P ≤ .015). The usage of the biological adhesive and dissolvent system was applicable for plaque surgery in an ex vivo animal model. Copyright 2016, SLACK Incorporated.

46 CFR 174.045 - Intact stability requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045...

46 CFR 174.045 - Intact stability requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability requirements. 174.045 Section 174.045 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Mobile Offshore Drilling Units § 174.045...

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization.

PubMed

Kwiatkowski, M; Wurlitzer, M; Krutilin, A; Kiani, P; Nimer, R; Omidi, M; Mannaa, A; Bussmann, T; Bartkowiak, K; Kruber, S; Uschold, S; Steffen, P; Lübberstedt, J; Küpker, N; Petersen, H; Knecht, R; Hansen, N O; Zarrine-Afsar, A; Robertson, W D; Miller, R J D; Schlüter, H

2016-02-16

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization

PubMed Central

Kwiatkowski, M.; Wurlitzer, M.; Krutilin, A.; Kiani, P.; Nimer, R.; Omidi, M.; Mannaa, A.; Bussmann, T.; Bartkowiak, K.; Kruber, S.; Uschold, S.; Steffen, P.; Lübberstedt, J.; Küpker, N.; Petersen, H.; Knecht, R.; Hansen, N.O.; Zarrine-Afsar, A.; Robertson, W.D.; Miller, R.J.D.; Schlüter, H.

2016-01-01

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Biological significance Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. PMID:26778141

Selenium Preferentially Accumulates in the Eye Lens Following Embryonic Exposure: A Confocal X-ray Fluorescence Imaging Study

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

Choudhury, Sanjukta; Thomas, Jith; Sylvain, Nicole J.

Maternal transfer of elevated selenium (Se) to offspring is an important route of Se exposure for fish in the natural environment. However, there is a lack of information on the tissue specific spatial distribution and speciation of Se in the early developmental stages of fish, which provide important information about Se toxicokinetics. The effect of maternal transfer of Se was studied by feeding adult zebrafish a Se-elevated or a control diet followed by collection of larvae from both groups. Novel confocal synchrotron-based techniques were used to investigate Se within intact preserved larvae. Confocal X-ray fluorescence imaging was used to comparemore » Se distributions within specific planes of an intact larva from each of the two groups. The elevated Se treatment showed substantially higher Se levels than the control; Se preferentially accumulated to highest levels in the eye lens, with lower levels in the retina, yolk and other tissues. Confocal X-ray absorption spectroscopy was used to determine that the speciation of Se within the eye lens of the intact larva was a selenomethionine-like species. Preferential accumulation of Se in the eye lens may suggest a direct cause-and-effect relationship between exposure to elevated Se and Se-induced ocular impairments reported previously. This study illustrates the effectiveness of confocal X-ray fluorescence methods for investigating trace element distribution and speciation in intact biological specimens« less

Potential clinical applications of photoacoustics

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

Rosencwaig, A.

1982-09-01

Photoacoustic spectroscopy offers the opportunity for extending the exact science of noninvasive spectral analysis to intact medical substances such as tissues. Thermal-wave imaging offers the potential for microscopic imaging of thermal features in biological matter.

The role of cryopreservation for women prior to treatment of malignancy.

PubMed

Donnez, Jacques; Dolmans, Marie-Madeleine; Martinez-Madrid, Belen; Demylle, Dominique; Van Langendonckt, Anne

2005-08-01

The purpose of this review is to investigate recent advances in xenografting, as well as in orthotopic and heterotopic autotransplantation of human cryopreserved ovarian tissue. The first livebirth after orthotopic transplantation of cryopreserved ovarian tissue was reported recently. We discuss this case and other cases of reimplantation of cryopreserved ovarian tissue, bearing in mind that many questions remain. Finally, we report the latest developments in research on the transplantation of an intact ovary and the reimplantation of isolated follicles.

Wound repair in Montipora capitata

USGS Publications Warehouse

Work, Thierry M.; Aeby, Greta S.

2010-01-01

We documented the microscopic morphology of tissue healing in Montipora capitata. Fragments from two healthy coral colonies were traumatized by scraping tissue and skeleton and monitored in flow-through seawater tables every 2-4. days for 40. days for gross and cellular changes. Grossly, corals appeared healed and repigmented by Day 40. Histologically, traumatized issues were undistinguishable from intact untraumatized tissues by Day 12. We suspect that the calicoblastic epidermis of basal body wall is pluripotential and can develop into surface epidermis when needed. ?? 2010.

pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

PubMed

Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

2017-09-05

The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

Quantification and Assessment of the Chemical Form of Residual Gadolinium in the Brain After Repeated Administration of Gadolinium-Based Contrast Agents: Comparative Study in Rats.

PubMed

Frenzel, Thomas; Apte, Chirag; Jost, Gregor; Schöckel, Laura; Lohrke, Jessica; Pietsch, Hubertus

2017-07-01

Multiple clinical and preclinical studies have reported a signal intensity increase and the presence of gadolinium (Gd) in the brain after repeated administration of Gd-based contrast agents (GBCAs). This bioanalytical study in rat brain tissue was initiated to investigate whether the residual Gd is present as intact GBCA or in other chemical forms by using tissue fractionation and chromatography. Rats were divided randomly in 6 groups of 10 animals each. They received 10 daily injections of 2.5 mmol/kg bodyweight of 1 of 5 different GBCAs: linear GBCAs such as gadodiamide (Omniscan; GE Healthcare), gadopentetate dimeglumine (Gd-DTPA, Magnevist; Bayer), or gadobenate dimeglumine (Multihance; Bracco) and macrocyclic GBCAs such as gadobutrol (Gadovist; Bayer) and gadoterate meglumine (Gd-DOTA, Dotarem; Guerbet) or saline. On days 3 and 24 after the last injection (p.i.), 5 randomly chosen animals of each group were killed by exsanguination, and their brains were excised and divided into cerebrum, pons, and cerebellum. The brain sections were homogenized by sonication in ice-cold buffer at pH 7.4. Soluble and insoluble fractions were separated by centrifugation, and the soluble fractions were further separated by gel permeation chromatography (GPC). The Gd concentration in all tissue fractions and in the GPC eluate was measured by inductively coupled plasma-mass spectrometry. In a recovery control experiment, all GBCAs were spiked to blank brain tissue and more than 94% recovery of Gd in the tissue fractions was demonstrated. Only traces of the administered Gd were found in the rat brain tissue on day 3 and day 24 p.i. In the animals treated with macrocyclic GBCAs, Gd was found only in the soluble brain fraction and was present solely as low molecular weight molecules, most likely the intact GBCA. In the animals treated with linear GBCAs Gd was found to a large extent in the insoluble tissue fraction. The Gd concentration in the soluble fraction was comparable to the macrocyclic agents. According to GPC, a smaller portion of the Gd in the soluble fraction of the linear GBCAs groups was bound to macromolecules larger than 250 to 300 kDa. The nature of the Gd-containing macromolecules and the insoluble species were not determined, but they appeared to be saturable with Gd. The excretion of the soluble Gd species in the linear and macrocyclic GBCA groups was still ongoing between days 3 and 24 p.i. This was also observed for the macromolecular Gd species in the linear GBCA groups, but at a slower rate. The residual Gd found in the rat brain after repeated administration of all 3 linear GBCAs was present in at least 3 distinctive forms-soluble small molecules, including the intact GBCA, soluble macromolecules, and to a large extent in insoluble form. The latter 2 are most likely responsible for the prolonged signal intensity enhancement in brain structures observed in magnetic resonance imaging. No relevant differences between the 3 linear GBCAs were observed. The Gd concentrations in the brain after administration of macrocyclic GBCAs are lower, and the Gd is only present in soluble small molecules, which were slowly excreted. This underlines the crucial importance of the kinetic inertness of macrocyclic agents in the prevention of potential retention of Gd in the brain compared with the 3 linear, kinetically less restricted GBCAs.

Quantification and Assessment of the Chemical Form of Residual Gadolinium in the Brain After Repeated Administration of Gadolinium-Based Contrast Agents

PubMed Central

Frenzel, Thomas; Apte, Chirag; Jost, Gregor; Schöckel, Laura; Lohrke, Jessica; Pietsch, Hubertus

2017-01-01

Objective Multiple clinical and preclinical studies have reported a signal intensity increase and the presence of gadolinium (Gd) in the brain after repeated administration of Gd-based contrast agents (GBCAs). This bioanalytical study in rat brain tissue was initiated to investigate whether the residual Gd is present as intact GBCA or in other chemical forms by using tissue fractionation and chromatography. Materials and Methods Rats were divided randomly in 6 groups of 10 animals each. They received 10 daily injections of 2.5 mmol/kg bodyweight of 1 of 5 different GBCAs: linear GBCAs such as gadodiamide (Omniscan; GE Healthcare), gadopentetate dimeglumine (Gd-DTPA, Magnevist; Bayer), or gadobenate dimeglumine (Multihance; Bracco) and macrocyclic GBCAs such as gadobutrol (Gadovist; Bayer) and gadoterate meglumine (Gd-DOTA, Dotarem; Guerbet) or saline. On days 3 and 24 after the last injection (p.i.), 5 randomly chosen animals of each group were killed by exsanguination, and their brains were excised and divided into cerebrum, pons, and cerebellum. The brain sections were homogenized by sonication in ice-cold buffer at pH 7.4. Soluble and insoluble fractions were separated by centrifugation, and the soluble fractions were further separated by gel permeation chromatography (GPC). The Gd concentration in all tissue fractions and in the GPC eluate was measured by inductively coupled plasma–mass spectrometry. In a recovery control experiment, all GBCAs were spiked to blank brain tissue and more than 94% recovery of Gd in the tissue fractions was demonstrated. Results Only traces of the administered Gd were found in the rat brain tissue on day 3 and day 24 p.i. In the animals treated with macrocyclic GBCAs, Gd was found only in the soluble brain fraction and was present solely as low molecular weight molecules, most likely the intact GBCA. In the animals treated with linear GBCAs Gd was found to a large extent in the insoluble tissue fraction. The Gd concentration in the soluble fraction was comparable to the macrocyclic agents. According to GPC, a smaller portion of the Gd in the soluble fraction of the linear GBCAs groups was bound to macromolecules larger than 250 to 300 kDa. The nature of the Gd-containing macromolecules and the insoluble species were not determined, but they appeared to be saturable with Gd. The excretion of the soluble Gd species in the linear and macrocyclic GBCA groups was still ongoing between days 3 and 24 p.i. This was also observed for the macromolecular Gd species in the linear GBCA groups, but at a slower rate. Conclusions The residual Gd found in the rat brain after repeated administration of all 3 linear GBCAs was present in at least 3 distinctive forms—soluble small molecules, including the intact GBCA, soluble macromolecules, and to a large extent in insoluble form. The latter 2 are most likely responsible for the prolonged signal intensity enhancement in brain structures observed in magnetic resonance imaging. No relevant differences between the 3 linear GBCAs were observed. The Gd concentrations in the brain after administration of macrocyclic GBCAs are lower, and the Gd is only present in soluble small molecules, which were slowly excreted. This underlines the crucial importance of the kinetic inertness of macrocyclic agents in the prevention of potential retention of Gd in the brain compared with the 3 linear, kinetically less restricted GBCAs. PMID:28125438

Load-unloading response of intact and artificially degraded articular cartilage correlated with near infrared (NIR) absorption spectra.

PubMed

Afara, I O; Singh, S; Oloyede, A

2013-04-01

The conventional mechanical properties of articular cartilage, such as compressive stiffness, have been demonstrated to be limited in their capacity to distinguish intact (visually normal) from degraded cartilage samples. In this paper, we explore the correlation between a new mechanical parameter, namely the reswelling of articular cartilage following unloading from a given compressive load, and the near infrared (NIR) spectrum. The capacity to distinguish mechanically intact from proteoglycan-depleted tissue relative to the "reswelling" characteristic was first established, and the result was subsequently correlated with the NIR spectral data of the respective tissue samples. To achieve this, normal intact and enzymatically degraded samples were subjected to both NIR probing and mechanical compression based on a load-unload-reswelling protocol. The parameter δr, characteristic of the osmotic "reswelling" of the matrix after unloading to a constant small load in the order of the osmotic pressure of cartilage, was obtained for the different sample types. Multivariate statistics was employed to determine the degree of correlation between δr and the NIR absorption spectrum of relevant specimens using Partial Least Squared (PLS) regression. The results show a strong relationship (R(2)=95.89%, p<0.0001) between the spectral data and δr. This correlation of δr with NIR spectral data suggests the potential for determining the reswelling characteristics non-destructively. It was also observed that δr values bear a significant relationship with the cartilage matrix integrity, indicated by its proteoglycan content, and can therefore differentiate between normal and artificially degraded proteoglycan-depleted cartilage samples. It is therefore argued that the reswelling of cartilage, which is both biochemical (osmotic) and mechanical (hydrostatic pressure) in origin, could be a strong candidate for characterizing the tissue, especially in regions surrounding focal cartilage defects in joints. Copyright © 2012 Elsevier Ltd. All rights reserved.

Visualization of femtosecond laser pulse-induced microincisions inside crystalline lens tissue.

PubMed

Stachs, Oliver; Schumacher, Silvia; Hovakimyan, Marine; Fromm, Michael; Heisterkamp, Alexander; Lubatschowski, Holger; Guthoff, Rudolf

2009-11-01

To evaluate a new method for visualizing femtosecond laser pulse-induced microincisions inside crystalline lens tissue. Laser Zentrum Hannover e.V., Hannover, Germany. Lenses removed from porcine eyes were modified ex vivo by femtosecond laser pulses (wavelength 1040 nm, pulse duration 306 femtoseconds, pulse energy 1.0 to 2.5 microJ, repetition rate 100 kHz) to create defined planes at which lens fibers separate. The femtosecond laser pulses were delivered by a 3-dimension (3-D) scanning unit and transmitted by focusing optics (numerical aperture 0.18) into the lens tissue. Lens fiber orientation and femtosecond laser-induced microincisions were examined using a confocal laser scanning microscope (CLSM) based on a Rostock Cornea Module attached to a Heidelberg Retina Tomograph II. Optical sections were analyzed in 3-D using Amira software (version 4.1.1). Normal lens fibers showed a parallel pattern with diameters between 3 microm and 9 microm, depending on scanning location. Microincision visualization showed different cutting effects depending on pulse energy of the femtosecond laser. The effects ranged from altered tissue-scattering properties with all fibers intact to definite fiber separation by a wide gap. Pulse energies that were too high or overlapped too tightly produced an incomplete cutting plane due to extensive microbubble generation. The 3-D CLSM method permitted visualization and analysis of femtosecond laser pulse-induced microincisions inside crystalline lens tissue. Thus, 3-D CLSM may help optimize femtosecond laser-based procedures in the treatment of presbyopia.

Hertwig's Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars.

PubMed

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-06-29

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis.

Hertwig’s Epithelial Root Sheath Fate during Initial Cellular Cementogenesis in Rat Molars

PubMed Central

Yamamoto, Tsuneyuki; Yamada, Tamaki; Yamamoto, Tomomaya; Hasegawa, Tomoka; Hongo, Hiromi; Oda, Kimimitsu; Amizuka, Norio

2015-01-01

To elucidate the fate of the epithelial root sheath during initial cellular cementogenesis, we examined developing maxillary first molars of rats by immunohistochemistry for keratin, vimentin, and tissue non-specific alkaline phosphatase (TNALP) and by TdT-mediated dUTP nick end labeling (TUNEL). The advancing root end was divided into three sections, which follow three distinct stages of initial cellular cementogenesis: section 1, where the epithelial sheath is intact; section 2, where the epithelial sheath becomes fragmented; and section 3, where initial cellular cementogenesis begins. After fragmentation of the epithelial sheath, many keratin-positive epithelial sheath cells were embedded in the rapidly growing cellular cementum. A few unembedded epithelial cells located on the cementum surface. Dental follicle cells, precementoblasts, and cementoblasts showed immunoreactivity for vimentin and TNALP. In all three sections, there were virtually no cells possessing double immunoreactivity for vimentin-keratin or TNALP-keratin and only embedded epithelial cells showed TUNEL reactivity. Taken together, these findings suggest that: (1) epithelial sheath cells divide into two groups; one group is embedded in the cementum and thereafter dies by apoptosis, and the other survives on the cementum surface as epithelial cell rests of Malassez; and (2) epithelial sheath cells do not undergo epithelial-mesenchymal transition during initial cellular cementogenesis. PMID:26160988

Ex-vivo imaging of excised tissue using vital dyes and confocal microscopy

PubMed Central

Johnson, Simon; Rabinovitch, Peter

2012-01-01

Vital dyes routinely used for staining cultured cells can also be used to stain and image live tissue slices ex-vivo. Staining tissue with vital dyes allows researchers to collect structural and functional data simultaneously and can be used for qualitative or quantitative fluorescent image collection. The protocols presented here are useful for structural and functional analysis of viable properties of cells in intact tissue slices, allowing for the collection of data in a structurally relevant environment. With these protocols, vital dyes can be applied as a research tool to disease processes and properties of tissue not amenable to cell culture based studies. PMID:22752953

Plant uptake of pentachlorophenol from sludge-amended soils

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

Bellin, C.A.; O'Connor, G.A.

A greenhouse study was conducted to determine the effects of sludge on plant uptake of {sup 14}C-pentachlorophenol (PCP). Plants included tall fescue (Festuca arundinacea Schreb.), lettuce (Latuca sativa L.), carrot (Daucus carota L.), and chile pepper (Capsicum annum L.). Minimal intact PCP was detected in the fescue and lettuce by gas chromatography/mass spectrometry (GC/MS) analysis. No intact PCP was detected in the carrot tissue extracts. Chile pepper was not analyzed for intact PCP because methylene chloride extracts contained minimal {sup 14}C. The GC/MS analysis of soil extracts at harvest suggests a half-life of PCP of about 10 d independent ofmore » sludge rate or PCP loading rate. Rapid degradation of PCP in the soil apparently limited PCP availability to the plant. Bioconcentration factors (dry plant wt./initial soil PCP concentration) based on intact PCP were <0.01 for all crops, suggesting little PCP uptake. Thus, food-chain crop PCP uptake in these alkaline soils should not limit land application of sludge.« less

Plant uptake of pentachlorophenol from sludge-amended soils

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

Bellin, C.A.; O'Connor, G.A.

A greenhouse study was conducted to determine the effects of sludge on plant uptake of {sup 14}C-pentachlorophenol (PCP). Plants included tall fescue (Festuca arundinacea Schreb.), lettuce (Latuca sativa L.), carrot (Daucus carota L.), and chile pepper (Capsicum annum L.). Minimal intact PCP was detected in the fescue and lettuce by gas chromatography/mass spectrometry (GC/MS) analysis. No intact PCP was detected in the carrot tissue extracts. Chile pepper was not analyzed for intact PCP because methylene chloride extracts contained minimal {sup 14}C. The GC/MS analysis of soil extracts at harvest suggests a half-life of PCP of about 10 d independent ofmore » sludge rate or PCP loading rate. Rapid degradation of PCP in the soil apparently limited PCP availability to the plant. Bioconcentration factors (dry plant wt./initial soil PCP concentration) based on intact PCP were < 0.01 for all crops, suggesting little PCP uptake. Thus, food-chain crop PCP uptake in these alkaline soils should not limit land application of sludge.« less

SU-F-T-220: Validation of Hounsfield Unit-To-Stopping Power Ratio Calibration Used for Dose Calculation in Proton Radiotherapy

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

Polf, J; Chung, H; Langen, K

Purpose: To validate the stoichiometric calibration of the Hounsfield Unit (HU) to Stopping Power Ratio (SPR) calibration used to commission a commercial treatment planning system (TPS) for proton radiotherapy dose calculation. Methods and Materials: The water equivalent thickness (WET) of several individual pig tissues (lung, fat, muscle, liver, intestine, rib, femur), mixed tissue samples (muscle/rib, ice/femur, rib/air cavity/muscle), and an intact pig head were measured with a multi-layer ionization chamber (MLIC). A CT scan of each sample was obtained and imported into a commercial TPS. The WET calculated by the TPS for each tissue sample was compared to the measuredmore » WET value to determine the accuracy of the HU-to-SPR calibration curve used by the TPS to calculate dose. Results: The WET values calculated by the TPS showed good agreement (< 2.0%) with the measured values for bone and all soft tissues except fat (3.1% difference). For the mixed tissue samples and the intact pig head measurements, the difference in the TPS and measured WET values all agreed to within 3.5%. In addition, SPR values were calculated from the measured WET of each tissue, and compared to SPR values of reference tissues from ICRU 46 used to generate the HU-to-SPR calibration for the TPS. Conclusion: For clinical scenarios where the beam passes through multiple tissue types and its path is dominated by soft tissues, we believe using an uncertainty of 3.5% of the planned beam range is acceptable to account for uncertainties in the TPS WET determination.« less

Isolation of tissues and preservation of RNA from intact, germinated barley grain.

PubMed

Betts, Natalie S; Berkowitz, Oliver; Liu, Ruijie; Collins, Helen M; Skadhauge, Birgitte; Dockter, Christoph; Burton, Rachel A; Whelan, James; Fincher, Geoffrey B

2017-08-01

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19 611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24 h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Quantitative real-time analysis of collective cancer invasion and dissemination

NASA Astrophysics Data System (ADS)

Ewald, Andrew J.

2015-05-01

A grand challenge in biology is to understand the cellular and molecular basis of tissue and organ level function in mammals. The ultimate goals of such efforts are to explain how organs arise in development from the coordinated actions of their constituent cells and to determine how molecularly regulated changes in cell behavior alter the structure and function of organs during disease processes. Two major barriers stand in the way of achieving these goals: the relative inaccessibility of cellular processes in mammals and the daunting complexity of the signaling environment inside an intact organ in vivo. To overcome these barriers, we have developed a suite of tissue isolation, three dimensional (3D) culture, genetic manipulation, nanobiomaterials, imaging, and molecular analysis techniques to enable the real-time study of cell biology within intact tissues in physiologically relevant 3D environments. This manuscript introduces the rationale for 3D culture, reviews challenges to optical imaging in these cultures, and identifies current limitations in the analysis of complex experimental designs that could be overcome with improved imaging, imaging analysis, and automated classification of the results of experimental interventions.

Phase Transformations in a Human Tooth Tissue at the Initial Stage of Caries

PubMed Central

Prutskij, Tatiana; Ippolitov, Yury

2015-01-01

The aim of the paper is to study phase transformations in solid tissues of the human teeth during the development of fissure caries by Raman and fluorescence microspectroscopy. The study of the areas with fissure caries confirmed the assumption of the formation of a weak interaction between phosphate apatite enamel and organic acids (products of microorganisms). The experimental results obtained with by Raman microspectroscopy showed the formation of dicalcium phosphate dihydrate - CaHPO4-2H2O in the area of mural demineralization of carious fissure. A comparative analysis of structural and spectroscopic data for the intact and carious enamel shows that emergence of a more soluble phase - carbonate-substituted hydroxyapatite - is typical for the initial stage of caries. It is shown that microareas of dental hard tissues in the carious fissure due to an emerging misorientation of apatite crystals have a higher fluorescence yield than the area of the intact enamel. These areas can be easily detected even prior to a deep demineralization (white spot stage) for the case of irreversibly changed organomineral complex and intensive removal of the mineral component. PMID:25901743

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection

PubMed Central

Pfaller, Kristian; Wagner, Johanna

2016-01-01

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 μm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 μm x 0.7 μm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris. PMID:27632365

Persistent Supercooling of Reproductive Shoots Is Enabled by Structural Ice Barriers Being Active Despite an Intact Xylem Connection.

PubMed

Kuprian, Edith; Tuong, Tan D; Pfaller, Kristian; Wagner, Johanna; Livingston, David P; Neuner, Gilbert

2016-01-01

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary to prevent the entry of ice from already frozen tissues. The reproductive shoot of Calluna vulgaris is able to supercool down to below -22°C throughout all developmental stages (shoot elongation, flowering, fruiting) despite an established xylem conductivity. After localization of the persistent ice barrier between the reproductive and vegetative shoot at the base of the pedicel by infrared differential thermal analysis, the currently unknown structural features of the ice barrier tissue were anatomically analyzed on cross and longitudinal sections. The ice barrier tissue was recognized as a 250 μm long constriction zone at the base of the pedicel that lacked pith tissue and intercellular spaces. Most cell walls in this region were thickened and contained hydrophobic substances (lignin, suberin, and cutin). A few cell walls had what appeared to be thicker cellulose inclusions. In the ice barrier tissue, the area of the xylem was as much as 5.7 times smaller than in vegetative shoots and consisted of tracheids only. The mean number of conducting units in the xylem per cross section was reduced to 3.5% of that in vegetative shoots. Diameter of conducting units and tracheid length were 70% and 60% (respectively) of that in vegetative shoots. From vegetative shoots water transport into the ice barrier must pass pit membranes that are likely impermeable to ice. Pit apertures were about 1.9 μm x 0.7 μm, which was significantly smaller than in the vegetative shoot. The peculiar anatomical features of the xylem at the base of the pedicel suggest that the diameter of pores in pit membranes could be the critical constriction for ice propagation into the persistently supercooled reproductive shoots of C. vulgaris.

Recovery of high-quality RNA from laser capture microdissected human and rodent pancreas.

PubMed

Butler, Alexandra E; Matveyenko, Aleksey V; Kirakossian, David; Park, Johanna; Gurlo, Tatyana; Butler, Peter C

Laser capture microdissection (LCM) is a powerful method to isolate specific populations of cells for subsequent analysis such as gene expression profiling, for example, microarrays or ribonucleic (RNA)-Seq. This technique has been applied to frozen as well as formalin-fixed, paraffin-embedded (FFPE) specimens with variable outcomes regarding quality and quantity of extracted RNA. The goal of the study was to develop the methods to isolate high-quality RNA from islets of Langerhans and pancreatic duct glands (PDG) isolated by LCM. We report an optimized protocol for frozen sections to minimize RNA degradation and maximize recovery of expected transcripts from the samples using quantitative real-time polymerase chain reaction (RT-PCR) by adding RNase inhibitors at multiple steps during the experiment. This technique reproducibly delivered intact RNA (RIN values 6-7). Using quantitative RT-PCR, the expected profiles of insulin, glucagon, mucin6 (Muc6), and cytokeratin-19 (CK-19) mRNA in PDGs and pancreatic islets were detected. The described experimental protocol for frozen pancreas tissue might also be useful for other tissues with moderate to high levels of intrinsic ribonuclease (RNase) activity.

Motion artifact and background noise suppression on optical microangiography frames using a naïve Bayes mask.

PubMed

Reif, Roberto; Baran, Utku; Wang, Ruikang K

2014-07-01

Optical coherence tomography (OCT) is a technique that allows for the three-dimensional (3D) imaging of small volumes of tissue (a few millimeters) with high resolution (∼10  μm). Optical microangiography (OMAG) is a method of processing OCT data, which allows for the extraction of the tissue vasculature with capillary resolution from the OCT images. Cross-sectional B-frame OMAG images present the location of the patent blood vessels; however, the signal-to-noise-ratio of these images can be affected by several factors such as the quality of the OCT system and the tissue motion artifact. This background noise can appear in the en face projection view image. In this work we propose to develop a binary mask that can be applied on the cross-sectional B-frame OMAG images, which will reduce the background noise while leaving the signal from the blood vessels intact. The mask is created by using a naïve Bayes (NB) classification algorithm trained with a gold standard image which is manually segmented by an expert. The masked OMAG images present better contrast for binarizing the image and quantifying the result without the influence of noise. The results are compared with a previously developed frequency rejection filter (FRF) method which is applied on the en face projection view image. It is demonstrated that both the NB and FRF methods provide similar vessel length fractions. The advantage of the NB method is that the results are applicable in 3D and that its use is not limited to periodic motion artifacts.

The bovine patella as a model of early osteoarthritis.

PubMed

Hargrave-Thomas, E J; Thambyah, A; McGlashan, S R; Broom, N D

2013-12-01

The bovine patella model has been used extensively for studying important structure-function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late-stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA. © 2013 Anatomical Society.

Transconjunctival penetration of mitomycin C

PubMed Central

Velpandian, T; Sihota, Ramanjit; Gupta, Viney

2008-01-01

Aims: The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon′s tissue following application over the intact conjunctiva before routine trabeculectomy. Settings and Design: Institution-based case series. Materials and Methods: In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon′s capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC). Statistical Analysis Used: Statistical analysis was performed using stata0 8.0 version software (STATA Corporation, Houston, TX, USA). In this study, P -values less than 0.05 were considered as statistically significant. Results: The average weight of the sample of Tenon′s tissue excised was 5.51 ± 4.42 mg (range: 0.9-17.1) and the average estimated MMC concentration found to be present in Tenon′s tissue using HPLC was 18.67 ± 32.36 × 10−6 moles/kg of the tissue (range: 0.38-197.05 x 10−6 ). In 36 of the 41 patients (87.80%), the MMC concentration reached above 2 x 10−6 moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts. Conclusions: Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs. PMID:18417819

The bovine patella as a model of early osteoarthritis

PubMed Central

Hargrave-Thomas, E J; Thambyah, A; McGlashan, S R; Broom, N D

2013-01-01

The bovine patella model has been used extensively for studying important structure–function aspects of articular cartilage, including its degeneration. However, the degeneration seen in this model has, to our knowledge, never been adequately compared with human osteoarthritis (OA). In this study, bovine patellae displaying normal to severely degenerate states were compared with human tissue displaying intact cartilage to severe OA. Comparisons of normal and OA features were made with histological scoring, morphometric measurements, and qualitative observations. Differential interference contrast microscopy was used to image early OA changes in the articular cartilage matrix and to investigate whether this method provided comparable quality of visualisation of key structural features with standard histology. The intact bovine cartilage was found to be similar to healthy human cartilage and the degenerate bovine cartilage resembled the human OA tissues with regard to structural disruption, cellularity changes, and staining loss. The extent of degeneration in the bovine tissues matched the mild to moderate range of human OA tissues; however, no bovine samples exhibited late-stage OA. Additionally, in both bovine and human tissues, cartilage degeneration was accompanied by calcified cartilage thickening, tidemark duplication, and the advancement of the cement line by protrusions of bony spicules into the calcified cartilage. This comparison of degeneration in the bovine and human tissues suggests a common pathway for the progression of OA and thus the bovine patella is proposed to be an appropriate model for investigating the structural changes associated with early OA. PMID:24111904

Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

PubMed

McCullough, D P; Gudla, P R; Harris, B S; Collins, J A; Meaburn, K J; Nakaya, M A; Yamaguchi, T P; Misteli, T; Lockett, S J

2008-05-01

Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

A low carbohydrate, high protein diet suppresses intratumoral androgen synthesis and slows castration-resistant prostate tumor growth in mice.

PubMed

Fokidis, H Bobby; Yieng Chin, Mei; Ho, Victor W; Adomat, Hans H; Soma, Kiran K; Fazli, Ladan; Nip, Ka Mun; Cox, Michael; Krystal, Gerald; Zoubeidi, Amina; Tomlinson Guns, Emma S

2015-06-01

Dietary factors continue to preside as dominant influences in prostate cancer prevalence and progression-free survival following primary treatment. We investigated the influence of a low carbohydrate diet, compared to a typical Western diet, on prostate cancer (PCa) tumor growth in vivo. LNCaP xenograft tumor growth was studied in both intact and castrated mice, representing a more advanced castration resistant PCa (CRPC). No differences in LNCaP tumor progression (total tumor volume) with diet was observed for intact mice (P = 0.471) however, castrated mice on the Low Carb diet saw a statistically significant reduction in tumor growth rate compared with Western diet fed mice (P = 0.017). No correlation with serum PSA was observed. Steroid profiles, alongside serum cholesterol and cholesteryl ester levels, were significantly altered by both diet and castration. Specifically, DHT concentration with the Low Carb diet was 58% that of the CRPC-bearing mice on the Western diet. Enzymes in the steroidogenesis pathway were directly impacted and tumors isolated from intact mice on the Low Carb diet had higher AKR1C3 protein levels and lower HSD17B2 protein levels than intact mice on the Western diet (ARK1C3: P = 0.074; HSD17B2: P = 0.091, with α = 0.1). In contrast, CRPC tumors from mice on Low Carb diets had higher concentrations of both HSD17B2 (P = 0.016) and SRD5A1 (P = 0.058 with α = 0.1) enzymes. There was no correlation between tumor growth in castrated mice for Low Carb diet versus Western diet and (a) serum insulin (b) GH serum levels (c) insulin receptor (IR) or (d) IGF-1R in tumor tissue. Intact mice fed Western diet had higher serum insulin which was associated with significantly higher blood glucose and tumor tissue IR. We conclude that both diet and castration have a significant impact on the endocrinology of mice bearing LNCaP xenograft tumors. The observed effects of diet on cholesterol and steroid regulation impact tumor tissue DHT specifically and are likely to be mechanistic drivers behind the observed tumor growth suppression. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells.

PubMed

Acerbi, Irene; Luque, Tomás; Giménez, Alícia; Puig, Marta; Reguart, Noemi; Farré, Ramon; Navajas, Daniel; Alcaraz, Jordi

2012-01-01

Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm(2) cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca(2+) signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis.

Effects of Tribulus terrestris on endocrine sensitive organs in male and female Wistar rats.

PubMed

Martino-Andrade, Anderson J; Morais, Rosana N; Spercoski, Katherinne M; Rossi, Stefani C; Vechi, Marina F; Golin, Munisa; Lombardi, Natália F; Greca, Cláudio S; Dalsenter, Paulo R

2010-01-08

Investigate the possible effects of Tribulus terrestris (TT) on endocrine sensitive organs in intact and castrated male rats as well as in a post-menopausal rat model using ovariectomized females. Three different dose levels of TT (11, 42 and 110 mg/kg/day) were administered to castrated males for 7 days and to intact males and castrated females for 28 days. In addition to TT treatment, all experiments also included a group of rats treated with dehydroepiandrosterone (DHEA). In experiments using castrated males and females we also used testosterone and 17 alpha-ethynylestradiol, respectively, as positive controls for androgenicity and estrogenicity. Neither DHEA nor TT was able to stimulate androgen sensitive tissues like the prostate and seminal vesicle in both intact and castrated male rats. In addition, administration of TT to intact male rats for 28 days did not change serum testosterone levels as well as did not produce any quantitative change in the fecal excretion of androgenic metabolites. However, a slight increase in the number of homogenization-resistant spermatids was observed in rats treated with 11 mg/kg/day of TT extract. In ovariectomized females, TT did not produce any stimulatory effects in uterine and vaginal epithelia. Tribulus terrestris was not able to stimulate endocrine sensitive tissues such as the prostate, seminal vesicle, uterus and vagina in Wistar rats, indicating lack of androgenic and estrogenic activity in vivo. We also showed a positive effect of TT administration on rat sperm production, associated with unchanged levels of circulating androgens. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

Magnetic resonance metabolomics of intact tissue: a biotechnological tool in cancer diagnostics and treatment evaluation.

PubMed

Bathen, Tone F; Sitter, Beathe; Sjøbakk, Torill E; Tessem, May-Britt; Gribbestad, Ingrid S

2010-09-01

Personalized medicine is increasingly important in cancer treatment for its role in staging and its potential to improve stratification of patients. Different types of molecules, genes, proteins, and metabolites are being extensively explored as potential biomarkers. This review discusses the major findings and potential of tissue metabolites determined by high-resolution magic angle spinning magnetic resonance spectroscopy for cancer detection, characterization, and treatment monitoring.

The variable clinical presentation of tuberculosis otitis media and the importance of early detection.

PubMed

Abes, Generoso T; Abes, Franco Louie L B; Jamir, Joselito C

2011-06-01

Tuberculosis (TB) is a rare cause of otitis media. This study aims to increase awareness on the clinical presentation of TB otitis media and illustrate how early detection affects treatment outcome. Chart review of 12 patients (13 ears) from a tertiary hospital in Manila, Philippines, seen from 2004 to 2009. Clinical predictors of the disease were summarized. Clinical, radiologic, and audiometric outcomes after treatment were compared between treatment groups. The 5 otoscopic presentations were multiple perforations, single perforation with refractory otorrhea and exuberant granulation tissue formation, single perforation with minimal otorrhea and no granulation tissue formation, intact tympanic membrane with middle ear effusion, and intact tympanic membrane with tumorlike tissue in the middle ear. Clinical predictors of the disease were history of pulmonary TB, work-related contamination of the infection, positive purified protein derivative test, positive chest radiographic finding and intraoperative granulation tissue with cheesy material, and temporal bone computed tomographic scan findings. Patients who had no middle ear surgery showed significantly better clinical, radiologic, and audiometric outcomes than those who were diagnosed late and had more complicated surgical procedure. The clinical presentation of TB otitis media is variable. Early detection of the early forms entail less surgical intervention and favors better treatment results.

Hypertrophic gene expression induced by chronic stretch of excised mouse heart muscle.

PubMed

Raskin, Anna M; Hoshijima, Masahiko; Swanson, Eric; McCulloch, Andrew D; Omens, Jeffrey H

2009-09-01

Altered mechanical stress and strain in cardiac myocytes induce modifications in gene expression that affects cardiac remodeling and myocyte contractile function. To study the mechanisms of mechanotransduction in cardiomyocytes, probing alterations in mechanics and gene expression has been an effective strategy. However, previous studies are self-limited due to the general use of isolated neonatal rodent myocytes or intact animals. The main goal of this study was to develop a novel tissue culture chamber system for mouse myocardium that facilitates loading of cardiac tissue, while measuring tissue stress and deformation within a physiological environment. Intact mouse right ventricular papillary muscles were cultured in controlled conditions with superfusate at 95% O2/ 5% CO2, and 34 degrees C, such that cell to extracellular matrix adhesions as well as cell to cell adhesions were undisturbed and both passive and active mechanical properties were maintained without significant changes. The system was able to measure the induction of hypertrophic markers (BNP, ANP) in tissue after 2 hrs and 5 hrs of stretch. ANP induction was highly correlated with the diastolic load of the muscle but not with developed systolic load. Load induced ANP expression was blunted in muscles from muscle-LIM protein knockout mice, in which defective mechanotransduction pathways have been predicted.

Biological and histopathological investigations of moclobemide on injured ovarian tissue following induction of ischemia-reperfusion in rats.

PubMed

Ingec, Metin; Calik, Muhammet; Gundogdu, Cemal; Kurt, Ali; Yilmaz, Mehmet; Isaoglu, Unal; Salman, Suleyman; Akcay, Fatih; Suleyman, Halis

2012-04-01

The effects of moclobemide on damaged ovarian tissue induced by ischemia- reperfusion and damaged contralateral ovarian tissue were investigated in rats, biochemically and histologically. In this experimental study, 40 rats were equally divided into four groups: 10 mg/kg moclobemide, 20 mg/kg moclobemide, ischemia/reperfusion control, and intact control groups. A 2-2.5-cm-long vertical incision was made in the lower abdomen of each rat in order to reach the ovaries, after which a vascular clip was placed on the lower side of the right ovary of each animal in the two treatment groups and the ischemia-reperfusion control group, but not in the healthy (intact control) animal group. The purpose of this procedure was to create ischemia over the course of three hours, then the clips were unclamped to provide reperfusion for the next two hours. At the end of the two hours of reperfusion, all the animals were killed by high-dose anaesthesia and their ovaries were taken and subjected to histological and biochemical (malondialdehyde, nitric oxide, glutathione) studies. The obtained results showed that moclobemide suppressed nitric oxide and malondialdehyde production in the ischemia-reperfusion damage area, and prevented the decrease in endogenous antioxidant levels (glutathione) in the rat ovarian tissue. Moclobemide also prevented infiltration of leukocytes to the ovarian tissue. These results showed that moclobemide protected ovarian tissue against ischemiareperfusion injury. This study shows that moclobemide represses malondialdehyde and nitric oxide production in the rat ovarian tissue subjected to ischemia-reperfusion injury and keeps the endogenous antioxidant glutathione level from decreasing. Moclobemide also inhibits leukocytic migration into ovarian tissue following ischemia-reperfusion injury. From these results, it is suggested that moclobemide can be used in the treatment of ovarian ischemia-reperfusion injury.

Iron Modifies Plasma FGF23 Differently in Autosomal Dominant Hypophosphatemic Rickets and Healthy Humans

PubMed Central

Peacock, Munro; Gray, Amie K.; Padgett, Leah R.; Hui, Siu L.; Econs, Michael J.

2011-01-01

Context: In autosomal dominant hypophosphatemic rickets (ADHR), fibroblast growth factor 23 (FGF23) resists cleavage, causing increased plasma FGF23 levels. The clinical phenotype includes variable onset during childhood or adulthood and waxing/waning of hypophosphatemia. Delayed onset after puberty in females suggests iron status may be important. Objective: Studies were performed to test the hypothesis that plasma C-terminal and intact FGF23 concentrations are related to serum iron concentrations in ADHR. Design and Setting: Cross-sectional and longitudinal studies of ADHR and a cross-sectional study in healthy subjects were conducted at an academic medical center. Participants: Participants included 37 subjects with ADHR mutations from four kindreds and 158 healthy adult controls. Main Outcome Measure: The relationships of serum iron concentrations with plasma C-terminal and intact FGF23 concentrations were evaluated. Results: Serum phosphate and 1,25-dihydroxyvitamin D correlated negatively with C-terminal FGF23 and intact FGF23 in ADHR but not in controls. Serum iron was negatively correlated to both C-terminal FGF23 (r = −0.386; P < 0.05) and intact FGF23 (r = −0.602; P < 0.0001) in ADHR. However, control subjects also demonstrated a negative relationship of serum iron with C-terminal FGF23 (r = −0.276; P < 0.001) but no relationship with intact FGF23. Longitudinally in ADHR subjects, C-terminal FGF23 and intact FGF23 concentrations changed negatively with iron concentrations (P < 0.001 and P = 0.055, respectively), serum phosphate changed negatively with C-terminal FGF23 and intact FGF23 (P < 0.001), and there was a positive relationship between serum iron and phosphate (P < 0.001). Conclusions: Low serum iron is associated with elevated FGF23 in ADHR. However, in controls, low serum iron was also associated with elevated C-terminal FGF23, but not intact FGF23, suggesting cleavage maintains homeostasis despite increased FGF23 expression. PMID:21880793

Iron modifies plasma FGF23 differently in autosomal dominant hypophosphatemic rickets and healthy humans.

PubMed

Imel, Erik A; Peacock, Munro; Gray, Amie K; Padgett, Leah R; Hui, Siu L; Econs, Michael J

2011-11-01

In autosomal dominant hypophosphatemic rickets (ADHR), fibroblast growth factor 23 (FGF23) resists cleavage, causing increased plasma FGF23 levels. The clinical phenotype includes variable onset during childhood or adulthood and waxing/waning of hypophosphatemia. Delayed onset after puberty in females suggests iron status may be important. Studies were performed to test the hypothesis that plasma C-terminal and intact FGF23 concentrations are related to serum iron concentrations in ADHR. Cross-sectional and longitudinal studies of ADHR and a cross-sectional study in healthy subjects were conducted at an academic medical center. Participants included 37 subjects with ADHR mutations from four kindreds and 158 healthy adult controls. The relationships of serum iron concentrations with plasma C-terminal and intact FGF23 concentrations were evaluated. Serum phosphate and 1,25-dihydroxyvitamin D correlated negatively with C-terminal FGF23 and intact FGF23 in ADHR but not in controls. Serum iron was negatively correlated to both C-terminal FGF23 (r = -0.386; P < 0.05) and intact FGF23 (r = -0.602; P < 0.0001) in ADHR. However, control subjects also demonstrated a negative relationship of serum iron with C-terminal FGF23 (r = -0.276; P < 0.001) but no relationship with intact FGF23. Longitudinally in ADHR subjects, C-terminal FGF23 and intact FGF23 concentrations changed negatively with iron concentrations (P < 0.001 and P = 0.055, respectively), serum phosphate changed negatively with C-terminal FGF23 and intact FGF23 (P < 0.001), and there was a positive relationship between serum iron and phosphate (P < 0.001). Low serum iron is associated with elevated FGF23 in ADHR. However, in controls, low serum iron was also associated with elevated C-terminal FGF23, but not intact FGF23, suggesting cleavage maintains homeostasis despite increased FGF23 expression.

Quantification of red myotomal muscle volume and geometry in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis) using T1-weighted magnetic resonance imaging.

PubMed

Perry, Cameron N; Cartamil, Daniel P; Bernal, Diego; Sepulveda, Chugey A; Theilmann, Rebecca J; Graham, Jeffrey B; Frank, Lawrence R

2007-04-01

T1-weighted magnetic resonance imaging (MRI) in conjunction with image and segmentation analysis (i.e., the process of digitally partitioning tissues based on specified MR image characteristics) was evaluated as a noninvasive alternative for differentiating muscle fiber types and quantifying the amounts of slow, red aerobic muscle in the shortfin mako shark (Isurus oxyrinchus) and the salmon shark (Lamna ditropis). MRI-determinations of red muscle quantity and position made for the mid-body sections of three mako sharks (73.5-110 cm fork length, FL) are in close agreement (within the 95% confidence intervals) with data obtained for the same sections by the conventional dissection method involving serial cross-sectioning and volumetric analyses, and with previously reported findings for this species. The overall distribution of salmon shark red muscle as a function of body fork length was also found to be consistent with previously acquired serial dissection data for this species; however, MR imaging revealed an anterior shift in peak red muscle cross-sectional area corresponding to an increase in body mass. Moreover, MRI facilitated visualization of the intact and anatomically correct relationship of tendon linking the red muscle and the caudal peduncle. This study thus demonstrates that MRI is effective in acquiring high-resolution three-dimensional digital data with high contrast between different fish tissue types. Relative to serial dissection, MRI allows more precise quantification of the position, volume, and other details about the types of muscle within the fish myotome, while conserving specimen structural integrity. Copyright (c) 2007 Wiley-Liss, Inc.

The immune system in children with malnutrition--a systematic review.

PubMed

Rytter, Maren Johanne Heilskov; Kolte, Lilian; Briend, André; Friis, Henrik; Christensen, Vibeke Brix

2014-01-01

Malnourished children have increased risk of dying, with most deaths caused by infectious diseases. One mechanism behind this may be impaired immune function. However, this immune deficiency of malnutrition has not previously been systematically reviewed. To review the scientific literature about immune function in children with malnutrition. A systematic literature search was done in PubMed, and additional articles identified in reference lists and by correspondence with experts in the field. The inclusion criteria were studies investigating immune parameters in children aged 1-60 months, in relation to malnutrition, defined as wasting, underweight, stunting, or oedematous malnutrition. The literature search yielded 3402 articles, of which 245 met the inclusion criteria. Most were published between 1970 and 1990, and only 33 after 2003. Malnutrition is associated with impaired gut-barrier function, reduced exocrine secretion of protective substances, and low levels of plasma complement. Lymphatic tissue, particularly the thymus, undergoes atrophy, and delayed-type hypersensitivity responses are reduced. Levels of antibodies produced after vaccination are reduced in severely malnourished children, but intact in moderate malnutrition. Cytokine patterns are skewed towards a Th2-response. Other immune parameters seem intact or elevated: leukocyte and lymphocyte counts are unaffected, and levels of immunoglobulins, particularly immunoglobulin A, are high. The acute phase response appears intact, and sometimes present in the absence of clinical infection. Limitations to the studies include their observational and often cross-sectional design and frequent confounding by infections in the children studied. The immunological alterations associated with malnutrition in children may contribute to increased mortality. However, the underlying mechanisms are still inadequately understood, as well as why different types of malnutrition are associated with different immunological alterations. Better designed prospective studies are needed, based on current understanding of immunology and with state-of-the-art methods.

Effect of Return to Overuse Activity Following an Isolated Supraspinatus Tendon Tear on Adjacent Intact Tendons and Glenoid Cartilage in a Rat Model

PubMed Central

Reuther, Katherine E.; Thomas, Stephen J.; Sarver, Joseph J.; Tucker, Jennica J.; Lee, Chang-Soo; Gray, Chancellor F.; Glaser, David L.; Soslowsky, Louis J.

2013-01-01

Rotator cuff tears are common conditions that can alter shoulder mechanics and may lead to damage of intact joint tissues. These injuries are of particular concern in populations who perform tasks requiring repetitive overhead activity (e.g., athletes and laborers) and who are likely to return to aggressive pre-injury activity levels despite limited understanding of the potentially damaging effects on the remaining tissues. Therefore, we investigated the effect of returning to overuse activity following a supraspinatus tear on shoulder function and the mechanical properties of the remaining intact tendons and glenoid cartilage. Forty rats underwent 4 weeks of overuse activity to create a tendinopathic condition followed by detachment of the supraspinatus tendon and were then randomized into two groups: continued overuse or cage activity. Ambulatory measurements were performed throughout the 8 weeks prior to euthaniasia, and properties of the adjacent tendons and cartilage were evaluated. Results demonstrated that shoulder function was not compromised in the return to overuse group. However, alterations of the glenoid cartilage and biceps tendon properties occurred. Our results help define the contributory roles of common mechanical injury mechanisms and provide a framework by which physicians could better prescribe long-term treatment strategies for patients. PMID:23280495

Effect of return to overuse activity following an isolated supraspinatus tendon tear on adjacent intact tendons and glenoid cartilage in a rat model.

PubMed

Reuther, Katherine E; Thomas, Stephen J; Sarver, Joseph J; Tucker, Jennica J; Lee, Chang-Soo; Gray, Chancellor F; Glaser, David L; Soslowsky, Louis J

2013-05-01

Rotator cuff tears are common conditions that can alter shoulder mechanics and may lead to damage of intact joint tissues. These injuries are of particular concern in populations who perform tasks requiring repetitive overhead activity (e.g., athletes and laborers) and who are likely to return to aggressive pre-injury activity levels despite limited understanding of the potentially damaging effects on the remaining tissues. Therefore, we investigated the effect of returning to overuse activity following a supraspinatus tear on shoulder function and the mechanical properties of the remaining intact tendons and glenoid cartilage. Forty rats underwent 4 weeks of overuse activity to create a tendinopathic condition followed by detachment of the supraspinatus tendon and were then randomized into two groups: continued overuse or cage activity. Ambulatory measurements were performed throughout the 8 weeks prior to euthaniasia, and properties of the adjacent tendons and cartilage were evaluated. Results demonstrated that shoulder function was not compromised in the return to overuse group. However, alterations of the glenoid cartilage and biceps tendon properties occurred. Our results help define the contributory roles of common mechanical injury mechanisms and provide a framework by which physicians could better prescribe long-term treatment strategies for patients. Copyright © 2012 Orthopaedic Research Society.

Interocular transfer of depth discrimination in intact and in DSO-sectioned pigeons.

PubMed

Musumeci, D; Lemeignan, M; Bloch, S

1991-07-22

Interocular transfer (IOT) of a depth discrimination task was studied in intact pigeons and with a section of the supraoptic decussation (DSO). Animals were trained to respond to the nearer of two small light emitting diodes placed at different depths in the left and right compartments of a black tunnel. The near stimulus (at 10.5 cm from the eyes) and the far one (at 21 cm) could only be seen one at a time. Though the task was difficult to learn monocularly, intact as well as lesioned animals had good transfer scores with the untrained eye. Success in transfer may be related to the presentation of the discriminanda which assured that their images impinged upon the retinal 'red field'. DSO-transection did not affect IOT possibly because differential oculomotor adjustments needed for focusing near or far targets provide central bilateral and/or binocular information which is not conveyed by the DSO.

Isolation and Oxidative Properties of Intact Mitochondria Isolated from Spinach Leaves 1

PubMed Central

Douce, Roland; Moore, Antony L.; Neuburger, Michel

1977-01-01

A procedure was described for preparing intact mitochondria from spinach (Spinacia oleracea L.) leaves. These mitochondria oxidized succinate, malate, pyruvate, α-ketoglutarate, and NADH with good respiratory control and ADP/O ratios comparable to those observed with mitochondria from other plant tissues. Glycine was oxidized by the preparations. This oxidation linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport and phosphorylation inhibitors. Cyanide completely inhibited the oxidation of NADH. The oxidation of succinate, malate, and glycine was only partially inhibited. Images PMID:16660151

Rationalisation and Validation of an Acrylamide-Free Procedure in Three-Dimensional Histological Imaging

PubMed Central

Lai, Hei Ming; Liu, Alan King Lun; Ng, Wai-Lung; DeFelice, John; Lee, Wing Sang; Li, Heng; Li, Wen; Ng, Ho Man; Chang, Raymond Chuen-Chung; Lin, Bin; Wu, Wutian; Gentleman, Steve M.

2016-01-01

Three-dimensional visualization of intact tissues is now being achieved by turning tissues transparent. CLARITY is a unique tissue clearing technique, which features the use of detergents to remove lipids from fixed tissues to achieve optical transparency. To preserve tissue integrity, an acrylamide-based hydrogel has been proposed to embed the tissue. In this study, we examined the rationale behind the use of acrylamide in CLARITY, and presented evidence to suggest that the omission of acrylamide-hydrogel embedding in CLARITY does not alter the preservation of tissue morphology and molecular information in fixed tissues. We therefore propose a novel and simplified workflow for formaldehyde-fixed tissue clearing, which will facilitate the laboratory implementation of this technique. Furthermore, we have investigated the basic tissue clearing process in detail and have highlighted some areas for targeted improvement of technologies essential for the emerging subject of three-dimensional histology. PMID:27359336

Interspecies Scaling in Blast Neurotrauma

DTIC Science & Technology

2015-08-27

shows increased force magnitude with similar relaxation form. ............................ 123 Figure 5-7: Relaxation test behavior for L1, Post L2...and Post L3 tests to assess progressive changes in material behavior for a) Mouse, b) Ferret, and c) Pig show changes in tissue behavior after higher...characterizations. Testing of brain tissue in vivo (in a living animal) or in situ (in a post -mortem intact skull) holds advantages of the common in vitro

Fabrication method, structure, mechanical, and biological properties of decellularized extracellular matrix for replacement of wide bone tissue defects.

PubMed

Anisimova, N Y; Kiselevsky, M V; Sukhorukova, I V; Shvindina, N V; Shtansky, D V

2015-09-01

The present paper was focused on the development of a new method of decellularized extracellular matrix (DECM) fabrication via a chemical treatment of a native bone tissue. Particular attention was paid to the influence of chemical treatment on the mechanical properties of native bones, sterility, and biological performance in vivo using the syngeneic heterotopic and orthotopic implantation models. The obtained data indicated that after a chemical decellularization treatment in 4% aqueous sodium chlorite, no noticeable signs of the erosion of compact cortical bone surface or destruction of trabeculae of spongy bone in spinal channel were observed. The histological studies showed that the chemical treatment resulted in the decellularization of both bone and cartilage tissues. The DECM samples demonstrated no signs of chemical and biological degradation in vivo. Thorough structural characterization revealed that after decellularization, the mineral frame retained its integrity with the organic phase; however clotting and destruction of organic molecules and fibers were observed. FTIR studies revealed several structural changes associated with the destruction of organic molecules, although all organic components typical of intact bone were preserved. The decellularization-induced structural changes in the collagen constituent resulted changed the deformation under compression mechanism: from the major fracture by crack propagation throughout the sample to the predominantly brittle fracture. Although the mechanical properties of radius bones subjected to decellularization were observed to degrade, the mechanical properties of ulna bones in compression and humerus bones in bending remained unchanged. The compressive strength of both the intact and decellularized ulna bones was 125-130 MPa and the flexural strength of humerus bones was 156 and 145 MPa for the intact and decellularized samples, respectively. These results open new avenues for the use of DECM samples as the replacement of wide bone tissue defects. Copyright © 2015 Elsevier Ltd. All rights reserved.

Dosimetric impact of the AeroForm tissue expander in postmastectomy radiation therapy: an ex vivo analysis.

PubMed

Moni, Janaki; Saleeby, Jonathan; Bannon, Elizabeth; Lo, Yuan-Chyuan; Fitzgerald, Thomas J

2015-01-01

To evaluate the effect of the AeroForm (AirXpanders Inc, Palo Alto, CA) tissue expander on the dose distribution in a phantom from a simulated postmastectomy radiation treatment for breast cancer. Experiments were conducted to determine the effect on the dose distribution with the metallic reservoir irradiated independently and with the entire AeroForm tissue expander placed on a RANDO phantom (The Phantom Laboratory, Salem, NY). The metallic reservoir was irradiated on a block of solid water with film at various depths ranging from 0 to 8.2 cm from the surface. The intact 400 cc AeroForm was inflated to full capacity and irradiated while positioned on a RANDO phantom, with 12 optically stimulated luminescent dosimeters (OSLDs) placed at clinically relevant expander-tissue interface points. Film dosimetry with the reservoir perpendicular to film reveals 40% transmission at a depth of 0.7 cm, which increases to 60% at a depth of 8.2 cm. In the parallel position, the results vary depending on which area under the reservoir is examined, indicating that the reservoir is not a uniformly dense object. Testing of the intact expander on the phantom revealed that the average percent difference (measured vs expected dose) was 2.7%, σ = 6.2% with heterogeneity correction and 3.7%, σ = 2.4% without heterogeneity correction. The only position where the OSLD readings were consistently higher than the calculated dose by >5% was at position 1, just deep to the canister at the expander-phantom interface. At this position, the readings varied from 5.2% to 14.5%, regardless of heterogeneity correction. Film dosimetry demonstrated beam attenuation in the shadow of the metallic reservoir in the expander. This decrease in dose was not reproduced on the intact expander on the phantom designed to replicate a clinical setup. Copyright © 2015 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.

Energetics of acclimation to NaCl by submerged, anoxic rice seedlings

PubMed Central

Kurniasih, Budiastuti; Greenway, Hank; Colmer, Timothy David

2017-01-01

Background and aims Our aim was to elucidate how plant tissues under a severe energy crisis cope with imposition of high NaCl, which greatly increases ion fluxes and hence energy demands. The energy requirements for ion regulation during combined salinity and anoxia were assessed to gain insights into ion transport processes in the anoxia-tolerant coleoptile of rice. Methods We studied the combined effects of anoxia plus 50 or 100 mm NaCl on tissue ions and growth of submerged rice (Oryza sativa) seedlings. Excised coleoptiles allowed measurements in aerated or anoxic conditions of ion net fluxes and O2 consumption or ethanol formation and by inference energy production. Key Results Over 80 h of anoxia, coleoptiles of submerged intact seedlings grew at 100 mm NaCl, but excised coleoptiles, with 50 mm exogenous glucose, survived only at 50 mm NaCl, possibly due to lower energy production with glucose than for intact coleoptiles with sucrose as substrate. Rates of net uptake of Na+ and Cl− by coleoptiles in anoxia were about half those in aerated solution. Ethanol formation in anoxia and O2 uptake in aerobic solution were each increased by 13–15 % at 50 mm NaCl, i.e. ATP formation was stimulated. For acclimation to 50 mm NaCl, the anoxic tissues used only 25 % of the energy that was expended by aerobic tissues. Following return of coleoptiles to aerated non-saline solution, rates of net K+ uptake recovered to those in continuously aerated solution, demonstrating there was little injury during anoxia with 50 mm NaCl. Conclusion Rice seedlings survive anoxia, without the coleoptile incurring significant injury, even with the additional energy demands imposed by NaCl (100 mm when intact, 50 mm when excised). Energy savings were achieved in saline anoxia by less coleoptile growth, reduced ion fluxes as compared to aerobic coleoptiles and apparent energy-economic ion transport systems. PMID:27694332

Fetal lacerations at caesarean section.

PubMed

Wiener, J J; Westwood, J

2002-01-01

Fetal lacerations occurred in 1.5% of caesarean sections carried out in our institution. The incidence was independent of type of caesarean section, fetal presentation, cervical dilatation, presence of intact membranes or operator grade. We advocate that this complication should be included in the preoperative counselling of all patients undergoing caesarean sections.

Ambient ionisation mass spectrometry for in situ analysis of intact proteins

PubMed Central

Kocurek, Klaudia I.; Griffiths, Rian L.

2018-01-01

Abstract Ambient surface mass spectrometry is an emerging field which shows great promise for the analysis of biomolecules directly from their biological substrate. In this article, we describe ambient ionisation mass spectrometry techniques for the in situ analysis of intact proteins. As a broad approach, the analysis of intact proteins offers unique advantages for the determination of primary sequence variations and posttranslational modifications, as well as interrogation of tertiary and quaternary structure and protein‐protein/ligand interactions. In situ analysis of intact proteins offers the potential to couple these advantages with information relating to their biological environment, for example, their spatial distributions within healthy and diseased tissues. Here, we describe the techniques most commonly applied to in situ protein analysis (liquid extraction surface analysis, continuous flow liquid microjunction surface sampling, nano desorption electrospray ionisation, and desorption electrospray ionisation), their advantages, and limitations and describe their applications to date. We also discuss the incorporation of ion mobility spectrometry techniques (high field asymmetric waveform ion mobility spectrometry and travelling wave ion mobility spectrometry) into ambient workflows. Finally, future directions for the field are discussed. PMID:29607564

Strategies for reducing body fat mass: effects of liposuction and exercise on cardiovascular risk factors and adiposity

PubMed Central

Benatti, Fabiana Braga; Lira, Fábio Santos; Oyama, Lila Missae; do Nascimento, Cláudia Maria da Penha Oller; Lancha, Antonio Herbert

2011-01-01

Liposuction is the most popular aesthetic surgery performed in Brazil and worldwide. Evidence showing that adipose tissue is a metabolically active tissue has led to the suggestion that liposuction could be a viable method for improving metabolic profile through the immediate loss of adipose tissue. However, the immediate liposuction-induced increase in the proportion of visceral to subcutaneous adipose tissue could be detrimental to metabolism, because a high proportion of visceral to subcutaneous adipose tissue is associated with risk factors for cardiovascular disease. The results of studies investigating the effects of liposuction on the metabolic profile are inconsistent, however, with most studies reporting either no change or improvements in one or more cardiovascular risk factors. In addition, animal studies have demonstrated a compensatory growth of intact adipose tissue in response to lipectomy, although studies with humans have reported inconsistent results. Exercise training improves insulin sensitivity, inflammatory balance, lipid oxidation, and adipose tissue distribution; increases or preserves the fat-free mass; and increases total energy expenditure. Thus, liposuction and exercise appear to directly affect metabolism in similar ways, which suggests a possible interaction between these two strategies. To our knowledge, no studies have reported the associated effects of liposuction and exercise in humans. Nonetheless, one could suggest that exercise training associated with liposuction could attenuate or even block the possible compensatory fat deposition in intact depots or regrowth of the fat mass and exert an additive or even a synergistic effect to liposuction on improving insulin sensitivity and the inflammatory balance, resulting in an improvement of cardiovascular risk factors. Consequently, one could suggest that liposuction and exercise appear to be safe and effective strategies for either the treatment of metabolic disorders or aesthetic purposes. PMID:21779146

The inhibitory effect of vitamin E on cigarette smoke-induced oxidative damage to the rat urothelium: can it prevent transitional cell carcinoma?

PubMed

Onol, Fikret Fatih; Demir, Aslan; Temiz, Yusuf; Yüksel, Meral; Eren, Funda; Türkeri, Levent N

2007-01-01

We aimed to detect reactive oxygen species (ROS) and assess subsequent carcinogenesis in terms of cellular proliferation in the bladder and kidney epithelial tissues of rats exposed to cigarette smoke (CS), and to investigate the changes following vitamin E treatment. Twenty-four male Sprague-Dawley rats were divided into 3 groups: group 1 was kept intact; group 2 was subjected to CS exposure for 8 weeks, and group 3 received intraperitoneal vitamin E injections (200 mg/kg/week) for 8 weeks in addition to CS exposure. Histological examination and Ki67 antigen expression measurements were made from bladder and renal pelvic tissue sections. Luminol-amplified chemiluminescence was used to measure ROS levels. All results were compared using a one-way ANOVA test. In CS-exposed rats, light microscopy of renal and bladder tissues revealed nonspecific epithelial changes; however, Ki67 expression was significantly increased in bladder tissues compared to other groups (17.5 +/- 4.7, 35 +/- 2.9 and 18.7 +/- 5.1% in groups 1, 2 and 3, respectively, p < 0.05). Chemiluminescence levels in bladder and renal tissues were also significantly higher in the CS-exposed animals (78.1 +/- 11.4, 148 +/- 13.3, 97.8 +/- 6.1 rlu/mg for the bladder, and 99.8 +/- 12.2, 176.1 +/- 27.9, 67.1 +/- 9 rlu/mg, for renal pelvic tissues, respectively, p < 0.05). Vitamin E can alleviate CS-induced oxidative damage in rat bladder and kidney epithelium suggesting a potential role for vitamin E in the prevention of CS-mediated carcinogenesis. 2007 S. Karger AG, Basel

Smooth muscle-protein translocation and tissue function.

PubMed

Eddinger, Thomas J

2014-09-01

Smooth muscle (SM) tissue is a complex organization of multiple cell types and is regulated by numerous signaling molecules (neurotransmitters, hormones, cytokines, etc.). SM contractile function can be regulated via expression and distribution of the contractile and cytoskeletal proteins, and activation of any of the second messenger pathways that regulate them. Spatial-temporal changes in the contractile, cytoskeletal or regulatory components of SM cells (SMCs) have been proposed to alter SM contractile activity. Ca(2+) sensitization/desensitization can occur as a result of changes at any of these levels, and specific pathways have been identified at all of these levels. Understanding when and how proteins can translocate within the cytoplasm, or to-and-from the plasmalemma and the cytoplasm to alter contractile activity is critical. Numerous studies have reported translocation of proteins associated with the adherens junction and G protein-coupled receptor activation pathways in isolated SMC systems. Specific examples of translocation of vinculin to and from the adherens junction and protein kinase C (PKC) and 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) to and from the plasmalemma in isolated SMC systems but not in intact SM tissues are discussed. Using both isolated SMC systems and SM tissues in parallel to pursue these studies will advance our understanding of both the role and mechanism of these pathways as well as their possible significance for Ca(2+) sensitization in intact SM tissues and organ systems. © 2014 Wiley Periodicals, Inc.

Shedding Light on the Molecular Pathology of Amyloid Plaques in Transgenic Alzheimer's Disease Mice Using Multimodal MALDI Imaging Mass Spectrometry.

PubMed

Kaya, Ibrahim; Zetterberg, Henrik; Blennow, Kaj; Hanrieder, Jörg

2018-05-04

Senile plaques formed by aggregated amyloid β peptides are one of the major pathological hallmarks of Alzheimer's disease (AD) which have been suggested to be the primary influence triggering the AD pathogenesis and the rest of the disease process. However, neurotoxic Aβ aggregation and progression are associated with a wide range of enigmatic biochemical, biophysical and genetic processes. MALDI imaging mass spectrometry (IMS) is a label-free method to elucidate the spatial distribution patterns of intact molecules in biological tissue sections. In this communication, we utilized multimodal MALDI-IMS analysis on 18 month old transgenic AD mice (tgArcSwe) brain tissue sections to enhance molecular information correlated to individual amyloid aggregates on the very same tissue section. Dual polarity MALDI-IMS analysis of lipids on the same pixel points revealed high throughput lipid molecular information including sphingolipids, phospholipids, and lysophospholipids which can be correlated to the ion images of individual amyloid β peptide isoforms at high spatial resolutions (10 μm). Further, multivariate image analysis was applied in order to probe the multimodal MALDI-IMS data in an unbiased way which verified the correlative accumulations of lipid species with dual polarity and Aβ peptides. This was followed by the lipid fragmentation obtained directly on plaque aggregates at higher laser pulse energies which provided tandem MS information useful for structural elucidation of several lipid species. Majority of the amyloid plaque-associated alterations of lipid species are for the first time reported here. The significance of this technique is that it allows correlating the biological discussion of all detected plaque-associated molecules to the very same individual amyloid plaques which can give novel insights into the molecular pathology of even a single amyloid plaque microenvironment in a specific brain region. Therefore, this allowed us to interpret the possible roles of lipids and amyloid peptides in amyloid plaque-associated pathological events such as focal demyelination, autophagic/lysosomal dysfunction, astrogliosis, inflammation, oxidative stress, and cell death.

Comparison of the effects of mechanical and osmotic pressures on the collagen fiber architecture of intact and proteoglycan-depleted articular cartilage.

PubMed

Saar, Galit; Shinar, Hadassah; Navon, Gil

2007-04-01

One of the functions of articular cartilage is to withstand recurrent pressure applied in everyday life. In previous studies, osmotic pressure has been used to mimic the effects of mechanical pressure. In the present study, the response of the collagen network of intact and proteoglycans (PG)-depleted cartilage to mechanical and osmotic pressures is compared. The technique used is one-dimensional (2)H double quantum filtered spectroscopic MRI, which gives information about the degree of order and the density of the collagen fibers at the different locations throughout the intact tissue. For the nonpressurized plugs, the depletion had no effect on these parameters. Major differences were found in the zones near the bone between the effects of the two types of application of pressure for both intact and depleted plugs. While the order is lost in these zones as a result of mechanical load, it is preserved under osmotic pressure. For both intact and PG-depleted plugs under osmotic stress most of the collagen fibers become disordered. Our results indicate that different modes of strain are produced by unidirectional mechanical load and the isotropic osmotic stress. Thus, osmotic stress cannot serve as a model for the effect of load on cartilage in vivo.

Familial Predictors of Sibling and Romantic-Partner Conflict Resolution: Comparing Late Adolescents from Intact and Divorced Families

ERIC Educational Resources Information Center

Reese-Weber, M.; Kahn, J.H.

2005-01-01

The present study examined whether predictors of romantic-partner conflict may vary as a function of family structure. Using a cross-sectional design, we tested a mediation model of conflict resolution behaviours among late adolescents from intact (n=185) and divorced (n=87) families. Adolescents rated conflict resolution behaviours in five dyadic…

The Axolotl Fibula as a Model for the Induction of Regeneration across Large Segment Defects in Long Bones of the Extremities

PubMed Central

Chen, Xiaoping; Song, Fengyu; Jhamb, Deepali; Li, Jiliang; Bottino, Marco C.; Palakal, Mathew J.; Stocum, David L.

2015-01-01

We tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process. PMID:26098852

The EPA Liver Project

EPA Science Inventory

The v-Liver is part of a broader EPA effort on Virtual Tissues (VT) aimed at reducing the magnitude and spectrum of animal testing by integrative in silico and in vitro models, which recapitulate the properties of intact organs. The other VT projects include the Virtual Embryo (...

Sensitivity to biomechanical limitations during postural decision-making depends on the integrity of posterior superior parietal cortex.

PubMed

Wood, Daniel K; Chouinard, Philippe A; Major, Alex J; Goodale, Melvyn A

2017-12-01

Most object-directed limb movements can be carried out with a comfortable grasp posture. However, the orientation of an object relative to our bodies can sometimes lead us to select an uncomfortable or awkward grasp posture due to limitations imposed by the biomechanics of the arm. In a series of experiments, we identified a network of cortical areas that are engaged during the selection of movement strategies. Neurologically intact participants and two brain-damaged patients with overlapping lesions in the right posterior superior parietal lobule (pSPL) performed a grasp posture selection task in which biomechanical constraints were the primary consideration for selecting an action. The task induced states of bistable actions whereby the same stimulus gave rise to categorically different grasp postures. In a behavioral experiment, the two patients displayed a large range of manual bistability with the contralesional hand, resulting in a higher incidence of awkward grasping postures. In neurologically intact participants, a separate functional magnetic resonance imaging (fMRI) experiment revealed activation of a parieto-frontal network, which included the posterior intraparietal sulcus (pIPS) along the banks of the pSPL that was parametrically modulated by the degree of bistability in grasp posture selection. Superimposing this activation over the patients' structural MRIs revealed that the pIPS/pSPL activation in the neurologically intact participants overlapped with lesioned cortical tissue in both patients; all other areas of activation overlapped with intact cortical tissue in the patients. These results provide converging evidence that the posterior parietal cortex plays a critical role in selecting biomechanically appropriate postures during reach-to-grasp behaviors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Novel GALNT3 mutations causing hyperostosis-hyperphosphatemia syndrome result in low intact fibroblast growth factor 23 concentrations.

PubMed

Ichikawa, Shoji; Guigonis, Vincent; Imel, Erik A; Courouble, Mélanie; Heissat, Sophie; Henley, John D; Sorenson, Andrea H; Petit, Barbara; Lienhardt, Anne; Econs, Michael J

2007-05-01

Hyperostosis-hyperphosphatemia syndrome (HHS) is a rare metabolic disorder characterized by hyperphosphatemia and localized hyperostosis. HHS is caused by mutations in GALNT3, which encodes UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 3. Familial tumoral calcinosis (TC), characterized by ectopic calcifications and hyperphosphatemia, is caused by mutations in the GALNT3 or fibroblast growth factor 23 (FGF23) genes. Our objective was to identify mutations in FGF23 or GALNT3 and determine serum FGF23 levels in an HHS patient. Mutation detection in FGF23 and GALNT3 was performed by DNA sequencing, and serum FGF23 concentrations were measured by ELISA. A 5-year-old French boy with HHS and his family members participated. The patient presented with painful cortical lesions in his leg. Radiographs of the affected bone showed diaphyseal hyperostosis. The lesional tissue comprised trabeculae of immature, woven bone surrounded by fibrous tissue. Biochemistry revealed elevated phosphate, tubular maximum rate for phosphate reabsorption per deciliter of glomerular filtrate, and 1,25-dihydroxyvitamin D levels. The patient was a compound heterozygote for two novel GALNT3 mutations. His parents and brother were heterozygous for one of the mutations and had no biochemical abnormalities. Intact FGF23 level in the patient was low normal, whereas C-terminal FGF23 was elevated, a pattern similar to TC. The presence of GALNT3 mutations and elevated C-terminal, but low intact serum FGF23, levels in HHS resemble those seen in TC, suggesting that HHS and TC are different manifestations of the same disorder. The absence of biochemical abnormalities in the heterozygous individuals suggests that one normal allele is sufficient for secretion of intact FGF23.

Effect of milk on somatostatin degradation in suckling rat jejunum in vivo.

PubMed

Rao, R K; Davis, T P; Williams, C; Koldovsky, O

1999-01-01

Somatostatin-14 is present in breast milk, and intact somatostatin-14 has been recovered from gastric lumen of infants. Studies have shown that somatostatin-14 is metabolized in the intestinal luminal contents in vitro, which could be prevented by the presence of breast milk. In this study, the effect of milk on stability of somatostatin-14 in suckling rat jejunum in vivo was examined. 125I-Somatostatin-14[Tyr 11] was administered to the isolated jejunal loops in anesthetized suckling rats in the absence or presence of milk, fractions of milk, or known protease-peptidase inhibitors. Structural integrity of 125I-somatostatin-14[Tyr 11] recovered from tissues at different intervals was analyzed by gel filtration and high-performance liquid chromatography. Radioactivity rapidly disappeared from the jejunal lumen with a 50% clearance achieved by 1.2 minutes. Gel filtration and high-performance liquid chromatography analyses showed that 125I-somatostatin- 14[Tyr 11] was rapidly degraded into smaller fragments. At 1 minute, jejunal luminal radioactivity was eluted in a major peak with retention time of 42.4 minutes, along with other minor peaks (retention time, 5.6, 8.0, 10.4, and 14.4 minutes); only a trace amount of intact 125I-somatostatin-14[Tyr 11] (retention time, 44.8 minutes) was present. Coadministration of rat's milk or its soluble fraction increased the level of intact 125I-somatostatin-14[Tyr 11] in the jejunal lumen and jejunal tissue. Presence of rat's milk-casein or peptidase inhibitors (bestatin, phosphoramidon, or Bowman-Birk inhibitor), however, failed to increase the level of intact 125I-somatostatin-14[Tyr 11]. These results suggest that somatostatin-14 is rapidly degraded in the jejunal lumen of suckling rats, and that milk-borne peptidase inhibitors prevent this somatostatin-14 degradation.

Understanding the Differences in Molecular Conformation of Carbohydrate and Protein in Endosperm Tissues of Grains with Different Biodegradation Kinetics Using Advanced Synchrotron Technology

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

Yu, P.; Block, H; Doiron, K

Conventional 'wet' chemical analyses rely heavily on the use of harsh chemicals and derivatization, thereby altering native seed structures leaving them unable to detect any original inherent structures within an intact tissue sample. A synchrotron is a giant particle accelerator that turns electrons into light (million times brighter than sunlight) which can be used to study the structure of materials at the molecular level. Synchrotron radiation-based Fourier transform IR microspectroscopy (SR-FTIRM) has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of the brightness of synchrotron light and a small effective source size, is capablemore » of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional 'wet' chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of plant seed tissue in relation to nutrient utilization. The objective of this study was to use novel synchrotron radiation-based technology (SR-FTIRM) to identify the differences in the molecular chemistry and conformation of carbohydrate and protein in various plant seed endosperms within intact tissues at cellular and subcellular level from grains with different biodegradation kinetics. Barley grain (cv. Harrington) with a high rate (31.3%/h) and extent (78%), corn grain (cv. Pioneer) with a low rate (9.6%/h) and extent of (57%), and wheat grain (cv. AC Barrie) with an intermediate rate (23%/h) and extent (72%) of ruminal DM degradation were selected for evaluation. SR-FTIRM evaluations were performed at the National Synchrotron Light Source at the Brookhaven National Laboratory (Brookhaven, NY). These results suggest that SR-FTIRM plus the multivariate analyses can be used to identify spectral features associated with the molecular structure of endosperm from grains with different biodegradation kinetics, especially in relation to protein structure. The Novel synchrotron radiation-based bioanalytical technique provides a new approach for plant seed structural molecular studies at ultraspatial resolution and within intact tissue in relation to nutrient availability.« less

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica.

PubMed

Jeong, J; Toida, T; Muneta, Y; Kosiishi, I; Imanari, T; Linhardt, R J; Choi, H S; Wu, S J; Kim, Y S

2001-12-01

Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

In vitro and biomechanical screening of polyethylene glycol and poly(trimethylene carbonate) block copolymers for annulus fibrosus repair.

PubMed

Long, Rose G; Rotman, Stijn G; Hom, Warren W; Assael, Dylan J; Illien-Jünger, Svenja; Grijpma, Dirk W; Iatridis, James C

2018-02-01

Herniated intervertebral discs (IVDs) are a common cause of back and neck pain. There is an unmet clinical need to seal annulus fibrosus (AF) defects, as discectomy surgeries address acute pain but are complicated by reherniation and recurrent pain. Copolymers of polyethylene glycol with trimethylene carbonate (TMC) and hexamethylene diisocyanate (HDI) end-groups were formulated as AF sealants as the HDI form covalent bonds with native AF tissue. TMC adhesives were evaluated and optimized using the design criteria: stable size, strong adherence to AF tissue, high cytocompatibility, restoration of IVD biomechanics to intact levels following in situ repair, and low extrusion risk. TMC adhesives had high adhesion strength as assessed with a pushout test (150 kPa), and low degradation rates over 3 weeks in vitro. Both TMC adhesives had shear moduli (220 and 490 kPa) similar to, but somewhat higher than, AF tissue. The adhesive with three TMC moieties per branch (TMC3) was selected for additional in situ testing because it best matched AF shear properties. TMC3 restored torsional stiffness, torsional hysteresis area and axial range of motion to intact states. However, in a failure test of compressive deformation under fixed 5 ° flexion, some herniation risk was observed with failure strength of 5.9 MPa compared with 13.5 MPa for intact samples; TMC3 herniated under cyclic organ culture testing. These TMC adhesives performed well during in vitro and in situ testing, but additional optimization to enhance failure strength is required to further this material to advanced screening tests, such as long-term degradation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Cascade Bioassay Evidence for the Existence of Urothelium-Derived Inhibitory Factor in Guinea Pig Urinary Bladder

PubMed Central

Guan, Na N.; Thor, Anna; Hallén, Katarina; Wiklund, N. Peter; Gustafsson, Lars E.

2014-01-01

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1–5 µM in the presence of scopolamine 5–30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor. PMID:25084114

Cascade bioassay evidence for the existence of urothelium-derived inhibitory factor in Guinea pig urinary bladder.

PubMed

Guan, Na N; Thor, Anna; Hallén, Katarina; Wiklund, N Peter; Gustafsson, Lars E

2014-01-01

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1-5 µM in the presence of scopolamine 5-30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.

Evidence does not support absorption of intact solid lipid nanoparticles via oral delivery

NASA Astrophysics Data System (ADS)

Hu, Xiongwei; Fan, Wufa; Yu, Zhou; Lu, Yi; Qi, Jianping; Zhang, Jian; Dong, Xiaochun; Zhao, Weili; Wu, Wei

2016-03-01

Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and simulated mixed micelles (SMMs) to intestinal mucus, but no evidence of penetration of integral nanocarriers. Both SLNs and SMMs exhibit significant cellular uptake, but fail to penetrate cell monolayers. Confocal laser scanning microscopy reveals that nanocarriers mainly concentrate on the surface of the monolayers, and no evidence of penetration of intact vehicles can be obtained. The mucous layer acts as a barrier to the penetration of both SLNs and SMMs. Both bile salt-decoration and SMM formulation help to strengthen the interaction with biomembranes. It is concluded that evidence does not support absorption of intact SLNs via oral delivery.Whether and to what extent solid lipid nanoparticles (SLNs) can be absorbed integrally via oral delivery should be clarified because it is the basis for elucidation of absorption mechanisms. To address this topic, the in vivo fate of SLNs as well as their interaction with biomembranes is investigated using water-quenching fluorescent probes that can signal structural variations of lipid-based nanocarriers. Live imaging indicates prolonged retention of SLNs in the stomach, whereas in the intestine, SLNs can be digested quickly. No translocation of intact SLNs to other organs or tissues can be observed. The in situ perfusion study shows bioadhesion of both SLNs and simulated mixed micelles (SMMs) to intestinal mucus, but no evidence of penetration of integral nanocarriers. Both SLNs and SMMs exhibit significant cellular uptake, but fail to penetrate cell monolayers. Confocal laser scanning microscopy reveals that nanocarriers mainly concentrate on the surface of the monolayers, and no evidence of penetration of intact vehicles can be obtained. The mucous layer acts as a barrier to the penetration of both SLNs and SMMs. Both bile salt-decoration and SMM formulation help to strengthen the interaction with biomembranes. It is concluded that evidence does not support absorption of intact SLNs via oral delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07474f

[18S-25S rDNA variation in tissue culture of some Gentiana L. species].

PubMed

Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

2007-01-01

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

All-near-infrared multiphoton microscopy interrogates intact tissues at deeper imaging depths than conventional single- and two-photon near-infrared excitation microscopes

PubMed Central

Sarder, Pinaki; Yazdanfar, Siavash; Akers, Walter J.; Tang, Rui; Sudlow, Gail P.; Egbulefu, Christopher

2013-01-01

Abstract. The era of molecular medicine has ushered in the development of microscopic methods that can report molecular processes in thick tissues with high spatial resolution. A commonality in deep-tissue microscopy is the use of near-infrared (NIR) lasers with single- or multiphoton excitations. However, the relationship between different NIR excitation microscopic techniques and the imaging depths in tissue has not been established. We compared such depth limits for three NIR excitation techniques: NIR single-photon confocal microscopy (NIR SPCM), NIR multiphoton excitation with visible detection (NIR/VIS MPM), and all-NIR multiphoton excitation with NIR detection (NIR/NIR MPM). Homologous cyanine dyes provided the fluorescence. Intact kidneys were harvested after administration of kidney-clearing cyanine dyes in mice. NIR SPCM and NIR/VIS MPM achieved similar maximum imaging depth of ∼100  μm. The NIR/NIR MPM enabled greater than fivefold imaging depth (>500  μm) using the harvested kidneys. Although the NIR/NIR MPM used 1550-nm excitation where water absorption is relatively high, cell viability and histology studies demonstrate that the laser did not induce photothermal damage at the low laser powers used for the kidney imaging. This study provides guidance on the imaging depth capabilities of NIR excitation-based microscopic techniques and reveals the potential to multiplex information using these platforms. PMID:24150231

Connexin43 contributes to electrotonic conduction across scar tissue in the intact heart

NASA Astrophysics Data System (ADS)

Mahoney, Vanessa M.; Mezzano, Valeria; Mirams, Gary R.; Maass, Karen; Li, Zhen; Cerrone, Marina; Vasquez, Carolina; Bapat, Aneesh; Delmar, Mario; Morley, Gregory E.

2016-05-01

Studies have demonstrated non-myocytes, including fibroblasts, can electrically couple to myocytes in culture. However, evidence demonstrating current can passively spread across scar tissue in the intact heart remains elusive. We hypothesize electrotonic conduction occurs across non-myocyte gaps in the heart and is partly mediated by Connexin43 (Cx43). We investigated whether non-myocytes in ventricular scar tissue are electrically connected to surrounding myocardial tissue in wild type and fibroblast-specific protein-1 driven conditional Cx43 knock-out mice (Cx43fsp1KO). Electrical coupling between the scar and uninjured myocardium was demonstrated by injecting current into the myocardium and recording depolarization in the scar through optical mapping. Coupling was significantly reduced in Cx43fsp1KO hearts. Voltage signals were recorded using microelectrodes from control scars but no signals were obtained from Cx43fsp1KO hearts. Recordings showed significantly decreased amplitude, depolarized resting membrane potential, increased duration and reduced upstroke velocity compared to surrounding myocytes, suggesting that the non-excitable cells in the scar closely follow myocyte action potentials. These results were further validated by mathematical simulations. Optical mapping demonstrated that current delivered within the scar could induce activation of the surrounding myocardium. These data demonstrate non-myocytes in the scar are electrically coupled to myocytes, and coupling depends on Cx43 expression.

An improved method of renal tissue engineering, by combining renal dissociation and reaggregation with a low-volume culture technique, results in development of engineered kidneys complete with loops of Henle.

PubMed

Chang, C-Hong; Davies, Jamie A

2012-01-01

Tissue engineering of functional kidney tissue is an important goal for clinical restoration of renal function in patients damaged by infectious, toxicological, or genetic disease. One promising approach is the use of the self-organizing abilities of embryonic kidney cells to arrange themselves, from a simply reaggregated cell suspension, into engineered organs similar to fetal kidneys. The previous state-of-the-art method for this results in the formation of a branched collecting duct tree, immature nephrons (S-shaped bodies) beside and connected to it, and supportive stroma. It does not, though, result in the significant formation of morphologically detectable loops of Henle - anatomical features of the nephron that are critical to physiological function. We have combined the best existing technique for renal tissue engineering from cell suspensions with a low-volume culture technique that allows intact kidney rudiments to make loops of Henle to test whether engineered kidneys can produce these loops. The result is the formation of loops of Henle in engineered cultured 'fetal kidneys', very similar in both morphology and in number to those formed by intact organ rudiments. This brings the engineering technique one important step closer to production of a fully realistic organ. Copyright © 2012 S. Karger AG, Basel.

Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy

PubMed Central

Wang, Mei; Tulman, David B.; Sholl, Andrew B.; Kimbrell, Hillary Z.; Mandava, Sree H.; Elfer, Katherine N.; Luethy, Samuel; Maddox, Michael M.; Lai, Weil; Lee, Benjamin R.; Brown, J. Quincy

2016-01-01

Achieving cancer-free surgical margins in oncologic surgery is critical to reduce the need for additional adjuvant treatments and minimize tumor recurrence; however, there is a delicate balance between completeness of tumor removal and preservation of adjacent tissues critical for normal post-operative function. We sought to establish the feasibility of video-rate structured illumination microscopy (VR-SIM) of the intact removed tumor surface as a practical and non-destructive alternative to intra-operative frozen section pathology, using prostate cancer as an initial target. We present the first images of the intact human prostate surface obtained with pathologically-relevant contrast and subcellular detail, obtained in 24 radical prostatectomy specimens immediately after excision. We demonstrate that it is feasible to routinely image the full prostate circumference, generating gigapixel panorama images of the surface that are readily interpreted by pathologists. VR-SIM confirmed detection of positive surgical margins in 3 out of 4 prostates with pathology-confirmed adenocarcinoma at the circumferential surgical margin, and furthermore detected extensive residual cancer at the circumferential margin in a case post-operatively classified by histopathology as having negative surgical margins. Our results suggest that the increased surface coverage of VR-SIM could also provide added value for detection and characterization of positive surgical margins over traditional histopathology. PMID:27257084

Intact Imaging of Human Heart Structure Using X-ray Phase-Contrast Tomography.

PubMed

Kaneko, Yukihiro; Shinohara, Gen; Hoshino, Masato; Morishita, Hiroyuki; Morita, Kiyozo; Oshima, Yoshihiro; Takahashi, Masashi; Yagi, Naoto; Okita, Yutaka; Tsukube, Takuro

2017-02-01

Structural examination of human heart specimens at the microscopic level is a prerequisite for understanding congenital heart diseases. It is desirable not to destroy or alter the properties of such specimens because of their scarcity. However, many of the currently available imaging techniques either destroy the specimen through sectioning or alter the chemical and mechanical properties of the specimen through staining and contrast agent injection. As a result, subsequent studies may not be possible. X-ray phase-contrast tomography is an imaging modality for biological soft tissues that does not destroy or alter the properties of the specimen. The feasibility of X-ray phase-contrast tomography for the structural examination of heart specimens was tested using infantile and fetal heart specimens without congenital diseases. X-ray phase-contrast tomography was carried out at the SPring-8 synchrotron radiation facility using the Talbot grating interferometer at the bending magnet beamline BL20B2 to visualize the structure of five non-pretreated whole heart specimens obtained by autopsy. High-resolution, three-dimensional images were obtained for all specimens. The images clearly showed the myocardial structure, coronary vessels, and conduction bundle. X-ray phase-contrast tomography allows high-resolution, three-dimensional imaging of human heart specimens. Intact imaging using X-ray phase-contrast tomography can contribute to further structural investigation of heart specimens with congenital heart diseases.

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

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

Zlokovic, B.V.; Segal, M.B.; Davson, H.

1988-05-01

Unidirectional flux of /sup 125/I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of /sup 125/I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternalmore » perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.« less

Porcine intact and wounded skin responses to atmospheric nonthermal plasma.

PubMed

Wu, Andrew S; Kalghatgi, Sameer; Dobrynin, Danil; Sensenig, Rachel; Cerchar, Ekaternia; Podolsky, Erica; Dulaimi, Essel; Paff, Michelle; Wasko, Kimberly; Arjunan, Krishna Priya; Garcia, Kristin; Fridman, Gregory; Balasubramanian, Manjula; Ownbey, Robert; Barbee, Kenneth A; Fridman, Alexander; Friedman, Gary; Joshi, Suresh G; Brooks, Ari D

2013-01-01

Thermal plasma is a valued tool in surgery for its coagulative and ablative properties. We suggested through in vitro studies that nonthermal plasma can sterilize tissues, inactive pathogens, promote coagulation, and potentiate wound healing. The present research was undertaken to study acute toxicity in porcine skin tissues. We demonstrate that floating electrode-discharge barrier discharge (FE-DBD) nonthermal plasma is electrically safe to apply to living organisms for short periods. We investigated the effects of FE-DBD plasma on Yorkshire pigs on intact and wounded skin immediately after treatment or 24h posttreatment. Macroscopic or microscopic histological changes were identified using histological and immunohistochemical techniques. The changes were classified into four groups for intact skin: normal features, minimal changes or congestive changes, epidermal layer damage, and full burn and into three groups for wounded skin: normal, clot or scab, and full burn-like features. Immunohistochemical staining for laminin layer integrity showed compromise over time. A marker for double-stranded DNA breaks, γ-H2AX, increased over plasma-exposure time. These findings identified a threshold for plasma exposure of up to 900s at low power and <120s at high power. Nonthermal FE-DBD plasma can be considered safe for future studies of external use under these threshold conditions for evaluation of sterilization, coagulation, and wound healing. Copyright © 2013 Elsevier Inc. All rights reserved.

Sex Steroid Metabolism in Benign and Malignant Intact Prostate Biopsies: Individual Profiling of Prostate Intracrinology

PubMed Central

Gianfrilli, Daniele; Pierotti, Silvia; Leonardo, Costantino; Ciccariello, Mauro

2014-01-01

In vitro studies reveal that androgens, oestrogens, and their metabolites play a crucial role in prostate homeostasis. Most of the studies evaluated intraprostatic hormone metabolism using cell lines or preprocessed specimens. Using an ex vivo model of intact tissue cultures with preserved architecture, we characterized the enzymatic profile of biopsies from patients with benign prostatic hyperplasia (BPH) or cancer (PC), focusing on 17β-hydroxy-steroid-dehydrogenases (17β-HSDs) and aromatase activities. Samples from 26 men who underwent prostate needle core biopsies (BPH n = 14; PC n = 12) were incubated with radiolabeled 3H-testosterone or 3H-androstenedione. Conversion was evaluated by TLC separation and beta-scanning of extracted supernatants. We identified three major patterns of conversion. The majority of BPHs revealed no active testosterone/oestradiol conversion as opposed to prostate cancer. Conversion correlated with histology and PSA, but not circulating hormones. Highest Gleason scores had a higher androstenedion-to-testosterone conversion and expression of 17β-HSD-isoenzymes-3/5. Conclusions. We developed an easy tool to profile individual intraprostatic enzymatic activity by characterizing conversion pathways in an intact tissue environment. In fresh biopsies we found that 17β-HSD-isoenzymes and aromatase activities correlate with biological behaviour allowing for morphofunctional phenotyping of pathology specimens and clinical monitoring of novel enzyme-targeting drugs. PMID:25184140

A fluorescent method for visualization of Nosema infection in whole-mount honey bee tissues.

PubMed

Snow, Jonathan W

2016-03-01

Honey bees are critical pollinators in both agricultural and ecological settings. The Nosema species, ceranae and apis, are microsporidian parasites that are pathogenic to honey bees. While current methods for detecting Nosema infection have key merits, additional techniques with novel properties for studying the cell biology of Nosema infection are highly desirable. We demonstrate that whole-mount staining of honey bee midgut tissue with chitin-binding agent Fluorescent Brightener 28 and DNA dye Propidium Iodide allows for observation of Nosema infection in structurally intact tissue, providing a new tool for increasing our understanding of Nosema infection at the cellular and tissue level. Copyright © 2016 Elsevier Inc. All rights reserved.

Mass loading of the upper airway extraluminal tissue space in rabbits: effects on tissue pressure and pharyngeal airway lumen geometry.

PubMed

Kairaitis, Kristina; Howitt, Lauren; Wheatley, John R; Amis, Terence C

2009-03-01

Lateral pharyngeal fat pad compression of the upper airway (UA) wall is thought to influence UA size in patients with obstructive sleep apnea. We examined interactions between acute mass/volume loading of the UA extra-luminal tissue space and UA patency. We studied 12 supine, anesthetized, spontaneously breathing, head position-controlled (50 degrees ), New Zealand White rabbits. Submucosal extraluminal tissue pressures (ETP) in the anterolateral (ETPlat) and anterior (ETPant) pharyngeal wall were monitored with surgically inserted pressure transducer-tipped catheters (Millar). Tracheal pressure (Ptr) and airflow (V) were measured via a pneumotachograph and pressure transducer inserted in series into the intact trachea, with hypopharyngeal cross-sectional area (CSA) measured via computed tomography, while graded saline inflation (0-1.5ml) of a compliant tissue expander balloon in the anterolateral subcutaneous tissue was performed. Inspiratory UA resistance (Rua) at 20 ml/s was calculated from a power function fitted to Ptr vs. V data. Graded expansion of the anterolateral balloon increased ETPlat from 2.3 +/- 0.5 cmH(2)O (n = 11, mean +/- SEM) to 5.0 +/- 1.1 cmH(2)O at 1.5-ml inflation (P < 0.05; ANOVA). However, ETPant was unchanged from 0.5 +/- 0.5 cmH(2)O (n = 9; P = 0.17). Concurrently, Rua increased to 119 +/- 4.2% of baseline value (n = 12; P < 0.001) associated with a significant reduction in CSA between 10 and 70% of airway length to a minimum of 82.2 +/- 4.4% of baseline CSA at 40% of airway length (P < 0.05). We conclude that anterolateral loading of the upper airway extraluminal tissue space decreases upper airway patency via an increase in ETPlat, but not ETPant. Lateral pharyngeal fat pad size may influence UA patency via increased tissue volume and pressure causing UA wall compression.

46 CFR 174.185 - Intact stability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... (b) and (d) of this section, at each angle of heel an OSV's righting arm may be calculated... vessel does not trim as it heels. (g) For the purpose of paragraphs (b) and (d) of this section, the...

MYOSITIS OSSIFICANS TRAUMATICA IN A VAMPIRE BAT (DESMODUS ROTUNDUS).

PubMed

Hausmann, Jennifer C; Manasse, Jorden; Churgin, Sarah; Steinberg, Howard; Clyde, Victoria L; Wallace, Roberta

2016-09-01

A 15-yr-old sexually intact female vampire bat ( Desmodus rotundus ) was diagnosed with myositis ossificans traumatica of the abdominal wall. The bat presented with a large ulcerated firm mass along the abdomen. Radiographs and cytology were performed, followed by surgical exploration. The mass was determined to be nonresectable and the bat was euthanized. Histopathology showed severe necrotizing, degenerative, and pyogranulomatous myositis with osseous and cartilaginous metaplasia, fibrosis, and ulceration, which were consistent with myositis ossificans traumatica. Myositis ossificans traumatica is commonly associated with previous trauma to skeletal muscle. Two years prior, this bat had an emergency Caesarian section at this site, which was postulated to elicit a marked tissue response leading to this condition. Myositis ossificans traumatica is infrequently reported in humans, dogs, cats, pigs, and horses. To the author's knowledge, this is the first report of this condition in a bat.

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed

Tweeten, K A; Bulla, L A; Consigli, R A

1977-09-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed Central

Tweeten, K A; Bulla, L A; Consigli, R A

1977-01-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid. Images PMID:334076

Imaging mass spectrometry of endogenous polypeptides and secondary metabolites from galls induced by root-knot nematodes in tomato roots.

PubMed

Barbosa, Eder Alves; Bonfim Junior, Mauro Ferreira; Bloch, Carlos; Rocha, Thales Lima; Engler, Gilbert; de Almeida Engler, Janice

2018-04-17

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation, mainly the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared to nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI-TOF-MS/MS and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

Non-Immunogenic Structurally and Biologically Intact Tissue Matrix Grafts for the Immediate Repair of Ballistic-Induced Vascular and Nerve Tissue Injury in Combat Casualty Care

DTIC Science & Technology

2005-07-01

as an access graft is addressed using statistical methods below. Graft consistency can be defined statistically as the variance associated with the...addressed using statistical methods below. Graft consistency can be defined statistically as the variance associated with the sample of grafts tested in...measured using a refractometer (Brix % method). The equilibration data are shown in Graph 1. The results suggest the following equilibration scheme: 40% v/v

In vivo multiphoton microscopy of deep tissue with gradient index lenses

NASA Astrophysics Data System (ADS)

Levene, Michael J.; Dombeck, Daniel A.; Williams, Rebecca M.; Skoch, Jesse; Hickey, Gregory A.; Kasischke, Karl A.; Molloy, Raymond P.; Ingelsson, Martin; Stern, Edward A.; Klucken, Jochen; Bacskai, Brian J.; Zipfel, Warren R.; Hyman, Bradley T.; Webb, Watt W.

2004-06-01

Gradient index lenses enable multiphoton microscopy of deep tissues in the intact animal. In order to assess their applicability to clinical research, we present in vivo multiphoton microscopy with gradient index lenses in brain regions associated with Alzheimer's disease and Parkinson's disease in both transgenic and wild-type mice. We also demonstrate microscopy of ovary in wild type mouse using only intrinsic fluorescence and second harmonic generation, signal sources which may prove useful for both the study and diagnosis of cancer.

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

PubMed

Parma, A E; Cerone, S I; Sansinanea, S A; Ghezzi, M

1992-10-01

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2014-02-01

maturation of MSC - laden DAPS at a length scale of the full intact tissue. This is the first study to our knowledge 5 showing beneficial effects of...was also published in the Journal of the Mechanical Behavior of Biomedical Materials in 2012. To carry out this study , MSCs were isolated from...in our preliminary studies . Cylindrical MSC -based NP constructs (Ø4 × 2.25mm) were cultured in a chemically defined medium (1mL/construct

Lineage plasticity and cell biology of fibrocartilage and hyaline cartilage: its significance in cartilage repair and replacement.

PubMed

Freemont, Anthony J; Hoyland, Judith

2006-01-01

Cartilage repair is a major goal of modern tissue engineering. To produce novel engineered implants requires a knowledge of the basic biology of the tissues that are to be replaced or reproduced. Hyaline articular cartilage and meniscal fibrocartilage are two tissues that have excited attention because of the frequency with which they are damaged. A basic strategy is to re-engineer these tissues ex vivo by stimulating stem cells to differentiate into the cells of the mature tissue capable of producing an intact functional matrix. In this brief review, the sources of cells for tissue engineering cartilage and the culture conditions that have promoted differentiation are discussed within the context of natural cartilage repair. In particular, the role of cell density, cytokines, load, matrices and oxygen tension are discussed.

Surgical reduction of adipose tissue in the male Sprague-Dawley rat.

PubMed

Kral, J G

1976-10-01

The lipostatic theory of regulation of adipose tissue mass was tested by a method for surgical reduction (adipectomy) of 24% of the total body fat of nonobese adult Sprague-Dawley rats, as judged from carcass analyses. The reduction persisted during an observation period of 12 wk without any evidence of altered food intake, weight gain, or compensatory hypertrophy or hyperplasia of adipose tissue compared with sham-operated controls. No changes were found in serum free fatty acids, glycerol, triglycerides, cholesterol, or insulin between adipectomized and control animals, implying an intact quantitative function of the remaining adipose tissue. It is concluded that the size of the adipocytes rather than the number is important for a presumed lipostatic regulation of adipose tissue mass in the adult male Sprague-Dawley rat.

Pathophysiological changes of the cerebellum and brain stem in a rabbit model after superior petrosal vein sacrifice.

PubMed

Cheng, Lei; Guo, Pin; Liao, Yi-Wei; Zhang, Hong-Liang; Li, Huan-Ting; Yuan, Xianrui

2017-11-13

In certain surgical procedures sacrifice of the superior petrosal vein (SPV) is required. Previous studies have reported transient cerebellar edema, venous infarction or hemorrhage might occur after sectioning of the SPV. This study investigated the pathophysiological changes of cerebellum and brain stem after SPV sacrifice. Rabbits were divided into the operation group where the SPV was sacrificed and the control group where the SPV remained intact. Each group was further subdivided into 4, 8, 12, 24, 48 and 72 hours groups which represented the time period from sacrifice of the SPV to sacrifice of the rabbits. The water content (WC), Na + content, K + content and pathophysiological changes of cerebellum and brain stem tissue were measured. In comparison to the control, the WC and Na + content of cerebellar tissue were increased in the 4h, 8h, 12h and 24h operation subgroups (p<0.05), but only increased in the 4h subgroup of the brain stem tissue (p<0.05). The K + content of the cerebellar tissue decreased in the 4h, 8h, 12h and 24h operation subgroups (p<0.05) but only decreased in the 4h subgroup of brain stem tissue (p<0.05). Nissl staining and transmission electron microscopy demonstrated that cerebellar edema occurred in the 4h, 8h, 12h and 24h operation subgroups but not in the 48h and 72h subgroups. Brain stem edema occurred in the 4h operation subgroup. In summary, cerebellum and brain stem edema can be observed at different time points after sacrifice of the SPV in the rabbit model. ©2017 The Author(s).

Label-Free 3D Visualization of Cellular and Tissue Structures in Intact Muscle with Second and Third Harmonic Generation Microscopy

PubMed Central

Rehberg, Markus; Krombach, Fritz; Pohl, Ulrich; Dietzel, Steffen

2011-01-01

Second and Third Harmonic Generation (SHG and THG) microscopy is based on optical effects which are induced by specific inherent physical properties of a specimen. As a multi-photon laser scanning approach which is not based on fluorescence it combines the advantages of a label-free technique with restriction of signal generation to the focal plane, thus allowing high resolution 3D reconstruction of image volumes without out-of-focus background several hundred micrometers deep into the tissue. While in mammalian soft tissues SHG is mostly restricted to collagen fibers and striated muscle myosin, THG is induced at a large variety of structures, since it is generated at interfaces such as refraction index changes within the focal volume of the excitation laser. Besides, colorants such as hemoglobin can cause resonance enhancement, leading to intense THG signals. We applied SHG and THG microscopy to murine (Mus musculus) muscles, an established model system for physiological research, to investigate their potential for label-free tissue imaging. In addition to collagen fibers and muscle fiber substructure, THG allowed us to visualize blood vessel walls and erythrocytes as well as white blood cells adhering to vessel walls, residing in or moving through the extravascular tissue. Moreover peripheral nerve fibers could be clearly identified. Structure down to the nuclear chromatin distribution was visualized in 3D and with more detail than obtainable by bright field microscopy. To our knowledge, most of these objects have not been visualized previously by THG or any label-free 3D approach. THG allows label-free microscopy with inherent optical sectioning and therefore may offer similar improvements compared to bright field microscopy as does confocal laser scanning microscopy compared to conventional fluorescence microscopy. PMID:22140560

Passive contribution of the rotator cuff to abduction and joint stability.

PubMed

Tétreault, Patrice; Levasseur, Annie; Lin, Jenny C; de Guise, Jacques; Nuño, Natalia; Hagemeister, Nicola

2011-11-01

The purpose of this study is to compare shoulder joint biomechanics during abduction with and without intact non-functioning rotator cuff tissue. A cadaver model was devised to simulate the clinical findings seen in patients with a massive cuff tear. Eight full upper limb shoulder specimens were studied. Initially, the rotator cuff tendons were left intact, representing a non-functional rotator cuff, as seen in suprascapular nerve paralysis or in cuff repair with a patch. Subsequently, a massive rotator cuff tear was re-created. Three-dimensional kinematics and force requirements for shoulder abduction were analyzed for each condition using ten abduction cycles in the plane of the scapula. Mediolateral displacements of the glenohumeral rotation center (GHRC) during abduction with an intact non-functioning cuff were minimal, but massive cuff tear resulted in significant lateral displacement of the GHRC (p < 0.013). Similarly, massive cuff tear caused increased superior migration of the GHRC during abduction compared with intact non-functional cuff (p < 0.01). From 5 to 30° of abduction, force requirements were significantly less with an intact non-functioning cuff than with massive cuff tear (p < 0.009). During abduction, an intact but non-functioning rotator cuff resulted in decreased GHRC displacement in two axes as well as lowered the force requirement for abduction from 5 to 30° as compared with the results following a massive rotator cuff tear. This provides insight into the potential biomechanical effect of repairing massive rotator cuff tears with a biological or synthetic "patch," which is a new treatment for massive cuff tear.

Lysosomal Changes in Renal Proximal Tubular Epithelial Cells of Male Sprague Dawley Rats Following Decalin Exposure

DTIC Science & Technology

1990-01-01

induced by decalin exposure are processes, accelerated apoptosis has been describedin renal tissue with hydronephrosis (6), during the clearly intact...experimental hydronephrosis in topathology and cell proliferation induced by 2,2.4- the rat. Lab. Invest. 56(3): 273-281. trimethylpentane in the

Vitamins E and C - effects on matrix components in the vascular system

USDA-ARS?s Scientific Manuscript database

The connective tissue in the vascular system, consisting mainly of vascular smooth muscle cells (VSMC) and the interstitial extracellular matrix (ECM), plays important roles in the maintenance of an intact vascular wall as well as in the repair of atherosclerotic lesions during disease development. ...

Defining the Role of Integrin Alpha 11 in Wound Healing and Fibrosis

DTIC Science & Technology

2009-09-01

Introduction Scleroderma is characterized by fibrosis, the replacement of healthy tissue with collagenous matrix. The collagen-binding integrins are...Itga11 and determine its role in the etiology of fibrosis and scleroderma . Body The heterozygous (Het) phenotype of one intact Itga11 allele and one

The mechanics of development: models and methods for tissue morphogenesis

PubMed Central

Gjorevski, Nikolce; Nelson, Celeste M.

2011-01-01

Embryonic development is a physical process during which masses of cells are sculpted into functional organs. The mechanical properties of tissues and the forces exerted on them serve as epigenetic regulators of morphogenesis. Understanding these mechanobiological effects in the embryo requires new experimental approaches. Here we focus on branching of the lung airways and bending of the heart tube to describe examples of mechanical and physical cues that guide cell fate decisions and organogenesis. We highlight recent technological advances to measure tissue elasticity and endogenous mechanical stresses in real time during organ development. We also discuss recent progress in manipulating forces in intact embryos. PMID:20860059

Life sciences research in space: The requirement for animal models

NASA Technical Reports Server (NTRS)

Fuller, C. A.; Philips, R. W.; Ballard, R. W.

1987-01-01

Use of animals in NASA space programs is reviewed. Animals are needed because life science experimentation frequently requires long-term controlled exposure to environments, statistical validation, invasive instrumentation or biological tissue sampling, tissue destruction, exposure to dangerous or unknown agents, or sacrifice of the subject. The availability and use of human subjects inflight is complicated by the multiple needs and demands upon crew time. Because only living organisms can sense, integrate and respond to the environment around them, the sole use of tissue culture and computer models is insufficient for understanding the influence of the space environment on intact organisms. Equipment for spaceborne experiments with animals is described.

Free tissue transfer of the rectus abdominis myoperitoneal flap for oral reconstruction in a dog.

PubMed

Lanz, O I

2001-12-01

A five-month-old intact/male Boxer dog was presented 5-days following bite wound trauma to the maxillary region resulting in an oronasal fistula extending from the maxillary canine teeth to the soft palate. Multiple surgical procedures using local, buccal mucosal flaps failed to repair the oronasal fistula. Free tissue transfer of the rectus abdominis myoperitoneal flap using microvascular surgical techniques was successful in providing soft tissue reconstruction of the hard palate area. Complications of these surgical techniques included muscle contraction and subsequent muzzle distortion. Small, refractory oronasal fistulae at the perimeter of the myoperitoneal flap were repaired by primary wound closure.

Static and dynamic stress heterogeneity in a multiscale model of the asthmatic airway wall

PubMed Central

Hiorns, J. E.

2016-01-01

Airway hyperresponsiveness (AHR) is a key characteristic of asthma that remains poorly understood. Tidal breathing and deep inspiration ordinarily cause rapid relaxation of airway smooth muscle (ASM) (as demonstrated via application of length fluctuations to tissue strips) and are therefore implicated in modulation of AHR, but in some cases (such as application of transmural pressure oscillations to isolated intact airways) this mechanism fails. Here we use a multiscale biomechanical model for intact airways that incorporates strain stiffening due to collagen recruitment and dynamic force generation by ASM cells to show that the geometry of the airway, together with interplay between dynamic active and passive forces, gives rise to large stress and compliance heterogeneities across the airway wall that are absent in tissue strips. We show further that these stress heterogeneities result in auxotonic loading conditions that are currently not replicated in tissue-strip experiments; stresses in the strip are similar to hoop stress only at the outer airway wall and are under- or overestimates of stresses at the lumen. Taken together these results suggest that a previously underappreciated factor, stress heterogeneities within the airway wall and consequent ASM cellular response to this micromechanical environment, could contribute to AHR and should be explored further both theoretically and experimentally. PMID:27197860

Research on optical properties of dental enamel for early caries diagnostics using a He-Ne laser

NASA Astrophysics Data System (ADS)

Tang, Jing; Liu, Li; Li, Song-zhan

2008-12-01

A new and non-invasive method adapted for optical diagnosis of early caries is proposed by researching on the interaction mechanism of laser with dental tissue and relations of remitted light with optical properties of the tissue. This method is based on simultaneous analyses of the following parameters: probing radiation, backscattering and auto-fluorescence. Investigation was performed on 104 dental samples in vitro by using He-Ne laser (λ=632.8nm, 2.0+/-0.1mW) as the probing. Spectrums of all samples were obtained. Characteristic spectrums of dental caries in various stages (intact, initial, moderate and deep) were given. Using the back-reflected light to normalize the intensity of back-scattering and fluorescence, a quantitative diagnosis standard for different stages of caries is proposed. In order to verify the test, comparison research was conducted among artificial caries, morphological damaged enamel, dental calculus and intact tooth. Results show that variations in backscattering characteristic changes in bio-tissue morphological and the quantity of auto-fluorescence is correlated with concentration of anaerobic microflora in hearth of caries lesion. This method poses a high potential of diagnosing various stages of dental caries, and is more reliability to detect early caries, surface damage of health enamel and dental calculus.

Wavelet-domain de-noising of OCT images of human brain malignant glioma

NASA Astrophysics Data System (ADS)

Dolganova, I. N.; Aleksandrova, P. V.; Beshplav, S.-I. T.; Chernomyrdin, N. V.; Dubyanskaya, E. N.; Goryaynov, S. A.; Kurlov, V. N.; Reshetov, I. V.; Potapov, A. A.; Tuchin, V. V.; Zaytsev, K. I.

2018-04-01

We have proposed a wavelet-domain de-noising technique for imaging of human brain malignant glioma by optical coherence tomography (OCT). It implies OCT image decomposition using the direct fast wavelet transform, thresholding of the obtained wavelet spectrum and further inverse fast wavelet transform for image reconstruction. By selecting both wavelet basis and thresholding procedure, we have found an optimal wavelet filter, which application improves differentiation of the considered brain tissue classes - i.e. malignant glioma and normal/intact tissue. Namely, it allows reducing the scattering noise in the OCT images and retaining signal decrement for each tissue class. Therefore, the observed results reveals the wavelet-domain de-noising as a prospective tool for improved characterization of biological tissue using the OCT.

Comparison of a modified mid-coronal sectioning technique and Wilson's technique when conducting eye and brain examinations in rabbit teratology studies.

PubMed

Ziejewski, Mary K; Solomon, Howard M; Rendemonti, Joyce; Stanislaus, Dinesh

2015-02-01

There are two methods used when examining fetal rabbit eyes and brain in teratology studies. One method employs prior fixation before serial sectioning (Wilson's technique) and the other uses fresh tissue (mid-coronal sectioning). We modified the mid-coronal sectioning technique to include removal of eyes and brain for closer examination and to increase the number of structures that can be evaluated and compared it to the Wilson's technique. We found that external examination of the head, in conjunction with either sectioning method, is equally sensitive in identifying developmental defects. We evaluated 40,401 New Zealand White (NZW) and Dutch-Belted (DB) rabbit fetuses for external head alterations, of which 28,538 fetuses were further examined for eye and brain alterations using the modified mid-coronal sectioning method (16,675 fetuses) or Wilson's technique (11,863 fetuses). The fetuses were from vehicle control or drug-treated pregnant rabbits in embryo-fetal development studies conducted to meet international regulatory requirements for the development of new drugs. Both methods detected the more common alterations (microphthalmia and dilated lateral cerebral ventricles) and other less common findings (changes in size and/or shape of eye and brain structures). While both methods are equally sensitive at detecting common and rare developmental defects, the modified mid-coronal sectioning technique eliminates the use of chemicals and concomitant fixation artifacts that occur with the Wilson's technique and allows for examination of 100% intact fetuses thereby increasing potential for detecting eye and brain alterations as these findings occur infrequently in rabbits. © 2015 Wiley Periodicals, Inc.

Technique: imaging earliest tooth development in 3D using a silver-based tissue contrast agent.

PubMed

Raj, Muhammad T; Prusinkiewicz, Martin; Cooper, David M L; George, Belev; Webb, M Adam; Boughner, Julia C

2014-02-01

Looking in microscopic detail at the 3D organization of initiating teeth within the embryonic jaw has long-proved technologically challenging because of the radio-translucency of these tiny un-mineralized oral tissues. Yet 3D image data showing changes in the physical relationships among developing tooth and jaw tissues are vital to understand the coordinated morphogenesis of vertebrate teeth and jaws as an animal grows and as species evolve. Here, we present a new synchrotron-based scanning solution to image odontogenesis in 3D and in histological detail using a silver-based contrast agent. We stained fixed, intact wild-type mice aged embryonic (E) day 10 to birth with 1% Protargol-S at 37°C for 12-32 hr. Specimens were scanned at 4-10 µm pixel size at 28 keV, just above the silver K-edge, using micro-computed tomography (µCT) at the Canadian Light Source synchrotron. Synchrotron µCT scans of silver-stained embryos showed even the earliest visible stages of tooth initiation, as well as many other tissue types and structures, in histological detail. Silver stain penetration was optimal for imaging structures in intact embryos E15 and younger. This silver stain method offers a powerful yet straightforward approach to visualize at high-resolution and in 3D the earliest stages of odontogenesis in situ, and demonstrates the important of studying the tooth organ in all three planes of view. Copyright © 2013 Wiley Periodicals, Inc.

Refractive errors and corrections for OCT images in an inflated lung phantom

PubMed Central

Golabchi, Ali; Faust, J.; Golabchi, F. N.; Brooks, D. H.; Gouldstone, A.; DiMarzio, C. A.

2012-01-01

Visualization and correct assessment of alveolar volume via intact lung imaging is important to study and assess respiratory mechanics. Optical Coherence Tomography (OCT), a real-time imaging technique based on near-infrared interferometry, can image several layers of distal alveoli in intact, ex vivo lung tissue. However optical effects associated with heterogeneity of lung tissue, including the refraction caused by air-tissue interfaces along alveoli and duct walls, and changes in speed of light as it travels through the tissue, result in inaccurate measurement of alveolar volume. Experimentally such errors have been difficult to analyze because of lack of ’ground truth,’ as the lung has a unique microstructure of liquid-coated thin walls surrounding relatively large airspaces, which is difficult to model with cellular foams. In addition, both lung and foams contain airspaces of highly irregular shape, further complicating quantitative measurement of optical artifacts and correction. To address this we have adapted the Bragg-Nye bubble raft, a crystalline two-dimensional arrangement of elements similar in geometry to alveoli (up to several hundred μm in diameter with thin walls) as an inflated lung phantom in order to understand, analyze and correct these errors. By applying exact optical ray tracing on OCT images of the bubble raft, the errors are predicted and corrected. The results are validated by imaging the bubble raft with OCT from one edge and with a charged coupled device (CCD) camera in transillumination from top, providing ground truth for the OCT. PMID:22567599

Characterization of the Influence of Semen-Derived Enhancer of Virus Infection on the Interaction of HIV-1 with Female Reproductive Tract Tissues

PubMed Central

Allen, Shannon A.; Carias, Ann M.; Anderson, Meegan R.; Okocha, Eneniziaogochukwu A.; Benning, Lorie; McRaven, Michael D.; Kelley, Z L.; Lurain, John; Veazey, Ronald S.

2015-01-01

ABSTRACT The majority of human immunodeficiency virus type 1 (HIV-1) transmission events occur in women when semen harboring infectious virus is deposited onto the mucosal barriers of the vaginal, ectocervical, and endocervical epithelia. Seminal factors such as semen-derived enhancer of virus infection (SEVI) fibrils were previously shown to greatly enhance the infectivity of HIV-1 in cell culture systems. However, when SEVI is intravaginally applied to living animals, there is no effect on vaginal transmission. To define how SEVI might function in the context of sexual transmission, we applied HIV-1 and SEVI to intact human and rhesus macaque reproductive tract tissues to determine how it influences virus interactions with these barriers. We show that SEVI binds HIV-1 and sequesters most virions to the luminal surface of the stratified squamous epithelium, significantly reducing the number of virions that penetrated the tissue. In the simple columnar epithelium, SEVI was no longer fibrillar in structure and was detached from virions but allowed significantly deeper epithelial virus penetration. These observations reveal that the action of SEVI in intact tissues is very different in the anatomical context of sexual transmission and begin to explain the lack of stimulation of infection observed in the highly relevant mucosal transmission model. IMPORTANCE The most common mode of HIV-1 transmission in women occurs via genital exposure to the semen of HIV-infected men. A productive infection requires the virus to penetrate female reproductive tract epithelial barriers to infect underlying target cells. Certain factors identified within semen, termed semen-derived enhancers of virus infection (SEVI), have been shown to significantly enhance HIV-1 infectivity in cell culture. However, when applied to the genital tracts of living female macaques, SEVI did not enhance virus transmission. Here we show that SEVI functions very differently in the context of intact mucosal tissues. SEVI decreases HIV-1 penetration of squamous epithelial barriers in humans and macaques. At the mucus-coated columnar epithelial barrier, the HIV-1/SEVI interaction is disrupted. These observations suggest that SEVI may not play a significant stimulatory role in the efficiency of male-to-female sexual transmission of HIV. PMID:25740984

Optimization of immunostaining on flat-mounted human corneas.

PubMed

Forest, Fabien; Thuret, Gilles; Gain, Philippe; Dumollard, Jean-Marc; Peoc'h, Michel; Perrache, Chantal; He, Zhiguo

2015-01-01

In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, -20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch's endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional -20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia.

Wrist kinetics after luno-triquetral dissociation: the changes in moment arms of the flexor carpi ulnaris tendon.

PubMed

Tang, Jin Bo; Xie, Ren Gou; Yu, Xiao Wei; Chen, Feng

2002-11-01

Wrist biomechanics after luno-triquetral (LT) dissociation is important for understanding the clinical sequelae of the disease and for determining its treatment options. The LT interosseous ligament plays an important role in stabilizing the joint and damage to the ligament would be expected to significantly increase moment arms of tendon of the flexor carpi ulnaris (FCU), the principal ulnar wrist flexor. We investigated the changes in moment arms of FCU tendon after various amounts of sectioning of the ligaments proven to be associated with LT dissociation. In six fresh frozen cadaveric upper extremities, excursions of the FCU tendon were recorded simultaneously with wrist joint angulation during wrist flexion-extension and radioulnar deviation. Tendon excursions were measured in intact wrists, in wrists with sectioning of the dorsal portion of the LT interosseous ligament, in wrists with sectioning of the entire LT interosseous ligament, and finally in wrists with further sectioning of the dorsal radiotriquetral and intercarpal ligaments. Moment arms of the tendon were calculated from tendon excursions and joint motion angulations and expressed as percentage changes from those in the intact wrist. During wrist flexion-extension, moment arms of the FCU tendon after sectioning of the entire LT interosseous ligament and after sectioning of the two capsular ligaments were 112 +/- 7% and 114 +/- 8%, respectively; these values were significantly greater than those in the intact wrist. During radioulnar deviation, the moment arms were 114 +/- 11% after sectioning of the dorsal portion of the LT interosseous ligament, 134 +/- 15% after sectioning of the entire ligament, and 153 +/- 18% after sectioning of the capsular ligaments, again being significantly greater than the normal wrist. Increase in moment arms of the FCU tendon after loss of integrity of the LT interosseous ligament and dorsal capsular ligaments may contribute to clinical sequelae of LT dissociation and difficulty in treating this disorder.

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

Ahmed, Raef S.; Shen, Sui; Ove, Roger

We wanted to describe a technique for the implementation of intensity-modulated radiotherapy (IMRT) with a real-time position monitor (RPM) respiratory gating system for the treatment of pleural space with intact lung. The technique is illustrated by a case of pediatric osteosarcoma, metastatic to the pleura of the right lung. The patient was simulated in the supine position where a breathing tracer and computed tomography (CT) scans synchronized at end expiration were acquired using the RPM system. The gated CT images were used to define target volumes and critical structures. Right pleural gated IMRT delivered at end expiration was prescribed tomore » a dose of 44 Gy, with 55 Gy delivered to areas of higher risk via simultaneous integrated boost (SIB) technique. IMRT was necessary to avoid exceeding the tolerance of intact lung. Although very good coverage of the target volume was achieved with a shell-shaped dose distribution, dose over the targets was relatively inhomogeneous. Portions of target volumes necessarily intruded into the right lung, the liver, and right kidney, limiting the degree of normal tissue sparing that could be achieved. The radiation doses to critical structures were acceptable and well tolerated. With intact lung, delivering a relatively high dose to the pleura with acceptable doses to surrounding normal tissues using respiratory gated pleural IMRT is feasible. Treatment delivery during a limited part of the respiratory cycle allows for reduced CT target volume motion errors, with reduction in the portion of the planning margin that accounts for respiratory motion, and subsequent increase in the therapeutic ratio.« less

MRI of plants and foods

NASA Astrophysics Data System (ADS)

Van As, Henk; van Duynhoven, John

2013-04-01

The importance and prospects for MRI as applied to intact plants and to foods are presented in view of one of humanity's most pressing concerns, the sustainable and healthy feeding of a worldwide increasing population. Intact plants and foods have in common that their functionality is determined by complex multiple length scale architectures. Intact plants have an additional level of complexity since they are living systems which critically depend on transport and signalling processes between and within tissues and organs. The combination of recent cutting-edge technical advances and integration of MRI accessible parameters has the perspective to contribute to breakthroughs in understanding complex regulatory plant performance mechanisms. In food science and technology MRI allows for quantitative multi-length scale structural assessment of food systems, non-invasive monitoring of heat and mass transport during shelf-life and processing, and for a unique view on food properties under shear. These MRI applications are powerful enablers of rationally (re)designed food formulations and processes. Limitations and bottlenecks of the present plant and food MRI methods are mainly related to short T2 values and susceptibility artefacts originating from small air spaces in tissues/materials. We envisage cross-fertilisation of solutions to overcome these hurdles in MRI applications in plants and foods. For both application areas we witness a development where MRI is moving from highly specialised equipment to mobile and downscaled versions to be used by a broad user base in the field, greenhouse, food laboratory or factory.

Tissue modification with feedback: the smart scalpel

NASA Astrophysics Data System (ADS)

Sebern, Elizabeth L.; Brenan, Colin J. H.; Anderson, R. Rox; Hunter, Ian W.

1998-10-01

While feedback control is widespread throughout many engineering fields, there are almost no examples of surgical instruments that utilize a real-time detection and intervention strategy. This concept of closed loop feedback can be applied to the development of autonomous or semi- autonomous minimally invasive robotic surgical systems for efficient excision or modification of diseased tissue. Spatially localized regions of the tissue are first probed to distinguish pathological from healthy tissue based on differences in histochemical and morphological properties. Energy is directed to only the diseased tissue, minimizing collateral damage by leaving the adjacent healthy tissue intact. Continuous monitoring determines treatment effectiveness and, if needed, enables real-time treatment modifications to produce optimal therapeutic outcomes. The present embodiment of this general concept is a microsurgical instrument we call the Smart Scalpel, designed to treat skin angiodysplasias such as port wine stains. Other potential Smart Scalpel applications include psoriasis treatment and early skin cancer detection and intervention.

Coral disease physiology: the impact of Acroporid white syndrome on Symbiodinium

NASA Astrophysics Data System (ADS)

Roff, G.; Kvennefors, E. C. E.; Ulstrup, K. E.; Fine, M.; Hoegh-Guldberg, O.

2008-06-01

Acroporid white syndrome, a disease-like syndrome from the Great Barrier Reef, results from degenerative host tissue at lesion borders. Tissue preceding lesion borders appears visually healthy, but it is currently unclear whether the endosymbiotic zooxanthellae ( Symbiodinium) are physiologically impacted. Compared to healthy colonies, this study found no significant differences in symbiont density, mitotic index or chlorophyll a content in tissue bordering (0 cm), and 8 cm away from white syndrome lesions. Using chlorophyll a fluorescence techniques, the border tissue did not appear to be photosynthetically compromised, and Symbiodinium extracted from this area were photosynthetically competent. Transmission electron microscopy revealed extensive degeneration of host tissues surrounding symbionts in affected areas, however, Symbiodinium cells were structurally intact with no sign of in situ degradation. Collectively, these results suggest that Symbiodinium at white syndrome lesion borders exist in a dynamic intra-cellular state during active host tissue loss, yet remain physiologically uncompromised.

The Immune System in Children with Malnutrition—A Systematic Review

PubMed Central

Rytter, Maren Johanne Heilskov; Kolte, Lilian; Briend, André; Friis, Henrik; Christensen, Vibeke Brix

2014-01-01

Background Malnourished children have increased risk of dying, with most deaths caused by infectious diseases. One mechanism behind this may be impaired immune function. However, this immune deficiency of malnutrition has not previously been systematically reviewed. Objectives To review the scientific literature about immune function in children with malnutrition. Methods A systematic literature search was done in PubMed, and additional articles identified in reference lists and by correspondence with experts in the field. The inclusion criteria were studies investigating immune parameters in children aged 1–60 months, in relation to malnutrition, defined as wasting, underweight, stunting, or oedematous malnutrition. Results The literature search yielded 3402 articles, of which 245 met the inclusion criteria. Most were published between 1970 and 1990, and only 33 after 2003. Malnutrition is associated with impaired gut-barrier function, reduced exocrine secretion of protective substances, and low levels of plasma complement. Lymphatic tissue, particularly the thymus, undergoes atrophy, and delayed-type hypersensitivity responses are reduced. Levels of antibodies produced after vaccination are reduced in severely malnourished children, but intact in moderate malnutrition. Cytokine patterns are skewed towards a Th2-response. Other immune parameters seem intact or elevated: leukocyte and lymphocyte counts are unaffected, and levels of immunoglobulins, particularly immunoglobulin A, are high. The acute phase response appears intact, and sometimes present in the absence of clinical infection. Limitations to the studies include their observational and often cross-sectional design and frequent confounding by infections in the children studied. Conclusion The immunological alterations associated with malnutrition in children may contribute to increased mortality. However, the underlying mechanisms are still inadequately understood, as well as why different types of malnutrition are associated with different immunological alterations. Better designed prospective studies are needed, based on current understanding of immunology and with state-of-the-art methods. PMID:25153531

Tibiofemoral Contact Mechanics with Horizontal Cleavage Tear and Resection of the Medial Meniscus in the Human Knee.

PubMed

Koh, Jason L; Yi, Seung Jin; Ren, Yupeng; Zimmerman, Todd A; Zhang, Li-Qun

2016-11-02

The meniscus is known to increase the contact area and decrease contact pressure in the tibiofemoral compartments of the knee. Radial tears of the meniscal root attachment along with partial resections of the torn meniscal tissue decrease the contact area and increase pressure; however, there is a lack of information on the effects of a horizontal cleavage tear (HCT) and partial leaf meniscectomy of such tears on tibiofemoral contact pressure and contact area. Twelve fresh-frozen human cadaveric knees were tested under 10 conditions: 5 serial conditions of posterior medial meniscectomy (intact meniscus, HCT, repaired HCT, inferior leaf resection, and resection of both inferior and superior leaves), each at 2 knee flexion angles (0° and 60°) under an 800-N axial load. Tekscan sensors (model 4000) were used to measure the contact pressure and contact area. HCT and HCT repair resulted in small changes in the contact area and an increase in contact pressure compared with the intact condition. Resection of the inferior leaf resulted in significantly decreased contact area (to a mean 82.3% of the intact condition at 0° of flexion and 81.8% at 60° of flexion; p < 0.05) and increased peak contact pressure (a mean 36.3% increase at 0° flexion and 43.2% increase at 60° flexion; p < 0.05) in the medial compartment. Further resection of the remaining superior leaf resulted in additional significant decreases in contact area (to a mean 60.1% of the intact condition at 0° of flexion and 49.7% at 60° of flexion; p < 0.05) and increases in peak contact pressure (a mean 79.2% increase at 0° of flexion and 74.9% increase at 60° of flexion; p < 0.05). Resection of meniscal tissue forming the inferior leaf of an HCT resulted in substantially decreased contact area and increased contact pressure. Additional resection of the superior leaf resulted in a further significant decrease in contact area and increase in contact pressure in the medial compartment. Repair or minimal resection of meniscal tissue of an HCT may be preferred to complete leaf resection to maintain knee tibiofemoral contact mechanics. Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.

BILIARY EXCRETION AND TISSUE DISTRIBUTION OF CADMIUM-109 ADMINISTERED TO RATS

EPA Science Inventory

The difference in the excretion of cadmium in urine and feces was measured in rats with either ligated or intact bile ducts. Three days following a single oral-administration of cadmium-109 plus stabe cadmium chloride, 0.004 percent of the dose was excreted in the urine of rats w...

GENOMIC COMPARISON OF IN VITRO AND IN VIVO EFFECTS OF ENDOCRINE DISRUPTORS ON FATHEAD MINNOW ( PIMEPHALES PROMELAS ) OVARIES REVEALS COMPENSATORY MECHANISMS

EPA Science Inventory

This study investigates compensatory mechanisms and feedback control within Fathead minnow (Pimephales promelas) by comparing genomic and biochemical responses of ovary tissue exposed in vitro to those of ovaries from intact fish after exposure to two model steroidogenesis...

Persistent supercooling of reproductive shoots is enabled by structural ice barriers being active despite an intact xylem connection

USDA-ARS?s Scientific Manuscript database

Extracellular ice nucleation usually occurs at mild subzero temperatures in most plants. For persistent supercooling of certain plant parts ice barriers are necessary that prevent the entry of ice from otherwise already frozen tissues. The reproductive shoot of the evergreen woody dwarf shrub Callun...

The multiple strategies of an insect herbivore to overcome plant cyanogenic glucoside defence.

PubMed

Pentzold, Stefan; Zagrobelny, Mika; Roelsgaard, Pernille Sølvhøj; Møller, Birger Lindberg; Bak, Søren

2014-01-01

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds that release toxic hydrogen cyanide by plant β-glucosidase activity after tissue damage. Specialised insect herbivores have evolved counter strategies and some sequester CNglcs, but the underlying mechanisms to keep CNglcs intact during feeding and digestion are unknown. We show that CNglc-sequestering Zygaena filipendulae larvae combine behavioural, morphological, physiological and biochemical strategies at different time points during feeding and digestion to avoid toxic hydrolysis of the CNglcs present in their Lotus food plant, i.e. cyanogenesis. We found that a high feeding rate limits the time for plant β-glucosidases to hydrolyse CNglcs. Larvae performed leaf-snipping, a minimal disruptive feeding mode that prevents mixing of plant β-glucosidases and CNglcs. Saliva extracts did not inhibit plant cyanogenesis. However, a highly alkaline midgut lumen inhibited the activity of ingested plant β-glucosidases significantly. Moreover, insect β-glucosidases from the saliva and gut tissue did not hydrolyse the CNglcs present in Lotus. The strategies disclosed may also be used by other insect species to overcome CNglc-based plant defence and to sequester these compounds intact.

Characterization of Cadmium Uptake by Plant Tissue 12

PubMed Central

Cutler, Jay M.; Rains, Donald W.

1974-01-01

The uptake of cadmium by excised root tissue of barley (Hordeum vulgare L. cv. Arivat) was investigated with respect to kinetics, concentration, and interactions with various cations. The role of metabolism in Cd absorption was examined using a range of temperatures, anaerobic treatments, and chemical inhibitors. The uptake and distribution of Cd in intact barley plants was also determined. A large fraction of the Cd taken up by excised barley roots was apparently the result of exchange adsorption and was displaced by subsequent desorption with unlabeled Cd, Zn, Cu, or Hg. Another fraction of Cd which could not be displaced by desorption in unlabeled Cd was thought to result from strong irreversible binding of Cd, perhaps on sites of the cell wall. The fraction of the Cd taken up beyond that by exchange adsorption by fresh roots was a linear function of temperature, and inhibited by conditions of low oxygen and by the presence of 2,4-dinitrophenol. It was concluded that this fraction of Cd entered excised barley roots by diffusion. Diffusion, when followed by sequestering, probably accounts for the accumulation of Cd observed in intact barley plants. PMID:16658840

Causal effect of disconnection lesions on interhemispheric functional connectivity in rhesus monkeys

PubMed Central

O’Reilly, Jill X.; Croxson, Paula L.; Jbabdi, Saad; Sallet, Jerome; Noonan, MaryAnn P.; Mars, Rogier B.; Browning, Philip G.F.; Wilson, Charles R. E.; Mitchell, Anna S.; Miller, Karla L.; Rushworth, Matthew F. S.; Baxter, Mark G.

2013-01-01

In the absence of external stimuli or task demands, correlations in spontaneous brain activity (functional connectivity) reflect patterns of anatomical connectivity. Hence, resting-state functional connectivity has been used as a proxy measure for structural connectivity and as a biomarker for brain changes in disease. To relate changes in functional connectivity to physiological changes in the brain, it is important to understand how correlations in functional connectivity depend on the physical integrity of brain tissue. The causal nature of this relationship has been called into question by patient data suggesting that decreased structural connectivity does not necessarily lead to decreased functional connectivity. Here we provide evidence for a causal but complex relationship between structural connectivity and functional connectivity: we tested interhemispheric functional connectivity before and after corpus callosum section in rhesus monkeys. We found that forebrain commissurotomy severely reduced interhemispheric functional connectivity, but surprisingly, this effect was greatly mitigated if the anterior commissure was left intact. Furthermore, intact structural connections increased their functional connectivity in line with the hypothesis that the inputs to each node are normalized. We conclude that functional connectivity is likely driven by corticocortical white matter connections but with complex network interactions such that a near-normal pattern of functional connectivity can be maintained by just a few indirect structural connections. These surprising results highlight the importance of network-level interactions in functional connectivity and may cast light on various paradoxical findings concerning changes in functional connectivity in disease states. PMID:23924609

Influence of Spinal Manipulative Therapy Force Magnitude and Application Site on Spinal Tissue Loading: A Biomechanical Robotic Serial Dissection Study in Porcine Motion Segments.

PubMed

Funabashi, Martha; Nougarou, François; Descarreaux, Martin; Prasad, Narasimha; Kawchuk, Greg

In order to define the relation between spinal manipulative therapy (SMT) input parameters and the distribution of load within spinal tissues, the aim of this study was to determine the influence of force magnitude and application site when SMT is applied to cadaveric spines. In 10 porcine cadavers, a servo-controlled linear actuator motor provided a standardized SMT simulation using 3 different force magnitudes (100N, 300N, and 500N) to 2 different cutaneous locations: L3/L4 facet joint (FJ), and L4 transverse processes (TVP). Vertebral kinematics were tracked optically using indwelling bone pins, the motion segment removed and mounted in a parallel robot equipped with a 6-axis load cell. The kinematics of each SMT application were replicated robotically. Serial dissection of spinal structures was conducted to quantify loading characteristics of discrete spinal tissues. Forces experienced by the L3/L4 segment and spinal structures during SMT replication were recorded and analyzed. Spinal manipulative therapy force magnitude and application site parameters influenced spinal tissues loading. A significant main effect (P < .05) of force magnitude was observed on the loads experienced by the intact specimen and supra- and interspinous ligaments. The main effect of application site was also significant (P < .05), influencing the loading of the intact specimen and facet joints, capsules, and ligamentum flavum (P < .05). Spinal manipulative therapy input parameters of force magnitude and application site significantly influence the distribution of forces within spinal tissues. By controlling these SMT parameters, clinical outcomes may potentially be manipulated. Copyright © 2017. Published by Elsevier Inc.

Isochoric and isobaric freezing of fish muscle

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

Năstase, Gabriel; Department of Building Services, University of Transilvania, Braşov, Braşov, 500152; Lyu, Chenang

We have recently shown that, a living organism, which succumbs to freezing to −4 °C in an isobaric thermodynamic system (constant atmospheric pressure), can survive freezing to −4 °C in an isochoric thermodynamic system (constant volume). It is known that the mechanism of cell damage in an isobaric system is the freezing caused increase in extracellular osmolality, and, the consequent cell dehydration. An explanation for the observed survival during isochoric freezing is the thermodynamic modeling supported hypothesis that, in the isochoric frozen solution the extracellular osmolality is comparable to the cell intracellular osmolality. Therefore, cells in the isochoric frozen organism do not dehydrate, andmore » the tissue maintains its morphological integrity. Comparing the histology of: a) fresh fish white muscle, b) fresh muscle frozen to −5 °C in an isobaric system and c) fresh muscle frozen to −5 °C I in an isochoric system, we find convincing evidence of the mechanism of cell dehydration during isobaric freezing. In contrast, the muscle tissue frozen to −5 °C in an isochoric system appears morphologically identical to fresh tissue, with no evidence of dehydration. This is the first experimental evidence in support of the hypothesis that in isochoric freezing there is no cellular dehydration and therefore the morphology of the frozen tissue remains intact. - Highlights: • Preservation of fish muscle at, subfreezing temperatures, in an isochoric system, is demonstrated. • Experiments were performed to an average pressure of 41.3 MPa and temperatures of −5 °C. • Isochoric subfreezing temperature is a new preservation method that does not require the.use of cryoprotectants. • No cellular dehydration and therefore the morphology of the frozen tissue remains intact.« less

Non-Immunogenic Structurally and Biologically Intact Tissue Matrix Grafts for the Immediate Repair of Ballistic-Induced Vascular and Nerve Tissue Injury in Combat

DTIC Science & Technology

2004-12-01

and -2. However, there was no product breakage. The product stabilizers breakage can be addressed by either using stronger Styrofoam or using the same...dry ice shipper is suitable for use as a 72hrs shipping container for the frozen UVG and that, if necessary, the shipping time can be extended to at...concentrations for two hours at room temperature under constant agitation (85 rpm). The step-wise equilibration was measured using a refractometer

Erosion of pelvicol used in sacrocolpopexy.

PubMed

Mukati, Marium S; Shobeiri, S Abbas

2013-01-01

Biologic graft materials are used more frequently in pelvic reconstructive surgeries. We describe here the complete process of removal of such a biologic graft in the office. We report a case of a 69-year-old woman with pig dermal graft erosion 1 year after placement. The patient presented with complaints of vaginal discharge. Upon examination, the graft material was seen eroding through the vaginal apex. The pig tissue was removed whole and intact in the office without complications. Transvaginal removal of pig tissue in the office relieved the patient's symptoms.

The use of injection-corrosive method in the study of extraorganic bloodstream of human intact stomach.

PubMed

Hryn, V H; Svintsytska, N L; Piliuhin, V; Ustenko, R L; Katsenko, A L

Functional and morphological state of the organs and tissues mainly depends on the adequate blood supply and lymph movement, function of which is integrated by the nervous system. A crucial link in the morphogenesis of the gastric lesions is the intensity of vascularization, as well as the fact that in its venous part the gastric bloodstream is almost entirely included into the portal vein system. Knowledge of the anatomy of the normal human stomach conditions is of indispensable practical value, since they are required for the proper interpretation of the pathological changes occurred in it. To obtain the spatial visual information about the angioarchitecture of the extraorganic bloodstream of human intact stomach deep in the gastric wall. 10 post-autopsy adult total stomach specimens of patients, died for the reasons not associated with manifested gastrointestinal diseases have been analyzed. The specimens were extracted during the dissection together with portions of lesser and greater omentum, and segment of aorta with celiac trunk. To neutralize the acidic contents of the stomach, its cavity was washed by 4% sodium bicarbonate solution with subsequent wash in warm running water. The vascular injection method with subsequent corrosion of soft tissues was used in investigation of gastric bloodstream. On the basis of the investigations the advantages of the countercurrent-crossing method of injection of extraorganic vessels to fill the bloodstream of human stomach have been discussed. Positive results of the suggested technique for morphological study of blood vessels have been noted. The three-dimensional spatial organization of the extraorganic bloodstream of the intact stomach can be studied on the basis of the injection-corrosive casts. Thus, the use of the suggested method enables to obtain the fine three-dimensional reproduction of extraorganic bloodstream of the human stomach. The obtained high-quality casts, in turn, are used for the subsequent morphological studies of the intact stomach.

Visualization of superparamagnetic nanoparticles in vascular tissue using XμCT and histology.

PubMed

Tietze, Rainer; Rahn, Helene; Lyer, Stefan; Schreiber, Eveline; Mann, Jenny; Odenbach, Stefan; Alexiou, Christoph

2011-02-01

In order to increase the dose of antineoplastic agents in the tumor area, the concept of magnetic drug targeting (MDT) has been developed. Magnetic nanoparticles consisting of iron oxide and a biocompatible cover layer suspended in an aqueous solution (ferrofluid) serve as carriers for chemotherapeutics being enriched by an external magnetic field after intra-arterial application in desired body compartments (i.e., tumor). We established an ex vivo model to simulate in vivo conditions in a circulating system consisting of magnetic iron oxide nanoparticles passing an intact bovine artery and being focused by an external magnetic field to study their distribution in the vessel. Micro-computed X-ray tomography (XμCT) and histology can elucidate the arrangement of these particles after application. XμCT-analysis has been performed on arterial sections after MDT in order to determine the distribution of the nanoparticles. These measurements have been carried out with a cone X-ray source and corresponding histological sections were stained with Prussian blue. It could be shown that combining XμCT and histology offers the opportunity for a better understanding of the mechanisms of nanoparticle deposition in the vascular system after MDT.

Three-dimensional micro-scale strain mapping in living biological soft tissues.

PubMed

Moo, Eng Kuan; Sibole, Scott C; Han, Sang Kuy; Herzog, Walter

2018-04-01

Non-invasive characterization of the mechanical micro-environment surrounding cells in biological tissues at multiple length scales is important for the understanding of the role of mechanics in regulating the biosynthesis and phenotype of cells. However, there is a lack of imaging methods that allow for characterization of the cell micro-environment in three-dimensional (3D) space. The aims of this study were (i) to develop a multi-photon laser microscopy protocol capable of imprinting 3D grid lines onto living tissue at a high spatial resolution, and (ii) to develop image processing software capable of analyzing the resulting microscopic images and performing high resolution 3D strain analyses. Using articular cartilage as the biological tissue of interest, we present a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning length scales from the tissue to the cell level. Using custom image processing software, we provide accurate and robust 3D micro-strain analysis that allows for detailed qualitative and quantitative assessment of the 3D tissue kinematics. This novel technique preserves tissue structural integrity post-scanning, therefore allowing for multiple strain measurements at different time points in the same specimen. The proposed technique is versatile and opens doors for experimental and theoretical investigations on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. We presented a novel two-photon excitation imaging technique for measuring the internal 3D kinematics in intact cartilage at sub-micrometer resolution, spanning from tissue length scale to cellular length scale. Using a custom image processing software (lsmgridtrack), we provide accurate and robust micro-strain analysis that allowed for detailed qualitative and quantitative assessment of the 3D tissue kinematics. The approach presented here can also be applied to other biological tissues such as meniscus and annulus fibrosus, as well as tissue-engineered tissues for the characterization of their mechanical properties. This imaging technique opens doors for experimental and theoretical investigation on the relationship between tissue deformation and cell biosynthesis. Studies of this nature may enhance our understanding of the mechanisms underlying cell mechano-transduction, and thus, adaptation and degeneration of soft connective tissues. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Noninvasive in situ evaluation of osteogenic differentiation by time-resolved laser-induced fluorescence spectroscopy.

PubMed

Ashjian, Peter; Elbarbary, Amir; Zuk, Patricia; DeUgarte, Daniel A; Benhaim, Prosper; Marcu, Laura; Hedrick, Marc H

2004-01-01

The clinical implantation of bioengineered tissues requires an in situ nondestructive evaluation of the quality of tissue constructs developed in vitro before transplantation. Time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) is demonstrated here to noninvasively monitor the formation of osteogenic extracellular matrix (ECM) produced by putative stem cells (PLA cells) derived from human adipose tissue. We show that this optical spectroscopy technique can assess the relative expression of collagens (types I, III, IV, and V) within newly forming osteogenic ECM. The results are consistent with those obtained by conventional histochemical techniques (immunofluorescence and Western blot) and demonstrate that TR-LIFS is a potential tool for monitoring the expression of distinct collagen types and the formation of collagen cross-links in intact tissue constructs.

Effect of demedullation on freezing injury in hind limbs of rats

NASA Astrophysics Data System (ADS)

Dhingra, Shashi; Bhatia, B.; Chhina, G. S.; Singh, Baldev

1987-09-01

Freezing incidence and tissue loss on exposure of hind limbs of female Wistar rats to freezing mixture was reduced by demedullation 6 days prior to cold exposure (p<0.01 and p<0.001 respectively); demedullation 1 h after freezing injury had no effect on tissue loss. Noradrenaline (1 mg/kg i.p.) 5 min before exposure increased the freezing incidence in intact (p<0.05) as well as in demedullated rats (p<0.01), with no effect on tissue loss. Adrenaline (500 mg/kg i.p.) had no effect on either. A sustained fall in plasma adrenaline after demedullation leading to reduced reactivity of the blood vessels to some vasoactive agents is postulated.

Histological, molecular and biochemical detection of renal injury after Echis pyramidum snake envenomation in rats

PubMed Central

Al-Johany, Awadh M.; Al-Sadoon, Mohamed K.; Abdel Moneim, Ahmed E.; Bauomy, Amira A.; Diab, Marwa S.M.

2014-01-01

Nephrotoxicity is a common sign of snake envenomation. The present work aimed to clarify the effect of intraperitoneal injection of 1/8 LD50 and 1/4 LD50 doses of Echis pyramidum snake venom on the renal tissue of rats after 2, 4 and 6 h from envenomation. Histopathological examination showed intense dose and time dependent abnormalities, including swelling glomerulus and tubular necrosis and damage as well as signs of intertubular medullary hemorrhage at early stages of envenomation. However, at late stages of envenomation by any of the doses under investigation, no intact renal corpuscles were recorded and complete lysis in renal corpuscles with ruptured Bowman’s capsules was observed. Immunohistochemistry by immunohistochemical staining was used to test the protein expression of Bax in renal tissue of rats. The result showed that the expression of Bax in renal tissue sections of envenomated rats was increased according to dose and time-dependant manner. The isolation of DNA from the renal cells of envenomed rats pointed out to the occurrence of DNA fragmentation, which is another indicator for renal tissue injury especially after 6 h of 1/4 LD50 of E. pyramidum envenomation. Oxidative stress biomarkers malondialdehyde and nitrite/nitrate levels, antioxidant parameters; glutathione, total antioxidant capacity and catalase were assayed in renal tissue homogenates. The venom induced significant increase in the levels of malondialdehyde and nitrite/nitrate while the levels of glutathione, total antioxidant capacity and catalase were significantly decreased, especially after 6 h of envenomation. The results revealed that the E. pyramidum induced dose and time-dependant significant disturbances in the physiological parameters in the kidney. We conclude that the use of the immunohistochemical techniques, the detection of DNA integrity and oxidative stress marker estimations are more specific tools that can clarify cellular injury and could point out to the defense activity of the renal tissue at envenomation. PMID:25972751

Histological, molecular and biochemical detection of renal injury after Echis pyramidum snake envenomation in rats.

PubMed

Al-Johany, Awadh M; Al-Sadoon, Mohamed K; Abdel Moneim, Ahmed E; Bauomy, Amira A; Diab, Marwa S M

2015-05-01

Nephrotoxicity is a common sign of snake envenomation. The present work aimed to clarify the effect of intraperitoneal injection of 1/8 LD50 and 1/4 LD50 doses of Echis pyramidum snake venom on the renal tissue of rats after 2, 4 and 6 h from envenomation. Histopathological examination showed intense dose and time dependent abnormalities, including swelling glomerulus and tubular necrosis and damage as well as signs of intertubular medullary hemorrhage at early stages of envenomation. However, at late stages of envenomation by any of the doses under investigation, no intact renal corpuscles were recorded and complete lysis in renal corpuscles with ruptured Bowman's capsules was observed. Immunohistochemistry by immunohistochemical staining was used to test the protein expression of Bax in renal tissue of rats. The result showed that the expression of Bax in renal tissue sections of envenomated rats was increased according to dose and time-dependant manner. The isolation of DNA from the renal cells of envenomed rats pointed out to the occurrence of DNA fragmentation, which is another indicator for renal tissue injury especially after 6 h of 1/4 LD50 of E. pyramidum envenomation. Oxidative stress biomarkers malondialdehyde and nitrite/nitrate levels, antioxidant parameters; glutathione, total antioxidant capacity and catalase were assayed in renal tissue homogenates. The venom induced significant increase in the levels of malondialdehyde and nitrite/nitrate while the levels of glutathione, total antioxidant capacity and catalase were significantly decreased, especially after 6 h of envenomation. The results revealed that the E. pyramidum induced dose and time-dependant significant disturbances in the physiological parameters in the kidney. We conclude that the use of the immunohistochemical techniques, the detection of DNA integrity and oxidative stress marker estimations are more specific tools that can clarify cellular injury and could point out to the defense activity of the renal tissue at envenomation.

In vitro terahertz spectroscopy of gelatin-embedded human brain tumors: a pilot study

NASA Astrophysics Data System (ADS)

Chernomyrdin, N. V.; Gavdush, A. A.; Beshplav, S.-I. T.; Malakhov, K. M.; Kucheryavenko, A. S.; Katyba, G. M.; Dolganova, I. N.; Goryaynov, S. A.; Karasik, V. E.; Spektor, I. E.; Kurlov, V. N.; Yurchenko, S. O.; Komandin, G. A.; Potapov, A. A.; Tuchin, V. V.; Zaytsev, K. I.

2018-04-01

We have performed the in vitro terahertz (THz) spectroscopy of human brain tumors. In order to fix tissues for the THz measurements, we have applied the gelatin embedding. It allows for preserving tissues from hydration/dehydration and sustaining their THz response similar to that of the freshly-excised tissues for a long time after resection. We have assembled an experimental setup for the reflection-mode measurements of human brain tissues based on the THz pulsed spectrometer. We have used this setup to study in vitro the refractive index and the amplitude absorption coefficient of 2 samples of malignant glioma (grade IV), 1 sample of meningioma (grade I), and samples of intact tissues. We have observed significant differences between the THz responses of normal and pathological tissues of the brain. The results of this paper highlight the potential of the THz technology in the intraoperative neurodiagnosis of tumors relying on the endogenous labels of tumorous tissues.

Microwave Therapy for Bone Tumors

NASA Astrophysics Data System (ADS)

Takakuda, Kazuo; Inaoka, Shuken; Saito, Hirokazu; Hassan, Moinuddin; Koyama, Yoshikazu; Kuroda, Hiroshi; Kanaya, Tomohiro; Kosaka, Toshifumi; Tanaka, Shigeo; Miyairi, Hiroo; Shinomiya, Kenichi

In vivo microwave treatments for bone tumor are designed, which enable us to conserve the activity and functionality of the matrix of living tissues. This treatment is composed of two steps. In the first step, the tumor was coagulated by the application of microwaves emitted from the antenna inserted into the tumor tissue, and then removed. In the second step, the surrounding tissue suspected to be invaded with transformed cells was covered with hydro gels and heated similarly. The tissue itself was heated by the conduction from the gels. The tissue temperature should be kept at 60°C for 30 minutes. This treatment should kill the whole cells within the tissues, but the mechanical strength and the biochemical activity of the matrix should be left intact. The matrix preserves the mechanical functions and ensures the maximum regeneration ability of the tissue. In this study, various hydro gels were examined and the most promising one was selected. Animal experiments were carried out and successful heating verified the applicability of the treatment.

Mycobacteria employ two different mechanisms to cross the blood-brain barrier.

PubMed

van Leeuwen, Lisanne M; Boot, Maikel; Kuijl, Coen; Picavet, Daisy I; van Stempvoort, Gunny; van der Pol, Susanne M A; de Vries, Helga E; van der Wel, Nicole N; van der Kuip, Martijn; van Furth, A Marceline; van der Sar, Astrid M; Bitter, Wilbert

2018-05-10

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle. © 2018 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Effect of supramolecular organization of a cartilaginous tissue on thermal stability of collagen II

NASA Astrophysics Data System (ADS)

Ignat'eva, N. Yu.; Averkiev, S. V.; Lunin, V. V.; Grokhovskaya, T. E.; Obrezkova, M. V.

2006-08-01

The thermal stability of collagen II in various cartilaginous tissues was studied. It was found that heating a tissue of nucleus pulposus results in collagen II melting within a temperature range of 60-70°C; an intact tissue of hyaline cartilage (of nasal septum and cartilage endplates) is a thermally stable system, where collagen II is not denatured completely up to 100°C. It was found that partial destruction of glycosaminoglycans in hyaline cartilage leads to an increase in the degree of denaturation of collagen II upon heating, although a significant fraction remains unchanged. It was shown that electrostatic interactions of proteoglycans and collagen only slightly affect the thermal stability of collagen II in the tissues. Evidently, proteoglycan aggregates play a key role: they create topological hindrances for moving polypeptide chains, thereby reducing the configurational entropy of collagen macromolecules in the state of a random coil.

Self-interference 3D super-resolution microscopy for deep tissue investigations.

PubMed

Bon, Pierre; Linarès-Loyez, Jeanne; Feyeux, Maxime; Alessandri, Kevin; Lounis, Brahim; Nassoy, Pierre; Cognet, Laurent

2018-06-01

Fluorescence localization microscopy has achieved near-molecular resolution capable of revealing ultra-structures, with a broad range of applications, especially in cellular biology. However, it remains challenging to attain such resolution in three dimensions and inside biological tissues beyond the first cell layer. Here we introduce SELFI, a framework for 3D single-molecule localization within multicellular specimens and tissues. The approach relies on self-interference generated within the microscope's point spread function (PSF) to simultaneously encode equiphase and intensity fluorescence signals, which together provide the 3D position of an emitter. We combined SELFI with conventional localization microscopy to visualize F-actin 3D filament networks and reveal the spatial distribution of the transcription factor OCT4 in human induced pluripotent stem cells at depths up to 50 µm inside uncleared tissue spheroids. SELFI paves the way to nanoscale investigations of native cellular processes in intact tissues.

Mueller matrix approach for probing multifractality in the underlying anisotropic connective tissue

NASA Astrophysics Data System (ADS)

Das, Nandan Kumar; Dey, Rajib; Ghosh, Nirmalya

2016-09-01

Spatial variation of refractive index (RI) in connective tissues exhibits multifractality, which encodes useful morphological and ultrastructural information about the disease. We present a spectral Mueller matrix (MM)-based approach in combination with multifractal detrended fluctuation analysis (MFDFA) to exclusively pick out the signature of the underlying connective tissue multifractality through the superficial epithelium layer. The method is based on inverse analysis on selected spectral scattering MM elements encoding the birefringence information on the anisotropic connective tissue. The light scattering spectra corresponding to the birefringence carrying MM elements are then subjected to the Born approximation-based Fourier domain preprocessing to extract ultrastructural RI fluctuations of anisotropic tissue. The extracted RI fluctuations are subsequently analyzed via MFDFA to yield the multifractal tissue parameters. The approach was experimentally validated on a simple tissue model comprising of TiO2 as scatterers of the superficial isotropic layer and rat tail collagen as an underlying anisotropic layer. Finally, the method enabled probing of precancer-related subtle alterations in underlying connective tissue ultrastructural multifractality from intact tissues.

In vivo tissue response and durability of five novel synthetic polymers in a rabbit model.

PubMed

Sahin, E; Cingi, C; Eskiizmir, G; Altintoprak, N; Calli, A; Calli, C; Yilgör, I; Yilgör, E

2016-04-01

Alloplastic materials are frequently used in facial plastic surgeries such as rhinoplasty and nasal reconstruction. Unfortunately, the ideal alloplastic material has not been found. This experimental study evaluates the tissue response and durability of five novel polymers developed as an alloplastic material. In this experimental study involving a tertiary university hospital, six subcuticular pockets were formed at the back of 10 rabbits for the implantation of each polymer and sham group. Each pocket was excised with its adjacent tissue after three months, and collected for histopathological examination. Semi-quantitative examination including neovascularisation, inflammation, fibrosis, abscess formation, multinucleated foreign body giant cells was performed, and integrity of polymer was evaluated. A statistical comparison was performed. No statically significant difference was detected in neovascularisation, inflammation, fibrosis, abscess formation and multinucleated foreign body giant cells when a paired comparison between sham and polymer II, III and IV groups was performed individually. Nevertheless, the degree of fibrosis was less than sham group in polymer I (p = .027) and V (p = .018), although the other variables were almost similar. The integrity of polymers III (9 intact, 1 fragmented) and IV (8 intact, 2 absent) was better than the other polymers. These novel synthetic polymers could be considered as good candidates for clinical applicability. All polymers provided satisfactory results in terms of tissue response; however, fibrovascular integration was higher in polymers II, III and IV. In addition, the durability of polymer III and IV was better than the others. © Copyright by Società Italiana di Otorinolaringologia e Chirurgia Cervico-Facciale, Rome, Italy.

L1 Peptide-Conjugated Fibrin Hydrogels Promote Salivary Gland Regeneration.

PubMed

Nam, K; Wang, C-S; Maruyama, C L M; Lei, P; Andreadis, S T; Baker, O J

2017-07-01

Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.

Revolutionizing orthopaedic biomaterials: The potential of biodegradable and bioresorbable magnesium-based materials for functional tissue engineering

PubMed Central

Farraro, Kathryn F.; Kim, Kwang E.; Woo, Savio L-Y.; Flowers, Jonquil R.; McCullough, Matthew B.

2014-01-01

In recent years, there has been a surge of interest in magnesium (Mg) and its alloys as biomaterials for orthopaedic applications, as they possess desirable mechanical properties, good biocompatibility, and biodegradability. Also shown to be osteoinductive, Mg-based materials could be particularly advantageous in functional tissue engineering to improve healing and serve as scaffolds for delivery of drugs, cells, and cytokines. In this paper, we will present two examples of Mg-based orthopaedic devices: an interference screw to accelerate ACL graft healing and a ring to aid in the healing of an injured ACL. In vitro tests using a robotic/UFS testing system showed that both devices could restore function of the goat stifle joint. Under a 67-N anterior tibial load, both the ACL graft fixed with the Mg-based interference screw and the Mg-based ring-repaired ACL could restore anterior tibial translation (ATT) to within 2 mm and 5 mm, respectively, of the intact joint at 301, 601, and 901 of flexion. In-situ forces in the replacement graft and Mg-based ring-repaired ACL were also similar to those of the intact ACL. Further, early in vivo data using the Mg-based interference screw showed that after 12 weeks, it was non-toxic and the joint stability and graft function reached similar levels as published data. Following these positive results, we will move forward in incorporating bioactive molecules and ECM bioscaffolds to these Mg-based biomaterials to test their potential for functional tissue engineering of musculoskeletal and other tissues. PMID:24373510

Revolutionizing orthopaedic biomaterials: The potential of biodegradable and bioresorbable magnesium-based materials for functional tissue engineering.

PubMed

Farraro, Kathryn F; Kim, Kwang E; Woo, Savio L-Y; Flowers, Jonquil R; McCullough, Matthew B

2014-06-27

In recent years, there has been a surge of interest in magnesium (Mg) and its alloys as biomaterials for orthopaedic applications, as they possess desirable mechanical properties, good biocompatibility, and biodegradability. Also shown to be osteoinductive, Mg-based materials could be particularly advantageous in functional tissue engineering to improve healing and serve as scaffolds for delivery of drugs, cells, and cytokines. In this paper, we will present two examples of Mg-based orthopaedic devices: an interference screw to accelerate ACL graft healing and a ring to aid in the healing of an injured ACL. In vitro tests using a robotic/UFS testing system showed that both devices could restore function of the goat stifle joint. Under a 67-N anterior tibial load, both the ACL graft fixed with the Mg-based interference screw and the Mg-based ring-repaired ACL could restore anterior tibial translation (ATT) to within 2mm and 5mm, respectively, of the intact joint at 30°, 60°, and 90° of flexion. In-situ forces in the replacement graft and Mg-based ring-repaired ACL were also similar to those of the intact ACL. Further, early in vivo data using the Mg-based interference screw showed that after 12 weeks, it was non-toxic and the joint stability and graft function reached similar levels as published data. Following these positive results, we will move forward in incorporating bioactive molecules and ECM bioscaffolds to these Mg-based biomaterials to test their potential for functional tissue engineering of musculoskeletal and other tissues. © 2013 Published by Elsevier Ltd.

Imaging Feedback of Histotripsy Treatments Using Ultrasound Shear Wave Elastography

PubMed Central

Wang, Tzu-Yin; Hall, Timothy L.; Xu, Zhen; Fowlkes, J. Brian; Cain, Charles A.

2013-01-01

Histotripsy is a cavitation-based ultrasound therapy that mechanically fractionates soft solid tissues into fluid-like homogenates. This paper investigates the feasibility of imaging the tissue elasticity change during the histotripsy process as a tool to provide feedback for the treatments. The treatments were performed on agar tissue phantoms and ex vivo kidneys using 3-cycle ultrasound pulses delivered by a 750-kHz therapeutic array at peak negative/positive pressure of 17/108 MPa and a repetition rate of 50 Hz. Lesions with different degrees of damage were created with increasing numbers of therapy pulses from 0 to 2000 pulses per treatment location. The elasticity of the lesions was measured with ultrasound shear wave elastography, in which a quasi-planar shear wave was induced by acoustic radiation force generated by the therapeutic array, and tracked with ultrasound imaging at 3000 frames per second. Based on the shear wave velocity calculated from the sequentially captured frames, the Young’s modulus was reconstructed. Results showed that the lesions were more easily identified on the shear wave velocity images than on B-mode images. As the number of therapy pulses increased from 0 to 2000 pulses/location, the Young’s modulus decreased exponentially from 22.1 ± 2.7 to 2.1 ± 1.1 kPa in the tissue phantoms (R2 = 0.99, N = 9 each), and from 33.0 ± 7.1 to 4.0 ± 2.5 kPa in the ex vivo kidneys (R2 = 0.99, N = 8 each). Correspondingly, the tissues transformed from completely intact to completely fractionated as examined via histology. A good correlation existed between the lesions’ Young’s modulus and the degree of tissue fractionation as examined with the percentage of remaining structurally intact cell nuclei (R2 = 0.91, N = 8 each). These results indicate that lesions produced by histotripsy can be detected with high sensitivity using shear wave elastography. Because the decrease in the tissue elasticity corresponded well with the morphological and histological change, this study provides a basis for predicting the local treatment outcomes from tissue elasticity change. PMID:22711412

Imaging feedback of histotripsy treatments using ultrasound shear wave elastography.

PubMed

Wang, Tzu-Yin; Hall, Timothy L; Xu, Zhen; Fowlkes, J Brian; Cain, Charles A

2012-06-01

Histotripsy is a cavitation-based ultrasound therapy that mechanically fractionates soft solid tissues into fluid-like homogenates. This paper investigates the feasibility of imaging the tissue elasticity change during the histotripsy process as a tool to provide feedback for the treatments. The treatments were performed on agar tissue phantoms and ex vivo kidneys using 3-cycle ultrasound pulses delivered by a 750-kHz therapeutic array at peak negative/positive pressure of 17/108 MPa and a repetition rate of 50 Hz. Lesions with different degrees of damage were created with increasing numbers of therapy pulses from 0 to 2000 pulses per treatment location. The elasticity of the lesions was measured with ultrasound shear wave elastography, in which a quasi-planar shear wave was induced by acoustic radiation force generated by the therapeutic array, and tracked with ultrasound imaging at 3000 frames per second. Based on the shear wave velocity calculated from the sequentially captured frames, the Young's modulus was reconstructed. Results showed that the lesions were more easily identified on the shear wave velocity images than on B-mode images. As the number of therapy pulses increased from 0 to 2000 pulses/location, the Young's modulus decreased exponentially from 22.1 ± 2.7 to 2.1 ± 1.1 kPa in the tissue phantoms (R2 = 0.99, N = 9 each), and from 33.0 ± 7.1 to 4.0 ± 2.5 kPa in the ex vivo kidneys (R2 = 0.99, N = 8 each). Correspondingly, the tissues transformed from completely intact to completely fractionated as examined via histology. A good correlation existed between the lesions' Young's modulus and the degree of tissue fractionation as examined with the percentage of remaining structurally intact cell nuclei (R2 = 0.91, N = 8 each). These results indicate that lesions produced by histotripsy can be detected with high sensitivity using shear wave elastography. Because the decrease in the tissue elasticity corresponded well with the morphological and histological change, this study provides a basis for predicting the local treatment outcomes from tissue elasticity change.

46 CFR 170.170 - Weather criteria.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 7 2014-10-01 2014-10-01 false Weather criteria. 170.170 Section 170.170 Shipping COAST... ALL INSPECTED VESSELS Intact Stability Criteria § 170.170 Weather criteria. (a) Each vessel must be... weather deck or abnormal sheer. (c) When doing the calculations required by paragraph (a) of this section...

46 CFR 170.170 - Weather criteria.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 7 2011-10-01 2011-10-01 false Weather criteria. 170.170 Section 170.170 Shipping COAST... ALL INSPECTED VESSELS Intact Stability Criteria § 170.170 Weather criteria. (a) Each vessel must be... weather deck or abnormal sheer. (c) When doing the calculations required by paragraph (a) of this section...

46 CFR 170.170 - Weather criteria.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 7 2012-10-01 2012-10-01 false Weather criteria. 170.170 Section 170.170 Shipping COAST... ALL INSPECTED VESSELS Intact Stability Criteria § 170.170 Weather criteria. (a) Each vessel must be... weather deck or abnormal sheer. (c) When doing the calculations required by paragraph (a) of this section...

46 CFR 170.170 - Weather criteria.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 7 2013-10-01 2013-10-01 false Weather criteria. 170.170 Section 170.170 Shipping COAST... ALL INSPECTED VESSELS Intact Stability Criteria § 170.170 Weather criteria. (a) Each vessel must be... weather deck or abnormal sheer. (c) When doing the calculations required by paragraph (a) of this section...

A raman microprobe investigation of the molecular architecture of loblolly pine tracheids

Treesearch

James S. Bond; Rajai H. Atalla

1999-01-01

Our understanding of the molecular architecture of intact, native plant cell walls is very limited. Traditional methods of investigation disturb the tissue to varying degrees and conclusions based on these methods may be intimately related to the technique used. A promising new technique to study native-state organization is polarized Raman spectroscopy. In this...

Prevalence of depressive symptoms in older nursing home residents with intact cognitive function in Taiwan.

PubMed

Hu, Sophia H; Chuang, Yeu-Hui; Ting, Yeh-Feng; Lin, Kuan-Yu; Hsieh, Chia-Jung

2018-03-25

The investigators aimed to explore the prevalence of depressive symptoms and associated factors among older residents with intact cognitive function in nursing homes in Taiwan. A cross-sectional descriptive and correlational research design was used. A convenience sample of 178 older residents without cognitive impairment was recruited from 36 nursing homes in Southern Taiwan. The questionnaires included demographic data; the Barthel Index, which assesses the ability to perform activities of daily living; and the Geriatric Depression Scale Short Form. Among older residents in nursing homes with intact cognitive function, 39.3% had depressive symptoms. Age, religion, previous living status, previous working status, being totally dependent in physical function, and being severely dependent in physical function were significant predictors of depressive symptoms among cognitively intact older residents. The findings highlight the critical mental healthcare issues among older residents with intact cognitive function in nursing homes. Practical strategies for preventing the occurrence of depressive symptoms and caring for those who have depressive symptoms should be developed, especially for younger or dependent older residents or residents who have never been employed, have no religious beliefs, or have lived alone before they moved into an institution. © 2018 Wiley Periodicals, Inc.

Ice formation in isolated human hepatocytes and human liver tissue.

PubMed

Bischof, J C; Ryan, C M; Tompkins, R G; Yarmush, M L; Toner, M

1997-01-01

Cryopreservation of isolated cells and tissue slices of human liver is required to furnish extracorporeal bioartificial liver devices with a ready supply of hepatocytes, and to create in vitro drug metabolism and toxicity models. Although both the bioartificial liver and many current biotoxicity models are based on reconstructing organ functions from single isolated hepatocytes, tissue slices offer an in vitro system that may more closely resemble the in vivo situation of the cells because of cell-cell and cell-extracellular matrix interactions. However, successful cryopreservation of both cellular and tissue level systems requires an increased understanding of the fundamental mechanisms involved in the response of the liver and its cells to freezing stress. This study investigates the biophysical mechanisms of water transport and intracellular ice formation during freezing in both isolated human hepatocytes and whole liver tissue. The effects of cooling rate on individual cells were measured using a cryomicroscope. Biophysical parameters governing water transport (Lpg = 2.8 microns/min-atm and ELp = 79 kcal/mole) and intracellular heterogeneous ice nucleation (omega het = 1.08 x 10(9) m-2s-1 and kappa het = 1.04 x 10(9) K5) were determined. These parameters were then incorporated into a theoretical Krogh cylinder model developed to simulate water transport and ice formation in intact liver tissue. Model simulations indicated that the cellular compartment of the Krogh model maintained more water than isolated cells under the same freezing conditions. As a result, intracellular ice nucleation occurred at lower cooling rates in the Krogh model than in isolated cells. Furthermore, very rapid cooling rates (1000 degrees C/min) showed a depression of heterogeneous nucleation and a shift toward homogeneous nucleation. The results of this study are in qualitative agreement with the findings of a previous experimental study of the response to freezing of intact human liver.

Inhibitory effect of the urothelium/lamina propria on female porcine urethral contractility & effect of age.

PubMed

Folasire, Oladayo S; Chess-Williams, Russ; Sellers, Donna J

2017-09-01

The urethral uroepithelium has been implicated in urethral sensation and maintenance of continence. However, relatively little is known about the function of the urethral urothelium compared with that of the bladder. The aim of the study was to examine the role of the urothelium/lamina propria on contractility of the porcine urethra, along with the influence of nitric oxide, prostaglandins and ageing. Porcine urethral tissues, intact and denuded of urothelium/lamina propria, were mounted in tissue baths and contractions to noradrenaline, phenylephrine and carbachol obtained. Contractions in the presence of Nώ-nitro-l-arginine (100 μmol/L) and indomethacin (10 μmol/L) were examined, along with contractions of tissues from young (6 months) and older (3 years) animals. The urothelium/lamina propria of the urethra significantly inhibited contractions to carbachol, noradrenaline and phenylephrine. This inhibitory effect was not significantly different for the three agonists (58.7±10.3%, 60.4±12.6% and 39.4±12.2% inhibition; n=4-7), and was also observed when denuded tissues were co-incubated with a second tissue with intact urothelium/lamina propria (40.6±7.5% inhibition; n=6). Inhibition of nitric oxide and prostaglandin production did not attenuate the inhibitory effect of the urothelium/lamina propria on noradrenaline contractions. In addition, ageing did not alter the inhibitory effect for either phenylephrine contractions (33.9±2.2% vs 41.0±9.7%, young vs older urethral tissues) or noradrenaline contractions (32.9±11.1% vs 53.7±11.0%). In conclusion the urothelium/lamina propria of the urethra has an inhibitory effect on receptor-mediated urethral contraction. This inhibition is due to the release of a diffusible factor, and the effect is not mediated by nitric oxide or prostaglandins, or affected by age. © 2017 John Wiley & Sons Australia, Ltd.

Pulp and apical tissue response to deep caries in immature teeth: A histologic and histobacteriologic study.

PubMed

Ricucci, Domenico; Siqueira, José F; Loghin, Simona; Lin, Louis M

2017-01-01

Descriptions of the pathologic changes in the pulp and associated apical structures of human immature teeth in response to deep caries are lacking in the literature. This article describes the histologic events associated with the radicular pulp and the apical tissues of human immature teeth following pulp inflammation and necrosis. Twelve immature teeth with destructive caries lesions were obtained from 8 patients. Two intact immature teeth served as controls. Teeth were extracted for reasons not related to this study and immediately processed for histopathologic and histobacteriologic analyses. Serial sections were examined for the pulp conditions and classified as reversible or irreversible pulp inflammation, or pulp necrosis. Other histologic parameters were also evaluated. In the 3 cases with reversible pulp inflammation, tissue in the pulp chamber showed mild to moderate inflammation and tertiary dentin formation related to tubules involved in the caries process. Overall, the radicular pulp tissue, apical papilla and Hertwig's epithelial root sheath (HERS) exhibited characteristics of normality. In the 3 cases with irreversible pulp inflammation, the pulps were exposed and severe inflammation occurred in the pulp chamber, with minor areas of necrosis and infection. Large areas of the canal walls were free from odontoblasts and lined by an atubular mineralized tissue. The apical papilla showed extremely reduced cellularity or lack of cells and HERS was discontinuous or absent. In the 6 cases with pulp necrosis, the coronal and radicular pulp tissue was necrotic and colonized by bacterial biofilms. The apical papilla could not be discerned, except for one case. HERS was absent in the necrotic cases. While immature teeth with reversible pulpitis showed histologic features almost similar to normal teeth in the canal and in the apical region, those with irreversible pulpitis and necrosis exhibited significant alterations not only in the radicular pulp but also in the apical tissues, including the apical papilla and HERS. Alterations in the radicular pulp and apical tissues help explain the outcome of current regenerative/reparative therapies and should be taken into account when devising more predictable therapeutic protocols for teeth with incomplete root formation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of foliar treatments on distribution of /sup 14/C-glyphosate in Convolvulus arvensis L

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

Lauridson, T.C.

1986-01-01

Field bindweed is a perennial weed which produces shoots from buds on its roots. Herbicides, such as glyphosate (N-(phosphonomethyl)glycine) used for control of field bindweed usually do not kill all shoot buds on the roots, thus field bindweed often reinfests areas within 3 to 6 weeks of treatment. This dissertation deals with the development of a technique to change glyphosate distribution in field bindweed roots and could result in less shoot regrowth after glyphosate application. In field studies eight plant growth regulators were applied in September, 3 days before 2.24 kg/ha of 2.4-D((2,4-dichlorophenoxy) acetic acid) or 1.68 kg/ha of glyphosate.more » Eight months later, regrowth of shoots was least where glyphosate was applied at 0.028 kg/ha as a pretreatment, followed by a standard rate of 1.68 kg/ha. In subsequent greenhouse studies, typical patterns of shoot growth and /sup 14/C-glyphosate distribution in isolated root sections taken from 15-week-old intact plants were determined. In subsequent growth chamber studies, plants were decapitated to observe the effect of shoot apical dominance on /sup 14/C-glyphosate translocation. After /sup 14/C-glyphosate was applied, intact plants had about twice as much /sup 14/C in distal root sections as in proximal or middle root sections. Decapitated plants had more /sup 14/C in proximal and middle root sections than in distal sections, and about twice as much /sup 14/C was translocated to roots of decapitated plants than intact plants. Eight concentrations of 2,4,-D or glyphosate from 1 to 5000 ppm were applied in logarithmic series to 6-week old plants.« less

Third harmonic generation imaging of intact human cerebral organoids to assess key components of early neurogenesis in Rett Syndrome (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yildirim, Murat; Feldman, Danielle; Wang, Tianyu; Ouzounov, Dimitre G.; Chou, Stephanie; Swaney, Justin; Chung, Kwanghun; Xu, Chris; So, Peter T. C.; Sur, Mriganka

2017-02-01

Rett Syndrome (RTT) is a pervasive, X-linked neurodevelopmental disorder that predominantly affects girls. It is mostly caused by a sporadic mutation in the gene encoding methyl CpG-binding protein 2 (MeCP2).The clinical features of RTT are most commonly reported to emerge between the ages of 6-18 months and implicating RTT as a disorder of postnatal development. However, a variety of recent evidence from our lab and others demonstrates that RTT phenotypes are present at the earliest stages of brain development including neurogenesis, migration, and patterning in addition to stages of synaptic and circuit development and plasticity. We have used RTT patient-derived induced pluripotent stem cells to generate 3D human cerebral organoids that can serve as a model for human neurogenesis in vitro. We aim to expand on our existing findings in order to determine aberrancies at individual stages of neurogenesis by performing structural and immunocytochemical staining in isogenic control and MeCP2-deficient organoids. In addition, we aim to use Third Harmonic Generation (THG) microscopy as a label-free, nondestructive 3D tissue visualization method in order to gain a complete understanding of the structural complexity that underlies human neurogenesis. As a proof of concept, we have performed THG imaging in healthy intact human cerebral organoids cleared with SWITCH. We acquired an intrinsic THG signal with the following laser configurations: 400 kHz repetition rate, 65 fs pulse width laser at 1350 nm wavelength. In these THG images, nuclei are clearly delineated and cross sections demonstrate the depth penetration capacity (< 1mm) that extends throughout the organoid. Imaging control and MeCP2-deficient human cerebral organoids in 2D sections reveals structural and protein expression-based alterations that we expect will be clearly elucidated via both THG and three-photon fluorescence microscopy.

Variable expression of GFP in different populations of peripheral cholinergic neurons of ChATBAC-eGFP transgenic mice.

PubMed

Brown, T Christopher; Bond, Cherie E; Hoover, Donald B

2018-03-01

Immunohistochemistry is used widely to identify cholinergic neurons, but this approach has some limitations. To address these problems, investigators developed transgenic mice that express enhanced green fluorescent protein (GFP) directed by the promoter for choline acetyltransferase (ChAT), the acetylcholine synthetic enzyme. Although, it was reported that these mice express GFP in all cholinergic neurons and non-neuronal cholinergic cells, we could not detect GFP in cardiac cholinergic nerves in preliminary experiments. Our goals for this study were to confirm our initial observation and perform a qualitative screen of other representative autonomic structures for the presences of GFP in cholinergic innervation of effector tissues. We evaluated GFP fluorescence of intact, unfixed tissues and the cellular localization of GFP and vesicular acetylcholine transporter (VAChT), a specific cholinergic marker, in tissue sections and intestinal whole mounts. Our experiments identified two major tissues where cholinergic neurons and/or nerve fibers lacked GFP: 1) most cholinergic neurons of the intrinsic cardiac ganglia and all cholinergic nerve fibers in the heart and 2) most cholinergic nerve fibers innervating airway smooth muscle. Most cholinergic neurons in airway ganglia stained for GFP. Cholinergic systems in the bladder and intestines were fully delineated by GFP staining. GFP labeling of input to ganglia with long preganglionic projections (vagal) was sparse or weak, while that to ganglia with short preganglionic projections (spinal) was strong. Total absence of GFP might be due to splicing out of the GFP gene. Lack of GFP in nerve projections from GFP-positive cell bodies might reflect a transport deficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

Minipig model of maxillary distraction osteogenesis: immunohistochemical and histomorphometric analysis of the sequence of osteogenesis.

PubMed

Papadaki, Maria E; Kaban, Leonard B; Troulis, Maria J

2012-11-01

To document the sequence of bone formation in a minipig model of Le Fort I distraction osteogenesis (DO) using immunohistochemistry and histomorphometry. Female Yucatan minipigs (N = 9) in the mixed-dentition stage underwent bilateral maxillary DO. The distraction protocol was 0 days of latency, with a distraction rate of 1 mm/d for 12 days and 24 days of fixation. Specimens were harvested and divided between the central incisors (18 hemi-maxillae) at the end of DO (n = 6), at mid-fixation (n = 6), and at the end of fixation (n = 6). Sections, including the advancement zone, were stained with hematoxylin-eosin, collagen II, CD34, proliferating cell nuclear antigen, and tartrate-resistant acid phosphatase. Light and fluorescence microscope images (original magnification ×200) were obtained, and percentage of surface area (PSA) of the advancement zone occupied by fibrous tissue, vessels, proliferating cells, osteoid, and bone was determined. An intact maxilla served as the control. At the end of DO, in the advancement zone, the PSA (mean values) of proliferating cells was 33.16%; fibrous tissue, 52%; vessels, 4.35%; and new bone, 5.45%. At the end of fixation, the PSA of proliferating cells decreased to 10.53%, fibrous tissue to 2.3%, and vessels to 1.5% whereas the PSA of new bone increased to 44.9%. The results of this study indicate that the progression of osteogenesis in the maxillary DO wound begins with intense cellular proliferation and vascular fibrous tissue formation and progresses to mature, cancellous bone by the end of fixation. The PSA occupied by mature bone is significantly less than in the control maxilla at the end of fixation. This is consistent with the sequence in the mandibular DO wound. Published by Elsevier Inc.

[Pathogen identification of 10 suspected cases of sparganosis mansoni].

PubMed

Zeng, Qing-Ren; He, Mei; Wang, Fang; Zhang, Zu-Ping; Su, Zhan-San; Zhou, Jun; Liu, Bao-An; Lan, Zhi-Hua; Hu, Mian-Juan; Cai, Li-Ting

2012-06-30

To diagnose 10 cases of clinically suspected cases of sparganosis mansoni by pathogen identification. In the period from August 2009 to August 2011, 10 biopsy specimens were obtained from 10 patients of four hospitals to identify the pathogen. Among the 10 cases, 4 cases showed abdominal subcutaneous mass, 3 showed eyelid swelling, 1 displayed brain lesions, 1 showed pulmonary mass, and 1 showed pleural effusion. There was one parasite each from three patients with eyelid swelling, and one patient with abdominal subcutaneous mass, which were observed by naked eye and microscope morphologically and histologically. Specimens from other six cases were examined by microscope after paraffin embedding, sectioning, and HE staining. For further identification, the parasite biopsy tissue specimens were detected by immunohistochemistry with Sparganum mansoni-immunized rabbit serum as the primary antibody. Three intact worms, from three patients with eyelid swelling, showed typical S. mansoni morphological characteristics. One residue parasite from the abdominal subcutaneous mass showed network structures and full of calcareous corpuscles in the body under microscope same as that of S. mansoni. The histological structure in three of the six sections showed typically the body wall with folds, which was dense, thick and deeply eosine stained, part of the tegument outside was covered by micro-hairs. In the worm body there was net-like loose structure and calcareous corpuscles without cavity. The structure of the other three worm sections was atypical. The six worm sections were positive by immunohistochemical detection. The 10 clinically suspected cases are diagnosed as sparganosis mansoni.

Deregulation of EZH2 expression in human spermatogenic disorders and testicular germ cell tumors.

PubMed

Hinz, Stefan; Magheli, Ahmed; Weikert, Steffen; Schulze, Wolfgang; Krause, Hans; Schrader, Mark; Miller, Kurt; Kempkensteffen, Carsten

2010-10-01

Enhancer of Zeste 2 (EZH2) is an epigenetic transcriptional repressor involved in cell cycle control and cell fate decisions. Since these processes play key roles during intact spermatogenesis, deregulation of EZH2 expression may contribute to the development and progression of benign and malignant testicular diseases. The objective of this study was to investigate the expression profile of EZH2 in testicular germ cell tumors (TGCT) and spermatogenic defects. Real-time RT-PCR was applied to quantify the m-RNA expression of EZH2 in 64 seminomas 36 non-seminomas, 4 carcinomas in situ (CIS), 40 samples harboring impaired spermatogenesis and 24 normal testicular reference biopsies. EZH2 was expressed in 99% of TGCT samples and in all biopsies with intact spermatogenesis. Its expression levels were highest in normal testicular tissue, and continuously decreased with malignant transformation to CIS and further progression to invasive TGCT (P < 0.001). EZH2 tumor levels were not related to the histological TGCT subtype or clinical tumor stage. Comparison of distinct stages of spermatogenic failure revealed an inverse association of EZH2 levels to the severity of the spermatogenic defect (P < 0.001). Our data strongly suggest that in TGCT EZH2 does not exert its often assumed oncogenic properties during malignant transformation and progression. High EZH2 levels in normal testicular tissue and the inverse association of its expression levels with the severity of spermatogenic failure point to its potential value as a molecular marker for spermatogenic defects and may indicate an important physiological role of EZH2 during intact spermatogenesis.

Mechanical characterization of stomach tissue under uniaxial tensile action.

PubMed

Jia, Z G; Li, W; Zhou, Z R

2015-02-26

In this article, the tensile properties of gastric wall were investigated by using biomechanical test and theoretical analysis. The samples of porcine stomach strips from smaller and greater curvature of the stomach were cut in longitudinal and circumferential direction, respectively. The loading-unloading, stress relaxation, strain creep, tensile fracture tests were performed at mucosa-submucosa, serosa-muscle and intact layer, respectively. Results showed that the biomechanical properties of the porcine stomach depended on the layers, orientations and locations of the gastric wall and presented typical viscoelastic, nonlinear and anisotropic mechanical properties. During loading-unloading test, the stress of serosa-muscle layer in the longitudinal direction was 15-20% more than that in the circumferential direction at 12% stretch ratio, while it could reach about 40% for the intact layer and 50% for the mucosa-submucosa layer. The results of stress relaxation and strain creep showed that the variation degree was obviously faster in the circumferential direction than that in the longitudinal direction, and the ultimate residual values were also different for the different layers, orientations and locations. In the process of fracture test, the serosa-muscle layer fractured firstly followed by the mucosa-submucosa layer when the intact layer was tested, the longitudinal strips firstly began to fracture and the required stress value was about twice as much as that in the circumferential strips. The anisotropy and heterogeneity of mechanical characterization of the porcine stomach were related to its complicated geometry, structure and functions. The results would help us to understand the biomechanics of soft organ tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advanced technique of infrared LED imaging of unstained cells and intracellular structures in isolated spinal cord, brainstem, ganglia and cerebellum.

PubMed

Szucs, Peter; Pinto, Vitor; Safronov, Boris V

2009-03-15

Light-emitting diodes (LEDs) have recently been used for the imaging of unstained living cells in the whole brain and spinal cord preparations, in which one cut was done to remove the overlying white matter. Here we show that in many cases the neurones can be visualized through the white matter in an intact nervous tissue (rats P0-P36 and mice P0-P2). We used an upright microscope with a water immersion objective and a powerful infrared LED (emission peak, 850 nm; maximum radiant intensity, 270 mW/sr) as a source of oblique illumination. In the isolated spinal cord, we were able to visualize lamina I and II neurones as well as motoneurones. In the brainstem, the neurones from the superficial nuclei were successfully viewed. In the sensory ganglion, we obtained images of unstained cells as well as intracellular structures, like endoplasmic reticulum, nucleus and nucleolus. In isolated cerebellum, parallel fibers, Purkinje and granule cells were viewed. Whole-cell recordings were done to fill spinal lamina I neurones, motoneurones and brainstem neurones with biocytin for detailed 2D-3D reconstruction of their dendritic and axonal arbores. Our imaging technique also allowed labelling individual intact neurones by injecting biocytin through the extracellular cell-attached pipette. This imaging technique opens broad possibilities for functional studies of neurones with completely preserved anatomical structures and synaptic inputs. We also show that the application of oblique infrared LED illumination allows a construction of a simple digital videomicroscope for the high-quality living cell imaging in intact nervous tissue.

Development of carbon-11 labeled acryl amides for selective PET imaging of active tissue transglutaminase.

PubMed

van der Wildt, Berend; Wilhelmus, Micha M M; Bijkerk, Jonne; Haveman, Lizeth Y F; Kooijman, Esther J M; Schuit, Robert C; Bol, John G J M; Jongenelen, Cornelis A M; Lammertsma, Adriaan A; Drukarch, Benjamin; Windhorst, Albert D

2016-04-01

Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme capable of forming metabolically and mechanically stable crosslinks between the γ-carboxamide of a glutamine acyl-acceptor substrate and the ε-amino functionality of a lysine acyl-donor substrate resulting in protein oligomers. High TG2 crosslinking activity has been implicated in the pathogenesis of various diseases including celiac disease, cancer and fibrotic and neurodegenerative diseases. Development of a PET tracer specific for active TG2 provides a novel tool to further investigate TG2 biology in vivo in disease states. Recently, potent irreversible active site TG2 inhibitors carrying an acrylamide warhead were synthesized and pharmacologically characterized. Three of these inhibitors, compound 1, 2 and 3, were successfully radiolabeled with carbon-11 on the acrylamide carbonyl position using a palladium mediated [(11)C]CO aminocarbonylation reaction. Ex vivo biodistribution and plasma stability were evaluated in healthy Wistar rats. Autoradiography was performed on MDA-MB-231 tumor sections. [(11)C]1, -2 and -3 were obtained in decay corrected radiochemical yields of 38-55%. Biodistribution showed low uptake in peripheral tissues, with the exception of liver and kidney. Low brain uptake of <0.05% ID/g was observed. Blood plasma analysis demonstrated that [(11)C]1 and [(11)C]2 were rapidly metabolized, whereas [(11)C]3 was metabolized at a more moderate rate (63.2 ± 6.8 and 28.7 ± 10.8% intact tracer after 15 and 45 min, respectively). Autoradiography with [(11)C]3 on MDA-MB-231 tumor sections showed selective and specific binding of the radiotracer to the active state of TG2. Taken together, these results identify [(11)C]3 as the most promising of the three compounds tested for development as PET radiotracer for the in vivo investigation of TG2 activity. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of the cavity margins after Er:YAG laser ablation of the enamel and dentin

NASA Astrophysics Data System (ADS)

Dostalova, Tatjana; Krejsa, Otakar; Jelinkova, Helena; Hamal, Karel

1994-12-01

This study investigates the checks of cavity margin after enamel and dentin ablation. The Er:YAG laser enamel and dentin ablation can be directly connected with the danger of cracks originating in the enamel near the cavity. This study evaluates the quality of the enamel edges after Er:YAG laser preparation. The enamel and dentin of buccal surfaces were ablated by the Er:YAG laser radiation. An Erbium:YAG laser system with the energy of 200 mJ was used to generate 200 microsecond(s) long pulses of mid-infrared 2.94 micrometers light in multimode configuration. The laser was operating in a free running mode, the repetition rate being 0.5 Hz with average laser power of 100 mW. Laser radiation was focused on the tooth tissue. Water cooling was used during the procedure in order to prevent tooth tissue destruction. The time of laser preparation was 5 minutes. A cavity of class V was prepared. The teeth were immersed into 0.5% basic fuchsin and then centrifuged at 6000 rev/min for 20 minutes. The microphotographs of the margins stained with 0.5% basic fuchsin were made and then the longitudinal section of the teeth were evaluated. The micrographs of the longitudinal section were checked and measured afterwards. The effect of the investigated laser irradiation on the origin of cracks was analyzed in the scanning electron microscope. Micrographs of each tooth before and after the laser ablation were compared. Micrographs of the intact teeth after extraction present the cracks of the enamel. They depend on the pressure exerted during extraction. The influence of the laser ablation proper is it bears no signs of new cracks. The conclusions of this study demonstrate the non-invasive nature of the Er:YAG laser ablation of the hard dental tissues.

Mineralization Induction of Gingival Fibroblasts and Construction of a Sandwich Tissue-Engineered Complex for Repairing Periodontal Defects

PubMed Central

Wu, Mingxuan; Zhang, Yanning; Liu, Huijuan; Dong, Fusheng

2018-01-01

Background The ideal healing technique for periodontal tissue defects would involve the functional regeneration of the alveolar bone, cementum, and periodontal ligament, with new periodontal attachment formation. In this study, gingival fibroblasts were induced and a “sandwich” tissue-engineered complex (a tissue-engineered periodontal membrane between 2 tissue-engineered mineralized membranes) was constructed to repair periodontal defects. We evaluated the effects of gingival fibroblasts used as seed cells on the repair of periodontal defects and periodontal regeneration. Material/Methods Primitively cultured gingival fibroblasts were seeded bilaterally on Bio-Gide collagen membrane (a tissue-engineered periodontal membrane) or unilaterally on small intestinal submucosa segments, and their mineralization was induced. A tissue-engineered sandwich was constructed, comprising the tissue-engineered periodontal membrane flanked by 2 mineralized membranes. Periodontal defects in premolar regions of Beagles were repaired using the tissue-engineered sandwich or periodontal membranes. Periodontal reconstruction was compared to normal and trauma controls 10 or 20 days postoperatively. Results Periodontal defects were completely repaired by the sandwich tissue-engineered complex, with intact new alveolar bone and cementum, and a new periodontal ligament, 10 days postoperatively. Conclusions The sandwich tissue-engineered complex can achieve ideal periodontal reconstruction rapidly. PMID:29470454

Characterization of dynamic physiology of the bladder by optical coherence tomography

NASA Astrophysics Data System (ADS)

Yuan, Zhijia; Keng, Kerri; Pan, Rubin; Ren, Hugang; Du, Congwu; Kim, Jason; Pan, Yingtian

2012-03-01

Because of its high spatial resolution and noninvasive imaging capabilities, optical coherence tomography has been used to characterize the morphological details of various biological tissues including urinary bladder and to diagnose their alternations (e.g., cancers). In addition to static morphology, the dynamic features of tissue morphology can provide important information that can be used to diagnose the physiological and functional characteristics of biological tissues. Here, we present the imaging studies based on optical coherence tomography to characterize motion related physiology and functions of rat bladder detrusor muscles and compared the results with traditional biomechanical measurements. Our results suggest that optical coherence tomography is capable of providing quantitative evaluation of contractile functions of intact bladder (without removing bladder epithelium and connective tissue), which is potentially of more clinical relevance for future clinical diagnosis - if incorporated with cystoscopic optical coherence tomography.

Equilibrium Passive Sampling of POP in Lipid-Rich and Lean Fish Tissue: Quality Control Using Performance Reference Compounds.

PubMed

Rusina, Tatsiana P; Carlsson, Pernilla; Vrana, Branislav; Smedes, Foppe

2017-10-03

Passive sampling is widely used to measure levels of contaminants in various environmental matrices, including fish tissue. Equilibrium passive sampling (EPS) of persistent organic pollutants (POP) in fish tissue has been hitherto limited to application in lipid-rich tissue. We tested several exposure methods to extend EPS applicability to lean tissue. Thin-film polydimethylsiloxane (PDMS) passive samplers were exposed statically to intact fillet and fish homogenate and dynamically by rolling with cut fillet cubes. The release of performance reference compounds (PRC) dosed to passive samplers prior to exposure was used to monitor the exchange process. The sampler-tissue exchange was isotropic, and PRC were shown to be good indicators of sampler-tissue equilibration status. The dynamic exposures demonstrated equilibrium attainment in less than 2 days for all three tested fish species, including lean fish containing 1% lipid. Lipid-based concentrations derived from EPS were in good agreement with lipid-normalized concentrations obtained using conventional solvent extraction. The developed in-tissue EPS method is robust and has potential for application in chemical monitoring of biota and bioaccumulation studies.

Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

PubMed

Marciniak, Anja; Cohrs, Christian M; Tsata, Vasiliki; Chouinard, Julie A; Selck, Claudia; Stertmann, Julia; Reichelt, Saskia; Rose, Tobias; Ehehalt, Florian; Weitz, Jürgen; Solimena, Michele; Slak Rupnik, Marjan; Speier, Stephan

2014-12-01

Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

An acidic microenvironment sets the humoral pattern recognition molecule PTX3 in a tissue repair mode

PubMed Central

Doni, Andrea; Musso, Tiziana; Morone, Diego; Bastone, Antonio; Zambelli, Vanessa; Sironi, Marina; Castagnoli, Carlotta; Cambieri, Irene; Stravalaci, Matteo; Pasqualini, Fabio; Laface, Ilaria; Valentino, Sonia; Tartari, Silvia; Ponzetta, Andrea; Maina, Virginia; Barbieri, Silvia S.; Tremoli, Elena; Catapano, Alberico L.; Norata, Giuseppe D.; Bottazzi, Barbara; Garlanda, Cecilia

2015-01-01

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity. PMID:25964372

Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient

PubMed Central

Klyen, Blake R.; Scolaro, Loretta; Shavlakadze, Tea; Grounds, Miranda D.; Sampson, David D.

2014-01-01

We present the assessment of ex vivo mouse muscle tissue by quantitative parametric imaging of the near-infrared attenuation coefficient µt using optical coherence tomography. The resulting values of the local total attenuation coefficient µt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice are higher (9.6 ± 0.3 mm−1) than regions from the same tissue containing only necrotic myofibers (7.0 ± 0.6 mm−1), and significantly higher than values from intact myofibers, whether from an adjacent region of the same sample (4.8 ± 0.3 mm−1) or from healthy tissue of the wild-type C57 mouse (3.9 ± 0.2 mm−1) used as a control. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy. PMID:24761302

THE FREEZING POINT DEPRESSION OF MAMMALIAN TISSUES IN RELATION TO THE QUESTION OF OSMOTIC ACTIVITY OF CELL FLUID

PubMed Central

Brodsky, William A.; Appelboom, Johannes W.; Dennis, Warren H.; Rehm, Warren S.; Miley, John F.; Diamond, Israel

1956-01-01

The freezing point depression of freshly excised frozen tissues, pulverized in a hydraulic press or in a mortar, is greater than that of plasma. Even at 0°C. the freezing point depression of such homogenates increases significantly with time. Dilution data indicate that such freezing point data are valid. The presence of intact cells has been shown in smears of tissues pulverized in a mortar, but not in smears of those crushed in a hydraulic press. The osmolarity of various diluent solutions affects the calculated osmotic activity of tissue homogenates presumably because of delayed diffusion between the diluent and cell fluid. With a hypertonic NaCl diluent, spuriously low values of tissue osmotic activity are found from calculations assuming instantaneous mixing between homogenates and diluents. The limitations of data from cryoscopic experiments and from tissue-swelling experiments are discussed in relation to the basic question of whether or not cell fluid is isotonic to extracellular fluid. PMID:13385447

Multiplexed and scalable super-resolution imaging of three-dimensional protein localization in size-adjustable tissues.

PubMed

Ku, Taeyun; Swaney, Justin; Park, Jeong-Yoon; Albanese, Alexandre; Murray, Evan; Cho, Jae Hun; Park, Young-Gyun; Mangena, Vamsi; Chen, Jiapei; Chung, Kwanghun

2016-09-01

The biology of multicellular organisms is coordinated across multiple size scales, from the subnanoscale of molecules to the macroscale, tissue-wide interconnectivity of cell populations. Here we introduce a method for super-resolution imaging of the multiscale organization of intact tissues. The method, called magnified analysis of the proteome (MAP), linearly expands entire organs fourfold while preserving their overall architecture and three-dimensional proteome organization. MAP is based on the observation that preventing crosslinking within and between endogenous proteins during hydrogel-tissue hybridization allows for natural expansion upon protein denaturation and dissociation. The expanded tissue preserves its protein content, its fine subcellular details, and its organ-scale intercellular connectivity. We use off-the-shelf antibodies for multiple rounds of immunolabeling and imaging of a tissue's magnified proteome, and our experiments demonstrate a success rate of 82% (100/122 antibodies tested). We show that specimen size can be reversibly modulated to image both inter-regional connections and fine synaptic architectures in the mouse brain.

In vivo labeling of cortical astrocytes with sulforhodamine 101 (SR101).

PubMed

Nimmerjahn, Axel; Helmchen, Fritjof

2012-03-01

Fluorescent markers that stain particular cell types in the intact brain are essential tools for fluorescence microscopy because they enable studies of structure and function of cells identified in this way. Although cell type-specific fluorescence staining can be achieved through promoter-driven expression of fluorescent proteins, this genetic approach is generally labor- and cost-intensive. Alternative viral approaches for targeted fluorophore expression are relatively invasive. For astrocytes, there is a simple alternative. This protocol describes an easy and robust method for rapid (within minutes) and high-contrast staining of astrocytes in defined regions of the intact rodent cortex using the synthetic, water-soluble but non-fixable red fluorescent dye sulforhodamine 101 (SR101). Selective staining is achieved through local uptake and gap junction-mediated spread of SR101 following its topical application or injection into tissue. Applications, technical pitfalls, and limitations of the SR101-staining technique are discussed. Given its simplicity and reliability, SR101 staining is a valuable tool for the study of astrocyte function in the intact brain and for in vivo fluorescence microscopy in general.

Structure-Property Relationships in Tough, Superabsorbent Thermoplastic Elastomers for Hemorrhage Control

NASA Astrophysics Data System (ADS)

Beyer, Frederick; Bain, Erich; Long, Tyler; Mrozek, Randy; Savage, Alice; Martin, Halie; Dadmun, Mark; Lenhart, Joseph

Between 2001 and 2009, uncontrolled hemorrhaging from major trauma accounted for the deaths of roughly 80% of wounded soldiers with potentially survivable injuries. Modern hemostatic materials are limited in their ability to deliver therapeutic agents, causing tissue damage themselves, or being difficult to remove intact. The goal of this study is to create a mechanically robust polymer that takes up as much as 1000 wt% water in seconds while maintaining sufficient toughness to be removed intact from the wound intact. A thermoplastic elastomer scaffold in which physical crosslinks provide mechanical toughness might provide an appropriate combination of fast swelling and excellent toughness if the matrix material can be engineered to be strongly hydrophilic and swell rapidly. In this work, a commercial SBS triblock copolymer has been modified with poly(acrylic acid) side chains, resulting in materials that are superabsorbent but retain good mechanical properties when saturated. Although SAXS experiments failed to show any significant changes in morphology, even with 800 wt% water uptake, preliminary SANS experiments using selectively deuterated materials and swelling with D2O show significant changes in morphology. Our most recent findings will be presented.

Effects of tissue mechanical properties on susceptibility to histotripsy-induced tissue damage

NASA Astrophysics Data System (ADS)

Vlaisavljevich, Eli; Kim, Yohan; Owens, Gabe; Roberts, William; Cain, Charles; Xu, Zhen

2014-01-01

Histotripsy is a non-invasive tissue ablation method capable of fractionating tissue by controlling acoustic cavitation. To determine the fractionation susceptibility of various tissues, we investigated histotripsy-induced damage on tissue phantoms and ex vivo tissues with different mechanical strengths. A histotripsy bubble cloud was formed at tissue phantom surfaces using 5-cycle long ultrasound pulses with peak negative pressure of 18 MPa and PRFs of 10, 100, and 1000 Hz. Results showed significantly smaller lesions were generated in tissue phantoms of higher mechanical strength. Histotripsy was also applied to 43 different ex vivo porcine tissues with a wide range of mechanical properties. Gross morphology demonstrated stronger tissues with higher ultimate stress, higher density, and lower water content were more resistant to histotripsy damage in comparison to weaker tissues. Based on these results, a self-limiting vessel-sparing treatment strategy was developed in an attempt to preserve major vessels while fractionating the surrounding target tissue. This strategy was tested in porcine liver in vivo. After treatment, major hepatic blood vessels and bile ducts remained intact within a completely fractionated liver volume. These results identify varying susceptibilities of tissues to histotripsy therapy and provide a rational basis to optimize histotripsy parameters for treatment of specific tissues.

Investigations of the Cavitation and Damage Thresholds of Histotripsy and Applications in Targeted Tissue Ablation

NASA Astrophysics Data System (ADS)

Vlaisavljevich, Eli

Histotripsy is a noninvasive ultrasound therapy that controls acoustic cavitation to mechanically fractionate soft tissue. This dissertation investigates the physical thresholds to initiate cavitation and produce tissue damage in histotripsy and factors affecting these thresholds in order to develop novel strategies for targeted tissue ablation. In the first part of this dissertation, the effects of tissue properties on histotripsy cavitation thresholds and damage thresholds were investigated. Results demonstrated that the histotripsy shock scattering threshold using multi-cycle pulses increases in stiffer tissues, while the histotripsy intrinsic threshold using single-cycle pulses is independent of tissue stiffness. Further, the intrinsic threshold slightly decreases with lower frequencies and significantly decreases with increasing temperature. The effects of tissue properties on the susceptibility to histotripsy-induced tissue damage were also investigated, demonstrating that stiffer tissues are more resistant to histotripsy. Two strategies were investigated for increasing the effectiveness of histotripsy for the treatment of stiffer tissues, with results showing that thermal preconditioning may be used to alter tissue susceptibility to histotripsy and that lower frequency treatments may increase the efficiency of histotripsy tissue ablation due to enhanced bubble expansion. In the second part of this dissertation, the feasibility of using histotripsy for targeted liver ablation was investigated in an intact in vivo porcine model, with results demonstrating that histotripsy was capable of non-invasively creating precise lesions throughout the entire liver. Additionally, a tissue selective ablation approach was developed, where histotripsy completely fractionated the liver tissue surrounding the major hepatic vessels and gallbladder while being self-limited at the boundaries of these critical structures. Finally, the long-term effects of histotripsy liver ablation were investigated in an intact in vivo rodent model, showing that the liver homogenate resulting from histotripsy-induced tissue fractionation was completely resorbed over the course of 28 days. In the final part of this dissertation, a novel ablation method combining histotripsy with acoustically sensitive nanodroplets was developed for targeted cancer cell ablation, demonstrating the potential of using nanodroplet-mediated histotripsy (NMH) for targeted, multi-focal ablation. Studies demonstrated that lower frequency and higher boiling point perfluorocarbon droplets can improve NMH therapy. The role of positive and negative pressure on cavitation nucleation in NMH was also investigated, showing that NMH cavitation nucleation is caused directly from the peak negative pressure of the incident wave, similar to histotripsy bubbles generated above the intrinsic threshold. Overall, the results of this dissertation provide significant insight into the physical mechanisms underlying histotripsy tissue ablation and will help to guide the future development of histotripsy for clinical applications such as the treatment of liver cancer.

The Anterolateral Capsule of the Knee Behaves Like a Sheet of Fibrous Tissue.

PubMed

Guenther, Daniel; Rahnemai-Azar, Amir A; Bell, Kevin M; Irarrázaval, Sebastián; Fu, Freddie H; Musahl, Volker; Debski, Richard E

2017-03-01

The function of the anterolateral capsule of the knee has not been clearly defined. However, the contribution of this region of the capsule to knee stability in comparison with other anterolateral structures can be determined by the relative force that each structure carries during loading of the knee. Purpose/Hypothesis: The purpose of this study was to determine the forces in the anterolateral structures of the intact and anterior cruciate ligament (ACL)-deficient knee in response to an anterior tibial load and internal tibial torque. It was hypothesized that the anterolateral capsule would not function like a traditional ligament (ie, transmitting forces only along its longitudinal axis). Controlled laboratory study. Loads (134-N anterior tibial load and 7-N·m internal tibial torque) were applied continuously during flexion to 7 fresh-frozen cadaveric knees in the intact and ACL-deficient state using a robotic testing system. The lateral collateral ligament (LCL) and the anterolateral capsule were separated from the surrounding tissue and from each other. This was done by performing 3 vertical incisions: lateral to the LCL, medial to the LCL, and lateral to the Gerdy tubercle. Attachments of the LCL and anterolateral capsule were detached from the underlying tissue (ie, meniscus), leaving the insertions and origins intact. The force distribution in the anterolateral capsule, ACL, and LCL was then determined at 30°, 60°, and 90° of knee flexion using the principle of superposition. In the intact knee, the force in the ACL in response to an anterior tibial load was greater than that in the other structures ( P < .001). However, in response to an internal tibial torque, no significant differences were found between the ACL, LCL, and forces transmitted between each region of the anterolateral capsule after capsule separation. The anterolateral capsule experienced smaller forces (~50% less) compared with the other structures ( P = .048). For the ACL-deficient knee in response to an anterior tibial load, the force transmitted between each region of the anterolateral capsule was 434% greater than was the force in the anterolateral capsule ( P < .001) and 54% greater than the force in the LCL ( P = .036) at 30° of flexion. In response to an internal tibial torque at 30°, 60°, or 90° of knee flexion, no significant differences were found between the force transmitted between each region of the anterolateral capsule and the LCL. The force in the anterolateral capsule was significantly smaller than that in the other structures at all knee flexion angles for both loading conditions ( P = .004 for anterior tibial load and P = .04 for internal tibial torque). The anterolateral capsule carries negligible forces in the longitudinal direction, and the forces transmitted between regions of the capsule were similar to the forces carried by the other structures at the knee, suggesting that it does not function as a traditional ligament. Thus, the anterolateral capsule should be considered a sheet of tissue. Surgical repair techniques for the anterolateral capsule should restore the ability of the tissue to transmit forces between adjacent regions of the capsule rather than along its longitudinal axis.

Pharmacologic basis for the enhanced efficacy of dutasteride against prostatic cancers.

PubMed

Xu, Yi; Dalrymple, Susan L; Becker, Robyn E; Denmeade, Samuel R; Isaacs, John T

2006-07-01

Prostatic dihydrotestosterone (DHT) concentration is regulated by precursors from systemic circulation and prostatic enzymes of androgen metabolism, particularly 5alpha-reductases (i.e., SRD5A1 and SRD5A2). Therefore, the levels of expression SRD5A1 and SRD5A2 and the antiprostatic cancer growth response to finasteride, a selective SRD5A2 inhibitor, versus the dual SRD5A1 and SRD5A2 inhibitor, dutasteride, were compared. Real-time PCR and enzymatic assays were used to determine the levels of SRD5A1 and SRD5A2 in normal versus malignant rat and human prostatic tissues. Rats bearing the Dunning R-3327H rat prostate cancer and nude mice bearing LNCaP or PC-3 human prostate cancer xenografts were used as model systems. Tissue levels of testosterone and DHT were determined using liquid chromatography-mass spectrometry. Prostate cancer cells express undetectable to low levels of SRD5A2 but elevated levels of SRD5A1 activity compared with nonmalignant prostatic tissue. Daily oral treatment of rats with the SRD5A2 selective inhibitor, finasteride, reduces prostate weight and DHT content but did not inhibit R-3327H rat prostate cancer growth or DHT content in intact (i.e., noncastrated) male rats. In contrast, daily oral treatment with even a low 1 mg/kg/d dose of the dual SRD5A1 and SRD5A2 inhibitor, dutasteride, reduces both normal prostate and H tumor DHT content and weight in intact rats while elevating tissue testosterone. Daily oral treatment with finasteride significantly (P < 0.05) inhibits growth of LNCaP human prostate cancer xenografts in intact male nude mice, but this inhibition is not as great as that by equimolar oral dosing with dutasteride. This anticancer efficacy is not equivalent, however, to that produced by castration. Only combination of dutasteride and castration produces a greater tumor inhibition (P < 0.05) than castration monotherapy against androgen-responsive LNCaP cancers. In contrast, no response was induced by dutasteride in nude mice bearing androgen-independent PC-3 human prostatic cancer xenografts. These results document that testosterone is not as potent as DHT but does stimulate prostate cancer growth, thus combining castration with dutasteride enhances therapeutic efficacy.

Reconstruction of Heel With Propeller Flap in Postfasciotomy and Popliteal Artery Revascularization State.

PubMed

Kang, Jin Seok; Choi, Hwan Jun; Tak, Min Sung

2016-06-01

Free flaps are still the gold standard for large defects of the lower limb, but propeller perforator flaps have become a simpler and faster alternative to free flaps because of some advantages such as reliable vascular pedicle, wide mobilization and rotation, great freedom in design, low donor site morbidity, and easy harvest with no requirement for anastomosis. But when the vessels show insufficient findings in preoperative evaluation using a Doppler probe or the vessel is injured, the surgeon should avoid performing free flap surgery to prevent flap failure and should select a propeller perforator flap as an alternative method on the condition that more than one perforator is intact. In this study, we report reconstruction of soft tissue defects of the heel with a pedicled propeller flap in postfasciotomy and popliteal artery revascularization state by making an incision on the central portion above the Achilles tendon, which can be covered by the posterior tibial artery perforator or the peroneal artery perforator based flaps. In conclusion, we showed that although the popliteal artery was injured, the soft tissue defect can be reconstructed using a perforator propeller flap if intact distal flow in the anastomosis site was confirmed. © The Author(s) 2015.

Light sheet theta microscopy for rapid high-resolution imaging of large biological samples.

PubMed

Migliori, Bianca; Datta, Malika S; Dupre, Christophe; Apak, Mehmet C; Asano, Shoh; Gao, Ruixuan; Boyden, Edward S; Hermanson, Ola; Yuste, Rafael; Tomer, Raju

2018-05-29

Advances in tissue clearing and molecular labeling methods are enabling unprecedented optical access to large intact biological systems. These developments fuel the need for high-speed microscopy approaches to image large samples quantitatively and at high resolution. While light sheet microscopy (LSM), with its high planar imaging speed and low photo-bleaching, can be effective, scaling up to larger imaging volumes has been hindered by the use of orthogonal light sheet illumination. To address this fundamental limitation, we have developed light sheet theta microscopy (LSTM), which uniformly illuminates samples from the same side as the detection objective, thereby eliminating limits on lateral dimensions without sacrificing the imaging resolution, depth, and speed. We present a detailed characterization of LSTM, and demonstrate its complementary advantages over LSM for rapid high-resolution quantitative imaging of large intact samples with high uniform quality. The reported LSTM approach is a significant step for the rapid high-resolution quantitative mapping of the structure and function of very large biological systems, such as a clarified thick coronal slab of human brain and uniformly expanded tissues, and also for rapid volumetric calcium imaging of highly motile animals, such as Hydra, undergoing non-isomorphic body shape changes.

Optical magnetic detection of single-neuron action potentials using quantum defects in diamond

PubMed Central

Barry, John F.; Turner, Matthew J.; Schloss, Jennifer M.; Glenn, David R.; Song, Yuyu; Lukin, Mikhail D.; Park, Hongkun; Walsworth, Ronald L.

2016-01-01

Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution—e.g., magnetic resonance imaging methods and magnetoencephalography—or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector. PMID:27911765

Optical magnetic detection of single-neuron action potentials using quantum defects in diamond.

PubMed

Barry, John F; Turner, Matthew J; Schloss, Jennifer M; Glenn, David R; Song, Yuyu; Lukin, Mikhail D; Park, Hongkun; Walsworth, Ronald L

2016-12-06

Magnetic fields from neuronal action potentials (APs) pass largely unperturbed through biological tissue, allowing magnetic measurements of AP dynamics to be performed extracellularly or even outside intact organisms. To date, however, magnetic techniques for sensing neuronal activity have either operated at the macroscale with coarse spatial and/or temporal resolution-e.g., magnetic resonance imaging methods and magnetoencephalography-or been restricted to biophysics studies of excised neurons probed with cryogenic or bulky detectors that do not provide single-neuron spatial resolution and are not scalable to functional networks or intact organisms. Here, we show that AP magnetic sensing can be realized with both single-neuron sensitivity and intact organism applicability using optically probed nitrogen-vacancy (NV) quantum defects in diamond, operated under ambient conditions and with the NV diamond sensor in close proximity (∼10 µm) to the biological sample. We demonstrate this method for excised single neurons from marine worm and squid, and then exterior to intact, optically opaque marine worms for extended periods and with no observed adverse effect on the animal. NV diamond magnetometry is noninvasive and label-free and does not cause photodamage. The method provides precise measurement of AP waveforms from individual neurons, as well as magnetic field correlates of the AP conduction velocity, and directly determines the AP propagation direction through the inherent sensitivity of NVs to the associated AP magnetic field vector.

Ovariectomy and overeating palatable, energy-dense food increase subcutaneous adipose tissue more than intra-abdominal adipose tissue in rats

PubMed Central

2011-01-01

Background Menopause is associated with increased adiposity, especially increased deposition of intra-abdominal (IA) adipose tissue (AT). This differs from common or 'dietary' obesity, i.e., obesity apparently due to environmentally stimulated overeating, in which IAAT and subcutaneous (S) AT increase in similar proportions. The effect of menopause on adiposity is thought to be due to the decreased secretion of ovarian estrogens. Ovariectomy in rats and other animals is a commonly used model of menopause. It is well known that ovariectomy increases adiposity and that this can be reversed by estradiol treatment, but whether ovariectomy selectively increases IAAT has not been measured directly. Therefore, we used micro-computed tomography (microCT) to investigate this question in both chow-fed and dietary-obese rats. Methods Ovariectomized, ovariectomized and estradiol treated, and sham-operated (intact) rats were fed chow or chow plus Ensure (Abbott Nutrition; n = 7/group). Total (T) AT, IAAT and SAT were measured periodically by microCT. Regional distribution of AT was expressed as IAAT as a percentage of TAT (%IAAT). Excesses in these measures were calculated with respect to chow-fed intact rats to control for normal maturational changes. Chemical analysis of fat was done in chow-fed intact and ovariectomized rats at study end. Data were analyzed by t-tests and planned comparisons. Results Body mass, TAT, total fat mass, fat-free body mass, and %IAAT all increased in chow-fed intact rats during the 41 d study. In chow-fed rats, ovariectomy increased excess body mass, TAT, fat mass, fat-free body mass, and SAT, but had little effect on IAAT, in chow-fed rats, leading to a decrease in %IAAT. Ensure feeding markedly increased SAT, IAAT and TAT and did not significantly affect %IAAT. Ovariectomy had similar effects in Ensure-fed rats as in chow-fed rats, although less statistically reliable. Estradiol treatment prevented all the effects of ovariectomy. Conclusions Both ovariectomy in rats and menopause are associated with increased TAT. After ovariectomy, fat is preferentially deposited as SAT and lean body mass increases, whereas after menopause fat is preferentially deposited as IAAT and lean body mass decreases. These opposite effects of ovariectomy and menopause on regional AT distribution and lean body mass indicate that ovariectomy in rats is not a homologous model of menopause-associated changes in body composition that should be used with great caution in investigations of adiposity-related diseases. PMID:21569336

Quantitative analysis of transcranial and intraparenchymal light penetration in human cadaver brain tissue.

PubMed

Tedford, Clark E; DeLapp, Scott; Jacques, Steven; Anders, Juanita

2015-04-01

Photobiomodulation (PBM) also known as low-level light therapy has been used successfully for the treatment of injury and disease of the nervous system. The use of PBM to treat injury and diseases of the brain requires an in-depth understanding of light propagation through tissues including scalp, skull, meninges, and brain. This study investigated the light penetration gradients in the human cadaver brain using a Transcranial Laser System with a 30 mm diameter beam of 808 nm wavelength light. In addition, the wavelength-dependence of light scatter and absorbance in intraparenchymal brain tissue using 660, 808, and 940 nm wavelengths was investigated. Intact human cadaver heads (n = 8) were obtained for measurement of light propagation through the scalp/skull/meninges and into brain tissue. The cadaver heads were sectioned in either the transverse or mid-sagittal. The sectioned head was mounted into a cranial fixture with an 808 nm wavelength laser system illuminating the head from beneath with either pulsed-wave (PW) or continuous-wave (CW) laser light. A linear array of nine isotropic optical fibers on a 5 mm pitch was inserted into the brain tissue along the optical axis of the beam. Light collected from each fiber was delivered to a multichannel power meter. As the array was lowered into the tissue, the power from each probe was recorded at 5 mm increments until the inner aspect of the dura mater was reached. Intraparenchymal light penetration measurements were made by delivering a series of wavelengths (660, 808, and 940 nm) through a separate optical fiber within the array, which was offset from the array line by 5 mm. Local light penetration was determined and compared across the selected wavelengths. Unfixed cadaver brains provide good anatomical localization and reliable measurements of light scatter and penetration in the CNS tissues. Transcranial application of 808 nm wavelength light penetrated the scalp, skull, meninges, and brain to a depth of approximately 40 mm with an effective attenuation coefficient for the system of 2.22 cm(-1) . No differences were observed in the results between the PW and CW laser light. The intraparenchymal studies demonstrated less absorption and scattering for the 808 nm wavelength light compared to the 660 or 940 nm wavelengths. Transcranial light measurements of unfixed human cadaver brains allowed for determinations of light penetration variables. While unfixed human cadaver studies do not reflect all the conditions seen in the living condition, comparisons of light scatter and penetration and estimates of fluence levels can be used to establish further clinical dosing. The 808 nm wavelength light demonstrated superior CNS tissue penetration. © 2015 Wiley Periodicals, Inc.

Protein vs electrolytes and all of the Starling forces.

PubMed

Peters, R M; Hargens, A R

1981-10-01

Hemodilution-induced reductions of the intravascular protein concentration in patients and experimental animals with intact capillaries do not lead to pulmonary edema, despite significant increases in the amount of extravascular water in the systemic interstitial space. The protective factors are a drop in the extravascular concentration of protein, a rise in interstitial tissue pressure, and an increase in lymph flow. If the capillary endothelium is damaged, protein leaks into the extravascular space, and protein infusion has a diminished effect on fluid exchange across the capillary. Whether capillaries are intact or injured, prevention of increases in capillary hydrostatic pressure is the most important factor in preventing pulmonary edema. Administration of hypertonic fluids may provide a useful method of limiting total fluid infusion and reducing cell swelling after blood loss.

Mineral Ion Contents and Cell Transmembrane Electropotentials of Pea and Oat Seedling Tissue 1

PubMed Central

Higinbotham, N.; Etherton, Bud; Foster, R. J.

1967-01-01

The relationships of concentration gradients to electropotential gradients resulting from passive diffusion processes, after equilibration, are described by the Nernst equation. The primary criterion for the hypothesis that any given ion is actively transported is to establish that it is not diffusing passively. A test was made of how closely the Nernst equation describes the electrochemical equilibrium in seedling tissues. Segments of roots and epicotyl internodes of pea (Pisum sativum var. Alaska) and of roots and coleoptiles of oat (Avena sativa var. Victory) seedlings were immersed and shaken in defined nutrient solutions containing eight major nutrients (K+, Na+, Ca2+, Mg2+, Cl−, NO3−, H2PO4− and SO42−) at 1-fold and 10-fold concentrations. The tissue content of each ion was assayed at 0, 8, 24, and 48 hours. A near-equilibrium condition was approached by roots for most ions; however, the segments of shoot tissue generally continued to show a net accumulation of some ions, mainly K+ and NO3−. Only K+ approached a reasonable fit to the Nernst equation and this was true for the 1-fold concentration but not the 10-fold. The data suggest that for Na+, Mg2+, and Ca2+ the electrochemical gradient is from the external solution to the cell interior; thus passive diffusion should be in an inward direction. Consequently, some mechanism must exist in plant tissue either to exclude these cations or to extrude them (e.g., by an active efflux pump). For each of the anions the electrochemical gradient is from the tissue to the solution; thus an active influx pump for anions seems required. Root segments approach ionic equilibrium with the solution concentration in which the seedlings were grown. Segments of shoot tissue, however, are far removed from such equilibration. Thus in the intact seedling the extracellular (wall space) fluid must be very different from that of the nutrient solution bathing the segments; it would appear that the root is the site of regulation of ion uptake in the intact plant although other correlative mechanisms may be involved. PMID:16656483

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue.

PubMed

Boyer, J S; Wu, G

1978-01-01

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials (ψ w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. ψ wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, ψ w was similar to the water potential of the environment when cell enlargement was not occurring. The low ψ w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (L'p). Despite the low L'p of growing cells, L'p decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and L'p increased when auxin was resupplied exogenously. The auxin-induced increase in L'p was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in L'p appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to L'p remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.

Characterization of Meteorites by Focused Ion Beam Sectioning: Recent Applications to CAIs and Primitive Meteorite Matrices

NASA Technical Reports Server (NTRS)

Christoffersen, Roy; Keller, Lindsay P.; Han, Jangmi; Rahman, Zia; Berger, Eve L.

2015-01-01

Focused ion beam (FIB) sectioning has revolutionized preparation of meteorite samples for characterization by analytical transmission electron microscopy (TEM) and other techniques. Although FIB is not "non-destructive" in the purest sense, each extracted section amounts to no more than nanograms (approximately 500 cubic microns) removed intact from locations precisely controlled by SEM imaging and analysis. Physical alteration of surrounding material by ion damage, fracture or sputter contamination effects is localized to within a few micrometers around the lift-out point. This leaves adjacent material intact for coordinate geochemical analysis by SIMS, microdrill extraction/TIMS and other techniques. After lift out, FIB sections can be quantitatively analyzed by electron microprobe prior to final thinning, synchrotron x-ray techniques, and by the full range of state-of-the-art analytical field-emission scanning transmission electron microscope (FE-STEM) techniques once thinning is complete. Multiple meteorite studies supported by FIB/FE-STEM are currently underway at NASA-JSC, including coordinated analysis of refractory phase assemblages in CAIs and fine-grained matrices in carbonaceous chondrites. FIB sectioning of CAIs has uncovered epitaxial and other overgrowth relations between corundum-hibonite-spinel consistent with hibonite preceding corundum and/or spinel in non-equilibrium condensation sequences at combinations of higher gas pressures, dust-gas enrichments or significant nebular transport. For all of these cases, the ability of FIB to allow for coordination with spatially-associated isotopic data by SIMS provides immense value for constraining the formation scenarios of the particular CAI assemblage. For carbonaceous chondrites matrix material, FIB has allowed us to obtain intact continuous sections of the immediate outer surface of Murchison (CM2) after it has been experimentally ion processed to simulate solar wind space weathering. The surface amorphization and loss of OH produced by the irradiation provides important clues regarding space weathering on primitive asteroids such as the OSIRIS-Rex target 101955 Bennu.

Benefits of Ilizarov automated bone distraction for nerves and articular cartilage in experimental leg lengthening.

PubMed

Shchudlo, Nathalia; Varsegova, Tatyana; Stupina, Tatyana; Shchudlo, Michael; Saifutdinov, Marat; Yemanov, Andrey

2017-09-18

To determine peculiarities of tissue responses to manual and automated Ilizarov bone distraction in nerves and articular cartilage. Twenty-nine dogs were divided in two experimental groups: Group M - leg lengthening with manual distraction (1 mm/d in 4 steps), Group A - automated distraction (1 mm/d in 60 steps) and intact group. Animals were euthanized at the end of distraction, at 30 th day of fixation in apparatus and 30 d after the fixator removal. M-responses in gastrocnemius and tibialis anterior muscles were recorded, numerical histology of peroneal and tibialis nerves and knee cartilage semi-thin sections, scanning electron microscopy and X-ray electron probe microanalysis were performed. Better restoration of M-response amplitudes in leg muscles was noted in A-group. Fibrosis of epineurium with adipocytes loss in peroneal nerve, subperineurial edema and fibrosis of endoneurium in some fascicles of both nerves were noted only in M-group, shares of nerve fibers with atrophic and degenerative changes were bigger in M-group than in A-group. At the end of experiment morphometric parameters of nerve fibers in peroneal nerve were comparable with intact nerve only in A-group. Quantitative parameters of articular cartilage (thickness, volumetric densities of chondrocytes, percentages of isogenic clusters and empty cellular lacunas, contents of sulfur and calcium) were badly changed in M-group and less changed in A-group. Automated Ilizarov distraction is more safe method of orthopedic leg lengthening than manual distraction in points of nervous fibers survival and articular cartilage arthrotic changes.

Benefits of Ilizarov automated bone distraction for nerves and articular cartilage in experimental leg lengthening

PubMed Central

Shchudlo, Nathalia; Varsegova, Tatyana; Stupina, Tatyana; Shchudlo, Michael; Saifutdinov, Marat; Yemanov, Andrey

2017-01-01

AIM To determine peculiarities of tissue responses to manual and automated Ilizarov bone distraction in nerves and articular cartilage. METHODS Twenty-nine dogs were divided in two experimental groups: Group M - leg lengthening with manual distraction (1 mm/d in 4 steps), Group A - automated distraction (1 mm/d in 60 steps) and intact group. Animals were euthanized at the end of distraction, at 30th day of fixation in apparatus and 30 d after the fixator removal. M-responses in gastrocnemius and tibialis anterior muscles were recorded, numerical histology of peroneal and tibialis nerves and knee cartilage semi-thin sections, scanning electron microscopy and X-ray electron probe microanalysis were performed. RESULTS Better restoration of M-response amplitudes in leg muscles was noted in A-group. Fibrosis of epineurium with adipocytes loss in peroneal nerve, subperineurial edema and fibrosis of endoneurium in some fascicles of both nerves were noted only in M-group, shares of nerve fibers with atrophic and degenerative changes were bigger in M-group than in A-group. At the end of experiment morphometric parameters of nerve fibers in peroneal nerve were comparable with intact nerve only in A-group. Quantitative parameters of articular cartilage (thickness, volumetric densities of chondrocytes, percentages of isogenic clusters and empty cellular lacunas, contents of sulfur and calcium) were badly changed in M-group and less changed in A-group. CONCLUSION Automated Ilizarov distraction is more safe method of orthopedic leg lengthening than manual distraction in points of nervous fibers survival and articular cartilage arthrotic changes. PMID:28979852

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa

PubMed Central

Thier, Katharina; Petermann, Philipp; Rahn, Elena; Rothamel, Daniel; Bloch, Wilhelm

2017-01-01

ABSTRACT Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo, we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor. PMID:28878080

Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa.

PubMed

Thier, Katharina; Petermann, Philipp; Rahn, Elena; Rothamel, Daniel; Bloch, Wilhelm; Knebel-Mörsdorf, Dagmar

2017-11-15

Oral mucosa is one of the main target tissues of the human pathogen herpes simplex virus 1 (HSV-1). How the virus overcomes the protective epithelial barriers and penetrates the tissue to reach its receptors and initiate infection is still unclear. Here, we established an ex vivo infection assay with human oral mucosa that allows viral entry studies in a natural target tissue. The focus was on the susceptibility of keratinocytes in the epithelium and the characterization of cellular receptors that mediate viral entry. Upon ex vivo infection of gingiva or vestibular mucosa, we observed that intact human mucosa samples were protected from viral invasion. In contrast, the basal layer of the oral epithelium was efficiently invaded once the connective tissue and the basement membrane were removed. Later during infection, HSV-1 spread from basal keratinocytes to upper layers, demonstrating the susceptibility of the stratified squamous epithelium to HSV-1. The analysis of potential receptors revealed nectin-1 on most mucosal keratinocytes, whereas herpesvirus entry mediator (HVEM) was found only on a subpopulation of cells, suggesting that nectin-1 acts as primary receptor for HSV-1 in human oral mucosa. To mimic the supposed entry route of HSV-1 via microlesions in vivo , we mechanically wounded the mucosa prior to infection. While we observed a limited number of infected keratinocytes in some wounded mucosa samples, other samples showed no infected cells. Thus, we conclude that mechanical wounding of mucosa is insufficient for the virus to efficiently overcome epithelial barriers and to make entry-mediating receptors accessible. IMPORTANCE To invade the target tissue of its human host during primary infection, herpes simplex virus (HSV) must overcome the epithelial barriers of mucosa, skin, or cornea. For most viruses, the mechanisms underlying the invasion into the target tissues of their host organism are still open. Here, we established an ex vivo infection model of human oral mucosa to explore how HSV can enter its target tissue. Our results demonstrate that intact mucosa samples and even compromised tissue allow only very limited access of HSV to keratinocytes. Detailed understanding of barrier functions is an essential precondition to unravel how HSV bypasses the barriers and approaches its receptors in tissue and why it is beneficial for the virus to use a cell-cell adhesion molecule, such as nectin-1, as a receptor. Copyright © 2017 American Society for Microbiology.

Fat Metabolism in Higher Plants XXVI. Biosynthesis of Fatty Acids in Tissues of Developing Seeds and Germinating Seedlings of Safflower (Carthamus tinctorius L.) 1

PubMed Central

McMahon, Vern; Stumpf, P. K.

1966-01-01

The capacity of both developing seeds and germinating seedlings of safflower for the incorporation of acetate-C14 into long-chain fatty acids is examined. Intact tissue of the developing seed shows a low rate of acetate incorporation into fatty acid initially but between the tenth and twenty-fifth day after flowering the tissue has a high rate of synthesis, in particular with respect to the unsaturated fatty acids. Centrifuged fractionation of homogenates of this developmental tissue yielded several active fractions, the most active being the PI fraction consisting mostly of plastids. Cofactor requirements and pH effects are examined. Germinating tissue shows a more uniform capacity for synthesis of fatty acids since there is no marked change in synthetic capacity. The newly synthesized fatty acids are consistently palmitic, stearic, and oleic acid. No linoleic synthesis could be detected. The most active fraction of cell-free preparation of germinating tissue is the plastid fraction (PI), similar to what was formed with developing tissue. PMID:5904587

Absorption, Distribution, Metabolism, and Excretion of the Androgen Receptor Inhibitor Enzalutamide in Rats and Dogs.

PubMed

Ohtsu, Yoshiaki; Gibbons, Jacqueline A; Suzuki, Katsuhiro; Fitzsimmons, Michael E; Nozawa, Kohei; Arai, Hiroshi

2017-08-01

Enzalutamide is an androgen receptor inhibitor that has been approved in several countries. Absorption, distribution, metabolism, and excretion (ADME) data in animals would facilitate understanding of the efficacy and safety profiles of enzalutamide, but little information has been reported in public. The purpose of this study was to clarify the missing ADME profile in animals. ADME of 14 C-enzalutamide after oral administration as Labrasol solution were investigated in non-fasted male Sprague-Dawley rats and beagle dogs. Plasma concentrations of 14 C-enzalutamide peaked in rats and dogs at 6-8 h after a single oral administration. In most tissues, radioactivity concentration peaked at 4 h after administration. Excluding the gastrointestinal tract, tissues with the highest concentration of radioactivity were liver, fat, and adrenal glands. The tissue concentrations of radioactivity declined below the limit of quantitation or <0.89 % of maximum concentration by 168 h post-dose. Two known metabolites (M1 and M2) and at least 15 novel possible metabolites were detected in this study. M1 was the most abundant metabolite in both rats and dogs. Unchanged drug was a minor component in excreta. In intact rats, the mean urinary and fecal excretion of radioactivity accounted for 44.20 and 49.80 % of administered radioactivity, respectively. In intact dogs, mean urinary and fecal excretion was 62.00 and 22.30 % of the administered radioactivity, respectively. Rapid oral absorption was observed in rats and dogs when 14 C-enzalutamide was administered as Labrasol solution. Tissue distribution in rats was clarified. The elimination of enzalutamide is mediated primarily by metabolism. Species differences were observed in excretion route.

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model.

PubMed

Bell, Angela D; Hurtig, Mark B; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D

2017-10-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan-NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro-computed tomography after 1 day ( n = 1) and 6 months ( n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage.

Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue

PubMed Central

Roy, Sashwati; Patel, Darshan; Khanna, Savita; Gordillo, Gayle M.; Biswas, Sabyasachi; Friedman, Avner; Sen, Chandan K.

2007-01-01

Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses. PMID:17728400

Effect of a Rapidly Degrading Presolidified 10 kDa Chitosan/Blood Implant and Subchondral Marrow Stimulation Surgical Approach on Cartilage Resurfacing in a Sheep Model

PubMed Central

Bell, Angela D.; Hurtig, Mark B.; Quenneville, Eric; Rivard, Georges-Étienne; Hoemann, Caroline D.

2016-01-01

Objective This study tested the hypothesis that presolidified chitosan-blood implants are retained in subchondral bone channels perforated in critical-size sheep cartilage defects, and promote bone repair and hyaline-like cartilage resurfacing versus blood implant. Design Cartilage defects (10 × 10 mm) with 3 bone channels (1 drill, 2 Jamshidi biopsy, 2 mm diameter), and 6 small microfracture holes were created bilaterally in n = 11 sheep knee medial condyles. In one knee, 10 kDa chitosan–NaCl/blood implant (presolidified using recombinant factor VIIa or tissue factor), was inserted into each drill and Jamshidi hole. Contralateral knee defects received presolidified whole blood clot. Repair tissues were assessed histologically, biochemically, biomechanically, and by micro–computed tomography after 1 day (n = 1) and 6 months (n = 10). Results Day 1 defects showed a 60% loss of subchondral bone plate volume fraction along with extensive subchondral hematoma. Chitosan implant was resident at day 1, but had no effect on any subsequent repair parameter compared with blood implant controls. At 6 months, bone defects exhibited remodeling and hypomineralized bone repair and were partly resurfaced with tissues containing collagen type II and scant collagen type I, 2-fold lower glycosaminoglycan and fibril modulus, and 4.5-fold higher permeability compared with intact cartilage. Microdrill holes elicited higher histological ICRS-II overall assessment scores than Jamshidi holes (50% vs. 30%, P = 0.041). Jamshidi biopsy holes provoked sporadic osteonecrosis in n = 3 debrided condyles. Conclusions Ten kilodalton chitosan was insufficient to improve repair. Microdrilling is a feasible subchondral marrow stimulation surgical approach with the potential to elicit poroelastic tissues with at least half the compressive modulus as intact articular cartilage. PMID:28934884

Are specific gene expressions of extracellular matrix and nucleus pulposus affected by primary cell cultures prepared from intact or degenerative intervertebral disc tissues?

PubMed

Karaarslan, Numan; Yilmaz, Ibrahim; Ozbek, Hanefi; Sirin Yasar, Duygu; Kaplan, Necati; Akyuva, Yener; Gonultas, Aylin; Ates, Ozkan

2018-01-22

In this scientific research project, the researchers aimed to determine the gene expression patterns of nucleus pulposus (NP) in cell cultures obtained from degenerated or intact tissues. Whereas 12 of the cases were diagnosed with lumbar disc hernia and had undergone lumbar microdiscectomy, 12 cases had undergone traumatic intervertebral discectomy and corpectomy, along with discectomy after spinal trauma. NP-specific markers and gene expressions of the reagents of the extracellular matrix in the experimental setup were tested at the 0th, 24th, and 48th hours by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Visual evaluations were simultaneously made in all samples using invert and fluorescence microscopy. Vitality and proliferation analyses were evaluated by UV spectrophotometer. As a method of statistical evaluation, Spearman was used for categorical variants, and the Pearson correlation was used for variants with numerical and plain distribution. No association was found either between the tissue type and times (r=0.000; p=1.000) or between the region that the tissue was obtained from and hypoxia transcription factor-1 alpha (HIF-1α) gene expression (r=0.098; p=0.245). There was no correlation between cell proliferation and chondroadherin (CHAD) expression or between type II collagen (COL2A1) and CHAD gene expressions. It was found that CHAD and HIF-1α gene expressions and HIF-1α and COL2A1 gene expressions affected cell proliferation. Cell culture setups are of paramount importance because they may influence the pattern of changes in the gene expressions of the cells used in these setups.

Growth of Listeria spp. in shredded cabbage is enhanced by a mild heat treatment.

PubMed

Ells, Timothy C; Truelstrup Hansen, Lisbeth

2010-03-01

Mild thermal processing can enhance the shelf life of cut fruits and vegetables by delaying the onset of spoilage and preserving the organoleptic properties of shredded cabbage. However, food safety issues related to this process have not been fully investigated. Therefore, the survival and growth of Listeria spp. on cabbage treated in this manner was examined. Experimentally, 24 strains of Listeria spp. (including L. monocytogenes) were inoculated onto cut and intact cabbage tissues and stored at 5 degrees C. All strains on intact tissues exhibited a moderate decline in numbers (up to 1.0 log CFU/cm(2)) over the 28-day storage period. Conversely, cut tissue supported growth of most strains during the first 7 to 14 days of incubation with maximum increases of 1.2 log CFU/cm(2). Subsequently, the survival or growth on heat-treated (50 degrees C for 3 min) and untreated shredded cabbage of four L. monocytogenes and four nonpathogenic Listeria spp. strains were compared during storage for 21 days at 5 degrees C. Growth on untreated shred for all strains was similar to the results observed on cut tissue with a maximum increase of approximately 1.0 log CFU/g. However, in the heat-treated cabbage shred all strains displayed a rapid increase in growth (up to 2.5 log CFU/g) during the first 7 days of incubation, which may be indicative of the destruction of an endogenous growth-inhibiting compound within the cabbage. In conclusion, this study shows that mild thermal treatments of cut cabbage may promote pathogen growth if other inimical barriers are not implemented downstream of the thermal treatment.

A factorial design to identify process parameters affecting whole mechanically disrupted rat pancreata in a perfusion bioreactor.

PubMed

Sharp, Jamie; Spitters, Tim Wgm; Vermette, Patrick

2018-03-01

Few studies report whole pancreatic tissue culture, as it is a difficult task using traditional culture methods. Here, a factorial design was used to investigate the singular and combinational effects of flow, dissolved oxygen concentration (D.O.) and pulsation on whole mechanically disrupted rat pancreata in a perfusion bioreactor. Whole rat pancreata were cultured for 72 h under defined bioreactor process conditions. Secreted insulin was measured and histological (haematoxylin and eosin (H&E)) as well as immunofluorescent insulin staining were performed and quantified. The combination of flow and D.O. had the most significant effect on secreted insulin at 5 h and 24 h. The D.O. had the biggest effect on tissue histological quality, and pulsation had the biggest effect on the number of insulin-positive structures. Based on the factorial design analysis, bioreactor conditions using high flow, low D.O., and pulsation were selected to further study glucose-stimulated insulin secretion. Here, mechanically disrupted rat pancreata were cultured for 24 h under these bioreactor conditions and were then challenged with high glucose concentration for 6 h and high glucose + IBMX (an insulin secretagogue) for a further 6 h. These cultures secreted insulin in response to high glucose concentration in the first 6 h, however stimulated-insulin secretion was markedly weaker in response to high glucose concentration + IBMX thereafter. After this bioreactor culture period, higher tissue metabolic activity was found compared to that of non-bioreacted static controls. More insulin- and glucagon-positive structures, and extensive intact endothelial structures were observed compared to non-bioreacted static cultures. H&E staining revealed more intact tissue compared to static cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:432-444, 2018. © 2017 American Institute of Chemical Engineers.

Voluntary wheel running improves adipose tissue immunometabolism in ovariectomized low-fit rats.

PubMed

Zidon, Terese M; Park, Young-Min; Welly, Rebecca J; Woodford, Makenzie L; Scroggins, Rebecca J; Britton, Steven L; Koch, Lauren G; Booth, Frank W; Padilla, Jaume; Kanaley, Jill A; Vieira-Potter, Victoria J

2018-01-02

Loss of ovarian hormones is associated with increased adiposity, white adipose tissue (WAT) inflammation, and insulin resistance (IR). Previous work demonstrated ovariectomized (OVX) rats bred for high aerobic fitness (HCR) are protected against weight gain and IR compared to rats bred for low aerobic fitness (LCR) yet wheel running prevents OVX-induced IR in LCR rats. The purpose of this study was to determine whether adipose tissue immunometabolic characteristics from female HCR and LCR rats differs before or after OVX, and whether wheel running mitigates OVX-induced adipose tissue immunometabolic changes in LCR rats. Female OVX HCR and LCR rats were all fed a high fat diet (HFD) (n = 7-8/group) and randomized to either a running wheel or remain sedentary for 11 weeks. Ovary-intact rats (n = 7-12/group) were fed a standard chow diet with no wheel. Ovary-intact LCR rats had a greater visceral WAT inflammatory profile compared to HCR. Following OVX, sedentary LCR rats had greater serum leptin (p<0.001) and WAT inflammation (p<0.05) than sedentary HCR. Wheel running normalized the elevated serum leptin and reduced both visceral (p<0.05) and subcutaneous (p<0.03) WAT inflammatory markers in the LCR rats. Paradoxically, wheel running increased some markers of WAT inflammation in OVX HCR rats (p<0.05), which correlated with observed weight gain. Taken together, HCR rats appear to have a healthier WAT immune and metabolic profile compared to LCR, even following OVX. Wheel running improves WAT health in previously sedentary LCR rats. On the other hand, increased WAT inflammation is associated with adiposity gain despite a high volume of wheel running in HCR rats.

Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

PubMed Central

Hanson, Laura K.; Slater, Jacquelyn S.; Karabekian, Zaruhi; Virgin, Herbert W.; Biron, Christine A.; Ruzek, Melanie C.; van Rooijen, Nico; Ciavarra, Richard P.; Stenberg, Richard M.; Campbell, Ann E.

1999-01-01

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection. PMID:10364349

Cell-level temperature distributions in skeletal muscle post spinal cord injury as related to deep tissue injury.

PubMed

Ruschkewitz, Yael; Gefen, Amit

2010-02-01

Deep tissue injury (DTI) is a severe pressure ulcer, which initiates in skeletal muscle tissue under intact skin. Patients with spinal cord injury (SCI) are especially vulnerable to DTI, due to their impaired motosensory capacities. The underlying mechanisms that lead to DTI are, however, still poorly understood. This study focuses on cell-level temperature distributions in muscles of patients with SCI, which typically contain thinner muscle fibers and fewer capillaries. It has been shown previously by our group that ischemic muscles of rat models of DTI cool down mildly and locally, which is very likely to slow the diffusivity of metabolites in the ischemic regions. However, it is unclear how these temperature decreases affect diffusivity at the scale of individual muscle cells in the microanatomy of SCI patients. We hypothesize that a 2 degrees C drop in the temperature of inflowing capillary blood, as shown in our animal studies, has a substantial effect on lowering the diffusivity of metabolites in skeletal muscle, but the pathological microanatomy in the chronic phase of SCI is less dominant in affecting the local temperatures in and around muscle cells. In order to test this hypothesis, two-dimensional finite element (FE) models of cross sections through the microanatomy of muscle tissue were developed using COMSOL Multiphysics software for normal and SCI muscles. The models included muscle cells, extracellular matrix (ECM), and capillaries, each with its own geometrical, thermal, and heat production properties. The SCI model configuration specifically included reduced cross section of myofibrils in favor of more ECM, less capillaries, and decreased blood inflow rate. After a 20-s heat transfer simulation, it was found that temperatures around the cells of the SCI muscle were approximately 2 degrees C lower than that in the normal muscle, that is, heat production from the muscle cell metabolism did not compensate for the lower inflowing blood temperature in the SCI model. We conclude that the temperature and rate of inflowing capillary blood are the dominant factors determining the localized temperatures in the microarchitecture of an ischemic SCI muscle tissue. The altered SCI microanatomy was shown to be less influential. Taken together with the Stokes-Einstein theory, our results indicate that diffusivity of metabolites would be approximately 50% less around the cells of SCI muscle due to local cooling, which is yet another factor compromising tissue viability in the patients with SCI.

Infection of lymphoid tissues in the macaque upper respiratory tract contributes to the emergence of transmissible measles virus.

PubMed

Ludlow, Martin; de Vries, Rory D; Lemon, Ken; McQuaid, Stephen; Millar, Emma; van Amerongen, Geert; Yüksel, Selma; Verburgh, R Joyce; Osterhaus, Albert D M E; de Swart, Rik L; Duprex, W Paul

2013-09-01

Measles virus (MV), a member of the family Paramyxoviridae, remains a major cause of morbidity and mortality in the developing world. MV is spread by aerosols but the mechanism(s) responsible for the high transmissibility of MV are largely unknown. We previously infected macaques with enhanced green fluorescent protein-expressing recombinant MV and euthanized them at a range of time points. In this study a comprehensive pathological analysis has been performed of tissues from the respiratory tract around the peak of virus replication. Isolation of virus from nose and throat swab samples showed that high levels of both cell-associated and cell-free virus were present in the upper respiratory tract. Analysis of tissue sections from lung and primary bronchus revealed localized infection of epithelial cells, concomitant infiltration of MV-infected immune cells into the epithelium and localized shedding of cells or cell debris into the lumen. While high numbers of MV-infected cells were present in the tongue, these were largely encapsulated by intact keratinocyte cell layers that likely limit virus transmission. In contrast, the integrity of tonsillar and adenoidal epithelia was disrupted with high numbers of MV-infected epithelial cells and infiltrating immune cells present throughout epithelial cell layers. Disruption was associated with large numbers of MV-infected cells or cell debris 'spilling' from epithelia into the respiratory tract. The coughing and sneezing response induced by disruption of the ciliated epithelium, leading to the expulsion of MV-infected cells, cell debris and cell-free virus, contributes to the highly infectious nature of MV.

Self-etching adhesive on intact enamel, with and without pre-etching.

PubMed

Devarasa, G M; Subba Reddy, V V; Chaitra, N L; Swarna, Y M

2012-05-01

Bond strengths of composite resin to enamel using self-etch adhesive (SEA) Clearfil SE bond system on intact enamel and enamel pre-etched with phosphoric acid were compared. The objective was to determine if the pre-etching would increase the bond strengths of the SEA systems to intact enamel and to evaluate the effect of pre-etching on bond formation of self-etch adhesives on intact enamel. Labial surfaces of 40 caries free permanent upper central and lateral incisors were cleaned, sectioned of their roots. All specimens were mounted on acrylic block and divided randomly into four groups. In two groups the application of self-etch adhesive, Clearfil SE bond was carried as per manufacturer's instructions, composite cylinders were built, whereas in the other two groups, 37% phosphoric acid etching was done before the application of self-etching adhesives. Then the resin tags were analyzed using scanning electron microscope and shear bond strength was measured using Instron universal testing machine. When phosphoric acid was used, there was significant increase in the depth of penetration of resin tags and in the Shear Bond Strength of composite to enamel. The results indicate that out of both treatment groups, pre-etching the intact enamel with 37% phosphoric acid resulted in formation of longer resin tags and higher depth of penetration of resin tags of the Clearfil SE bond, and attaining higher bond strength of the Clearfil SE bond to intact enamel. Copyright © 2011 Wiley Periodicals, Inc.

Full face transplant: the first case report.

PubMed

Barret, Juan P; Gavaldà, Joan; Bueno, Javier; Nuvials, Xavier; Pont, Teresa; Masnou, Nuria; Colomina, Maria J; Serracanta, Jordi; Arno, Anna; Huguet, Pere; Collado, Jose M; Salamero, Pere; Moreno, Carlos; Deulofeu, Roser; Martínez-Ibáñez, Vicenç

2011-08-01

Since 2005, 11 human face transplants have been performed. In each, varying amounts of tissue have been transplanted. Herein we report a "full" face transplant including all intact aesthetic and functional units. On March 27, 2010, we performed a full face transplant, including all the soft tissues and part of the underlaying bony structure, at the University Hospital Vall d'Hebron, Barcelona, Spain. The donor was a 41-year-old male, who died from a massive brain hemorrhage. The recipient was a 30-year-old male with a severe facial deformity caused by a ballistic trauma in 2005. Harvest and subsequent implant took 24 hours. The patient received initial induction (Thymoglobulin 2 mg/kg/iv; Prednisone 1 gm/iv) and maintenance (Prednisone 1 mg/kg/24hours, Tacrolimus 10-15 ng/mL/PO, and Mycophenolate mofetil 2g/daily/PO) immunosuppression and Infection prophylaxis (Valganciclovir and Co-trimoxazole). There were no intraoperative complications. Postoperative complications included; venous anastomoses thrombosis, acute oro-cutaneous fistula, right parotid sialocele and 2 acute rejection episodes, which were resolved by revision of the anastomosis, profuse irrigation and immunotherapy adjustment, respectively. The patient was discharged from the hospital at 4 months posttransplant with; near-total sensation and partial-motor recovery, no psychological complications and excellent acceptance of his new facial appearance. The early success described in this case report demonstrates the technical and clinical feasibility of transplanting all the tissues of the with all its aesthetic and functional units intact.

MALDI FTICR IMS of Intact Proteins: Using Mass Accuracy to Link Protein Images with Proteomics Data

NASA Astrophysics Data System (ADS)

Spraggins, Jeffrey M.; Rizzo, David G.; Moore, Jessica L.; Rose, Kristie L.; Hammer, Neal D.; Skaar, Eric P.; Caprioli, Richard M.

2015-06-01

MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here, we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (~75,000 at m/z 5000) and accuracy (<5ppm) for proteins up to ~12kDa, enabling identification based on correlation with LC-MS/MS proteomics data. Analysis of rat brain tissue was performed as a proof-of-concept highlighting the capabilities of this approach by imaging and identifying a number of proteins including N-terminally acetylated thymosin β4 ( m/z 4,963.502, 0.6ppm) and ATP synthase subunit ɛ ( m/z 5,636.074, -2.3ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 ( m/z 10,164.03, -2.1ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 - M37O/C42O3 ( m/z 10228.00, -2.6ppm) was found to co-localize with bacterial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin's roll in nutritional immunity.

Laparoscopic retroperitoneal lymph-node dissection with the waterjet is technically feasible and safe in testis-cancer patient.

PubMed

Corvin, Stefan; Sturm, Wolfgang; Schlatter, Evelin; Anastasiadis, Aristotelis; Kuczyk, Markus; Stenzl, Arnulf

2005-09-01

The acceptance of open retroperitoneal lymph node dissection (RPLND) for stage I and II nonseminomatous testicular cancer has decreased because of the intraoperative and postoperative morbidity of the procedure. Laparoscopic RPLND is a minimally invasive and safe alternative for low-stage germ-cell tumors. It is, however, technically demanding and should therefore be performed only in experienced centers. The purpose of the present study was to evaluate the waterjet technique for laparoscopic RPLND. A series of 18 patients with clinical stage I testis cancer (group A) and 7 patients who had received chemotherapy for stage II disease (group B) underwent laparoscopic RPLND at our institution. The procedure was performed identically to the open approach using the modified template according to Weissbach and associates. The waterjet was used for removal of lymphatic tissue from the aorta and the vena cava, as well as from the sympathetic trunk. The operation was completed in all patients without conversion to open surgery. The mean operating time was 232 +/- 48 minutes. The waterjet was able to remove lymphatic tissue easily and atraumatically. At pressures of 20 bar, the lymph-node capsule remained completely intact, thus avoiding tumor-cell spread. Antegrade ejaculation could be preserved in all patients, who, to date, show no evidence of disease. The waterjet allows the safe and complete removal of lymphatic tissue, leaving vulnerable anatomic structures intact. It can decrease the learning curve of laparoscopic RPLND and contribute to better acceptance of this procedure.

An open randomized controlled clinical trial to evaluate ridge preservation and repair using SocketKAP(™) and SocketKAGE(™) : part 2 - three-dimensional alveolar bone volumetric analysis of CBCT imaging.

PubMed

Abdelhamid, Alaa; Omran, Mostafa; Bakhshalian, Neema; Tarnow, Dennis; Zadeh, Homayoun H

2016-06-01

The aims of this study were (i) to evaluate the efficacy of ridge preservation and repair procedures involving the application of SocketKAP(™) and SocketKAGE(™) devices following tooth removal and (ii) to evaluate alveolar bone volumetric changes at 6 months post-extraction in intact sockets or those with facial wall dehiscence defects using 3-dimensional pre- and postoperative CBCT data. Thirty-six patients required 61 teeth extracted. Five cohorts were established: Group A: Intact Socket Negative Control Group B: Intact Socket + SocketKAP(™) Group C: Intact Socket Filled with Anorganic Bovine Bone Mineral (ABBM) + SocketKAP(™) Group D: Facial Dehiscence Socket Negative Control Group E: Facial Dehiscence Socket Filled with ABBM + SocketKAP(™) + SocketKAGE(™) . Preoperative CBCT scans were obtained followed by digital subtraction of the test teeth. At 6 months post-extraction, another CBCT scan was obtained. The pre- and postoperative scans were then superimposed, allowing highly accurate quantitative determination of the 3D volumetric alveolar bone volume changes from baseline through 6 months. Significant volumetric bone loss occurred in all sockets, localized mainly in the 0-3 mm zone apical to the ridge crest. For intact sockets, SocketKAP(™) + ABBM treatment led to a statistically significant greater percentage of remaining mineralized tissue volume when compared to negative control group. A significant difference favoring SocketKAP(™) + SocketKAGE(™) + ABBM treatment was observed for sockets with facial dehiscence defects compared to the negative control group. SocketKAP(™) , with ABBM, appears effective in limiting post-extraction volumetric bone loss in intact sockets, while SocketKAP(™) + SocketKAGE + ABBM appears effective in limiting post-extraction bone loss in sockets with dehiscence defects. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantification of oral palatine Langerhans cells in HIV/AIDS associated oral Kaposi sarcoma with and without oral candidiasis.

PubMed

Jivan, Vibha; Meer, Shabnum

2016-01-01

Langerhans cells (LCs) are effective antigen-presenting cells that function as "custodians" of mucosa, modifying the immune system to pathogen entry, and tolerance to self-antigen and commensal microbes. A reduction in number of LCs in human immunodeficiency virus (HIV)-positive individuals may predispose to local mucosal infections. To quantitatively determine the number of oral mucosal LCs in HIV/acquired immunodeficiency syndrome HIV/acquired immunodeficiency syndrome (AIDS) associated oral Kaposi sarcoma (KS) with/without oral candidiasis (OC) and to define in situ interrelationships between the cells, OC, and HIV infection. Thirty-two periodic acid-Schiff. (PAS) stained histologic sections of palatal HIV/AIDS associated KS with intact oral epithelium were examined for Candida and divided into two groups: . (1) KS coinfected with Candida and. (2) KS noninfected with Candida. Sections were immunohistochemically stained with CD1a. The standard length of surface epithelium was measured and number of positively stained LCs counted per unit length. Control cases included non-Candida infected palatal mucosa overlying pleomorphic adenoma. (PA) and oral mucosa infected with Candida in otherwise healthy individuals. LC number per unit length of surface epithelium was statistically significantly greatest in uninfected PA mucosa and lowest in KS coinfected with Candida (P = 0.0001). A statistically significant difference was also noted between uninfected PA mucosa and non-Candida infected KS (P = 0.0014), in KS coinfected with Candida and non-infected KS (P = 0.0035), between OC and PA (P = 0.0001), and OC and KS coinfected with Candida (P = 0.0247). LC numbers are significantly reduced in oral tissues of HIV/AIDS infected patients by Candida infection when compared to oral tissues without.

Irreversible Electroporation in a Swine Lung Model

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

Dupuy, Damian E., E-mail: ddupuy@lifespan.org; Aswad, Bassam, E-mail: baswad@lifespan.org; Ng, Thomas, E-mail: tng@usasurg.org

2011-04-15

Purpose: This study was designed to evaluate the safety and tissue effects of IRE in a swine lung model. Methods: This study was approved by the institutional animal care committee. Nine anesthetized domestic swine underwent 15 percutaneous irreversible electroporation (IRE) lesion creations (6 with bipolar and 3 with 3-4 monopolar electrodes) under fluoroscopic guidance and with pancuronium neuromuscular blockade and EKG gating. IRE electrodes were placed into the central and middle third of the right mid and lower lobes in all animals. Postprocedure PA and lateral chest radiographs were obtained to evaluate for pneumothorax. Three animals were sacrificed at 2more » weeks and six at 4 weeks. Animals underwent high-resolution CT scanning and PA and lateral radiographs 1 h before sacrifice. The treated lungs were removed en bloc, perfused with formalin, and sectioned. Gross pathologic and microscopic changes after standard hematoxylin and eosin staining were analyzed within the areas of IRE lesion creation. Results: No significant adverse events were identified. CT showed focal areas of spiculated high density ranging in greatest diameter from 1.1-2.2 cm. On gross inspection of the sectioned lung, focal areas of tan discoloration and increased density were palpated in the areas of IRE. Histological analysis revealed focal areas of diffuse alveolar damage with fibrosis and inflammatory infiltration that respected the boundaries of the interlobular septae. No pathological difference could be discerned between the 2- and 4-week time points. The bronchioles and blood vessels within the areas of IRE were intact and did not show signs of tissue injury. Conclusion: IRE creates focal areas of diffuse alveolar damage without creating damage to the bronchioles or blood vessels. Short-term safety in a swine model appears to be satisfactory.« less

Human papillomavirus detection in paraffin-embedded colorectal cancer tissues.

PubMed

Tanzi, Elisabetta; Bianchi, Silvia; Frati, Elena R; Amicizia, Daniela; Martinelli, Marianna; Bragazzi, Nicola L; Brisigotti, Maria Pia; Colzani, Daniela; Fasoli, Ester; Zehender, Gianguglielmo; Panatto, Donatella; Gasparini, Roberto

2015-01-01

Human papillomavirus (HPV) has a well-recognized aetiological role in the development of cervical cancer and other anogenital tumours. Recently, an association between colorectal cancer and HPV infection has been suggested, although this is still controversial. This study aimed at detecting and characterizing HPV infection in 57 paired biopsies from colorectal cancers and adjacent intact tissues using a degenerate PCR approach. All amplified fragments were genotyped by means of sequencing. Overall, HPV prevalence was 12.3 %. In particular, 15.8 % of tumour tissues and 8.8 % of non-cancerous tissue samples were HPV DNA-positive. Of these samples, 85.7 % were genotyped successfully, with 41.7 % of sequences identifying four genotypes of the HR (high oncogenic risk) clade Group 1; the remaining 58.3 % of HPV-genotyped specimens had an unclassified β-HPV. Examining additional cases and analysing whole genomes will help to outline the significance of these findings.

Extravasation of paclitaxel into breast tissue from central catheter port.

PubMed

Barutca, Sabri; Kadikoylu, Gurhan; Bolaman, Zahit; Meydan, Nezih; Yavasoglu, Irfan

2002-10-01

A 53-year-old woman with advanced-stage ovarian cancer experienced extravasation of paclitaxel into the breast tissue as a result of inappropriate needle insertion and/or dislodgement; it came from a central catheter port (CCP) that was found to be intact under radiological examination with contrast material. The breast became tender and oedematous with erythema, and local warming was observed within a few hours. The patient improved in the next few days during nonsteroidal anti-inflammatory medication and close observation, and the breast healed with thickened and darkened skin and central scarring in the 6th month of follow-up. To the best of our knowledge, extravasation into breast tissue is rare in the literature. Extravasation of vesicant drugs from CCP can cause tissue necrosis; it is therefore essential that ports be carefully assessed and used by experienced staff to lessen the likelihood of such an unpleasant complication.

Hyperforin accumulates in the translucent glands of Hypericum perforatum.

PubMed

Soelberg, Jens; Jørgensen, Lise Bolt; Jäger, Anna K

2007-06-01

Hypericum perforatum contains the therapeutically important compounds hypericin and hyperforin. Hypericin is known to accumulate in the dark glands. This investigation aimed to determine the accumulation site of hyperforin. Dark and translucent glands as well as non-secretory tissue in leaves were manually isolated under the microscope. Hyperforin content was quantified by UV HPLC. Secretory structures were surveyed anatomically. The hyperforin content of intact leaves was found to be about 3 mg g(-1) fresh tissue, whereas a content of about 7 mg g(-1) fresh material was found in isolated translucent glands. Hyperforin was found only to occur in minute amounts in dark glands (approx. 0.4 mg g(-1) fresh tissue). In non-secretory tissue no hyperforin was detected. The accumulation of hyperforin detected in the translucent glands supports the proposed hypothesis that hyperforin is synthesized by the same biosynthetic machinery as monoterpenes in the chloroplasts of cells delimiting the gland.

High-resolution magic angle spinning 1H-NMR spectroscopy studies on the renal biochemistry in the bank vole (Clethrionomys glareolus) and the effects of arsenic (As3+) toxicity.

PubMed

Griffin, J L; Walker, L; Shore, R F; Nicholson, J K

2001-06-01

1. High-resolution magic angle spinning (MAS) 1H-NMR spectroscopy was used to study renal metabolism and the toxicity of As3+, a common environmental contaminant, in the bank vole (Clethrionomys glareolus), a wild species of rodent. 2. Following a 14-day exposure to an environmentally relevant dose of As2O3 (28 mg kg(-1) feed), voles displayed tissue damage at autopsy. MAS 1H spectra indicated abnormal lipid profiles in these samples. 3. Tissue necrosis was also evident from measurements of the apparent diffusion coefficient of water in the intact tissue using MAS 1H diffusion-weighted spectroscopy, its first application to toxicology. 4. Comparison of renal tissue from the wood mouse (Apodemus sylvaticus) exposed to identical exposure levels of As3+ suggested that the bank vole is particularly vulnerable to As3+ toxicity.

Pharmacological characterization of an imidazolopyrazole as novel selective androgen receptor modulator.

PubMed

Zhang, Xuqing; Allan, George F; Tannenbaum, Pamela; Sbriscia, Tifanie; Linton, Olivia; Lai, Muh-Tsann; Haynes-Johnson, Donna; Bhattacharjee, Sheela; Lundeen, Scott G; Sui, Zhihua

2013-03-01

Selective androgen receptor modulators (SARMs) are androgens with tissue-selective activity. SARMs that have anabolic activity on muscle while having minimal stimulatory activity on prostate are classified as SARM agonists. They can be used to prevent the loss of lean body mass that is associated with cancer, immunodeficiency, renal disease and aging. They may also have anabolic activity on bone; thus, unlike estrogens, they may reverse the loss of bone strength associated with aging or hypogonadism. Our in-house effort on SARM program discovers a nonsteroidal androgen receptor ligand with a unique imidazolopyrazole moiety in its structure. In vitro, this compound is a weak androgen receptor binder and a weak androgen agonist. Despite this, in orchidectomized mature rats it is an effective SARM agonist, with an ED(50) on levator ani muscle of 3.3mg/kg and an ED(50) on ventral prostate of >30mg/kg. It has its maximal effect on muscle at the dose of 10mg/kg. In addition, this compound has mixed agonistic and antagonistic activities on prostate, reducing the weight of that tissue in intact rats by 22% at 10mg/kg. The compound does not have significant effect on gonadotropin levels or testosterone levels in both orchidectomized and intact male rats. It does not have notable progestin, estrogen or glucocorticoid agonistic or antagonistic activity in rats. In a female sexual behavior model, it improves the sexual desire of ovariectomized female rats for sexually mature intact males over nonsexually ovariectomized females. Overall, the imidazolopyrazole is a potent prostate-sparing candidate for development as a SARM agonist with an appropriate pharmacological profile for clinical benefit in muscle-wasting conditions and female sexual function disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

Effect of unfocused extracorporeal shock wave therapy on growth factor gene expression in wounds and intact skin of horses.

PubMed

Link, Kaitlyn A; Koenig, Judith B; Silveira, Andressa; Plattner, Brandon L; Lillie, Brandon N

2013-02-01

To compare the effect of extracorporeal shock wave therapy (ESWT) on expression of fibroblast growth factor-7 (FGF-7), transforming growth factor-β1 (TGF-β1), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-A (PDGF), and vascular endothelial growth factor-A (VEGF) in skin with surgically created skin wounds and intact skin in horses. 14 healthy horses. 8 horses were treated with ESWT at 6 locations along the neck at 36, 24, 12, 6, 2, or 1 hour prior to collection of full-thickness biopsy specimens from each location; a control specimen was collected from a sham-treated location. In 6 horses, 5 full-thickness wounds were created in each forelimb. Wounds in 1 forelimb/horse received ESWT immediately after creation and subsequently on days 7, 14, and 21; wounds in the contralateral forelimb remained untreated. Biopsy specimens were collected from 1 wound on each forelimb on days 7, 14, 21, 28, and 35. Expression levels of FGF-7, TGF-β1, IGF-1, PDGF, and VEGF were assessed in tissue samples from the horses' necks and forelimbs. In surgically created wounds, ESWT treatment was associated with reduced TGF-β1 expression, compared with expression in control wounds, during the entire study period. At 28 days following wound creation, IGF-1 expression was significantly increased for treated and untreated wounds, compared with findings on days 7, 14, 21, and 35. There was no significant effect of treatment on FGF-7, TGF-β1, IGF-1, PDGF, or VEGF expression in intact skin. Intervention with ESWT to suppress TGF-β1 may decrease granulation tissue production, resulting in improved wound healing on the distal portion of horses' limbs.

Ameliorative Effects of Endurance Exercise with Two Different Intensities on Nandrolone Decanoate-Induced Neurodegeneration in Rats: Involving Redox and Apoptotic Systems.

PubMed

Joukar, Siyavash; Vahidi, Reza; Farsinejad, Alireza; Asadi-Shekaari, Majid; Shahouzehi, Beydolah

2017-07-01

Despite the importance of this issue, less has been paid to the influence of exercise on the neural side effects of anabolic androgenic steroids and mechanisms. We investigated the effects of two levels of endurance exercise on neurodegeneration side effects of nandrolone. The study period was 8 weeks. Wistar rats were divided into nine groups including the control (CTL) group, mild exercise (mEx) group, and vehicle (Arach) group which received arachis oil intramuscularly, nandrolone (Nan) group which received nandrolone decanoate 5 mg/kg two times weekly, mEx+Arach group which treated with arachis oil along with mild exercise, mEx+Nan group which treated with nandrolone along with mild exercise, severe exercise (sEx) group, sEx+Arach, and sEx+Nan groups. Finally, brain samples were taken for histopathological, biochemical, and western blot analysis. Nandrolone significantly decreased the intact cells of the hippocampus, total antioxidant capacity (TAC) (P < 0.05 versus CTL and Arach groups), TAC to malondialdehyde ratio (TAC/MDA), and Bcl-2. Nandrolone increased the Bax/Bcl-2 ratio of the brain tissue (P < 0.01 versus CTL and Arach groups). Combination of mild exercise and nandrolone rescued the intact cells to some extent, and this effect was associated with the improvement of Bcl-2 level and Bax/Bcl-2 ratio of brain tissue. Combination of severe exercise and nandrolone rescued the intact cells and improved the TAC, TAC/MDA, and Bax/Bcl-2 ratios. The findings suggest that low- and high-intensity endurance exercise decreased the risk of neurodegeneration effect of nandrolone in the hippocampus of rats. This effect can be explained by the regulation of the redox system and cell homeostasis.

46 CFR 173.020 - Intact stability standards: Counterballasted and non-counterballasted vessels.

Code of Federal Regulations, 2010 CFR

2010-10-01

... not heel beyond the limits specified in paragraph (d) of this section: (1) Beam to depth—3.40 to 4.75...) of this section, the following limits of heel apply with the vessel at its deepest operating draft: (1) Protected and partially protected waters and Great Lakes in summer—heel to main deck immersion or...

46 CFR 173.020 - Intact stability standards: Counterballasted and non-counterballasted vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... not heel beyond the limits specified in paragraph (d) of this section: (1) Beam to depth—3.40 to 4.75...) of this section, the following limits of heel apply with the vessel at its deepest operating draft: (1) Protected and partially protected waters and Great Lakes in summer—heel to main deck immersion or...

Using Games in Online Education: Is It a Winning Strategy?

ERIC Educational Resources Information Center

Vu, Phu; Crow, Sherry R.; Fredrickson, Scott

2014-01-01

This study examined the impact of adding game elements on students' performances in an online learning setting. Two intact online graduate level course sections were chosen for this study. Each course sections had 18 students. The results of the study showed that there were significant differences in the time students spent in their courses and…

Differential attractiveness of potato tuber volatiles to Phthorimaea operculella (Gelechiidae) and the predator Orius insidiosus (Anthocoridae).

PubMed

Arab, Alberto; Trigo, José Roberto; Lourenção, André Luiz; Peixoto, Aiane Michele; Ramos, Fernanda; Bento, José Mauricio Simões

2007-10-01

The behavioral responses of the potato tuberworm moth Phthorimaea operculella and the polyphagous predator Orius insidiosus to volatiles emanating from exposed tubers were studied by four-arm olfactometer bioassays. Mated females of P. operculella distinguished volatiles released by intact potato tubers from volatiles damaged mechanically or by conspecific larvae. Volatiles from intact potato tubers were attractive to them. On the other hand, unmated females of P. operculella did not respond to tuber volatiles. Adults of O. insidiosus were attracted to volatiles from tubers damaged by P. operculella larvae, but did not respond to intact or mechanically damaged tubers. Methyl jasmonate (MeJA) was the only compound identified from the headspace of potato tubers (GC-MS of direct headspace sampling). The amount varied with the type of induction, being 0.001 +/- 0.0003 ng g(-1) in tissues of intact fresh tubers, 0.002 +/- 0.0007 ng g(-1) in mechanically damaged tubers, and showing a six- to tenfold increase in P. operculella damaged tubers (0.090 +/- 0.006 ng g(-1)). Behavioral bioassays with synthetic MeJA confirmed that the response of the insects is dependent on MeJA concentration. Mated females of P. operculella showed the highest response at 0.001 ng g(-1) (concentration released by intact tubers), whereas O. insidiosus showed the highest response, between 0.01 and 0.05 ng g(-1), which is close to the concentration released by P. operculella damaged tubers. Based on these results, we postulate that P. operculella and O. insidiosus have adapted their responses to plant volatiles differently, enabling them to locate suitable hosts or prey.

Increased fat deposition in injured skeletal muscle is regulated by sex-specific hormones

PubMed Central

McHale, Matthew J.; Sarwar, Zaheer U.; Cardenas, Damon P.; Porter, Laurel; Salinas, Anna S.; Michalek, Joel E.; McManus, Linda M.

2012-01-01

Sex differences in skeletal muscle regeneration are controversial; comparisons of regenerative events between sexes have not been rigorously defined in severe injury models. We comprehensively quantified inflammation and muscle regeneration between sexes and manipulated sex-specific hormones to determine effects on regeneration. Cardiotoxin injury was induced in intact, castrated and ovariectomized female and male mice; ovariectomized mice were replaced with low- or high-dose 17-β estradiol (E2) or progesterone (P4). Extent of injury was comparable between intact mice, but females were more efficient in removal of necrotic debris, despite similar tissue levels of inflammatory cells and chemokines. Myofiber size during regeneration was equivalent between intact mice and after castration or ovariectomy (OVX) but was decreased (P < 0.001) in ovariectomized mice with high-dose E2 replacement. Intermuscular adipocytes were absent in uninjured muscle, whereas adipocyte area was increased among regenerated myofibers in all groups. Interestingly, intermuscular fat was greater (P = 0.03) in intact females at day 14 compared with intact males. Furthermore, castration increased (P = 0.01) and OVX decreased adipocyte accumulation. After OVX, E2, but not P4, replacement decreased (P ≤ 0.03) fat accumulation. In conclusion, sex-dependent differences in regeneration consisted of more efficient removal of necrosis and increased fat deposition in females with similar injury, inflammation, and regenerated myofiber size; high-dose E2 decreased myofiber size and fat deposition. Adipocyte accumulation in regenerating muscle was influenced by sex-specific hormones. Recovery following muscle injury was different between males and females, and sex-specific hormones contributed to these differences, suggesting that sex-specific treatments could be beneficial after injury. PMID:22116509

Mitochondrial motility and vascular smooth muscle proliferation.

PubMed

Chalmers, Susan; Saunter, Christopher; Wilson, Calum; Coats, Paul; Girkin, John M; McCarron, John G

2012-12-01

Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells. High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (≈ 2 μm by 0.5 μm), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery. These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Wall extensibility and cell hydraulic conductivity decrease in enlarging stem tissues at low water potentials.

PubMed

Nonami, H; Boyer, J S

1990-08-01

Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low psi(w)) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low psi(w) by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low psi(w). It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low psi(w) but that the elastic properties of the walls were of little consequence in this response.

Wall Extensibility and Cell Hydraulic Conductivity Decrease in Enlarging Stem Tissues at Low Water Potentials 1

PubMed Central

Nonami, Hiroshi; Boyer, John S.

1990-01-01

Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low ψw) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low ψw by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low ψw. It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low ψw but that the elastic properties of the walls were of little consequence in this response. PMID:16667664

Deterioration of autonomic neuronal receptor signaling and mechanisms intrinsic to heart pacemaker cells contribute to age-associated alterations in heart rate variability in vivo.

PubMed

Yaniv, Yael; Ahmet, Ismayil; Tsutsui, Kenta; Behar, Joachim; Moen, Jack M; Okamoto, Yosuke; Guiriba, Toni-Rose; Liu, Jie; Bychkov, Rostislav; Lakatta, Edward G

2016-08-01

We aimed to determine how age-associated changes in mechanisms extrinsic and intrinsic to pacemaker cells relate to basal beating interval variability (BIV) reduction in vivo. Beating intervals (BIs) were measured in aged (23-25 months) and adult (3-4 months) C57BL/6 male mice (i) via ECG in vivo during light anesthesia in the basal state, or in the presence of 0.5 mg mL(-1) atropine + 1 mg mL(-1) propranolol (in vivo intrinsic conditions), and (ii) via a surface electrogram, in intact isolated pacemaker tissue. BIV was quantified in both time and frequency domains using linear and nonlinear indices. Although the average basal BI did not significantly change with age under intrinsic conditions in vivo and in the intact isolated pacemaker tissue, the average BI was prolonged in advanced age. In vivo basal BIV indices were found to be reduced with age, but this reduction diminished in the intrinsic state. However, in pacemaker tissue BIV indices increased in advanced age vs. adults. In the isolated pacemaker tissue, the sensitivity of the average BI and BIV in response to autonomic receptor stimulation or activation of mechanisms intrinsic to pacemaker cells by broad-spectrum phosphodiesterase inhibition declined in advanced age. Thus, changes in mechanisms intrinsic to pacemaker cells increase the average BIs and BIV in the mice of advanced age. Autonomic neural input to pacemaker tissue compensates for failure of molecular intrinsic mechanisms to preserve average BI. But this compensation reduces the BIV due to both the imbalance of autonomic neural input to the pacemaker cells and altered pacemaker cell responses to neural input. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Scanning ion conductance microscopy: a convergent high-resolution technology for multi-parametric analysis of living cardiovascular cells

PubMed Central

Miragoli, Michele; Moshkov, Alexey; Novak, Pavel; Shevchuk, Andrew; Nikolaev, Viacheslav O.; El-Hamamsy, Ismail; Potter, Claire M. F.; Wright, Peter; Kadir, S.H. Sheikh Abdul; Lyon, Alexander R.; Mitchell, Jane A.; Chester, Adrian H.; Klenerman, David; Lab, Max J.; Korchev, Yuri E.; Harding, Sian E.; Gorelik, Julia

2011-01-01

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies. PMID:21325316

Quantifying growing versus non-growing ovarian follicles in the mouse.

PubMed

Uslu, Bahar; Dioguardi, Carola Conca; Haynes, Monique; Miao, De-Qiang; Kurus, Meltem; Hoffman, Gloria; Johnson, Joshua

2017-01-13

A standard histomorphometric approach has been used for nearly 40 years that identifies atretic (e.g., dying) follicles by counting the number of pyknotic granulosa cells (GC) in the largest follicle cross-section. This method holds that if one pyknotic granulosa nucleus is seen in the largest cross section of a primary follicle, or three pyknotic cells are found in a larger follicle, it should be categorized as atretic. Many studies have used these criteria to estimate the fraction of atretic follicles that result from genetic manipulation or environmental insult. During an analysis of follicle development in a mouse model of Fragile X premutation, we asked whether these 'historical' criteria could correctly identify follicles that were not growing (and could thus confirmed to be dying). Reasoning that the fraction of mitotic GC reveals whether the GC population was increasing at the time of sample fixation, we compared the number of pyknotic nuclei to the number of mitotic figures in follicles within a set of age-matched ovaries. We found that, by itself, pyknotic nuclei quantification resulted in high numbers of false positives (improperly categorized as atretic) and false negatives (improperly categorized intact). For preantral follicles, scoring mitotic and pyknotic GC nuclei allowed rapid, accurate identification of non-growing follicles with 98% accuracy. This method most often required the evaluation of one follicle section, and at most two serial follicle sections to correctly categorize follicle status. For antral follicles, we show that a rapid evaluation of follicle shape reveals which are intact and likely to survive to ovulation. Combined, these improved, non-arbitrary methods will greatly improve our ability to estimate the fractions of growing/intact and non-growing/atretic follicles in mouse ovaries.

Prolonged Allograft Survival in a Patient With Chronic Immunosuppression: A Case Report and Systematic Review.

PubMed

Vyas, Krishna S; Burns, Chase; Ryan, Dylan T; Wong, Lesley

2017-06-01

A 41-year-old man with past medical history of kidney-liver transplantation requiring chronic immunosuppression presented 2 years posttransplant with a necrotizing soft tissue infection of his right thigh. Serial debridement to remove necrotic tissue was performed, and a Matrix HD Allograft Fenestrated (RTI Surgical, Alachua, FL) was applied. At 5-months post grafting, the patient demonstrated fully vascularized and intact skin. Under normal circumstances, a cadaveric allograft sloughs over several weeks and is not usually considered a permanent solution for wound closure. A systematic review of transplant patients on chronic immunosuppression with skin allografts demonstrates the potential for the indefinite survival of an allograft. Necrotizing soft tissue infections can definitively be treated using serial debridement and allograft transplantation in the chronically immunosuppressed.

Characterizing lamina propria of human gastric mucosa by multiphoton microscopy

NASA Astrophysics Data System (ADS)

Liu, Y. C.; Yang, H. Q.; Chen, G.; Zhuo, S. M.; Chen, J. X.; Yan, J.

2011-01-01

Lamina propria (LP) of gastric mucosa plays an important role in progression of gastric cancer because of the site at where inflammatory reactions occur. Multiphoton imaging has been recently employed for microscopic examination of intact tissue. In this paper, using multiphoton microscopy (MPM) based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), high resolution multiphoton microscopic images of lamina propria (LP) are obtained in normal human gastric mucosa at excitation wavelength λex = 800 nm. The main source of tissue TPEF originated from the cells of gastric glands, and loose connective tissue, collagen, produced SHG signals. Our results demonstrated that MPM can be effective for characterizing the microstructure of LP in human gastric mucosa. The findings will be helpful for diagnosing and staging early gastric cancer in the clinics.

Long gap esophageal atresia: lengthening technique and primary anastomosis.

PubMed

Hadidi, Ahmed T; Hosie, Stuart; Waag, Karl-Ludwig

2007-10-01

The treatment of long gap esophageal atresia remains a major surgical challenge. The authors describe a modification of a lengthening technique based on tissue expansion to avoid sutures cutting through the esophagus. Between January 2004 and August 2006, 4 patients did not respond to stretching, and underwent this modified esophageal lengthening technique using silastic tubes. RESULTS AND FOLLOW-UP: All infants recovered and have an intact esophagus. All infants developed gastroesophageal reflux. Thal antireflux procedure was performed in the first infant. The other 3 patients were managed conservatively. Follow-up ranged between 6 and 34 months. The tissue expansion principle can be successfully applied in the esophagus through external traction. Silastic tube fixation at esophageal ends may help to apply even traction and avoid sutures cutting through the esophageal tissue.

Desensitization and Down Regulation of Muscarinic Acetylcholine Receptors

DTIC Science & Technology

1988-06-22

function, in vitro. This technique offers an easy method to obtain intact differentiated brain cells with minimal diffusion barriers. Preincubation of...neuroblastoma cells (clone NIE- 115 ). This treatment demonstrated that the muscarinic receptors in this neuronal clone can be divided into two types; one...mouse neuroblastoma NlE- 115 cells, and in other tissues, mediated an increase in phosphoinositide hydrolysis. Diacylglycerol is one of the important

Case study: fatal poisoning by malathion.

PubMed

Thompson, T S; Treble, R G; Magliocco, A; Roettger, J R; Eichhorst, J C

1998-07-20

A case involving a fatal poisoning (suicide) by the insecticide malathion is described. The intact insecticide was found in the post-mortem blood and gastric contents at concentrations of 1.8 and 978 micrograms/ml, respectively. None of the insecticide was found in the autopsied liver tissue. Gas chromatography-mass spectrometry (GC-MS) techniques were used for the identification and quantification of malathion in the body fluids.

Impact of epidermal leaf mining by the aspen leaf miner (Phyllocnistis populiella) on the growth, physiology, and leaf longevity of quaking aspen.

Treesearch

Diane L. Wagner; Linda DeFoliart; Patricia Doak; Jenny Schneiderheinze

2008-01-01

The aspen leaf miner, Phyllocnistis populiella, feeds on the contents of epidermal cells on both top (adaxial) and bottom (abaxial) surfaces of quaking aspen leaves, leaving the photosynthetic tissue of the mesophyll intact. This type of feeding is taxonomically restricted to a small subset of leaf mining insects but can cause widespread plant...

Estimation of the viscous properties of skin and subcutaneous tissue in uniaxial stress relaxation tests.

PubMed

Wu, John Z; Cutlip, Robert G; Welcome, Daniel; Dong, Ren G

2006-01-01

Knowledge of viscoelastic properties of soft tissues is essential for the finite element modelling of the stress/strain distributions in finger-pad during vibratory loading, which is important in exploring the mechanism of hand-arm vibration syndrome. In conventional procedures, skin and subcutaneous tissue have to be separated for testing the viscoelastic properties. In this study, a novel method has been proposed to simultaneously determine the viscoelastic properties of skin and subcutaneous tissue in uniaxial stress relaxation tests. A mathematical approach has been derived to obtain the creep and relaxation characteristics of skin and subcutaneous tissue using uniaxial stress relaxation data of skin/subcutaneous composite specimens. The micro-structures of collagen fiber networks in the soft tissue, which underline the tissue mechanical characteristics, will be intact in the proposed method. Therefore, the viscoelastic properties of soft tissues obtained using the proposed method would be more physiologically relevant than those obtained using the conventional method. The proposed approach has been utilized to measure the viscoelastic properties of soft tissues of pig. The relaxation curves of pig skin and subcutaneous tissue obtained in the current study agree well with those in literature. Using the proposed approach, reliable material properties of soft tissues can be obtained in a cost- and time-efficient manner, which simultaneously improves the physiological relevance.

The PAXgene® Tissue System Preserves Phosphoproteins in Human Tissue Specimens and Enables Comprehensive Protein Biomarker Research

PubMed Central

Gündisch, Sibylle; Schott, Christina; Wolff, Claudia; Tran, Kai; Beese, Christian; Viertler, Christian; Zatloukal, Kurt; Becker, Karl-Friedrich

2013-01-01

Precise quantitation of protein biomarkers in clinical tissue specimens is a prerequisite for accurate and effective diagnosis, prognosis, and personalized medicine. Although progress is being made, protein analysis from formalin-fixed and paraffin-embedded tissues is still challenging. In previous reports, we showed that the novel formalin-free tissue preservation technology, the PAXgene Tissue System, allows the extraction of intact and immunoreactive proteins from PAXgene-fixed and paraffin-embedded (PFPE) tissues. In the current study, we focused on the analysis of phosphoproteins and the applicability of two-dimensional gel electrophoresis (2D-PAGE) and enzyme-linked immunosorbent assay (ELISA) to the analysis of a variety of malignant and non-malignant human tissues. Using western blot analysis, we found that phosphoproteins are quantitatively preserved in PFPE tissues, and signal intensities are comparable to that in paired, frozen tissues. Furthermore, proteins extracted from PFPE samples are suitable for 2D-PAGE and can be quantified by ELISA specific for denatured proteins. In summary, the PAXgene Tissue System reliably preserves phosphoproteins in human tissue samples, even after prolonged fixation or stabilization times, and is compatible with methods for protein analysis such as 2D-PAGE and ELISA. We conclude that the PAXgene Tissue System has the potential to serve as a versatile tissue fixative for modern pathology. PMID:23555997

Normal keratinized mucosa transplants in nude mice.

PubMed

Holmstrup, P; Dabelsteen, E; Reibel, J; Harder, F

1981-01-01

Two types of normal keratinized mucosa were transplanted to subcutaneous sites of nude mice of two different strains. 24 intact specimens of clinically normal human palatal mucosa were transplanted to nude mice of the strain nu/nu NC. The transplants were recovered after 42 d with a recovery rate of 96%. Moreover, 22 intact specimens of normal rat forestomach mucosa were transplanted to nude mice of the strain nu/nu BALB/c/BOM. These transplants were recovered after 21 d with a recovery rate of 63%. The histologic features of the transplants were essentially the same as those of the original tissues. However, epithelial outgrowths from the transplants differed with respect to the pattern of keratinization. The outgrowths of human palatal mucosa transplants were essentially unkeratinized, while the outgrowths of the rat forestomach transplants showed continued keratinization.

Proliferative enteritis in leopard geckos (Eublepharis macularius) associated with Cryptosporidium sp. infection.

PubMed

Terrell, Scott P; Uhl, Elizabeth W; Funk, Richard S

2003-03-01

Twenty-three leopard geckos (Eublepharis macularius) with various clinical histories of weight loss, anorexia, lethargy, and diarrhea were submitted either intact or as biopsy specimens to the University of Florida Anatomic Pathology Service. Gross necropsy findings in the intact geckos included marked reduction of subcutaneous adipose tissue stores at the tail base and mild thickening and reddening of the small intestine. Histologic examination revealed Cryptosporidium sp. infection associated with hyperplasia and mononuclear inflammation of the small intestine in all geckos. Parasites and lesions were only rarely observed in the stomach and large intestine of geckos. The histologic and ultrastructural lesions in the small intestine of leopard geckos infected with Cryptosporidium sp. have not been well characterized previously. This report implicates Cryptosporidium sp. as the cause of disease in the geckos and describes the range of histologic lesions observed.

Facile synthesis of titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles for highly specific enrichment and analysis of intact phosphoproteins.

PubMed

Wang, Hao; Tian, Zhixin

2018-06-06

Analysis of phosphoproteins always faces the challenge of low stoichiometry, which demands highly selective and efficient enrichment in the initial sample preparation. Here we report our synthesis of the novel titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles (Ti 4+ -ATP-NPs) for efficient enrichment of intact phosphoproteins. The average diameter of Ti 4+ -ATP-NPs was about 128 nm with good dispersibility and the saturated adsorption capacity for β-casein was 1046.5 mg/g. In addition, Ti 4+ -ATP-NPs exhibited high specificity and selectivity in enriching phosphoproteins from both standard protein mixtures and complex biological samples (non-fat milk, chicken egg white and mouse heart tissue extract) as demonstrated by SDS-PAGE. Copyright © 2018 Elsevier B.V. All rights reserved.

Selective two-photon collagen crosslinking in situ measured by Brillouin microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kwok, Sheldon J. J.; Kuznetsov, Ivan A.; Kim, Moonseok; Choi, Myunghwan; Scarcelli, Giuliano; Yun, Seok-Hyun

2017-02-01

Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively. Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.

Cell delivery in regenerative medicine: the cell sheet engineering approach.

PubMed

Yang, Joseph; Yamato, Masayuki; Nishida, Kohji; Ohki, Takeshi; Kanzaki, Masato; Sekine, Hidekazu; Shimizu, Tatsuya; Okano, Teruo

2006-11-28

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

A curated collection of tissue microarray images and clinical outcome data of prostate cancer patients

PubMed Central

Zhong, Qing; Guo, Tiannan; Rechsteiner, Markus; Rüschoff, Jan H.; Rupp, Niels; Fankhauser, Christian; Saba, Karim; Mortezavi, Ashkan; Poyet, Cédric; Hermanns, Thomas; Zhu, Yi; Moch, Holger; Aebersold, Ruedi; Wild, Peter J.

2017-01-01

Microscopy image data of human cancers provide detailed phenotypes of spatially and morphologically intact tissues at single-cell resolution, thus complementing large-scale molecular analyses, e.g., next generation sequencing or proteomic profiling. Here we describe a high-resolution tissue microarray (TMA) image dataset from a cohort of 71 prostate tissue samples, which was hybridized with bright-field dual colour chromogenic and silver in situ hybridization probes for the tumour suppressor gene PTEN. These tissue samples were digitized and supplemented with expert annotations, clinical information, statistical models of PTEN genetic status, and computer source codes. For validation, we constructed an additional TMA dataset for 424 prostate tissues, hybridized with FISH probes for PTEN, and performed survival analysis on a subset of 339 radical prostatectomy specimens with overall, disease-specific and recurrence-free survival (maximum 167 months). For application, we further produced 6,036 image patches derived from two whole slides. Our curated collection of prostate cancer data sets provides reuse potential for both biomedical and computational studies. PMID:28291248

Long-Term Implanted cOFM Probe Causes Minimal Tissue Reaction in the Brain

PubMed Central

Hochmeister, Sonja; Asslaber, Martin; Kroath, Thomas; Pieber, Thomas R.; Sinner, Frank

2014-01-01

This study investigated the histological tissue reaction to long-term implanted cerebral open flow microperfusion (cOFM) probes in the frontal lobe of the rat brain. Most probe-based cerebral fluid sampling techniques are limited in application time due to the formation of a glial scar that hinders substance exchange between brain tissue and the probe. A glial scar not only functions as a diffusion barrier but also alters metabolism and signaling in extracellular brain fluid. cOFM is a recently developed probe-based technique to continuously sample extracellular brain fluid with an intact blood-brain barrier. After probe implantation, a 2 week healing period is needed for blood-brain barrier reestablishment. Therefore, cOFM probes need to stay in place and functional for at least 15 days after implantation to ensure functionality. Probe design and probe materials are optimized to evoke minimal tissue reaction even after a long implantation period. Qualitative and quantitative histological tissue analysis revealed no continuous glial scar formation around the cOFM probe 30 days after implantation and only a minor tissue reaction regardless of perfusion of the probe. PMID:24621608

Effect of Electrothermal Treatment on Nerve Tissue Within the Triangular Fibrocartilage Complex, Scapholunate, and Lunotriquetral Interosseous Ligaments.

PubMed

Pirolo, Joseph M; Le, Wei; Yao, Jeffrey

2016-05-01

To evaluate the effect of thermal treatment on neural tissue in the triangular fibrocartilage complex (TFCC), scapholunate interosseous ligament (SLIL), and lunotriquetral interosseous ligament (LTIL). The intact TFCC, SLIL, and LTIL were harvested from cadaveric specimens and treated with a radiofrequency probe as would be performed intraoperatively. Slides were stained using a triple-stain technique for neurotrophin receptor p75, pan-neuronal marker protein gene product 9.5 (PGP 9.5), and 4',6-diamidino-2-phenylindole for neural identification. Five TFCC, 5 SLIL, and 4 LTIL specimens were imaged with fluorescence microscopy. Imaging software was used to measure fluorescence signals and compare thermally treated areas with adjacent untreated areas. A paired t test was used to compare treated versus untreated areas. P < .05 was considered significant. For the TFCC, a mean of 94.9% ± 2.7% of PGP 9.5-positive neural tissue was ablated within a mean area of 11.7 ± 2.5 mm(2) (P = .02). For the SLIL treated from the radiocarpal surface, 97.4% ± 1.0% was ablated to a mean depth of 2.4 ± 0.3 mm from the surface and a mean horizontal spread of 3.4 ± 0.5 mm (P = .01). For the LTIL, 96.0% ± 1.5% was ablated to a mean depth of 1.7 ± 0.7 mm and a mean horizontal spread of 2.6 ± 1.0 mm (P = .02). Differences in the presence of neural tissue between treated areas and adjacent untreated areas were statistically significant for all specimens. Our study confirms elimination of neuronal markers after thermal treatment of the TFCC, SLIL, and LTIL in cadaveric specimens. This effect penetrates below the surface to innervated collagen tissue that is left structurally intact after treatment. Electrothermal treatment as commonly performed to treat symptomatic SLIL, LTIL, and TFCC tears eliminates neuronal tissue in treated areas and may function to relieve pain through a denervation effect. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.

Ocular biodistribution of bevasiranib following a single intravitreal injection to rabbit eyes

PubMed Central

Dejneka, Nadine S.; Wan, Shanhong; Bond, Ottrina S.; Kornbrust, Douglas J.

2008-01-01

Purpose The primary objective of these investigations was to determine the ocular biodistribution of bevasiranib, a small interfering RNA (siRNA) targeting vascular endothelial growth factor A (VEGF-A), following a single intravitreal injection to rabbit eyes. Methods A tissue distribution and pharmacokinetic study was conducted with 3H-bevasiranib prepared in balanced-salt solution (BSS). Single doses of either 0.5 mg/eye or 2.0 mg/eye of 3H-bevasiranib were given by intravitreal injection to Dutch-Belted rabbits (both eyes were treated). Subgroups of rabbits were serially-sacrificed at various times up to 7 days following dosing for collection of tissue samples. The right eye of each rabbit was collected whole, and the left eye was dissected to isolate five ocular tissues. All samples were analyzed by liquid scintillation counting to determine the concentrations of bevasiranib equivalents. An ocular disposition study was also performed with non-radiolabeled bevasiranib, which was administered to Dutch-Belted rabbit eyes via intravitreal injection at a dose of 2.0 mg/eye. Twenty-four hours post-dose, the eyes were enucleated and dissected into eight individual ocular structures that were analyzed for intact bevasiranib using a locked nuleic acid (LNA) noncompetitive hybridization-ligation enzyme-linked immunosorbent assay. Results Following intravitreal injection of 0.5 mg or 2.0 mg radiolabeled bevasiranib to Dutch-Belted rabbits, bevasiranib was detected in the vitreous, iris, retina, retinal pigment epithelium (RPE), and sclera (+choroid). As expected, the highest concentrations were found in the vitreous, and vitreous levels steadily decreased over time, while concentrations of radioactivity in the other ocular tissues increased to maximum values between 24 h and 72 h after dosing. Of these tissues, the highest concentration of radioactivity was detected in the retina. The LNA assay further confirmed the presence of intact bevasiranib in these tissues 24 h following intravitreal injection of non-radiolabeled bevasiranib (2 mg/eye). Conclusions These studies demonstrate distribution of bevasiranib throughout the eye following intravitreal injection, including extensive uptake into the retina. PMID:18523657

Cellular responses to various levels of sustained delivery of testosterone in the ventral prostate.

PubMed

Cavett, W; Tucci, M; Cason, Z; Lemos, L; England, B; Tsao, A; Benghuzzi, H

1997-01-01

The objective of this study was to evaluate the effect of various dosages of testosterone (T) delivered in a sustained manner by means of tricalcium phosphate-lysine (TCPL) delivery system on morphological changes of prostatic tissue using adult male rats as a model. In this experiment, adult male rats (250-300 g BW) were randomly divided into five equal groups (n = 8). Rats in group I, II, and III were castrated and implanted subcutaneously with TCPL loaded with three different dosages (10, 100 and 200 mg T, respectively) of T. Rats in group IV were castrated and implanted with sharm TCPL capsules, and rats in group V served as intact unimplanted controls. Surgical aseptic techniques were performed according to standard laboratory procedures. At the end of 4 and 12 weeks post implantation, four animals from each group were sacrificed and the prostate tissues were collected, weighted, and embedded for histo-pathological evaluations. Data collected from this study have shown that exogenous intake of T delivered in a sustained manner for twelve weeks induced several pathophysiological conditions in ventral prostatic tissue in comparison to the control and sham operated groups. This phenomenon was found to be directly proportional to the dose or the level of sustained delivery. The results demonstrated that the use of 10 mg filled TCPL implants decreased the total mass weight of ventral prostate. Light microscopic evaluation of this group (Group I) revealed a cellular adaptation through an atrophy in the epithelium component. Cytopathological observations such as low cuboidal and thin glands, pleomorphism, and occasional presence of connective tissue stroma were detected. In contrast, ventral prostate collected from animals implanted with TCPL filled with 200 mg T (Group III) showed a significant increase in weights of the wet prostatic tissues in comparison to all groups. Histopathological evaluations demonstrated the following. (i) prostatic hypertrophy alone, or in conjunction with hyperplasia of the epithelial cells, (ii) less connective tissue stroma in comparison to the control group, (iii) occasional involvement of mitotic figures, and (iv) increased angiogenesis. No significant change was observed in those animals implanted with TCPL capsules containing 100 mg T compared to the intact control animals.

Fluorescence Molecular Tomography: Principles and Potential for Pharmaceutical Research

PubMed Central

Stuker, Florian; Ripoll, Jorge; Rudin, Markus

2011-01-01

Fluorescence microscopic imaging is widely used in biomedical research to study molecular and cellular processes in cell culture or tissue samples. This is motivated by the high inherent sensitivity of fluorescence techniques, the spatial resolution that compares favorably with cellular dimensions, the stability of the fluorescent labels used and the sophisticated labeling strategies that have been developed for selectively labeling target molecules. More recently, two and three-dimensional optical imaging methods have also been applied to monitor biological processes in intact biological organisms such as animals or even humans. These whole body optical imaging approaches have to cope with the fact that biological tissue is a highly scattering and absorbing medium. As a consequence, light propagation in tissue is well described by a diffusion approximation and accurate reconstruction of spatial information is demanding. While in vivo optical imaging is a highly sensitive method, the signal is strongly surface weighted, i.e., the signal detected from the same light source will become weaker the deeper it is embedded in tissue, and strongly depends on the optical properties of the surrounding tissue. Derivation of quantitative information, therefore, requires tomographic techniques such as fluorescence molecular tomography (FMT), which maps the three-dimensional distribution of a fluorescent probe or protein concentration. The combination of FMT with a structural imaging method such as X-ray computed tomography (CT) or Magnetic Resonance Imaging (MRI) will allow mapping molecular information on a high definition anatomical reference and enable the use of prior information on tissue's optical properties to enhance both resolution and sensitivity. Today many of the fluorescent assays originally developed for studies in cellular systems have been successfully translated for experimental studies in animals. The opportunity of monitoring molecular processes non-invasively in the intact organism is highly attractive from a diagnostic point of view but even more so for the drug developer, who can use the techniques for proof-of-mechanism and proof-of-efficacy studies. This review shall elucidate the current status and potential of fluorescence tomography including recent advances in multimodality imaging approaches for preclinical and clinical drug development. PMID:24310495

Clinical applications of in vivo fluorescence confocal laser scanning microscopy

NASA Astrophysics Data System (ADS)

Oh, Chilhwan; Park, Sangyong; Kim, Junhyung; Ha, Seunghan; Park, Gyuman; Lee, Gunwoo; Lee, Onseok; Chun, Byungseon; Gweon, Daegab

2008-02-01

Living skin for basic and clinical research can be evaluated by Confocal Laser Scanning Microscope (CLSM) non-invasively. CLSM imaging system can achieve skin image its native state either "in vivo" or "fresh biopsy (ex vivo)" without fixation, sectioning and staining that is necessary for routine histology. This study examines the potential fluorescent CLSM with a various exogenous fluorescent contrast agent, to provide with more resolution images in skin. In addition, in vivo fluorescent CLSM researchers will be extended a range of potential clinical application. The prototype of our CLSM system has been developed by Prof. Gweon's group. The operating parameters are composed of some units, such as illuminated wavelength 488 nm, argon illumination power up to 20mW on the skin, objective lens, 0.9NA oil immersion, axial resolution 1.0μm, field of view 200μm x 100μm (lateral resolution , 0.3μm). In human volunteer, fluorescein sodium was administrated topically and intradermally. Animal studies were done in GFP transgenic mouse, IRC mouse and pig skin. For imaging of animal skin, fluorescein sodium, acridine orange, and curcumine were used for fluorescein contrast agent. We also used the GFP transgenic mouse for fluorescein CLSM imaging. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. Acridin Orange can be highlight nuclei in viable keratinocyte. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, hair and eccrine gland. In intact skin, absorption of fluorescein sodium by individual corneocyte and hair. Intradermal administrated the fluorescein sodium, distinct outline of keratinocyte cell border could be seen. In papillary dermis, fluorescein distribution is more homogeneous. Curcumin is a yellow food dye that has similar fluorescent properties to fluorescein sodium. In vivo CLSM of transgenic GFP mouse enable on in vivo, high resolution view of GFP expressing skin tissue. GFP signals are brightest in corneocyte, kertinocyte, skin appendage and blood vessels. In conclusion, this study demonstrates the usefulness of CLSM as technique for imaging skin in vivo. In addition, CLSM is non-invasive, the same tissue site may be imaged over a period of time to monitor the various change such as wound healing, severity of skin diseases and effect of therapeutic management.

Understanding the differences in molecular conformation of carbohydrate and protein in endosperm tissues of grains with different biodegradation kinetics using advanced synchrotron technology

NASA Astrophysics Data System (ADS)

Yu, P.; Block, H. C.; Doiron, K.

2009-01-01

Conventional "wet" chemical analyses rely heavily on the use of harsh chemicals and derivatization, thereby altering native seed structures leaving them unable to detect any original inherent structures within an intact tissue sample. A synchrotron is a giant particle accelerator that turns electrons into light (million times brighter than sunlight) which can be used to study the structure of materials at the molecular level. Synchrotron radiation-based Fourier transform IR microspectroscopy (SR-FTIRM) has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of the brightness of synchrotron light and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional 'wet' chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of plant seed tissue in relation to nutrient utilization. The objective of this study was to use novel synchrotron radiation-based technology (SR-FTIRM) to identify the differences in the molecular chemistry and conformation of carbohydrate and protein in various plant seed endosperms within intact tissues at cellular and subcellular level from grains with different biodegradation kinetics. Barley grain (cv. Harrington) with a high rate (31.3%/h) and extent (78%), corn grain (cv. Pioneer) with a low rate (9.6%/h) and extent of (57%), and wheat grain (cv. AC Barrie) with an intermediate rate (23%/h) and extent (72%) of ruminal DM degradation were selected for evaluation. SR-FTIRM evaluations were performed at the National Synchrotron Light Source at the Brookhaven National Laboratory (Brookhaven, NY). The molecular structure spectral analysis involved the fingerprint regions of ca. 1720-1485 cm -1 (attributed to protein amide I C dbnd O and C sbnd N stretching; amide II N sbnd H bending and C sbnd N stretching), ca. 1650-950 cm -1 (non-structural CHO starch in endosperms), and ca. 1185-800 cm -1 (attributed to total CHO C sbnd O stretching vibrations) together with agglomerative hierarchical cluster and principal component analyses. Analyses involving the protein amide I features consistently identified differences between all three grains. Other analyses involving carbohydrate features were able to differentiate between wheat and barley but failed however to differentiate between wheat and corn. These results suggest that SR-FTIRM plus the multivariate analyses can be used to identify spectral features associated with the molecular structure of endosperm from grains with different biodegradation kinetics, especially in relation to protein structure. The Novel synchrotron radiation-based bioanalytical technique provides a new approach for plant seed structural molecular studies at ultraspatial resolution and within intact tissue in relation to nutrient availability.

Limited Associations between Keel Bone Damage and Bone Properties Measured with Computer Tomography, Three-Point Bending Test, and Analysis of Minerals in Swiss Laying Hens

PubMed Central

Gebhardt-Henrich, Sabine G.; Pfulg, Andreas; Fröhlich, Ernst K. F.; Käppeli, Susanna; Guggisberg, Dominik; Liesegang, Annette; Stoffel, Michael H.

2017-01-01

Keel bone damage is a wide-spread welfare problem in laying hens. It is unclear so far whether bone quality relates to keel bone damage. The goal of the present study was to detect possible associations between keel bone damage and bone properties of intact and damaged keel bones and of tibias in end-of-lay hens raised in loose housing systems. Bones were palpated and examined by peripheral quantitative computer tomography (PQCT), a three-point bending test, and analyses of bone ash. Contrary to our expectations, PQCT revealed higher cortical and trabecular contents in fractured than in intact keel bones. This might be due to structural bone repair after fractures. Density measurements of cortical and trabecular tissues of keel bones did not differ between individuals with and without fractures. In the three-point bending test of the tibias, ultimate shear strength was significantly higher in birds with intact vs. fractured keel bones. Likewise, birds with intact or slightly deviated keel bones had higher mineral and calcium contents of the keel bone than birds with fractured keel bones. Calcium content in keel bones was correlated with calcium content in tibias. Although there were some associations between bone traits related to bone strength and keel bone damage, other factors such as stochastic events related to housing such as falls and collisions seem to be at least as important for the prevalence of keel bone damage. PMID:28848740

Dimensional changes in soft tissues around dental implants following free gingival grafting: an experimental study in dogs.

PubMed

Bengazi, Franco; Lang, Niklaus P; Caroprese, Marino; Urbizo Velez, Joaquin; Favero, Vittorio; Botticelli, Daniele

2015-02-01

To study the buccal dimensional tissue changes at oral implants following free gingival grafting, with or without including the keratin layer, performed at the time of implant installation into alveolar mucosa. The mandibular premolars and first molars were extracted bilaterally in six Beagle dogs. In the right side of the mandible (Test), flaps were first elevated, and the buccal as well as part of the lingual masticatory mucosa was removed. An incision of the periosteum at the buccal aspect was performed to allow the flap to be coronally repositioned. Primary wound closure was obtained. In the left side, the masticatory (keratinized) mucosa was left in situ, and no sutures were applied (Control). After 3 months of healing, absence of keratinized mucosa was confirmed at the test sites. Two recipient sites were prepared at each side of the mandible in the region of the third and fourth premolars. All implants were installed with the shoulder placed flush with the buccal alveolar bony crest, and abutments were connected to allow a non-submerged healing. Two free gingival mucosal grafts were harvested from the buccal region of the maxillary canines. One graft was left intact (gingival mucosal graft), while for the second, the epithelial layer was removed (gingival connective tissue graft). Subsequently, the grafts were fixed around the test implants in position of the third and fourth premolars, respectively. After 3 months, the animals were euthanized and ground sections obtained. Similar bony crest resorption and coronal extension of osseointegration were found at test and control sites. Moreover, similar dimensions of the peri-implant soft tissues were obtained at test and control sites. The increase in the alveolar mucosal thickness by means of a gingival graft affected the peri-implant marginal bone resorption and soft tissue recession around implants. This resulted in outcomes that were similar to those at implants surrounded by masticatory mucosa, indicating that gingival grafting in the absence of keratinized mucosa around implants may reduce the resorption of the marginal crest and soft tissue recession. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

46 CFR 178.410 - Drainage of flush deck vessels.

Code of Federal Regulations, 2011 CFR

2011-10-01

... TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.410 Drainage of flush deck vessels. (a) Except as provided in paragraph (b) of this section, the weather deck on a flush deck vessel...

46 CFR 178.410 - Drainage of flush deck vessels.

Code of Federal Regulations, 2012 CFR

2012-10-01

... TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.410 Drainage of flush deck vessels. (a) Except as provided in paragraph (b) of this section, the weather deck on a flush deck vessel...

46 CFR 178.410 - Drainage of flush deck vessels.

Code of Federal Regulations, 2013 CFR

2013-10-01

... TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.410 Drainage of flush deck vessels. (a) Except as provided in paragraph (b) of this section, the weather deck on a flush deck vessel...

46 CFR 178.410 - Drainage of flush deck vessels.

Code of Federal Regulations, 2014 CFR

2014-10-01

... TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.410 Drainage of flush deck vessels. (a) Except as provided in paragraph (b) of this section, the weather deck on a flush deck vessel...

46 CFR 178.410 - Drainage of flush deck vessels.

Code of Federal Regulations, 2010 CFR

2010-10-01

... TONS) INTACT STABILITY AND SEAWORTHINESS Drainage of Weather Decks § 178.410 Drainage of flush deck vessels. (a) Except as provided in paragraph (b) of this section, the weather deck on a flush deck vessel...

Histological methods to determine blood flow distribution with fluorescent microspheres.

PubMed

Luchtel, D L; Boykin, J C; Bernard, S L; Glenny, R W

1998-11-01

We evaluated several histological methods and determined their advantages and disadvantages for histological studies of tissues and organs perfused with fluorescent microspheres. Microspheres retained their fluorescence in 7-10 microm serial sections with a change in the antimedium from toluene when samples were fixed in formalin and embedded in paraffin. Several antimedia allowed both wax infiltration of tissue and preservation of microsphere fluorescence. Histoclear II was the best substitute for toluene. When samples were fixed in formalin and embedded in glycol methacrylate, thinner (3-5 microm) sections provided greater histological detail but had fewer microspheres per section. Air dried lung tissue followed by Vibratome sectioning provided thick sections (100 microm) that facilitated rapid survey of large volumes of tissue for microspheres but limited histological detail, and the air drying procedure was restricted to lung tissue. Samples fixed in formalin followed by Vibratome sectioning of unembedded tissue provided better histological detail of lung tissue and was also useful for other organs. These sections were more difficult to handle and to mount on slides compared to air dried tissue, whereas fixed tissue embedded in gelatin provided better tissue support for Vibratome sectioning. Rapid freezing followed by cryo-microtome sectioning resulted in frozen sections that were relatively difficult to handle compared to embedded or unembedded tissue; they also deteriorated relatively rapidly with time. Paraffin sections were stained with hematoxylin and eosin or with aqueous methyl green, although tissue autofluorescence by itself was usually sufficient to identify histological features. Methacrylate sections quenched tissue autofluorescence, and Lee's stain or Richardson's stain were used for staining sections. Toluene based mountants such as Cytoseal quenched fluorescence, particularly the red fluorescent microspheres. Aqueous based mountants such as Aquamount, Crystal/Mount, Fluoromount-G were substituted, although such preparations were not as permanent as Cytoseal mounted coverglasses and tended to cause fading of stained sections.

Role of Tumor Collagenase Stimulating Factor in Breast Cancer Invasion and Metastasis.

DTIC Science & Technology

1997-12-01

propose that in physiologic processes, the presence of an intact basement membrane separating the normal/benign epithelium from underlying stromal...second Ig domain is a junctional exon encoding the transmembrane domain and part of the cytoplasmic domain as well. Most members of the Ig...produce EMMPRIN. A role for EMMPRIN at epithelial dermal junctions in tissue repair during wound healing seems highly plausible. Taken together

The Morphology of Smoke Inhalation Injury in Sheep,

DTIC Science & Technology

1991-01-01

exposure. Segments of intact epithelium (E) areethane. The specimens were dried by the critical point method adjacent to necrotic areas ( N ). The tracheal...magnification: X325. essentially normal lung ( N ). Original magnification: x125. 1484 The Journal of Trauma November 1991 The extent and severity of the injury...obstruction by desqapate netic endofronclia n tise acuand esulant ypoxa.’by desquamated necrotic endobronchial tissue. Accu- and resultant hypoxia.5 mulation

Gravitational effects on body composition in birds

NASA Technical Reports Server (NTRS)

Smith, A. H.; Sanchez P., O.; Burton, R. R.

1975-01-01

Gallinaceous birds, presenting a wide range of body size, were adapted physiologically to hyperdynamic environments, provided by chronic centrifugation. Chemical composition was measured directly on prepared carcasses, which were anatomically comparable, and more amenable to analysis than the intact body. Body mass and body fat decreased arithmetically with increasing field strength and also with increasing body mass. Water content of lean tissue increased in hyperdynamic environments, but irrespectively of body size.

Optimization of Evans blue quantitation in limited rat tissue samples

PubMed Central

Wang, Hwai-Lee; Lai, Ted Weita

2014-01-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting. PMID:25300427

The European wool-carder bee (Anthidium manicatum) eavesdrops on plant volatile organic compounds (VOCs) during trichome collection.

PubMed

Graham, Kelsey K; Brown, Steve; Clarke, Stephanie; Röse, Ursula S R; Starks, Philip T

2017-11-01

The plant-pollinator relationship is generally considered mutualistic. This relationship is less clear, however, when pollinators also cause tissue damage. Some Megachilidae bees collect plant material for nests from the plants they pollinate. In this study, we examined the relationship between Anthidium manicatum, the European wool-carder bee, and the source of its preferred nesting material - Stachys byzantina, lamb's ear. Female A. manicatum use their mandibles to trim trichomes from plants for nesting material (a behaviour dubbed "carding"). Using volatile organic compound (VOC) headspace analysis and behavioural observations, we explored (a) how carding effects S. byzantina and (b) how A. manicatum may choose specific S. byzantina plants. We found that removal of trichomes leads to a dissimilar VOC bouquet compared to intact leaves, with a significant increase in VOC detection following damage. A. manicatum also visit S. byzantina plants with trichomes removed at a greater frequency compared to plants with trichomes intact. Our data suggest that A. manicatum eavesdrop on VOCs produced by damaged plants, leading to more carding damage for individual plants due to increased detectability by A. manicatum. Accordingly, visitation by A. manicatum to S. byzantina may incur both a benefit (pollination) and cost (tissue damage) to the plant. Copyright © 2017 Elsevier B.V. All rights reserved.

[Accelerated senescence of fresh-cut Chinese water chestnut tissues in relation to hydrogen peroxide accumulation].

PubMed

Peng, Li-Tao; Jiang, Yue-Ming; Yang, Shu-Zhen; Pan, Si-Yi

2005-10-01

Accelerated senescence of fresh-cut Chinese water chestnut (CWC) tissues in relation to active oxygen species (AOS) metabolism was investigated. Fresh-cut CWC (2 mm thick) and intact CWC were stored at 4 degrees C in trays wrapped with plastic films. Changes in superoxide anion production rate, activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were monitored, while contents of hydrogen peroxide, ascorbic acid, MDA as well as electrolyte leakage were measured. Fresh-cutting of CWC induced activities of SOD, CAT and APX to a certain extent (Fig. 2B and Fig. 3), but simultaneously stimulated superoxide anion production markedly (Fig. 2A), enhanced hydrogen peroxide accumulation and accelerated loss in ascorbic acid (Figs. 4 and 5), which resulted in increased lipid peroxidation indicated by malondialdehyde (MDA) content and electrolyte leakage (Fig. 1). Statistics analysis indicated that there was a significantly positive correlation among hydrogen peroxide accumulation, MDA content and electrolyte leakage (Table 1). Histochemical detection with 3, 3'-diaminobenzidine further demonstrated that hydrogen peroxide accumulation increased in fresh-cut CWC during storage (Fig. 5). AOS production rate and activities of SOD, CAT and APX changed little while no obvious hydrogen peroxide accumulation was observed, in intact CWC during storage.

Preliminary observations on whole-ovary xenotransplantation as an experimental model for fertility preservation.

PubMed

Nichols-Burns, Stephanie M; Lotz, Laura; Schneider, Heike; Adamek, Edyta; Daniel, Christoph; Stief, Andrea; Grigo, Christina; Klump, Dorothee; Hoffmann, Inge; Beckmann, Matthias W; Dittrich, Ralf

2014-11-01

Ovarian tissue preservation and retransplantation is a promising strategy to restore fertility in cancer survivors. Ischaemia accompanying ovarian tissue grafting, however, can lead to significant follicle loss. Transplantation of the whole ovary by vascular anastomosis has been considered as an alternative to prevent widespread ischaemic damage. In this study, the feasibility and function of transplanting whole ovary with intact vasculature were evaluated, with the goal of developing a xenograft model for studies using donated human ovaries. Whole-swine ovaries with vascular pedicles were perfused and transplanted as intact ovaries by anastomosis into irradiated ovariectomized nude rats (n = 10). The observation period was between 1 and 4 weeks. Fresh swine ovaries served as controls (n = 10). Ovarian stroma and follicle populations were assessed through histological examination in both transplanted and control ovaries. Most of the transplanted whole ovaries (n = 6) maintained stromal quality and all preantral follicle classes were represented, although follicle numbers decreased compared with fresh control. Four transplanted ovaries were fibrotic after 1-4 weeks within the nude rat. Our results demonstrate transplantation of whole-pig ovary into nude rats is possible and support development of this xenograft model system for human studies. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Optimization of Evans blue quantitation in limited rat tissue samples

NASA Astrophysics Data System (ADS)

Wang, Hwai-Lee; Lai, Ted Weita

2014-10-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Down-regulate of Djrfc2 causes tissues hypertrophy during planarian regeneration.

PubMed

Guo, Qi; Zhao, Guixia; Ni, Jiajia; Guo, Yanan; Zhang, Yizhe; Tian, Qingnan; Zhang, Shoutao

2017-11-25

Planarians are an ideal model organism for regeneration research due to their amazing ability to regenerate. DNA replication is crucial for genome stability. Replication factor C (RFC), which is a replication factor C-like complex and plays an important role during DNA replication in eukaryotes, has been reported as a wound response factor during planarian regeneration. However, how RFC controls regeneration in planarians by regulating DNA replication remains to be explained. Here, we used a two-dimensional electrophoresis (2-DE) proteomic approach to identify differentially expressed proteins in intact and regenerated planarians. Approximately 132 protein spots showed differences between intact and regenerative tissues. We selected 21 significantly expressed protein spots and processed them using TOF MS analysis. Finally, we cloned three of these candidate genes (Djhsp70, Djrfc2, Djfaim), focusing on the function of Djrfc2 during regeneration. We found that the distribution of Djrfc2 tends toward the wound site. RNA interference (RNAi) of Djrfc2 increases the number of dividing cells and the expression level of planarian neoblast marker genes, which may result in hyper-proliferation. Our studies use an available approach to directly study the regeneration dynamic at the protein level and provide further evidence to support a function of Djrfc2 in planarian regeneration. Copyright © 2017. Published by Elsevier Inc.

The Subunit Principle in Scar Face Revision.

PubMed

Elshahat, Ahmed; Lashin, Riham

2017-06-01

Facial scaring is considered one of the most difficult cosmetic problems for any plastic surgeon to solve. The condition is more difficult if the direction of the scar is not parallel to relaxed skin tension lines. Attempts to manage this difficult situation included revisions using geometric designs, Z plasties or W plasties to camouflage the straight line visible scaring. The use of long-lasting resorbable sutures was tried too. Recently, the use of botulinum toxin during revision improved the results. Fractional CO2 lasers, microfat grafts, and platelet-rich plasma were added to the armamentarium. The scar is least visible if placed in the junction between the facial subunits. The aim of this study is to investigate the use of the subunit principle to improve the results of scar revision. Four patients were included in this study. Tissue expansion of the intact part of the subunit allowed shifting the scar to the junction between the affected subunit and the adjacent one. Tissue expansion, delivery of the expanders, and advancement of the flaps were successful in all patients. The fact that this is a 2-stage procedure and sacrifices some of the intact skin from the affected facial subunit, makes this technique reserved to patients with ugly facial scars who are ambitious to improve their appearance.

NanoLuc reporter for dual luciferase imaging in living animals.

PubMed

Stacer, Amanda C; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E; Rehemtulla, Alnawaz; Luker, Gary D

2013-10-01

Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc [NL]) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.

NanoLuc Reporter for Dual Luciferase Imaging in Living Animals

PubMed Central

Stacer, Amanda C.; Nyati, Shyam; Moudgil, Pranav; Iyengar, Rahul; Luker, Kathryn E.; Rehemtulla, Alnawaz; Luker, Gary D.

2014-01-01

Bioluminescence imaging is utilized widely for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on high signal-to-background of this technique. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events as done commonly with fluorescence imaging. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, ATP-independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc, NL) as a reporter for animal imaging. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We combined NL and firefly luciferase reporters to quantify two key steps in TGF-β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Our results establish NL as new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development. PMID:24371848

Chemically rich seaweeds poison corals when not controlled by herbivores

PubMed Central

Rasher, Douglas B.; Hay, Mark E.

2010-01-01

Coral reefs are in dramatic global decline, with seaweeds commonly replacing corals. It is unclear, however, whether seaweeds harm corals directly or colonize opportunistically following their decline and then suppress coral recruitment. In the Caribbean and tropical Pacific, we show that, when protected from herbivores, ~40 to 70% of common seaweeds cause bleaching and death of coral tissue when in direct contact. For seaweeds that harmed coral tissues, their lipid-soluble extracts also produced rapid bleaching. Coral bleaching and mortality was limited to areas of direct contact with seaweeds or their extracts. These patterns suggest that allelopathic seaweed-coral interactions can be important on reefs lacking herbivore control of seaweeds, and that these interactions involve lipid-soluble metabolites transferred via direct contact. Seaweeds were rapidly consumed when placed on a Pacific reef protected from fishing but were left intact or consumed at slower rates on an adjacent fished reef, indicating that herbivory will suppress seaweeds and lower frequency of allelopathic damage to corals if reefs retain intact food webs. With continued removal of herbivores from coral reefs, seaweeds are becoming more common. This occurrence will lead to increasing frequency of seaweed-coral contacts, increasing allelopathic suppression of remaining corals, and continuing decline of reef corals. PMID:20457927

Chemically rich seaweeds poison corals when not controlled by herbivores.

PubMed

Rasher, Douglas B; Hay, Mark E

2010-05-25

Coral reefs are in dramatic global decline, with seaweeds commonly replacing corals. It is unclear, however, whether seaweeds harm corals directly or colonize opportunistically following their decline and then suppress coral recruitment. In the Caribbean and tropical Pacific, we show that, when protected from herbivores, approximately 40 to 70% of common seaweeds cause bleaching and death of coral tissue when in direct contact. For seaweeds that harmed coral tissues, their lipid-soluble extracts also produced rapid bleaching. Coral bleaching and mortality was limited to areas of direct contact with seaweeds or their extracts. These patterns suggest that allelopathic seaweed-coral interactions can be important on reefs lacking herbivore control of seaweeds, and that these interactions involve lipid-soluble metabolites transferred via direct contact. Seaweeds were rapidly consumed when placed on a Pacific reef protected from fishing but were left intact or consumed at slower rates on an adjacent fished reef, indicating that herbivory will suppress seaweeds and lower frequency of allelopathic damage to corals if reefs retain intact food webs. With continued removal of herbivores from coral reefs, seaweeds are becoming more common. This occurrence will lead to increasing frequency of seaweed-coral contacts, increasing allelopathic suppression of remaining corals, and continuing decline of reef corals.

Status quo of management of the human tissue banks in Taiwan.

PubMed

Chou, Ching-Pang; Chou, Szu-Cheng; Chen, Ying-Hua; Chen, Yu-Hsuan; Lee, Ming-Shin

2017-03-01

As the technologies associated with transplantation and biological tissue engineering continue to advance, human cells and tissues form an integral part to the practice of regenerative medicine. The patient's use of tissues entails the risk of introducing, transmitting and spreading communicable diseases. To prevent such risk and to ensure that the human organs, tissues and cells remain intact and functional after being handled and processed, the transplanted tissues must be subject to good management standards through all stages of collection, screening, processing, storage and distribution as the safety of the users is of the utmost importance. On February 2009, the government of Taiwan promulgated the Regulations for Administration on Human Organ Bank that requires all human tissues banks to adhere to the Good Tissue Practice for Human Organ, Tissue and Cell in terms of establishment and operation in order to cope with the international management trend and the development and management need of the domestic industry. Six years have passed since the law became effective. This article seeks to introduce the current management mechanism and status quo of management of human tissue banks in Taiwan. We also conducted statistical analysis of the data relating to the tissue banks to identify potential risks and the room for improvement. The study concludes that human tissue banks in Taiwan are on the right track with their management practice, leading to a state of steady development and progress.

Functional plasticity of regenerated and intact taste receptors in adult rats unmasked by dietary sodium restriction.

PubMed

Hill, D L; Phillips, L M

1994-05-01

Unilateral chorda tympani nerve sectioning was combined with institution of a sodium-restricted diet in adult rats to determine the role that environment has on the functional properties of regenerating taste receptor cells. Rats receiving chorda tympani sectioning but no dietary manipulation (cut controls) and rats receiving only the dietary manipulation (diet controls) had normal responses to a concentration series of NaCl, sodium acetate (NaAc), and NH4Cl. However, responses from the regenerated nerve in NaCl-restricted rats (40-120 d postsectioning) to NaCl and NaAc were reduced by as much as 30% compared to controls, indicating that regenerating taste receptors are influenced by environmental (dietary) factors. Responses to NH4Cl were normal; therefore, the effect appears specific to sodium salts. Surprisingly, in the same rats, NaCl responses from the contralateral, intact chorda tympani were up to 40% greater than controls. Thus, in the same rat, there was over a twofold difference in sodium responses between the right and left chorda tympani nerves. A study of the time course of the functional alterations in the intact nerve revealed that responses to NaCl were extremely low immediately following sectioning (about 20% of the normal response), and then increased monotonically during the following 50 d until relative response magnitudes became supersensitive. This function occurred even when the cut chorda tympani was prevented from reinnervating lingual epithelia, demonstrating that events related to regeneration do not play a role in the functional properties of the contralateral side of the tongue.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-Specific Regulation of Chromatin Insulator Function

PubMed Central

Matzat, Leah H.; Dale, Ryan K.; Moshkovich, Nellie; Lei, Elissa P.

2012-01-01

Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type–specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. Here we identify the RNA recognition motif-containing protein Shep as a direct interactor of two individual components of the gypsy insulator complex in Drosophila. Mutation of shep improves gypsy-dependent enhancer blocking, indicating a role as a negative regulator of insulator activity. Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP–seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator. PMID:23209434

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

Varma, S.D.; Mooney, J.M.

Studies have been conducted to examine the implications of photochemical generation of O2- and its derivatization to H/sub 2/O/sub 2/ and OH in the physiology of the lens in vitro. Physiological status was determined by measuring the uptake of rubidium by the intact tissue when cultured in riboflavin-containing medium, in dark and light, and in the presence and absence of various scavengers. In the presence of light, the uptake of rubidium in the lens was greatly diminished; this suggests photodamage to the tissue. MnSOD and ferricyanide protected against this photochemical damage. The damaging process was thus initiated by the generationmore » of O2-. The tissue damage was also attenuated by catalase, ferrocyanide, and mannitol. These results, therefore, suggest the participation of hydrogen peroxide and the subsequent Haber-Weiss reaction in the photodamaging process.« less

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting.

PubMed

Ong, Chin Siang; Fukunishi, Takuma; Nashed, Andrew; Blazeski, Adriana; Zhang, Huaitao; Hardy, Samantha; DiSilvestre, Deborah; Vricella, Luca; Conte, John; Tung, Leslie; Tomaselli, Gordon; Hibino, Narutoshi

2017-07-02

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions.

PubMed

Wagner, J M; Franco, M; Kephart, G M; Gleich, G J

1998-07-01

Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis. Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied. The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation. To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P. brasiliensis, we used an immunofluorescence technique to localize the P. brasiliensis organisms and eosinophils and major basic protein. Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P. brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed. Nine biopsy tissues from seven patients were analyzed. All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P. brasiliensis. Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils). The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P. brasiliensis had extracellular major basic protein deposited on the organisms. These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Quantitative, Structural and Image-based Mechanical Analysis of Nonunion Fracture Repaired by Genetically Engineered Mesenchymal Stem Cells

PubMed Central

Kallai, Ilan; van Lenthe, G. Harry; Ruffoni, Davide; Zilberman, Yoram; Müller, Ralph; Pelled, Gadi; Gazit, Dan

2010-01-01

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High resolution micro computed tomography (Micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro finite element (Micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2 to 1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post treatment. These results could be attributed to the fusion that occurred between in the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair. PMID:20471652

Odors influencing foraging behavior of the California spiny lobster, Panulirus interruptus, and other decapod crustacea

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

Zimmer-Faust, R.K.; Case, J.F.

1982-01-01

Trapping experiments were conducted in the More Mesa coastal area of Santa Barbara, California, 4 km east of the U.C. Santa Barbara campus. Live intact and injured prey and excised tissues were placed in traps, in containers allowing odor release but preventing contacts with entering animals. Individuals of six prey species failed to attract lobsters when alive and intact, but some became attractive once injured. Excised tissues were the most effective baits. Abalone and mackerel muscle were attractive to lobsters but relatively nonattractive to crabs, while angel shark muscle was attractive to crabs but not to lobsters. Shrimp cephalothoraces weremore » repellant to lobsters. Naturally occurring attractant and repellent tissues are thus identified and chemosensory abilities of lobsters and sympatric crabs are demonstrated to differ. Abalone muscle increased in attractivity following 1-2 days field exposure. Molecular weights of stimulants released by both weathered and fresh abalone were < 10,000 daltons with evidence suggesting that the 1000-10,000 dalton fraction may contribute significantly to attraction. Concentrations of total primary amines released from abalone muscle failed to differ from background levels, following an initial three (0-3h) period. Primary amines thus appear not to contribute directly to captures of lobsters, since animals were usually caught greater than or equal to 7 h after baits were positioned. Amino acids were the dominant contributors to present measurements of total primary amines, suggesting that these molecules may not direct lobster foraging behavior in the present experiments. 41 references, 4 figures, 8 tables.« less

Characterization of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) as an inhibitor of brain glycogen shunt activity.

PubMed

Walls, Anne B; Sickmann, Helle M; Brown, Angus; Bouman, Stephan D; Ransom, Bruce; Schousboe, Arne; Waagepetersen, Helle S

2008-05-01

The pharmacological properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), a potent inhibitor of glycogen phosphorylase and synthase activity in liver preparations, were characterized in different brain tissue preparations as a prerequisite for using it as a tool to investigate brain glycogen metabolism. Its inhibitory effect on glycogen phosphorylase was studied in homogenates of brain tissue and astrocytes and IC50-values close to 400 nM were found. However, the concentration of DAB needed for inhibition of glycogen shunt activity, i.e. glucose metabolism via glycogen, in intact astrocytes was almost three orders of magnitude higher. Additionally, such complete inhibition required a pre-incubation period, a finding possibly reflecting a limited permeability of the astrocytic membrane. DAB did not affect the accumulation of 2-deoxyglucose-6-phosphate indicating that the transport of DAB is not mediated by the glucose transporter. DAB had no effect on enzymes involving glucose-6-phosphate, i.e. glucose-6-phosphate dehydrogenase, phosphoglucoisomerase and hexokinase. Furthermore, DAB was evaluated in a functional preparation of the isolated mouse optic nerve, in which its presence severely reduced the ability to sustain evoked compound action potentials in the absence of glucose, a condition in which glycogen serves as an important energy substrate. Based on the experimental findings, DAB can be used to evaluate glycogen shunt activity and its functional importance in intact brain tissue and cells at a concentration of 300-1000 muM and a pre-incubation period of 1 h.

Cadaver embalming fluid for surgical training courses: modified Larssen solution.

PubMed

Bilge, Okan; Celik, Servet

2017-11-01

10% Formalin (F10)-fixed cadavers have disadvantages such as disturbing smell, mucosal irritation, discoloration and rigidity. We aimed to determine a suitable, simple and cost-effective embalming method that preserves color, texture, pliability and flexibility of the tissues for a long time without a disturbing smell and mucosal irritation. The embalmed cadavers were expected to be durable against environmental effects, utilizable for multiple and repetitive surgical trainings and instrumentations. Eight male (six intact, two autopsied bodies) and four female (three intact and one imported trunk) human cadavers were preserved with modified Larssen solution (MLS). Preserved bodies were kept in the deep freezers at -18/-20 °C. Bodies were allowed to thaw at room temperature 3 days prior to use. They were used in postgraduate hands-on courses for several medical disciplines. Each course lasted at least 1 day and during this period the bodies were stayed at room temperature. Assessments of 30 trainers and 252 trainees were collected during the courses. Additionally, the organoleptic characteristics of the fresh frozen (FF), preserved with MLS and F10-fixed cadavers were compared. The colors of muscles, fasciae, fatty tissue, nerves and vessels were evaluated and life-like tissues of MLS cadavers were impressive. There were no obvious or disturbing smell and sign of putrefaction of the MLS cadavers. MLS is a sustainable and relatively affordable soft cadaver embalming method. Its application is same as in other conventional methods and does not need new equipment. This article indicates the success of the MLS method in human cadavers.

Cardiac activation heat remains inversely dependent on temperature over the range 27-37°C.

PubMed

Johnston, Callum M; Han, June-Chiew; Loiselle, Denis S; Nielsen, Poul M F; Taberner, Andrew J

2016-06-01

The relation between heat output and stress production (force per cross-sectional area) of isolated cardiac tissue is a key metric that provides insight into muscle energetic performance. The heat intercept of the relation, termed "activation heat," reflects the metabolic cost of restoring transmembrane gradients of Na(+) and K(+) following electrical excitation, and myoplasmic Ca(2+) concentration following its release from the sarcoplasmic reticulum. At subphysiological temperatures, activation heat is inversely dependent on temperature. Thus one may presume that activation heat would decrease even further at body temperature. However, this assumption is prima facie inconsistent with a study, using intact hearts, which revealed no apparent change in the combination of activation and basal metabolism between 27 and 37°C. It is thus desired to directly determine the change in activation heat between 27 and 37°C. In this study, we use our recently constructed high-thermal resolution muscle calorimeter to determine the first heat-stress relation of isolated cardiac muscle at 37°C. We compare the relation at 37°C to that at 27°C to examine whether the inverse temperature dependence of activation heat, observed under hypothermic conditions, prevails at body temperature. Our results show that activation heat was reduced (from 3.5 ± 0.3 to 2.3 ± 0.3 kJ/m(3)) at the higher temperature. This leads us to conclude that activation metabolism continues to decline as temperature is increased from hypothermia to normothermia and allows us to comment on results obtained from the intact heart by previous investigators. Copyright © 2016 the American Physiological Society.

Laser-ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia.

PubMed

Stopka, Sylwia A; Agtuca, Beverly J; Koppenaal, David W; Paša-Tolić, Ljiljana; Stacey, Gary; Vertes, Akos; Anderton, Christopher R

2017-07-01

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen-fixing ability). The symbiotic interaction between legumes and nitrogen-fixing soil bacteria results in a specialized plant organ (i.e., root nodule) where the exchange of nutrients between host and endosymbiont occurs. Laser-ablation electrospray ionization mass spectrometry (LAESI-MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI-MS to explore the well characterized symbiosis between soybean (Glycine max L. Merr.) and its compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 differentially abundant spectral features in the nodule samples. The data presented demonstrate the advantages of using LAESI-IMS-MS for the rapid analysis of intact root nodules, uninfected root segments, and free-living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI-IMS-MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean-rhizobial symbiosis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Effect of Bioactive Glass air Abrasion on Shear Bond Strength of Two Adhesive Resins to Decalcified Enamel

PubMed Central

Eshghi, Alireza; Khoroushi, Maryam; Rezvani, Alireza

2014-01-01

Objective: Bioactive glass air abrasion is a conservative technique to remove initial decalcified tissue and caries. This study examined the shear bond strength of composite resin to sound and decalcified enamel air-abraded by bioactive glass (BAG) or alumina using etch-and-rinse and self-etch adhesives. Materials and Methods: Forty-eight permanent molars were root-amputated and sectioned mesiodistally. The obtained 96 specimens were mounted in acrylic resin; the buccal and lingual surfaces remained exposed. A demineralizing solution was used to decalcify half the specimens. Both sound and decalcified specimens were divided into two groups of alumina and bioactive glass air abrasion. In each group, the specimens were subdivided into two subgroups of Clearfil SE Bond or OptiBond FL adhesives (n=12). Composite resin cylinders were bonded on enamel surfaces cured and underwent thermocycling. The specimens were tested for shear bond strength. Data were analyzed using SPSS 16.0 and three-way ANOVA (α=0.05). Similar to the experimental groups, the enamel surface of one specimen underwent SEM evaluation. Results: No significant differences were observed in composite resin bond strength subsequent to alumina or bioactive glass air abrasion preparation techniques (P=0.987). There were no statistically significant differences between the bond strength of etch-and-rinse and self-etch adhesive groups (P=1). Also, decalcified or intact enamel groups had no significant difference (P=0.918). However, SEM analysis showed much less enamel irregularities with BAG air abrasion compared to alumina air abrasion. Conclusion: Under the limitations of this study, preparation of both intact and decalcified enamel surfaces with bioactive glass air abrasion results in similar bond strength of composite resin in comparison with alumina air abrasion using etch-&-rinse or self-etch adhesives. PMID:25628694

Laser-ablation electrospray ionization mass spectrometry with ion mobility separation reveals metabolites in the symbiotic interactions of soybean roots and rhizobia

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

Stopka, Sylwia A.; Agtuca, Beverly J.; Koppenaal, David W.

Technologies enabling in situ metabolic profiling of living plant systems are invaluable for understanding physiological processes and could be used for rapid phenotypic screening (e.g., to produce plants with superior biological nitrogen fixing ability). The symbiotic interaction between legumes and nitrogen-fixing soil bacteria results in a specialized plant organ (i.e., root nodule), where the exchange of nutrients between host and endosymbiont occurs. Laser ablation electrospray ionization mass spectrometry (LAESI-MS) is a method that can be performed under ambient conditions requiring minimal sample preparation. Here, we employed LAESI-MS to explore the well-characterized symbiosis between soybean (Glycine max L. Merr.) and itsmore » compatible symbiont, Bradyrhizobium japonicum. The utilization of ion mobility separation (IMS) improved the molecular coverage, selectivity, and identification of the detected biomolecules. Specifically, incorporation of IMS resulted in an increase of 153 detected metabolites in the nodule samples. The data presented demonstrates the advantages of using LAESI-IMS-MS for the rapid analysis of intact root nodules, uninfected root segments, and free-living rhizobia. Untargeted pathway analysis revealed several metabolic processes within the nodule (e.g., zeatin, riboflavin, and purine synthesis). Compounds specific to the uninfected root and bacteria were also detected. Lastly, we performed depth-profiling of intact nodules to reveal the location of metabolites to the cortex and inside the infected region, and lateral profiling of sectioned nodules confirmed these molecular distributions. Our results established the feasibility of LAESI-IMS-MS for the analysis and spatial mapping of plant tissues, with its specific demonstration to improve our understanding of the soybean-rhizobial symbiosis.« less

Handling Golgi-impregnated tissue for light microscopy.

PubMed

Berbel, P J; Fairén, A

1983-08-08

The use of cyanocrylic glue to fix pieces of Golgi-stained nervous tissue on a paraffin blank is proposed for obtaining thick sections of unembedded tissue with a sliding microtome. This procedure makes correct orientation of the tissue easy during sectioning and makes it possible to obtain tissue sections quickly. The sections are flat-mounted using epoxy resin, resulting in permanent preparations with excellent optical properties and enabling further thin-sectioning for light and electron microscopic studies.

A pilot to examine the logistical and feasibility issues in testing deceased tissue donors for vCJD using tonsil as the analyte.

PubMed

Warwick, Ruth M; Armitage, W John; Chandrasekar, Akila; Mallinson, Gary; Poniatowski, Stefan; Clarkson, Anthony

2012-03-01

Transplanted tissues have transmitted transmissible spongiform encephalopathies and in the UK there have been more cases of variant Creutzfeldt-Jakob disease (vCJD) than elsewhere in the world. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. This pilot showed that obtaining consent for removal and testing tonsil tissue was feasible. Donor eligibility for inclusion in the pilot was limited to tissue donors from the National Health Service Blood and Transplant, Tissue Services and to donors shared with the Corneal Transplant Service Eye Banks. Obtaining tonsillar tissue in the immediate post-mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the presence of prion associated with vCJD in deceased tissue donors. Production and processing of tissue was straightforward and a low assay background was obtained from most samples. Since palatine and lingual tonsil tissue can be obtained in pairs it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required. In one instance a sample was reactive by Western blot. However, the pattern of reactivity was not typical for that obtained from vCJD patients. Unfortunately the sample was not of sufficient quality for the confirmatory test to provide a conclusive result.

Mapping a multiplexed zoo of mRNA expression.

PubMed

Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A

2016-10-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.

Mapping a multiplexed zoo of mRNA expression

PubMed Central

Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.

2016-01-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788

Control of the rate of cell enlargement: Excision, wall relaxation, and growth-induced water potentials.

PubMed

Boyer, J S; Cavalieri, A J; Schulze, E D

1985-04-01

A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential (Ψ s ), turgor pressure (Ψ p ) could be calculated. In the intact plant, Ψ s and Ψ p were essentially constant for the entire 22 h measurement, but Ψ s was lower and Ψ p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, Ψ s and Ψ p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue Ψ p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The Ψ p decreased because the cell walls relaxed as extension, caused by Ψ p , continued briefly without water uptake. The Ψ p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or Ψ p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in Ψ p.

The collagen structure of equine articular cartilage, characterized using polarization-sensitive optical coherence tomography

NASA Astrophysics Data System (ADS)

Ugryumova, Nadya; Attenburrow, Don P.; Winlove, C. Peter; Matcher, Stephen J.

2005-08-01

Optical coherence tomography and polarization-sensitive optical coherence tomography images of equine articular cartilage are presented. Measurements were made on intact joint surfaces. Significant (e.g. × 2) variations in the intrinsic birefringence were found over spatial scales of a few millimetres, even on samples taken from young (18 month) animals that appeared visually homogeneous. A comparison of data obtained on a control tissue (equine flexor tendon) further suggests that significant variations in the orientation of the collagen fibres relative to the plane of the joint surface exist. Images of visually damaged cartilage tissue show characteristic features both in terms of the distribution of optical scatterers and of the birefringent components.

Two Photon Intravital Microscopy of Lyme Borrelia in Mice.

PubMed

Belperron, Alexia A; Mao, Jialing; Bockenstedt, Linda K

2018-01-01

Two-photon intravital microscopy is a powerful tool that allows visualization of cells in intact tissues in a live animal in real time. In recent years, this advanced technology has been applied to understand pathogen-host interactions using fluorescently labeled bacteria. In particular, infectious fluorescent transformants of the Lyme disease spirochete Borrelia burgdorferi, an Ixodes tick-transmitted pathogen, have been imaged by two-photon intravital microscopy to study bacterial motility and interactions of the pathogen with feeding ticks and host tissues. Here, we describe the techniques and equipment used to image mammalian-adapted spirochetes in the skin of living mice in vivo and in joints ex vivo using two-photon intravital microscopy.

The significance of amperometric detection of alkaline phosphatase in colorectal cancer diagnostics

NASA Astrophysics Data System (ADS)

Belkin, Anton; Freynd, Genrietta; Katsnelson, Mikhail

2016-08-01

Colorectal cancer (CRC) is one of the most common cancers in the world; it takes the second place in oncological morbidity. The ALP activity of intestinal epithelial differentiation marker (ALP) was investigated in surgical material and colon biopsies of 47 patients with the electrochemical method using biosensors (biochips). The average current obtained in the studies of colorectal cancer tissues was lower than in the studies of intact colon mucosa. Histology of tumors matched the differentiation types/stages of adenocarcinomas. The study of ALP activity in the surgical material and biopsies of colon tumors can become one of the most useful methods for evaluating the functional atypia in tumor tissue.

A method to detect transfected chloramphenicol acetyltransferase gene expression in intact animals

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

Narayanan, R.; Jastreboff, M.M.; Chiu, Chang Fang

1988-01-01

A rapid procedure is described for assaying chloramphenicol acetyltransferase enzyme activity in intact animals following transfection of the RSV CAT plasmid into mouse bone marrow cells by electroporation. The reconstituted mice were injected with ({sup 14}C)chloramphenicol and ethyl acetate extracts of 24-h urine samples were analyzed by TLC autoradiography for the excretion of {sup 14}C-labeled metabolites. CAT expression in vivo can be detected by the presence of acetylated {sup 14}C-labeled metabolites in the urine within 1 week after bone marrow transplantation and, under the conditions described, these metabolites can be detected for at least 3 months. CAT expression in intactmore » mice as monitored by the urine assay correlates with the CAT expression in the hematopoietic tissues assayed in vitro. This method offers a quick mode of screening for introduced CAT gene expression in vivo without sacrificing the mice.« less

Label-free in vivo optical micro-angiography imaging of cerebral capillary blood flow within meninges and cortex in mice with the skull left intact

NASA Astrophysics Data System (ADS)

Jia, Yali; Wang, Ruikang K.

2011-03-01

Abnormal microcirculation within meninges is common in many neurological diseases. There is a need for an imaging method that is capable of visualizing functional meningeal microcirculations alone, preferably decoupled from the cortical blood flow. Optical microangiography (OMAG) is a recently developed label-free imaging method capable of producing 3D images of dynamic blood perfusion within micro-circulatory tissue beds at an imaging depth up to ~2 mm, with an unprecedented imaging sensitivity to the blood flow at ~4 μm/s. In this study, we demonstrate the utility of ultra-high sensitive OMAG in imaging the detailed blood flow distributions, at a capillary level resolution, within meninges and cortex in mice with the cranium left intact. The results indicate that OMAG can be a valuable tool for the study of meningeal circulations.

Functional anatomy and ultrasound examination of the canine penis.

PubMed

Goericke-Pesch, Sandra; Hölscher, Catharina; Failing, Klaus; Wehrend, Axel

2013-07-01

The aim of this study was to identify the functional-anatomical structures of the canine penis during and after erection to demonstrate the respective changes to provide a basis for further examinations of pathological conditions like priapism. Additionally, a gray-scale analysis was performed to quantify results from the ultrasound examination. In total, 80 dogs were examined. In group (Gr.) A, 44 intact or castrated dogs were examined, and in Gr. B, 36 dogs were examined during erection and after complete detumescence of the penis. The following parameters were assessed: (1) using physical measurements: length of the Pars longa glandis [Plg] and length of the Bulbus glandis [Bg]; and (2) using ultrasound: total penile diameter, width of the erectile tissue of the Plg, diameter of the Corpus spongiosum [Cs] including the penile bone and urethra, vertical diameter, circumference of the penis, cross-sectional area, and area of the Cs including the urethra. The mentioned parameters could be assessed in all dogs of Gr. A and Gr. B with the only exception being the urethra that could be visualized using ultrasound in some dogs only and predominantly in the erected penis (Gr. B). Concomitantly, the erectile tissue of the Plg and the Cs was more heterogenous and hypo- to anechoic during erection compared with dogs in Gr. A and Gr. B after detumescence. Comparing the results in Gr. B, the length of the Plg and the Bg were decreased approximately 40.6% and 38.0%, the total width of the penis 40.5%, the total width of the erectile tissue of the Plg 48.0%, and the width of the Cs 15.6% during detumescence compared with erection. Comparing the decrease in size at the different locations (apex penis, middle of Plg, middle of Bg) for vertical diameter, total circumference, and cross-section area, it was largest at the Bg. B-mode ultrasound is a suitable tool to investigate not only the morpho-functional structures of the resting canine penis, but also of the erected and detumescent penis, and to investigate the underlying changes during erection and detumenscence. Copyright © 2013 Elsevier Inc. All rights reserved.

Hyperforin Accumulates in the Translucent Glands of Hypericum perforatum

PubMed Central

Soelberg, Jens; Jørgensen, Lise Bolt; Jäger, Anna K.

2007-01-01

Background and Aims Hypericum perforatum contains the therapeutically important compounds hypericin and hyperforin. Hypericin is known to accumulate in the dark glands. This investigation aimed to determine the accumulation site of hyperforin. Methods Dark and translucent glands as well as non-secretory tissue in leaves were manually isolated under the microscope. Hyperforin content was quantified by UV HPLC. Secretory structures were surveyed anatomically. Key Results The hyperforin content of intact leaves was found to be about 3 mg g−1 fresh tissue, whereas a content of about 7 mg g−1 fresh material was found in isolated translucent glands. Hyperforin was found only to occur in minute amounts in dark glands (approx. 0·4 mg g−1 fresh tissue). In non-secretory tissue no hyperforin was detected. Conclusions The accumulation of hyperforin detected in the translucent glands supports the proposed hypothesis that hyperforin is synthesized by the same biosynthetic machinery as monoterpenes in the chloroplasts of cells delimiting the gland. PMID:17468113

Transcriptome In Vivo Analysis (TIVA) of spatially defined single cells in intact live mouse and human brain tissue

PubMed Central

Lovatt, Ditte; Ruble, Brittani K.; Lee, Jaehee; Dueck, Hannah; Kim, Tae Kyung; Fisher, Stephen; Francis, Chantal; Spaethling, Jennifer M.; Wolf, John A.; Grady, M. Sean; Ulyanova, Alexandra V.; Yeldell, Sean B.; Griepenburg, Julianne C.; Buckley, Peter T.; Kim, Junhyong; Sul, Jai-Yoon; Dmochowski, Ivan J.; Eberwine, James

2014-01-01

Transcriptome profiling is an indispensable tool in advancing the understanding of single cell biology, but depends upon methods capable of isolating mRNA at the spatial resolution of a single cell. Current capture methods lack sufficient spatial resolution to isolate mRNA from individual in vivo resident cells without damaging adjacent tissue. Because of this limitation, it has been difficult to assess the influence of the microenvironment on the transcriptome of individual neurons. Here, we engineered a Transcriptome In Vivo Analysis (TIVA)-tag, which upon photoactivation enables mRNA capture from single cells in live tissue. Using the TIVA-tag in combination with RNA-seq to analyze transcriptome variance among single dispersed cells and in vivo resident mouse and human neurons, we show that the tissue microenvironment shapes the transcriptomic landscape of individual cells. The TIVA methodology provides the first noninvasive approach for capturing mRNA from single cells in their natural microenvironment. PMID:24412976

Tissue vibration pulsatility for arterial bleeding detection using Doppler ultrasound.

PubMed

Xie, Zhiyong; Kim, Eung-Hun; Kim, Yongmin

2009-01-01

Trauma is the number one cause of death among Americans between 1 and 44 years old, and exsanguination due to internal bleeding resulting from arterial injuries is a major factor in trauma deaths. We have evaluated the feasibility of using tissue vibration pulsatility in arterial bleeding detection. Eight femoral arteries from four juvenile pigs were punctured transcutaneously with a 6 or 9-French catheter. Also, 11 silicone vessels wrapped with turkey breast were placed in a pulsatile flow phantom and penetrated with an 18-gauge needle. The tissue vibration pulsatility was derived as a ratio of the maximum spectral energy from 200 to 2500 Hz of tissue vibration in systole over a baseline value in diastole. Then, the tissue vibration pulsatility index (TVPI) was defined as the maximum tissue vibration pulsatility value for each experimental condition. Both in vitro and in vivo results showed that the TVPI from injured vessels is significantly higher (p<0.005) than that of intact vessels. In addition, we constructed the 2D map of tissue vibration pulsatility during in vitro studies and found that it could be used for spatial localization of the puncture site. Our preliminary results indicate that the tissue vibration pulsatility may be useful for detecting arterial bleeding and localizing the bleeding site.

An accurate homogenized tissue phantom for broad spectrum autofluorescence studies: a tool for optimizing quantum dot-based contrast agents

NASA Astrophysics Data System (ADS)

Roy, Mathieu; Wilson, Brian C.

2008-02-01

We are investigating the use of ZnS-capped CdSe quantum dot (QD) bioconjugates combined with fluorescence endoscopy for improved early cancer detection in the esophagus, colon and lung. A major challenge in using fluorescent contrast agents in vivo is to extract the relevant signal from the tissue autofluorescence (AF). The present studies are aimed at maximizing the QD signal to AF background ratio (SBR) to facilitate detection. These contrast optimization studies require optical phantoms that simulate tissue autofluorescence, absorption and scattering over the entire visible spectrum, while allowing us to control the optical thickness. We present an optical phantom made of fresh homogenized tissue diluted in water. The homogenized tissue is poured into a clear polymer tank designed to hold a QD-loaded silica capillary in its center. Because of the non-linear effects of absorption and scattering on measured autofluorescence, direct comparison between results obtained using tissue phantoms of different concentration is not possible. We introduce mathematical models that make it possible to perform measurements on diluted tissue homogenates and subsequently extrapolate the results to intact (non-diluted) tissue. Finally, we present preliminary QD contrast data showing that the 380-420 nm spectral window is optimal for surface QD imaging.

Effect of heat treatment on the n-3/n-6 ratio and content of polyunsaturated fatty acids in fish tissues.

PubMed

Schneedorferová, Ivana; Tomčala, Aleš; Valterová, Irena

2015-06-01

The aim of this study was to compare the effect of different heat treatments (pan-frying, oven-baking, and grilling) on the contents of polyunsaturated fatty acids (PUFAs) in fish tissue. Four fish species were examined: pike, carp, cod, and herring. High performance liquid chromatography, coupled with electrospray ionization and mass spectrometric detection (HPLC/ESI/MS), was employed for determination of intact lipid molecules containing n-3 and n-6 PUFAs. Although mostly non-polar lipids (triacylglycerols, TGs) were present in the fish tissue, the PUFAs were present preferentially in the phospholipid fraction. Omnivorous fish species (carp, herring) contained more TGs than did predatory ones (pike, cod). Higher amounts of PUFAs were detected in the marine species than in the freshwater ones. The impact of heat treatments on the lipid composition in the fish tissue seems to be species-specific, as indicated by multivariate data analysis. Herring tissue is most heat-stable, and the mildest heat treatment for PUFA preservation was oven-baking. Copyright © 2014 Elsevier Ltd. All rights reserved.

A standardized procedure for using human corpus cavernosum strips to evaluate drug activity.

PubMed

Mirone, V; Sorrentino, R; di Villa Bianca, R; Imbimbo, C; Palmieri, A; Fusco, F; Tajana, G; Cirino, G

2000-01-01

The main problem of using human corpus cavernosum (HCC) tissue to perform bioassay is linked to its limited availability further complicated by the heterogeneous source of the tissues used. Here, we show that gender reassignment is a reliable source of human tissue without major ethical problems. Indeed, the entire corpus cavernosum is obtained from the surgery procedure, which allows creating a standardized procedure to prepare HCC strip. In addition, human tissue, if kept in the fridge in the condition described, does not loose its ability to contract to phenylephrine (PE; alpha agonist), angiotensin II (AG II) and KCl up to 4 days. Furthermore, once contracted with PE, HCC relaxes to acetylcholine (endothelium-dependent mechanism); sodium nitroprusside (endothelium-independent mechanism); cromakalim (CRK), a K(ATP) channel opener; or alprostadil, a synthetic PGE2 (ALPR). In conclusion, we have standardized a procedure that allows the use of HCC strips to evaluate drug activity and/or to study pathophysiological mechanisms with an intact functional human tissue up to 4 days from the surgery procedure.

Protocol for Monitoring Gulf War Veterans with Imbedded Fragments of Depleted Uranium

DTIC Science & Technology

1993-03-01

interest is evidence of total or partial fibrotic encapsulation ; local tissue necrosis; growing granuloma; or if there is evidence of a breakdown, a...formed fibrotic capsule. - If the fragment is encapsulated , remove and save the intact capsule (with the fragment still inside) if possible. If the...0704-0188 Public reporng burden for this owllectlon of infotmation iS estimated to average 1 hour par response , ircluding the time for revewing

A Clinically Realistic Large Animal Model of Intra-Articular Fracture

DTIC Science & Technology

2012-10-01

articular bone intact. The distal impact face is anchored to the talus using three “tripod” pins, for direct (i.e., no soft tissue intervention) delivery of...pilon) fractures. In this technique, the porcine hock joint (human ankle analogue) is subjected to an injurious transarticular compressive force pulse...fracture, to create fractures morphologically similar to human ankle anterior malleolar fractures. This cut was made on the anterior distal tibial cortex

Laboratory study on leachability of five herbicides in South Australian soils.

PubMed

Ying, G G; Williams, B

2000-03-01

Norflurazon, oxadiazon, oxyfluorfen, trifluralin and simazine are herbicides widely used in the vineyards of the Barossa Valley, South Australia. The leaching behaviour of norflurazon, oxadiazon, oxyfluorfen and trifluralin was investigated on four key soils in the Barossa Valley. Leaching potential on packed soil columns and actual mobility using intact soil columns were investigated. On the packed soil columns, norflurazon was the most leachable herbicide. More of the herbicides were detected in the leachates from the sandy soils (Mountadam and Nuriootpa) than from the clayey soils (Lyndoch and Tanunda). Organic matter is generally low in soils in the Barossa region. Porosity and saturated conductivity significantly affect herbicide movement and in the sandy Mountadam and Nuriootpa soils, the water flux is greater than for the higher clay content Lyndoch and Tanunda soils. Increasing the time interval between herbicide application and the incidence of "rainfall" reduced the amounts of herbicides found in the leachates. The use of intact soil columns and including simazine for comparison showed that both norflurazon and simazine were present in the leachates. Simazine was the first herbicide to appear in leachates. Sectioning of the intact soil columns after leaching clearly demonstrated that norflurazon and simazine reached the bottom of the soil columns for all soils studied. Greater amounts of norflurazon were retained in the soil columns compared with simazine. The other herbicides were mostly retained in the initial sections of the soil columns.

Decomposition of water-soluble mononitrosyl iron complexes with dithiocarbamates and of dinitrosyl iron complexes with thiol ligands in animal organisms.

PubMed

Serezhenkov, Vladimir A; Timoshin, Alexander A; Orlova, Tsvetina R; Mikoyan, Vasak D; Kubrina, Lioudmila N; Poltorakov, Alexander P; Ruuge, Enno K; Sanina, Natalia A; Vanin, Anatoly F

2008-05-01

EPR studies have shown that water-soluble mononitrosyl iron complexes with N-methyl-d-glucamine dithiocarbamate (MNIC-MGD) (3 micromol) injected to intact mice were decomposed virtually completely within 1h. The total content of MNIC-MGD in animal urine did not exceed 30 nmol/ml. In the liver, a small amount of MNIC-MGD were converted into dinitrosyl iron complexes (30 nmol/g of liver tissue). The same was observed in intact rabbits in which MNIC-MGD formation was induced by endogenous or exogenous NO binding to NO traps, viz., iron complexes with MGD. In mice, the content of MNIC-MGD in urine samples did not change after bacterial lipopolysaccharide-induced expression of iNOS. It was supposed that MNIC-MGD decomposition in intact animals was largely due to the release of NO from the complexes and its further transfer to other specific acceptors. In mice with iNOS expression, the main contribution to MNIC-MGD decomposition was made by superoxide ions whose destructive effect is mediated by an oxidative mechanism. This effect could fully compensate the augmented synthesis of MNIC-MGD involving endogenous NO whose production was supported by iNOS. Water-soluble dinitrosyl iron complexes (DNIC) with various thiol-containing ligands and thiosulfate injected to intact mice were also decomposed; however, in this case the effect was less pronounced than in the case of MNIC-MGD. It was concluded that DNIC decomposition was largely due to the oxidative effect of superoxide ions on these complexes.

An Intact Dorsomedial Posterior Arcuate Nucleus is Not Necessary for Photoperiodic Responses in Siberian Hamsters1

PubMed Central

Teubner, Brett J.W.; Leitner, Claudia; Thomas, Michael A.; Ryu, Vitaly; Bartness, Timothy J.

2015-01-01

Seasonal responses of many animal species are triggered by changes in daylength and its transduction into a neuroendocrine signal by the pineal gland through the nocturnal duration of melatonin (MEL) release. The precise central sites necessary to receive, transduce, and relay the short day (SD) fall-winter MEL signals into seasonal responses and changes in physiology and behavior are unclear. In Siberian hamsters, SDs trigger decreases in body and lipid mass, testicular regression and pelage color changes. Several candidate genes and their central sites of expression have been proposed as components of the MEL transduction system with considerable recent focus on the arcuate nucleus (ARC) and its component, the dorsomedial posterior arcuate nucleus (dmpARC). This site has been postulated as a critical relay of SD information through the modulation of a variety of neurochemicals/receptors important for the control of energy balance. Here the necessity of an intact dmpARC for SD responses was tested by making electrolytic lesions of the Siberian hamster dmpARC and then exposing them to either long days (LD) or SDs for 12 weeks. The SD typical decreases in body and fat mass, food intake, testicular volume, serum testosterone concentrations, pelage color change and increased UCP-1 protein expression (a proxy for brown adipose tissue thermogenesis) all occurred despite the lack of an intact dmpARC. Although the Siberian hamster dmpARC contains photoperiod-modulated constituents, these data demonstrate that an intact dmpARC is not necessary for SD responses and not integral to the seasonal energy- and reproductive-related responses measured here. PMID:25647158

Sympathetic denervation of one white fat depot changes norepinephrine content and turnover in intact white and brown fat depots

PubMed Central

Harris, Ruth B.S.

2013-01-01

It is well established that the sympathetic nervous system regulates adipocyte metabolism and recently it has been reported that sensory afferents from white fat overlap anatomically with sympathetic efferents to white fat. The studies described here characterize the response of intact fat pads to selective sympathectomy (local 6-hydroxydopamine injections) of inguinal (ING) or epididymal (EPI) fat in male NIH Swiss mice and provide in vivo evidence for communication between individual white and brown fat depots. The contralateral ING pad, both EPI pads, perirenal and mesenteric pads were significantly enlarged four weeks after denervating one ING pad, but only intrascapular brown fat (IBAT) increased when both ING pads were denervated. Denervation of one or both EPI pad had no effect on fat depot weights. In an additional experiment, NE turnover was inhibited in ING, retroperitoneal, mesenteric and IBAT two days after denervation of both EPI or of both ING pads. NE content was reduced to 10-30% of control values in all fat depots. There was no relation between early changes in NE turnover and fat pad weight 4 weeks after denervation, even though the reduction in NE content of intact fat pads was maintained. These data demonstrate that there is communication among individual fat pads, presumably through central integration of activity of sensory afferent and sympathetic efferent fibers,that changes sympathetic drive to white adipose tissue in a unified manner. In specific situations, removal of sympathetic efferents to one pad induces a compensatory enlargement of other intact depots. PMID:22513494

Release of low molecular weight silicones and platinum from silicone breast implants.

PubMed

Lykissa, E D; Kala, S V; Hurley, J B; Lebovitz, R M

1997-12-01

We have conducted a series of studies addressing the chemical composition of silicone gels from breast implants as well as the diffusion of low molecular weight silicones (LM-silicones) and heavy metals from intact implants into various surrounding media, namely, lipid-rich medium (soy oil), aqueous tissue culture medium (modified Dulbecco's medium, DMEM), or an emulsion consisting of DMEM plus 10% soy oil. LM-silicones in both implants and surrounding media were detected and quantitated using gas chromatography (GC) coupled with atomic emission (GC-AED) as well as mass spectrometric (GC/MS) detectors, which can detect silicones in the nanogram range. Platinum, a catalyst used in the preparation of silicone gels, was detected and quantitated using inductive argon-coupled plasma/mass spectrometry (ICP-MS), which can detect platinum in the parts per trillion range. Our results indicate that GC-detectable low molecular weight silicones contribute approximately 1-2% to the total gel mass and consist predominantly of cyclic and linear poly-(dimethylsiloxanes) ranging from 3 to 20 siloxane [(CH3)2-Si-O] units (molecular weight 200-1500). Platinum can be detected in implant gels at levels of approximately 700 micrograms/kg by ICP-MS. The major component of implant gels appears to be high molecular weight silicone polymers (HM-silicones) too large to be detected by GC. However, these HM-silicones can be converted almost quantitatively (80% by mass) to LM-silicones by heating implant gels at 150-180 degrees C for several hours. We also studied the rates at which LM-silicones and platinum leak through the intact implant outer shell into the surrounding media under a variety of conditions. Leakage of silicones was greatest when the surrounding medium was lipid-rich, and up to 10 mg/day LM-silicones was observed to diffuse into a lipid-rich medium per 250 g of implant at 37 degrees C. This rate of leakage was maintained over a 7-day experimental period. Similarly, platinum was also observed to leak through intact implants into lipid-containing media at rates of approximately 20-25 micrograms/day/250 g of implant at 37 degrees C. The rates at which both LM-silicones and platinum have been observed to leak from intact implants could lead to significant accumulation within lipid-rich tissues and should be investigated more fully in vivo.

The mechanical fingerprint of murine excisional wounds.

PubMed

Pensalfini, Marco; Haertel, Eric; Hopf, Raoul; Wietecha, Mateusz; Werner, Sabine; Mazza, Edoardo

2018-01-01

A multiscale mechanics approach to the characterization of murine excisional wounds subjected to uniaxial tensile loading is presented. Local strain analysis at a physiological level of tension uncovers the presence of two distinct regions within the wound: i) a very compliant peripheral cushion and ii) a core area undergoing modest deformation. Microstructural visualizations of stretched wound specimens show negligible engagement of the collagen located in the center of a 7-day old wound; fibers remain coiled despite the applied tension, confirming the existence of a mechanically isolated wound core. The compliant cushion located at the wound periphery appears to protect the newly-formed tissue from excessive deformation during the phase of new tissue formation. The early remodeling phase (day 14) is characterized by a restored mechanical connection between far field and wound center. The latter remains less deformable, a characteristic possibly required for cell activities during tissue remodeling. The distribution of fibrillary collagens at these two time points corresponds well to the identified heterogeneity of mechanical properties of the wound region. This novel approach provides new insight into the mechanical properties of wounded skin and will be applicable to the analysis of compound-treated wounds or wounds in genetically modified tissue. Biophysical characterization of healing wounds is crucial to assess the recovery of the skin barrier function and the associated mechanobiological processes. For the first time, we performed highly resolved local deformation analysis to identify mechanical characteristics of the wound and its periphery. Our results reveal the presence of a compliant cushion surrounding a stiffer wound core; we refer to this heterogeneous mechanical behavior as "mechanical fingerprint" of the wound. The mechanical response is shown to progress towards that of the intact skin as healing takes place. Histology and multiphoton microscopy suggest that wounded skin recovers its mechanical function via progressive reconnection of the newly-deposited collagen fibers with the surrounding intact matrix. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats.

PubMed

Enami, Yuta; Joseph, Brigid; Bandi, Sriram; Lin, Juan; Gupta, Sanjeev

2012-04-01

Organs from non-heart-beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non-heart-beating donor cells. We studied non-heart-beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV-deficient rats. In non-heart-beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart-beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non-heart-beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart-beating donors (P < 0.05). Similarly, although hepatocytes from non-heart-beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart-beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non-heart-beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix-receptor interactions, multiple ligand-receptor interactions, and signaling in insulin, calcium, wnt, Jak-Stat, or other cascades. Liver tissue remained intact over prolonged periods after death in non-heart-beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non-heart-beating donors offer opportunities for improving donor cell viability, which will advance the utility of non-heart-beating donor organs for cell therapy or other applications. Copyright © 2012 American Association for the Study of Liver Diseases.