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Sample records for interventions phantom ex-vivo

  1. Shear Wave Velocity Imaging Using Transient Electrode Perturbation: Phantom and ex vivo Validation

    PubMed Central

    Varghese, Tomy; Madsen, Ernest L.

    2011-01-01

    This paper presents a new shear wave velocity imaging technique to monitor radio-frequency and microwave ablation procedures, coined electrode vibration elastography. A piezoelectric actuator attached to an ablation needle is transiently vibrated to generate shear waves that are tracked at high frame rates. The time-to-peak algorithm is used to reconstruct the shear wave velocity and thereby the shear modulus variations. The feasibility of electrode vibration elastography is demonstrated using finite element models and ultrasound simulations, tissue-mimicking phantoms simulating fully (phantom 1) and partially ablated (phantom 2) regions, and an ex vivo bovine liver ablation experiment. In phantom experiments, good boundary delineation was observed. Shear wave velocity estimates were within 7% of mechanical measurements in phantom 1 and within 17% in phantom 2. Good boundary delineation was also demonstrated in the ex vivo experiment. The shear wave velocity estimates inside the ablated region were higher than mechanical testing estimates, but estimates in the untreated tissue were within 20% of mechanical measurements. A comparison of electrode vibration elastography and electrode displacement elastography showed the complementary information that they can provide. Electrode vibration elastography shows promise as an imaging modality that provides ablation boundary delineation and quantitative information during ablation procedures. PMID:21075719

  2. Pulmonary ultrasound elastography: a feasibility study with phantoms and ex-vivo tissue

    NASA Astrophysics Data System (ADS)

    Nguyen, Man Minh; Xie, Hua; Paluch, Kamila; Stanton, Douglas; Ramachandran, Bharat

    2013-03-01

    Elastography has become widely used for minimally invasive diagnosis in many tumors as seen with breast, liver and prostate. Among different modalities, ultrasound-based elastography stands out due to its advantages including being safe, real-time, and relatively low-cost. While lung cancer is the leading cause of cancer mortality among both men and women, the use of ultrasound elastography for lung cancer diagnosis has hardly been investigated due to the limitations of ultrasound in air. In this work, we investigate the use of static-compression based endobronchial ultrasound elastography by a 3D trans-oesophageal echocardiography (TEE) transducer for lung cancer diagnosis. A water-filled balloon was designed to 1) improve the visualization of endobronchial ultrasound and 2) to induce compression via pumping motion inside the trachea and bronchiole. In a phantom study, we have successfully generated strain images indicating the stiffness difference between the gelatin background and agar inclusion. A similar strain ratio was confirmed with Philips ultrasound strain-based elastography product. For ex-vivo porcine lung study, different tissue ablation methods including chemical injection, Radio Frequency (RF) ablation, and direct heating were implemented to achieve tumor-mimicking tissue. Stiff ablated lung tissues were obtained and detected with our proposed method. These results suggest the feasibility of pulmonary elastography to differentiate stiff tumor tissue from normal tissue.

  3. Validation of diffusion spectrum magnetic resonance imaging with manganese-enhanced rat optic tracts and ex vivo phantoms.

    PubMed

    Lin, Ching-Po; Wedeen, Van Jay; Chen, Jyh-Horng; Yao, Ching; Tseng, Wen-Yih Isaac

    2003-07-01

    Diffusion spectrum imaging (DSI) has been demonstrated to resolve crossing axonal fibers by mapping the probability density function of water molecules diffusion at each voxel. However, the accuracy of DSI in defining individual fiber orientation and the validity of Fourier relation under finite gradient pulse widths are not assessed yet. We developed an ex vivo and an in vivo model to evaluate the error of DSI with gradient pulse widths being relatively short and long, respectively. The ex vivo model was a phantom comprising sheets of parallel capillaries filled with water. Sheets were stacked on each other with capillaries crossed at 45 degrees or 90 degrees. High-resolution T2-weighted images (T2WI) of the phantom served as a reference for the orientation of intersecting capillaries. In the in vivo model, manganese ions were infused into rats' optic tracts. The optic tracts were enhanced on T1-weighted images (T1WI) and served as a reference for the tract orientation. By comparing DSI with T2WI, the deviation angles between the primary orientation of diffusion spectrum and the 90 degrees and 45 degrees phantoms were 1.19 degrees +/- 4.82 degrees and -0.71 degrees +/- 4.91 degrees, respectively. By comparing DSI with the T1WI of rat optic tracts, the deviation angle between primary orientation of diffusion spectrum and optic tracts was -0.41 degrees +/- 6.18 degrees. In addition, two sequences of DSI using short and long gradient pulses were performed in a rat brain. The bias of the primary orientation between these two sequences was approximately 10 degrees. In conclusion, DSI can resolve crossing fiber orientation accurately. The effect of finite gradient pulse widths on the primary orientation is not critical. PMID:12880782

  4. Porcine Ex Vivo Liver Phantom for Dynamic Contrast-Enhanced Computed Tomography: Development and Initial Results

    PubMed Central

    Thompson, Scott M.; Giraldo, Juan C. Ramirez; Knudsen, Bruce; Grande, Joseph P.; Christner, Jodie A.; Xu, Man; Woodrum, David A.; McCollough, Cynthia H.; Callstrom, Matthew R.

    2011-01-01

    Objectives To demonstrate the feasibility of developing a fixed, dual-input, biological liver phantom for dynamic contrast-enhanced computed tomography (CT) imaging and to report initial results of use of the phantom for quantitative CT perfusion imaging. Materials and Methods Porcine livers were obtained from completed surgical studies and perfused with saline and fixative. The phantom was placed in a body-shaped, CT-compatible acrylic container and connected to a perfusion circuit fitted with a contrast injection port. Flow-controlled contrast-enhanced imaging experiments were performed using a 128-slice and 64 slice, dual-source multidetector CT scanners. CT angiography protocols were employed to obtain portal venous and hepatic arterial vascular enhancement, reproduced over a period of four to six months. CT perfusion protocols were employed at different input flow rates to correlate input flow with calculated tissue perfusion, to test reproducibility and demonstrate the feasibility of simultaneous dual input liver perfusion. Histologic analysis of the liver phantom was also performed. Results CT angiogram 3D reconstructions demonstrated homogenous tertiary and quaternary branching of the portal venous system out to the periphery of all lobes of the liver as well as enhancement of the hepatic arterial system to all lobes of the liver and gallbladder throughout the study period. For perfusion CT, the correlation between the calculated mean tissue perfusion in a volume of interest and input pump flow rate was excellent (R2 = 0.996) and color blood flow maps demonstrated variations in regional perfusion in a narrow range. Repeat perfusion CT experiments demonstrated reproducible time-attenuation curves and dual-input perfusion CT experiments demonstrated that simultaneous dual input liver perfusion is feasible. Histologic analysis demonstrated that the hepatic microvasculature and architecture appeared intact and well preserved at the completion of four to six

  5. Measurements of optical parameters of phantom solution and bulk animal tissues ex vivo at 650 nm

    NASA Astrophysics Data System (ADS)

    Sun, Ping; Wang, Yu; Liu, Jian

    2008-12-01

    Optical parameters of biological tissues, including absorption coefficient (μa), reduced scattering coefficient (μs') or scattering coefficient (μs), anisotropy factor (g) and refractive index (n) are investigated extensively and systemically at wavelength of 650 nm. Intralipid solution was selected to be the tissue phantom in order to test the validity of measurements. Considering the factors of fiber orientation and haemoglobin content, we chose some fresh bulk animal tissues in vitro which were bovine adipose, bovine muscle, porcine adipose, porcine muscle, porcine kidney, porcine liver, mutton and chicken breast. The basic assumption is that in vitro samples are a reasonable representation of the in vivo situation. We have gained numbers of experimental data of Intralipid and some tissues. Particularly, we have set up the close relationships among six optical parameters involving μa, μs', μs, g, n and μt. The experimental results show that for animal tissues, μa, μs' or μs and n rely deeply on muscle fiber orientations. Both of μs and μt range from 10mm-1 to 20mm-1. μa ranges from 10-2 mm-1 to 10-3 mm-1 and g from 0.95 to 0.99. The results of this study will be helpful in further understanding of optical properties of tissues.

  6. Interventional multispectral photoacoustic imaging with a clinical ultrasound probe for discriminating nerves and tendons: an ex vivo pilot study.

    PubMed

    Mari, Jean Martial; Xia, Wenfeng; West, Simeon J; Desjardins, Adrien E

    2015-11-01

    Accurate and efficient identification of nerves is an essential component of peripheral nerve blocks. While ultrasound (US) imaging is increasingly used as a guidance modality, it often provides insufficient contrast for identifying nerves from surrounding tissues such as tendons. Electrical nerve stimulators can be used in conjunction with US imaging for discriminating nerves from surrounding tissues, but they are insufficient to reliably prevent neural punctures, so that alternative methods are highly desirable. In this study, an interventional multispectral photoacoustic (PA) imaging system was used to directly compare the signal amplitudes and spectra acquired from nerves and tendons ex vivo, for the first time. The results indicate that the system can provide significantly higher image contrast for discriminating nerves and tendons than that provided by US imaging. As such, photoacoustic imaging could be valuable as an adjunct to US for guiding peripheral nerve blocks. PMID:26580699

  7. Interventional multispectral photoacoustic imaging with a clinical ultrasound probe for discriminating nerves and tendons: an ex vivo pilot study

    NASA Astrophysics Data System (ADS)

    Mari, Jean Martial; Xia, Wenfeng; West, Simeon J.; Desjardins, Adrien E.

    2015-11-01

    Accurate and efficient identification of nerves is an essential component of peripheral nerve blocks. While ultrasound (US) imaging is increasingly used as a guidance modality, it often provides insufficient contrast for identifying nerves from surrounding tissues such as tendons. Electrical nerve stimulators can be used in conjunction with US imaging for discriminating nerves from surrounding tissues, but they are insufficient to reliably prevent neural punctures, so that alternative methods are highly desirable. In this study, an interventional multispectral photoacoustic (PA) imaging system was used to directly compare the signal amplitudes and spectra acquired from nerves and tendons ex vivo, for the first time. The results indicate that the system can provide significantly higher image contrast for discriminating nerves and tendons than that provided by US imaging. As such, photoacoustic imaging could be valuable as an adjunct to US for guiding peripheral nerve blocks.

  8. Transscleral visible/near-infrared spectroscopy for quantitative assessment of melanin in a uveal melanoma phantom of ex vivo porcine eyes.

    PubMed

    Krohn, Jørgen; Xu, Can T; Svenmarker, Pontus; Khoptyar, Dmitry; Andersson-Engels, Stefan

    2010-02-01

    Optical spectroscopy has been used as a supplement to conventional techniques for analyzing and diagnosing cancer in many human organs. Because ocular tumors may be characterized by their different melanin content, we investigated the feasibility of using transscleral visible/near-infrared spectroscopy (Vis/NIRS) to estimate the quantity of melanin in a novel uveal melanoma phantom of ex vivo porcine eyes. The phantoms were made by injecting a freshly prepared suspension of 15% (wt/vol) gelatin, 10 mg/ml titanium dioxide (TiO(2)), and natural melanin, isolated from the ink sac of cuttlefish (Sepia officinalis), into the suprachoroidal space of 30 enucleated porcine eyes. The melanin concentrations used were 1 mg/ml, 2 mg/ml, and 3 mg/ml, with 10 eyes in each group. After gelation, the size and location of the phantoms were documented by B-scan ultrasonography and transillumination. Vis/NIRS recordings, covering the wavelength region from 550 to 1000 nm, were performed with two optical fibers separated by 6 mm to deliver and collect the light through the sclera. During all measurements, the exact pressure exerted by the fiber probe on the scleral surface was monitored by placing the eye on an electronic scale. Transscleral Vis/NIRS was performed across the phantom inclusion, as well as on the opposite (normal) side of each eye. A total of three consecutive measurements were carried out alternately on each side of the globe. The spectral data were analyzed using partial least squares regression. In the melanin concentration groups of 1 mg/ml (n = 10), 2 mg/ml (n = 10), and 3 mg/ml (n = 10), the largest basal phantom diameters (mean +/- SD) were 14.9 +/- 1.6 mm, 14.6 +/- 1.5 mm, and 14.3 +/- 1.0 mm, respectively (p > 0.05). The largest phantom thicknesses (mean +/- SD) were 4.0 +/- 0.5 mm, 4.4 +/- 0.7 mm, and 4.5 +/- 0.5 mm, respectively (p > 0.05). Statistical regression modeling of the Vis/NIRS data revealed that it was possible to correctly classify the phantoms

  9. Optical coherence tomography detection of shear wave propagation in inhomogeneous tissue equivalent phantoms and ex-vivo carotid artery samples

    PubMed Central

    Razani, Marjan; Luk, Timothy W.H.; Mariampillai, Adrian; Siegler, Peter; Kiehl, Tim-Rasmus; Kolios, Michael C.; Yang, Victor X.D.

    2014-01-01

    In this work, we explored the potential of measuring shear wave propagation using optical coherence elastography (OCE) in an inhomogeneous phantom and carotid artery samples based on a swept-source optical coherence tomography (OCT) system. Shear waves were generated using a piezoelectric transducer transmitting sine-wave bursts of 400 μs duration, applying acoustic radiation force (ARF) to inhomogeneous phantoms and carotid artery samples, synchronized with a swept-source OCT (SS-OCT) imaging system. The phantoms were composed of gelatin and titanium dioxide whereas the carotid artery samples were embedded in gel. Differential OCT phase maps, measured with and without the ARF, detected the microscopic displacement generated by shear wave propagation in these phantoms and samples of different stiffness. We present the technique for calculating tissue mechanical properties by propagating shear waves in inhomogeneous tissue equivalent phantoms and carotid artery samples using the ARF of an ultrasound transducer, and measuring the shear wave speed and its associated properties in the different layers with OCT phase maps. This method lays the foundation for future in-vitro and in-vivo studies of mechanical property measurements of biological tissues such as vascular tissues, where normal and pathological structures may exhibit significant contrast in the shear modulus. PMID:24688822

  10. Robotic system for MRI-guided prostate biopsy: feasibility of teleoperated needle insertion and ex vivo phantom study

    PubMed Central

    Seifabadi, Reza; Song, Sang-Eun; Krieger, Axel; Cho, Nathan Bongjoon; Tokuda, Junichi; Fichtinger, Gabor; Iordachita, Iulian

    2012-01-01

    Purpose Magnetic Resonance Imaging (MRI) combined with robotic assistance has the potential to improve on clinical outcomes of biopsy and local treatment of prostate cancer. Methods We report the workspace optimization and phantom evaluation of a five Degree of Freedom (DOF) parallel pneumatically actuated modular robot for MRI-guided prostate biopsy. To shorten procedure time and consequently increase patient comfort and system accuracy, a prototype of a MRI-compatible master–slave needle driver module using piezo motors was also added to the base robot. Results Variable size workspace was achieved using appropriate link length, compared with the previous design. The 5-DOF targeting accuracy demonstrated an average error of 2.5mm (STD=1.37mm) in a realistic phantom inside a 3T magnet with a bevel-tip 18G needle. The average position tracking error of the master–slave needle driver was always below 0.1mm. Conclusion Phantom experiments showed sufficient accuracy for manual prostate biopsy. Also, the implementation of teleoperated needle insertion was feasible and accurate. These two together suggest the feasibility of accurate fully actuated needle placement into prostate while keeping the clinician supervision over the task. PMID:21698389

  11. 20 MHz Forward-imaging Single-element Beam Steering with an Internal Rotating Variable-Angle Reflecting Surface: Wire phantom and Ex vivo pilot study

    PubMed Central

    Raphael, David T.; Li, Xiang; Park, Jinhyoung; Chen, Ruimin; Chabok, Hamid; Barukh, Arthur; Zhou, Qifa; Elgazery, Mahmoud; Shung, K. Kirk

    2012-01-01

    Feasibility is demonstrated for a forward-imaging beam steering system involving a single-element 20 MHz angled-face acoustic transducer combined with an internal rotating variable-angle reflecting surface (VARS). Rotation of the VARS structure, for a fixed position of the transducer, generates a 2-D angular sector scan. If these VARS revolutions were to be accompanied by successive rotations of the single-element transducer, 3-D imaging would be achieved. In the design of this device, a single-element 20 MHz PMN-PT press-focused angled-face transducer is focused on the circle of midpoints of a micro-machined VARS within the distal end of an endoscope. The 2-D imaging system was tested in water bath experiments with phantom wire structures at a depth of 10 mm, and exhibited an axial resolution of 66 μm and a lateral resolution of 520 μm. Chirp coded excitation was used to enhance the signal-to-noise ratio, and to increase the depth of penetration. Images of an ex vivo cow eye were obtained. This VARS-based approach offers a novel forward-looking beam-steering method, which could be useful in intra-cavity imaging. PMID:23122968

  12. 20 MHz forward-imaging single-element beam steering with an internal rotating variable-angle reflecting surface: Wire phantom and ex vivo pilot study.

    PubMed

    Raphael, David T; Li, Xiang; Park, Jinhyoung; Chen, Ruimin; Chabok, Hamid; Barukh, Arthur; Zhou, Qifa; Elgazery, Mahmoud; Shung, K Kirk

    2013-02-01

    Feasibility is demonstrated for a forward-imaging beam steering system involving a single-element 20MHz angled-face acoustic transducer combined with an internal rotating variable-angle reflecting surface (VARS). Rotation of the VARS structure, for a fixed position of the transducer, generates a 2-D angular sector scan. If these VARS revolutions were to be accompanied by successive rotations of the single-element transducer, 3-D imaging would be achieved. In the design of this device, a single-element 20MHz PMN-PT press-focused angled-face transducer is focused on the circle of midpoints of a micro-machined VARS within the distal end of an endoscope. The 2-D imaging system was tested in water bath experiments with phantom wire structures at a depth of 10mm, and exhibited an axial resolution of 66μm and a lateral resolution of 520μm. Chirp coded excitation was used to enhance the signal-to-noise ratio, and to increase the depth of penetration. Images of an ex vivo cow eye were obtained. This VARS-based approach offers a novel forward-looking beam-steering method, which could be useful in intra-cavity imaging. PMID:23122968

  13. Automated continuous quantitative measurement of proximal airways on dynamic ventilation CT: initial experience using an ex vivo porcine lung phantom

    PubMed Central

    Yamashiro, Tsuneo; Tsubakimoto, Maho; Nagatani, Yukihiro; Moriya, Hiroshi; Sakuma, Kotaro; Tsukagoshi, Shinsuke; Inokawa, Hiroyasu; Kimoto, Tatsuya; Teramoto, Ryuichi; Murayama, Sadayuki

    2015-01-01

    Background The purpose of this study was to evaluate the feasibility of continuous quantitative measurement of the proximal airways, using dynamic ventilation computed tomography (CT) and our research software. Methods A porcine lung that was removed during meat processing was ventilated inside a chest phantom by a negative pressure cylinder (eight times per minute). This chest phantom with imitated respiratory movement was scanned by a 320-row area-detector CT scanner for approximately 9 seconds as dynamic ventilatory scanning. Obtained volume data were reconstructed every 0.35 seconds (total 8.4 seconds with 24 frames) as three-dimensional images and stored in our research software. The software automatically traced a designated airway point in all frames and measured the cross-sectional luminal area and wall area percent (WA%). The cross-sectional luminal area and WA% of the trachea and right main bronchus (RMB) were measured for this study. Two radiologists evaluated the traceability of all measurable airway points of the trachea and RMB using a three-point scale. Results It was judged that the software satisfactorily traced airway points throughout the dynamic ventilation CT (mean score, 2.64 at the trachea and 2.84 at the RMB). From the maximum inspiratory frame to the maximum expiratory frame, the cross-sectional luminal area of the trachea decreased 17.7% and that of the RMB 29.0%, whereas the WA% of the trachea increased 6.6% and that of the RMB 11.1%. Conclusion It is feasible to measure airway dimensions automatically at designated points on dynamic ventilation CT using research software. This technique can be applied to various airway and obstructive diseases. PMID:26445535

  14. Spectra from 2.5-15 microm of tissue phantom materials, optical clearing agents and ex vivo human skin: implications for depth profiling of human skin.

    PubMed

    Viator, John A; Choi, Bernard; Peavy, George M; Kimel, Sol; Nelson, J Stuart

    2003-01-21

    Infrared measurements have been used to profile or image biological tissue, including human skin. Usually, analysis of such measurements has assumed that infrared absorption is due to water and collagen. Such an assumption may be reasonable for soft tissue, but introduction of exogenous agents into skin or the measurement of tissue phantoms has raised the question of their infrared absorption spectrum. We used Fourier transform infrared spectroscopy in attenuated total reflection mode to measure the infrared absorption spectra, in the range of 2-15 microm, of water, polyacrylamide, Intralipid, collagen gels, four hyperosmotic clearing agents (glycerol, 1,3-butylene glycol, trimethylolpropane, Topicare), and ex vivo human stratum corneum and dermis. The absorption spectra of the phantom materials were similar to that of water, although additional structure was noted in the range of 6-10 microm. The absorption spectra of the clearing agents were more complex, with molecular absorption bands dominating between 6 and 12 microm. Dermis was similar to water, with collagen structure evident in the 6-10 microm range. Stratum corneum had a significantly lower absorption than dermis due to a lower content of water. These results suggest that the assumption of water-dominated absorption in the 2.5-6 microm range is valid. At longer wavelengths, clearing agent absorption spectra differ significantly from the water spectrum. This spectral information can be used in pulsed photothermal radiometry or utilized in the interpretation of reconstructions in which a constant mu(ir) is used. In such cases, overestimating mu(ir) will underestimate chromophore depth and vice versa, although the effect is dependent on actual chromophore depth. PMID:12587910

  15. Ex vivo lung perfusion.

    PubMed

    Reeb, Jeremie; Cypel, Marcelo

    2016-03-01

    Lung transplantation is an established life-saving therapy for patients with end-stage lung disease. Unfortunately, greater success in lung transplantation is hindered by a shortage of lung donors and the relatively poor early-, mid-, and long-term outcomes associated with severe primary graft dysfunction. Ex vivo lung perfusion has emerged as a modern preservation technique that allows for a more accurate lung assessment and improvement in lung quality. This review outlines the: (i) rationale behind the method; (ii) techniques and protocols; (iii) Toronto ex vivo lung perfusion method; (iv) devices available; and (v) clinical experience worldwide. We also highlight the potential of ex vivo lung perfusion in leading a new era of lung preservation. PMID:26700566

  16. Ex vivo lung perfusion.

    PubMed

    Machuca, Tiago N; Cypel, Marcelo

    2014-08-01

    Lung transplantation (LTx) is an established treatment option for eligible patients with end-stage lung disease. Nevertheless, the imbalance between suitable donor lungs available and the increasing number of patients considered for LTx reflects in considerable waitlist mortality. Among potential alternatives to address this issue, ex vivo lung perfusion (EVLP) has emerged as a modern preservation technique that allows for more accurate lung assessment and also improvement of lung function. Its application in high-risk donor lungs has been successful and resulted in safe expansion of the donor pool. This article will: (I) review the technical details of EVLP; (II) the rationale behind the method; (III) report the worldwide clinical experience with the EVLP, including the Toronto technique and others; (IV) finally, discuss the growing literature on EVLP application for donation after cardiac death (DCD) lungs. PMID:25132972

  17. Ex vivo lung perfusion

    PubMed Central

    Machuca, Tiago N.

    2014-01-01

    Lung transplantation (LTx) is an established treatment option for eligible patients with end-stage lung disease. Nevertheless, the imbalance between suitable donor lungs available and the increasing number of patients considered for LTx reflects in considerable waitlist mortality. Among potential alternatives to address this issue, ex vivo lung perfusion (EVLP) has emerged as a modern preservation technique that allows for more accurate lung assessment and also improvement of lung function. Its application in high-risk donor lungs has been successful and resulted in safe expansion of the donor pool. This article will: (I) review the technical details of EVLP; (II) the rationale behind the method; (III) report the worldwide clinical experience with the EVLP, including the Toronto technique and others; (IV) finally, discuss the growing literature on EVLP application for donation after cardiac death (DCD) lungs. PMID:25132972

  18. A Wearable Goggle Navigation System for Dual-Mode Optical and Ultrasound Localization of Suspicious Lesions: Validation Studies Using Tissue-Simulating Phantoms and an Ex Vivo Human Breast Tissue Model.

    PubMed

    Zhang, Zeshu; Pei, Jing; Wang, Dong; Gan, Qi; Ye, Jian; Yue, Jian; Wang, Benzhong; Povoski, Stephen P; Martin, Edward W; Hitchcock, Charles L; Yilmaz, Alper; Tweedle, Michael F; Shao, Pengfei; Xu, Ronald X

    2016-01-01

    Surgical resection remains the primary curative treatment for many early-stage cancers, including breast cancer. The development of intraoperative guidance systems for identifying all sites of disease and improving the likelihood of complete surgical resection is an area of active ongoing research, as this can lead to a decrease in the need of subsequent additional surgical procedures. We develop a wearable goggle navigation system for dual-mode optical and ultrasound imaging of suspicious lesions. The system consists of a light source module, a monochromatic CCD camera, an ultrasound system, a Google Glass, and a host computer. It is tested in tissue-simulating phantoms and an ex vivo human breast tissue model. Our experiments demonstrate that the surgical navigation system provides useful guidance for localization and core needle biopsy of simulated tumor within the tissue-simulating phantom, as well as a core needle biopsy and subsequent excision of Indocyanine Green (ICG)-fluorescing sentinel lymph nodes. Our experiments support the contention that this wearable goggle navigation system can be potentially very useful and fully integrated by the surgeon for optimizing many aspects of oncologic surgery. Further engineering optimization and additional in vivo clinical validation work is necessary before such a surgical navigation system can be fully realized in the everyday clinical setting. PMID:27367051

  19. A Wearable Goggle Navigation System for Dual-Mode Optical and Ultrasound Localization of Suspicious Lesions: Validation Studies Using Tissue-Simulating Phantoms and an Ex Vivo Human Breast Tissue Model

    PubMed Central

    Wang, Dong; Gan, Qi; Ye, Jian; Yue, Jian; Wang, Benzhong; Povoski, Stephen P.; Martin, Edward W.; Hitchcock, Charles L.; Yilmaz, Alper; Tweedle, Michael F.; Shao, Pengfei; Xu, Ronald X.

    2016-01-01

    Surgical resection remains the primary curative treatment for many early-stage cancers, including breast cancer. The development of intraoperative guidance systems for identifying all sites of disease and improving the likelihood of complete surgical resection is an area of active ongoing research, as this can lead to a decrease in the need of subsequent additional surgical procedures. We develop a wearable goggle navigation system for dual-mode optical and ultrasound imaging of suspicious lesions. The system consists of a light source module, a monochromatic CCD camera, an ultrasound system, a Google Glass, and a host computer. It is tested in tissue-simulating phantoms and an ex vivo human breast tissue model. Our experiments demonstrate that the surgical navigation system provides useful guidance for localization and core needle biopsy of simulated tumor within the tissue-simulating phantom, as well as a core needle biopsy and subsequent excision of Indocyanine Green (ICG)—fluorescing sentinel lymph nodes. Our experiments support the contention that this wearable goggle navigation system can be potentially very useful and fully integrated by the surgeon for optimizing many aspects of oncologic surgery. Further engineering optimization and additional in vivo clinical validation work is necessary before such a surgical navigation system can be fully realized in the everyday clinical setting. PMID:27367051

  20. Real-Time Integrated Photoacoustic and Ultrasound (PAUS) Imaging System to Guide Interventional Procedures: Ex Vivo Study

    PubMed Central

    Wei, Chen-Wei; Nguyen, Thu-Mai; Xia, Jinjun; Arnal, Bastien; Wong, Emily Y.; Pelivanov, Ivan M.; O’Donnell, Matthew

    2015-01-01

    Because of depth-dependent light attenuation, bulky, low-repetition-rate lasers are usually used in most photoacoustic (PA) systems to provide sufficient pulse energies to image at depth within the body. However, integrating these lasers with real-time clinical ultrasound (US) scanners has been problematic because of their size and cost. In this paper, an integrated PA/US (PAUS) imaging system is presented operating at frame rates >30 Hz. By employing a portable, low-cost, low-pulse-energy (~2 mJ/pulse), high-repetition-rate (~1 kHz), 1053-nm laser, and a rotating galvo-mirror system enabling rapid laser beam scanning over the imaging area, the approach is demonstrated for potential applications requiring a few centimeters of penetration. In particular, we demonstrate here real-time (30 Hz frame rate) imaging (by combining multiple single-shot sub-images covering the scan region) of an 18-gauge needle inserted into a piece of chicken breast with subsequent delivery of an absorptive agent at more than 1-cm depth to mimic PAUS guidance of an interventional procedure. A signal-to-noise ratio of more than 35 dB is obtained for the needle in an imaging area 2.8 × 2.8 cm (depth × lateral). Higher frame rate operation is envisioned with an optimized scanning scheme. PMID:25643081

  1. Ex vivo lung graft perfusion.

    PubMed

    Briot, Raphaël; Gennai, Stéphane; Maignan, Maxime; Souilamas, Redha; Pison, Christophe

    2016-04-01

    This review proposes an update of the state of the art and the ongoing clinical trials of ex vivo lung perfusion for lung transplantation in patients. Ex vivo lung perfusion techniques (EVLP) can be used to evaluate a lung graft outside of the body. The goal of EVLP is to study the functional status of lung grafts that were first rejected for transplantation because they did not match all criteria for a conventional transplantation. After an EVLP evaluation, some of these lungs may be requalified for a possible transplantation in patients. This article proposes an overview of the developments of EVLP techniques. During EVLP, the perfusion and ventilation of the isolated lung preparation are very progressive in order to avoid oedema due to ischaemia-reperfusion injuries. Lung evaluation is mainly based on gasometric (PaO2/FiO2) and rheological criteria (low pulmonary arterial resistance). Several series of patients transplanted with EVLP evaluated lungs have been recently published with promising results. EVLP preparations also allow a better understanding of the physiopathology and treatments of ischaemia-reperfusion injuries. Organ procurements from "non-heart-beating" donors will probably require a wider application of these ex vivo techniques. The development of semi-automated systems might facilitate the clinical use of EVLP techniques. PMID:26746565

  2. Ex vivo expansion of mesenchymal stromal cells.

    PubMed

    Bernardo, Maria Ester; Cometa, Angela Maria; Pagliara, Daria; Vinti, Luciana; Rossi, Francesca; Cristantielli, Rosaria; Palumbo, Giuseppe; Locatelli, Franco

    2011-03-01

    Mesenchymal stromal cells (MSCs) are adult multipotent cells that can be isolated from several human tissues. MSCs represent a novel and attractive tool in strategies of cellular therapy. For in vivo use, MSCs have to be ex vivo expanded in order to reach the numbers suitable for their clinical application. Despite being efficacious, the use of fetal calf serum for MSC ex vivo expansion for clinical purposes raises concerns related to immunization and transmission of zoonoses; the standardization of expansion methods, possibly devoid of animal components, such as those based on platelet lysate, are discussed in this paper. Moreover, this review focuses on the search of novel markers for the prospective identification/isolation of MSCs and on the potential risks connected with ex vivo expansion of MSCs, in particular that of their malignant transformation. Available tests to study the genetic stability of ex vivo expanded MSCs are also analyzed. PMID:21396595

  3. Intrathoracic airway measurement: ex-vivo validation

    NASA Astrophysics Data System (ADS)

    Reinhardt, Joseph M.; Raab, Stephen A.; D'Souza, Neil D.; Hoffman, Eric A.

    1997-05-01

    High-resolution x-ray CT (HRCT) provides detailed images of the lungs and bronchial tree. HRCT-based imaging and quantitation of peripheral bronchial airway geometry provides a valuable tool for assessing regional airway physiology. Such measurements have been sued to address physiological questions related to the mechanics of airway collapse in sleep apnea, the measurement of airway response to broncho-constriction agents, and to evaluate and track the progression of disease affecting the airways, such as asthma and cystic fibrosis. Significant attention has been paid to the measurements of extra- and intra-thoracic airways in 2D sections from volumetric x-ray CT. A variety of manual and semi-automatic techniques have been proposed for airway geometry measurement, including the use of standardized display window and level settings for caliper measurements, methods based on manual or semi-automatic border tracing, and more objective, quantitative approaches such as the use of the 'half-max' criteria. A recently proposed measurements technique uses a model-based deconvolution to estimate the location of the inner and outer airway walls. Validation using a plexiglass phantom indicates that the model-based method is more accurate than the half-max approach for thin-walled structures. In vivo validation of these airway measurement techniques is difficult because of the problems in identifying a reliable measurement 'gold standard.' In this paper we report on ex vivo validation of the half-max and model-based methods using an excised pig lung. The lung is sliced into thin sections of tissue and scanned using an electron beam CT scanner. Airways of interest are measured from the CT images, and also measured with using a microscope and micrometer to obtain a measurement gold standard. The result show no significant difference between the model-based measurements and the gold standard; while the half-max estimates exhibited a measurement bias and were significantly

  4. Ebola Virus Persistence in Semen Ex Vivo

    PubMed Central

    Fischer, Robert J.; Judson, Seth; Miazgowicz, Kerri; Bushmaker, Trent

    2016-01-01

    On March 20, 2015, a case of Ebola virus disease was identified in Liberia that most likely was transmitted through sexual contact. We assessed the efficiency of detecting Ebola virus in semen samples by molecular diagnostics and the stability of Ebola virus in ex vivo semen under simulated tropical conditions. PMID:26811984

  5. Manipulating megakaryocytes to manufacture platelets ex vivo

    PubMed Central

    Karagiannis, P; Eto, K

    2015-01-01

    Historically, platelet transfusion has proven a reliable way to treat patients suffering from thrombocytopenia or similar ailments. An undersupply of donors, however, has demanded alternative platelet sources. Scientists have therefore sought to recapitulate the biological events that convert hematopoietic stem cells into platelets in the laboratory. Such platelets have shown good function and potential for treatment. Yet the number manufactured ex vivo falls well short of clinical application. Part of the reason is the remarkable gaps in our understanding of the molecular mechanisms driving platelet formation. Using several stem cell sources, scientists have progressively clarified the chemical signaling and physical microenvironment that optimize ex vivo platelets and reconstituted them in synthetic environments. Key advances in cell reprogramming and the ability to propagate self-renewal have extended the lifetime of megakaryocytes to increase the pool of platelet progenitors. PMID:26149050

  6. Analysis of elastography methods using mathematical and ex vivo data

    NASA Astrophysics Data System (ADS)

    Byram, Brett C.; Wahl, Michael R.; Holmes, David R., III; Lerman, Amir; Robb, Richard A.

    2003-05-01

    Intravascular ultrasound (IVUS) currently has a limited ability to characterize endovascular anatomic properties. IVUS elastography enhances the ability to characterize the biomechanical properties of arterial walls. A mathematical phantom generator was developed based on the characteristics of 30MHz, 64 element IVUS catheter images from excised canine femoral arteries. The difference between high and low-pressure intra-arterial images was modeled using phase shifts. The increase in phase shift occurred randomly, generally at every three pixels in our images. Using mathematical phantoms, different methods for calculating elastograms were quantitatively analyzed. Specifically, the effect of standard cross correlation versus cross correlation of the integral of the inflection characteristics for a given set of data, and the effect of an algorithm utilizing a non-constant kernel, were assessed. The specific methods found to be most accurate on the mathematical phantom data were then applied to ex vivo canine data of a scarred and a healthy artery. The algorithm detected significant differences between these two sets of arterial data. It will be necessary to obtain and analyze several more sets of canine arterial data in order to determine the accuracy and reproducibility of the algorithm.

  7. Electromechanical Reshaping of Ex Vivo Porcine Trachea

    PubMed Central

    Hussain, Syed; Manuel, Cyrus T.; Protsenko, Dmitriy E.; Wong, Brian J. F.

    2015-01-01

    Objectives The trachea is a composite cartilaginous structure particularly prone to various forms of convexities. Electromechanical reshaping (EMR) is an emerging technique used to reshape cartilaginous tissues by applying electric current in tandem with imposed mechanical deformation to achieve shape change. In this study, EMR was used to reshape tracheal cartilage rings to demonstrate the feasibility of this technology as a potentially minimally invasive procedure to alter tracheal structure. Study Design Controlled laboratory study using ex vivo porcine tracheae. Methods The natural concavity of each porcine tracheal ring was reversed around a cork mandrel. Two pairs of electrodes were inserted along the long axis of the tracheal ring and placed 1.5 millimeters from the midline. Current was applied over a range of voltages (3 volts [V], 4V, and 5V) for either 2 or 3 minutes. The degree of EMR-induced reshaping was quantified from photographs using digital techniques. Confocal imaging with fluorescent live and dead assays was conducted to determine viability of the tissue after EMR. Results Specimens that underwent EMR for 2 or 3 minutes at 4V or 5V were observed to have undergone significant (P <.05) reshaping relative to the control. Viability results demonstrated that EMR reshaping occurs at the expense of tissue injury, although the extent of injury is modest relative to conventional techniques. Conclusion EMR reshapes tracheal cartilage rings as a function of voltage and application time. It has potential as a minimally invasive and cost-efficient endoscopic technology to treat pathologic tracheal convexities. Given our findings, consideration of EMR for use in larger ex vivo tracheal segments and animal studies is now plausible. Level of Evidence N/A. PMID:25692713

  8. Comprehensive phantom for interventional fluorescence molecular imaging.

    PubMed

    Anastasopoulou, Maria; Koch, Maximilian; Gorpas, Dimitris; Karlas, Angelos; Klemm, Uwe; Garcia-Allende, Pilar Beatriz; Ntziachristos, Vasilis

    2016-09-01

    Fluorescence imaging has been considered for over a half-century as a modality that could assist surgical guidance and visualization. The administration of fluorescent molecules with sensitivity to disease biomarkers and their imaging using a fluorescence camera can outline pathophysiological parameters of tissue invisible to the human eye during operation. The advent of fluorescent agents that target specific cellular responses and molecular pathways of disease has facilitated the intraoperative identification of cancer with improved sensitivity and specificity over nonspecific fluorescent dyes that only outline the vascular system and enhanced permeability effects. With these new abilities come unique requirements for developing phantoms to calibrate imaging systems and algorithms. We briefly review herein progress with fluorescence phantoms employed to validate fluorescence imaging systems and results. We identify current limitations and discuss the level of phantom complexity that may be required for developing a universal strategy for fluorescence imaging calibration. Finally, we present a phantom design that could be used as a tool for interlaboratory system performance evaluation. PMID:27304578

  9. Ex vivo rheology of spider silk.

    PubMed

    Kojić, N; Bico, J; Clasen, C; McKinley, G H

    2006-11-01

    We investigate the rheological properties of microliter quantities of the spinning material extracted ex vivo from the major ampullate gland of a Nephila clavipes spider using two new micro-rheometric devices. A sliding plate micro-rheometer is employed to measure the steady-state shear viscosity of approximately 1 microl samples of silk dope from individual biological specimens. The steady shear viscosity of the spinning solution is found to be highly shear-thinning, with a power-law index consistent with values expected for liquid crystalline solutions. Calculations show that the viscosity of the fluid decreases 10-fold as it flows through the narrow spinning canals of the spider. By contrast, measurements in a microcapillary extensional rheometer show that the transient extensional viscosity (i.e. the viscoelastic resistance to stretching) of the spinning fluid increases more than 100-fold during the spinning process. Quantifying the properties of native spinning solutions provides new guidance for adjusting the spinning processes of synthetic or genetically engineered silks to match those of the spider. PMID:17050850

  10. Ex vivo lung perfusion in Brazil

    PubMed Central

    Abdalla, Luis Gustavo; Braga, Karina Andrighetti de Oliveira; Nepomuceno, Natalia Aparecida; Fernandes, Lucas Matos; Samano, Marcos Naoyuki; Pêgo-Fernandes, Paulo Manuel

    2016-01-01

    Objective: To evaluate the use of ex vivo lung perfusion (EVLP) clinically to prepare donor lungs for transplantation. Methods: A prospective study involving EVLP for the reconditioning of extended-criteria donor lungs, the criteria for which include aspects such as a PaO2/FiO2 ratio < 300 mmHg. Between February of 2013 and February of 2014, the lungs of five donors were submitted to EVLP for up to 4 h each. During EVLP, respiratory mechanics were continuously evaluated. Once every hour during the procedure, samples of the perfusate were collected and the function of the lungs was evaluated. Results: The mean PaO2 of the recovered lungs was 262.9 ± 119.7 mmHg at baseline, compared with 357.0 ± 108.5 mmHg after 3 h of EVLP. The mean oxygenation capacity of the lungs improved slightly over the first 3 h of EVLP-246.1 ± 35.1, 257.9 ± 48.9, and 288.8 ± 120.5 mmHg after 1, 2, and 3 h, respectively-without significant differences among the time points (p = 0.508). The mean static compliance was 63.0 ± 18.7 mmHg, 75.6 ± 25.4 mmHg, and 70.4 ± 28.0 mmHg after 1, 2, and 3 h, respectively, with a significant improvement from hour 1 to hour 2 (p = 0.029) but not from hour 2 to hour 3 (p = 0.059). Pulmonary vascular resistance remained stable during EVLP, with no differences among time points (p = 0.284). Conclusions: Although the lungs evaluated remained under physiological conditions, the EVLP protocol did not effectively improve lung function, thus precluding transplantation. PMID:27167429

  11. Fibre optic sensors for temperature and pressure monitoring in laser ablation: experiments on ex-vivo animal model

    NASA Astrophysics Data System (ADS)

    Tosi, Daniele; Saccomandi, Paola; Schena, Emiliano; Duraibabu, Dinesh B.; Poeggel, Sven; Adilzhan, Abzal; Aliakhmet, Kamilla; Silvestri, Sergio; Leen, Gabriel; Lewis, Elfed

    2016-05-01

    Optical fibre sensors have been applied to perform biophysical measurement in ex-vivo laser ablation (LA), on pancreas animal phantom. Experiments have been performed using Fibre Bragg Grating (FBG) arrays for spatially resolved temperature detection, and an all-glass Extrinsic Fabry-Perot Interferometer (EFPI) for pressure measurement. Results using a Nd:YAG laser source as ablation device, are presented and discussed.

  12. Ex vivo effect of gold nanoparticles on porcine synovial membrane

    PubMed Central

    Labens, Raphael; Lascelles, B. Duncan X.; Charlton, Anna N.; Ferrero, Nicole R.; Van Wettere, Arnaud J.; Xia, Xin-Riu; Blikslager, Anthony T.

    2013-01-01

    Gold nanoparticles (AuNPs) have great potential as carriers for local drug delivery and as a primary therapeutic for treatment of inflammation. Here we report on the AuNP-synovium interaction in an ex vivo model of intra-articular application for treatment of joint inflammation. Sheets of porcine femoropatellar synovium were obtained post mortem and each side of the tissue samples was maintained in a separate fluid environment. Permeability to AuNPs of different sizes (5−52 nm) and biomarker levels of inflammation were determined to characterize the ex vivo particle interaction with the synovium. Lipopolysaccharide or recombinant human interleukin-1β were added to fluid environments to assess the ex vivo effect of pro-inflammatory factors on permeability and biomarker levels. The synovium showed size selective permeability with only 5 nm AuNPs effectively permeating the entire tissues’ width. This process was further governed by particle stability in the fluid environment. AuNPs reduced matrix metalloproteinase and lactate dehydrogenase activity and hyaluronic acid concentrations but had no effect on prostaglandin E2 levels. Exposure to pro-inflammatory factors did not significantly affect AuNP permeation or biomarker levels in this model. Results with ex vivo tissue modeling of porcine synovium support an anti-inflammatory effect of AuNPs warranting further investigation. PMID:24665389

  13. Ex vivo production of platelets from stem cells.

    PubMed

    Avanzi, Mauro P; Mitchell, William Beau

    2014-04-01

    Stem cell technology holds great promise for transfusion medicine, and generation of platelets from stem cells would be transformative. Platelet transfusions are life saving for millions of people and the clinical demand for platelets continues to increase: there is a real need to increase the supply of platelets. Accordingly, there is great interest in the potential of producing platelets from stem cells for clinical use. There has been initial success in ex vivo generation of platelets from stem cells using cord blood stem cells, embryonic stem cells and induced pluripotent stem cells. However, the platelet yields achieved by these strategies have not been sufficient for clinical purposes. This review provides updated information about the current strategies of ex vivo generation of platelets. Megakaryocytopoiesis and platelet generation, along with the importance of genetic determinants of these processes, are reviewed in the context of efforts to generate these products from stem cells. Current challenges and rate-limiting steps in ex vivo platelet generation are discussed, together with strategies to overcome them. While much work remains, great progress has been made, moving ex vivo generation of platelets ever closer to the clinic. PMID:24521452

  14. Influence of ultrasonic scattering in the calculation of thermal dose in ex-vivo bovine muscular tissues.

    PubMed

    Cortela, Guillermo A; von Krüger, Marco A; Negreira, Carlos A; Pereira, Wagner C A

    2016-02-01

    This study explores the effect of ultrasound scattering on the temperature increase in phantoms and in samples of ex-vivo biological tissue through the calculation of the thermal dose (TD). Phantoms with different weight percentages of graphite powder (0-1%w/w, different scattering mean free paths, ℓS) and ex-vivo bovine muscle tissue were isonified by therapeutic ultrasound (1 MHz). The TD values were calculated from the first 4 min of experimental temperature curves obtained at several depths and were compared with those acquired from the numerical solution of the bio-heat transfer equation (simulated with 1 MHz and 0.5-2.0 W cm(-2)). The temperature curves suggested that scattering had an important role because the temperature increments were found to be higher for higher percentages of graphite powder (lower ℓS). For example, at a 30-mm depth and a 4-min therapeutic ultrasound application (0.5 W cm(-2)), the TDs (in equivalent minutes at 43 °C) were 7.2, 17.8, and 58.3 for the phantom with ℓS of 4.35, 3.85, and 3.03 mm, respectively. In tissue, the inclusion of only absorption or full attenuation in the bio-heat transfer equation (BHTE) heat source term of the simulation leads to under- or overestimation of the TD, respectively, as compared to the TD calculated from experimental data. The experiments with phantoms (with different scatterer concentrations) and ex-vivo samples show that the high values of TD were caused by the increase of energy absorption due to the lengthening of the propagation path caused by the changing in the propagation regime. PMID:26522957

  15. Susceptibility of anthocyanins to ex vivo degradation in human saliva

    PubMed Central

    Kamonpatana, Kom; Giusti, M. Mónica; Chitchumroonchokchai, Chureeporn; MorenoCruz, Maria; Riedl, Ken M.; Kumar, Purnima; Failla, Mark L.

    2013-01-01

    Some fruits and their anthocyanin-rich extracts have been reported to exhibit chemopreventive activity in the oral cavity. Insights regarding oral metabolism of anthocyanins remain limited. Anthocyanin-rich extracts from blueberry, chokeberry, black raspberry, red grape, and strawberry were incubated ex vivo with human saliva from 14 healthy subjects. All anthocyanins were partially degraded in saliva. Degradation of chokeberry anthocyanins in saliva was temperature dependent and decreased by heating saliva to 80 °C and after removal of cells. Glycosides of delphinidin and petunidin were more susceptible to degradation than those of cyanidin, pelargonidin, peonidin and malvidin in both intact and artificial saliva. Stability of di- and tri-saccharide conjugates of anthocyanidins slightly, but significantly, exceeded that of monosaccharide compounds. Ex vivo degradation of anthocyanins in saliva was significantly decreased after oral rinsing with antibacterial chlorhexidine. These results suggest that anthocyanin degradation in the mouth is structure-dependent and largely mediated by oral microbiota. PMID:22868153

  16. Isolation of Cardiomyocytes and Cardiofibroblasts for Ex Vivo Analysis.

    PubMed

    Mbogo, George Williams; Nedeva, Christina; Puthalakath, Hamsa

    2016-01-01

    Heart failure (HF) is a common clinical endpoint to several underlying causes including aging, hypertension, stress, and cardiomyopathy. It is characterized by a significant decline in the cardiac output. Cardiomyocytes are terminally differentiated cells and therefore, apoptotic death due to beta adrenergic (β-AR) signaling contributes to high attrition rate of these cells. Past treatments of HF offer some survival benefit to patients (e.g., the beta blockers), but at the expense of blocking the compensatory beta-adrenergic signaling in surviving cells. One prerequisite for developing new therapeutics is to be able to grow cardiomyocytes ex vivo, and test their apoptotic response to drugs. Here we describe methods for isolation and culturing of neonatal and adult calcium tolerant cardiomyocytes. Similarly, cardiofibroblasts can also be isolated using the same protocol and subsequently, immortalized with SV40 T-Antigen for ex vivo studies. PMID:27108436

  17. Portable system for continuous ex vivo measurements of lactate.

    PubMed

    Håkanson, H; Kyröläinen, M; Mattiasson, B

    1993-01-01

    The lactate profile in serum was monitored continuously in volunteers under physical exercise on a test bicycle. The assay was operated ex vivo by means of continuous sampling through a coaxial catheter. After dialysis online, lactate was converted by means of lactate oxidase immobilized to porous glass, and the depletion of oxygen was registered by means of a Clark electrode. Good agreement between the continuous monitoring and off-line analyses was found. PMID:8357576

  18. The impact of anthropometric patient-phantom matching on organ dose: A hybrid phantom study for fluoroscopy guided interventions

    SciTech Connect

    Johnson, Perry B.; Geyer, Amy; Borrego, David; Ficarrotta, Kayla; Johnson, Kevin; Bolch, Wesley E.

    2011-02-15

    Purpose: To investigate the benefits and limitations of patient-phantom matching for determining organ dose during fluoroscopy guided interventions. Methods: In this study, 27 CT datasets representing patients of different sizes and genders were contoured and converted into patient-specific computational models. Each model was matched, based on height and weight, to computational phantoms selected from the UF hybrid patient-dependent series. In order to investigate the influence of phantom type on patient organ dose, Monte Carlo methods were used to simulate two cardiac projections (PA/left lateral) and two abdominal projections (RAO/LPO). Organ dose conversion coefficients were then calculated for each patient-specific and patient-dependent phantom and also for a reference stylized and reference hybrid phantom. The coefficients were subsequently analyzed for any correlation between patient-specificity and the accuracy of the dose estimate. Accuracy was quantified by calculating an absolute percent difference using the patient-specific dose conversion coefficients as the reference. Results: Patient-phantom matching was shown most beneficial for estimating the dose to heavy patients. In these cases, the improvement over using a reference stylized phantom ranged from approximately 50% to 120% for abdominal projections and for a reference hybrid phantom from 20% to 60% for all projections. For lighter individuals, patient-phantom matching was clearly superior to using a reference stylized phantom, but not significantly better than using a reference hybrid phantom for certain fields and projections. Conclusions: The results indicate two sources of error when patients are matched with phantoms: Anatomical error, which is inherent due to differences in organ size and location, and error attributed to differences in the total soft tissue attenuation. For small patients, differences in soft tissue attenuation are minimal and are exceeded by inherent anatomical differences

  19. Trans-cranial focused ultrasound without hair shaving: feasibility study in an ex vivo cadaver model

    PubMed Central

    2013-01-01

    In preparing a patient for a trans-cranial magnetic resonance (MR)-guided focused ultrasound procedure, current practice is to shave the patient’s head on treatment day. Here we present an initial attempt to evaluate the feasibility of trans-cranial focused ultrasound in an unshaved, ex vivo human head model. A human skull filled with tissue-mimicking phantom and covered with a wig made of human hair was sonicated using 220- and 710-kHz head transducers to evaluate the feasibility of acoustic energy transfer. Heating at the focal point was measured by MR proton resonance shift thermometry. Results showed that the hair had a negligible effect on focal spot thermal rise at 220 kHz and a 17% drop in temperature elevation when using 710 kHz. PMID:25512865

  20. Real-time vascular mechanosensation through ex vivo artery perfusion

    PubMed Central

    2014-01-01

    Background Cell-based perfusion studies have provided great insight into fluid-sensing mechanisms, such as primary cilia in the renal and vascular systems. However, the intrinsic limitations of in vitro cell culture, such as the inability to reflect cellular organization within tissues, has distanced observed paradigms from possible clinical developments. Here we describe a protocol that applies ex vivo artery perfusion and calcium imaging to observe real-time cellular responses to fluid-shear stress. Results Through our ex vivo artery perfusion method, we were able to simulate physiological flow and initiate distinct fluid shear stress mechanosensory responses, as well as induced acetylcholine responses in mouse aortic tissue. The observed calcium profiles confirm results found through previous in vitro cell culture experiments. The overall procedure, including dissection, sample preparation and perfusion, takes around 3 hours to complete. Conclusion Through our unique method, we are able to induce laminar flow within intact mouse aortic tissue and illicit subsequent cellular responses. This method of ex vivo artery perfusion provides the opportunity to bridge the novel findings of in vitro studies with subsequent physiological models of fluid-shear stress mechanosensation in vascular tissues. PMID:24685068

  1. MRI parcellation of ex vivo medial temporal lobe.

    PubMed

    Augustinack, Jean C; Magnain, Caroline; Reuter, Martin; van der Kouwe, André J W; Boas, David; Fischl, Bruce

    2014-06-01

    Recent advancements in radio frequency coils, field strength and sophisticated pulse sequences have propelled modern brain mapping and have made validation to biological standards - histology and pathology - possible. The medial temporal lobe has long been established as a pivotal brain region for connectivity, function and unique structure in the human brain, and reveals disconnection in mild Alzheimer's disease. Specific brain mapping of mesocortical areas affected with neurofibrillary tangle pathology early in disease progression provides not only an accurate description for location of these areas but also supplies spherical coordinates that allow comparison between other ex vivo cases and larger in vivo datasets. We have identified several cytoarchitectonic features in the medial temporal lobe with high resolution ex vivo MRI, including gray matter structures such as the entorhinal layer II 'islands', perirhinal layer II-III columns, presubicular 'clouds', granule cell layer of the dentate gyrus as well as lamina of the hippocampus. Localization of Brodmann areas 28 and 35 (entorhinal and perirhinal, respectively) demonstrates MRI based area boundaries validated with multiple methods and histological stains. Based on our findings, both myelin and Nissl staining relate to contrast in ex vivo MRI. Precise brain mapping serves to create modern atlases for cortical areas, allowing accurate localization with important applications to detecting early disease processes. PMID:23702414

  2. Preliminary Study of Open Quotient in an Ex-Vivo Perfused Human Larynx Model

    PubMed Central

    Mendelsohn, Abie H.; Zhang, Zhaoyan; Luegmair, Georg; Orestes, Michael; Berke, Gerald S.

    2016-01-01

    Importance Scientific understanding human voice production to date is a product of indirect investigations including animal models, cadaveric tissue study, or computational modeling. Due to its invasive nature, direct experimentation of human voice production has previously not been possible. The feasibility of an ex-vivo perfused human phonatory model has recently allowed systematic investigation in virtually living human larynges with parametric laryngeal muscle stimulation. Objective In this study, the relationship between adductor muscle group stimulation and the open quotient (OQ) of vocal fold vibration was investigated using an ex-vivo perfused human larynx. Design Human perfused tissue study. Setting Physiology Laboratory. Participants Human larynx is recovered from research-consented organ donors within two hours of cardiac death. Interventions, Main Outcomes and Measures Perfusion with donated human blood is re-established shortly after cardiac death. Human perfused phonation is achieved by providing subglottal airflow under graded neuromuscular electrical stimulation bilaterally to the intrinsic adductor groups and cricothyroid muscles. The phonation resulting from the graded states of neuromuscular stimulations are evaluated through high-speed vibratory imaging. OQ is derived through digital kymography and glottal area waveform analysis. Results Under constant glottal flow, step-wise increase in adductor muscle group stimulation decreased OQ. Quantitatively, OQ values reached a lower limit of 0.42. Increased stimulation above maximal muscle deformation was unable to affect OQ beyond this lower limit. Conclusions and Relevance For the first time in a neuromuscularly activated human larynx, a negative relationship between adductor muscle group stimulation and phonatory OQ was demonstrated. Further experience with the ex-vivo perfused human phonatory model will work to systematically define this causal relationship. PMID:26181642

  3. Comparison of in vivo and ex vivo imaging of the microvasculature with 2-photon fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Steinman, Joe; Koletar, Margaret; Stefanovic, Bojana; Sled, John G.

    2016-03-01

    This study evaluates 2-Photon fluorescence microscopy of in vivo and ex vivo cleared samples for visualizing cortical vasculature. Four mice brains were imaged with in vivo 2PFM. Mice were then perfused with a FITC gel and cleared in fructose. The same regions imaged in vivo were imaged ex vivo. Vessels were segmented automatically in both images using an in-house developed algorithm that accounts for the anisotropic and spatially varying PSF ex vivo. Through non-linear warping, the ex vivo image and tracing were aligned to the in vivo image. The corresponding vessels were identified through a local search algorithm. This enabled comparison of identical vessels in vivo/ex vivo. A similar process was conducted on the in vivo tracing to determine the percentage of vessels perfused. Of all the vessels identified over the four brains in vivo, 98% were present ex vivo. There was a trend towards reduced vessel diameter ex vivo by 12.7%, and the shrinkage varied between specimens (0% to 26%). Large diameter surface vessels, through a process termed 'shadowing', attenuated in vivo signal from deeper cortical vessels by 40% at 300 μm below the cortical surface, which does not occur ex vivo. In summary, though there is a mean diameter shrinkage ex vivo, ex vivo imaging has a reduced shadowing artifact. Additionally, since imaging depths are only limited by the working distance of the microscope objective, ex vivo imaging is more suitable for imaging large portions of the brain.

  4. CT Fluoroscopy-Guided Lung Biopsy with Novel Steerable Biopsy Canula: Ex-Vivo Evaluation in Ventilated Porcine Lung Explants

    SciTech Connect

    Schaefer, Philipp J. Fabel, Michael; Bolte, Hendrik; Schaefer, Fritz K. W.; Jahnke, Thomas; Heller, Martin; Lammer, Johannes; Biederer, Juergen

    2010-08-15

    The purpose was to evaluate ex-vivo a prototype of a novel biopsy canula under CT fluoroscopy-guidance in ventilated porcine lung explants in respiratory motion simulations. Using an established chest phantom for porcine lung explants, n = 24 artificial lesions consisting of a fat-wax-Lipiodol mixture (approx. 70HU) were placed adjacent to sensible structures such as aorta, pericardium, diaphragm, bronchus and pulmonary artery. A piston pump connected to a reservoir beneath a flexible silicone reconstruction of a diaphragm simulated respiratory motion by rhythmic inflation and deflation of 1.5 L water. As biopsy device an 18-gauge prototype biopsy canula with a lancet-like, helically bended cutting edge was used. The artificial lesions were punctured under CT fluoroscopy-guidance (SOMATOM Sensation 64, Siemens, Erlangen, Germany; 30mAs/120 kV/5 mm slice thickness) implementing a dedicated protocol for CT fluoroscopy-guided lung biopsy. The mean-diameter of the artificial lesions was 8.3 {+-} 2.6 mm, and the mean-distance of the phantom wall to the lesions was 54.1 {+-} 13.5 mm. The mean-displacement of the lesions by respiratory motion was 14.1 {+-} 4.0 mm. The mean-duration of CT fluoroscopy was 9.6 {+-} 5.1 s. On a 4-point scale (1 = central; 2 = peripheral; 3 = marginal; 4 = off target), the mean-targeted precision was 1.9 {+-} 0.9. No misplacement of the biopsy canula affecting adjacent structures could be detected. The novel steerable biopsy canula proved to be efficient in the ex-vivo set-up. The chest phantom enabling respiratory motion and the steerable biopsy canula offer a feasible ex-vivo system for evaluating and training CT fluoroscopy-guided lung biopsy adapted to respiratory motion.

  5. Development and clinical translation of OTIS: a wide-field OCT imaging device for ex-vivo tissue characterization

    NASA Astrophysics Data System (ADS)

    Munro, Elizabeth A.; Rempel, David; Danner, Christine; Atchia, Yaaseen; Valic, Michael S.; Berkeley, Andrew; Davoudi, Bahar; Magnin, Paul A.; Akens, Margarete; Done, Susan J.; Kulkarni, Supriya; Leong, Wey-Liang; Wilson, Brian C.

    2016-03-01

    We have developed an automated, wide-field optical coherence tomography (OCT)-based imaging device (OTISTM Perimeter Medical Imaging) for peri-operative, ex-vivo tissue imaging. This device features automated image acquisition, enabling rapid capture of high-resolution (15 μm) OCT images from samples up to 10 cm in diameter. We report on the iterative progression of device development from phantom and pre-clinical (tumor xenograft) models through to initial clinical results. We discuss the challenges associated with proving a novel imaging technology against the clinical "gold standard" of conventional post-operative pathology.

  6. Functional Genetic Targeting of Embryonic Kidney Progenitor Cells Ex Vivo

    PubMed Central

    Junttila, Sanna; Saarela, Ulla; Halt, Kimmo; Manninen, Aki; Pärssinen, Heikki; Lecca, M. Rita; Brändli, André W.; Sims-Lucas, Sunder; Skovorodkin, Ilya

    2015-01-01

    The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor–treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting. PMID:25201883

  7. Ex Vivo Fluorescence Molecular Tomography of the Spine

    PubMed Central

    Pimpalkhare, Monish; Chen, Jin; Venugopal, Vivek; Intes, Xavier

    2012-01-01

    We investigated the potential of fluorescence molecular tomography to image ex vivo samples collected from a large animal model, in this case, a dog spine. Wide-field time-gated fluorescence tomography was employed to assess the impact of multiview acquisition, data type, and intrinsic optical properties on the localization and quantification accuracy in imaging a fluorescent inclusion in the intervertebral disk. As expected, the TG data sets, when combining early and late gates, provide significantly better performances than the CW data sets in terms of localization and quantification. Moreover, the use of multiview imaging protocols led to more accurate localization. Additionally, the incorporation of the heterogeneous nature of the tissue in the model to compute the Jacobians led to improved imaging performances. This preliminary imaging study provides a proof of concept of the feasibility of quantitatively imaging complex ex vivo samples nondestructively and with short acquisition times. This work is the first step towards employing optical molecular imaging of the spine to detect and characterize disc degeneration based on targeted fluorescent probes. PMID:23197973

  8. Generation and Expansion of T Helper 17 Lymphocytes Ex Vivo.

    PubMed

    Alizadeh, Darya; Larmonier, Nicolas

    2016-01-01

    CD4(+) T helper (Th) lymphocytes are essential elements of the complex cellular networks regulating the initiation, development, and termination of adaptive immune responses. Different independent and specialized subsets of Th cells can be distinguished based on their dedicated transcription factor and cytokine expression profiles. Th17 lymphocytes have been described about a decade ago as CD4(+) Th cells producing high quantity of IL-17A as a signature cytokine. Since their initial discovery, Th17 have drawn intense scrutiny for their dominant role in the pathogenesis of multiple autoimmune, infectious diseases and allergy. The influence of Th17 lymphocytes in cancer remains however ambiguous. The plethoric functions of Th17 may rely on the remarkable plasticity of these cells, endowed with the ability to trans-differentiate into other Th subpopulations depending on the environmental cytokine context. The possibility to generate Th17 ex vivo has facilitated the elucidation of the signals and transcription factors required for their differentiation and functions and has allowed for the evaluation of their functions following adoptive transfer in vivo. Several protocols have been developed to produce Th17 in vitro. The intent of this chapter is to provide examples of procedures for generating and expanding Th17 ex vivo. PMID:26530797

  9. Fluorescent probes concentration estimation in vitro and ex vivo as a model for early detection of Alzheimer's disease

    NASA Astrophysics Data System (ADS)

    Harbater, Osnat; Gannot, Israel

    2014-12-01

    The pathogenic process of Alzheimer's disease (AD) begins years before clinical diagnosis. Here, we suggest a method that may detect AD several years earlier than current exams. The method is based on previous reports that relate the concentration ratio of biomarkers (amyloid-beta and tau) in the cerebrospinal fluid (CSF) to the development of AD. Our method replaces the lumbar puncture process required for CSF drawing by using fluorescence measurements. The system uses an optical fiber coupled to a laser source and a detector. The laser radiation excites two fluorescent probes which may bond to the CSF biomarkers. Their concentration ratio is extracted from the fluorescence intensities and can be used for future AD detection. First, we present a theoretical model for fluorescence concentration ratio estimation. The method's feasibility was validated using Monte Carlo simulations. Its accuracy was then tested using multilayered tissue phantoms simulating the epidural fat, CSF, and bone. These phantoms have various optical properties, thicknesses, and fluorescence concentrations in order to simulate human anatomy variations and different fiber locations. The method was further tested using ex vivo chicken tissue. The average errors of the estimated concentration ratios were low both in vitro (4.4%) and ex vivo (10.9%), demonstrating high accuracy.

  10. Validation of NIRS in measuring tissue hemoglobin concentration and oxygen saturation on ex vivo and isolated limb models

    NASA Astrophysics Data System (ADS)

    Xu, Xiaorong; Zhu, Wen; Padival, Vikram; Xia, Mengna; Cheng, Xuefeng; Bush, Robin; Christenson, Linda; Chan, Tim; Doherty, Tim; Iatridis, Angelo

    2003-07-01

    Photonify"s tissue spectrometer uses Near-Infrared Spectroscopy for real-time, noninvasive measurement of hemoglobin concentration and oxygen saturation [SO2] of biological tissues. The technology was validated by a series of ex vivo and animal studies. In the ex vivo experiment, a close loop blood circulation system was built, precisely controlling the oxygen saturation and the hemoglobin concentration of a liquid phantom. Photonify"s tissue spectrometer was placed on the surface of the liquid phantom for real time measurement and compared with a gas analyzer, considered the gold standard to measure oxygen saturation and hemoglobin concentration. In the animal experiment, the right hind limb of each dog accepted onto the study was surgically removed. The limb was kept viable by connecting the femoral vein and artery to a blood-primed extracorporeal circuit. Different concentrations of hemoglobin were obtained by adding designated amount of saline solution into the perfusion circuit. Photonify"s tissue spectrometers measured oxygen saturation and hemoglobin concentration at various locations on the limb and compared with gas analyzer results. The test results demonstrated that Photonify"s tissue spectrometers were able to detect the relative changes in tissue oxygen saturation and hemoglobin concentration with a high linear correlation compared to the gas analyzer

  11. Radioprotective effects of ATP in human blood ex vivo

    SciTech Connect

    Swennen, Els L.R. Dagnelie, Pieter C.; Van den Beucken, Twan; Bast, Aalt

    2008-03-07

    Damage to healthy tissue is a major limitation of radiotherapy treatment of cancer patients, leading to several side effects and complications. Radiation-induced release of pro-inflammatory cytokines is thought to be partially responsible for the radiation-associated complications. The aim of the present study was to investigate the protective effects of extracellular ATP on markers of oxidative stress, radiation-induced inflammation and DNA damage in irradiated blood ex vivo. ATP inhibited radiation-induced TNF-{alpha} release and increased IL-10 release. The inhibitory effect of ATP on TNF- {alpha} release was completely reversed by adenosine 5'-O-thiomonophosphate, indicating a P2Y{sub 11} mediated effect. Furthermore, ATP attenuated radiation-induced DNA damage immediate, 3 and 6 h after irradiation. Our study indicates that ATP administration alleviates radiation-toxicity to blood cells, mainly by inhibiting radiation-induced inflammation and DNA damage.

  12. Polyethylene glycol diffusion in ex vivo skin tissue

    NASA Astrophysics Data System (ADS)

    Genin, V. D.; Tuchina, D. K.; Bashkatov, A. N.; Genina, E. A.; Tuchin, V. V.

    2015-11-01

    Optical clearing of the rat skin under the action of polyethylene glycol (PEG) with molecular weight 300 and 400 Dalton was studied ex vivo. The collimated transmittance was measured at the wavelength range 500-900 nm. It was found that collimated transmittance of skin samples increased, whereas weight, thickness and area of the samples decreased during PEG penetration in skin tissue. A mechanism of the optical clearing under the action of PEG is discussed. Taking into account the kinetics of volume and thickness of the skin samples, diffusion coefficient of PEGs in skin tissue has been estimated as (1.83±2.22)×10-6 cm2/s and (1.70±1.47)×10-6 cm2/s for PEG-300 and PEG-400, respectively. The presented results can be useful for enhancement of many methods of laser therapy and optical diagnostics of skin diseases and localization of subcutaneous neoplasms.

  13. Altered T Lymphocyte Proliferation upon Lipopolysaccharide Challenge Ex Vivo

    PubMed Central

    Poujol, Fanny; Monneret, Guillaume; Pachot, Alexandre; Textoris, Julien; Venet, Fabienne

    2015-01-01

    Context Sepsis is characterized by the development of adaptive immune cell alterations, which intensity and duration are associated with increased risk of health-care associated infections and mortality. However, pathophysiological mechanisms leading to such lymphocyte dysfunctions are not completely understood, although both intrinsic lymphocyte alterations and antigen-presenting cells (APCs) dysfunctions are most likely involved. Study The aim of the current study was to evaluate whether lipopolysaccharide (LPS, mimicking initial Gram negative bacterial challenge) could directly impact lymphocyte function after sepsis. Therefore, we explored ex-vivo the effect of LPS priming on human T lymphocyte proliferation induced by different stimuli. Results We showed that LPS priming of PBMCs reduced T cell proliferative response and altered IFNγ secretion after stimulation with OKT3 but not with phytohaemagglutinin or anti-CD2/CD3/CD28-coated beads stimulations. Interestingly only LPS priming of monocytes led to decreased T cell proliferative response as opposed to LPS priming of lymphocytes. Importantly, LPS priming was associated with reduced expression of HLA-DR, CD86 and CD64 on monocytes but not with the modification of CD3, CTLA4, PD-1 and CD28 expressions on lymphocytes. Finally, IFNγ stimulation restored monocytes accessory functions and T cell proliferative response to OKT3. Conclusion We conclude that LPS priming does not directly impact lymphocyte functions but reduces APC’s capacity to activate T cells. This recapitulates ex vivo indirect mechanisms participating in sepsis-induced lymphocyte alterations and suggests that monocyte-targeting immunoadjuvant therapies in sepsis may also help to improve adaptive immune dysfunctions. Direct mechanisms impacting lymphocytes being also at play during sepsis, the respective parts of direct versus indirect sepsis-induced lymphocyte alterations remain to be evaluated in clinic. PMID:26642057

  14. Artifacts of vena cava filters ex vivo on MR angiography.

    PubMed

    Honda, Minoru; Obuchi, Masao; Sugimoto, Hideharu

    2003-07-01

    We evaluated magnetic susceptibility artifacts of nine types of vena cava filters in MR angiography (MRA) at 1.0T ex vivo in order to assess the filters' compatibility with MRA. Each filter (tulip filter, tulip MReye filter, stainless Greenfield filter, titanium Greenfield filter, TrapEase filter, Simon filter, LGM Vena-Tech filter, Antheor temporary filter, and Bird's nest filter) was inserted into an acrylic tube (20 or 25 mm in diameter, 15 or 30 cm in length). Gd-DTPA was poured into each tube at a concentration of 1/500 and each was placed in a water-filled container for imaging. We evaluated artifacts of the filters according to the following criteria: signal void beyond the tube, 3+; signal void within the tube but at more than one-half the diameter of the tube, 2+; and signal void within the tube but at less than one-half the diameter of the tube, 1+. We evaluated artifacts originating at the tip, intermediate portion, and distal end of the filters. We judged the artifacts as follows: tulip (3+, 3+, 3+); tulip MReye (2+, 1+, 1+); stainless Greenfield (2+, 1+, 2+); titanium Greenfield (1+, 1+, 1+); TrapEase (1+, 2+, 1+); Simon (2+, 2+, 1+); LGM (2+, 2+, 1+); Antheor (2+, 2+, 2+); and Bird's nest (3+, 3+, 3+). The numbers in parentheses refer to the degree of signal void at the tip, intermediate portion, and distal end of the filter, respectively. The tulip filter and Bird's nest filter made of 304 stainless steel caused extensive signal voids beyond the areas defined by the filters. The signal voids in the remaining seven filters were limited to within the tube. We concluded that seven of the nine filters were compatible with MRA ex vivo. PMID:16210823

  15. Influence of water dilution on percutaneous absorption of N-vinyl-2-pyrrolidone in vivo and ex vivo in rats and ex vivo in humans.

    PubMed

    Marquet, Fabrice; Payan, Jean-Paul; Beydon, Dominique; Wathier, Ludivine; Ferrari, Elisabeth; Grandclaude, Marie-Christine

    2015-11-01

    N-vinyl-2-pyrrolidone (NVP) is mainly used as a monomer in the production of polyvinylpyrrolidone or copolymers. Percutaneous absorption is an important source of exposure in the work environment. However, few studies have investigated this route of absorption. In this study, percutaneous absorption of neat or aqueous NVP solutions was measured in vivo and ex vivo in rats, and ex vivo in humans. Penetration and absorption fluxes were very similar following in vivo exposure to neat NVP (0.54 and 0.43 mg/cm(2)/h, respectively). Exposing rats to a 50% aqueous solution of NVP increased both fluxes threefold (to 1.48 and 1.55 mg/cm(2)/h, respectively). Ex vivo, the absorption flux increased with solutions from 10 to 25% of NVP, reached a plateau (between 25 and 50% in rat, 25 and 75% in human) and then decreased with neat NVP. In vivo and ex vivo absorption fluxes measured using rat skin were similar, supporting the hypothesis that the ex vivo measurements were a good representation of what was observed in vivo. Thus, for humans, the ex vivo measurements are likely the same as would be determined in vivo. PMID:25160662

  16. Vesicoureteral reflux in young children: a study of radiometric thermometry as detection modality using an ex vivo porcine model

    NASA Astrophysics Data System (ADS)

    Jacobsen, Svein; Klemetsen, Øystein; Birkelund, Yngve

    2012-09-01

    Microwave radiometry is evaluated for renal thermometry tailored to detect the pediatric condition of vesicoureteral urine reflux (VUR) from the bladder through the ureter into the kidney. Prior to a potential reflux event, the urine is heated within the bladder by an external body contacting a hyperthermia applicator to generate a fluidic contrast temperature relative to normal body temperature. A single band, miniaturized radiometer (operating at 3.5 GHz) is connected to an electromagnetic-interference-shielded and suction-coupled elliptical antenna to receive thermal radiation from an ex vivo porcine phantom model. Brightness (radiometric) and fiberoptic temperature data are recorded for varying urine phantom reflux volumes (20-40 mL) and contrast temperatures ranging from 2 to 10 °C within the kidney phantom. The kidney phantom itself is located at 40 mm depth (skin-to-kidney center distance) and surrounded by the porcine phantom. Radiometric step responses to injection of urine simulant by a syringe are shown to be highly correlated with in situ kidney temperatures measured by fiberoptic probes. Statistically, the performance of the VUR detecting scheme is evaluated by error probabilities of making a wrong decision. Laboratory testing of the radiometric system supports the feasibility of passive non-invasive kidney thermometry for the detection of VUR classified within the two highest grades

  17. Vesicoureteral reflux in young children: a study of radiometric thermometry as detection modality using an ex vivo porcine model.

    PubMed

    Jacobsen, Svein; Klemetsen, Øystein; Birkelund, Yngve

    2012-09-01

    Microwave radiometry is evaluated for renal thermometry tailored to detect the pediatric condition of vesicoureteral urine reflux (VUR) from the bladder through the ureter into the kidney. Prior to a potential reflux event, the urine is heated within the bladder by an external body contacting a hyperthermia applicator to generate a fluidic contrast temperature relative to normal body temperature. A single band, miniaturized radiometer (operating at 3.5 GHz) is connected to an electromagnetic-interference-shielded and suction-coupled elliptical antenna to receive thermal radiation from an ex vivo porcine phantom model. Brightness (radiometric) and fiberoptic temperature data are recorded for varying urine phantom reflux volumes (20-40 mL) and contrast temperatures ranging from 2 to 10 °C within the kidney phantom. The kidney phantom itself is located at 40 mm depth (skin-to-kidney center distance) and surrounded by the porcine phantom. Radiometric step responses to injection of urine simulant by a syringe are shown to be highly correlated with in situ kidney temperatures measured by fiberoptic probes. Statistically, the performance of the VUR detecting scheme is evaluated by error probabilities of making a wrong decision. Laboratory testing of the radiometric system supports the feasibility of passive non-invasive kidney thermometry for the detection of VUR classified within the two highest grades. PMID:22892477

  18. MR elastography and diffusion-weighted imaging of ex vivo prostate cancer: quantitative comparison to histopathology.

    PubMed

    Sahebjavaher, Ramin S; Nir, Guy; Gagnon, Louis O; Ischia, Joseph; Jones, Edward C; Chang, Silvia D; Yung, Andrew; Honarvar, Mohammad; Fazli, Ladan; Goldenberg, S Larry; Rohling, Robert; Sinkus, Ralph; Kozlowski, Piotr; Salcudean, Septimiu E

    2015-01-01

    The purpose of this work was (1) to develop a magnetic resonance elastography (MRE) system for imaging of the ex vivo human prostate and (2) to assess the diagnostic power of mono-frequency and multi-frequency MRE and diffusion weighted imaging (DWI) alone and combined as correlated with histopathology in a patient study. An electromagnetic driver was designed specifically for MRE studies in small-bore MR scanners. Ex vivo prostate specimens (post-fixation) of 14 patients who underwent radical prostatectomy were imaged with MRE at 7 T (nine cases had DWI). In six patients, the MRE examination was performed at three frequencies (600, 800, 1000 Hz) to extract the power-law exponent Gamma. The images were registered to wholemount pathology slides marked with the Gleason score. The areas under the receiver-operator-characteristic curves (AUC) were calculated. The methods were validated in a phantom study and it was demonstrated that (i) the driver does not interfere with the acquisition process and (ii) the driver can generate amplitudes greater than 100 µm for frequencies less than 1 kHz. In the quantitative study, cancerous tissue with Gleason score at least 3 + 3 was distinguished from normal tissue in the peripheral zone (PZ) with an average AUC of 0.75 (Gd ), 0.75 (Gl ), 0.70 (Gamma-Gd ), 0.68 (apparent diffusion coefficient, ADC), and 0.82 (Gd  + Gl  + ADC). The differentiation between PZ and central gland was modest for Gd (p < 0.07), Gl (p < 0.06) but not significant for Gamma (p < 0.2). A correlation of 0.4 kPa/h was found between the fixation time of the prostate specimen and the stiffness of the tissue, which could affect the diagnostic power results. DWI and MRE may provide complementary information; in fact MRE performed better than ADC in distinguishing normal from cancerous tissue in some cases. Multi-frequency (Gamma) analysis did not appear to improve the results. However, in light of the effect of tissue fixation, the

  19. Photodynamic diagnosis of bladder cancer in ex vivo urine cytology

    NASA Astrophysics Data System (ADS)

    Fu, C. Y.; Ng, B. K.; Razul, S. Gulam; Olivo, Malini C.; Lau, Weber K. O.; Tan, P. H.; Chin, William

    2006-02-01

    Bladder cancer is the fourth common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate can be reduced by early treatment following precancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimen, which is both invasive and time-consuming. In this study ex vivo urine fluorescence cytology is investigated to offer a timely and biopsy-free means for detecting bladder cancers. Sediments in patients' urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cells from the other cells based on the cellular size. Intensity histogram of each targeted cell was plotted and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cells was observed. Diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this paper. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.

  20. Recellularized human dermis for testing gene electrotransfer ex vivo.

    PubMed

    Bulysheva, Anna A; Burcus, Nina; Lundberg, Cathryn; Edelblute, Chelsea M; Francis, Michael P; Heller, Richard

    2016-01-01

    Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies. PMID:27121769

  1. Photoacoustic tomography of pathological tissue in ex vivo mouse hearts

    NASA Astrophysics Data System (ADS)

    Holotta, Markus; Grossauer, Harald; Kremser, Christian; Torbica, Pavle; Völkl, Jakob; Degenhart, Gerald; Esterhammer, Regina; Nuster, Robert; Paltauf, Günther; Jaschke, Werner

    2010-02-01

    In the present study, we evaluate the applicability of ex-vivo photoacoustic imaging (PAI) in organs of small animals. We used photoacoustic tomography (PAT) to visualize infarcted areas within mouse hearts and compared it to other imaging techniques (MRI and μCT). In order to induce ischemia an in-vivo ligation of the Ramus interventricularis anterior (RIVA, left anterior descending, LAD) was performed on nine wild type C41 mice. After varying survival periods the mice were sacrificed. The hearts were excised and immediately transferred into a formaldehyde solution for conservation. Various wavelengths in the visible and near infrared region (500 nm - 1000 nm) had been tested to find the best representation of the ischemic regions. Samples were illuminated with nanosecond laser pulses delivered by an Nd:YAG pumped optical parametric oscillator. Ultrasound detection was achieved by an optical Mach-Zehnder interferometer working as an integrating line detector. For acoustic coupling the samples were located inside a water tank. The voxel data are computed from the measurement data by a Fourier-domain based reconstruction algorithm, followed by a sequence of inverse Radon transforms. Results clearly show the capability of PAI to detect pathological tissue and the possibility to produce three-dimensional images with resolutions well below 100 μm. Different wavelengths allow the representation of structure inside an organ or on the surface even without contrast enhancing tracers.

  2. Photoacoustic tomography of ex vivo mouse hearts with myocardial infarction

    NASA Astrophysics Data System (ADS)

    Holotta, Markus; Grossauer, Harald; Kremser, Christian; Torbica, Pavle; Völkl, Jakob; Degenhart, Gerald; Esterhammer, Regina; Nuster, Robert; Paltauf, Günther; Jaschke, Werner

    2011-03-01

    In the present study, we evaluated the applicability of ex vivo photoacoustic imaging (PAI) on small animal organs. We used photoacoustic tomography (PAT) to visualize infarcted areas within murine hearts and compared these data to other imaging techniques [magnetic resonance imaging (MRI), micro-computed tomography] and histological slices. In order to induce ischemia, an in vivo ligation of the left anterior descending artery was performed on nine wild-type mice. After varying survival periods, the hearts were excised and fixed in formaldehyde. Samples were illuminated with nanosecond laser pulses delivered by a Nd:YAG pumped optical parametric oscillator. Ultrasound detection was achieved using a Mach-Zehnder interferometer (MZI) working as an integrating line detector. The voxel data were computed using a Fourier-domain based reconstruction algorithm, followed by inverse Radon transforms. The results clearly showed the capability of PAI to visualize myocardial infarction and to produce three-dimensional images with a spatial resolution of approximately 120 μm. Regions of affected muscle tissue in PAI corresponded well with the results of MRI and histology. Photoacoustic tomography utilizing a MZI for ultrasound detection allows for imaging of small tissue samples. Due to its high spatial resolution, good soft tissue contrast and comparatively low cost, PAT offers great potentials for imaging.

  3. Impact of Hydration Media on Ex Vivo Corneal Elasticity Measurements

    PubMed Central

    Dias, Janice; Ziebarth, Noël M.

    2014-01-01

    Objectives To determine the effect of hydration media on ex vivo corneal elasticity. Methods Experiments were conducted on forty porcine eyes retrieved from an abattoir (10 eyes each for PBS, BSS, Optisol, 15% Dextran). The epithelium was removed and the cornea was excised with an intact scleral rim and placed in 20% Dextran overnight to restore its physiological thickness. For each hydration media, corneas were evenly divided into two groups: one with an intact scleral rim and the other without. Corneas were mounted onto a custom chamber and immersed in a hydration medium for elasticity testing. While in each medium, corneal elasticity measurements were performed for 2 hours: at 5-minute intervals for the first 30 minutes and then 15-minute intervals for the remaining 90 minutes. Elasticity testing was performed using nanoindentation with spherical indenters and Young’s modulus was calculated using the Hertz model. Thickness measurements were taken before and after elasticity testing. Results The percentage change in corneal thickness and elasticity was calculated for each hydration media group. BSS, PBS, and Optisol showed an increase in thickness and Young’s moduli for corneas with and without an intact scleral rim. 15% Dextran exhibited a dehydrating effect on corneal thickness and provided stable maintenance of corneal elasticity for both groups. Conclusions Hydration media affects the stability of corneal thickness and elasticity measurements over time. 15% Dextran was most effective in maintaining corneal hydration and elasticity, followed by Optisol. PMID:25603443

  4. Optical clearing of skin tissue ex vivo with polyethylene glycol

    NASA Astrophysics Data System (ADS)

    Tuchina, D. K.; Genin, V. D.; Bashkatov, A. N.; Genina, E. A.; Tuchin, V. V.

    2016-01-01

    Alterations of the optical and structural (weight, thickness, and square) parameters of skin caused by polyethylene glycol (PEG) with molecular weights of 300 and 400 Da were studied experimentally. The objects of the study were ex vivo skin samples of albino laboratory rats. Collimated transmittance of the skin was measured in the wavelength range 500-900 nm. As a result of exposure to the agents, an increase in the collimated transmittance and a decrease in weight, thickness, and square of skin samples were observed. Analysis of the kinetics of parameters alterations allowed us to measure the diffusion coefficient of the agents in the skin as (1.83 ± 2.22) × 10-6 and (1.70 ± 1.47) × 10-6 cm2/s for PEG-300 and PEG-400, respectively, and the rate of alterations of the structural parameters. The results obtained in this study can be used for the improvement of existing and development of new methods of noninvasive diagnostics and therapy of subcutaneous diseases.

  5. Brain-derived neurotrophic factor expression ex vivo in obesity.

    PubMed

    Huang, Chun-Jung; Mari, David C; Whitehurst, Michael; Slusher, Aaron; Wilson, Alan; Shibata, Yoshimi

    2014-01-17

    Obesity is associated with an increased risk in neurodegenerative diseases. To counteract the neuronal damage, the human body increases brain-derived neurotrophic factor (BDNF) expression, leading to neuronal survival and plasticity. Recently, peripheral blood mononuclear cells (PBMCs) have been found to release BDNF as a potential neuroprotective role of inflammation. Therefore, the purpose of this study was to examine whether lipopolysaccharide (LPS)-induced PBMC activation would lead to differences in BDNF and inflammatory responses between obese and non-obese subjects. Thirty-one subjects (14 obese and 17 non-obese), ages 18 to 30years, were recruited. PBMCs were cultured for 24h with 10ng/mL LPS. BDNF, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured in both plasma and cell culture supernatants. Our results did not illustrate any differences in plasma BDNF levels between obese and non-obese groups. However, obese subjects elicited a greater plasma IL-6 production, which was positively associated with plasma BDNF. Furthermore, LPS-induced PBMCs expressed significantly higher BDNF and IL-6 levels in obese subjects compared to the non-obese subjects. Finally, these BDNF levels were positively correlated with IL-6 response ex vivo. These findings suggest that under a high inflammatory state, PBMCs produce greater BDNF and IL-6 expression which may play a collaborative role to protect against neuronal damage associated with obesity. PMID:24140987

  6. Ex vivo laser lipolysis assisted with radially diffusing optical applicator

    NASA Astrophysics Data System (ADS)

    Hwang, Jieun; Hau, Nguyen Trung; Park, Sung Yeon; Rhee, Yun-Hee; Ahn, Jin-Chul; Kang, Hyun Wook

    2016-05-01

    Laser-assisted lipolysis has been implemented to reduce body fat in light of thermal interactions with adipose tissue. However, using a flat fiber with high irradiance often needs rapid cannula movements and even undesirable thermal injury due to direct tissue contact. The aim of the current study was to explore the feasibility of a radially diffusing optical applicator to liquefy the adipose tissue for effective laser lipolysis. The proposed diffuser was evaluated with a flat fiber in terms of temperature elevation and tissue liquefaction after laser lipolysis with a 980-nm wavelength. Given the same power (20 W), the diffusing applicator generated a 30% slower temperature increase with a 25% lower maximum temperature (84±3.2°C in 1 min p<0.001) in the tissue, compared with the flat fiber. Under the equivalent temperature development, the diffuser induced up to fivefold larger area of the adipose liquefaction due to radial light emission than the flat fiber. Ex vivo tissue tests for 5-min irradiation demonstrated that the diffuser (1.24±0.15 g) liquefied 66% more adipose tissue than the flat fiber (0.75±0.05 g). The proposed diffusing applicator can be a feasible therapeutic device for laser lipolysis due to low temperature development and wide coverage of thermal treatment.

  7. Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs.

    PubMed

    Nakajima, D; Cypel, M; Bonato, R; Machuca, T N; Iskender, I; Hashimoto, K; Linacre, V; Chen, M; Coutinho, R; Azad, S; Martinu, T; Waddell, T K; Hwang, D M; Husain, S; Liu, M; Keshavjee, S

    2016-04-01

    Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function. PMID:26730551

  8. Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment.

    PubMed

    van den Bijgaart, Renske J E; Kong, Niwen; Maynard, Carrie; Plaks, Vicki

    2016-01-01

    Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment. PMID:26862704

  9. Ex vivo expansion of human peripheral blood progenitors.

    PubMed

    Chabannon, C; Herrera-Rodriguez, D; Bardin, F; Mouren, M; Novakovitch, G; Blaise, D; Maraninchi, D; Mannoni, P

    1995-01-01

    Culture of human hematopoietic progenitors on a large scale could lead to several clinical applications within the near future, including the production of differentiated and functional cells, the increase in the number of early progenitors, especially stem cells, with such use as gene transfer, or the improvement of grafts used to limit the hematological toxicity associated with high-dose chemotherapy. In this case, one can still distinguish different objectives: improvement of grafts that contain low numbers of progenitors because of prior chemotherapies or because of marrow involvement for example, and qualitative changes in the graft content that would allow to envision the disappearance, or the further reduction, in the duration of absolute neutropenia that follows delivery of high dose chemotherapy ("nadir rescue"), despite substitution of mobilized blood cells to marrow cells and the in vivo use of hematopoietic growth factors. Additional advantages may be related to tumor purging in autologous expanded cells, and to the change in the ratio between hematopoietic progenitors and immunocompetent cells in allogeneic expanded populations. Therefore it appears that in vitro expansion currently raises two types of questions: the first ones are related to the definition of clinical or biological endpoints to be achieved, the second ones are related to "bioengineering", and deal with the efficiency and safety of progenitor cell cultures to be used for clinical applications. We here present preliminary results preparing future pilot clinical studies with ex vivo cultured human hematopoietic cells. PMID:8907631

  10. A superfusion apparatus for ex vivo human eye irritation investigations.

    PubMed

    Elbadawy, Hossein Mostafa; Salvalaio, Gianni; Parekh, Mohit; Ruzza, Alessandro; Baruzzo, Mattia; Cagini, Carlo; Ponzin, Diego; Ferrari, Stefano

    2015-10-01

    A superfusion apparatus (SA) was developed to maintain isolated human corneas ex vivo under conditions which mimic the natural eye environment in vivo, including controlled temperature, tear flow and intraocular pressure. The SA was designed, developed and tested for use in ophthalmic pre-clinical research and to test new pharmaceutical formulations. Corneas undergo an equilibration process in the new physiological environment for one day. The test was then initiated by the application of the test substance, incubation, and temporal assessment of corneal damage using various parameters. The effects of mild and severe irritant concentrations of NaOH (2% and 8%, respectively) on corneal opacity, swelling and epithelial integrity were studied, and the inflammatory status assessed using F4/80 and MPO as macrophages and neutrophils markers, respectively. The SA was then used to test new artificial tear formulations supplemented with silver ions as an active constituent, showing different degrees of inflammatory responses as indicated by the migration of MPO and F4/80 positive cells towards the epithelium. The human cornea superfusion apparatus was proposed as a model for acute eye irritation research. PMID:26100225

  11. Photodynamic inactivation of somatic frog nerve ex vivo

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Seliverstov, George A.; Akchurin, George G.; Kudryashova, Svetlana Y.

    2004-06-01

    New techniques research mechanisms of photdynamic reactions at somatic frog nerve was approved. Dosimetry PDT with minimum time resolution ~1ms determined by changing the amplitude of compound action potential of somatic frog nerve. Light-induced inactivation of dynamic response of somatic frog nerve on electrical pulsed excitation was study ex vivo. The light-sensitive dyes: methylene blue (Mb), Indocianin green and eryhtrocin-B has been used on photodynamic induced inactivation of the processes generation nerve pulses. Inactivation of consequence action potential of somatic frog nerve using excitation of electical pulsed was achieved by irradiation with He-Ne laser light in a red spectral region (λ=633 nm, power level 2-20 mW), diode laser (λ=805 nm, P<0.1-1 W/cm2) in the case of Indocianin green and YAG:Nd laser (λ=532 nm, P~1mW) for eryhtrocin-B. It was discovered that methylene blue, Indocainine green and erytrocin-B decrease of the amplitude compound action potential of the ensemble neurons. The possible cell death mechanism was connected with damage of the sodium potassium adenosine triphosphatase (K-Na ATP) active transport which decrease of amplitude of compound action potential and decrease lifetime ionic channel of membrane nerve.

  12. Effect of ticlopidine ex vivo on platelet intracellular calcium mobilization

    SciTech Connect

    Derian, C.K.; Friedman, P.A.

    1988-04-01

    The antiplatelet compound ticlopidine exerts its potent inhibitory activity through an as yet undetermined mechanism(s). The goal of this study was to determine the effect, if any, of ticlopidine ex vivo on platelet calcium mobilization. Ticlopidine inhibited ADP-induced platelet aggregation by 50-80%. In the presence of 1 mM EGTA, ticlopidine inhibited ADP- and thrombin-stimulated increases in (Ca2+)i in fura-2 loaded platelets. We evaluated further the effect of ticlopidine on calcium mobilization by examining both agonist-stimulated formation of inositol trisphosphate in intact platelets and the ability of inositol trisphosphate to release /sup 45/Ca from intracellular sites in permeabilized cells. We show here that while ticlopidine significantly affected agonist-induced intracellular calcium mobilization in intact platelets, the drug was without effect on agonist-stimulated formation of inositol trisphosphate in intact platelets and on inositol trisphosphate-induced /sup 45/Ca release in saponin-permeabilized platelets. Our study demonstrates that ticlopidine exerts at least part of its effect via inhibition of intracellular calcium mobilization but that its site of action remains to be determined.

  13. Effects of human hair on trans-cranial focused ultrasound efficacy in an ex-vivo cadaver model

    NASA Astrophysics Data System (ADS)

    Hananel, Arik; Snell, John W.; Kassell, Neal F.; Eames, Matthew D. C.

    2012-11-01

    Current practice before a trans-cranial MR guided Focused ultrasound procedure is shaving the patient head on treatment day. Here we present an initial attempt to evaluate the feasibility of trans-cranial FUS, in an unshaved, ex-vivo cadaver skull. We have sonicated using 220kHz and 710kHz head transducers, a cadaver skull filled with tissue mimicking phantom and covered with a wig made of human hair to evaluate feasibility of acoustic energy transfer in a full size model. Heating at focal point was measured using MR proton resonance shift thermometry. Results showed negligible effect of hair in 220kHz, and an 18% drop in temperature elevation when using 710kHz.

  14. Angiotensin II regulates growth of the developing papillas ex vivo

    PubMed Central

    Song, Renfang; Preston, Graeme; Khalili, Ali; El-Dahr, Samir S.

    2012-01-01

    We tested the hypothesis that lack of angiotensin (ANG) II production in angiotensinogen (AGT)-deficient mice or pharmacologic antagonism of ANG II AT1 receptor (AT1R) impairs growth of the developing papillas ex vivo, thus contributing to the hypoplastic renal medulla phenotype observed in AGT- or AT1R-null mice. Papillas were dissected from Hoxb7GFP+ or AGT+/+, +/−, −/− mouse metanephroi on postnatal day P3 and grown in three-dimentional collagen matrix gels in the presence of media (control), ANG II (10−5 M), or the specific AT1R antagonist candesartan (10−6 M) for 24 h. Percent reduction in papillary length was attenuated in AGT+/+ and in AGT+/− compared with AGT−/− (−18.4 ± 1.3 vs. −32.2 ± 1.6%, P < 0.05, −22.8 ± 1.3 vs. −32.2 ± 1.6%, P < 0.05, respectively). ANG II blunted the decrease in papilla length observed in respective media-treated controls in Hoxb7GFP+ (−1.5 ± 0.3 vs. −10.0 ± 1.4%, P < 0.05) or AGT+/+, +/−, and −/− papillas (−12.8 ± 0.7 vs. −18.4 ± 1.3%, P < 0.05, −16.8 ± 1.1 vs. −23 ± 1.2%, P < 0.05; −26.2 ± 1.6 vs. −32.2 ± 1.6%, P < 0.05, respectively). In contrast, percent decrease in the length of Hoxb7GFP+ papillas in the presence of the AT1R antagonist candesartan was higher compared with control (−24.3 ± 2.1 vs. −10.5 ± 1.8%, P < 0.05). The number of proliferating phospho-histone H3 (pH3)-positive collecting duct cells was lower, whereas the number of caspase 3-positive cells undergoing apoptosis was higher in candesartan- vs. media-treated papillas (pH3: 12 ± 1.4 vs. 21 ± 2.1, P < 0.01; caspase 3: 3.8 ± 0.5 vs. 1.7 ± 0.2, P < 0.01). Using quantitative RT-PCR, we demonstrate that AT1R signaling regulates the expression of genes implicated in morphogenesis of the renal medulla. We conclude that AT1R prevents shrinkage of the developing papillas observed ex vivo via control of Wnt7b, FGF7, β-catenin, calcineurin B1, and α3 integrin gene expression, collecting duct cell

  15. Ex Vivo Estimation of Photoacoustic Imaging for Detecting Thyroid Microcalcifications

    PubMed Central

    Kang, Jeeun; Chung, Woong Youn; Kang, Sang-Wook; Kwon, Hyeong Ju; Yoo, Jaeheung; Kim, Eun-Kyung; Chang, Jin Ho; Song, Tai-kyong; Lee, Sohee; Kwak, Jin Young

    2014-01-01

    Background The aim of this study was to evaluate the diagnostic utility of PAI at detecting thyroid microcalcifications at 700 nm laser wavelengths. Methods This study included 36 resected samples in 18 patients. To evaluate the PA manifestation of microcalcifications in PAI, gray level histogram and co-occurrence matrix (COM) texture parameters were extracted from the 3 fixed ROI US and PA images, respectively, per sample. We compared the textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without microcalcifications. Results On specimen US, the mean value (2748.4±862.5) of samples with microcalcifications on specimen radiography was higher than that (1961.9±780.2) of those without microcalcifications (P = 0.007). However, there were no significant differences in textural parameters obtained from specimen PAIs between samples with punctate microcalcifications on specimen radiography and those without when applying both the mean value of the three slices of thyroid specimens and the value of the thyroid specimen slice which had the highest value of the mean values in specimen US. Conclusion PAI did not show significant PA contrast on thyroid microcalcifications indicating that the experimental setup and protocols should be enhanced, e.g., method of complete blood rejection from ex vivo specimens, the multi-wavelength spectroscopic PA imaging method which can solely extract the PA signal from microcalcifications even with high spectral interferences, or PA imaging with narrower slice thickness using 2-dimensional array transducer, etc. PMID:25415564

  16. Choroid Sprouting Assay: An Ex Vivo Model of Microvascular Angiogenesis

    PubMed Central

    Shao, Zhuo; Friedlander, Mollie; Hurst, Christian G.; Cui, Zhenghao; Pei, Dorothy T.; Evans, Lucy P.; Juan, Aimee M.; Tahir, Houda; Duhamel, François; Chen, Jing; Sapieha, Przemyslaw; Chemtob, Sylvain; Joyal, Jean-Sébastien; Smith, Lois E. H.

    2013-01-01

    Angiogenesis of the microvasculature is central to the etiology of many diseases including proliferative retinopathy, age-related macular degeneration and cancer. A mouse model of microvascular angiogenesis would be very valuable and enable access to a wide range of genetically manipulated tissues that closely approximate small blood vessel growth in vivo. Vascular endothelial cells cultured in vitro are widely used, however, isolating pure vascular murine endothelial cells is technically challenging. A microvascular mouse explant model that is robust, quantitative and can be reproduced without difficulty would overcome these limitations. Here we characterized and optimized for reproducibility an organotypic microvascular angiogenesis mouse and rat model from the choroid, a microvascular bed in the posterior of eye. The choroidal tissues from C57BL/6J and 129S6/SvEvTac mice and Sprague Dawley rats were isolated and incubated in Matrigel. Vascular sprouting was comparable between choroid samples obtained from different animals of the same genetic background. The sprouting area, normalized to controls, was highly reproducible between independent experiments. We developed a semi-automated macro in ImageJ software to allow for more efficient quantification of sprouting area. Isolated choroid explants responded to manipulation of the external environment while maintaining the local interactions of endothelial cells with neighboring cells, including pericytes and macrophages as evidenced by immunohistochemistry and fluorescence-activated cell sorting (FACS) analysis. This reproducible ex vivo angiogenesis assay can be used to evaluate angiogenic potential of pharmacologic compounds on microvessels and can take advantage of genetically manipulated mouse tissue for microvascular disease research. PMID:23922736

  17. An Ex vivo Culture System to Study Thyroid Development

    PubMed Central

    Delmarcelle, Anne-Sophie; Villacorte, Mylah

    2014-01-01

    The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the “angio-follicular” units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood. This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo. Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR. In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis. PMID:24961920

  18. In and ex-vivo Myocardial Tissue Temperature Monitoring by Combined Infrared and Ultrasonic Thermometries

    NASA Astrophysics Data System (ADS)

    Engrand, C.; Laux, D.; Ferrandis, J.-Y.; Sinquet, J.-C.; Demaria, R.; Le Clézio, E.

    The success of cardiac surgery essentially depends on tissue preservation during intervention. Consequently a hypothermic cardio-plegia is applied in order to avoid ischemia. However, myocardial temperature is not monitored during operation. The aim of this study is then to find a relevant and simple method for myocardial global temperature estimation in real time using both ultrasounds and infra-red thermography. In order to quantify the sensitivity of ultrasonic velocity to temperature, a 2.25 MHz ultrasonic probe was used for ex-vivo tests. Pig myocards (n=25) were placed in a thermostatically-controlled water bath and measurements of the ultrasound velocity were realized from 10 to 30 ˚C. The results of this study indicate that the specificity and sensitivity of the ultrasonic echo delay induced by the modification of temperature can be exploited for in-depth thermometry. In parallel, for TIR experiments, a bolometer was used to detect the myocardium surface thermal evolution during in-vivo pig heart experiments. Hypothermic cardioplegic solutions were injected and infra-red surface imaging was performed during one hour. In the near futur, the correlation of the ultrasound and the infrared measurements should allow the real time estimation of the global temperature of the heart. The final objective being to realize in vivo measurements on human hearts, this information may have a very high importance in terms of per-operation inspection as well as decision making process during medical interventions.

  19. Identification of ex-vivo confocal scanning microscopic features and their histological correlates in human skin.

    PubMed

    Hartmann, Daniela; Ruini, Cristel; Mathemeier, Leonie; Dietrich, Andreas; Ruzicka, Thomas; von Braunmühl, Tanja

    2016-04-01

    Ex-vivo confocal laser scanning microscopy (CLSM) is an emerging diagnostic tool allowing fast and easy microscopic tissue examination. The first generation of ex-vivo devices have already shown promising results in the ex-vivo evaluation of basal cell carcinoma compared to Mohs surgery. Nevertheless, for the diagnostics of pathological skin lesions the knowledge of normal skin features is essential. Therefore we examined 50 samples of healthy skin from various donor sites including head and neck (n = 25), trunk (n = 10), upper (n = 10) and lower extremities (n = 5) using a new generation ex-vivo CLSM device offering three different laser wavelengths and compared the findings to the corresponding histological sections. In correlation with the histopathology we identified different layers of the epidermis, differentiated keratinocytes from melanocytes and described in detail skin appendages including hair follicle, sebaceous and sweat glands. Furthermore, structures of the dermis and subcutis were illustrated. Additionally, artefacts and pitfalls occurring with the use of ex-vivo CLSM have been documented. The study offers an overview of the main ex-vivo CLSM skin characteristics in comparison to the standard histological examination and helps to recognize and avoid common artefacts. Anatomy of a hair follicle in the reflectance mode (RM) CLSM, fluorescence mode (FM) CLSM and in a routine hematoxylin-eosin stained histological section (H). PMID:25996548

  20. Ex Vivo Growth of Bioengineered Ligaments and Other Tissues

    NASA Technical Reports Server (NTRS)

    Altman, Gregory; Kaplan, David L.; Martin, Ivan; Vunjak-Novakovic, Gordana

    2005-01-01

    A method of growing bioengineered tissues for use in surgical replacement of damaged anterior cruciate ligaments has been invented. An anterior cruciate ligament is one of two ligaments (the other being the posterior cruciate ligament) that cross in the middle of a knee joint and act to prevent the bones in the knee from sliding forward and backward relative to each other. Anterior cruciate ligaments are frequently torn in sports injuries and traffic accidents, resulting in pain and severe limitations on mobility. By making it possible to grow replacement anterior cruciate ligaments that structurally and functionally resemble natural ones more closely than do totally synthetic replacements, the method could create new opportunities for full or nearly full restoration of functionality in injured knees. The method is also adaptable to the growth of bioengineered replacements for other ligaments (e.g., other knee ligaments as well as those in the hands, wrists, and elbows) and to the production of tissues other than ligaments, including cartilage, bones, muscles, and blood vessels. The method is based on the finding that the histomorphological properties of a bioengineered tissue grown in vitro from pluripotent cells within a matrix are affected by the direct application of mechanical force to the matrix during growth generation. This finding provides important new insights into the relationships among mechanical stress, biochemical and cell-immobilization methods, and cell differentiation, and is applicable to the production of the variety of tissues mentioned above. Moreover, this finding can be generalized to nonmechanical (e.g., chemical and electromagnetic) stimuli that are experienced in vivo by tissues of interest and, hence, the method can be modified to incorporate such stimuli in the ex vivo growth of replacements for the various tissues mentioned above. In this method, a three-dimensional matrix made of a suitable material is seeded with pluripotent stem

  1. Effects of Ex-Vivo and In-Vivo Treatment with Probiotics on the Inflammasome in Dogs with Chronic Enteropathy

    PubMed Central

    Schmitz, Silke; Werling, Dirk; Allenspach, Karin

    2015-01-01

    Inflammasomes coordinate the maturation of IL-1β and IL-18 in response to danger signals. They are vital for maintenance of intestinal homeostasis and have been linked to chronic intestinal inflammation in humans. Probiotics have been advocated as treatment in intestinal inflammation. So far, no study has investigated the role of the inflammasome in canine chronic enteropathy (CE). In this study the intestinal expression of inflammasome components was assessed in CE dogs compared to controls, when treated with probiotic Enterococcus faecium (EF) ex-vivo and in-vivo. RNA extraction from endoscopic biopsies and reverse-transcriptase quantitative PCR was performed for NLRP3, casp-1, IL-1β and IL-18. Immunohistochemistry was performed to investigate protein expression in tissues. Gene expression of casp-1 and NLRP3 was lower in CE samples than controls. Ex-vivo treatment with EF reduced NLRP3 expression in control samples. Treatment of CE dogs with EF alongside dietary intervention had no effect on gene expression. In contrast, IL-1β protein expression in CE decreased with dietary treatment (but not with probiotics). The results of this study suggest that the inflammasome or its components may be partially involved in the inflammatory process seen in CE, but distinct from intestinal inflammation in humans. PMID:25799280

  2. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo

    PubMed Central

    Scott, Yanille M.; Park, Seo Young

    2015-01-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection. PMID:26596954

  3. Visualizing Oxazine 4 nerve-specific fluorescence ex vivo in frozen tissue sections

    NASA Astrophysics Data System (ADS)

    Barth, Connor W.; Gibbs, Summer L.

    2016-03-01

    Nerve damage plagues surgical outcomes and remains a major burden for patients, surgeons, and the healthcare system. Fluorescence image-guided surgery using nerve specific small molecule fluorophores offers a solution to diminish surgical nerve damage through improved intraoperative nerve identification and visualization. Oxazine 4 has shown superior nerve specificity in initial testing in vivo, while exhibiting a red shifted excitation and emission spectra compared to other nerve-specific fluorophores. However, Oxazine 4 does not exhibit near-infrared (NIR) excitation and emission, which would be ideal to improve penetration depth and nerve signal to background ratios for in vivo imaging. Successful development of a NIR nerve-specific fluorophore will require understanding of the molecular target of fluorophore nerve specificity. While previous small molecule nerve-specific fluorophores have demonstrated excellent ex vivo nerve specificity, Oxazine 4 ex vivo nerve specific fluorescence has been difficult to visualize. In the present study, we examined each step of the ex vivo fluorescence microscopy sample preparation procedure to discover how in vivo nerve-specific fluorescence is changed during ex vivo tissue sample preparation. Through step-by-step examination we found that Oxazine 4 fluorescence was significantly diminished by washing and mounting tissue sections for microscopy. A method to preserve Oxazine 4 nerve specific fluorescence ex vivo was determined, which can be utilized for visualization by fluorescence microscopy.

  4. Correlation of histological and ex-vivo confocal tumor thickness in malignant melanoma.

    PubMed

    Hartmann, Daniela; Krammer, Sebastian; Ruini, Cristel; Ruzicka, Thomas; von Braunmühl, Tanja

    2016-07-01

    The ex-vivo confocal laser scanning microscopy (ex-vivo CLSM) is a novel diagnostic method for fresh tissue examination, which has already shown promising results in the evaluation of healthy skin and different skin tumors. In malignant melanoma, the histological tumor thickness plays an essential role for further treatment strategies. The immediate perioperative measurement of tumor thickness by means of ex-vivo CLSM might accelerate the decision for further operating procedures in malignant melanoma. Ten histologically confirmed malignant melanomas from various donor sites were blindly examined by two investigators via ex-vivo CLSM and conventional light microscopy. The histopathological tumor thickness (HTT) and confocal tumor thickness (CTT) were measured independently and evaluated using correlation curves, Spearman's correlation coefficient, and Bland-Altman plots. Bland-Altman plots for HTT and reflectance-mode CTT, as well as for fluorescence-mode CTT, showed high correlations. Spearman's correlation coefficient of HTT and CTT was 1.00 in FM and RM. The mean difference of RM-CTT and FM-CTT versus HTT was 0.09 ± 0.30 mm and 0.19 ± 0.35 mm. In one case, the HTT was identical to the CTT in both modes. This pilot study shows high conformity of CTT and HTT measured in malignant melanoma underlining the potential of ex-vivo CLSM for perioperative decisions on safety margin excisions of malignant melanoma in the future. PMID:27056706

  5. Ex Vivo Lung Perfusion and Transplant: State of the Art and View to the Future.

    PubMed

    Mohamed, Mohamed S A

    2015-12-01

    After the first clinical application of ex vivo lung perfusion in 2001, the technique has been used in many lung transplant centers worldwide. In addition, many modifications have been tested, leading to the development of various ex vivo lung perfusion systems and application protocols. Currently, the Lund protocol, the Toronto protocol, and Organ Care System Lung protocol are the clinically applied ex vivo lung perfusion protocols, based on the favorable results of the safety studies. Accordingly, the comparison among these EVLP systems and protocols should be an important research target, in order to provide the evidence based medical data that would recommend one protocol over the others. In this manuscript, the current experience with EVLP is reviewed and some molecular and clinical targets, that could be used to compare the various protocols of the technique, are introduced. PMID:26643670

  6. Intervention Planning Using a Laser Navigation System for CT-Guided Interventions: A Phantom and Patient Study

    PubMed Central

    Lee, Clara; Bolck, Jan; Naguib, Nagy N.N.; Schulz, Boris; Eichler, Katrin; Aschenbach, Rene; Wichmann, Julian L.; Vogl, Thomas. J.; Zangos, Stephan

    2015-01-01

    Objective To investigate the accuracy, efficiency and radiation dose of a novel laser navigation system (LNS) compared to those of free-handed punctures on computed tomography (CT). Materials and Methods Sixty punctures were performed using a phantom body to compare accuracy, timely effort, and radiation dose of the conventional free-handed procedure to those of the LNS-guided method. An additional 20 LNS-guided interventions were performed on another phantom to confirm accuracy. Ten patients subsequently underwent LNS-guided punctures. Results The phantom 1-LNS group showed a target point accuracy of 4.0 ± 2.7 mm (freehand, 6.3 ± 3.6 mm; p = 0.008), entrance point accuracy of 0.8 ± 0.6 mm (freehand, 6.1 ± 4.7 mm), needle angulation accuracy of 1.3 ± 0.9° (freehand, 3.4 ± 3.1°; p < 0.001), intervention time of 7.03 ± 5.18 minutes (freehand, 8.38 ± 4.09 minutes; p = 0.006), and 4.2 ± 3.6 CT images (freehand, 7.9 ± 5.1; p < 0.001). These results show significant improvement in 60 punctures compared to freehand. The phantom 2-LNS group showed a target point accuracy of 3.6 ± 2.5 mm, entrance point accuracy of 1.4 ± 2.0 mm, needle angulation accuracy of 1.0 ± 1.2°, intervention time of 1.44 ± 0.22 minutes, and 3.4 ± 1.7 CT images. The LNS group achieved target point accuracy of 5.0 ± 1.2 mm, entrance point accuracy of 2.0 ± 1.5 mm, needle angulation accuracy of 1.5 ± 0.3°, intervention time of 12.08 ± 3.07 minutes, and used 5.7 ± 1.6 CT-images for the first experience with patients. Conclusion Laser navigation system improved accuracy, duration of intervention, and radiation dose of CT-guided interventions. PMID:26175571

  7. Biomonitoring and Hormone-Disrupting Effect Biomarkers of Persistent Organic Pollutants In Vitro and Ex Vivo

    PubMed Central

    Bonefeld-Jørgensen, Eva C; Ghisari, Mandana; Wielsøe, Maria; Bjerregaard-Olesen, Christian; Kjeldsen, Lisbeth S; Long, Manhai

    2014-01-01

    Persistent organic pollutants (POPs) include lipophilic legacy POPs and the amphiphilic perfluorinated alkyl acids (PFAAs). They have long half-lives and bioaccumulate in the environment, animals and human beings. POPs possess toxic, carcinogenic and endocrine-disrupting potentials. Endocrine-disrupting chemicals (EDCs) are compounds that either mimic or block endogenous hormones and thus disrupt the normal hormone homeostasis. Biomonitoring assesses the internal doses of a person to provide information about chemical exposures. Effect biomarkers assess chemicals potential to affect cellular functions in vivo/ex vivo. Human beings are exposed to complex mixtures of chemicals, having individually very different biological potentials and effects. Therefore, the assessment of the combined, integrated biological effect of the actual chemical mixture in human blood is important. In vitro and ex vivo cell systems have been introduced for the assessment of the integrated level of xenobiotic cellular effects in human beings. Ex vivo studies have shown geographical differences in bioaccumulated POP serum levels, being reflected by the combined biomarker effects of the complex mixture extracted from human serum. Xenohormone receptor transactivities can be used as an ex vivo integrated biomarker of POP exposure and effects. Epidemiological and in vitro/ex vivo studies have supported the potential impact of the combined effect of serum POPs on the activity of hormone and/or dioxin receptors as a risk factor for human health. With focus on hormone disruption, this MiniReview will give an update on recent POP-related endocrine-disrupting effects in vitro/ex vivo/in vivo and some related genetic data. PMID:24797035

  8. Novel Use of Ex Vivo Uretero-Pyeloscopy in Autotransplantation: A Systematic Review and Case Report

    PubMed Central

    Elmer, Sandra; Bolton, Damien M.; Lawrentschuk, Nathan

    2015-01-01

    Abstract Background: Autotransplant has been practiced for decades but is regaining popularity in the nephron-sparing era. Initially for benign disease, autotransplantation has a select role in malignant processes that warrants new techniques and ideas to ensure patient safety. We review the use of ex vivo uretero-pyeloscopy and frozen section to ensure kidneys may be utilized in a patient with suspected malignancy. Case Presentation: A systematic review (PRISMA standard) of ex vivo uretreo–pyeloscopy was undertaken. We then present the case of a 37-year-old Caucasian female who was suspected of having ureteral obstructing malignancy; she had previous treatment of the bladder with bacillus Calmette–Guerin (BCG) for recurrent urothelial malignancy. The lesion biopsies and cytology were suspicious but inconclusive, indicating nephroureterectomy was a likely course of management. Results: On reviewing the literature, we found that the use of ex vivo uretero-pyeloscopy has been described for urolithiasis to remove stones before transplantation but not specifically to exclude malignancy. Ultimately, in this case, the patient underwent a renal autotransplantation for obstruction that was caused by a granuloma on the background of the previous BCG treatment. Intraoperatively, ex vivo uretero-pyeloscopy and frozen section were crucial in guiding this case by allowing for appropriate identification and resection of the ureteral lesion. In addition, the preservation of ureteral length allowed for autotransplantation, which remains effective at follow-up. Conclusion: Ex vivo urteroscopy has been used effectively in donor kidneys to treat urolithiasis with minimal complications. We believe that this is the first documented case of ex vivo uretero-pyeloscopy being used effectively in renal autotransplantation to exclude urothelial malignancy.

  9. Organotypic slice co-culture systems to study axon regeneration in the dopaminergic system ex vivo.

    PubMed

    Heine, Claudia; Franke, Heike

    2014-01-01

    Organotypic slice co-cultures are suitable tools to study axonal regeneration and development (growth or regrowth) of different projection systems of the CNS under ex vivo conditions.In this chapter, we describe in detail the reconstruction of the mesocortical and nigrostriatal dopaminergic projection system culturing tissue slices from the ventral tegmental area/substantia nigra (VTA/SN) with the prefrontal cortex (PFC) or the striatum (STR). The protocol includes the detailed slice preparation and incubation. Moreover, different application possibilities of the ex vivo model are mentioned; as an example, the substance treatment procedure and biocytin tracing are described to reveal the effect of applied substances on fiber outgrowth. PMID:24838961

  10. Optical clearing of skin under action of glycerol: Ex vivo and in vivo investigations

    NASA Astrophysics Data System (ADS)

    Genina, E. A.; Bashkatov, A. N.; Sinichkin, Yu. P.; Tuchin, V. V.

    2010-08-01

    The behavior of optical parameters of the skin of a laboratory rat under the action of an aqueous solution of glycerol is studied ex vivo and in vivo. It is found that the collimated transmission coefficient of ex vivo skin samples increases by a factor of 20-40-fold depending on the wavelength in the studied spectral range, and the diffuse reflection coefficient of skin in vivo decreases on the average by 16%. The results presented can be useful for many methods of laser therapy and optical diagnostics of skin diseases and localization of subcutaneous neoplasms.

  11. Assessing patient dose in interventional fluoroscopy using patient-dependent hybrid phantoms

    NASA Astrophysics Data System (ADS)

    Johnson, Perry Barnett

    Interventional fluoroscopy uses ionizing radiation to guide small instruments through blood vessels or other body pathways to sites of clinical interest. The technique represents a tremendous advantage over invasive surgical procedures, as it requires only a small incision, thus reducing the risk of infection and providing for shorter recovery times. The growing use and increasing complexity of interventional procedures, however, has resulted in public health concerns regarding radiation exposures, particularly with respect to localized skin dose. Tracking and documenting patient-specific skin and internal organ dose has been specifically identified for interventional fluoroscopy where extended irradiation times, multiple projections, and repeat procedures can lead to some of the largest doses encountered in radiology. Furthermore, inprocedure knowledge of localized skin doses can be of significant clinical importance to managing patient risk and in training radiology residents. In this dissertation, a framework is presented for monitoring the radiation dose delivered to patients undergoing interventional procedures. The framework is built around two key points, developing better anthropomorphic models, and designing clinically relevant software systems for dose estimation. To begin, a library of 50 hybrid patient-dependent computational phantoms was developed based on the UF hybrid male and female reference phantoms. These phantoms represent a different type of anthropomorphic model whereby anthropometric parameters from an individual patient are used during phantom selection. The patient-dependent library was first validated and then used in two patient-phantom matching studies focused on cumulative organ and local skin dose. In terms of organ dose, patient-phantom matching was shown most beneficial for estimating the dose to large patients where error associated with soft tissue attenuation differences could be minimized. For small patients, inherent difference

  12. Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

    PubMed Central

    Guarnieri, Adriano; Moreno-Montañés, Javier; Alfonso-Bartolozzi, Belén; Sabater, Alfonso L.; García-Guzmán, María; Andreu, Enrique J.; Prosper, Felipe

    2014-01-01

    AIM To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization. METHODS This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animal-free culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization, vessel caliber (VC), and invasive area (IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity (BCVA), epithelial transparency, and impression cytology were also measured. RESULTS One year after surgery, successful cases showed a reduction (improvement) of all three parameters of corneal neovascularization [neovascular area (NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31% (P=0.035), invasion area 29.37% (P=0.018) and VC 14.29% (P=0.072). BCVA improved in all eyes (mean follow-up, 76±21mo). Epithelial transparency improved significantly from 2.00±0.93 to 0.88±1.25 (P=0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up. CONCLUSION This method of analyzing and monitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method, successful cases could be differentiated from failed cases. PMID:25540752

  13. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  14. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  15. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  16. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  17. 21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture processing applications. (a) Identification. Tissue culture media for human ex vivo tissue and cell...

  18. Variation of glucoraphanin metabolism in vivo and ex vivo by human gut bacteria

    PubMed Central

    Li, Fei; Hullar, Meredith AJ; Beresford, Shirley AA; Lampe, Johanna W

    2011-01-01

    Glucosinolates, phytochemicals found in cruciferous vegetables, are metabolized to bioactive isothiocyanates (ITC) by certain bacteria in the human gut. Substantial individual variation in urinary ITC excretion has been observed in previous cruciferous-vegetable feeding studies. We hypothesized that individual differences in gut microbial community contribute to the observed variation in glucosinolate metabolism, i.e., gut microbiota composition between high- and low-ITC excreters differ. We recruited 23 healthy individuals and fed them a standardized meal containing 200 g cooked broccoli. 24-h urinary ITC excretion was measured after the meal. Study participants with the highest (n=5) and the lowest (n=5) ITC excretion provided fecal samples for ex vivo bacterial cultivation with 50 μM glucoraphanin, the major glucosinolate found in broccoli. When grown ex vivo, fecal bacteria from the selected high ITC excreters were able to degrade more glucoraphanin than those from the low excreters (P=0.05). However, bacterial fingerprints of fecal and ex vivo culture microbiota revealed no statistically significant differences between the high and low ITC excreters in terminal restriction fragment length polymorphism analysis of the bacterial 16S rRNA gene. In conclusion, glucosinolate degradation by fecal bacteria ex vivo may be associated with in vivo bacterial glucosinolate metabolism capacity but no direct link to specific bacterial species could be established, possibly due to the complexity and functional redundancy of the gut microbiota. PMID:21342607

  19. Transdermal delivery system for zidovudine: in vitro, ex vivo and in vivo evaluation.

    PubMed

    Narishetty, Sunil Thomas Kumar; Panchagnula, Ramesh

    2004-01-01

    The objective of this study was to prepare a transdermal delivery system (TDS) for zidovudine (AZT) with a combination of menthol and oleic acid as penetration enhancers incorporated in hydroxypropyl methylcellulose, and to evaluate ex vivo as well as in vivo permeation across rat skin. It was found that AZT in gel formulation was stable in both refrigerated as well as accelerated stability conditions for 3 months and further, the gel did not significantly retard the permeability of AZT across the skin in comparison with solution formulation. Ex vivo steady state flux of AZT across rat skin from gel was 2.26 mg cm(-2) h(-1), which is sufficient to achieve therapeutic plasma concentrations. Intravenous pharmacokinetic parameters of AZT in rats were determined and used together with ex vivo flux data to generate theoretical plasma profiles of AZT and compared with plasma concentrations achieved after application of TDS. Further, steady state plasma concentrations of drug following multiple applications of TDS were determined and good correlations between ex vivo and in vivo data were observed. In addition, the combination of penetration enhancers used at 2.5% w/w in this study proved efficient in achieving sufficient enhancement in the transdermal permeability of AZT across rat skin with reduced skin irritation potential when compared with individual penetration enhancers at higher concentrations. PMID:14716748

  20. Polydimethylsiloxane embedded mouse aorta ex vivo perfusion model: proof-of-concept study focusing on atherosclerosis

    NASA Astrophysics Data System (ADS)

    Wang, Xueya; Wolf, Marc P.; Keel, Rahel Bänziger; Lehner, Roman; Hunziker, Patrick R.

    2012-07-01

    Existing mouse artery ex vivo perfusion models have utilized arteries such as carotid, uterine, and mesenteric arteries, but not the aorta. However, the aorta is the principal vessel analyzed for atherosclerosis studies in vivo. We have devised a mouse aorta ex vivo perfusion model that can bridge this gap. Aortas from apoE(-/-) mice are embedded in a transparent, gas-permeable, and elastic polymer matrix [polydimethylsiloxane (PDMS)] and artificially perfused with cell culture medium under cell culture conditions. After 24 h of artificial ex vivo perfusion, no evidence of cellular apoptosis is detected. Utilizing a standard confocal microscope, it is possible to image specific receptor targeting of cells in atherosclerotic plaques during 24 h. Imaging motion artifacts are minimal due to the polymer matrix embedding. Re-embedding of the aorta enables tissue sectioning and immuno-histochemical analysis. The ex vivo data are validated by comparison with in vivo experiments. This model can save animal lives via production of multiple endpoints in a single experiment, is easy to apply, and enables straightforward comparability with pre-existing atherosclerosis in vivo data. It is suited to investigate atherosclerotic disease in particular and vascular biology in general.

  1. Development and characterization of an ex-vivo brain slice culture model of chronic wasting disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Prion diseases have long incubation times in vivo, therefore, modeling the diseases ex-vivo will advance the development of rationale-based therapeutic strategies. An organotypic slice culture assay (POSCA) was recently developed for scrapie prions by inoculating mouse cerebellar brain slices with R...

  2. Use of an Ex Vivo Porcine Mucosal Model to Study Superantigen Penetration.

    PubMed

    Squier, Christopher A; Mantz, Mary J

    2016-01-01

    In vitro perfusion studies are frequently used to determine the penetration of compounds through skin and mucosa. Porcine tissue has been shown to be an excellent model for human tissue in terms of structure, function, and reactivity. We describe the use of porcine tissue ex-vivo in a continuous flow perfusion system to study the behavior of superantigens in this model. PMID:26676044

  3. Antioxidant properties of carp (Cyprinus carpio L.) protein ex vivo and in vitro hydrolysates.

    PubMed

    Borawska, Justyna; Darewicz, Małgorzata; Vegarud, Gerd E; Minkiewicz, Piotr

    2016-03-01

    The presence of specific peptides with antioxidant properties released during carp protein ex vivo and in vitro hydrolysis by human/porcine digestive enzymes, respectively, was examined. Based on the results of the in silico data analysis, antioxidant peptides were selected for subsequent identification in the digests/hydrolysates. Carp proteins were more resistant to hydrolysis by porcine enzymes than by human digestive juices. The sarcoplasmic proteins were hydrolyzed faster than the myofibrillar ones by both human/porcine enzymes. The in vitro myofibrillar protein hydrolysate showed the highest ABTS(+) scavenging activity (∼232.3 TEAC, μM Trolox/g), whereas the ex vivo hydrolysate of sarcoplasmic proteins showed the highest DPPH scavenging activity (∼88μM/g) and reducing power. Five antioxidant peptides were identified in carp protein ex vivo and in vitro hydrolysates: FIKK, HL, IY, PW, VY. The peptide HL from myofibrillar proteins was identified only in the ex vivo hydrolysate, whereas the peptide PW from sarcoplasmic proteins was identified only in the in vitro hydrolysate. PMID:26471617

  4. Otospheres derived from neonatal mouse cochleae retain the progenitor cell phenotype after ex vivo expansions.

    PubMed

    Lou, Xiang-Xin; Nakagawa, Takayuki; Ohnishi, Hiroe; Nishimura, Koji; Ito, Juichi

    2013-02-01

    Because of their limited regenerative potential, cochlear hair cell loss is one of the major causes of permanent hearing loss in mammals. However, recent studies have shown that postnatal cochlear epithelia retain the progenitor cells that form otospheres. Otospheres are capable of self-renewing and differentiating into inner ear cell lineages, thereby suggesting a promising source for hair cell regeneration. We investigated retention of the progenitor cell phenotype in otospheres after ex vivo expansion, which is crucial for transplantation approaches. Reverse transcriptase-polymerase chain reaction and immunocytochemical analyses showed that otospheres derived from neonatal mice retained expression of stem and cochlear cell markers. After in vitro differentiation, otosphere-consisting cells differentiated into hair cell phenotypes after ex vivo expansion. However, the capacity of otospheres for self-renewal weakened with subsequent generations of ex vivo expansion. Our results indicate that ex vivo expanded-otospheres are useful experimental tools for studying hair cell regeneration in transplantation approaches and that the mechanisms for retention of the progenitor cell phenotype in otospheres should be investigated. PMID:23238450

  5. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT DURING PREGNANCY

    EPA Science Inventory

    Effects of Bromodichloromethane (BDCM) on Ex Vivo Luteal Function In the Pregnant F344 Rat

    Susan R. Bielmeier1, Ashley S. Murr2, Deborah S. Best2, Jerome M. Goldman2, and Michael G. Narotsky2

    1Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC 27599,...

  6. Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

    PubMed Central

    Vinberg, Frans; Kefalov, Vladimir

    2015-01-01

    An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types. PMID:25992809

  7. Electrical recordings from the accessory olfactory bulb in VNO-AOB ex vivo preparations.

    PubMed

    Meeks, Julian P; Holy, Timothy E

    2013-01-01

    Electrical recordings from individual accessory olfactory bulb neurons allow exploration of the functional properties of this important pheromonal processing circuit. Several approaches to performing such recordings have been used. Here, we describe ex vivo methods that we have found useful for recording from accessory olfactory bulb neurons using simple extracellular glass electrodes. PMID:24014366

  8. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT

    EPA Science Inventory

    EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT.

    S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2

    1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA
    2 Reproductive T...

  9. EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE F344 RAT

    EPA Science Inventory

    EFFECTS OF BROMODICHLOROMETHANE (BDCM) ON EX VIVO LUTEAL FUNCTION IN THE PREGNANT F344 RAT.

    S. R. Bielmeier1, A. S. Murr2, D. S. Best2, J. M. Goldman2, and M. G. Narotsky2

    1 Curriculum in Toxicology, Univ. of North Carolina, Chapel Hill, NC, USA
    2 Reproductive T...

  10. Results of the TOP Study: Prospectively Randomized Multicenter Trial of an Ex Vivo Tacrolimus Rinse Before Transplantation in EDC Livers

    PubMed Central

    Pratschke, Sebastian; Arnold, Hannah; Zollner, Alfred; Heise, Michael; Pascher, Andreas; Schemmer, Peter; Scherer, Marcus N.; Bauer, Andreas; Jauch, Karl-Walter; Werner, Jens; Guba, Markus; Angele, Martin K.

    2016-01-01

    Background Organ shortage results in the transplantation of extended donor criteria (EDC) livers which is associated with increased ischemia-reperfusion injury (IRI). Experimental studies indicate that an organ rinse with the calcineurin inhibitor tacrolimus before implantation protects against IRI. The tacrolimus organ perfusion study was initiated to examine the effects of ex vivo tacrolimus perfusion on IRI in transplantation of EDC livers. Methods A prospective randomized multicenter trial comparing ex vivo perfusion of marginal liver grafts (≥2 EDC according to Eurotransplant manual) with tacrolimus (20 ng/mL) or histidine-tryptophane-ketoglutarate solution (control) was carried out at 5 German liver transplant centers (Munich Ludwig-Maximilians University, Berlin, Heidelberg, Mainz, Regensburg) between October 2011 and July 2013. Primary endpoint was the maximum alanine transaminase (ALT) level within 48 hours after transplantation. Secondary endpoints were aspartate transaminase (AST), prothrombine ratio, and graft-patient survival within an observation period of 1 week. After an interim analysis, the study was terminated by the scientific committee after the treatment of 24 patients (tacrolimus n = 11, Control n = 13). Results Tacrolimus rinse did not reduce postoperative ALT peaks compared with control (P = 0.207; tacrolimus: median, 812; range, 362-3403 vs control: median, 652; range, 147-2034). Moreover, ALT (P = 0.100), prothrombine ratio (P = 0.553), and bilirubin (P = 0.815) did not differ between the groups. AST was higher in patients treated with tacrolimus (P = 0.011). Survival was comparable in both groups (P > 0.05). Conclusions Contrary to experimental findings, tacrolimus rinse failed to improve the primary endpoint of the study (ALT). Because 1 secondary endpoint (AST) was even higher in the intervention group, the study was terminated prematurely. Thus, tacrolimus rinse cannot be recommended in transplantation of EDC livers. PMID:27500266

  11. Augmented reality system for MR-guided interventions: phantom studies and first animal test

    NASA Astrophysics Data System (ADS)

    Vogt, Sebastian; Wacker, Frank; Khamene, Ali; Elgort, Daniel R.; Sielhorst, Tobias; Niemann, Heinrich; Duerk, Jeff; Lewin, Jonathan S.; Sauer, Frank

    2004-05-01

    We developed an augmented reality navigation system for MR-guided interventions. A head-mounted display provides in real-time a stereoscopic video-view of the patient, which is augmented with three-dimensional medical information to perform MR-guided needle placement procedures. Besides with the MR image information, we augment the scene with 3D graphics representing a forward extension of the needle and the needle itself. During insertion, the needle can be observed virtually at its actual location in real-time, supporting the interventional procedure in an efficient and intuitive way. In this paper we report on quantitative results of AR guided needle placement procedures on gel phantoms with embedded targets of 12mm and 6mm diameter; we furthermore evaluate our first animal experiment involving needle insertion into deep lying anatomical structures of a pig.

  12. Spatially matched in vivo and ex vivo MR metabolic profiles of prostate cancer -- investigation of a correlation with Gleason score.

    PubMed

    Selnaes, Kirsten M; Gribbestad, Ingrid S; Bertilsson, Helena; Wright, Alan; Angelsen, Anders; Heerschap, Arend; Tessem, May-Britt

    2013-05-01

    MR metabolic profiling of the prostate is promising as an additional diagnostic approach to separate indolent from aggressive prostate cancer. The objective of this study was to assess the relationship between the Gleason score and the metabolic biomarker (choline + creatine + spermine)/citrate (CCS/C) measured by ex vivo high-resolution magic angle spinning MRS (HR-MAS MRS) and in vivo MRSI, and to evaluate the correlation between in vivo- and ex vivo-measured metabolite ratios from spatially matched prostate regions. Patients (n = 13) underwent in vivo MRSI prior to radical prostatectomy. A prostate tissue slice was snap-frozen shortly after surgery and the locations of tissue samples (n = 40) collected for ex vivo HR-MAS were matched to in vivo MRSI voxels (n = 40). In vivo MRSI was performed on a 3T clinical MR system and ex vivo HR-MAS on a 14.1T magnet. Relative metabolite concentrations were calculated by LCModel fitting of in vivo spectra and by peak integration of ex vivo spectra. Spearman's rank correlations (ρ) between CCS/C from in vivo and ex vivo MR spectra, and with their corresponding Gleason score, were calculated. There was a strong positive correlation between the Gleason score and CCS/C measured both in vivo and ex vivo (ρ = 0.77 and ρ = 0.69, respectively; p < 0.001), and between in vivo and ex vivo metabolite ratios from spatially matched regions (ρ = 0.67, p < 0.001). Our data indicate that MR metabolic profiling is a potentially useful tool for the assessment of cancer aggressiveness. Moreover, the good correlation between in vivo- and ex vivo-measured CCS/C demonstrates that our method is able to bridge MRSI and HR-MAS molecular analysis. PMID:23280546

  13. Economically affordable anatomical kidney phantom with calyxes for puncture and drainage training in interventional urology and radiology

    PubMed Central

    Ross, Peeter; Gavšin, Juri; Semjonov, Eero; Kruusmaa, Maarja

    2014-01-01

    Background Trends in interventional radiology and urology training are orientated towards reducing costs and increasing efficiency. In order to comply with the trends, we propose training on inexpensive patient-specific kidney phantoms. Purpose To develop a new kidney phantom for puncture and drainage training in interventional urology and radiology, and to evaluate their anatomical correctness and suitability for training compared to the traditional way of training on home-made phantoms. Material and Methods A case study for validation of kidney phantoms was conducted with nine radiology students divided into two groups: one trained on standard home-made training phantom (n = 4) and the other on our kidney phantoms (n = 5). Another test phantom was used to evaluate the effectiveness of the training of the two groups. The tests were video recorded and analyzed. Duration of the procedure was used as the primary indicator of procedure’s quality. Comparison tests were also conducted with professional radiologists. Anatomical correctness of the kidney phantom was evaluated by comparing the post mortem kidney scans with reconstructed models from CT scans. Subjective feedback was also collected from the participants. Wider use of kidney phantoms was analyzed. Results The average volumetric difference between post mortem kidney scans and reconstructed CT kidney models was 4.70 ± 3.25%. All five students practicing on the kidney phantom improved their performance and the results were almost equal to the results of the professional radiologist while in the other group two students out of four trained on standard home-made training phantoms failed to improve their performance. However, the small number of test subjects prevents us from drawing general conclusions about the efficiency of the new practice. The kidney phantoms were found usable also for nephrostomy catheter placement training under fluoroscopy. Conclusion The feedback from radiologists showed

  14. Comparing methods for ex vivo characterization of human monocyte phenotypes and in vitro responses.

    PubMed

    Johnston, Lisa; Harding, Scott A; La Flamme, Anne Camille

    2015-12-01

    Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC)(2) isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses. Furthermore, purification of monocytes using CD14(+) microbeads resulted in a loss of CD14(low)CD16(+) monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized. PMID:26256247

  15. Myelin contrast across lamina at 7T, ex-vivo and in-vivo dataset.

    PubMed

    Fracasso, Alessio; van Veluw, Susanne J; Visser, Fredy; Luijten, Peter R; Spliet, Wim; Zwanenburg, Jaco J M; Dumoulin, Serge O; Petridou, Natalia

    2016-09-01

    In this article we report the complete data obtained in-vivo for the paper: "Lines of Baillarger in vivo and ex-vivo: myelin contrast across lamina at 7T MRI and histology" (Fracasso et al., 2015) [1]. Single participant data (4 participants) from the occipital lobe acquisition are reported for axial, coronal and sagittal slices; early visual area functional localization and laminar profiles are reported. Data from whole brain images are reported and described (5 participants), for axial, coronal and sagittal slices. Laminar profiles from occipital, parietal and frontal lobes are reported. The data reported in this manuscript complements the paper (Fracasso et al., 2015) [1] by providing the full set of results from the complete pool of participants, on a single-participant basis. Moreover, we provide histological images from the ex-vivo sample reported in Fracasso et al. (2015) [1]. PMID:27508254

  16. Monitoring changes in tissue optical properties following interstitial photothermal therapy of ex vivo human prostate tissue

    NASA Astrophysics Data System (ADS)

    Weersink, Robert A.; He, Jie; Veilleux, Israel; Trachtenberg, John; Wilson, Brian C.

    2013-03-01

    We are developing a method of monitoring treatment progression of interstitial photothermal therapy of focal prostate cancer using transrectal diffuse optical tomography (TRDOT) combined with transrectal 3D ultrasound (3D-TRUS). Measurements of prostate tissue optical properties were made on ex vivo human prostate samples prior to and post coagulation. Interstitial photothermal treatments were delivered to the ex vivo samples and monitored using an interstitial probe near the treatment fiber. After treatment, bulk optical properties were measured on native and coagulated zones of tissue. Changes in optical properties across the boundary between native and coagulated tissues were spatially mapped using a small diffuse reflectance probe. The optical property estimates and spatial information obtained using each method was compared.

  17. T 1 Relaxation Measurement of Ex-Vivo Breast Cancer Tissues at Ultralow Magnetic Fields

    PubMed Central

    Lee, Seong-Joo; Shim, Jeong Hyun; Kim, Kiwoong; Hwang, Seong-min; Yu, Kwon Kyu; Lim, Sanghyun; Han, Jae Ho; Yim, Hyunee; Kim, Jang-Hee; Jung, Yong Sik; Kim, Ku Sang

    2015-01-01

    We investigated T1 relaxations of ex-vivo cancer tissues at low magnetic fields in order to check the possibility of achieving a T1 contrast higher than those obtained at high fields. The T1 relaxations of fifteen pairs (normal and cancerous) of breast tissue samples were measured at three magnetic fields, 37, 62, and 122 μT, using our superconducting quantum interference device-based ultralow field nuclear magnetic resonance setup, optimally developed for ex-vivo tissue studies. A signal reconstruction based on Bayesian statistics for noise reduction was exploited to overcome the low signal-to-noise ratio. The ductal and lobular-type tissues did not exhibit meaningful T1 contrast values between normal and cancerous tissues at the three different fields. On the other hand, an enhanced T1 contrast was obtained for the mucinous cancer tissue. PMID:25705658

  18. Ex Vivo (Fluorescence) Confocal Microscopy in Surgical Pathology: State of the Art.

    PubMed

    Ragazzi, Moira; Longo, Caterina; Piana, Simonetta

    2016-05-01

    First developed in 1957, confocal microscopy is a powerful imaging tool that can be used to obtain near real-time reflected light images of untreated human tissue with nearly histologic resolution. Besides its research applications, in the last decades, confocal microscopy technology has been proposed as a useful device to improve clinical diagnosis, especially in ophthalmology, dermatology, and endomicroscopy settings, thanks to advances in instrument development. Compared with the wider use of the in vivo tissue assessment, ex vivo applications of confocal microscopy are not fully explored. A comprehensive review of the current literature was performed here, focusing on the reliable applications of ex vivo confocal microscopy in surgical pathology and on some potential evolutions of this new technique from pathologists' viewpoint. PMID:27058244

  19. In vivo and ex vivo imaging with ultrahigh resolution full-field OCT

    NASA Astrophysics Data System (ADS)

    Grieve, Kate; Moneron, Gael; Schwartz, Wilfrid; Boccara, Albert C.; Dubois, Arnaud

    2005-08-01

    Imaging of in vivo and ex vivo biological samples using full-field optical coherence tomography is demonstrated. Three variations on the original full-field optical coherence tomography instrument are presented, and evaluated in terms of performance. The instruments are based on the Linnik interferometer illuminated by a white light source. Images in the en face orientation are obtained in real-time without scanning by using a two-dimensional parallel detector array. An isotropic resolution capability better than 1 μm is achieved thanks to the use of a broad spectrum source and high numerical aperture microscope objectives. Detection sensitivity up to 90 dB is demonstrated. Image acquisition times as short as 10 μs per en face image are possible. A variety of in vivo and ex vivo imaging applications is explored, particularly in the fields of embryology, ophthalmology and botany.

  20. Microwave Ablation With a Triaxial Antenna: Results in ex vivo Bovine Liver.

    PubMed

    Brace, Christopher L; Laeseke, Paul F; van der Weide, Daniel W; Lee, Fred T

    2005-01-01

    We apply a new triaxial antenna for microwave ablation procedures to an ex vivo bovine liver. The antenna consists of a coaxial monopole inserted through a biopsy needle positioned one quarter-wavelength from the antenna base. The insertion needle creates a triaxial structure, which enhances return loss more than 10 dB, maximizing energy transfer to the tissue while minimizing feed cable heating and invasiveness. Numerical electromagnetic and thermal simulations are used to optimize the antenna design and predict heating patterns. Numerical and ex vivo experimental results show that the lesion size depends strongly on ablation time and average input power, but not on peak power. Pulsing algorithms are also explored. We were able to measure a 3.8-cm lesion using 50 W for 7 min, which we believe to be the largest lesion reported thus far using a 17-gauge insertion needle. PMID:18079981

  1. Direct Visualization of the Perforant Pathway in the Human Brain with Ex Vivo Diffusion Tensor Imaging

    PubMed Central

    Augustinack, Jean C.; Helmer, Karl; Huber, Kristen E.; Kakunoori, Sita; Zöllei, Lilla; Fischl, Bruce

    2010-01-01

    Ex vivo magnetic resonance imaging yields high resolution images that reveal detailed cerebral anatomy and explicit cytoarchitecture in the cerebral cortex, subcortical structures, and white matter in the human brain. Our data illustrate neuroanatomical correlates of limbic circuitry with high resolution images at high field. In this report, we have studied ex vivo medial temporal lobe samples in high resolution structural MRI and high resolution diffusion MRI. Structural and diffusion MRIs were registered to each other and to histological sections stained for myelin for validation of the perforant pathway. We demonstrate probability maps and fiber tracking from diffusion tensor data that allows the direct visualization of the perforant pathway. Although it is not possible to validate the DTI data with invasive measures, results described here provide an additional line of evidence of the perforant pathway trajectory in the human brain and that the perforant pathway may cross the hippocampal sulcus. PMID:20577631

  2. Thermal effect of endoscopic thermal vapour ablation on the lung surface in human ex vivo tissue

    PubMed Central

    Henne, Erik; Anderson, Joseph C.; Barry, Robert; Kesten, Steven

    2012-01-01

    Purpose: An investigation of the thermal effect and the potential for injury at the lung surface following thermal vapour ablation (InterVapor), an energy-based method of achieving endoscopic lung volume reduction. Methods: Heated water vapour was delivered to fifteen ex vivo human lungs using standard clinical procedure, and the thermal effect at the visceral pleura was monitored with an infrared camera. The time–temperature response was analysed mathematically to determine a cumulative injury quotient, which was compared to published thresholds. Results: The cumulative injury quotients for all 71 treatments of ex vivo tissue were found to be below the threshold for first degree burn and no other markers of tissue injury at the lung surface were observed. Conclusion: The safety profile for thermal vapour ablation is further supported by the demonstration that the thermal effect in a worst-case model is not expected to cause injury at the lung surface. PMID:22690896

  3. Comparative Ex Vivo Activity of Novel Endoperoxides in Multidrug-Resistant Plasmodium falciparum and P. vivax

    PubMed Central

    Chalfein, Ferryanto; Prayoga, Pak; Wabiser, Frans; Wirjanata, Grennady; Sebayang, Boni; Piera, Kim A.; Wittlin, Sergio; Haynes, Richard K.; Möhrle, Jörg J.; Anstey, Nicholas M.; Kenangalem, Enny; Price, Ric N.

    2012-01-01

    The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC50s) in both species (median IC50s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity. PMID:22850522

  4. The use of ex vivo human skin tissue for genotoxicity testing

    SciTech Connect

    Reus, Astrid A.; Usta, Mustafa; Krul, Cyrille A.M.

    2012-06-01

    As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air–liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. -- Highlights: ► We use human skin obtained from surgery for genotoxicity evaluation of chemicals. ► We use the comet assay as parameter for genotoxicity in ex vivo human skin. ► Sensitivity, specificity and accuracy to predict in vivo genotoxins are determined. ► Sensitivity, specificity and accuracy are 89%, 90% and 90%, respectively. ► The method

  5. IL-12 directs further maturation of ex vivo differentiated NK cells with improved therapeutic potential.

    PubMed

    Lehmann, Dorit; Spanholtz, Jan; Sturtzel, Caterina; Tordoir, Marleen; Schlechta, Bernhard; Groenewegen, Dirk; Hofer, Erhard

    2014-01-01

    The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34(+) stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56(dim) peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56(dim) than CD56(bright) peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33(+)NKG2A(+) NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies. PMID:24498025

  6. Depleted uranium disturbs immune parameters in zebrafish, Danio rerio: an ex vivo/in vivo experiment.

    PubMed

    Gagnaire, Béatrice; Bado-Nilles, Anne; Sanchez, Wilfried

    2014-10-01

    In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 µg DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 µg DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 µg DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments. Cell mortality was also decreased at 20 µg DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases. PMID:24723161

  7. Utilization of the organ care system as ex-vivo lung perfusion after cold storage transportation.

    PubMed

    Mohite, P N; Maunz, O; Popov, A-F; Zych, B; Patil, N P; Simon, A R

    2015-11-01

    The Organ Care System (OCS) allows perfusion and ventilation of the donor lungs under physiological conditions. Ongoing trials to compare preservation with OCS Lung with standard cold storage do not include donor lungs with suboptimal gas exchange and donor lungs treated with OCS following cold storage transportation. We present a case of a 48-yr-old man who received such lungs after cold storage transportation treated with ex-vivo lung perfusion utilizing OCS. PMID:25662732

  8. Ex vivo evaluation of acellular and cellular collagen-glycosaminoglycan flowable matrices.

    PubMed

    Hodgkinson, Tom; Bayat, Ardeshir

    2015-08-01

    Collagen-glycosaminoglycan flowable matrices (CGFM) are increasingly finding utility in a diversifying number of cutaneous surgical procedures. Cellular in-growth and vascularisation of CGFM remain rate-limiting steps, increasing cost and decreasing efficacy. Through in vitro and ex vivo culture methods, this study investigated the improvement of injectable CGFM by the incorporation of hyaluronan (HA) and viable human cells (primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs)). Ex vivo investigations included the development and evaluation of a human cutaneous wound healing model for the comparison of dermal substitutes. Cells mixed into the Integra Flowable Wound Matrix (IFWM), a commercially available CGFM, were confirmed to be viable and proliferative through MTT assays (p  <  0.05). PHDFs proliferated with greater rapidity than BM-MSCs up to 1 week in culture (p  <  0.05), with PHDF proliferation further enhanced by HA supplementation (p  <  0.05). After scaffold mixing, gene expression was not significantly altered (qRT-PCR). PHDF and BM-MSC incorporation into ex vivo wound models significantly increased re-epithelialisation rate, with maximal effects observed for BM-MSC supplemented IFWM. HA supplementation to PHDF populated IFWM increased re-epithelialisation but had no significant effect on BM-MSC populated IFWM. In conclusion, when combined with PHDF, HA increased re-epithelialisation in IFWM. BM-MSC incorporation significantly improved re-epithelialisation in ex vivo models over acellular and PHDF populated scaffolds. Viable cell incorporation into IFWM has potential to significantly benefit wound healing in chronic and acute cutaneous injuries by allowing a point-of-care matrix to be formed from autologous or allogenic cells and bioactive molecules. PMID:26181360

  9. MCNP simulation of radiation doses distributions in a water phantoms simulating interventional radiology patients

    NASA Astrophysics Data System (ADS)

    He, Wenjun; Mah, Eugene; Huda, Walter; Selby, Bayne; Yao, Hai

    2011-03-01

    Purpose: To investigate the dose distributions in water cylinders simulating patients undergoing Interventional Radiological examinations. Method: The irradiation geometry consisted of an x-ray source, dose-area-product chamber, and image intensifier as currently used in Interventional Radiology. Water cylinders of diameters ranging between 17 and 30 cm were used to simulate patients weighing between 20 and 90 kg. X-ray spectra data with peak x-ray tube voltages ranging from 60 to 120 kV were generated using XCOMP3R. Radiation dose distributions inside the water cylinder (Dw) were obtained using MCNP5. The depth dose distribution along the x-ray beam central axis was normalized to free-in-air air kerma (AK) that is incident on the phantom. Scattered radiation within the water cylinders but outside the directly irradiated region was normalized to the dose at the edge of the radiation field. The total absorbed energy to the directly irradiated volume (Ep) and indirectly irradiated volume (Es) were also determined and investigated as a function of x-ray tube voltage and phantom size. Results: At 80 kV, the average Dw/AK near the x-ray entrance point was 1.3. The ratio of Dw near the entrance point to Dw near the exit point increased from ~ 26 for the 17 cm water cylinder to ~ 290 for the 30 cm water cylinder. At 80 kV, the relative dose for a 17 cm water cylinder fell to 0.1% at 49 cm away from the central ray of the x-ray beam. For a 30 cm water cylinder, the relative dose fell to 0.1% at 53 cm away from the central ray of the x-ray beam. At a fixed x-ray tube voltage of 80 kV, increasing the water cylinder diameter from 17 to 30 cm increased the Es/(Ep+Es) ratio by about 50%. At a fixed water cylinder diameter of 24 cm, increasing the tube voltage from 60 kV to 120 kV increased the Es/(Ep+Es) ratio by about 12%. The absorbed energy from scattered radiation was between 20-30% of the total energy absorbed by the water cylinder, and was affected more by patient size

  10. Independent component analysis for three-dimensional optical imaging and localization of a fluorescent contrast agent target embedded in a slab of ex vivo human breast tissue

    NASA Astrophysics Data System (ADS)

    Alrubaiee, M.; Xu, M.; Gayen, S. K.; Alfano, R. R.

    2005-08-01

    An innovative approach for three-dimensional localization and characterization of a fluorescent target embedded in a turbid medium is presented. The target was a ~4-mm diameter glass sphere with a solution of indocyanine green placed within a 50-mm thick tissuelike phantom with mean free path of ~1-mm at 784-nm and a ~ 26-mm thick ex vivo breast tissue slab. The experimental approach uses a multi-source illumination, and a multi-detector signal acquisition scheme. An analysis scheme based on the independent component analysis from information theory is used for target localization and characterization. Independent component analysis of the perturbation in the spatial intensity distribution of the fluorescent signal measured on the exit plane of the turbid medium locates the embedded objects. The location and size, of the embedded objects are obtained from a Green's function analysis and back-projection Fourier transform of the retrieved independent components.

  11. Ex vivo determination of bone tissue strains for an in vivo mouse tibial loading model.

    PubMed

    Carriero, Alessandra; Abela, Lisa; Pitsillides, Andrew A; Shefelbine, Sandra J

    2014-07-18

    Previous studies introduced the digital image correlation (DIC) as a viable technique for measuring bone strain during loading. In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations on the strain contour maps measured with a DIC system. We loaded tibiae of C57BL/6 mice (12 and 18 weeks old male) up to 12 N at 8 N/min. Images of speckles on the bone surface were recorded at 1N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps. This work provides a better understanding of parameters affecting full field strain measurements from DIC in ex vivo murine tibial loading tests. PMID:24835472

  12. Toxoplasma gondii inhibits R5 HIV-1 replication in human lymphoid tissues ex vivo

    PubMed Central

    Sassi, Atfa; Brichacek, Beda; Hieny, Sara; Yarovinsky, Felix; Golding, Hana; Grivel, Jean-Charles; Sher, Alan; Margolis, Leonid

    2016-01-01

    Critical events of HIV-1 pathogenesis occur in lymphoid tissues where HIV-1 is typically accompanied by infections with other pathogens (HIV co-pathogens). Co-pathogens greatly affect the clinical course of the disease and the transmission of HIV. The apicomplexan parasite Toxoplasma gondii is a common HIV co-pathogen associated with AIDS development. Here, we examined the interaction of T. gondii and HIV in coinfected human lymphoid tissue ex vivo. Both pathogens readily replicate in ex vivo infected blocks of human tonsillar tissue. Surprisingly, we found that live T. gondii preferentially inhibits R5 HIV-1 replication in coinfected tissues. This effect is reproduced by treatment of the tissue blocks with recombinant C-18, a T. gondii -encoded cyclophilin that binds to CCR5. These ex vivo findings raise the possibility that, in addition to being a co-factor in HIV disease, T. gondii may influence the outcome of viral infection by preferentially suppressing R5 variants. PMID:19671446

  13. The effect of vocal fold adduction on the acoustic quality of phonation: ex vivo investigations

    PubMed Central

    Regner, Michael F.; Tao, Chao; Ying, Di; Olszewski, Aleksandra; Zhang, Yu; Jiang, Jack J.

    2011-01-01

    OBJECTIVES The purpose of this study was to investigate the effect of vocal fold adduction on voice quality in an ex vivo larynx model. STUDY DESIGN Prospective, repeated-measures experiments. METHODS Ten excised canine larynges were mounted on an excised larynx phonation system and measurements were recorded for three different vocal fold adduction levels. Acoustic perturbation measurements of jitter, shimmer, and signal-to-noise ratio (SNR) were calculated from recorded radiated sound histories. RESULTS Ex vivo experiments indicated that statistically significant increases in the means of jitter (p=0.005), shimmer (p=0.002), and SNR (p=0.011) measures decreased with respect to vocal fold adduction as the independent variable. Theoretical results showed that the DC and AC component of glottal area increased monotonically with prephonatory glottal area. CONCLUSIONS Acoustic perturbation increased with the degree of vocal fold abduction. Ex vivo larynx measurements suggested that a hyperadducted state may be acoustically best. This may be explained theoretically by an increase in DC/AC ratio as the prephonatory area is increased. PMID:22578437

  14. Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia

    PubMed Central

    Sarkar, Debalina; Biswas, Moanaro; Liao, Gongxian; Seay, Howard R; Perrin, George Q; Markusic, David M; Hoffman, Brad E; Brusko, Todd M; Terhorst, Cox; Herzog, Roland W

    2014-01-01

    Adoptive cell therapy utilizing ex vivo expanded polyclonal CD4+CD25+FOXP3+ regulatory T cells (Treg) is in use in clinical trials for the treatment of type 1 diabetes and prevention of graft versus host disease in bone marrow transplantation. Here, we seek to evaluate this approach in the treatment of inherited protein deficiencies, i.e., hemophilia, which is often complicated by antibody formation against the therapeutic protein. Treg from mice that express green fluorescent protein–marked FoxP3 were highly purified by two-step magnetic/flow sorting and ex vivo expanded 50- to 100-fold over a 2-week culture period upon stimulation with antibody-coated microbeads. FoxP3 expression was maintained in >80% of expanded Treg, which also expressed high levels of CD62L and CTLA-4. Transplanted Treg suppressed inhibitory antibody formation against coagulation factors VIII and IX in protein and gene therapies in strain-matched hemophilia A and B mice, including in mice with pre-existing antibodies. Although transplanted Treg became undetectable within 2 weeks, suppression persisted for >2 months. Additional studies suggested that antigen-specific suppression emerged due to induction of endogenous Treg. The outcomes of these studies support the concept that cell therapy with ex vivo expanded autologous Treg can be used successfully to minimize immune responses in gene and protein replacement therapies. PMID:25364772

  15. Compound ex vivo and in silico method for hemodynamic analysis of stented arteries.

    PubMed

    Rikhtegar, Farhad; Pacheco, Fernando; Wyss, Christophe; Stok, Kathryn S; Ge, Heng; Choo, Ryan J; Ferrari, Aldo; Poulikakos, Dimos; Müller, Ralph; Kurtcuoglu, Vartan

    2013-01-01

    Hemodynamic factors such as low wall shear stress have been shown to influence endothelial healing and atherogenesis in stent-free vessels. However, in stented vessels, a reliable quantitative analysis of such relations has not been possible due to the lack of a suitable method for the accurate acquisition of blood flow. The objective of this work was to develop a method for the precise reconstruction of hemodynamics and quantification of wall shear stress in stented vessels. We have developed such a method that can be applied to vessels stented in or ex vivo and processed ex vivo. Here we stented the coronary arteries of ex vivo porcine hearts, performed vascular corrosion casting, acquired the vessel geometry using micro-computed tomography and reconstructed blood flow and shear stress using computational fluid dynamics. The method yields accurate local flow information through anatomic fidelity, capturing in detail the stent geometry, arterial tissue prolapse, radial and axial arterial deformation as well as strut malapposition. This novel compound method may serve as a unique tool for spatially resolved analysis of the relationship between hemodynamic factors and vascular biology. It can further be employed to optimize stent design and stenting strategies. PMID:23516442

  16. Ex vivo differential phase contrast and magnetic resonance imaging for characterization of human carotid atherosclerotic plaques.

    PubMed

    Meletta, Romana; Borel, Nicole; Stolzmann, Paul; Astolfo, Alberto; Klohs, Jan; Stampanoni, Marco; Rudin, Markus; Schibli, Roger; Krämer, Stefanie D; Herde, Adrienne Müller

    2015-10-01

    Non-invasive detection of specific atherosclerotic plaque components related to vulnerability is of high clinical relevance to prevent cerebrovascular events. The feasibility of magnetic resonance imaging (MRI) for characterization of plaque components was already demonstrated. We aimed to evaluate the potential of ex vivo differential phase contrast X-ray tomography (DPC) to accurately characterize human carotid plaque components in comparison to high field multicontrast MRI and histopathology. Two human plaque segments, obtained from carotid endarterectomy, classified according to criteria of the American Heart Association as stable and unstable plaque, were examined by ex vivo DPC tomography and multicontrast MRI (T1-, T2-, and proton density-weighted imaging, magnetization transfer contrast, diffusion-weighted imaging). To identify specific plaque components, the plaques were subsequently sectioned and stained for fibrous and cellular components, smooth muscle cells, hemosiderin, and fibrin. Histological data were then matched with DPC and MR images to define signal criteria for atherosclerotic plaque components. Characteristic structures, such as the lipid and necrotic core covered by a fibrous cap, calcification and hemosiderin deposits were delineated by histology and found with excellent sensitivity, resolution and accuracy in both imaging modalities. DPC tomography was superior to MRI regarding resolution and soft tissue contrast. Ex vivo DPC tomography allowed accurate identification of structures and components of atherosclerotic plaques at different lesion stages, in good correlation with histopathological findings. PMID:26179860

  17. Model-based registration of ex vivo and in vivo MRI of the prostate using elastography.

    PubMed

    Nir, Guy; Sahebjavaher, Ramin S; Kozlowski, Piotr; Chang, Silvia D; Sinkus, Ralph

    2013-06-01

    Registration of histopathology to in vivo magnetic resonance imaging (MRI) of the prostate is an important task that can be used to optimize in vivo imaging for cancer detection. Such registration is challenging due to the change in volume and deformation of the prostate during excision and fixation. One approach towards this problem involves the use of an ex vivo MRI of the excised prostate specimen, followed by in vivo to ex vivo MRI registration of the prostate. We propose a novel registration method that uses a patient-specific biomechanical model acquired using magnetic resonance elastography to deform the in vivo volume and match it to the surface of the ex vivo specimen. The forces that drive the deformations are derived from a region-based energy, with the elastic potential used for regularization. The incorporation of elastography data into the registration framework allows inhomogeneous elasticity to be assigned to the in vivo volume. We show that such inhomogeneity improves the registration results by providing a physical regularization of the deformation map. The method is demonstrated and evaluated on six clinical cases. PMID:23475353

  18. Lung transplantation from donors after circulatory death using portable ex vivo lung perfusion

    PubMed Central

    Bozso, Sabin; Vasanthan, Vishnu; Luc, Jessica GY; Kinaschuk, Katie; Freed, Darren; Nagendran, Jayan

    2015-01-01

    BACKGROUND: Donation after circulatory death is a novel method of increasing the number of donor lungs available for transplantation. Using organs from donors after circulatory death has the potential to increase the number of transplants performed. METHODS: Three bilateral lung transplants from donors after circulatory death were performed over a six-month period. Following organ retrieval, all sets of lungs were placed on a portable ex vivo lung perfusion device for evaluation and preservation. RESULTS: Lung function remained stable during portable ex vivo perfusion, with improvement in partial pressure of oxygen/fraction of inspired oxygen ratios. Mechanical ventilation was discontinued within 48 h for each recipient and no patient stayed in the intensive care unit longer than eight days. There was no postgraft dysfunction at 72 h in two of the three recipients. Ninety-day mortality for all recipients was 0% and all maintain excellent forced expiratory volume in 1 s and forced vital capacity values post-transplantation. CONCLUSION: The authors report excellent results with their initial experience using donors after circulatory death after portable ex vivo lung perfusion. It is hoped this will allow for the most efficient use of available donor lungs, leading to more transplants and fewer deaths for potential recipients on wait lists. PMID:25379654

  19. The use of ex vivo human skin tissue for genotoxicity testing.

    PubMed

    Reus, Astrid A; Usta, Mustafa; Krul, Cyrille A M

    2012-06-01

    As a result of the chemical legislation concerning the registration, evaluation, authorization and restriction of chemicals (REACH), and the Seventh Amendment to the Cosmetics Directive, which prohibits animal testing in Europe for cosmetics, alternative methods for safety evaluation of chemicals are urgently needed. Current in vitro genotoxicity assays are not sufficiently predictive for the in vivo situation, resulting in an unacceptably high number of misleading positives. For many chemicals and ingredients of personal care products the skin is the first site of contact, but there are no in vitro genotoxicity assays available in the skin for additional evaluation of positive or equivocal responses observed in regulatory in vitro genotoxicity assays. In the present study ex vivo human skin tissue obtained from surgery was used for genotoxicity evaluation of chemicals by using the comet assay. Fresh ex vivo human skin tissue was cultured in an air-liquid interface and topically exposed to 20 chemicals, including true positive, misleading positive and true negative genotoxins. Based on the results obtained in the present study, the sensitivity, specificity and accuracy of the ex vivo skin comet assay to predict in vivo genotoxicity were 89%, 90% and 89%, respectively. Donor and experimental variability were mainly reflected in the magnitude of the response and not the difference between the presence and absence of a genotoxic response. The present study indicates that human skin obtained from surgery is a promising and robust model for safety evaluation of chemicals that are in direct contact with the skin. PMID:22507867

  20. Digital Radiography for Determination of Primary Tooth Length: In Vivo and Ex Vivo Studies

    PubMed Central

    Basso, Maria D.; Jeremias, Fabiano; Cordeiro, Rita C. L.; Santos-Pinto, Lourdes

    2015-01-01

    Background. Methods for determining the root canal length of the primary tooth should yield accurate and reproducible results. In vitro studies show some limitations, which do not allow their findings to be directly transferred to a clinical situation. Aim. To compare the accuracy of radiographic tooth length obtained from in vivo digital radiograph with that obtained from ex vivo digital radiograph. Method. Direct digital radiographs of 20 upper primary incisors were performed in teeth (2/3 radicular resorption) that were radiographed by an intraoral sensor, according to the long-cone technique. Teeth were extracted, measured, and mounted in a resin block, and then radiographic template was used to standardise the sensor-target distance (30 cm). The apparent tooth length (APTL) was obtained from the computer screen by means of an electronic ruler accompanying the digital radiography software (CDR 2.0), whereas the actual tooth length (ACTL) was obtained by means of a digital calliper following extraction. Data were compared to the ACTL by variance analysis and Pearson's correlation test. Results. The values for APTL obtained from in vivo radiography were slightly underestimated, whereas those values obtained from ex vivo were slightly overestimated. No significance was observed (P ≤ 0.48) between APTL and ACTL. Conclusion. The length of primary teeth estimated by in vivo and ex vivo comparisons using digital radiography was found to be similar to the actual tooth length. PMID:25802894

  1. Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

    PubMed Central

    Kaths, J. Moritz; Spetzler, Vinzent N.; Goldaracena, Nicolas; Echeverri, Juan; Louis, Kristine S.; Foltys, Daniel B.; Strempel, Mari; Yip, Paul; John, Rohan; Mucsi, Istvan; Ghanekar, Anand; Bagli, Darius; Robinson, Lisa; Selzner, Markus

    2015-01-01

    Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods. Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes. In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated. PMID:26275014

  2. Improved Recellularization of Ex Vivo Vascular Scaffolds using Directed Transport Gradients to Modulate ECM Remodeling

    PubMed Central

    Tosun, Zehra; McFetridge, Peter S.

    2015-01-01

    The regeneration of functional, clinically viable, tissues from acellular ex vivo tissues has been problematic largely due to poor nutrient transport conditions that limit cell migration and integration. Compounding these issues are subcellular pore sizes that necessarily requires extracellular matrix (ECM) remodeling in order for cells to migrate and regenerate the tissue. The aim of the present work was to create a directed growth environment that allows cells to fully populate an ex vivo-derived vascular scaffold and maintain viability over extended periods. Three different culture conditions using single (one nutrient source) or dual perfusion bioreactor systems (two nutrients sources) were designed to assess the effect of pressure and nutrient gradients under either low (50/30 mmHg) or high (120/80) relative pressure conditions. Human myofibroblasts were seeded to the ablumenal periphery of an ex vivo-derived vascular scaffold using a collagen/hydrogel cell delivery system. After 30 days culture, total cell density was consistent between groups; however, significant variation was noted in cell distribution and construct mechanics as a result of differing perfusion conditions. The most aggressive transport gradient was developed by the single perfusion low-pressure circuits and resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source). These investigations illustrate the influence of directed nutrient gradients where precisely controlled perfusion conditions significantly affects cell migration, distribution and function, resulting in pronounced effects on construct mechanics during early remodeling events. PMID:23613430

  3. The Antipsychotics Olanzapine, Risperidone, Clozapine, and Haloperidol Are D2-Selective Ex Vivo but Not In Vitro

    PubMed Central

    McCormick, Patrick N; Kapur, Shitij; Graff-Guerrero, Ariel; Raymond, Roger; Nobrega, José N; Wilson, Alan A

    2010-01-01

    In a recent human [11C]-(+)-PHNO positron emission tomography study, olanzapine, clozapine, and risperidone occupied D2 receptors in striatum (STR), but, despite their similar in vitro D2 and D3 affinities, failed to occupy D3 receptors in globus pallidus. This study had two aims: (1) to characterize the regional D2/D3 pharmacology of in vitro and ex vivo [3H]-(+)-PHNO binding sites in rat brain and (2) to compare, using [3H]-(+)-PHNO autoradiography, the ex vivo and in vitro pharmacology of olanzapine, clozapine, risperidone, and haloperidol. Using the D3-selective drug SB277011, we found that ex vivo and in vitro [3H]-(+)-PHNO binding in STR is exclusively due to D2, whereas that in cerebellar lobes 9 and 10 is exclusively due to D3. Surprisingly, the D3 contribution to [3H]-(+)-PHNO binding in the islands of Calleja, ventral pallidum, substantia nigra, and nucleus accumbens was greater ex vivo than in vitro. Ex vivo, systemically administered olanzapine, risperidone, and haloperidol, at doses occupying ∼80% D2, did not occupy D3 receptors. Clozapine, which also occupied ∼80% of D2 receptors ex vivo, occupied a smaller percentage of D3 receptors than predicted by its in vitro pharmacology. Across brain regions, ex vivo occupancy by antipsychotics was inversely related to the D3 contribution to [3H]-(+)-PHNO binding. In contrast, in vitro occupancy was similar across brain regions, independent of the regional D3 contribution. These data indicate that at clinically relevant doses, olanzapine, clozapine, risperidone, and haloperidol are D2-selective ex vivo. This unforeseen finding suggests that their clinical effects cannot be attributed to D3 receptor blockade. PMID:20410873

  4. Novel Sensor-Enabled Ex Vivo Bioreactor: A New Approach towards Physiological Parameters and Porcine Artery Viability

    PubMed Central

    Mundargi, Raghavendra; Venkataraman, Divya; Kumar, Saranya; Mogal, Vishal; Ortiz, Raphael; Loo, Joachim; Venkatraman, Subbu; Steele, Terry

    2015-01-01

    The aim of the present work is to design and construct an ex vivo bioreactor system to assess the real time viability of vascular tissue. Porcine carotid artery as a model tissue was used in the ex vivo bioreactor setup to monitor its viability under physiological conditions such as oxygen, pressure, temperature, and flow. The real time tissue viability was evaluated by monitoring tissue metabolism through a fluorescent indicator “resorufin.” Our ex vivo bioreactor allows real time monitoring of tissue responses along with physiological conditions. These ex vivo parameters were vital in determining the tissue viability in sensor-enabled bioreactor and our initial investigations suggest that, porcine tissue viability is considerably affected by high shear forces and low oxygen levels. Histological evaluations with hematoxylin and eosin and Masson's trichrome staining show intact endothelium with fresh porcine tissue whereas tissues after incubation in ex vivo bioreactor studies indicate denuded endothelium supporting the viability results from real time measurements. Hence, this novel viability sensor-enabled ex vivo bioreactor acts as model to mimic in vivo system and record vascular responses to biopharmaceutical molecules and biomedical devices. PMID:26609536

  5. Treatment planning for image-guided neuro-vascular interventions using patient-specific 3D printed phantoms

    NASA Astrophysics Data System (ADS)

    Russ, M.; O'Hara, R.; Setlur Nagesh, S. V.; Mokin, M.; Jimenez, C.; Siddiqui, A.; Bednarek, D.; Rudin, S.; Ionita, C.

    2015-03-01

    Minimally invasive endovascular image-guided interventions (EIGIs) are the preferred procedures for treatment of a wide range of vascular disorders. Despite benefits including reduced trauma and recovery time, EIGIs have their own challenges. Remote catheter actuation and challenging anatomical morphology may lead to erroneous endovascular device selections, delays or even complications such as vessel injury. EIGI planning using 3D phantoms would allow interventionists to become familiarized with the patient vessel anatomy by first performing the planned treatment on a phantom under standard operating protocols. In this study the optimal workflow to obtain such phantoms from 3D data for interventionist to practice on prior to an actual procedure was investigated. Patientspecific phantoms and phantoms presenting a wide range of challenging geometries were created. Computed Tomographic Angiography (CTA) data was uploaded into a Vitrea 3D station which allows segmentation and resulting stereo-lithographic files to be exported. The files were uploaded using processing software where preloaded vessel structures were included to create a closed-flow vasculature having structural support. The final file was printed, cleaned, connected to a flow loop and placed in an angiographic room for EIGI practice. Various Circle of Willis and cardiac arterial geometries were used. The phantoms were tested for ischemic stroke treatment, distal catheter navigation, aneurysm stenting and cardiac imaging under angiographic guidance. This method should allow for adjustments to treatment plans to be made before the patient is actually in the procedure room and enabling reduced risk of peri-operative complications or delays.

  6. Informing participants in clinical trials with ex vivo human tissue-engineered products: what to tell and how to tell it?

    PubMed

    Trommelmans, Leen; Selling, Joseph; Dierickx, Kris

    2008-06-01

    Ex vivo tissue-engineered products are increasingly entered into clinical trials. To allow prospective participants to make a fully informed, autonomous decision on their participation, we have to adapt the informed consent process by taking the specific aspects of tissue engineering into consideration. New elements in ex vivo tissue engineering are the source and manipulation of the cells in the product, the implantation of the product and the additional risks and benefits due to the construction of the product and its activity in the body. They are the result of the delicate nature of some cell types and of the complexity of the tissue engineering process. The process of informing the participant should be designed in such a way that the participant's capacity to understand the intervention and its implications is enhanced. Crucial issues, such as the aim and procedure of the trial, the risks and benefits involved and the role of the investigator, have to be clarified. We suggest that participants' understanding of the trial can be enhanced through the use of audiovisual material, by developing a simple questionnaire to direct the information process further, and by the assistance of informed third parties to help participants in their decision-making processes. PMID:18493918

  7. Dissection of tumour and host cells from target organs of metastasis for testing gene expression directly ex vivo.

    PubMed Central

    Rocha, M.; Hexel, K.; Bucur, M.; Schirrmacher, V.; Umansky, V.

    1996-01-01

    We report on a new methodology which allows the direct analysis ex vivo of tumour cells and host cells (lymphocytes, macrophages, endothelial cells) from a metastasised organ (liver or spleen) at any time point during the metastatic process and without any further in vitro culture. First, we used a tumour cell line transduced with the bacterial gene lacZ, which permits the detection of the procaryotic enzyme beta-galactosidase in eukaryotic cells at the single cell level thus allowing flow adhesion cell sorting (FACS) analysis of tumour cells from metastasised target organs. Second, we established a method for the separation and enrichment of tumour and host cells from target organs of metastasis with a high viability and reproducibility. As exemplified with the murine lymphoma ESb, this new methodology permits the study of molecules of importance for metastasis or anti-tumour immunity (adhesion, costimulatory and cytotoxic molecules, cytokines, etc.) at the RNA or protein level in tumour and host cells during the whole process of metastasis. This novel approach may open new possibilities of developing strategies for intervention in tumour progression, since it allows the determination of the optimal window in time for successful treatments. The possibility of direct analysis of tumour and host cell properties also provides a new method for the evaluation of the effects of immunisation with tumour vaccines or of gene therapy. Images Figure 3 PMID:8883407

  8. Ex Vivo Propagation of Human Corneal Stromal "Activated Keratocytes" for Tissue Engineering.

    PubMed

    Yam, Gary Hin-Fai; Yusoff, Nur Zahirah Binte M; Kadaba, Aishwarya; Tian, Dechao; Myint, Htoon Hla; Beuerman, Roger W; Zhou, Lei; Mehta, Jodhbir S

    2015-01-01

    Keratoconus is a corneal disorder characterized by a thinning of stromal tissue, and the affected patients have induced astigmatism and visual impairment. It is associated with a loss of corneal stromal keratocytes (CSKs). Hence, reconstructing stromal tissue with autologous CSK replacement can be a viable alternative to corneal transplantation, which is restricted by the global donor material shortage and graft rejection. Human CSKs are normally quiescent and express unique markers, like aldehyde dehydrogenases and keratocan. In serum culture, they proliferate, but lose their characteristic phenotype and become stromal fibroblasts. Here we report a novel culture cocktail to ex vivo propagate and maintain CSKs. Primary human CSKs were obtained from adult donors and cultured with soluble human amnion stromal extract (ASE), rho-associated coiled-coil-forming protein serine/threonine kinase inhibitor Y-27632, and insulin-like growth factor-1 (collectively named as ERI). Protein profiling using mass spectrometry followed by MetaCore™ pathway analysis predicted that ASE proteins might participate in transforming growth factor-β (TGF-β) signaling and fibroblast development, cell adhesion, extracellular matrix remodeling, and immune response. In culture with 0.5% fetal bovine serum and ERI, the population of "activated keratocytes" was expanded. They had much lowered expression of both keratocyte and fibroblast markers, suppressed TGF-β-mediated Smad2/3 activation, and lacked fibroblast-mediated collagen contractibility. These "activated keratoctyes" could be propagated for six to eight passages ex vivo, and they regained CSK-specific dendritic morphology and gene marker expression, including aldehyde dehydrogenases, lumican, and keratocan biosynthesis, expression, and secretion when returned to serum-depleted ERI condition. This novel cocktail maintained human CSKs in both adherent and suspension cultures with proper keratocyte features and without the

  9. Application of Gold Nanorods for Photothermal Therapy in Ex Vivo Human Oesophagogastric Adenocarcinoma.

    PubMed

    Singh, Mohan; Harris-Birtill, David C C; Zhou, Yu; Gallina, Maria E; Cass, Anthony E G; Hanna, George B; Elson, Daniel S

    2016-03-01

    Gold nanoparticles are chemically fabricated and tuned to strongly absorb near infrared (NIR) light, enabling deep optical penetration and therapy within human tissues, where sufficient heating induces tumour necrosis. In our studies we aim to establish the optimal gold nanorod (GNR) concentration and laser power for inducing hyperthermic effects in tissues and test this photothermal effect on ex vivo human oesophagogastric adenocarcinoma. The ideal GNR concentration and NIR laser power that would elicit sufficient hyperthermia for tumour necrosis was pre-determined on porcine oesophageal tissues. Human ex vivo oesophageal and gastric adenocarcinoma tissues were incubated with GNR solutions and a GNR-free control solution with corresponding healthy tissues for comparison, then irradiated with NIR light for 10 minutes. Temperature rise was found to vary linearly with both the concentration of GNRs and the laser power. Human ex vivo oesophageal and gastric tissues consistently demonstrated a significant temperature rise when incubated in an optimally concentrated GNR solution (3 x 10(10) GNRs/ml) prior to NIR irradiation delivered at an optimal power (2 W/cm2). A mean temperature rise of 27 degrees C was observed in tissues incubated with GNRs, whereas only a modest 2 degrees C rise in tissues not exposed to any GNRs. This study evaluates the photothermal effects of GNRs on oesophagogastric tissue examines their application in the minimally invasive therapeutics of oesophageal and gastric adenocarcinomas. This could potentially be an effective method of clinically inducing irreversible oesophagogastric tumour photodestruction, with minimal collateral damage expected in (healthy) tissues free from GNRs. PMID:27280246

  10. Using computed tomography scans to develop an ex-vivo gastric model

    PubMed Central

    Henry, Jerome A; O’Sullivan, Gerard; Pandit, Abhay S

    2007-01-01

    The objective of this research was to use abdominal computed tomography (CT) scans to non-invasively quantify anthropometrical data of the human stomach and to concomitantly create an anatomically correct and distensible ex-vivo gastric model. Thirty-three abdominal CT scans of human subjects were obtained and were imported into reconstruction software to generate 3D models of the stomachs. Anthropometrical data such as gastric wall thickness, gastric surface area and gastric volume were subsequently quantified. A representative 3D computer model was exported into a selective laser sintering (SLS) rapid prototyping machine to create an anatomically correct solid gastric model. Subsequently, a replica wax template of the SLS model was created. A negative mould was offset around the wax template such that the offset distance was equivalent to that of the gastric wall thickness. A silicone with similar mechanical properties to the human stomach was poured into the offset. The lost wax manufacturing technique was employed to create a hollow distensible stomach model. 3D computer gastric models were generated from the CT scans. A hollow distensible silicone ex-vivo gastric model with similar compliance to that of the human stomach was created. The anthropometrical data indicated that there is no significant relationship between BMI and gastric surface area or gastric volume. There were inter- and intra-group differences between groups with respect to gastric wall thickness. This study demonstrates that abdominal CT scans can be used to both non-invasively determine gastric anthropometrical data as well as create realistic ex-vivo stomach models. PMID:17457968

  11. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    SciTech Connect

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  12. Ex vivo expansion marginally amplifies repopulating cells from baboon peripheral blood mobilized CD34+ cells.

    PubMed

    Norol, Françoise; Drouet, Michel; Pflumio, Françoise; Léonardi, Marjorie; Mourcin, Frédéric; Debili, Najet; Job, Agnès; Vainchenker, William; Kuentz, Mathieu; Hérodin, Francis

    2002-06-01

    The ability of ex vivo expansion to increase the long-term repopulating capacity of a graft is still unknown. One problem is the most reliable way to quantify transplantable cells. We addressed this point in a baboon model based on autologous transplantation of serial limiting doses of non-manipulated or ex vivo-expanded mobilized CD34+ cells and determined the threshold doses of non-manipulated and expanded cells which supported long-term multilineage engraftment. In the expansion group, CD34+ cells were cultured for 6 d with a combination of early acting cytokines (Flt3-ligand, stem cell factor, thrombopoietin and interleukin 3). Grafted cells were characterized by their surface antigens and biological properties [semisolid assays, long-term culture-initiating cells (LTC-IC) and non-obese diabetic severe combined immunodeficient reconstituting cells (SRC)]. Animals were followed for at least 12 months post transplantation. The expansion protocol yielded 12.3-fold, 16.9-fold, 3.7-fold, 3.5-fold and 2.2-fold increases in CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte CFU (CFU-MK), LTC-IC and SRC respectively. It induced a modest increase in the long term reconstitutive ability of the graft; the threshold value for long-term engraftment was 0.5 x 10(6)/kg CD34+ cells in the control group and 0.3 x 10(6)/kg CD34+ cells in the expansion group, although one animal in this latter group remained hypoplastic. Frequencies of SRC had a high predictive value of long-term engraftment (r > 0.80). The main advantage of the protocol was the acceleration of granulocyte recovery, achieved at the different doses tested. In conclusion, these experiments suggest that this ex vivo expansion protocol marginally amplifies long-term reconstituting cells. PMID:12060132

  13. Recombinant Human Elastase Alters the Compliance of Atherosclerotic Tibial Arteries After Ex Vivo Angioplasty

    PubMed Central

    Bingham, Karen; Moss, Emma; Gottlieb, Daniel P.; Wong, Marco D.; Bland, Kimberly S.; Franano, F. Nicholas

    2016-01-01

    Purpose: This study was designed to determine whether vonapanitase (formerly PRT-201), a recombinant human elastase, treatment can fragment the protein elastin in elastic fibers and cause dilation of atherosclerotic human peripheral arteries subjected to ex vivo balloon angioplasty. Materials and Methods: Seven patients undergoing lower limb amputation for peripheral artery disease or who died and donated their bodies to science donated 11 tibial arteries (5 anterior, 6 posterior) for this study. All arteries were atherosclerotic by visual inspection. The arteries underwent ex vivo balloon angioplasty and thereafter were cut into rings and studied on wire myographs where the rings were stretched and tension was recorded. After treatment with vonapanitase 2 mg/mL or vehicle control, myography was repeated and the rings were then subject to elastin content measurement using a desmosine radioimmunoassay and elastic fiber visualization by histology. The wire myography data were used to derive compliance, stress-strain, and incremental elastic modulus curves. Results: Vonapanitase treatment reduced elastin (desmosine) content by 60% and decreased elastic fiber histologic staining. Vonapanitase-treated rings experienced less tension at any level of stretch and as a result had shifts in the compliance and stress-strain curves relative to vehicle-treated rings. Vonapanitase treatment did not alter the incremental elastic modulus curve. Conclusions: Vonapanitase treatment of atherosclerotic human peripheral arteries after ex vivo balloon angioplasty fragmented elastin in elastic fibers, decreased tension in the rings at any level of stretch, and altered the compliance and stress-strain curves in a manner predicting arterial dilation in vivo. Based on this result, local treatment of balloon angioplasty sites may increase blood vessel diameter and thereby improve the success of balloon angioplasty in peripheral artery disease. PMID:26745001

  14. Characterization of micro-invasive trabecular bypass stents by ex vivo perfusion and computational flow modeling

    PubMed Central

    Hunter, Kendall S; Fjield, Todd; Heitzmann, Hal; Shandas, Robin; Kahook, Malik Y

    2014-01-01

    Micro-invasive glaucoma surgery with the Glaukos iStent® or iStent inject® (Glaukos Corporation, Laguna Hills, CA, USA) is intended to create a bypass through the trabecular meshwork to Schlemm’s canal to improve aqueous outflow through the natural physiologic pathway. While the iStent devices have been evaluated in ex vivo anterior segment models, they have not previously been evaluated in whole eye perfusion models nor characterized by computational fluid dynamics. Intraocular pressure (IOP) reduction with the iStent was evaluated in an ex vivo whole human eye perfusion model. Numerical modeling, including computational fluid dynamics, was used to evaluate the flow through the stents over physiologically relevant boundary conditions. In the ex vivo model, a single iStent reduced IOP by 6.0 mmHg from baseline, and addition of a second iStent further lowered IOP by 2.9 mmHg, for a total IOP reduction of 8.9 mmHg. Computational modeling showed that simulated flow through the iStent or iStent inject is smooth and laminar at physiological flow rates. Each stent was computed to have a negligible flow resistance consistent with an expected significant decrease in IOP. The present perfusion results agree with prior clinical and laboratory studies to show that both iStent and iStent inject therapies are potentially titratable, providing clinicians with the opportunity to achieve lower target IOPs by implanting additional stents. PMID:24648713

  15. Relationship between wave aberrations and histological features in ex vivo porcine crystalline lenses

    NASA Astrophysics Data System (ADS)

    Acosta, Eva; Bueno, Juan M.; Schwarz, Christina; Artal, Pablo

    2010-09-01

    Wave aberrations of isolated ex vivo porcine crystalline lenses were measured by using a point-diffraction interferometer. This method allowed us to gain greater insight into the detailed aberration structure of eye lenses showing systematic presence of some dominant aberrations. In order of significance, astigmatism together with spherical aberration, coma, and trefoil are the main aberrations present in all lenses. We found a high correlation between the axis of both astigmatism and trefoil with the Y-shaped suture planes of the lens, revealing a subtle relationship between the induced aberrations and the histological features.

  16. Long-term ex vivo and in vivo monitoring of tumor progression by using dual luciferases.

    PubMed

    Morita, Naoki; Haga, Sanae; Ohmiya, Yoshihiro; Ozaki, Michitaka

    2016-03-15

    We propose a new concept of tumor progression monitoring using dual luciferases in living animals to reduce stress for small animals and the cost of luciferin. The secreted Cypridina luciferase (CLuc) was used as an ex vivo indicator to continuously monitor tumor progression. On the other hand, the non-secreted firefly luciferase was used as an in vivo indicator to analyze the spatial distribution of the tumor at suitable time points indicated by CLuc. Thus, the new monitoring systems that use dual luciferases are available, allowing long-term bioluminescence imaging under minimal stress for the experimental animals. PMID:26717897

  17. Ex-vivo assessment of tissue viability using dynamic laser speckle

    NASA Astrophysics Data System (ADS)

    Ramírez-Miquet, E. E.; Miquet Romero, L. M.; Darias, J. G.; Martínez-Celorio, R. A.

    2015-08-01

    Dynamic laser speckle is a non-destructive contactless sensing method useful for exploring activity inherent to biological samples. We present an ex-vivo analysis of dermal and epidermal tissue with different degrees of activity in healthy and burned tissue. Pseudocolor images obtained after processing biospeckle stacks with the generalized differences reveal a correlation between cellular lysis and speckle pattern activity. Epidermis shows higher activity than dermal tissue, which is attributable to the number of cells and each tissue. The analysis presented here could be employed in assessment of viability of tissues for graft and burns treatments.

  18. An ex vivo approach to botanical-drug interactions: A proof of concept study

    PubMed Central

    Wang, Xinwen; Zhu, Hao-Jie; Munoz, Juliana; Gurley, Bill J.; Markowitz, John S.

    2015-01-01

    Ethnopharmacological relevance Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodoloy in applied ethnopharmacological research. Materials and Methods This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems. Results Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal’s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybinA, silybin B and hydrastine and berberine

  19. In vivo and ex vivo magnetic resonance spectroscopy in the characterization of hemangioblastoma cyst fluid.

    PubMed

    Crisi, Girolamo; Filice, Silvano; Pertinhez, Thelma A; Ventura, Elisa; Servadei, Franco

    2014-01-01

    Peritumoral cyst formation is commonly associated with hemangioblastomas of the central nervous system. Results of a proteomic profiling of hemangioblastoma cyst fluid suggested that cyst formation, whether intratumoral or peritumoral, is a consequence of vascular leakage because protein profiles of cyst fluid and blood serum were similar. To the best of our knowledge, this is the first report of in vivo and ex vivo magnetic resonance spectroscopy analyses of hemangioblastoma cyst fluid that investigates on the mechanism leading to peritumoral cyst formation. PMID:24424553

  20. Ex vivo models to evaluate the role of ocular melanin in trans-scleral drug delivery.

    PubMed

    Pescina, Silvia; Santi, Patrizia; Ferrari, Giulio; Padula, Cristina; Cavallini, Pierugo; Govoni, Paolo; Nicoli, Sara

    2012-08-15

    Trans-scleral delivery is nowadays considered as a possible way to deliver drugs to the posterior segment of the eye. Despite the potentiality of this administration route, there is a lack of fundamental knowledge on the role of the numerous barriers involved. The aim of this work was to develop an easy and cheap ex vivo method to evaluate the barrier properties of the choroid-Bruch's layer and in particular to estimate the role of melanin in drug diffusion through ocular tissues. In vitro binding studies were performed to estimate drug affinity for melanin; model molecules used were methylene blue, propranolol, levofloxacin and methylprednisolone sodium succinate. The ex vivo model set up is based on porcine eye bulbs with light blue iris or brown iris. While the choroid of brown eyes is dark, the choroid of blue eyes is transparent, due to the absence of melanin. Permeation experiments using pigmented and not-pigmented porcine tissues gave the opportunity to discriminate between the barrier role of choroid-Bruch's membrane as such and the barrier role of melanin. Ex vivo permeation experiments can be performed using isolated choroid-Bruch's or the sclera-choroid-Bruch's layer. In this last case, it is possible to take into account also the barrier role of the sclera that tends to decrease the drug concentration at the sclera/choroid interface, thus amplifying the effect of melanin. The data obtained in this paper indicate that for some drugs melanin can really represent a barrier and the effect can imply a lower drug flux or simply a longer lag time depending on the kind of drug and the concentration applied. However, it is a saturable barrier, thus its effect can probably be overtaken by high doses or multiple administrations. The ex vivo model set up can help to refine computational models, to better evaluate the interplay among static, dynamic and metabolic barriers. Additionally, since human eyes display a full range of pigmentation, the model could also be

  1. Ex-vivo liver perfusion for organ preservation: Recent advances in the field.

    PubMed

    Barbas, A S; Goldaracena, N; Dib, M J; Selzner, M

    2016-07-01

    Liver transplantation is the optimal treatment for end-stage liver disease but is limited by the severe shortage of donor organs. This shortage has prompted increased utilization of marginal grafts from DCD and extended criteria donors, which poorly tolerate cold storage in comparison to standard criteria grafts. Ex-vivo liver perfusion (EVLP) technology has emerged as a potential alternative to cold storage for organ preservation, but there is no consensus regarding the optimal temperature or conditions for EVLP. Herein, we review recent advances in both pre-clinical and clinical studies, organized by perfusion temperature (hypothermic, subnormothermic, normothermic). PMID:27158081

  2. Optical controling dynamic and fluctuation processes in ensemble of neurons at pulsed electrical excitation ex vivo

    NASA Astrophysics Data System (ADS)

    Akchurin, Garif G.; Seliverstov, George A.; Akchurin, Alexander G.; Akchurin, George G.

    2004-05-01

    Dynamic response of the somatic frog nerve on electrical pulsed excitation was investigated ex vivo. Strong fluctuation of consequence compound action potential in ensemble of neurons near-threshold was discovered. The nonlinear response of the Hodgkin-Huxley model neurons with external electrical pulsed was investigated and numeral results correlation with experiments. Complex dynamic of compound action potential was discovered when on-line time of stimulatory electrical pulses comparable with nerve refractory period. New techniques research nonlinear behavior using photodynamic reactions or UV-A radiation at somatic frog nerve was approved. This nonlinear dynamic regime was controlling laser induced inactivation of processes in membrane of nerve.

  3. Modification of the NEMA XR21-2000 cardiac phantom for testing of imaging systems used in endovascular image guided interventions

    NASA Astrophysics Data System (ADS)

    Ionita, C. N.; Dohatcu, A.; Jain, A.; Keleshis, C.; Hoffmann, K. R.; Bednarek, D. R.; Rudin, S.

    2009-02-01

    X-ray equipment testing using phantoms that mimic the specific human anatomy, morphology, and structure is a very important step in the research, development, and routine quality assurance for such equipment. Although the NEMA XR21 phantom exists for cardiac applications, there is no such standard phantom for neuro-, peripheral and cardiovascular angiographic applications. We have extended the application of the NEMA XR21-2000 phantom to evaluate neurovascular x-ray imaging systems by structuring it to be head-equivalent; two aluminum plates shaped to fit into the NEMA phantom geometry were added to a 15 cm thick section. Also, to enable digital subtraction angiography (DSA) testing, two replaceable central plates with a hollow slot were made so that various angiographic sections could be inserted into the phantom. We tested the new modified phantom using a flat panel C-arm unit dedicated for endovascular image-guided interventions. All NEMA XR21-2000 standard test sections were used in evaluations with the new "headequivalent" phantom. DSA and DA are able to be tested using two standard removable blocks having simulated arteries of various thickness and iodine concentrations (AAPM Report 15). The new phantom modifications have the benefits of enabling use of the standard NEMA phantom for angiography in both neuro- and cardio-vascular applications, with the convenience of needing only one versatile phantom for multiple applications. Additional benefits compared to using multiple phantoms are increased portability and lower cost.

  4. Exposures in interventional radiology using Monte Carlo simulation coupled with virtual anthropomorphic phantoms.

    PubMed

    Santos, William S; Neves, Lucio P; Perini, Ana P; Belinato, Walmir; Caldas, Linda V E; Carvalho, Albérico B; Maia, Ana F

    2015-12-01

    In this work we investigated the way in which conversion coefficients from air kerma-area product for effective doses (CCE) and entrance skin doses (CCESD) in interventional radiology (IR) are affected by variations in the filtration, projection angle of the X-ray beam, lead curtain attached to the surgical table, and suspended shield lead glass in regular conditions of medical practice. Computer simulations were used to model an exposure scenario similar to a real IR room. The patient and the physician were represented by MASH virtual anthropomorphic phantoms, inserted in the MCNPX 2.7.0 radiation transport code. In all cases, the addition of copper filtration also increased the CCE and CCESD values. The highest CCE values were obtained for lateral, cranial and caudal projections. In these projections, the X-ray tube was located above the table, and more scattered radiation reached the middle and upper portions of the physician trunk, where most of the radiosensitive organs are located. Another important result of this study was to show that the physician's protection is 358% higher when the lead curtain and suspended shield lead glasses are used. The values of CCE and CCESD, presented in this study, are an important resource for calculation of effective doses and entrance skin doses in clinical practice. PMID:26160701

  5. In vivo and ex vivo evaluation of cosmetic properties of seedcakes.

    PubMed

    Ratz-Łyko, Anna; Arct, Jacek; Pytkowska, Katarzyna; Majewski, Sławomir

    2015-04-01

    The seedcakes are a potential source of natural bioactive substances: antioxidants, protein, and carbohydrates. Thus, they may scavenge free radicals and have an effect on the stratum corneum hydration and epidermal barrier function. The aim of the study was to evaluate the in vivo and ex vivo properties of emulsions with the seedcake extracts using the pH meter, corneometer, tewameter, methyl nicotinate model of micro-inflammation in human skin, and tape stripping of the stratum corneum. The in vivo and ex vivo studies showed that the emulsions with Oenothera biennis, Borago officinalis, and Nigella sativa seedcake extracts have anti-inflammatory and antioxidant activity. The 6-week topical application of the emulsions with the B. officinalis and N. sativa seedcakes significantly reduced skin irritation and influenced the improvement of the skin hydration and epidermal barrier function compared with placebo. The seedcakes due to their antioxidant and anti-inflammatory activities have potential application in anti-aging, moisturizing, mitigating, and protective cosmetics. PMID:25415370

  6. Recognition algorithm for assisting ovarian cancer diagnosis from coregistered ultrasound and photoacoustic images: ex vivo study

    NASA Astrophysics Data System (ADS)

    Alqasemi, Umar; Kumavor, Patrick; Aguirre, Andres; Zhu, Quing

    2012-12-01

    Unique features and the underlining hypotheses of how these features may relate to the tumor physiology in coregistered ultrasound and photoacoustic images of ex vivo ovarian tissue are introduced. The images were first compressed with wavelet transform. The mean Radon transform of photoacoustic images was then computed and fitted with a Gaussian function to find the centroid of a suspicious area for shift-invariant recognition process. Twenty-four features were extracted from a training set by several methods, including Fourier transform, image statistics, and different composite filters. The features were chosen from more than 400 training images obtained from 33 ex vivo ovaries of 24 patients, and used to train three classifiers, including generalized linear model, neural network, and support vector machine (SVM). The SVM achieved the best training performance and was able to exclusively separate cancerous from non-cancerous cases with 100% sensitivity and specificity. At the end, the classifiers were used to test 95 new images obtained from 37 ovaries of 20 additional patients. The SVM classifier achieved 76.92% sensitivity and 95.12% specificity. Furthermore, if we assume that recognizing one image as a cancer is sufficient to consider an ovary as malignant, the SVM classifier achieves 100% sensitivity and 87.88% specificity.

  7. Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

    PubMed

    Laird, Gregory M; Bullen, C Korin; Rosenbloom, Daniel I S; Martin, Alyssa R; Hill, Alison L; Durand, Christine M; Siliciano, Janet D; Siliciano, Robert F

    2015-05-01

    Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs. PMID:25822022

  8. Multiphoton gradient index endoscopy for evaluation of diseased human prostatic tissue ex vivo

    NASA Astrophysics Data System (ADS)

    Huland, David M.; Jain, Manu; Ouzounov, Dimitre G.; Robinson, Brian D.; Harya, Diana S.; Shevchuk, Maria M.; Singhal, Paras; Xu, Chris; Tewari, Ashutosh K.

    2014-11-01

    Multiphoton microscopy can instantly visualize cellular details in unstained tissues. Multiphoton probes with clinical potential have been developed. This study evaluates the suitability of multiphoton gradient index (GRIN) endoscopy as a diagnostic tool for prostatic tissue. A portable and compact multiphoton endoscope based on a 1-mm diameter, 8-cm length GRIN lens system probe was used. Fresh ex vivo samples were obtained from 14 radical prostatectomy patients and benign and malignant areas were imaged and correlated with subsequent H&E sections. Multiphoton GRIN endoscopy images of unfixed and unprocessed prostate tissue at a subcellular resolution are presented. We note several differences and identifying features of benign versus low-grade versus high-grade tumors and are able to identify periprostatic tissues such as adipocytes, periprostatic nerves, and blood vessels. Multiphoton GRIN endoscopy can be used to identify both benign and malignant lesions in ex vivo human prostate tissue and may be a valuable diagnostic tool for real-time visualization of suspicious areas of the prostate.

  9. Clinical study of ex vivo photoacoustic imaging in endoscopic mucosal resection tissues

    NASA Astrophysics Data System (ADS)

    Lim, Liang; Streutker, Catherine J.; Marcon, Norman; Cirocco, Maria; Lakovlev, Vladimir V.; DaCosta, Ralph; Foster, F. S.; Wilson, Brian C.

    2015-03-01

    Accurate endoscopic detection and dysplasia in patients with Barrett's esophagus (BE) remains a major unmet clinical need. Current diagnosis use multiple biopsies under endoscopic image guidance, where up to 99% of the tissue remains unsampled, leading to significant risk of missing dysplasia. We conducted an ex vivo clinical trial using photoacoustic imaging (PAI) in patients undergoing endoscopic mucosal resection (EMR) with known high-grade dysplasia for the purpose of characterizing the esophageal microvascular pattern, with the long-term goal of performing in vivo endoscopic PAI for dysplasia detection and therapeutic guidance. EMR tissues were mounted immediately on an agar layer and covered with ultrasound gel. Digital photography guided the placement of the PAI transducer (40 MHz center frequency). The luminal side of the specimen was scanned over a field of view of 14 mm (width) by 15 mm (depth) at 680, 750, 824, 850 and 970 nm. Acoustic images were simultaneously acquired. Tissues were then sliced and fixed in formalin for histopathology with H and E staining. Analysis consisted of co-registration and correlation between the intrinsic PAI features and the histological images. The initial PAI + ultrasound images from 8 BE patients have demonstrated the technical feasibility of this approach and point to the potential of PAI to reveal the microvascular pattern within EMR specimens. There are several technical factors to be considered in rigorous interpretation of the PAI characteristics, including the loss of blood from the ex vivo specimens and the limited depth penetration of the photoacoustic signal.

  10. Ex vivo expansion of circulating CD34(+) cells enhances the regenerative effect on rat liver cirrhosis.

    PubMed

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34(+) cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34(+) cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34(+) cells were injected via the spleen. After 7 days, CD34(+) cells were effectively expanded in a serum-free culture medium. Expanded CD34(+) cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34(+) cells increased compared with nonexpanded CD34(+) cells. Expanded CD34(+) cell transplantation reduced liver fibrosis, with a decrease of αSMA(+) cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34(+) cells over non-expanded CD34(+) cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34(+) cells and aggravated liver fibrosis. These findings suggest that expanded CD34(+) cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  11. Role of Transgene Regulation in Ex Vivo Lentiviral Correction of Artemis Deficiency

    PubMed Central

    Multhaup, Megan M.; Podetz-Pedersen, Kelly M.; Karlen, Andrea D.; Olson, Erik R.; Gunther, Roland; Somia, Nikunj V.; Blazar, Bruce R.; Cowan, Morton J.

    2015-01-01

    Abstract Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  12. Preparation and evaluation of SEDDS of simvastatin by in vivo, in vitro and ex vivo technique.

    PubMed

    Karim, Fahim Tamzeedul; Kalam, Azad; Anwar, Rafi; Miah, Muhammad Masum; Rahman, Md Shamim; Islam, S M Ashraful

    2015-01-01

    The objective of this work was to formulate a Self Emulsifying Drug Delivery System (SEDDS) of simvastatin, a poorly soluble drug and to evaluate by in vivo, in vitro and ex vivo techniques. Oils and surfactants were screened out depending upon their solubilizing capacity. Among all of the solvents, Capryol 90 showed good solubilizing capacity. It dissolved 105 mg/ml of simvastatin. Tween-80 also showed good solubilizing capacity which was 117 mg/ml. The two excipients were used to prepare simvastatin SEDDS. Formulations were initially checked for the color, clarity and sedimentation. The SEDDS formulations were transparent and clear. Formulation F2 containing 7:3 (m/m) mixture of Capryol 90/Tween-80 produced smallest micro-emulsion with particles size of 0.074 µm and drug release was higher than other formulation (102% within 20 min). Ex vivo study of the SEDDS formulation was evaluated using guinea pig intestinal sac. Drug diffused from F2 formulation was significantly higher than pure drug (p < 0.001). In vivo study of SEDDS was performed in albino mice using plasma cholesterol level as a pharmacodynamic marker parameter. The test formulation (F2) appeared remarkable reduction in plasma cholesterol level, after oral administration which showed that SEDDS may be an effective technique for the oral administration of simvastatin. PMID:25138349

  13. Assessment of thermal sensitivity of CT during heating of liver: an ex vivo study

    PubMed Central

    Pandeya, G D; Greuter, M J W; Schmidt, B; Flohr, T; Oudkerk, M

    2012-01-01

    Objectives The purpose of this study was to assess the thermal sensitivity of CT during heating of ex-vivo animal liver. Methods Pig liver was indirectly heated from 20 to 90 °C by passage of hot air through a plastic tube. The temperature in the heated liver was measured using calibrated thermocouples. In addition, image acquisition was performed with a multislice CT scanner before and during heating of the liver sample. The reconstructed CT images were then analysed to assess the change of CT number as a function of temperature. Results During heating, a decrease in CT numbers was observed as a hypodense area on the CT images. In addition, the hypodense area extended outward from the heat source during heating. The analysis showed a linear decrease of CT number as a function of temperature. From this relationship, we derived a thermal sensitivity of CT for pig liver tissue of −0.54±0.03 HU °C−1 with an r2 value of 0.91. Conclusions The assessment of the thermal sensitivity of CT in ex-vivo pig liver tissue showed a linear dependency on temperature ≤90 °C. This result may be beneficial for the application of isotherms or thermal maps in CT images of liver tissue. PMID:22919016

  14. Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment

    PubMed Central

    Parker, Maura H.; Loretz, Carol; Tyler, Ashlee E.; Duddy, William J.; Hall, John K.; Olwin, Bradley B.; Bernstein, Irwin D.; Storb, Rainer; Tapscott, Stephen J.

    2012-01-01

    Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. However, a single donor muscle biopsy is unlikely to provide enough cells to effectively transplant the muscle mass of a patient affected by muscular dystrophy. Expansion of cells ex vivo using traditional culture techniques significantly reduces engraftment potential. We hypothesized that activation of Notch signaling during ex vivo expansion would maintain donor cell engraftment potential. In this study, we expanded freshly isolated canine muscle-derived cells on tissue culture plates coated with Delta-1ext-IgG to activate Notch signaling or with human IgG as a control. A model of canine-to-murine xenotransplantation was used to quantitatively compare canine muscle cell engraftment, and determine if engrafted donor cells could function as satellite cells in vivo. We show that Delta-1ext-IgG inhibited differentiation of canine muscle-derived cells, and increased the level of genes normally expressed in myogenic precursors. Moreover, cells expanded on Delta-1ext-IgG resulted in a significant increase in the number of donor-derived fibers, as compared to cells expanded on human IgG, reaching engraftment levels similar to freshly isolated cells. Importantly, cells expanded on Delta-1ext-IgG engrafted to the recipient satellite cell niche, and contributed to further regeneration. A similar strategy of expanding human muscle-derived cells on Notch ligand might facilitate engraftment and muscle regeneration for patients affected with muscular dystrophy. PMID:22865615

  15. Ex vivo expansion of circulating CD34+ cells enhances the regenerative effect on rat liver cirrhosis

    PubMed Central

    Nakamura, Toru; Koga, Hironori; Iwamoto, Hideki; Tsutsumi, Victor; Imamura, Yasuko; Naitou, Masako; Masuda, Atsutaka; Ikezono, Yu; Abe, Mitsuhiko; Wada, Fumitaka; Sakaue, Takahiko; Ueno, Takato; Ii, Masaaki; Alev, Cantas; Kawamoto, Atsuhiko; Asahara, Takayuki; Torimura, Takuji

    2016-01-01

    Ex vivo expansion of autologous cells is indispensable for cell transplantation therapy of patients with liver cirrhosis. The aim of this study was to investigate the efficacy of human ex vivo-expanded CD34+ cells for treatment of cirrhotic rat liver. Recipient rats were intraperitoneally injected with CCl4 twice weekly for 3 weeks before administration of CD34+ cells. CCl4 was then re-administered twice weekly for 3 more weeks, and the rats were sacrificed. Saline, nonexpanded or expanded CD34+ cells were injected via the spleen. After 7 days, CD34+ cells were effectively expanded in a serum-free culture medium. Expanded CD34+ cells were also increasingly positive for cell surface markers of VE-cadherin, VEGF receptor-2, and Tie-2. The expression of proangiogenic growth factors and adhesion molecules in expanded CD34+ cells increased compared with nonexpanded CD34+ cells. Expanded CD34+ cell transplantation reduced liver fibrosis, with a decrease of αSMA+ cells. Assessments of hepatocyte and sinusoidal endothelial cell proliferative activity indicated the superior potency of expanded CD34+ cells over non-expanded CD34+ cells. The inhibition of integrin αvβ3 and αvβ5 disturbed the engraftment of transplanted CD34+ cells and aggravated liver fibrosis. These findings suggest that expanded CD34+ cells enhanced the preventive efficacy of cell transplantation in a cirrhotic model. PMID:27162932

  16. Recognition algorithm for assisting ovarian cancer diagnosis from coregistered ultrasound and photoacoustic images: ex vivo study

    PubMed Central

    Kumavor, Patrick; Aguirre, Andres; Zhu, Quing

    2012-01-01

    Abstract. Unique features and the underlining hypotheses of how these features may relate to the tumor physiology in coregistered ultrasound and photoacoustic images of ex vivo ovarian tissue are introduced. The images were first compressed with wavelet transform. The mean Radon transform of photoacoustic images was then computed and fitted with a Gaussian function to find the centroid of a suspicious area for shift-invariant recognition process. Twenty-four features were extracted from a training set by several methods, including Fourier transform, image statistics, and different composite filters. The features were chosen from more than 400 training images obtained from 33 ex vivo ovaries of 24 patients, and used to train three classifiers, including generalized linear model, neural network, and support vector machine (SVM). The SVM achieved the best training performance and was able to exclusively separate cancerous from non-cancerous cases with 100% sensitivity and specificity. At the end, the classifiers were used to test 95 new images obtained from 37 ovaries of 20 additional patients. The SVM classifier achieved 76.92% sensitivity and 95.12% specificity. Furthermore, if we assume that recognizing one image as a cancer is sufficient to consider an ovary as malignant, the SVM classifier achieves 100% sensitivity and 87.88% specificity. PMID:23208214

  17. Multicolor core/shell silica nanoparticles for in vivo and ex vivo imaging

    NASA Astrophysics Data System (ADS)

    Rampazzo, Enrico; Boschi, Federico; Bonacchi, Sara; Juris, Riccardo; Montalti, Marco; Zaccheroni, Nelsi; Prodi, Luca; Calderan, Laura; Rossi, Barbara; Becchi, Serena; Sbarbati, Andrea

    2012-01-01

    Biocompatible highly bright silica nanoparticles were designed, prepared and tested in small living organisms for both in vivo and ex vivo imaging. The results that we report here demonstrate that they are suitable for optical imaging applications as a possible alternative to commercially available fluorescent materials including quantum dots. Moreover, the tunability of their photophysical properties, which was enhanced by the use of different dyes as doping agents, constitutes a very important added value in the field of medical diagnostics.Biocompatible highly bright silica nanoparticles were designed, prepared and tested in small living organisms for both in vivo and ex vivo imaging. The results that we report here demonstrate that they are suitable for optical imaging applications as a possible alternative to commercially available fluorescent materials including quantum dots. Moreover, the tunability of their photophysical properties, which was enhanced by the use of different dyes as doping agents, constitutes a very important added value in the field of medical diagnostics. Electronic supplementary information (ESI) available: Particle size distribution by DLS and TEM images. See DOI: 10.1039/c1nr11401h

  18. In situ and ex vivo evaluation of a wireless magnetoelastic biliary stent monitoring system.

    PubMed

    Green, Scott Ryan; Kwon, Richard S; Elta, Grace H; Gianchandani, Yogesh B

    2010-06-01

    This paper presents the in situ and ex vivo evaluation of a system that wirelessly monitors the accumulation of intimal tissue and sludge in a biliary stent. The sensing element, located within the stent, is a magnetoelastic resonator that is queried by a wireless radio frequency signal. The in situ testing uses a commercially-available self-expanding biliary stent enhanced with a 1 mm x 25 mm magnetoelastic ribbon sensor (formed from Metglas 2605SA1). The stent has a conformal magnetic layer (consisting of strontium ferrite particles suspended in polydimethylsiloxane) that biases the sensor. The external interrogation module is able to acquire a signal from the sensor from a distance of at least 5 cm while the sensor is implanted in a porcine carcass and loaded with biological fluids. The ex vivo testing uses bile harvested from the porcine carcass. The response of a 1 mm x 25 mm magnetoelastic ribbon sensor is first calibrated with fluids of known density and viscosity, and the calibrated sensor is used to estimate that the viscosity of the harvested bile is 2.7-3.7 cP. The test results presented in this paper illustrate the fundamental usability of the system when the sensor is implanted, loaded by biological fluids, and interrogated in a surgical setup. PMID:20180152

  19. Temperature distribution during RF ablation on ex vivo liver tissue: IR measurements and simulations

    NASA Astrophysics Data System (ADS)

    Macchi, Edoardo Gino; Gallati, Mario; Braschi, Giovanni; Cigada, Alfredo; Comolli, Lorenzo

    2015-05-01

    Radiofrequency thermal ablation is the first therapeutic option for the minimally invasive treatment of liver tumors. This medical procedure employs the Joule heat produced by a RF electromagnetic field to kill tumor cells. The outcome of the procedure is strongly affected by the temperature distribution near the RF applicator, however the measurement of this distribution, even in ex vivo experiments, is not straightforward since most traditional local temperature measurement techniques are not well-suited, due to both electromagnetic interferences and the sensor heat sink effect. Given the importance of the temperature field knowledge, in this paper special care was devoted to its measurement employing both infrared thermal imaging and NTC thermistors. Several RF ablation tests on ex vivo porcine liver tissue were carried out measuring the space-time evolution of temperature during the procedure (with spatial resolution ≤1 mm) and producing useful data for the design and the calibration of a numerical model. Electro-thermal numerical simulations of the experimental tests were performed using a mathematical model suitable for the heating phase of the procedure (up to 95 °C). The simulations results allowed to check the physical consistency of the measured data and suggested that a constant thermal conductivity is satisfactory for modeling the temperature evolution during RF ablation.

  20. Ex vivo reconstruction of the epidermis with melanocytes and the influence of UVB.

    PubMed

    Bessou, S; Surlève-Bazeille, J E; Sorbier, E; Taïeb, A

    1995-10-01

    To study pigmentation, we have reconstructed an epidermis ex vivo with keratinocytes and melanocytes. Keratinocytes and melanocytes were grown first in primary cocultures and separately in secondary cultures, then seeded on a dead deepidermized dermis (Pruniéras type) at a 1:20 melanocyte/keratinocyte ratio. Reconstructed epidermis were grown in a special medium enriched with calcium and fetal bovine serum lifted for 15 days at the air-liquid interface. Using histology, immunohistochemistry and electron microscopy we have shown an excellent level of differentiation of the reconstructed epidermis and a physiologic distribution of dendritic melanocytes in the basal layer capable of melanosome transfer to keratinocytes. UVB irradiation 0.15 J/cm2 x 5 consecutive days increased melanocyte numbers and stimulated pigmentation as evidenced macroscopically and microscopically and at the biochemical level. Following UVB irradiation melanosome transfer was markedly increased and isolated or clumps of melanosomes were seen in the basal layers as well as in the stratum corneum. This model allows the study of the physiology of pigmentation ex vivo. PMID:8789198

  1. Ex Vivo Sentinel Node Mapping in Colon Cancer Combining Blue Dye Staining and Fluorescence Imaging

    PubMed Central

    Schaafsma, Boudewijn E.; Verbeek, Floris P.R.; van der Vorst, Joost R.; Hutteman, Merlijn; Kuppen, Peter J.K.; Frangioni, John V.; van de Velde, Cornelis J.H.; Vahrmeijer, Alexander L.

    2013-01-01

    Background The sentinel lymph node procedure has been proposed to improve nodal staging in colon cancer patients. The aim of this study was to assess the added value of near-infrared fluorescence imaging to conventional blue dye staining for ex vivo sentinel lymph node mapping. Materials and Methods Twenty-two consecutive patients undergoing surgery for colon cancer were included. After tumor resection, a premixed cocktail of the near-infrared lymphatic tracer HSA800 and blue dye was submucosally injected around the tumor for detection of sentinel lymph nodes. The Mini-FLARE imaging system was used for fluorescence imaging. Results In 95% of the patients, at least one sentinel lymph node was identified. Overall, a total of 77 sentinel lymph nodes were identified, of which 77 were fluorescent (100%) and 70 (91%) were blue. Sentinel lymph nodes that were located deeper in the mesenteric fat could easily be located by NIR fluorescence. In 4 out of 5 patients with lymph node metastases, tumor cells were present in at least 1 of the sentinel lymph nodes. Conclusions This study shows the successful use and added value of the near-infrared fluorescence tracer HSA800 to conventional blue dye for the ex vivo sentinel lymph node procedure in colon cancer. PMID:23391167

  2. Maintenance of mouse hematopoietic stem cells ex vivo by reprogramming cellular metabolism.

    PubMed

    Liu, Xia; Zheng, Hong; Yu, Wen-Mei; Cooper, Todd M; Bunting, Kevin D; Qu, Cheng-Kui

    2015-03-01

    The difficulty in maintaining the reconstituting capabilities of hematopoietic stem cells (HSCs) in culture outside of the bone marrow microenvironment has severely limited their utilization for clinical therapy. This hurdle is largely due to the differentiation of long-term stem cells. Emerging evidence suggests that energy metabolism plays an important role in coordinating HSC self-renewal and differentiation. Here, we show that treatment with alexidine dihydrochloride, an antibiotic and a selective inhibitor of the mitochondrial phosphatase Ptpmt1, which is crucial for the differentiation of HSCs, reprogrammed cellular metabolism from mitochondrial aerobic metabolism to glycolysis, resulting in a remarkable preservation of long-term HSCs ex vivo in part through hyperactivation of adenosine 5'-monophosphate-activated protein kinase (AMPK). In addition, inhibition of mitochondrial metabolism and activation of AMPK by metformin, a diabetes drug, also decreased differentiation and helped maintain stem cells in culture. Thus, manipulating metabolic pathways represents an effective new strategy for ex vivo maintenance of HSCs. PMID:25593337

  3. Dynamic holographic endoscopy--ex vivo investigations of malignant tumors in the human stomach.

    PubMed

    Avenhaus, Wolfgang; Kemper, Björn; Knoche, Sabine; Domagk, Dirk; Poremba, Christopher; von Bally, Gert; Domschke, Wolfram

    2005-01-01

    Laser holographic interferometry is based on the superimposition of the holograms of different motional states of an object on a single holographic storing medium. Using a combination of holographic interferometry and endoscopic imaging, we tried to detect areas of focally disturbed tissue elasticity in gastric cancer preparations. By connecting a mobile electronic speckle pattern interferometry (ESPI) camera system (light source: double frequency Nd:YAG laser, lambda = 532 nm) to different types of endoscopes, ex vivo experiments were performed on ten formalin fixed human stomachs, nine containing adenocarcinomas and one with a gastric lymphoma. Linking the endoscopic ESPI camera complex to a fast image processing system, the method of double pulse exposure image subtraction was applied at a video frame rate of 12.5 Hz. Speckle correlation patterns and corresponding phase difference distributions resulting from gastric wall deformation by gentle touch with a guide wire were analyzed. Tumor-free gastric areas showed high-contrast concentric fringes around the point of stimulation. In contrast, fringe patterns and filtered phase difference distributions corresponding to the areas of malignancy in all the cases were characterized by largely parallel lines, indicating that stimulation of rigid tumor tissue primarily led to tilting. Our ex vivo investigations of malignant gastric tumors show that the application of dynamic holographic endoscopy makes it possible to distinguish areas of malignancy from surrounding healthy tissue based on the differences in tissue elasticity. PMID:15726298

  4. Linarin Inhibits the Acetylcholinesterase Activity In-vitro and Ex-vivo.

    PubMed

    Feng, Xinchi; Wang, Xin; Liu, Youping; Di, Xin

    2015-01-01

    Linarin is a flavone glycoside in the plants Flos chrysanthemi indici, Buddleja officinalis, Cirsium setosum, Mentha arvensis and Buddleja davidii, and has been reported to possess analgesic, antipyretic, anti-inflammatory and neuroprotective activities. In this paper, linarin was investigated for its AChE inhibitory potential both in-vitro and ex-vivo. Ellman's colorimetric method was used for the determination of AChE inhibitory activity in mouse brain. In-vitro assays revealed that linarin inhibited AChE activity with an IC50 of 3.801 ± 1.149 μM. Ex-vivo study showed that the AChE activity was significantly reduced in both the cortex and hippocampus of mice treated intraperitoneally with various doses of linarin (35, 70 and 140 mg/Kg). The inhibition effects produced by high dose of linarin were the same as that obtained after huperzine A treatment (0.5 mg/Kg). Molecular docking study revealed that both 4'-methoxyl group and 7-O-sugar moiety of linarin played important roles in ligand-receptor binding and thus they are mainly responsible for AChE inhibitory activity. In view of its potent AChE inhibitory activity, linarin may be a promising therapeutic agent for the treatment of some diseases associated with AChE, such as glaucoma, myasthenia gravis, gastric motility and Alzheimer's disease. PMID:26330885

  5. Linarin Inhibits the Acetylcholinesterase Activity In-vitro and Ex-vivo

    PubMed Central

    Feng, Xinchi; Wang, Xin; Liu, Youping; Di, Xin

    2015-01-01

    Linarin is a flavone glycoside in the plants Flos chrysanthemi indici, Buddleja officinalis, Cirsium setosum, Mentha arvensis and Buddleja davidii, and has been reported to possess analgesic, antipyretic, anti-inflammatory and neuroprotective activities. In this paper, linarin was investigated for its AChE inhibitory potential both in-vitro and ex-vivo. Ellman’s colorimetric method was used for the determination of AChE inhibitory activity in mouse brain. In-vitro assays revealed that linarin inhibited AChE activity with an IC50 of 3.801 ± 1.149 μM. Ex-vivo study showed that the AChE activity was significantly reduced in both the cortex and hippocampus of mice treated intraperitoneally with various doses of linarin (35, 70 and 140 mg/Kg). The inhibition effects produced by high dose of linarin were the same as that obtained after huperzine A treatment (0.5 mg/Kg). Molecular docking study revealed that both 4’-methoxyl group and 7-O-sugar moiety of linarin played important roles in ligand-receptor binding and thus they are mainly responsible for AChE inhibitory activity. In view of its potent AChE inhibitory activity, linarin may be a promising therapeutic agent for the treatment of some diseases associated with AChE, such as glaucoma, myasthenia gravis, gastric motility and Alzheimer’s disease. PMID:26330885

  6. Subnormothermic Machine Perfusion for ex vivo Preservation and Recovery of the Human Liver for Transplantation

    PubMed Central

    Bruinsma, B.G.; Yeh, H.; Özer, S; Martins, P.N.; Farmer, A.; Wu, W.; Saeidi, N.; op den Dries, S.; Berendsen, T.A.; Smith, R.N.; Markmann, J.F.; Porte, R.; Yarmush, M.L.; Uygun, K.; Izamis, M.L.

    2015-01-01

    To reduce widespread shortages, attempts are made to use more marginal livers for transplantation. Many of these grafts are discarded for fear of inferior survival rates or biliary complications. Recent advances in organ preservation have shown that ex vivo subnormothermic machine perfusion has the potential to improve preservation and recover marginal livers pre- transplantation. To determine the feasibility in human livers, we assessed the effect of 3 hours of oxygenated subnormothermic machine perfusion (21 °C) on seven livers discarded for transplantation. Biochemical and microscopic assessment revealed minimal injury sustained during perfusion. Improved oxygen uptake (1.30 [1.11–1.94] to 6.74 [4.15–8.16] mL O2/min.kg liver), lactate levels (4.04 [3.70–6.00] to 2.29 [1.20–3.42] mmol/L) and adenosine triphosphate content (45.0 [70.6–87.5] pre-perfusion to 167.5 [151.5–237.2] pmol/mg after perfusion) were observed. Liver function, reflected by urea, albumin and bile production was seen during perfusion. Bile production increased and the composition of bile (bile salts/phospholipid ratio, pH and bicarbonate concentration) became more favorable. In conclusion, ex vivo subnormothermic machine perfusion effectively maintains liver function with minimal injury and sustains or improves various hepatobiliary parameters post-ischemia. PMID:24758155

  7. Ex-Vivo percutaneous absorption of enrofloxacin: Comparison of LMOG organogel vs. pentravan cream.

    PubMed

    Kirilov, Plamen; Tran, Van Hung; Ducrotté-Tassel, Alban; Salvi, Jean-Paul; Perrot, Sébastien; Haftek, Marek; Boulieu, Roselyne; Pirot, Fabrice

    2016-02-10

    The objective of this study was to investigate the percutaneous absorption of enrofloxacin from two base formulations, Pentravan cream and LMOG organogel. Ex-vivo experiments were carried out on pig ear skin. The percutaneous permeation through pig skin of two formulations containing 5 wt% of enrofloxacin was measured and compared using Franz diffusion cells. At appropriate intervals up to 120 h, diffusion samples were taken and analyzed using HPLC assays. Permeation profiles were established and the parameters Tlag and flux values were calculated. In this ex-vivo study, the flux values were 0.35 μgcm(-2)h(-1) for Pentravan and 1.22 μgcm(-2)h(-1) for LMOG organogel, corresponding respectively to 7.9 % and 29.3 % of enrofloxacin absorbed after 120 h by these formulations. The lag time (T lag) of Pentravan and organogel were 6.32 and 0.015 h respectively. The absorption time to reach the antibiotic concentration of enrofloxacin (2 μgmL(-1)) in the receptor was 60 h with Pentravan and 30 h with the organogel, suggesting more effective treatment by the latter. Enrofloxacin contained in organogel could be absorbed through pig ear skin 3.7 times greater than that in Pentravan (commercial formulation). This study demonstrates the perspective of organogel formulations as potential drug delivery systems. PMID:26688043

  8. Investigating detrusor muscle concentrations of oxybutynin after intravesical delivery in an ex vivo porcine model.

    PubMed

    Williams, Nicholas A; Lee, Kay M; Allender, Chris J; Bowen, Jenna L; Gumbleton, Mark; Harrah, Tim; Raja, Aditya; Joshi, Hrishi B

    2015-07-01

    Intravesical oxybutynin is highly effective in the treatment of overactive bladder. Traditionally the mechanism of action was explained by antagonism of muscarinic receptors located in the detrusor, however evidence now suggests antimuscarinics may elicit their effect by modifying afferent pathways in the mucosal region. This study aimed to investigate the bladder wall distribution of oxybutynin in an ex vivo setting providing tissue - layer specific concentrations of drug achieved after intravesical delivery. Whole ex vivo porcine bladders were intravesically instilled with 0.167 mg mL(-1) oxybutynin solution. After 60 min, tissue samples were excised, serially sectioned parallel to the urothelial surface and extracted drug quantified. Drug distribution into the urothelium, lamina propria and detrusor was determined. Oxybutynin permeated into the bladder wall at a higher rate than other drugs previously investigated (apparent transurothelial Kp = 1.36 × 10(-5) cm s(-1) ). After 60 min intravesical instillation, concentrations achieved in the urothelium (298.69 μg g(-1) ) and lamina propria (43.65 μg g(-1) ) but not the detrusor (0.93 μg g(-1) ) were greater than reported IC50 values for oxybutynin. This work adds to the increasing body of evidence suggesting antimuscarinics elicit their effects via mechanisms other than direct inhibition of detrusor contraction. PMID:25989054

  9. Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

    PubMed

    Multhaup, Megan M; Podetz-Pedersen, Kelly M; Karlen, Andrea D; Olson, Erik R; Gunther, Roland; Somia, Nikunj V; Blazar, Bruce R; Cowan, Morton J; McIvor, R Scott

    2015-04-01

    Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A. Recipient animals treated with the PGK-Artemis vector exhibited moderate repopulation of their immune compartment, yet demonstrated a defective proliferative T lymphocyte response to in vitro antigen stimulation. Animals treated with the EF1α-Artemis vector displayed high levels of T lymphocytes but an absence of B lymphocytes and deficient lymphocyte function. In contrast, ex vivo transduction with the APro-Artemis vector supported effective immune reconstitution to wild-type levels, resulting in fully functional T and B lymphocyte responses. These results demonstrate the importance of regulated Artemis expression in immune reconstitution of Artemis-deficient SCID. PMID:25738323

  10. Activation of Notch signaling during ex vivo expansion maintains donor muscle cell engraftment.

    PubMed

    Parker, Maura H; Loretz, Carol; Tyler, Ashlee E; Duddy, William J; Hall, John K; Olwin, Bradley B; Bernstein, Irwin D; Storb, Rainer; Tapscott, Stephen J

    2012-10-01

    Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. However, a single donor muscle biopsy is unlikely to provide enough cells to effectively transplant the muscle mass of a patient affected by muscular dystrophy. Expansion of cells ex vivo using traditional culture techniques significantly reduces engraftment potential. We hypothesized that activation of Notch signaling during ex vivo expansion would maintain donor cell engraftment potential. In this study, we expanded freshly isolated canine muscle-derived cells on tissue culture plates coated with Delta-1(ext) -IgG to activate Notch signaling or with human IgG as a control. A model of canine-to-murine xenotransplantation was used to quantitatively compare canine muscle cell engraftment and determine whether engrafted donor cells could function as satellite cells in vivo. We show that Delta-1(ext) -IgG inhibited differentiation of canine muscle-derived cells and increased the level of genes normally expressed in myogenic precursors. Moreover, cells expanded on Delta-1(ext) -IgG resulted in a significant increase in the number of donor-derived fibers, as compared to cells expanded on human IgG, reaching engraftment levels similar to freshly isolated cells. Importantly, cells expanded on Delta-1(ext) -IgG engrafted to the recipient satellite cell niche and contributed to further regeneration. A similar strategy of expanding human muscle-derived cells on Notch ligand might facilitate engraftment and muscle regeneration for patients affected with muscular dystrophy. PMID:22865615

  11. Ex Vivo Prefabricated Rat Skin Flap Using Cell Sheets and an Arteriovenous Vascular Bundle

    PubMed Central

    Fujisawa, Daisuke; Sekine, Hidekazu; Okano, Teruo; Sakurai, Hiroyuki

    2015-01-01

    Background: Recently, research on tissue-engineered skin substitutes have been active in plastic surgery, and significant development has been made in this area over the past several decades. However, a regenerative skin flap has not been developed that could provide immediate blood flow after transplantation. Here, we make a regenerative skin flap ex vivo that is potentially suitable for microsurgical transplantation in future clinical applications. Methods: In rats, for preparing a stable vascular carrier, a femoral vascular pedicle was sandwiched between collagen sponges and inserted into a porous chamber in the abdomen. The vascular bed was harvested 3 weeks later, and extracorporeal perfusion was performed. A green fluorescent protein positive epidermal cell sheet was placed on the vascular bed. After perfusion culture, the whole construct was harvested and fixed for morphological analyses. Results: After approximately 10 days perfusion, the epidermal cell sheet cornified sufficiently. The desquamated corneum was positive for filaggrin. The basement membrane protein laminin 332 and type 4 collagen were deposited on the interface area between the vascular bed and the epidermal cell sheet. Moreover, an electron microscopic image showed anchoring junctions and keratohyalin granules. These results show that we were able to produce native-like skin. Conclusions: We have succeeded in creating regenerative skin flap ex vivo that is similar to native skin, and this technique could be applied to create various tissues in the future. PMID:26180725

  12. Automated segmentation of in vivo and ex vivo mouse brain magnetic resonance images.

    PubMed

    Scheenstra, Alize E H; van de Ven, Rob C G; van der Weerd, Louise; van den Maagdenberg, Arn M J M; Dijkstra, Jouke; Reiber, Johan H C

    2009-01-01

    Segmentation of magnetic resonance imaging (MRI) data is required for many applications, such as the comparison of different structures or time points, and for annotation purposes. Currently, the gold standard for automated image segmentation is nonlinear atlas-based segmentation. However, these methods are either not sufficient or highly time consuming for mouse brains, owing to the low signal to noise ratio and low contrast between structures compared with other applications. We present a novel generic approach to reduce processing time for segmentation of various structures of mouse brains, in vivo and ex vivo. The segmentation consists of a rough affine registration to a template followed by a clustering approach to refine the rough segmentation near the edges. Compared with manual segmentations, the presented segmentation method has an average kappa index of 0.7 for 7 of 12 structures in in vivo MRI and 11 of 12 structures in ex vivo MRI. Furthermore, we found that these results were equal to the performance of a nonlinear segmentation method, but with the advantage of being 8 times faster. The presented automatic segmentation method is quick and intuitive and can be used for image registration, volume quantification of structures, and annotation. PMID:19344574

  13. Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry.

    PubMed

    Demange, Elise; Kassim, Yusra; Petit, Cyrille; Buquet, Catherine; Dulong, Virginie; Cerf, Didier Le; Buchonnet, Gérard; Vannier, Jean-Pierre

    2013-11-01

    Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so-called 'haematopoietic niche'. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long-term cultures of CD34⁺ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34⁺ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum-free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280-fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion. PMID:22473677

  14. Temperature distribution during RF ablation on ex vivo liver tissue: IR measurements and simulations

    NASA Astrophysics Data System (ADS)

    Macchi, Edoardo Gino; Gallati, Mario; Braschi, Giovanni; Cigada, Alfredo; Comolli, Lorenzo

    2014-09-01

    Radiofrequency thermal ablation is the first therapeutic option for the minimally invasive treatment of liver tumors. This medical procedure employs the Joule heat produced by a RF electromagnetic field to kill tumor cells. The outcome of the procedure is strongly affected by the temperature distribution near the RF applicator, however the measurement of this distribution, even in ex vivo experiments, is not straightforward since most traditional local temperature measurement techniques are not well-suited, due to both electromagnetic interferences and the sensor heat sink effect. Given the importance of the temperature field knowledge, in this paper special care was devoted to its measurement employing both infrared thermal imaging and NTC thermistors. Several RF ablation tests on ex vivo porcine liver tissue were carried out measuring the space-time evolution of temperature during the procedure (with spatial resolution ≤1 mm) and producing useful data for the design and the calibration of a numerical model. Electro-thermal numerical simulations of the experimental tests were performed using a mathematical model suitable for the heating phase of the procedure (up to 95 °C). The simulations results allowed to check the physical consistency of the measured data and suggested that a constant thermal conductivity is satisfactory for modeling the temperature evolution during RF ablation.

  15. Chemiluminescence response induced by mesenteric ischaemia/reperfusion: effect of antioxidative compounds ex vivo

    PubMed Central

    Nosál'ová, Viera; Sotníková, Ružena; Drábiková, Katarína; Fialová, Silvia; Košťálová, Daniela; Banášová, Silvia; Navarová, Jana

    2010-01-01

    Ischaemia and reperfusion (I/R) play an important role in human pathophysiology as they occur in many clinical conditions and are associated with high morbidity and mortality. Interruption of blood supply rapidly damages metabolically active tissues. Restoration of blood flow after a period of ischaemia may further worsen cell injury due to an increased formation of free radicals. The aim of our work was to assess macroscopically the extent of intestinal pathological changes caused by mesenteric I/R, and to study free radical production by luminol enhanced chemiluminescence (CL) of ileal samples. In further experiments, the antioxidative activity of the drugs tested was evaluated spectrophotometrically by the use of the DPPH radical. We studied the potential protective ex vivo effect of the plant origin compound arbutin as well as of the pyridoindole stobadine and its derivative SMe1EC2. I/R induced pronounced haemorrhagic intestinal injury accompanied by increase of myeloperoxidase (MPO) and N-acetyl-β-D-glucosaminidase (NAGA) activity. Compared to sham operated (control) rats, there was only a slight increase of CL response after I/R, probably in association with neutrophil increase, indicated by enhanced MPO activity. All compounds significantly reduced the peak values of CL responses of the ileal samples ex vivo, thus reducing the I/R induced increase of free radical production. The antioxidants studied showed a similar inhibitory effect on the CL response influenced by mesenteric I/R. If proved in vivo, these compounds would represent potentially useful therapeutic antioxidants. PMID:21217883

  16. Adrenergic Effect on Cytokine Release After Ex Vivo Healthy Volunteers' Whole Blood LPS Stimulation.

    PubMed

    Papandreou, Vasiliki; Kavrochorianou, Nadia; Katsoulas, Theodoros; Myrianthefs, Pavlos; Venetsanou, Kyriaki; Baltopoulos, George

    2016-06-01

    Catecholamines are molecules with immunomodulatory properties in health and disease. Several studies showed the effect of catecholamines when administered to restore hemodynamic stability in septic patients. This study investigates the effect of norepinephrine and dobutamine on whole blood cytokine release after ex vivo lipopolysaccharide (LPS) stimulation. Whole blood collected from healthy individuals was stimulated with LPS, in the presence of norepinephrine or dobutamine at different concentrations, with or without metoprolol, a β1 receptor antagonist. Cytokine measurement was performed in isolated cell culture supernatants with ELISA. Results are expressed as mean ± SEM and compared with Mann-Whitney rank-sum test. Both norepinephrine and dobutamine significantly reduced TNF-α and IL-6 production after ex vivo LPS stimulation of whole blood in a dose-dependent manner, and this effect was partially reversed by the presence of metoprolol. Norepinephrine and dobutamine reduce the LPS-induced production of pro-inflammatory cytokines, thus possibly contributing to altered balance between the inflammatory and anti-inflammatory responses, which are vital for a successful host response to severe disease, shock, and sepsis. PMID:27037808

  17. Ex vivo reconstruction of the donor renal artery in renal transplantation: a case-control study.

    PubMed

    McLoughlin, Louise C; Davis, Niall F; Dowling, Catherine M; Power, Richard E; Mohan, Ponnusamy; Hickey, David P; Smyth, Gordon P; Eng, Molly M P; Little, Dilly M

    2014-05-01

    Transplantation of renal allografts with anatomic variability or injured vasculature poses a challenge to the transplanting surgeon but can be salvaged for transplantation with ex vivo bench reconstruction of the vasculature. We investigated whether renal allograft function is impaired in these reconstructed allografts; compared to the donor-matched, un-reconstructed allograft. Reconstructed allografts were transplanted into 60 patients at our institution between 1986 and 2012. A control group was selected from the matched pair of the recipient in deceased donor transplantation. We found no significant difference in the overall graft and patient survival rates (P = 1.0, P = 0.178). Serum creatinine levels were not significantly higher in the study group at 1, 3 and 12 months postoperatively. There were two cases of vascular thrombosis in the study group that were not related to the ex vivo reconstruction. A significantly greater proportion of reconstructed patients were investigated with a colour duplex ultrasound postoperatively (0.007). Although we have demonstrated a higher index of suspicion of transplant failure in patients with a reconstructed allograft, this practice has proven to be a safe and useful technique with equivocal outcome when compared to normal grafts; increasing the organ pool available for transplantation. PMID:24851246

  18. Histopathology and ex vivo insulin secretion of pancreatic islets in gestational diabetes: A case report.

    PubMed

    Tancredi, Mariella; Marselli, Lorella; Lencioni, Cristina; Masini, Matilde; Bugliani, Marco; Suleiman, Mara; Masiello, Pellegrino; Boggi, Ugo; Filipponi, Franco; Dotta, Francesco; Marchetti, Piero; Di Cianni, Graziano

    2011-01-01

    Gestational diabetes (GD) results from insufficient endogenous insulin supply. No information is available on features of islet cells in human GD. Herein, we describe several properties of islets from a woman with GD. Immunohistochemical stainings and EM analyses were performed on pancreatic samples. Islet isolation was achieved by enzymatic dissociation and density gradient centrifugation. Ex vivo insulin secretion was studied in response to fuel secretagogues. Control islets were obtained from matched non-pregnant, non-diabetic women. Total insulin positive area was lower in GD, mainly due to the presence of smaller islets. β-cell apoptosis and the presence of Ki67 positive islet cells were similar in GD and controls, whereas the amount of insulin positive cells in or close to the ducts was decreased in GD. Ex vivo insulin secretion did not differ between GD and non-pregnant, non-diabetic islets. These findings suggest that in this case of human GD there might mainly be a defect of β-cell amount, not due to increased apoptosis, but possibly to insufficient regeneration. PMID:21765242

  19. Pharmacokinetics of buspirone as determined by ex vivo (/sup 3/H)-DPAT binding

    SciTech Connect

    Sethy, V.H.; Francis, J.W.

    1988-01-01

    Ex vivo (/sup 3/H)-8-hydroxy-2-(di-n-propylamino)-tetraline ((/sup 3/H)-DPAT) binding to the hippocampus has been utilized to determine the pharmacokinetic parameters of buspirone after i.v. and oral administration of this drug to rats. Intravenous buspirone rapidly penetrated the brain as demonstrated by a maximum inhibition of (/sup 3/H)-DPAT binding at 1 min. Elimination of drug from the brain was biphasic, with a first component half-life of 24.8 min and a second component half-life of 96 min. Oral buspirone at 3 times the i.v. dose produced less than one-third the maximum inhibition of (/sup 3/H)-DPAT binding compared to that observed with i.v. buspirone. The pharmacokinetic parameters of buspirone observed in the present study are in agreement with those reported previously. Thus, the ex vivo binding assay could be utilized to determine the bioavailability of the drug to the brain, and its duration of action. 20 references, 2 figures, 5 tables.

  20. Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells.

    PubMed

    Charville, Gregory W; Cheung, Tom H; Yoo, Bryan; Santos, Pauline J; Lee, Gordon K; Shrager, Joseph B; Rando, Thomas A

    2015-10-13

    Adult skeletal muscle stem cells, or satellite cells (SCs), regenerate functional muscle following transplantation into injured or diseased tissue. To gain insight into human SC (huSC) biology, we analyzed transcriptome dynamics by RNA sequencing of prospectively isolated quiescent and activated huSCs. This analysis indicated that huSCs differentiate and lose proliferative potential when maintained in high-mitogen conditions ex vivo. Further analysis of gene expression revealed that p38 MAPK acts in a transcriptional network underlying huSC self-renewal. Activation of p38 signaling correlated with huSC differentiation, while inhibition of p38 reversibly prevented differentiation, enabling expansion of huSCs. When transplanted, expanded huSCs differentiated to generate chimeric muscle and engrafted as SCs in the sublaminar niche with a greater frequency than freshly isolated cells or cells cultured without p38 inhibition. These studies indicate characteristics of the huSC transcriptome that promote expansion ex vivo to allow enhanced functional engraftment of a defined population of self-renewing huSCs. PMID:26344908

  1. The ex vivo purge of cancer cells using oncolytic viruses: recent advances and clinical implications

    PubMed Central

    Tsang, Jovian J; Atkins, Harold L

    2015-01-01

    Hematological malignancies are treated with intensive high-dose chemotherapy, with or without radiation. This is followed by hematopoietic stem cell (HSC) transplantation (HSCT) to rescue or reconstitute hematopoiesis damaged by the anticancer therapy. Autologous HSC grafts may contain cancer cells and purging could further improve treatment outcomes. Similarly, allogeneic HSCT may be improved by selectively purging alloreactive effector cells from the graft rather than wholesale immune cell depletion. Viral agents that selectively replicate in specific cell populations are being studied in experimental models of cancer and immunological diseases and have potential applications in the context of HSC graft engineering. This review describes preclinical studies involving oncolytic virus strains of adenovirus, herpes simplex virus type 1, myxoma virus, and reovirus as ex vivo purging agents for HSC grafts, as well as in vitro and in vivo experimental studies using oncolytic coxsackievirus, measles virus, parvovirus, vaccinia virus, and vesicular stomatitis virus to eradicate hematopoietic malignancies. Alternative ex vivo oncolytic virus strategies are also outlined that aim to reduce the risk of relapse following autologous HSCT and mitigate morbidity and mortality due to graft-versus-host disease in allogeneic HSCT. PMID:27512666

  2. Ex vivo evaluation of a microneedle array device for transdermal application.

    PubMed

    Indermun, Sunaina; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Modi, Girish; van Vuuren, Sandy; Luttge, Regina; Pillay, Viness

    2015-12-30

    A new approach of transdermal drug delivery is the use of microneedles. This promising technique offers the potential to be broadly used for drug administration as it enables the dramatic increase in permeation of medicaments across the stratum corneum. The potential of microneedles has evolved to spawn a plethora of potential transdermal applications. In order to advance the microneedle capabilities and possibly revolutionize advanced drug delivery, this study introduces a novel transdermal electro-modulated hydrogel-microneedle array (EMH-MNA) device composed of a nano-porous, embeddable ceramic microneedle array as well as an optimized EMH for the electro-responsive delivery of indomethacin through the skin. The ex vivo permeation as well as drug release experiments were performed on porcine skin tissue to ascertain the electro-responsive capabilities of the device. In addition, the microbial permeation ability of the microneedles across the viable epidermis in both microneedle-punctured skin as well as hypodermic needle-punctured skin was determined. Ex vivo evaluation of the EMH-MNA device across porcine skin demonstrated that without electro-stimulation, significantly less drug release was obtained (±0.4540mg) as compared to electro-stimulation (±2.93mg). PMID:26453791

  3. Activity ex vivo of cytotoxic drugs in patient samples of peritoneal carcinomatosis with special focus on colorectal cancer

    PubMed Central

    2013-01-01

    Background The optimal choice of cytotoxic drugs for intraperitoneal chemotherapy (IPC) in conjunction with cytoreductive surgery (CRS) for treatment of peritoneal carcinomatosis (PC) is poorly defined. We investigated drug sensitivity ex vivo in patient samples of various PC tumor types and correlated clinical outcome to drug sensitivity within the subset of PC from colorectal cancer (CRC). Methods PC tissue samples (n = 174) from mesothelioma, pseudomyxoma peritonei (PMP), ovarian cancer, CRC or appendix cancer were analyzed ex vivo for sensitivity to oxaliplatin, cisplatin, mitomycin C, melphalan, irinotecan, docetaxel, doxorubicin and 5-FU. Clinicopathological variables and outcome data were collected for the CRC subset. Results Mesothelioma and ovarian cancer were generally more drug sensitive than CRC, appendix cancer and PMP. Oxaliplatin showed the most favorable ratio between achievable IPC concentration and ex vivo drug sensitivity. Drug sensitivity in CRC varied considerably between individual samples. Ex vivo drug sensitivity did not obviously correlate to time-to-progression (TTP) in individual patients. Conclusions Drug-sensitivity varies considerably between PC diagnoses and individual patients arguing for individualized therapy in IPC rather than standard diagnosis-specific therapy. However, in the current paradigm of treatment according to diagnosis, oxaliplatin is seemingly the preferred drug for IPC from a drug sensitivity and concentration perspective. In the CRC subset, analysis of correlation between ex vivo drug sensitivity and TTP was inconclusive due to the heterogeneous nature of the data. PMID:24063788

  4. In vivo and ex vivo measurements: noninvasive assessment of alcoholic fatty liver using 1H-MR spectroscopy

    PubMed Central

    Keese, Daniel; Korkusuz, Hüdayi; Huebner, Frank; Namgaladze, Dmitry; Raschidi, Bahram; Vogl, Thomas J.

    2016-01-01

    PURPOSE We aimed to evaluate the ability of 1H-magnetic resonance spectroscopy (1H-MRS) to detect and quantify hepatic fat content in vivo and ex vivo in an experimental rat model of alcoholic fatty liver using histopathology, biochemistry, and laboratory analyses as reference. METHODS Alcoholic fatty liver was induced within 48 hours in 20 Lewis rats; 10 rats served as control. Intrahepatic fat content determined by 1H-MRS was expressed as the percent ratio of the lipid and water peaks and was correlated with intrahepatic fat content determined histologically and biochemically. Liver enzymes were measured in serum. RESULTS Fatty liver could be detected in vivo as well as ex vivo using 1H-MRS, in all 20 animals. Histologic analysis showed a fatty liver in 16 of 20 animals. Histology and 1H-MRS results were highly correlated (in vivo, r=0.93, P = 0.0005; ex vivo, r=0.92, P = 0.0006). Also a strong correlation was noted between in vivo 1H-MRS measurements and the fat content determined biochemically (r=0.96, P = 0.0003). Ex vivo results showed a similarly strong correlation between 1H-MRS and biochemistry (r=0.89, P = 0.0011). CONCLUSION 1H-MRS can be carried out in ex vivo models, as well as in vivo, to detect and quantify intrahepatic fat content in the acute fatty liver. PMID:26627137

  5. An Eye-adapted Beamforming for Axial B-scans Free from Crystalline Lens Aberration: In vitro and ex vivo Results with a 20 MHz Linear Array

    NASA Astrophysics Data System (ADS)

    Matéo, Tony; Mofid, Yassine; Grégoire, Jean-Marc; Ossant, Frédéric

    In ophtalmic ultrasonography, axial B-scans are seriously deteriorated owing to the presence of the crystalline lens. This strongly aberrating medium affects both spatial and contrast resolution and causes important distortions. To deal with this issue, an adapted beamforming (BF) has been developed and experimented with a 20 MHz linear array working with a custom US research scanner. The adapted BF computes focusing delays that compensate for crystalline phase aberration, including refraction effects. This BF was tested in vitro by imaging a wire phantom through an eye phantom consisting of a synthetic gelatin lens, shaped according to the unaccommodated state of an adult human crystalline lens, anatomically set up in an appropriate liquid (turpentine) to approach the in vivo velocity ratio. Both image quality and fidelity from the adapted BF were assessed and compared with conventional delay-and-sum BF over the aberrating medium. Results showed 2-fold improvement of the lateral resolution, greater sensitivity and 90% reduction of the spatial error (from 758 μm to 76 μm) with adapted BF compared to conventional BF. Finally, promising first ex vivo axial B-scans of a human eye are presented.

  6. Fluorophore-labeling of core-crosslinked polymeric micelles for multimodal in vivo and ex vivo optical imaging

    PubMed Central

    Shi, Yang; Kunjachan, Sijumon; Wu, Zhuojun; Gremse, Felix; Moeckel, Diana; van Zandvoort, Marc; Kiessling, Fabian; Storm, Gert; van Nostrum, Cornelus F.; Hennink, Wim E.; Lammers, Twan

    2015-01-01

    Aim To enable multimodal in vivo and ex vivo optical imaging of the biodistribution and tumor accumulation of core-crosslinked polymeric micelles (CCPM). Materials & Methods mPEG-b-p(HPMAm-Lac)-based polymeric micelles, core-crosslinked via cystamine and covalently labeled with two fluorophores (Dy-676/488) were synthesized. The CCPM were intravenously injected in CT26 tumor-bearing mice. Results Upon intravenous injection, the CCPM accumulated in CT26 tumors reasonably efficiently, with values reaching ~4 %ID at 24 hours. Ex vivo TPLSM confirmed efficient extravasation of the iCCPM out of tumor blood vessels and deep penetration into the tumor interstitium. Conclusions CCPM were labeled with multiple fluorophores, and they exemplify that combining different in vivo and ex vivo optical imaging techniques is highly useful for analyzing the biodistribution and tumor accumulation of nanomedicines. PMID:25929568

  7. Treatment Planning for Image-Guided Neuro-Vascular Interventions Using Patient-Specific 3D Printed Phantoms

    PubMed Central

    Russ, M.; O’Hara, R.; Setlur Nagesh, S.V.; Mokin, M.; Jimenez, C.; Siddiqui, A.; Bednarek, D.; Rudin, S.; Ionita, C.

    2015-01-01

    Minimally invasive endovascular image-guided interventions (EIGIs) are the preferred procedures for treatment of a wide range of vascular disorders. Despite benefits including reduced trauma and recovery time, EIGIs have their own challenges. Remote catheter actuation and challenging anatomical morphology may lead to erroneous endovascular device selections, delays or even complications such as vessel injury. EIGI planning using 3D phantoms would allow interventionists to become familiarized with the patient vessel anatomy by first performing the planned treatment on a phantom under standard operating protocols. In this study the optimal workflow to obtain such phantoms from 3D data for interventionist to practice on prior to an actual procedure was investigated. Patient-specific phantoms and phantoms presenting a wide range of challenging geometries were created. Computed Tomographic Angiography (CTA) data was uploaded into a Vitrea 3D station which allows segmentation and resulting stereo-lithographic files to be exported. The files were uploaded using processing software where preloaded vessel structures were included to create a closed-flow vasculature having structural support. The final file was printed, cleaned, connected to a flow loop and placed in an angiographic room for EIGI practice. Various Circle of Willis and cardiac arterial geometries were used. The phantoms were tested for ischemic stroke treatment, distal catheter navigation, aneurysm stenting and cardiac imaging under angiographic guidance. This method should allow for adjustments to treatment plans to be made before the patient is actually in the procedure room and enabling reduced risk of peri-operative complications or delays. PMID:26778878

  8. In Vitro and Ex Vivo Evaluations on Transdermal Delivery of the HIV Inhibitor IQP-0410

    PubMed Central

    Ham, Anthony S.; Lustig, William; Yang, Lu; Boczar, Ashlee; Buckheit, Karen W.; Buckheit Jr, Robert W.

    2013-01-01

    The aim of this study was to investigate the physicochemical and in vitro/ex vivo characteristics of the pyrmidinedione IQP-0410 formulated into transdermal films. IQP-0410 is a potent therapeutic anti-HIV nonnucleoside reverse transcriptase inhibitor that would be subjected to extensive first pass metabolism, through conventional oral administration. Therefore, IQP-0410 was formulated into ethyl cellulose/HPMC-based transdermal films via solvent casting. In mano evaluations were performed to evaluate gross physical characteristics. In vitro release studies were performed in both Franz cells and USP-4 dissolution vessels. Ex vivo release and permeability assays were performed on human epidermal tissue models, and the permeated IQP-0410 was collected for in vitro HIV-1 efficacy assays in CEM-SS cells and PBMCs. Film formulation D3 resulted in pliable, strong transdermal films that were loaded with 2% (w/w) IQP-0410. Composed of 60% (w/w) ethyl cellulose and 20% (w/w) HPMC, the films contained < 1.2% (w/w) of water and were hygroscopic resulting in significant swelling under humid conditions. The water permeable nature of the film resulted in complete in vitro dissolution and drug release in 26 hours. When applied to ex vivo epidermal tissues, the films were non-toxic to the tissue and also were non-toxic to HIV target cells used in the in vitro efficacy assays. Over a 3 day application, the films delivered IQP-0410 through the skin tissue at a zero-order rate of 0.94 ± 0.06 µg/cm2/hr with 134 ± 14.7 µM collected in the basal media. The delivered IQP-0410 resulted in in vitro EC50 values against HIV-1 of 2.56 ± 0.40 nM (CEM-SS) and 0.58 ± 0.03 nM (PBMC). The film formulation demonstrated no significant deviation from target values when packaged in foil pouches under standard and accelerated environmental conditions. It was concluded that the transdermal film formulation was a potentially viable method of administering IQP-0410 that warrants further development

  9. HLA-Ig Based Artificial Antigen Presenting Cells for Efficient ex vivo Expansion of Human CTL

    PubMed Central

    Chiu, Yen-Ling; Schneck, Jonathan P.; Oelke, Mathias

    2011-01-01

    CTL with optimal effector function play critical roles in mediating protection against various intracellular infections and cancer. However, individuals may exhibit suppressive immune microenvironment and, in contrast to activating CTL, their autologous antigen presenting cells may tend to tolerize or anergize antigen specific CTL. As a result, although still in the experimental phase, CTL-based adoptive immunotherapy has evolved to become a promising treatment for various diseases such as cancer and virus infections. In initial experiments ex vivo expanded CMV (cytomegalovirus) specific CTL have been used for treatment of CMV infection in immunocompromised allogeneic bone marrow transplant patients. While it is common to have life-threatening CMV viremia in these patients, none of the patients receiving expanded CTL develop CMV related illness, implying the anti-CMV immunity is established by the adoptively transferred CTL1. Promising results have also been observed for melanoma and may be extended to other types of cancer2. While there are many ways to ex vivo stimulate and expand human CTL, current approaches are restricted by the cost and technical limitations. For example, the current gold standard is based on the use of autologous DC. This requires each patient to donate a significant number of leukocytes and is also very expensive and laborious. Moreover, detailed in vitro characterization of DC expanded CTL has revealed that these have only suboptimal effector function 3. Here we present a highly efficient aAPC based system for ex vivo expansion of human CMV specific CTL for adoptive immunotherapy (Figure 1). The aAPC were made by coupling cell sized magnetic beads with human HLA-A2-Ig dimer and anti-CD28mAb4. Once aAPC are made, they can be loaded with various peptides of interest, and remain functional for months. In this report, aAPC were loaded with a dominant peptide from CMV, pp65 (NLVPMVATV). After culturing purified human CD8+ CTL from a healthy

  10. Ho:YAG laser irradiation in blood vessel as a vasodilator: ex vivo study

    NASA Astrophysics Data System (ADS)

    Nakatani, E.; Iwasaki, T.; Kaneko, K.; Shimazaki, N.; Arai, T.

    2007-02-01

    We studied Ho:YAG laser irradiation in blood vessel as a vasodilator ex vivo. We thought that the Ho:YAG laser-induced bubble expansion might be able to dilate the vessel because we found the vessel wall expansion after the Ho:YAG laser irradiation, that is steady deformation, in the vessel ex vivo. There have been many reports regarding to the Ho:YAG laser irradiation in the vessel. Most of studies concentrated on the interaction between Ho:YAG laser irradiation and vessel wall to investigate side effect on Ho:YAG laser angioplasty. We proposed to use the Ho:YAG laser-induced bubble expansion as a vasodilator. We studied vasodilation effect of the Ho:YAG laser-induced bubble ex vivo. The flash lamp excited Ho:YAG laser surgical unit (IH102, NIIC, Japan) (λ=2.1μm) was used. The laser energy was delivered by a silica glass fiber (outer diameter: 1000μm, core diameter: 600μm). The laser-induced bubble was generated in the extracted fresh porcine carotid artery with the warmed saline perfusion. The laser energy at the fiber tip was ranging from 170-1300mJ per pulse. Number of the laser irradiation was ranged from 20pulses to 100pulses. The outer diameter of the vessel was observed. To examine the change in mechanical properties of the vessel wall, the stress-strain curve of the laser-irradiated vessel was measured. Birefringence observation and microscopic observation of staining specimen were performed. When the laser energy was set to 1300mJ per pulse, the outer diameter of the vessel after the laser irradiation was expanded by 1.4 times comparing with that of before the laser irradiation and the dilatation effect was kept even at 10minutes after the irradiation. The elasticity modulus of the artery by collagen was changed by the laser irradiation. In the polarized microscopic observation, the brightness of the intimal side of the vessel is increased comparing with that of the normal. We think this brightness increasing may be attributed to birefringence change

  11. Late Effects of Heavy Ion Irradiation on Ex Vivo Osteoblastogenesis and Cancellous Bone Microarchitecture

    NASA Technical Reports Server (NTRS)

    Tran, Luan Hoang; Alwood, Joshua; Kumar, Akhilesh; Limoli, C. L.; Globus, Ruth

    2012-01-01

    Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even after return to Earth. We hypothesize that heavy ion irradiation, a component of Galactic Cosmic Radiation, damages osteoblast progenitors and may contribute to bone loss during long duration space travel beyond the protection of the Earth's magnetosphere. Male, 16 week old C57BL6/J mice were exposed to high LET (56 Fe, 600MeV) radiation using either low (5 or 10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 3 - 4, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 micron pixel size) and marrow cell redox assessed using membrane permeable, free radical sensitive fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo, then mineralized nodule formation quantified by imaging and gene expression analyzed by RT PCR. Interestingly, 3 - 4 days post exposure, fluorogenic dyes that reflect cytoplasmic generation of reactive nitrogen/oxygen species (DAF FM Diacetate or CM H2DCFDA) revealed irradiation (50cGy) reduced free radical generation (20-45%) compared to sham irradiated controls. Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation (MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive oxygen/nitrogen levels remained lower(24%) relative to sham irradiated controls. After one month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy) compared to controls. High dose irradiation (200cGy) up regulated expression of a late osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure or ex vivo nodule formation, but did down regulate iNOS by 0.54 - 0.58 fold

  12. Evaluation of hybrid algorithm for analysis of scattered light using ex vivo nuclear morphology measurements of cervical epithelium.

    PubMed

    Ho, Derek; Drake, Tyler K; Bentley, Rex C; Valea, Fidel A; Wax, Adam

    2015-08-01

    We evaluate a new hybrid algorithm for determining nuclear morphology using angle-resolved low coherence interferometry (a/LCI) measurements in ex vivo cervical tissue. The algorithm combines Mie theory based and continuous wavelet transform inverse light scattering analysis. The hybrid algorithm was validated and compared to traditional Mie theory based analysis using an ex vivo tissue data set. The hybrid algorithm achieved 100% agreement with pathology in distinguishing dysplastic and non-dysplastic biopsy sites in the pilot study. Significantly, the new algorithm performed over four times faster than traditional Mie theory based analysis. PMID:26309741

  13. Use of Extended-Criteria Lungs on a Lobe-by-Lobe Basis Through Ex Vivo Lung Perfusion Assessment.

    PubMed

    Miyoshi, Kentaroh; Oto, Takahiro; Konishi, Yusuke; Hirano, Yutaka; Okada, Masanori; Iga, Norichika; Hirayama, Shin; Sugimoto, Seiichiro; Yamane, Masaomi; Kobayashi, Motomu; Miyoshi, Shinichiro

    2015-01-01

    Initially rejected and extended-criteria lungs were partially used through an ex vivo lung perfusion (EVLP) assessment that was first clinically applied in Asia. The truly injured lobe (left lower lobe) was identified during 89-minute normothermic EVLP and was excised, and the remaining lobes were successfully transplanted into a patient with lymphangioleiomyomatosis. The lung lobes showed heterogeneous changes on the ex vivo rig, and a brief duration of EVLP helped differentiate lung quality on a lobe-by-lobe basis. PMID:25952220

  14. Mind-body interventions for treatment of phantom limb pain in persons with amputation.

    PubMed

    Moura, Vera Lucia; Faurot, Keturah R; Gaylord, Susan A; Mann, J Douglas; Sill, Morgan; Lynch, Chanee; Lee, Michael Y

    2012-08-01

    Phantom limb pain (PLP) is a significant source of chronic pain in most persons with amputation at some time in their clinical course. Pharmacologic therapies for this condition are often only moderately effective and may produce unwanted adverse effects. There is growing empirical evidence of the therapeutic effectiveness of mind-body therapies for the relief of chronic pain; therefore, an exploration of their role in relieving amputation-related chronic pain is warranted. We undertook a focused literature review on mind-body interventions for patients with amputation who experience PLP. Because of study heterogeneity, only descriptive presentations of the studies are presented. Only studies of hypnosis, imagery, and biofeedback, including visual mirror feedback, were found; studies on meditation, yoga, and tai chi/qigong were missing from the literature. Few studies of specific mind-body therapies were dedicated to management of PLP, with the exception of mirror visual therapy. Overall, studies were largely exploratory and reflect considerable variability in the application of mind-body techniques, making definitive conclusions inadvisable. Nevertheless, the weight of existing findings indicates that a mind-body approach to PLP pain management is promising and that specific methods may offer either temporary or long-term relief, either alone or in combination with conventional therapies. The authors discuss the potential for usefulness of specific mind-body therapies and the relevance of their mechanisms of action to those of PLP, including targeting cortical reorganization, autonomic nervous system deregulation, stress management, coping ability, and quality-of-life. The authors recommend more and better quality research exploring the efficacy and mechanisms of action. PMID:22286895

  15. Fusing in vivo and ex vivo NMR sources of information for brain tumor classification

    NASA Astrophysics Data System (ADS)

    Croitor-Sava, A. R.; Martinez-Bisbal, M. C.; Laudadio, T.; Piquer, J.; Celda, B.; Heerschap, A.; Sima, D. M.; Van Huffel, S.

    2011-11-01

    In this study we classify short echo-time brain magnetic resonance spectroscopic imaging (MRSI) data by applying a model-based canonical correlation analyses algorithm and by using, as prior knowledge, multimodal sources of information coming from high-resolution magic angle spinning (HR-MAS), MRSI and magnetic resonance imaging. The potential and limitations of fusing in vivo and ex vivo nuclear magnetic resonance sources to detect brain tumors is investigated. We present various modalities for multimodal data fusion, study the effect and the impact of using multimodal information for classifying MRSI brain glial tumors data and analyze which parameters influence the classification results by means of extensive simulation and in vivo studies. Special attention is drawn to the possibility of considering HR-MAS data as a complementary dataset when dealing with a lack of MRSI data needed to build a classifier. Results show that HR-MAS information can have added value in the process of classifying MRSI data.

  16. Release of rosmarinic acid from semisolid formulations and its penetration through human skin ex vivo.

    PubMed

    Stelmakienė, Ada; Ramanauskienė, Kristina; Briedis, Vitalis

    2015-06-01

    The aim of this study was to evaluate the release of rosmarinic acid (RA) from the experimental topical formulations with the Melissa officinalis L. extract and to evaluate its penetration through undamaged human skin ex vivo. The results of the in vitro release study showed that higher amounts of RA were released from the emulsion vehicle when lemon balm extract was added in its dry form. An inverse correlation was detected between the released amount of RA and the consistency index of the formulation. Different penetration of RA into the skin may be influenced by the characteristics of the vehicle as well as by the form of the extract. The results of penetration assessment showed that the intensity of RA penetration was influenced by its lipophilic properties: RA was accumulating in the epidermis, while the dermis served as a barrier, impeding its deeper penetration. PMID:26011936

  17. Isolation of an in vitro and ex vivo antiradical melanoidin from roasted barley.

    PubMed

    Papetti, Adele; Daglia, Maria; Aceti, Camilla; Quaglia, Milena; Gregotti, Cesarina; Gazzani, Gabriella

    2006-02-22

    The antiradical properties of water-soluble components of both natural and roasted barley were determined in vitro, by means of DPPH* assay and the linoleic acid-beta-carotene system, and ex vivo, in rat liver hepatocyte microsomes against lipid peroxidation induced by CCl4. The results show the occurrence in natural barley of weak antioxidant components. These are able to react against low reactive peroxyl radicals, but offer little protection against stable DPPH radicals deriving from peroxidation in microsomal lipids. Conversely, roasted barley yielded strong antioxidant components that are able to efficiently scavenge free radicals in any system used. The results show that the barley grain roasting process induces the formation of soluble Maillard reaction products with powerful antiradical activity. From roasted barley solution (barley coffee) was isolated a brown high molecular mass melanoidinic component, resistant to acidic hydrolysis, that is responsible for most of the barley coffee antioxidant activity in the biosystem. PMID:16478238

  18. Ex vivo Characterization of Blast Wave Impact and Spinal Cord Tissue Deformation

    NASA Astrophysics Data System (ADS)

    Chen, Jun; Gao, Jian; Connell, Sean; Shi, Riyi

    2010-11-01

    Primary blast injury on central nervous system is responsible for many of the war related casualties and mortalities. An ex vivo model system is developed to introduce a blast wave, generated from a shock tube, directly to spinal cord tissue sample. A high-speed shadowgraph system is utilized to visualize the development of the blast wave and its interaction with tissue sample. Surface deformation of the tissue sample is also measured for the analysis of internal stress and possible injury occurred within the tissue sample. Understanding the temporal development of the blast-tissue interaction provides valuable input for modeling blast-induced neurotrauma. Tracking the sample surface deformation as a function of time provides realistic boundary conditions for numerical simulation of injury process.

  19. [INVITED] Time reversal optical tomography: Detecting and locating tumors in an ex vivo model human breast

    NASA Astrophysics Data System (ADS)

    Wu, Binlin; Alrubaiee, Mohammad; Gayen, S. K.

    2016-03-01

    Time reversal optical tomography (TROT), a recently introduced diffuse optical imaging approach, is used to detect, locate, and obtain cross-section images of tumors inside a "model human breast." The model cancerous breast is assembled as a semi-cylindrical slab of uniform thickness using ex vivo human breast tissues with two pieces of tumors embedded in it. The experimental arrangement used a 750-nm light beam from a Ti:sapphire laser to illuminate an end face (source plane) of the sample in a multi-source probing scheme. A multi-detector signal acquisition scheme measured transmitted light intensity distribution on the other end face (detector plane). The perturbations in light intensity distribution in the detector plane were analyzed using TROT to obtain locations of the tumor pieces in three dimensions and estimate their cross sections. The estimated locations and dimensions of targets are in good agreement with the results of a corroborating magnetic resonance imaging experiment.

  20. Ex Vivo Lung Perfusion – State of the Art in Lung Donor Pool Expansion

    PubMed Central

    Popov, Aron-Frederik; Sabashnikov, Anton; Patil, Nikhil P.; Zeriouh, Mohamed; Mohite, Prashant N.; Zych, Bartlomiej; Saez, Diana Garcia; Schmack, Bastian; Ruhparwar, Arjang; Dohmen, Pascal M.; Karck, Matthias; Simon, Andre R.; Weymann, Alexander

    2015-01-01

    Lung transplantation remains the gold standard for patients with end-stage lung disease. Nevertheless, the number of suitable donor lungs for the increasing number of patients on the waiting list necessitates alternative tools to expand the lung donor pool. Modern preservation and lung assessment techniques could contribute to improved function in previously rejected lungs. Ex vivo lung perfusion (EVLP) already demonstrated its value in identification of transplantable grafts from the higher risk donor pool. Moreover, lungs from EVLP did not show significantly different postoperative results compared to standard criteria lungs. This could be explained by the reduction of the ischemia-reperfusion injury through EVLP application. The aim of this article is to review technical characteristics and the growing clinical EVLP experience with special attention to EVLP application for donation after cardiac death (DCD) lungs. PMID:25644463

  1. Peptides that anneal to natural collagen in vitro and ex vivo

    PubMed Central

    Chattopadhyay, Sayani; Murphy, Christopher J.; McAnulty, Jonathan F.

    2012-01-01

    Collagen comprises ¼ of the protein in humans and ¾ of the dry weight of human skin. Here, we implement recent discoveries about the structure and stability of the collagen triple helix to design new chemical modalities that anchor to natural collagen. The key components are collagen mimetic peptides (CMPs) that are incapable of self-assembly into homotrimeric triple helices, but are able to anneal spontaneously to natural collagen. We show that such CMPs containing 4-fluoroproline residues, in particular, bind tightly to mammalian collagen in vitro and to a mouse wound ex vivo. These synthetic peptides, coupled to dyes or growth factors, could herald a new era in assessing or treating wounds. PMID:22522497

  2. Efficient measurement of total tumor microvascularity ex vivo using a mathematical model to optimize volume subsampling

    PubMed Central

    Spring, Bryan Q.; Palanisami, Akilan; Zheng, Lei Zak; Blatt, Amy E.; Bryan Sears, R.

    2013-01-01

    Abstract. We introduce immunofluorescence and automated image processing protocols for serial tumor sections to objectively and efficiently quantify tumor microvasculature following antivascular therapy. To determine the trade-off between tumor subsampling and throughput versus microvessel quantification accuracy, we provide a mathematical model that accounts for tumor-specific vascular heterogeneity. This mathematical model can be applied broadly to define tumor volume samplings needed to reach statistical significance, depending on the biomarker in question and the number of subjects. Here, we demonstrate these concepts for tumor microvessel density and total microvascularity (TMV) quantification in whole pancreatic ductal adenocarcinoma tumors ex vivo. The results suggest that TMV is a more sensitive biomarker for detecting reductions in tumor vasculature following antivascular treatment. TMV imaging is a broadly accessible technique that offers robust assessment of antivascular therapies, and it offers promise as a tool for developing high-throughput assays to quantify treatment-induced microvascular alterations for therapeutic screening and development.

  3. Dependence of ultrasound echo decorrelation on local tissue temperature during ex vivo radiofrequency ablation

    NASA Astrophysics Data System (ADS)

    Subramanian, Swetha; Schmidt, Daniel T.; Rao, Marepalli B.; Mast, T. Douglas

    2016-03-01

    This study investigates echo decorrelation imaging, an ultrasound method for thermal ablation monitoring. The effect of tissue temperature on the mapped echo decorrelation parameter was assessed in radiofrequency ablation experiments performed on ex vivo bovine liver tissue. Echo decorrelation maps were compared with corresponding tissue temperatures simulated using the finite element method. For both echo decorrelation imaging and integrated backscatter imaging, the mapped tissue parameters correlated significantly but weakly with local tissue temperature. Receiver operating characteristic (ROC) curves were used to assess the ability of echo decorrelation and integrated backscatter to predict tissue temperature greater than 40, 60, and 80 °C. Significantly higher area under the ROC curve (AUROC) values were obtained for prediction of tissue temperatures greater than 40, 60, and 80 ° C using echo decorrelation imaging (AUROC =0.871,~0.948 and 0.966) compared to integrated backscatter imaging (AUROC =0.865,~0.877 and 0.832).

  4. Ex vivo method for high resolution imaging of cilia motility in rodent airway epithelia.

    PubMed

    Francis, Richard; Lo, Cecilia

    2013-01-01

    An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy. PMID:23963287

  5. Ex Vivo 3D Diffusion Tensor Imaging and Quantification of Cardiac Laminar Structure

    PubMed Central

    Helm, Patrick A.; Tseng, Hsiang-Jer; Younes, Laurent; McVeigh, Elliot R.; Winslow, Raimond L.

    2007-01-01

    A three-dimensional (3D) diffusion-weighted imaging (DWI) method for measuring cardiac fiber structure at high spatial resolution is presented. The method was applied to the ex vivo reconstruction of the fiber architecture of seven canine hearts. A novel hypothesis-testing method was developed and used to show that distinct populations of secondary and tertiary eigenvalues may be distinguished at reasonable confidence levels (P ≤ 0.01) within the canine ventricle. Fiber inclination and sheet angles are reported as a function of transmural depth through the anterior, lateral, and posterior left ventricle (LV) free wall. Within anisotropic regions, two consistent and dominant orientations were identified, supporting published results from histological studies and providing strong evidence that the tertiary eigenvector of the diffusion tensor (DT) defines the sheet normal. PMID:16149057

  6. Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants

    PubMed Central

    Kerschensteiner, Martin; Reuter, Miriam S; Lichtman, Jeff W; Misgeld, Thomas

    2008-01-01

    We provide a protocol that describes an explant system that allows the dynamics of motor axons to be imaged. This method is based on nerve–muscle explants prepared from the triangularis sterni muscle of mice, a thin muscle that covers the inside of the thorax. These explants, which can be maintained alive for several hours, contain long stretches of peripheral motor axons including their terminal arborizations and neuromuscular junctions. Explants can be prepared from transgenic mouse lines that express fluorescent proteins in neurons or glial cells, which enables direct visualization of their cellular and subcellular morphology by fluorescence microscopy. Time-lapse imaging then provides a convenient and reliable approach to follow the dynamic behavior of motor axons, their surrounding glial cells and their intracellular organelles with high temporal and spatial resolution. Triangularis sterni explants can be prepared in 15 min, imaged ex vivo for several hours and processed for immunohistochemistry in about 2 h. PMID:18833201

  7. Percutaneous dilatational tracheostomy using a tracheoscopic ventilation tube in an experimental ex vivo animal model.

    PubMed

    Fiorelli, A F; Ferraro, F F; Milione, R M; Scarumuzzi, R S; Imitazione, P I; Marulli, L M; Orsini, A O; Santini, M S

    2016-05-01

    The ETView® tube is a standard endotracheal tube with an embedded miniature video camera that permits real-time video imaging of the tracheal lumen. We evaluated its use when performing percutaneous dilatational tracheostomy (PDT) in an ex vivo animal model. The model consisted of a pig larynyx and trachea. The ETView tube was used as an alternative to bronchoscopy, to see all manoeuvres of PDT in real time. At the end of the PDT, operative time and any complications such as trauma to the cricoid cartilage, tracheal wall or tube cuff were assessed. Nine PDT procedures were performed by two experienced operators. The mean operative time was 7.1 ± 0.9 minutes. No complications were observed. Our study supports the use of the ETView tube as a suitable alternative to bronchoscopy when performing PDT, although clinical studies are required to confirm our experimental results. . PMID:27246937

  8. Ex vivo lung perfusion in clinical lung transplantation--state of the art.

    PubMed

    Andreasson, Anders S I; Dark, John H; Fisher, Andrew J

    2014-11-01

    Ex vivo lung perfusion (EVLP) has emerged as a new technique for assessing and potentially reconditioning human donor lungs previously unacceptable for clinical transplantation with the potential to dramatically push the limits of organ acceptability. With the recent introduction of portable EVLP, a new era in lung preservation may be upon us with the opportunity to also limit organ ischaemic times and potentially improve the outcome of donor lungs already deemed acceptable for transplantation. It took over half a century for the technique to evolve from basic theory to semi-automated circuits fit for clinical use that are now rapidly being adopted in transplant centres across the globe. With this field in constant evolution and many unanswered questions remaining, our review serves as an update on the state of the art of EVLP in clinical lung transplantation. PMID:25061215

  9. Ex vivo lung perfusion - state of the art in lung donor pool expansion.

    PubMed

    Popov, Aron-Frederik; Sabashnikov, Anton; Patil, Nikhil P; Zeriouh, Mohamed; Mohite, Prashant N; Zych, Bartlomiej; Saez, Diana Garcia; Schmack, Bastian; Ruhparwar, Arjang; Dohmen, Pascal M; Karck, Matthias; Simon, Andre R; Weymann, Alexander

    2015-01-01

    Lung transplantation remains the gold standard for patients with end-stage lung disease. Nevertheless, the number of suitable donor lungs for the increasing number of patients on the waiting list necessitates alternative tools to expand the lung donor pool. Modern preservation and lung assessment techniques could contribute to improved function in previously rejected lungs. Ex vivo lung perfusion (EVLP) already demonstrated its value in identification of transplantable grafts from the higher risk donor pool. Moreover, lungs from EVLP did not show significantly different postoperative results compared to standard criteria lungs. This could be explained by the reduction of the ischemia-reperfusion injury through EVLP application. The aim of this article is to review technical characteristics and the growing clinical EVLP experience with special attention to EVLP application for donation after cardiac death (DCD) lungs. PMID:25644463

  10. Altered Immunogenicity of Donor Lungs via Removal of Passenger Leukocytes Using Ex Vivo Lung Perfusion.

    PubMed

    Stone, J P; Critchley, W R; Major, T; Rajan, G; Risnes, I; Scott, H; Liao, Q; Wohlfart, B; Sjöberg, T; Yonan, N; Steen, S; Fildes, J E

    2016-01-01

    Passenger leukocyte transfer from the donor lung to the recipient is intrinsically involved in acute rejection. Direct presentation of alloantigen expressed on donor leukocytes is recognized by recipient T cells, promoting acute cellular rejection. We utilized ex vivo lung perfusion (EVLP) to study passenger leukocyte migration from donor lungs into the recipient and to evaluate the effects of donor leukocyte depletion prior to transplantation. For this purpose, female pigs received male left lungs either following 3 h of EVLP or retrieved using standard protocols. Recipients were monitored for 24 h and sequential samples were collected. EVLP-reduced donor leukocyte transfer into the recipient and migration to recipient lymph nodes was markedly reduced. Recipient T cell infiltration of the donor lung was significantly diminished via EVLP. Donor leukocyte removal during EVLP reduces direct allorecognition and T cell priming, diminishing recipient T cell infiltration, the hallmark of acute rejection. PMID:26366523

  11. Animal models of ex vivo lung perfusion as a platform for transplantation research

    PubMed Central

    Nelson, Kevin; Bobba, Christopher; Ghadiali, Samir; Jr, Don Hayes; Black, Sylvester M; Whitson, Bryan A

    2014-01-01

    Ex vivo lung perfusion (EVLP) is a powerful experimental model for isolated lung research. EVLP allows for the lungs to be manipulated and characterized in an external environment so that the effect of specific ventilation/perfusion variables can be studied independent of other confounding physiologic contributions. At the same time, EVLP allows for normal organ level function and real-time monitoring of pulmonary physiology and mechanics. As a result, this technique provides unique advantages over in vivo and in vitro models. Small and large animal models of EVLP have been developed and each of these models has their strengths and weaknesses. In this manuscript, we provide insight into the relative strengths of each model and describe how the development of advanced EVLP protocols is leading to a novel experimental platform that can be used to answer critical questions in pulmonary physiology and transplant medicine. PMID:24977117

  12. Animal models of ex vivo lung perfusion as a platform for transplantation research.

    PubMed

    Nelson, Kevin; Bobba, Christopher; Ghadiali, Samir; Hayes, Don; Black, Sylvester M; Whitson, Bryan A

    2014-05-20

    Ex vivo lung perfusion (EVLP) is a powerful experimental model for isolated lung research. EVLP allows for the lungs to be manipulated and characterized in an external environment so that the effect of specific ventilation/perfusion variables can be studied independent of other confounding physiologic contributions. At the same time, EVLP allows for normal organ level function and real-time monitoring of pulmonary physiology and mechanics. As a result, this technique provides unique advantages over in vivo and in vitro models. Small and large animal models of EVLP have been developed and each of these models has their strengths and weaknesses. In this manuscript, we provide insight into the relative strengths of each model and describe how the development of advanced EVLP protocols is leading to a novel experimental platform that can be used to answer critical questions in pulmonary physiology and transplant medicine. PMID:24977117

  13. Surveying the Delivery Methods of CRISPR/Cas9 for ex vivo Mammalian Cell Engineering.

    PubMed

    Kelton, William J; Pesch, Theresa; Matile, Stefan; Reddy, Sai T

    2016-01-01

    The simplicity of the CRISPR/Cas9 technology has been transformative in making targeted genome editing accessible for laboratories around the world. However, due to the sheer volume of literature generated in the past five years, determining the best format and delivery method of CRISPR/Cas9 components can be challenging. Here, we provide a brief overview of the progress that has been made in the ex vivo genome editing of mammalian cells and summarize the key advances made for improving efficiency and delivery of CRISPR/Cas9 in DNA, RNA, and protein form. In particular, we highlight the delivery of Cas9 components to human cells for advanced genome editing applications such as large gene insertion. PMID:27363374

  14. Corneal injury to ex vivo eyes exposed to a 3.8-micron laser

    NASA Astrophysics Data System (ADS)

    Fyffe, James G.; Randolph, Donald Q.; Winston, Golda C. H.; Johnson, Thomas E.

    2005-04-01

    As a consequence of the enormous expansion of laser use in medicine, industry and research, specific safety standards must be developed that appropriately address eye protection. The purpose of this study is to establish injury thresholds to the cornea for 3.8 micron 8 microsecond laser light pulses and to investigate a possible replacement model to live animal testing. Previous studies of pulsed energy absorption at 3.8 microns were performed using rhesus monkey cornea and were at pulse durations two orders of magnitude different than the 8 microsecond pulses used in this study. Ex-vivo pig eyes were exposed at varying energies and evaluated to establish the statistical threshold for corneal damage. Histology was used to determine the extent of damage to the cornea. It is expected that the results will be used to assist in the establishment of safety standards for laser use and offer an alternative to future animal use in establishment of safety standards.

  15. Ex vivo evaluation of the percutaneous penetration of proanthocyanidin extracts from Guazuma ulmifolia using photoacoustic spectroscopy.

    PubMed

    Rocha, J C B; Pedrochi, F; Hernandes, L; de Mello, J C P; Baesso, M L

    2007-03-21

    In this work photoacoustic spectroscopy has been applied to determine ex vivo the percutaneous penetration of proanthocyanidins present in extracts obtained from Guazuma ulmifolia, in rats. Lotion formulations containing 0.0663 mg of procyanidin B2 day(-1)animal(-1) were topically applied during 7, 10 and 13 days in each group of the animals. After the end of treatment the animals were killed, the skin dissected to remove the basal content, and the measurements were carried out as a function of the period of time of treatment. The results showed that despite the very low concentration of the active principle (procyanidin B2) in the lotion, the photoacoustic method was able to show the presence of optical absorption bands from this substance in the dermis region, evidencing once again that this method may be useful for studies of topically applied formulations of interest in the pharmacokinetic area. PMID:17386764

  16. Ex vivo discrimination between normal and pathological tissues in human breast surgical biopsies using bioimpedance spectroscopy.

    PubMed

    Chauveau, N; Hamzaoui, L; Rochaix, P; Rigaud, B; Voigt, J J; Morucci, J P

    1999-04-20

    Ex vivo bioimpedance data measured on normal and cancerous female breast tissues are reported. They clearly show that the electrical properties of normal tissues, surrounding tissues, and carcinoma are different. These differences lie in the conductivity, in the characteristic frequency (frequency of the maximum of the imaginary part of the bioimpedance), and also in the shape of the Bode plots. Modeling using an R-S-Zcpe model is reported as well as indexes extracted from the real and imaginary parts of the bioimpedance. Even if a classification of the different types of tissues remains a difficult task and leads to much less precise diagnosis than microscopic examination, the electrical behavior of mammary tissue could be used to develop a noninvasive technique for early breast cancer detection. PMID:10372148

  17. Multipurpose nonlinear optical imaging system for in vivo and ex vivo multimodal histology

    PubMed Central

    Weinigel, Martin; Breunig, Hans Georg; Uchugonova, Aisada; König, Karsten

    2015-01-01

    Abstract. We report on a flexible multipurpose nonlinear microscopic imaging system based on a femtosecond excitation source and a photonic crystal fiber with multiple miniaturized time-correlated single-photon counting detectors. The system provides the simultaneous acquisition of e.g., two-photon autofluorescence, second-harmonic generation, and coherent anti-Stokes Raman scattering images. Its flexible scan head permits ex vivo biological imaging with subcellular resolution such as rapid biopsy examination during surgery as well as imaging on small as well as large animals. Above all, such an arrangement perfectly matches the needs for the clinical investigation of human skin in vivo where knowledge about the distribution of endogenous fluorophores, second-harmonic generation–active collagen as well as nonfluorescent lipids is of high interest. PMID:26158089

  18. Dependence of ultrasound echo decorrelation on local tissue temperature during ex vivo radiofrequency ablation.

    PubMed

    Subramanian, Swetha; Schmidt, Daniel T; Rao, Marepalli B; Mast, T Douglas

    2016-03-21

    This study investigates echo decorrelation imaging, an ultrasound method for thermal ablation monitoring. The effect of tissue temperature on the mapped echo decorrelation parameter was assessed in radiofrequency ablation experiments performed on ex vivo bovine liver tissue. Echo decorrelation maps were compared with corresponding tissue temperatures simulated using the finite element method. For both echo decorrelation imaging and integrated backscatter imaging, the mapped tissue parameters correlated significantly but weakly with local tissue temperature. Receiver operating characteristic (ROC) curves were used to assess the ability of echo decorrelation and integrated backscatter to predict tissue temperature greater than 40, 60, and 80 °C. Significantly higher area under the ROC curve (AUROC) values were obtained for prediction of tissue temperatures greater than 40, 60, and 80 ° C using echo decorrelation imaging (AUROC [Formula: see text] and 0.966) compared to integrated backscatter imaging (AUROC [Formula: see text] and 0.832). PMID:26943026

  19. Gene therapy approaches against cancer using in vivo and ex vivo gene transfer of interleukin-12.

    PubMed

    Hernandez-Alcoceba, Ruben; Poutou, Joanna; Ballesteros-Briones, María Cristina; Smerdou, Cristian

    2016-02-01

    IL-12 is an immunostimulatory cytokine with strong antitumor properties. Systemic administration of IL-12 in cancer patients led to severe toxic effects, prompting the development of gene therapy vectors able to express this cytokine locally in tumors. Both nonviral and viral vectors have demonstrated a high antitumor efficacy in preclinical tumor models. Some of these vectors, including DNA electroporation, adenovirus and ex vivo transduced dendritic cells, were tested in patients, showing low toxicity and moderate antitumor efficacy. IL-12 activity can be potentiated by molecules with immunostimulatory, antiangiogenic or cytotoxic activity. These combination therapies are of clinical interest because they could lower the threshold for IL-12 efficacy, increasing the therapeutic potential of gene therapy and preventing the toxicity mediated by this cytokine. PMID:26786809

  20. Ex vivo label-free microscopy of head and neck cancer patient tissues

    NASA Astrophysics Data System (ADS)

    Shah, Amy T.; Skala, Melissa C.

    2015-03-01

    Standard methods to characterize patient tissue rely on histology. This technique provides only anatomical information, so complementary imaging methods could provide beneficial phenotypic information. Cancer cells exhibit altered metabolism, and metabolic imaging could be applied to better understand cancer tissue. This study applies redox ratio, fluorescence lifetime, and second harmonic generation (SHG) imaging to ex vivo tissue from head and neck cancer patients. This high-resolution imaging technique has unique advantages of utilizing intrinsic tissue contrast, which eliminates the need for sample processing or staining, and multiphoton microscopy, which provides depth sectioning in intact tissue. This study demonstrates feasibility of these measurements in patient tissue from multiple anatomical sites and carcinoma types of head and neck cancer.

  1. CD80 antigen expression as a predictor of ex vivo chemosensitivity in chronic lymphocytic leukemia.

    PubMed

    Kivekäs, Ilkka; Hulkkonen, Janne; Hurme, Mikko; Vilpo, Leena; Vilpo, Juhani

    2002-05-01

    We investigated the correlation between expression of 31 surface membrane antigens and chemosensitivity of peripheral blood mononuclear cells from 36 patients with CLL. The sensitivity of CLL cells to nine drugs (2'-chlorodeoxyadenosine, cisplatin, chlorambucil, cyclosporin A, doxorubicin, fludarabine, prednisolone, verapamil and vincristine) and two types of irradiation (gamma and UV-irradiation) was determined from dose-response curves of 4-day cultures ex vivo. The results indicated that the CLL cases responding to purine analogs (2'-chlorodeoxyadenosine and fludarabine) can be identified according to CD80 expression: all resistant cases had low or negative CD80 expression. No other correlations were revealed. CD80 may be a surrogate chemosensitivity marker for purine analogs. PMID:11916516

  2. Enhancement of bone formation ex vivo and in vivo by a helioxanthin-derivative.

    PubMed

    Nakajima, Keiji; Komiyama, Yusuke; Hojo, Hironori; Ohba, Shinsuke; Yano, Fumiko; Nishikawa, Naoko; Ihara, Sigeo; Aburatani, Hiroyuki; Takato, Tsuyoshi; Chung, Ung-Il

    2010-05-14

    To effectively treat serious bone defects using bone-regenerative medicine, a small chemical compound that potently induces bone formation must be developed. We previously reported on the osteogenic effect of 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin-derivative, in vitro. Here, we report on TH's osteogenic effects ex vivo and in vivo. TH-induced new bone formation in both calvarial and metatarsal organ cultures. A novel monitoring system of osteoblastic differentiation using MC3T3-E1 cells revealed that TH was released from alpha-TCP bone cement and this release continued for more than one month. Lastly, the implantation of the alpha-TCP carrier containing TH into defects in mouse skull resulted in increased new bone areas within the defects after 4 weeks. A TH-containing scaffold may help establish a more efficient bone regeneration system. PMID:20382113

  3. Human Ex-Vivo Liver Model for Acetaminophen-induced Liver Damage

    PubMed Central

    Schreiter, Thomas; Sowa, Jan-Peter; Schlattjan, Martin; Treckmann, Jürgen; Paul, Andreas; Strucksberg, Karl-Heinz; Baba, Hideo A.; Odenthal, Margarete; Gieseler, Robert K.; Gerken, Guido; Arteel, Gavin E.; Canbay, Ali

    2016-01-01

    Reliable test systems to identify hepatotoxicity are essential to predict unexpected drug-related liver injury. Here we present a human ex-vivo liver model to investigate acetaminophen-induced liver injury. Human liver tissue was perfused over a 30 hour period with hourly sampling from the perfusate for measurement of general metabolism and clinical parameters. Liver function was assessed by clearance of indocyanine green (ICG) at 4, 20 and 28 hours. Six pieces of untreated human liver specimen maintained stable liver function over the entire perfusion period. Three liver sections incubated with low-dose acetaminophen revealed strong damage, with ICG half-lives significantly higher than in non-treated livers. In addition, the release of microRNA-122 was significantly higher in acetaminophen-treated than in non-treated livers. Thus, this model allows for investigation of hepatotoxicity in human liver tissue upon applying drug concentrations relevant in patients. PMID:27550092

  4. Optical spectroscopy for differentiation of liver tissue under distinct stages of fibrosis: an ex vivo study

    NASA Astrophysics Data System (ADS)

    Fabila, D. A.; Hernández, L. F.; de la Rosa, J.; Stolik, S.; Arroyo-Camarena, U. D.; López-Vancell, M. D.; Escobedo, G.

    2013-11-01

    Liver fibrosis is the decisive step towards the development of cirrhosis; its early detection affects crucially the diagnosis of liver disease, its prognosis and therapeutic decision making. Nowadays, several techniques are employed to this task. However, they have the limitation in estimating different stages of the pathology. In this paper we present a preliminary study to evaluate if optical spectroscopy can be employed as an auxiliary tool of diagnosis of biopsies of human liver tissue to differentiate the fibrosis stages. Ex vivo fluorescence and diffuse reflectance spectra were acquired from biopsies using a portable fiber-optic system. Empirical discrimination algorithms based on fluorescence intensity ratio at 500 nm and 680 nm as well as diffuse reflectance intensity at 650 nm were developed. Sensitivity and specificity of around 80% and 85% were respectively achieved. The obtained results show that combined use of fluorescence and diffuse reflectance spectroscopy could represent a novel and useful tool in the early evaluation of liver fibrosis.

  5. Diagnostic Accuracy of Renal Mass Biopsy: An Ex Vivo Study of 100 Nephrectomy Specimens.

    PubMed

    von Rundstedt, Friedrich-Carl; Mata, Douglas Alexander; Kryvenko, Oleksandr N; Roth, Stephan; Degener, Stephan; Dreger, Nici Markus; Goedde, Daniel; Assaid, Ahmed; Kamper, Lars; Haage, Patrick; Stoerkel, Stephan; Lazica, David A

    2016-05-01

    We investigated the diagnostic accuracy of renal mass biopsy in an ex vivo model, as well as compared the agreement of the preoperative radiological diagnosis with the final pathologic diagnosis. Two 18-gauge needle-core and 2 vacuum-needle biopsies were performed ex vivo from the tumors of 100 consecutive patients undergoing radical nephrectomy between 2006 and 2010. The median tumor size was 5.5 cm. There was no significant difference with regard to cylinder length or tissue quality between the sampling methods. At least 1 of 4 needle cores contained diagnostic tissue in 88% of patients. Biopsy specimens identified clear cell (54%), papillary (13%), or chromophobe (5%) renal cell carcinoma; urothelial carcinoma (6%); oncocytoma (5%); liposarcoma (1%); metastatic colorectal carcinoma (1%); squamous cell carcinoma (1%); unclassified renal cell neoplasm (1%); and no tumor sampled (12%). The sensitivity of the biopsy for accurately determining the diagnosis was 88% (95% CI: 79% to 93%). The specificity was 100% (95% CI: 17% to 100%). Biopsy grade correlated strongly with final pathology (83.5% agreement). There was no difference in average tumor size in cases with the same versus higher grade on final pathology (5.87 vs 5.97; P = .87). Appraisal of tumor histology by radiology agreed with the pathologic diagnosis in 68% of cases. Provided that the biopsy samples the tumor tissue in a renal mass, pathologic analysis is of great diagnostic value in respect of grade and tumor type and correlates well with excisional pathology. This constitutes strong ground for increasingly used renal mass biopsy in patients considering active surveillance or ablation therapy. PMID:26811388

  6. Photothermal stimulation of chondrocyte proliferation in ex-vivo cartilage grafts

    NASA Astrophysics Data System (ADS)

    Truong, Mai T.; Gardener, David; Pandoh, Nidhi S.; Wong, Brian J.

    2001-07-01

    In vivo, laser radiation has been shown to stimulate cartilage repair and proliferation, which is of clinical relevance as light can be delivered using minimally invasive techniques. However, dosimetry and temperature dependence of this phenomenon have neither been determined nor have these findings been conclusively demonstrated ex vivo. In this study, we detected the presence of proliferating chondrocytes in intact laser irradiated rabbit septal cartilage using a novel whole mount Bromodeoxyuridine (BrdU) assay, and determined the dependence of this phenomenon on laser dosimetry. Cartilage specimens were irradiated with light from an Nd:YAG laser (λ= 1.32 μm, 3-16 sec, 10-45 W/cm2) and placed in tissue culture with BrdU for 7-9 days. BrdU (a thymidine analogue) is incorporated into DNA during replication. Specimens were then fixed and treated with an enzyme-linked double antibody system providing a color change to indicate the presence of BrdU in dividing cells. The samples were analyzed in whole mount and with conventional histology. Proliferation was clearly identified for laser exposures greater than 6 seconds at (25 W/cm2), and was observed only on the periphery of the laser spot. This study clearly demonstrates that laser heating of ex vivo cartilage tissue results in chondrocyte proliferation. Inasmuch as this phenomenon was observed in tissue culture, the non-specific cellular and humoral responses present an intact organism were eliminated. Cell division likely results form either changes in the fine structure of the tissue matrix or direct stimulation of chondrocyte metabolism.

  7. Cyclooxygenase selectivity of non-steroid anti-inflammatory drugs in humans: ex vivo evaluation.

    PubMed

    Giuliano, F; Ferraz, J G; Pereira, R; de Nucci, G; Warner, T D

    2001-08-24

    We have recently described a novel assay to assess ex vivo the activity and selectivity on cyclooxygenase-1 and -2 (EC 1.14.99.1) of non-steroid anti-inflammatory drugs (NSAID) administered to rats [Br. J. Pharmacol. 126 (1999) 1824.]. Here, we have extended these studies to humans. Healthy male volunteers were given orally one of the following drugs (mg) for 5 days: etodolac (200 or 400 b.i.d.), meloxicam (7.5 or 15 q.d.), nimesulide (100 or 200 b.i.d.), nabumetone (500 or 1000 b.i.d.) or naproxen (500 b.i.d.). Blood samples were withdrawn from the volunteers before and up to 24 h after the last dose. Plasma obtained from the blood was tested for its ability to inhibit prostanoid formation in interleukin-1beta-treated A549 cells (cyclooxygenase-2 system) and human washed platelets (cyclooxygenase-1 system). Plasma from etodolac-treated subjects demonstrated a slight selectivity towards the inhibition of cyclooxygenase-2. This effect was more prominent in plasma from subjects receiving meloxicam or nimesulide. Plasma from nabumetone-treated subjects showed no or little selectivity towards cyclooxygenase-1 depending on the dose of drug administered, while plasma taken from subjects receiving naproxen was more active at inhibiting cyclooxygenase-1 than cyclooxygenase-2. In conclusion, we have demonstrated that this assay can be used to assess ex vivo the relative activity against cyclooxygenase-1 and cyclooxygenase-2 of NSAIDs consumed by human volunteers. It is to be hoped that data from such systems will aid in our understanding of the relationships between the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2 by NSAIDs and their reported efficacies and (gastrointestinal) toxicities. PMID:11525777

  8. Manufacturing blood ex vivo: a futuristic approach to deal with the supply and safety concerns

    PubMed Central

    Singh, Vimal K.; Saini, Abhishek; Tsuji, Kohichiro; Sharma, P. B.; Chandra, Ramesh

    2014-01-01

    Blood transfusions are routinely done in every medical regimen and a worldwide established collection, processing/storage centers provide their services for the same. There have been extreme global demands for both raising the current collections and supply of safe/adequate blood due to increasingly demanding population. With, various risks remain associated with the donor derived blood, and a number of post collection blood screening and processing methods put extreme constraints on supply system especially in the underdeveloped countries. A logistic approach to manufacture erythrocytes ex-vivo by using modern tissue culture techniques have surfaced in the past few years. There are several reports showing the possibilities of RBCs (and even platelets/neutrophils) expansion under tightly regulated conditions. In fact, ex vivo synthesis of the few units of clinical grade RBCs from a single dose of starting material such as umbilical cord blood (CB) has been well established. Similarly, many different sources are also being explored for the same purpose, such as embryonic stem cells, induced pluripotent stem cells. However, the major concerns remain elusive before the manufacture and clinical use of different blood components may be used to successfully replace the present system of donor derived blood transfusion. The most important factor shall include the large scale of RBCs production from each donated unit within a limited time period and cost of their production, both of these issues need to be handled carefully since many of the recipients among developing countries are unable to pay even for the freely available donor derived blood. Anyways, keeping these issues in mind, present article shall be focused on the possibilities of blood production and their use in the near future. PMID:25364733

  9. Micro CT imaging assessment for spatial distribution of magnetic nanoparticles in an ex vivo thrombolysis model

    NASA Astrophysics Data System (ADS)

    Wang, Fu-Sheng; Chao, Tsi-Chian; Tu, Shu-Ju

    2012-03-01

    In recent nanotechnology development, iron-based magnetic nanoparticles (MNPs) have been used in several investigations on biomedical research for small animal experiments. Their important applications include targeted drug delivery for therapeutic purpose, contrast agent for magnetic resonance imaging, and hyperthermia treatment for tumors. These MNPs can be guided by an external magnetic field due to their physical characteristics of superparamagnetism. In a recent report, authors indicated that covalently bound recombinant tissue plasminogen activator (rtPA) to MNP (MNPrtPA) with preserved enzyme activity may be guided by a bar magnet and induce target thrombolysis in an embolic model in rats. Delivery of rtPA by binding the thrombolytic drug to MNPs will improve the possibility of the drug to be delivered under magnetic guidance and retained in a local targeted area in the circulation system. In this work, an ex vivo intravascular thrombolysis model was developed to study the impact of external magnetic field on the penetration of MNP-rtPA in the blood clot samples. The samples were then scanned by a micro CT system for quantification. Images of MNPs show strong contrast with their surrounding blood clot materials. The optimum drug loading was found when 0.5 mg/ml rtPA is conjugated with 10 mg SiO2-MNP where 98% drug was attached to the carrier with full retention of its thrombolytic activity. Effective thrombolysis with tPA bound to SiO2-MNP under magnetic guidance was demonstrated in our ex vivo model where substantial reduction in time for blood clot lysis was observed compared with control groups without magnetic field application.

  10. Theoretical versus Ex Vivo Assessment of Radiation Damage Repair: An Investigation in Normal Breast Tissue.

    PubMed

    Ebert, Martin A; Dhal, Bipina; Prunster, Janelle; McLaren, Sally; Zeps, Nikolajs; House, Michael; Reniers, Brigitte; Verhaegen, Frank; Corica, Tammy; Saunders, Christobel; Joseph, David J

    2016-04-01

    In vivo validation of models of DNA damage repair will enable their use for optimizing clinical radiotherapy. In this study, a theoretical assessment was made of DNA double-strand break (DSB) induction in normal breast tissue after intraoperative radiation therapy (IORT), which is now an accepted form of adjuvant radiotherapy for selected patients with early breast cancer. DSB rates and relative biological effectiveness (RBE) were calculated as a function of dose, radiation quality and dose rate, each varying based on the applicator size used during IORT. The spectra of primary electrons in breast tissue adjacent to each applicator were calculated using measured X-ray spectra and Monte Carlo methods, and were used to inform a Monte Carlo damage simulation code. In the absence of repair, asymptotic RBE values (relative to (60)Co) were approximately 1.5. Beam-quality changes led to only minor variations in RBE among applicators, though differences in dose rate and overall dose delivery time led to larger variations and a rapid decrease in RBE. An experimental assessment of DSB induction was performed ex vivo using pre- and postirradiation tissue samples from patients receiving breast intraoperative radiation therapy. Relative DSB rates were assessed via γ-H2AX immunohistochemistry using proportional staining. Maximum-likelihood parameter estimation yielded a DSB repair halftime of 25.9 min (95% CI, 21.5-30.4 min), although the resulting model was not statistically distinguishable from one where there was no change in DSB yield among patients. Although the model yielded an in vivo repair halftime of the order of previous estimates for in vitro repair halftimes, we cannot conclude that it is valid in this context. This study highlights some of the uncertainties inherent in population analysis of ex vivo samples, and of the quantitative limitations of immunohistochemistry for assessment of DSB repair. PMID:27023258

  11. A tyrosine-rich amelogenin peptide promotes neovasculogenesis in vitro and ex vivo.

    PubMed

    Amin, Harsh D; Olsen, Irwin; Knowles, Jonathan; Dard, Michel; Donos, Nikolaos

    2014-05-01

    The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo. PMID:24321350

  12. Differential ex vivo responses of primary leukocytes from turkey pedigree lines to Salmonella Heidelberg.

    PubMed

    Potter, Tiffany D; Glover, Paige K; Evans, Nicholas P; Dalloul, Rami A

    2016-02-01

    Escalating product recalls as a consequence of Salmonella-contaminated poultry products have resulted in detrimental economic impacts in the poultry industry. One potential long-term alternative method to Salmonella prevention is genetic selection to improve innate resistance. This study evaluated the ex vivo effects of Salmonella Heidelberg (SH) on phagocytic and bactericidal leukocyte function in turkeys from six pedigree lines (A-F). Day-of-hatch poults (n = 48) were placed and raised in cages (2 birds/gender/genetic line/cage) to 35 d when heterophils and peripheral blood mononuclear cells (PBMCs) were extracted from males and females of each line. Cells were used in phagocytic and bactericidal assays to determine the ex vivo effects of SH on turkey leukocyte activity. Data were analyzed using the Fit Model platform in JMP Pro 10.0 (SAS Institute Inc.) with differences considered significant at P ≤ 0.05 and data reported as LS Means with SEM. Although genetic line had no significant effect on phagocytosis of SH by heterophils and PBMCs, cumulatively, female cells exhibited higher phagocytosis potential than those from males. The main effect of gender was significant on bactericidal activity of PBMCs when incubated at a 1:10 and 1:100 PBMC to SH ratio. Genetic line also had a significant effect on bactericidal activity of PBMCs with cells from line F exhibiting the best activity. These results suggest that gender had a marked cumulative effect on phagocytosis of SH by heterophils and PBMCs while both genetic line and gender had a prominent effect on bacterial killing of SH by turkey PBMCs. Once able to determine genetic markers associated with these immune responses to Salmonella, genetic selection for increased resistance may become feasible in turkeys. PMID:26706359

  13. In vitro, ex vivo and in vivo anti-hypertensive activity of Chrysophyllum cainito L. extract

    PubMed Central

    Mao, Li-Mei; Qi, Xue-Wen; Hao, Ji-Heng; Liu, Hai-Feng; Xu, Qing-Hua; Bu, Pei-Li

    2015-01-01

    Chrysophyllum cainito L., a traditional herbal medicine, could have the potential for management of hypertension due to presence of polyphenolic compounds. The extracts and fractions of the pulp of plant were evaluated for in vitro (inhibition of angiotensin I converting enzyme/ACE assay), ex vivo (isolated aorta relaxation assay) and in vivo (salt induced hypertensive rat assay). The alcoholic and aqueous extract (ALE and AQE respectively) of fruit of plant C. cainito was having 14.8 and 9.2% yield respectively. The fractionation with ethyl alcohol (EAF) and butanol (BTF) yielded 2.52 & 2.17% respectively from ALE and 0.46 & 0.31% respectively from AQE with respect to fruit pulp dry weight. More phenolic content was found in ALE (3.75±0.15 mg gallic acid equivalent or GAE g-1 of dry power of fruit pulp) compared to AQE and maximum in ethyl acetate fraction of ALE (ALE-EAF) (2.32±0.21 mg GAE g-1 of dry power of fruit pulp) among all fractions. ACE inhibition activity was found to be maximum in ALE-EAF 62.5±7.34%. While ex vivo study using isolated tissue of aorta showed again showed maximum activity (62.82±6.19 and 46.47±8.32% relaxation with 50 µg mL-1 and 10 µg mL-1 GAE concentration respectively). ALE-EAF reduced the elevated arterial pressure of salt induced hypertensive rat significantly to the level of normotensive animal group. Results of ALE-EAF have shown its potential as a source for novel constituent for the treatment hypertension and should further be studied for isolation of specific constituent for more effectiveness. PMID:26770385

  14. Fiber optic microneedles for transdermal light delivery: ex vivo porcine skin penetration experiments.

    PubMed

    Kosoglu, Mehmet A; Hood, Robert L; Chen, Ye; Xu, Yong; Rylander, Marissa Nichole; Rylander, Christopher G

    2010-09-01

    Shallow light penetration in tissue has been a technical barrier to the development of light-based methods for in vivo diagnosis and treatment of epithelial carcinomas. This problem can potentially be solved by utilizing minimally invasive probes to deliver light directly to target areas. To develop this solution, fiber optic microneedles capable of delivering light for either imaging or therapy were manufactured by tapering step-index silica-based optical fibers employing a melt-drawing process. Some of the microneedles were manufactured to have sharper tips by changing the heat source during the melt-drawing process. All of the microneedles were individually inserted into ex vivo pig skin samples to demonstrate the feasibility of their application in human tissues. The force on each microneedle was measured during insertion in order to determine the effects of sharper tips on the peak force and the steadiness of the increase in force. Skin penetration experiments showed that sharp fiber optic microneedles that are 3 mm long penetrate through 2 mm of ex vivo pig skin specimens. These sharp microneedles had a minimum average diameter of 73 mum and a maximum tip diameter of 8 mum. Flat microneedles, which had larger tip diameters, required a minimum average diameter of 125 mum in order to penetrate through pig skin samples. Force versus displacement plots showed that a sharp tip on a fiber optic microneedle decreased the skin's resistance during insertion. Also, the force acting on a sharp microneedle increased more steadily compared with a microneedle with a flat tip. However, many of the sharp microneedles sustained damage during skin penetration. Two designs that did not accrue damage were identified and will provide a basis of more robust microneedles. Developing resilient microneedles with smaller diameters will lead to transformative, novel modes of transdermal imaging and treatment that are less invasive and less painful for the patient. PMID:20815648

  15. Effects of Ex Vivo Transduction of Mesencephalic Reaggregates with Bcl-2 on Grafted Dopamine Neuron Survival

    PubMed Central

    Sortwell, Caryl E.; Bowers, William J.; Counts, Scott E.; Pitzer, Mark R.; Fleming, Matthew F.; McGuire, Susan O.; Maguire-Zeiss, Kathleen A.; Federoff, Howard J.; Collier, Timothy J.

    2007-01-01

    Survival rates of dopamine (DA) neurons grafted to the denervated striatum are extremely poor (5-20%). Gene transfer of survival promoting factors, such as the anti-apoptotic protein bcl-2, to mesencephalic DA neurons prior to transplantation (ex vivo transduction) offers a novel approach to increase graft survival. However, specific criteria to assess the efficacy of various vectors must be adhered to in order to reasonably predict successful gene transfer with appropriate timing and levels of protein expression. Cell culture results utilizing three different herpes simplex virus (HSV) vectors to deliver the reporter ß-galactosidase gene (lacZ) indicate that transduction of mesencephalic cells with a helper virus-free HSV amplicon (HF HSVTH9lac) that harbors the 9-kb tyrosine hydroxylase (TH) promoter to drive lacZ gene expression elicits the transduction of the highest percentage (≈50%) of TH-immunoreactive (THir) neurons without significant cytotoxic effects. This transduction efficiency and limited cytotoxicity was superior to that observed following transduction with helper virus-containing HSV (HC HSVlac) and helper virus-free HSV amplicons (HF HSVlac) expressing lacZ under the transcriptional control of the HSV immediate-early 4/5 gene promoter. Subsequently, we assessed the ability of HSV-TH9lac and the bcl-2 expressing HSV-TH9bcl-2 amplicon to transduce mesencephalic reaggregates. Although an increase in bcl-2 and ß-galactosidase protein was induced by transduction, amplicon-mediated overexpression of bcl-2 did not lead to an increase in grafted THir neuron number. Even with highly efficient viral vector-mediated transduction, our results demonstrate that ex vivo gene transfer of bcl-2 to mesencephalic reaggregates is ineffective in increasing grafted DA neuron survival. PMID:17196186

  16. Ex vivo identification of protein-protein interactions involving the dopamine transporter.

    PubMed

    Hadlock, Gregory C; Nelson, Chad C; Baucum, Anthony J; Hanson, Glen R; Fleckenstein, Annette E

    2011-03-30

    The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT. PMID:21291912

  17. Evaluation of the VE1 Antibody in Thyroid Cytology Using Ex Vivo Papillary Thyroid Carcinoma Specimens

    PubMed Central

    Kim, Yon Hee; Yim, Hyunee; Lee, Yong-Hee; Han, Jae Ho; Lee, Kyi Beom; Lee, Jeonghun; Soh, Euy Young; Jeong, Seon-Yong; Kim, Jang-Hee

    2016-01-01

    Background: Recently, VE1, a monoclonal antibody against the BRAFV600E mutant protein, has been investigated in terms of its detection of the BRAFV600E mutation. Although VE1 immunostaining and molecular methods used to assess papillary thyroid carcinoma in surgical specimens are in good agreement, evaluation of VE1 in thyroid cytology samples is rarely performed, and its diagnostic value in cytology has not been well established. In present study, we explored VE1 immunoexpression in cytology samples from ex vivo papillary thyroid carcinoma specimens in order to minimize limitations of low cellularity and sampling/targeting errors originated from thyroid fineneedle aspiration and compared our results with those obtained using the corresponding papillary thyroid carcinoma tissues. Methods: The VE1 antibody was evaluated in 21 cases of thyroid cytology obtained directly from ex vivo thyroid specimens. VE1 immunostaining was performed using liquid-based cytology, and the results were compared with those obtained using the corresponding tissues. Results: Of 21 cases, 19 classic papillary thyroid carcinomas had BRAFV600E mutations, whereas two follicular variants expressed wild-type BRAF. VE1 immunoexpression varied according to specimen type. In detection of the BRAFV600E mutation, VE1 immunostaining of the surgical specimen exhibited 100% sensitivity and 100% specificity, whereas VE1 immunostaining of the cytology specimen exhibited only 94.7% sensitivity and 0% specificity. Conclusions: Our data suggest that VE1 immunostaining of a cytology specimen is less specific than that of a surgical specimen for detection of the BRAFV600E mutation, and that VE1 immunostaining of a cytology specimen should be further evaluated and optimized for clinical use. PMID:26657312

  18. Direct Ex-Vivo Evaluation of Pneumococcal Specific T-Cells in Healthy Adults

    PubMed Central

    Aslam, Aamir; Chapel, Helen; Ogg, Graham

    2011-01-01

    Streptococcus pneumoniae is an encapsulated bacterium that causes significant global morbidity and mortality. The nasopharynxes of children are believed to be the natural reservoir of pneumococcus and by adulthood nasopharyngeal carriage is infrequent; such infrequency may be due to demonstrable pneumococcal specific T and B-cell responses. HLA Class 2 tetrameric complexes have been used to characterise antigen specific T-cell responses in a variety of models of infection. We therefore sought to determine the frequency and phenotype of pneumococcal specific T-cells, using a novel HLA-DRB1*1501 tetramer complex incorporating a recently defined T-cell epitope derived from the conserved pneumococcal serine/threonine kinase (StkP). We were able to detect direct ex-vivo StkP446–60-tetramer binding in HLA-DRB1*1501 adults. These StkP446–60-tetramer binding T-cells had increased CD38 expression and were enriched in CCR7- CD45RA+ expression indicating recent and on-going activation and differentiation. Furthermore, these StkP446–60-tetramer binding T-cells demonstrated rapid effector function by secreting interferon-gamma on stimulation with recombinant StkP. This is the first study to directly enumerate and characterise pneumococcal specific T-cells using HLA class 2 tetrameric complexes. We found that ex-vivo pneumococcal-specific T cells were detectable in healthy adults and that they were enriched with cell surface markers associated with recent antigen exposure and later stages of antigen-driven differentiation. It is likely that these activated pneumococcal specific T-cells reflect recent immunostimulatory pneumococcal exposure in the nasopharynx and it is possible that they may be preventing subsequent colonisation and disease. PMID:22039412

  19. Ex vivo testing of immune responses in precision-cut lung slices

    SciTech Connect

    Henjakovic, M.; Sewald, K.; Switalla, S.; Kaiser, D.; Mueller, M.; Veres, T.Z.; Martin, C.; Uhlig, S.; Krug, N.; Braun, A.

    2008-08-15

    The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon {gamma} (IFN{gamma}), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1{alpha}, TNF{alpha}, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFN{gamma} resulting in increased levels of TNF{alpha}, IL-12(p40), RANTES, and IL-1{alpha}. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.

  20. Microwaves create larger ablations than radiofrequency when controlled for power in ex vivo tissue

    PubMed Central

    Andreano, A.; Huang, Yu; Meloni, M. Franca; Lee, Fred T.; Brace, Christopher

    2010-01-01

    Purpose: To compare ablation zones created with equal amounts of 2.45 GHz microwave and 480 kHz radiofrequency (RF) energy in ex vivo liver and lung. Methods: A total of 38 ablations were performed in ex vivo liver and lung for 10 min each. Nineteen RF ablations (nine liver, ten lung) were performed with a 480 kHz system (200 W max, impedance-based pulsing) and cooled electrode while measuring the average RF power applied. Nineteen microwave ablations (nine liver, ten lung) were then created using a cooled triaxial antenna to deliver 2.45 GHz at the same power level as in RF experiments. Ablation zones were then sectioned and measured for minimum, maximum and mean diameters, and circularity. Measurements were compared using t-tests, with P<0.05 indicating statistical significance. Results: Mean diameters of microwave ablations were greater than RF ablations in both liver and lung (4.4±0.3 vs 3.3±0.2 cm in liver; 2.45±0.3 vs 1.6±0.5 cm in lungs; P<0.0005 all comparisons). There was no significant difference in the mean power applied during microwave or RF ablations in either organ (54.44±1.71 W vs 56.4±6.7 W in liver, P>0.05; 40±0.95 W vs 44.9±7.1 W in lung, P>0.05). Conclusions: Using a single cooled applicator, microwave energy at 2.45 GHz produces larger ablations than an equivalent amount of 480 kHz RF energy in normal liver and lung. This was more apparent in lung, likely due to the high baseline impedance which limits RF, but not microwave power delivery. PMID:20632609

  1. Safety and feasibility of liver-directed ex vivo gene therapy for homozygous familial hypercholesterolemia.

    PubMed Central

    Raper, S E; Grossman, M; Rader, D J; Thoene, J G; Clark, B J; Kolansky, D M; Muller, D W; Wilson, J M

    1996-01-01

    OBJECTIVE: The purpose of this report was to provide detailed information on the safety and feasibility of surgical procedures associated with the first ex vivo liver-directed gene therapy trial for the treatment of vivo gene therapy for homozygous familial hypercholesterolemia (FH). SUMMARY BACKGROUND DATA: Familial hypercholesterolemia is an autosomal dominant disease in which the gene encoding the low density lipoprotein receptor is defective. Patients homozygous for this mutation have extraordinarily high levels of cholesterol and accelerated atherosclerosis and die prematurely of myocardial infarction. The concept of liver-directed gene therapy was based on the report of normalization of cholesterol levels by orthotopic cardiac/liver transplant in a child with homozygous FH. METHODS: Five patients with homozygous FH were selected for inclusion in this trial. The patients underwent hepatic resection and placement of a portal venous catheter. Primary hepatocytes cultures were prepared from the resected liver and transduced with a recombinant retrovirus encoding the gene for the human low density lipoprotein receptor. The genetically modified cells were then transplanted into the liver through the portal venous catheter. RESULTS: Numerous clinical, laboratory, and radiologic parameters were analyzed. Elevations of the hepatic transaminases and leukocyte counts and a decline in hematocrit count were noted. Transient elevations of the portal pressure were observed during cell infusion. No major perioperative morbidity--specifically, myocardial infarct, perioperative hemorrhage, or portal vein thrombosis--or death occurred as a result of this protocol. CONCLUSION: Liver-directed ex vivo gene therapy can be accomplished safely in humans and is appropriate for selected patients. Images Figure 5. PMID:8597504

  2. Ex Vivo Apoptosis in CD8+ Lymphocytes Predicts Rectal Cancer Patient Outcome

    PubMed Central

    Haderlein, Marlen

    2016-01-01

    Background. Apoptotic rates in peripheral blood lymphocytes can predict radiation induced normal tissue toxicity. We studied whether apoptosis in lymphocytes has a prognostic value for therapy outcome. Methods. Lymphocytes of 87 rectal cancer patients were ex vivo irradiated with 2 Gy, 8 Gy, or a combination of 2 Gy ionizing radiation and Oxaliplatin. Cells were stained with Annexin V and 7-Aminoactinomycin D and apoptotic and necrotic rates were analyzed by multicolor flow cytometry. Results. After treatment, apoptotic and necrotic rates in CD8+ cells are consistently higher than in CD4+ cells, with lower corresponding necrotic rates. Apoptotic and necrotic rates of CD4+ cells and CD8+ cells correlated well within the 2 Gy, 8 Gy, and 2 Gy and Oxaliplatin arrangements (p ≤ 0.009). High apoptotic CD8+ rates after 2 Gy, 8 Gy, and 2 Gy + Oxaliplatin treatment were prognostically favorable for metastasis-free survival (p = 0.009, p = 0.038, and p = 0.009) and disease-free survival (p = 0.013, p = 0.098, and p = 0.013). Conclusions. Ex vivo CD8+ apoptotic rates are able to predict the patient outcome in regard to metastasis-free or disease-free survival. Patients with higher CD8+ apoptotic rates in the peripheral blood have a more favorable prognosis. In addition to the prediction of late-toxicity by utilization of CD4+ apoptotic rates, the therapy outcome can be predicted by CD8+ apoptotic rates. PMID:27340400

  3. A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

    PubMed Central

    Akhavanpoor, Mohammadreza; Zhao, Li; Wangler, Susanne; Hakimi, Maani; Doesch, Andreas; Dengler, Thomas J.; Katus, Hugo A.; Gleissner, Christian A.

    2014-01-01

    Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets. PMID:24836700

  4. A human ex vivo atherosclerotic plaque model to study lesion biology.

    PubMed

    Erbel, Christian; Okuyucu, Deniz; Akhavanpoor, Mohammadreza; Zhao, Li; Wangler, Susanne; Hakimi, Maani; Doesch, Andreas; Dengler, Thomas J; Katus, Hugo A; Gleissner, Christian A

    2014-01-01

    Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets. PMID:24836700

  5. Rapid evaluation of fresh ex vivo kidney tissue with full-field optical coherence tomography

    PubMed Central

    Jain, Manu; Robinson, Brian D.; Salamoon, Bekheit; Thouvenin, Olivier; Boccara, Claude; Mukherjee, Sushmita

    2015-01-01

    Background: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that rapidly generates images reminiscent of histology without any tissue processing, warranting its exploration for evaluation of ex vivo kidney tissue. Methods: Fresh tissue sections from tumor and adjacent nonneoplastic kidney (n = 25 nephrectomy specimens; clear cell renal cell carcinoma (CCRCC) = 12, papillary RCC (PRCC) = 4, chromophobe RCC (ChRCC) = 4, papillary urothelial carcinoma (PUC) = 1, angiomyolipoma (AML) = 2 and cystic nephroma = 2) were imaged with a commercial FFOCT device. Sections were submitted for routine histopathological diagnosis. Results: Glomeruli, tubules, interstitium, and blood vessels were identified in nonneoplastic tissue. In tumor sections, the normal architecture was completely replaced by either sheets of cells/trabeculae or papillary structures. The former pattern was seen predominantly in CCRCC/ChRCC and the latter in PRCC/PUC (as confirmed on H&E). Although the cellular details were not very prominent at this resolution, we could identify unique cytoplasmic signatures in some kidney tumors. For example, the hyper-intense punctate signal in the cytoplasm of CRCC represents glycogen/lipid, large cells with abundant hyper-intense cytoplasm representing histiocytes in PRCC, and signal-void large polygonal cell representing adipocytes in AML. According to a blinded analysis was performed by an uropathologist, all nonneoplastic tissues were differentiated from neoplastic tissues. Further, all benign tumors were called benign and malignant were called malignant. A diagnostic accuracy of 80% was obtained in subtyping the tumors. Conclusion: The ability of FFOCT to reliably differentiate nonneoplastic from neoplastic tissue and identify some tumor types makes it a valuable tool for rapid evaluation of ex vivo kidney tissue e.g. for intraoperative margin assessment and kidney biopsy adequacy. Recently, higher resolution images were achieved

  6. The effect of different root canal medicaments on the elimination of Enterococcus faecalis ex vivo

    PubMed Central

    Dammaschke, Till; Jung, Nina; Harks, Inga; Schafer, Edgar

    2013-01-01

    Objective: The aim of this study was to evaluate the antimicrobial effect of chlorhexidine gel (CHX-G) 2%, chlorhexidine powder (CHX-P) 1%, povidone-iodine (PVP-I), polyhexanide and camphorated-and-mentholated chlorophenol (ChKM) ex vivo. Materials and Methods: For every medicament group 10 root segments (15 mm long) of extracted human teeth were prepared to ISO-size 45 and sterilized (n = 50). The root segments were then inoculated with Enterococcus faecalis and aerobically incubated at 37°C. After 1 week, ten root canals were filled with one of the medicaments, respectively and aerobically incubated at 37°C for another week. Ten teeth served as positive controls and were filled with sterile saline solution. After 7 days, the medicaments were inactivated and all root canals were instrumented to ISO-size 50. The obtained dentin samples were dispersed in Ringer solution followed by the preparation of serial dilutions. 10 μl per sample were applied to an agar plate and incubated at 37°C for 48 h. The colony forming units were counted and the reduction factors (RFs) were calculated and statistically analyzed. Results: Compared with the positive controls all medicaments exhibited an antibacterial effect against E. faecalis. The RFs for CHX-G, CHX-P and ChKM were significantly higher compared to PVP-I and polyhexanide (P < 0.05). In contrast to PVP-I and polyhexanide, CHX-G, CHX-P and ChKM were able to eliminate E. faecalis from all dentin samples. Conclusions: Within the limitations of this ex vivo investigation, 2% CHX-G and CHX-P were as effective as ChKM against E. faecalis. Thus, when choosing a root canal medicament the better biocompatibility of CHX compared with ChKM should be taken in consideration. PMID:24932119

  7. Multispectral Photoacoustic Imaging of Prostate Cancer: Preliminary Ex-vivo Results

    PubMed Central

    Dogra, Vikram S.; Chinni, Bhargava K.; Valluru, Keerthi S.; Joseph, Jean V.; Ghazi, Ahmed; Yao, Jorge L.; Evans, Katie; Messing, Edward M.; Rao, Navalgund A.

    2013-01-01

    Objective: The objective of this study is to validate if ex-vivo multispectral photoacoustic (PA) imaging can differentiate between malignant prostate tissue, benign prostatic hyperplasia (BPH), and normal human prostate tissue. Materials and Methods: Institutional Review Board's approval was obtained for this study. A total of 30 patients undergoing prostatectomy for biopsy-confirmed prostate cancer were included in this study with informed consent. Multispectral PA imaging was performed on surgically excised prostate tissue and chromophore images that represent optical absorption of deoxyhemoglobin (dHb), oxyhemoglobin (HbO2), lipid, and water were reconstructed. After the imaging procedure is completed, malignant prostate, BPH and normal prostate regions were marked by the genitourinary pathologist on histopathology slides and digital images of marked histopathology slides were obtained. The histopathology images were co-registered with chromophore images. Region of interest (ROI) corresponding to malignant prostate, BPH and normal prostate were defined on the chromophore images. Pixel values within each ROI were then averaged to determine mean intensities of dHb, HbO2, lipid, and water. Results: Our preliminary results show that there is statistically significant difference in mean intensity of dHb (P < 0.0001) and lipid (P = 0.0251) between malignant prostate and normal prostate tissue. There was difference in mean intensity of dHb (P < 0.0001) between malignant prostate and BPH. Sensitivity, specificity, positive predictive value, and negative predictive value of our imaging system were found to be 81.3%, 96.2%, 92.9% and 89.3% respectively. Conclusion: Our preliminary results of ex-vivo human prostate study suggest that multispectral PA imaging can differentiate between malignant prostate, BPH and normal prostate tissue. PMID:24228210

  8. Microwave Ablation Compared with Radiofrequency Ablation for Breast Tissue in an Ex Vivo Bovine Udder Model

    SciTech Connect

    Tanaka, Toshihiro; Westphal, Saskia; Isfort, Peter; Braunschweig, Till; Penzkofer, Tobias Bruners, Philipp; Kichikawa, Kimihiko; Schmitz-Rode, Thomas Mahnken, Andreas H.

    2012-08-15

    Purpose: To compare the effectiveness of microwave (MW) ablation with radiofrequency (RF) ablation for treating breast tissue in a nonperfused ex vivo model of healthy bovine udder tissue. Materials and Methods: MW ablations were performed at power outputs of 25W, 35W, and 45W using a 915-MHz frequency generator and a 2-cm active tip antenna. RF ablations were performed with a bipolar RF system with 2- and 3-cm active tip electrodes. Tissue temperatures were continuously monitored during ablation. Results: The mean short-axis diameters of the coagulation zones were 1.34 {+-} 0.14, 1.45 {+-} 0.13, and 1.74 {+-} 0.11 cm for MW ablation at outputs of 25W, 35W, and 45W. For RF ablation, the corresponding values were 1.16 {+-} 0.09 and 1.26 {+-} 0.14 cm with electrodes having 2- and 3-cm active tips, respectively. The mean coagulation volumes were 2.27 {+-} 0.65, 2.85 {+-} 0.72, and 4.45 {+-} 0.47 cm{sup 3} for MW ablation at outputs of 25W, 35W, and 45W and 1.18 {+-} 0.30 and 2.29 {+-} 0.55 cm{sup 3} got RF ablation with 2- and 3-cm electrodes, respectively. MW ablations at 35W and 45W achieved significantly longer short-axis diameters than RF ablations (P < 0.05). The highest tissue temperature was achieved with MW ablation at 45W (P < 0.05). On histological examination, the extent of the ablation zone in MW ablations was less affected by tissue heterogeneity than that in RF ablations. Conclusion: MW ablation appears to be advantageous with respect to the volume of ablation and the shape of the margin of necrosis compared with RF ablation in an ex vivo bovine udder.

  9. Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture.

    PubMed

    Peeters, Mirte; van Rijn, Sjoerd; Vergroesen, Pieter-Paul A; Paul, Cornelis P L; Noske, David P; Vandertop, W Peter; Wurdinger, Thomas; Helder, Marco N

    2015-01-01

    Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges. PMID:26350622

  10. Current status of ex vivo gene therapy for hematological disorders: a review of clinical trials in Japan around the world.

    PubMed

    Tani, Kenzaburo

    2016-07-01

    Gene therapies are classified into two major categories, namely, in vivo and ex vivo. Clinical trials of human gene therapy began with the ex vivo techniques. Based on the initial successes of gene-therapy clinical trials, these approaches have spread worldwide. The number of gene therapy trials approved worldwide increased gradually starting in 1989, reaching 116 protocols per year in 1999, and a total of 2210 protocols had been approved by 2015. Accumulating clinical evidence has demonstrated the safety and benefits of several types of gene therapy, with the exception of serious adverse events in several clinical trials. These painful experiences were translated backward to basic science, resulting in the development of several new technologies that have influenced the recent development of ex vivo gene therapy in this field. To date, six gene therapies have been approved in a limited number of countries worldwide. In Japan, clinical trials of gene therapy have developed under the strong influence of trials in the US and Europe. Since the initial stages, 50 clinical trials have been approved by the Japanese government. In this review, the history and current status of clinical trials of ex vivo gene therapy for hematological disorders are introduced and discussed. PMID:27289360

  11. Pharmacological preconditioning for short-term ex vivo expansion of human umbilical cord blood hematopoietic stem cells by filgrastim

    PubMed Central

    Grigoriadis, Nikolaos G; Grigoriadis, Ioannis G; Markoula, Sofia; Paschopoulos, Minas; Zikopoulos, Konstantinos; Apostolakopoulos, Panagiotis Gr; Vizirianakis, Ioannis S; Georgiou, Ioannis

    2016-01-01

    Although umbilical cord blood (UCB) hematopoietic stem cell transplantation (UCBT) has emerged as a promising haematological reconstitution therapy for leukemias and other related disorders, the insufficient UCB stem cell dosage still hinders better clinical outcomes. Previous research efforts, by focusing on ex vivo UCB expansion capabilities have sought to benefit from well-known mechanisms of self-renewal characteristics of UCB stem cells. However, the long-term (> 21 days) in vitro culture period and the low neutrophil recovery significantly reduce the transplantability of such ex vivo expanded UCB stem cells. To overcome the latter hurdles in this study, a post-thaw, short-term ex vivo expansion methodology of UCB mononuclear (UCB-MN) and CD34+ cells has been established. Notably, such effort was achieved through pharmacological preconditioned of UCB cultures by filgrastim agent already used in the clinical setting. In crucial cell populations implicated in the promotion of functional engraftment, the progression of free survival rates (PFS), a marked increase of 6.65 to 9.34 fold for UCB-MN and 35 to 49 fold for CD34+ cells has been noticed. Overall, these results indicate that transplantation of pharmacologically-preconditioned ex vivo expansion of UCB stem and progenitor cells keep high promise upon transplantation to enhance therapeutic potential in everyday clinical practice. PMID:27335700

  12. Functional and fine structural changes in isolated rat lungs challenged with endotoxin ex vivo and in vitro.

    PubMed Central

    Uhlig, S.; Brasch, F.; Wollin, L.; Fehrenbach, H.; Richter, J.; Wendel, A.

    1995-01-01

    The aim of this study was to relate changes in rat lung functions caused by the endotoxin lipopolysaccharide (LPS) to alterations in structure. The following four experimental groups were used: 1), control in vitro, perfusion for 150 minutes; 2), LPS in vitro, perfusion for 150 minutes and infusion of 5 mg of LPS after 40 minutes; 3), control ex vivo, perfusion for 10 minutes; and 4), LPS ex vivo, lungs perfused for 10 minutes from rats treated for 110 minutes with 20 mg/kg LPS intraperitoneally. Histologically, blood-derived leukocytes were detectable only in lungs from group 4, where neutrophils were found in capillaries, interstitium, and endothelial pouches. LPS treatment increased pulmonary resistance and decreased pulmonary compliance in group 4 (ex vivo), and, to a greater extent, in group 2 (in vitro). In these two groups, formation of giant lamellar bodies in the type II pneumocytes was observed. By histological examination, the bronchoconstriction induced by LPS in vitro was localized to the terminal bronchioles. At 2 hours after LPS treatment, no edema and no change in precapillary and postcapillary resistance, capillary pressure, vascular compliance, capillary permeability, and the wet/dry ratio was observed. Thus, our major findings are that LPS induced constriction of the terminal bronchioles in vitro, formation of giant lamellar bodies in type II pneumocytes ex vivo and in vitro, and trapping of neutrophils in endothelial pouches in vivo. Images Figure 2 Figure 3 Figure 4 Figure 6 PMID:7747816

  13. “Same Day” Ex-vivo Regional Gene Therapy: A Novel Strategy to Enhance Bone Repair

    PubMed Central

    Virk, Mandeep S; Sugiyama, Osamu; Park, Sang H; Gambhir, Sanjiv S; Adams, Douglas J; Drissi, Hicham; Lieberman, Jay R

    2011-01-01

    Ex-vivo regional gene therapy with bone marrow cells (BMCs) overexpressing bone morphogenetic protein-2 (BMP-2) has demonstrated efficacy in healing critical sized bone defects in preclinical studies. The purpose of this preclinical study was to compare the osteoinductive potential of a novel “same day” ex-vivo regional gene therapy versus a traditional two-step approach, which involves culture expansion of the donor cells before implantation. In the “same day” strategy buffy coat cells were harvested from the rat bone marrow, transduced with a lentiviral vector-expressing BMP-2 for 1 hour and implanted into a rat femoral defect in the same sitting. There was no significant difference (P = 0.22) with respect to the radiographic healing rates between the femoral defects treated with the “same day” strategy (13/13; 100%) versus the traditional two-step approach (11/14; 78%). However, the femoral defects treated with the “same day” strategy induced earlier radiographic bone healing (P = 0.004) and higher bone volume (BV) [micro-computed tomography (micro-CT); P < 0.001]. The “same day” strategy represents a significant advance in the field of ex-vivo regional gene therapy because it offers a solution to limitations associated with the culture expansion process required in the traditional ex vivo approach. This strategy should be cost-effective when adapted for human use. PMID:21343916

  14. Bioluminescence-mediated longitudinal monitoring of adipose-derived stem cells in a large mammal ex vivo organ culture

    PubMed Central

    Peeters, Mirte; van Rijn, Sjoerd; Vergroesen, Pieter-Paul A.; Paul, Cornelis P. L.; Noske, David P.; Peter Vandertop, W.; Wurdinger, Thomas; Helder, Marco N.

    2015-01-01

    Recently, ex vivo three-dimensional organ culture systems have emerged to study the physiology and pathophysiology of human organs. These systems also have potential as a translational tool in tissue engineering; however, this potential is limited by our ability to longitudinally monitor the fate and action of cells used in regenerative therapies. Therefore, we investigated luciferase-mediated bioluminescence imaging (BLI) as a non-invasive technique to continuously monitor cellular behavior in ex vivo whole organ culture. Goat adipose-derived stem cells (gADSCs) were transduced with either Firefly luciferase (Fluc) or Gaussia luciferase (Gluc) reporter genes and injected in isolated goat intervertebral discs (IVD). Luciferase activity was monitored by BLI for at least seven days of culture. Additionally, possible confounders specific to avascular organ culture were investigated. Gluc imaging proved to be more suitable compared to Fluc in monitoring gADSCs in goat IVDs. We conclude that BLI is a promising tool to monitor spatial and temporal cellular behavior in ex vivo organ culture. Hence, ex vivo organ culture systems allow pre-screening and pre-validation of novel therapeutic concepts prior to in vivo large animal experimentation. Thereby, organ culture systems can reduce animal use, and improve the speed of innovation by overcoming technological, ethical and financial challenges. PMID:26350622

  15. Photonic Characteristics and Ex Vivo Imaging of Escherichia coli-Xen14 Within the Bovine Reproductive Tract

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or with...

  16. Design and evaluation of a transesophageal HIFU probe for ultrasound-guided cardiac ablation: simulation of a HIFU mini-maze procedure and preliminary ex vivo trials.

    PubMed

    Constanciel, Elodie; N'Djin, W Apoutou; Bessière, Francis; Chavrier, Françoise; Grinberg, Daniel; Vignot, Alexandre; Chevalier, Philippe; Chapelon, Jean Yves; Lafon, Cyril

    2013-09-01

    Atrial fibrillation (AF) is the most frequent cardiac arrhythmia. Left atrial catheter ablation is currently performed to treat this disease. Several energy sources are used, such as radio-frequency or cryotherapy. The main target of this procedure is to isolate the pulmonary veins. However, significant complications caused by the invasive procedure are described, such as stroke, tamponade, and atrioesophageal fistula, and a second intervention is often needed to avoid atrial fibrillation recurrence. For these reasons, a minimally-invasive device allowing performance of more complex treatments is still needed. High-intensity focused ultrasound (HIFU) can cause deep tissue lesions without damaging intervening tissues. Left atrial ultrasound-guided transesophageal HIFU ablation could have the potential to become a new ablation technique. The goal of this study was to design and test a minimally-invasive ultrasound-guided transesophageal HIFU probe under realistic treatment conditions. First, numerical simulations were conducted to determine the probe geometry, and to validate the feasibility of performing an AF treatment using a HIFU mini-maze (HIFUMM) procedure. Then, a prototype was manufactured and characterized. The 18-mm-diameter probe head housing contained a 3-MHz spherical truncated HIFU transducer divided into 8 rings, with a 5-MHz commercial transesophageal echocardiography (TEE) transducer integrated in the center. Finally, ex vivo experiments were performed to test the impact of the esophagus layer between the probe and the tissue to treat, and also the influence of the lungs and the vascularization on lesion formation. First results show that this prototype successfully created ex vivo transmural myocardial lesions under ultrasound guidance, while preserving intervening tissues (such as the esophagus). Ultrasound-guided transesophageal HIFU can be a good candidate for treatment of AF in the future. PMID:24658718

  17. Separate and combined effects of a 10-d exposure to hypoxia and inactivity on oxidative function in vivo and mitochondrial respiration ex vivo in humans.

    PubMed

    Salvadego, Desy; Keramidas, Michail E; Brocca, Lorenza; Domenis, Rossana; Mavelli, Irene; Rittweger, Jörn; Eiken, Ola; Mekjavic, Igor B; Grassi, Bruno

    2016-07-01

    An integrative evaluation of oxidative metabolism was carried out in 9 healthy young men (age, 24.1 ± 1.7 yr mean ± SD) before (CTRL) and after a 10-day horizontal bed rest carried out in normoxia (N-BR) or hypoxia (H-BR, FiO2 = 0.147). H-BR was designed to simulate planetary habitats. Pulmonary O2 uptake (V̇o2) and vastus lateralis fractional O2 extraction (changes in deoxygenated hemoglobin+myoglobin concentration, Δ[deoxy(Hb+Mb)] evaluated using near-infrared spectroscopy) were evaluated in normoxia and during an incremental cycle ergometer (CE) and one-leg knee extension (KE) exercise (aimed at reducing cardiovascular constraints to oxidative function). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in permeabilized vastus lateralis fibers. During CE V̇o2peak and Δ[deoxy(Hb+Mb)]peak were lower (P < 0.05) after both N-BR and H-BR than during CTRL; during KE the variables were lower after N-BR but not after H-BR. During CE the overshoot of Δ[deoxy(Hb+Mb)] during constant work rate exercise was greater in N-BR and H-BR than CTRL, whereas during KE a significant difference vs. CTRL was observed only after N-BR. Maximal mitochondrial respiration determined ex vivo was not affected by either intervention. In N-BR, a significant impairment of oxidative metabolism occurred downstream of central cardiovascular O2 delivery and upstream of mitochondrial function, possibly at the level of the intramuscular matching between O2 supply and utilization and peripheral O2 diffusion. Superposition of hypoxia on bed rest did not aggravate, and partially reversed, the impairment of muscle oxidative function in vivo induced by bed rest. The effects of longer exposures will have to be determined. PMID:27197861

  18. SU-E-I-22: Dependence On Calibration Phantom and Field Area of the Conversion Factor Used to Calculate Skin Dose During Neuro-Interventional Fluoroscopic Procedures

    SciTech Connect

    Rana, V K; Vijayan, S; Rudin, S R; Bednarek, D R

    2014-06-01

    Purpose: To determine the appropriate calibration factor to use when calculating skin dose with our real-time dose-tracking system (DTS) during neuro-interventional fluoroscopic procedures by evaluating the difference in backscatter from different phantoms and as a function of entrance-skin field area. Methods: We developed a dose-tracking system to calculate and graphically display the cumulative skin-dose distribution in real time. To calibrate the DTS for neuro-interventional procedures, a phantom is needed that closely approximates the scattering properties of the head. We compared the x-ray backscatter from eight phantoms: 20-cm-thick solid water, 16-cm diameter water-filled container, 16-cm CTDI phantom, modified-ANSI head phantom, 20-cm-thick PMMA, Kyoto-Kagaku PBU- 50 head, Phantom-Labs SK-150 head, and RSD RS-240T head. The phantoms were placed on the patient table with the entrance surface at 15 cm tube-side from the isocenter of a Toshiba Infinix C-arm, and the entrance-skin exposure was measured with a calibrated 6-cc PTW ionization chamber. The measurement included primary radiation, backscatter from the phantom and forward scatter from the table and pad. The variation in entrance-skin exposure was also measured as a function of the skin-entrance area for a 30x30 cm by 20-cm-thick PMMA phantom and the SK-150 head phantom using four different added beam filters. Results: The entranceskin exposure values measured for eight different phantoms differed by up to 12%, while the ratio of entrance exposure of all phantoms relative to solid water showed less than 3% variation with kVp. The change in entrance-skin exposure with entrance-skin area was found to differ for the SK-150 head compared to the 20-cm PMMA phantom and the variation with field area was dependent on the added beam filtration. Conclusion: To accurately calculate skin dose for neuro-interventional procedures with the DTS, the phantom for calibration should be carefully chosen since different

  19. Lactobacillus rhamnosus GG increases Toll-like receptor 3 gene expression in murine small intestine ex vivo and in vivo.

    PubMed

    Aoki-Yoshida, A; Saito, S; Fukiya, S; Aoki, R; Takayama, Y; Suzuki, C; Sonoyama, K

    2016-06-01

    Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo. We employed cultured intestinal organoids derived from small intestinal crypts as an ex vivo tissue model. LGG supplementation increased TLR3 mRNA levels in the intestinal organoids, as estimated by quantitative real-time polymerase chain reaction. Likewise, single and 7-day consecutive daily administrations of LGG increased TLR3 mRNA levels in the small intestine of C57BL/6N mice. The mRNA levels of other TLRs were not substantially altered both ex vivo and in vivo. In addition, LGG supplementation increased the mRNA levels of an antiviral type 1 interferon, interferon-α (IFN-α), and a neutrophil chemokine, CXCL1, upon stimulation with a synthetic TLR3 ligand, poly(I:C) in the intestinal organoids. LGG administration did not alter IFN-α and CXCL1 mRNA levels in the small intestine in vivo. Supplementation of other bacterial strains, Bifidobacterium bifidum and Lactobacillus paracasei, failed to increase TLR3 and poly(I:C)-stimulated CXCL1 mRNA levels ex vivo. We propose that upregulation of TLR3 gene expression may play a pivotal role in the therapeutic efficacy of LGG against rotavirus-associated diarrhoea. In addition, we demonstrated that intestinal organoids may be a promising ex vivo tissue model for investigating host-pathogen interactions and the antiviral action of probiotics in the intestinal epithelium. PMID:27013459

  20. [Anti-platelet actions of salicylates: in vivo, ex vivo and in vitro effects of choline salicylate].

    PubMed

    Irino, O; Saitoh, K; Ohkubo, K

    1985-07-01

    Effects of choline salicylate, sodium salicylate, choline chloride and acetylsalicylic acid on platelet aggregation in vivo, ex vivo and in vitro in mice were studied. These drugs all inhibited adenosine diphosphate (ADP)-induced respiratory depression, which is closely related to platelet aggregation in vivo, with choline salicylate showing the strongest inhibitory effect. Choline salicylate had a tendency to reduce the mortality of animals injected intravenously with endotoxin, but the other drugs had no such effect. The inhibitory effects of these drugs on ADP-induced platelet aggregation ex vivo were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate greater than choline chloride congruent to no effect, and plasma concentrations of protein-unbound salicylic acid at 1 hr after oral administration of drugs were in the order of choline salicylate greater than acetylsalicylic acid congruent to sodium salicylate. The in vitro effects of these drugs were in the order of choline salicylate congruent to sodium salicylate greater than choline chloride congruent to acetylsalicylic acid congruent to no effect. Therefore, it was considered that salicylic acid played an important role on the in vivo, ex vivo and in vitro effects of choline salicylate and that choline increased plasma concentrations of salicylic acid and consequently enhanced the in vivo and ex vivo effects of salicylic acid. Furthermore, the ex vivo effects of choline salicylate were found when ADP-induced platelet aggregation was measured with platelet-rich plasma prepared from blood collected with heparin as anti-coagulant, but not when blood was collected with citrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:4043866

  1. Optimized magnetic resonance diffusion protocol for ex-vivo whole human brain imaging with a clinical scanner

    NASA Astrophysics Data System (ADS)

    Scherrer, Benoit; Afacan, Onur; Stamm, Aymeric; Singh, Jolene; Warfield, Simon K.

    2015-03-01

    Diffusion-weighted magnetic resonance imaging (DW-MRI) provides a novel insight into the brain to facilitate our understanding of the brain connectivity and microstructure. While in-vivo DW-MRI enables imaging of living patients and longitudinal studies of brain changes, post-mortem ex-vivo DW-MRI has numerous advantages. Ex-vivo imaging benefits from greater resolution and sensitivity due to the lack of imaging time constraints; the use of tighter fitting coils; and the lack of movement artifacts. This allows characterization of normal and abnormal tissues with unprecedented resolution and sensitivity, facilitating our ability to investigate anatomical structures that are inaccessible in-vivo. This also offers the opportunity to develop today novel imaging biomarkers that will, with tomorrow's MR technology, enable improved in-vivo assessment of the risk of disease in an individual. Post-mortem studies, however, generally rely on the fixation of specimen to inhibit tissue decay which starts as soon as tissue is deprived from its blood supply. Unfortunately, fixation of tissues substantially alters tissue diffusivity profiles. In addition, ex-vivo DW-MRI requires particular care when packaging the specimen because the presence of microscopic air bubbles gives rise to geometric and intensity image distortion. In this work, we considered the specific requirements of post-mortem imaging and designed an optimized protocol for ex-vivo whole brain DW-MRI using a human clinical 3T scanner. Human clinical 3T scanners are available to a large number of researchers and, unlike most animal scanners, have a bore diameter large enough to image a whole human brain. Our optimized protocol will facilitate widespread ex-vivo investigations of large specimen.

  2. Development and application of an ex vivo fosphenytoin nasal bioconversion/permeability evaluation method.

    PubMed

    Antunes Viegas, Daniel; Rodrigues, Márcio; Francisco, Joana; Falcão, Amílcar; Alves, Gilberto; Santos, Adriana O

    2016-06-30

    There is an increasing interest in the intranasal delivery of central nervous system-active drugs due to the existence of a direct nose-to-brain connection. However, poor solubility limits the amount of drug that can be administered within an aqueous solution. In the present work, the objectives were to develop an ex vivo bioconversion/permeability evaluation method and to study the ex vivo bioconversion of the hydrophilic phosphate ester prodrug fosphenytoin (FOS) to the active drug phenytoin (PHT) and their comparative nasal permeation. Bioconversion/permeability studies were performed in excised porcine nasal mucosa mounted in Ussing chambers. The physical integrity of the tissues was evaluated by measurement of the transepithelial electrical resistance (TEER). The simultaneous quantitative assay of FOS, PHT and its major metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) was developed and validated according to international guidelines using a liquid chromatography analytical method. The FOS bioconversion rate and PHT and FOS apparent permeability coefficients (Papp) were determined at different time points. FOS bioconversion was also qualitatively investigated in human nasal mucus. The developed liquid chromatography method combines a fast and inexpensive sample preparation with inactivation of the enzymatic metabolism of the prodrug during sample manipulation and storage. It was linear, precise, accurate, and presented a high analyte recovery. FOS was converted ex vivo to PHT but the metabolite HPPH was not detected. The bioconversion rate increased with FOS concentration and with time, which suggests a diffusion-limited process. FOS was also converted to its active drug by human nasal mucus. A novel mathematical data analysis method was developed to reduce the bias introduced by variable mucosal TEER in the permeability results. At comparable FOS and PHT concentrations the ln(Papp(PHT)) of both compounds showed little difference, which indicates that

  3. Lung flooding enables efficient lung sonography and tumour imaging in human ex vivo and porcine in vivo lung cancer model

    PubMed Central

    2013-01-01

    Background Sonography has become the imaging technique of choice for guiding intraoperative interventions in abdominal surgery. Due to artefacts from residual air content, however, videothoracoscopic and open intraoperative ultrasound-guided thermoablation of lung malignancies are impossible. Lung flooding is a new method that allows complete ultrasound imaging of lungs and their tumours. Methods Fourteen resected tumourous human lung lobes were examined transpleurally with B-mode ultrasound before (in atelectasis) and after lung flooding with isotonic saline solution. In two swine, the left lung was filled with 15 ml/kg isotonic saline solution through the left side of a double-lumen tube. Lung tumours were simulated by transthoracic ultrasound-guided injection of 5 ml of purified bovine serum albumin in glutaraldehyde, centrally into the left lower lung lobe. The rate of tumour detection, the severity of disability caused by residual gas, and sonomorphology of the lungs and tumours were assessed. Results The ex vivo tumour detection rate was 100% in flooded human lung lobes and 43% (6/14) in atelectatic lungs. In all cases of atelectasis, sonographic tumour imaging was impaired by residual gas. Tumours and atelectatic tissue were isoechoic. In 28% of flooded lungs, a little residual gas was observed that did not impair sonographic tumour imaging. In contrast to tumours, flooded lung tissue was hyperechoic, homogeneous, and of fine-grained structure. Because of the bronchial wall three-laminar structure, sonographic differentiation of vessels and bronchi was possible. In all cases, malignant tumours in the flooded lung appeared well-demarcated from the lung parenchyma. Adenocarcinoma, squamous, and large cell carcinomas were hypoechoic. Bronchioloalveolar cell carcinoma was slightly hyperechoic. Transpleural sonography identifies endobronchial tumour growth and bronchial wall destruction. With transthoracic sonography, the flooded animal lung can be completely

  4. Granulocyte-Colony Stimulating Factor Related Pathways Tested on an Endometrial Ex-Vivo Model

    PubMed Central

    Rahmati, Mona; Petitbarat, Marie; Dubanchet, Sylvie; Bensussan, Armand; Chaouat, Gerard; Ledee, Nathalie

    2014-01-01

    Introduction Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. Materials and Methods Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. Results At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G-CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. Conclusion RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early

  5. Ex-vivo assessment of chronic toxicity of low levels of cadmium on testicular meiotic cells

    SciTech Connect

    Geoffroy-Siraudin, Cendrine; Perrard, Marie-Hélène; Ghalamoun-Slaimi, Rahma; Ali, Sazan; Chaspoul, Florence; Lanteaume, André; Achard, Vincent; Gallice, Philippe; Durand, Philippe; and others

    2012-08-01

    Using a validated model of culture of rat seminiferous tubules, we assessed the effects of 0.1, 1 and 10 μg/L cadmium (Cd) on spermatogenic cells over a 2‐week culture period. With concentrations of 1 and 10 μg/L in the culture medium, the Cd concentration in the cells, determined by ICP-MS, increased with concentration in the medium and the day of culture. Flow cytometric analysis enabled us to evaluate changes in the number of Sertoli cells and germ cells during the culture period. The number of Sertoli cells did not appear to be affected by Cd. By contrast, spermatogonia and meiotic cells were decreased by 1 and 10 μg/L Cd in a time and dose dependent manner. Stage distribution of the meiotic prophase I and qualitative study of the synaptonemal complexes (SC) at the pachytene stage were performed by immunocytochemistry with an anti SCP3 antibody. Cd caused a time-and-dose-dependent increase of total abnormalities, of fragmented SC and of asynapsis from concentration of 0.1 μg/L. Additionally, we observed a new SC abnormality, the “motheaten” SC. This abnormality is frequently associated with asynapsis and SC widening which increased with both the Cd concentration and the duration of exposure. This abnormality suggests that Cd disrupts the structure and function of proteins involved in pairing and/or meiotic recombination. These results show that Cd induces dose-and-time-dependent alterations of the meiotic process of spermatogenesis ex-vivo, and that the lowest metal concentration, which induces an adverse effect, may vary with the cell parameter studied. -- Highlights: ► Cadmium induces ex-vivo severe time- and dose-dependent germ cell abnormalities. ► Cadmium at very low concentration (0.1 µg/l) induces synaptonemal complex abnormalities. ► The lowest concentration inducing adverse effect varied with the cell parameter studied. ► Cadmium alters proteins involved in pairing and recombination. ► Cadmium leads to achiasmate univalents and

  6. Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model

    PubMed Central

    Medeiros, Israel L.; Pêgo-Fernandes, Paulo M.; Mariani, Alessandro W.; Fernandes, Flávio G.; Unterpertinger, Fernando V.; Canzian, Mauro; Jatene, Fabio B.

    2012-01-01

    OBJECTIVE: Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice. METHOD: Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex® was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters. RESULTS: After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p = 0.98). The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn·s·cm-5, respectively (p = 0.035). The mean pulmonary compliance was 46.8 cm H2O in Group 1 and 49.3 ml/cm H2O in Group 2 (p = 0.816). The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p = 0.87). The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p = 1.0), and the apoptotic cell counts were 118.75/mm2 and 137.50/mm2, respectively (p = 0.71). CONCLUSION: The locally produced preservation solution proved to be as good as Perfadex®. The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation. PMID:23018310

  7. Modeling the Human Tibiofemoral Joint Using Ex Vivo Determined Compliance Matrices.

    PubMed

    Lamberto, Giuliano; Richard, Vincent; Dumas, Raphaël; Valentini, Pier Paolo; Pennestrì, Ettore; Lu, Tung-Wu; Camomilla, Valentina; Cappozzo, Aurelio

    2016-06-01

    Several approaches have been used to devise a model of the human tibiofemoral joint for embedment in lower limb musculoskeletal models. However, no study has considered the use of cadaveric 6 × 6 compliance (or stiffness) matrices to model the tibiofemoral joint under normal or pathological conditions. The aim of this paper is to present a method to determine the compliance matrix of an ex vivo tibiofemoral joint for any given equilibrium pose. Experiments were carried out on a single ex vivo knee, first intact and, then, with the anterior cruciate ligament (ACL) transected. Controlled linear and angular displacements were imposed in single degree-of-freedom (DoF) tests to the specimen, and the resulting forces and moments were measured using an instrumented robotic arm. This was done starting from seven equilibrium poses characterized by the following flexion angles: 0 deg, 15 deg, 30 deg, 45 deg, 60 deg, 75 deg, and 90 deg. A compliance matrix for each of the selected equilibrium poses and for both the intact and ACL-deficient specimen was calculated. The matrix, embedding the experimental load-displacement relationship of the examined DoFs, was calculated using a linear least squares inversion based on a QR decomposition, assuming symmetric and positive-defined matrices. Single compliance matrix terms were in agreement with the literature. Results showed an overall increase of the compliance matrix terms due to the ACL transection (2.6 ratio for rotational terms at full extension) confirming its role in the joint stabilization. Validation experiments were carried out by performing a Lachman test (the tibia is pulled forward) under load control on both the intact and ACL-deficient knee and assessing the difference (error) between measured linear and angular displacements and those estimated using the appropriate compliance matrix. This error increased nonlinearly with respect to the values of the load. In particular, when an incremental posterior-anterior force

  8. The ex vivo antiplatelet activation potential of fruit phenolic metabolite hippuric acid.

    PubMed

    Santhakumar, Abishek Bommannan; Stanley, Roger; Singh, Indu

    2015-08-01

    Polyphenol-rich fruit and vegetable intake has been associated with reduction in platelet hyperactivity, a significant contributor to thrombus formation. This study was undertaken to investigate the possible role of hippuric acid, a predominant metabolite of plant cyclic polyols, phenolic acids and polyphenols, in reduction of platelet activation-related thrombogenesis. Fasting blood samples were collected from 13 healthy subjects to analyse the effect of varying concentrations of hippuric acid (100 μM, 200 μM, 500 μM, 1 mM and 2 mM) on activation-dependant platelet surface-marker expression. Procaspase activating compound-1 (PAC-1) and P-selectin/CD62P monoclonal antibodies were used to evaluate platelet activation-related conformational changes and α-granule release respectively using flow cytometry. Platelets were stimulated ex vivo via the P2Y1/P2Y12- adenosine diphosphate (ADP) pathway of platelet activation. Hippuric acid at a concentration of 1 mM and 2 mM significantly reduced P-selectin/CD62P expression (p = 0.03 and p < 0.001 respectively) induced by ADP. Hippuric acid at 2 mM concentration also inhibited PAC-1 activation-dependant antibody expression (p = 0.03). High ex vivo concentrations of hippuric acid can therefore significantly reduce P-selectin and PAC-1 expression thus reducing platelet activation and clotting potential. However, although up to 11 mM of hippuric acid can be excreted in the urine per day following consumption of fruit, hippuric acid is actively excreted with a recorded Cmax for hippuric acid in human plasma at 250-300 μM. This is lower than the blood concentration of 1-2 mM shown to be bioactive in this research. The contribution of hippuric acid to the protective effects of fruit and vegetable intake against vascular disorders by the pathways measured is therefore low but could be synergistic with lowered doses of antiplatelet drugs and help reduce risk of thrombosis in current antiplatelet drug sensitive populations. PMID

  9. Sensitivity and ex vivo validation of finite element models of the domestic pig cranium

    PubMed Central

    Bright, Jen A; Rayfield, Emily J

    2011-01-01

    A finite element (FE) validation and sensitivity study was undertaken on a modern domestic pig cranium. Bone strain data were collected ex vivo from strain gauges, and compared with results from specimen-specific FE models. An isotropic, homogeneous model was created, then input parameters were altered to investigate model sensitivity. Heterogeneous, isotropic models investigated the effects of a constant-thickness, stiffer outer layer (representing cortical bone) atop a more compliant interior (representing cancellous bone). Loading direction and placement of strain gauges were also varied, and the use of 2D membrane elements at strain gauge locations as a method of projecting 3D model strains into the plane of the gauge was investigated. The models correctly estimate the loading conditions of the experiment, yet at some locations fail to reproduce correct principal strain magnitudes, and hence strain ratios. Principal strain orientations are predicted well. The initial model was too stiff by approximately an order of magnitude. Introducing a compliant interior reported strain magnitudes more similar to the ex vivo results without notably affecting strain orientations, ratios or contour patterns, suggesting that this simple heterogeneity was the equivalent of reducing the overall stiffness of the model. Models were generally insensitive to moderate changes in loading direction or strain gauge placement, except in the squamosal portion of the zygomatic arch. The use of membrane elements made negligible differences to the reported strains. The models therefore seem most sensitive to changes in material properties, and suggest that failure to model local heterogeneity in material properties and structure of the bone may be responsible for discrepancies between the experimental and model results. This is partially attributable to a lack of resolution in the CT scans from which the model was built, and partially due to an absence of detailed material properties data

  10. Mathematical analysis of the temperature field during ex-vivo and in-vivo experimental laser interstitial thermotherapy (LITT) in breast tissue models

    NASA Astrophysics Data System (ADS)

    Manns, Fabrice; Milne, Peter J.; Salas, Nelson, Jr.; Pandya, Nish; Denham, David B.; Parel, Jean-Marie A.; Robinson, David S.

    1999-06-01

    Purpose: Laser interstitial thermotherapy is a promising minimally- invasive technique for the treatment of small cancers of the breast that are currently removed surgically lumpectomy. The purpose of this work was to analyze in situ temperature fields recorded with stainless-steel thermocoupled probes during experimental laser interstitial thermo-therapy (LITT). Methods: Both a CW Nd:YAG laser system emitting 20W for 25 to 30s and a 980 nm diode laser emitting 10 to 20 W for up to 1200s delivered through a fiber-optic probe were used to create localized heating in fatty cadaver pig tissue and milk as phantoms. To quantify an artifact due to direct heating of the thermocouple probes by laser radiation, experiments were also performed in air, water and intralipid solution. The temperature field around the fiber-optic probe during laser irradiation was measured every 0.3 s or 1 s with an array of up to fifteen needle thermocoupled probes. The effect of light absorption by the thermocouples probes was quantified and the time-dependence of the temperature distribution was analyzed. Results: After removal of the thermocouple artifact, the temperature was found to vary exponentially with time with a time constant of 600 to 700 s. Conclusions:The time-dependence of the interstitial temperature can be modeled by exponential functions both during ex vivo and in vivo experiments.

  11. Multi-Angle Switched HIFU: A New Ultrasound Device for Controlled Non-Invasive Induction of Small Spherical Ablation Zones—Simulation and Ex-Vivo Results

    NASA Astrophysics Data System (ADS)

    Novák, Petr; Jamshidi-Parsian, Azemat; Benson, Donny G.; Webber, Jessica S.; Moros, Eduardo G.; Shafirstein, Gal; Griffin, Robert J.

    2009-04-01

    Current HIFU devices produce elongated elliptical lesions (cigar shaped) in a single energy deposition. This prohibits the effective use of HIFU in small animal research as well as in clinical treatment where small volumes of tissue surrounded by critical structures need to be destroyed. We developed an ultrasound ablation device that non-invasively creates spheroidal lesions of an arbitrary diameter of up to 1 cm in a depth of up to 5 cm. The device consists of two focused ultrasound transducers aimed to the ablation target volume from two directions at a 90 degree angle. The operation of the transducers is switched back and forth so that only one transducer is energized at a time. A transient analysis of this ablation approach was performed using coupled simulations of acoustical pressure distributions, resulting temperature distributions, and thermal dose deposited to soft tissue. A prototype of the device was developed and tested in-vitro in a phantom and later in ex-vivo experiments in pig liver. The experimental results agreed with the numerical simulations and confirmed the ability of the multi-angle switched HIFU (MASH) device to create small spheroidal lesions in soft tissue within 2 minutes without significantly affecting the surrounding tissues.

  12. Photoacoustic tomography of joints aided by an Etanercept-conjugated gold nanoparticle contrast agent—an ex vivo preliminary rat study

    NASA Astrophysics Data System (ADS)

    Chamberland, David L.; Agarwal, Ashish; Kotov, Nicholas; Fowlkes, J. Brian; Carson, Paul L.; Wang, Xueding

    2008-03-01

    Monitoring of anti-rheumatic drug delivery in experimental models and in human diseases would undoubtedly be very helpful for both basic research and clinical management of inflammatory diseases. In this study, we have investigated the potential of an emerging hybrid imaging technology—photoacoustic tomography—in noninvasive monitoring of anti-TNF drug delivery. After the contrast agent composed of gold nanorods conjugated with Etanercept molecules was produced, ELISA experiments were performed to prove the conjugation and to show that the conjugated anti-TNF-α drug was biologically active. PAT of ex vivo rat tail joints with the joint connective tissue enhanced by intra-articularly injected contrast agent was conducted to examine the performance of PAT in visualizing the distribution of the gold-nanorod-conjugated drug in articular tissues. By using the described system, gold nanorods with a concentration down to 1 pM in phantoms or 10 pM in biological tissues can be imaged with good signal-to-noise ratio and high spatial resolution. This study demonstrates the feasibility of conjugating TNF antagonist pharmaceutical preparations with gold nanorods, preservation of the mechanism of action of TNF antagonist along with preliminary evaluation of novel PAT technology in imaging optical contrast agents conjugated with anti-rheumatic drugs. Further in vivo studies on animals are warranted to test the specific binding between such conjugates and targeted antigen in joint tissues affected by inflammation.

  13. A new era of thoracic oncology? Ex-vivo stereotactic ablative radiosurgery within Ex-vivo Lung Treatment System as a hybrid therapy for unresectable locally advanced pulmonary malignancies.

    PubMed

    Henkenberens, C; Zinne, N; Biancosino, C; Höffler, K; Schmitto, J D; Bremer, M; Haverich, A; Krüger, M

    2016-07-01

    The concept of oligometastases is the medical rationale for a local treatment of a limited number of metastatic tumor manifestations. Patients with pulmonary oligometastases are candidates for surgery or radiotherapy, however there are a number of technical issues that limit treatment. Technical issues relating to radiotherapy include organs at risk of irradiation, chest wall toxicity and decreased precision of tumor targeting because of breathing movements. Technical issues relating to surgery include loss of lung parenchyma and unresectability. We propose the hypothesis that ex-vivo radiosurgery as new hybrid technique in thoracic oncology has the capability to overcome these technical issues and will expand the medical spectrum in thoracic oncology. The proposed - highly complex - technique consists of surgical lung explantation, followed by stereotactic radiotherapy during ex-vivo perfusion followed by surgical re-implantation. PMID:27241251

  14. Metabolic profiling during ex vivo machine perfusion of the human liver

    PubMed Central

    Bruinsma, Bote G.; Sridharan, Gautham V.; Weeder, Pepijn D.; Avruch, James H.; Saeidi, Nima; Özer, Sinan; Geerts, Sharon; Porte, Robert J.; Heger, Michal; van Gulik, Thomas M.; Martins, Paulo N.; Markmann, James F.; Yeh, Heidi; Uygun, Korkut

    2016-01-01

    As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from ‘transplantable’ DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers. PMID:26935866

  15. Urinary concentrations and urine ex-vivo effect of mecillinam and sulphamethizole.

    PubMed

    Kerrn, M B; Frimodt-Møller, N; Espersen, F

    2004-01-01

    Healthy adult volunteers received 1 g of sulphamethizole orally (n = 10) and later 400 mg of pivmecillinam (274 mg of mecillinam) (n = 9). All urine was collected in defined periods over 24 h, and the drug concentrations in urine were determined. For sulphamethizole, the maximum urine concentration for seven subjects was reached in 0-3 h, and for the remaining three in 3-6 h. For mecillinam, eight of the nine subjects attained a maximum urine concentration in 0-3 h, after which the concentration declined rapidly for six subjects in 3-6 h. Strains of Escherichia coli with different MICs for sulphamethizole and mecillinam were exposed to collected urine for 2.5 h and 5 h. The results indicated that a sensitive E. coli population should be suppressed by sulphamethizole in urine for two-thirds of the time (with 1 g twice-daily) and by mecillinam in urine throughout the 24-h period (with 400 mg three times a day). There was a slight but significant correlation between the ex-vivo effect (Delta log10 CFU/mL) and the log10 concentration/MIC ratio after exposure to sulphamethizole for 5 h (r2 = 0.27, p < 0.0001), and a significant correlation between the variables with mecillinam (r2 = 0.66, p < 0.0001). PMID:14706087

  16. Real-time motion compensation for EM bronchoscope tracking with smooth output - ex-vivo validation

    NASA Astrophysics Data System (ADS)

    Reichl, Tobias; Gergel, Ingmar; Menzel, Manuela; Hautmann, Hubert; Wegner, Ingmar; Meinzer, Hans-Peter; Navab, Nassir

    2012-02-01

    Navigated bronchoscopy provides benefits for endoscopists and patients, but accurate tracking information is needed. We present a novel real-time approach for bronchoscope tracking combining electromagnetic (EM) tracking, airway segmentation, and a continuous model of output. We augment a previously published approach by including segmentation information in the tracking optimization instead of image similarity. Thus, the new approach is feasible in real-time. Since the true bronchoscope trajectory is continuous, the output is modeled using splines and the control points are optimized with respect to displacement from EM tracking measurements and spatial relation to segmented airways. Accuracy of the proposed method and its components is evaluated on a ventilated porcine ex-vivo lung with respect to ground truth data acquired from a human expert. We demonstrate the robustness of the output of the proposed method against added artificial noise in the input data. Smoothness in terms of inter-frame distance is shown to remain below 2 mm, even when up to 5 mm of Gaussian noise are added to the input. The approach is shown to be easily extensible to include other measures like image similarity.

  17. Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli.

    PubMed

    Lewis, Steven B; Prior, Alison; Ellis, Samuel J; Cook, Vivienne; Chan, Simon S M; Gelson, William; Schüller, Stephanie

    2016-01-01

    Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8. PMID:27446815

  18. Hybrid System for Ex Vivo Hemorheological and Hemodynamic Analysis: A Feasibility Study

    PubMed Central

    Yeom, Eunseop; Jun Kang, Yang; Joon Lee, Sang

    2015-01-01

    Precise measurement of biophysical properties is important to understand the relation between these properties and the outbreak of cardiovascular diseases (CVDs). However, a systematic measurement for these biophysical parameters under in vivo conditions is nearly impossible because of complex vessel shape and limited practicality. In vitro measurements can provide more biophysical information, but in vitro exposure changes hemorheological properties. In this study, a hybrid system composed of an ultrasound system and microfluidic device is proposed for monitoring hemorheological and hemodynamic properties under more reasonable experimental conditions. Biophysical properties including RBC aggregation, viscosity, velocity, and pressure of blood flows are simultaneously measured under various conditions to demonstrate the feasibility and performance of this measurement system. The proposed technique is applied to a rat extracorporeal loop which connects the aorta and jugular vein directly. As a result, the proposed system is found to measure biophysical parameters reasonably without blood collection from the rat and provided more detailed information. This hybrid system, combining ultrasound imaging and microfluidic techniques to ex vivo animal models, would be useful for monitoring the variations of biophysical properties induced by chemical agents. It can be used to understand the relation between biophysical parameters and CVDs. PMID:26090816

  19. Ex vivo evaluation of caries infiltration after different application times in primary molars.

    PubMed

    Soviero, V M; Paris, S; Leal, S C; Azevedo, R B; Meyer-Lueckel, H

    2013-01-01

    Low viscosity resins (infiltrants) have been shown to penetrate the lesion body of natural caries lesions almost completely in vitro. However, penetration depths (PD) have not been evaluated in vivo. Therefore, the aim of the present study was to evaluate the penetration of an infiltrant into proximal caries lesions in primary molars after different application times using an ex vivo model. 59 proximal lesions from 34 children were randomly allocated to one of the application times and were infiltrated under clinical conditions for 1, 3, or 5 min. After extraction or exfoliation (n = 48), teeth were sectioned perpendicular to their surfaces and lesion depths (LD) as well as lesion areas (LA) were evaluated using polarized light microscopy. PD and penetration areas (PA) were measured on scanning electron microscopic images. Percentage penetration depth (PPD) and percentage penetration area (PPA) were calculated. The mean (±SD) LD and LA were 596 ± 203 µm and 4.03 ± 2.75 × 10(5) µm(2), respectively. PPD ranged from 70 to 80% and PPA from 54 to 60%. Longer application times did not result in significantly deeper or more complete penetration (p > 0.05; ANOVA). In conclusion, proximal caries lesions in primary molars can be infiltrated in vivo to a similar extent as observed previously in vitro. Moreover, 1-min application of the infiltrant led to PD and homogeneity similar to those observed with longer application times up to 5 min. PMID:23207512

  20. Ex vivo study of bacterial coronal leakage in indirect pulp treatment

    PubMed Central

    Baca, Pilar; Pardo-Ridao, Maria M.; Arias-Moliz, Maria T.; Ferrer-Luque, Carmen M.

    2013-01-01

    Objective: The aim of this study was to evaluate, ex vivo, bacterial coronal leakage with different antimicrobial agents applied to the dentine for indirect pulp treatment (IPT). Study Design: Sixty extracted teeth were prepared and randomly distributed into 5 groups (n=10): Group 1: no antimicrobial dentine treatment; group 2: 1% chlorhexidine (CHX)+1% thymol varnish (Cervitec®); group 3: 2 % CHX solution; group 4: 40% CHX varnish (EC40™) and group 5: Clearfil™ Protect Bond (CPB). Ten teeth served as controls. The teeth were restored using a resin-modified glass ionomer cement (GIC) and then mounted in a two-chamber device. The coronal access was exposed to Streptococcus mutans for 45 days. The appearance of turbidity in the BHI broth of the lower chamber was considered as specimen leakage. Results: Survival analysis, determined by non parametric Kaplan-Meier and log-rank tests, showed that the best results were for groups EC40™+GIC and GIC alone; yet there were not statistically significant differences between them. All specimens of CPB+GIC and 2% CHX+GIC, leaked at 45 days. Conclusions: In IPT the use of GIC without pretreatment of the dentine and pretreatment with 40% CHX varnish resulted in a significant delay of bacterial coronal leakage. Key words:Streptococcus mutans, bacterial leakage, resin-modified glass ionomer cement, indirect pulp treatment. PMID:23229261

  1. Ex vivo micro-CT imaging of murine brain models using non-ionic iodinated contrast

    NASA Astrophysics Data System (ADS)

    Salas Bautista, N.; Martínez-Dávalos, A.; Rodríguez-Villafuerte, M.; Murrieta-Rodríguez, T.; Manjarrez-Marmolejo, J.; Franco-Pérez, J.; Calvillo-Velasco, M. E.

    2014-11-01

    Preclinical investigation of brain tumors is frequently carried out by means of intracranial implantation of brain tumor xenografts or allografts, with subsequent analysis of tumor growth using conventional histopathology. However, very little has been reported on the use contrast-enhanced techniques in micro-CT imaging for the study of malignant brain tumors in small animal models. The aim of this study has been to test a protocol for ex vivo imaging of murine brain models of glioblastoma multiforme (GBM) after treatment with non-ionic iodinated solution, using an in-house developed laboratory micro-CT. We have found that the best compromise between acquisition time and image quality is obtained using a 50 kVp, 0.5 mAs, 1° angular step on a 360 degree orbit acquisition protocol, with 70 μm reconstructed voxel size using the Feldkamp algorithm. With this parameters up to 4 murine brains can be scanned in tandem in less than 15 minutes. Image segmentation and analysis of three sample brains allowed identifying tumor volumes as small as 0.4 mm3.

  2. Stimulation of chondrocyte proliferation following photothermal, thermal, and mechanical injury in ex-vivo cartilage grafts

    NASA Astrophysics Data System (ADS)

    Pandoh, Nidhi S.; Truong, Mai T.; Diaz-Valdes, Sergio H.; Gardiner, David M.; Wong, Brian J.

    2002-06-01

    Laser irradiation may stimulate chondrocytes proliferation in the peripheral region surrounding a photothermally-heated area in rabbit nasal septal cartilage. In this study, ex- vivo rabbit nasal septal cartilages maintained in culture were irradiated with an Nd:YAG laser ((lambda) equals1.32 micrometers , 4-16 sec, 10-45 W/cm2) to examine the relationship between the diameter of replicating cells and irradiation time. Also, this study investigated whether proliferation occurs following heating (by immersion in hot saline baths, with a heated metal rod, and a soldering iron) and mechanical modification (crushing with a metal stamp and scoring with a scalpel). Replicating chondrocytes were identified using a Bromodeoxyuridine (BrdU) double antibody detection system in whole mount tissue. Light microscopy was used to confirm the presence of BrdU stained chondrocytes. The mechanical and thermal stressors used failed to produce a proliferative response in chondrocytes as previously seen with laser irradiation. We suspect that chondrocyte proliferation may be induced as a response to alteration in matrix structure produced by photothermal, thermal, or mechanical modification of the matrix. Heat generated by a laser to stimulate chondrocyte proliferation may lead to new treatment options for degenerative articular diseases and disorders. Laser technology can be adapted for use with minimally invasive surgical instrumentation to deliver light into otherwise inaccessible regions of the body.

  3. Radiation absorption in different kinds of tissue analysis: ex vivo study with supercontinuum laser source

    NASA Astrophysics Data System (ADS)

    Fornaini, Carlo; Merigo, Elisabetta; Selleri, Stefano; Cucinotta, Annamaria

    2016-03-01

    With the introduction of more and more new wavelengths, one of the main problems of medical laser users was centered on the study of laser-tissue interactions with the aim of determining the ideal wavelength for their treatments. The aim of this ex vivo study was to determine, by means of the utilization of a supercontinuum source, the amount of transmitted energy of different wavelengths in different organ samples obtained by Sprague Dawley rats. Supercontinuum light is generated by exploiting high optical non-linearity in a material and it combines the broadband attributes of a lamp with the spatial coherence and high brightness of laser. Even if the single transmission measurement does not allow us to separate out the respective contribution of scattering and absorption, it gives us an evaluation of the wavelengths not interacting with the tissue. In this way, being possible to determine what of the laser wavelengths are not useful or active in the different kinds of tissue, physicians may choose the proper device for his clinical treatments.

  4. Ex Vivo Assay of Electrical Stimulation to Rat Sciatic Nerves: Cell Behaviors and Growth Factor Expression.

    PubMed

    Du, Zhiyong; Bondarenko, Olexandr; Wang, Dingkun; Rouabhia, Mahmoud; Zhang, Ze

    2016-06-01

    Neurite outgrowth and axon regeneration are known to benefit from electrical stimulation. However, how neuritis and their surroundings react to electrical field is difficult to replicate by monolayer cell culture. In this work freshly harvested rat sciatic nerves were cultured and exposed to two types of electrical field, after which time the nerve tissues were immunohistologically stained and the expression of neurotrophic factors and cytokines were evaluated. ELISA assay was used to confirm the production of specific proteins. All cell populations survived the 48 h culture with little necrosis. Electrical stimulation was found to accelerate Wallerian degeneration and help Schwann cells to switch into migratory phenotype. Inductive electrical stimulation was shown to upregulate the secretion of multiple neurotrophic factors. Cellular distribution in nerve tissue was altered upon the application of an electrical field. This work thus presents an ex vivo model to study denervated axon in well controlled electrical field, bridging monolayer cell culture and animal experiment. It also demonstrated the critical role of electrical field distribution in regulating cellular activities. PMID:26516696

  5. Long-term ex vivo maintenance of testis tissues producing fertile sperm in a microfluidic device

    PubMed Central

    Komeya, Mitsuru; Kimura, Hiroshi; Nakamura, Hiroko; Yokonishi, Tetsuhiro; Sato, Takuya; Kojima, Kazuaki; Hayashi, Kazuaki; Katagiri, Kumiko; Yamanaka, Hiroyuki; Sanjo, Hiroyuki; Yao, Masahiro; Kamimura, Satoshi; Inoue, Kimiko; Ogonuki, Narumi; Ogura, Atsuo; Fujii, Teruo; Ogawa, Takehiko

    2016-01-01

    In contrast to cell cultures, particularly to cell lines, tissues or organs removed from the body cannot be maintained for long in any culture conditions. Although it is apparent that in vivo regional homeostasis is facilitated by the microvascular system, mimicking such a system ex vivo is difficult and has not been proved effective. Using the culture system of mouse spermatogenesis, we addressed this issue and devised a simple microfluidic device in which a porous membrane separates a tissue from the flowing medium, conceptually imitating the in vivo relationship between the microvascular flow and surrounding tissue. Testis tissues cultured in this device successfully maintained spermatogenesis for 6 months. The produced sperm were functional to generate healthy offspring with micro-insemination. In addition, the tissue kept producing testosterone and responded to stimulation by luteinizing hormone. These data suggest that the microfluidic device successfully created in vivo-like conditions, in which testis tissue maintained its physiologic functions and homeostasis. The present model of the device, therefore, would provide a valuable foundation of future improvement of culture conditions for various tissues and organs, and revolutionize the organ culture method as a whole. PMID:26892171

  6. Bridging the gap: functional healing of embryonic small intestine ex vivo.

    PubMed

    Coletta, Riccardo; Roberts, Neil A; Oltrabella, Francesca; Khalil, Basem A; Morabito, Antonino; Woolf, Adrian S

    2016-02-01

    The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2-3 mm tubes, which were placed in pairs, separated by a small gap, on semi-permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum-free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. PMID:26234729

  7. Interleukin-3 and ex vivo maintenance of hematopoietic stem cells: facts and controversies.

    PubMed

    Ivanovic, Zoran

    2004-01-01

    Although the utilization of IL-3 in the ex vivo expansion of hematopoietic stem cells has been considered as an attractive possibility, its mode of action remains unclear and controversial. Some reports show that IL-3 maintains or even enhances primitive stem cell activity, whereas others show the opposite. The presence of serum in culture media enhances the pro-differentiating effect of IL-3 on stem cells. Conversely, addition of IL-3 to serum-free cultures improves the capacity of TPO, SCF and Flt3-ligand to promote the self-renewal of primitive stem cells. The presence or absence of serum or of some serum substitutes (in serum-free cultures), as well as other culture parameters are probably responsible for these contrasting effects of IL-3 on stem cells. However, none of the data presently evaluated bring a clear, definitive explanation to this apparent paradox. Those data that appear to be the most informative are presented and discussed in this "technical review". PMID:15217747

  8. Spectral domain optical coherence tomography for ex vivo brain tumor analysis

    NASA Astrophysics Data System (ADS)

    Lenz, Marcel; Krug, Robin; Jaedicke, Volker; Stroop, Ralf; Schmieder, Kirsten; Hofmann, Martin R.

    2015-07-01

    Non-contact imaging methods to distinguish between healthy tissue and brain tumor tissue during surgery would be highly desirable but are not yet available. Optical Coherence Tomography (OCT) is a non-invasive imaging technology with a resolution around 1-15 μm and a penetration depth of 1-2 mm that may satisfy the demands. To analyze its potential, we measured ex vivo human brain tumor tissue samples from 10 patients with a Spectral Domain OCT system (Thorlabs Callisto: center wavelength of 930 nm) and compared the results with standard histology. In detail, three different measurements were made for each sample. First the sample was measured directly after surgery. Then it was embedded in paraffin (also H and E staining) and examined for the second time. At last, the slices of each paraffin block cut by the pathology were measured. Each time a B-scan was created and for a better comparison with the histology a 3D image was generated, in order to get the corresponding en face images. In both, histopathological diagnosis and the analysis of the OCT images, different types of brain tumor showed difference in structure. This has been affirmed by two blinded investigators. Nevertheless the difference between two images of samples taken directly after surgery is less distinct. To enhance the contrast in the images further, we employ Spectroscopic OCT and pattern recognition algorithms and compare these results to the histopathological standard.

  9. Transcostal high-intensity-focused ultrasound: ex vivo adaptive focusing feasibility study

    PubMed Central

    Aubry, Jean-François; Pernot, Mathieu; Marquet, Fabrice; Tanter, Mickaël; Fink, Mathias

    2008-01-01

    Ex vivo experiments have been conducted through excised pork rib with bone, cartilage, muscle and skin. The aberrating effect of the ribcage has been experimentally evaluated. Adaptive ultrasonic focusing through ribs has been studied at low power. Without any correction, the pressure fields in the focal plane were both affected by inhomogeneous attenuation and phase distortion and three main effects were observed: a mean 2 mm shift of the main lobe, a mean 1.25 mm spreading of the half width of the main lobe and up to 20 dB increase of the secondary lobe level. Thanks to time reversal focusing, a 5-dB decrease in the secondary lobes was obtained and the ratio between the energy deposited at the target location and the total amount of energy emitted by the therapeutic array was 6 times higher than without correction. Time reversal minimizes the heating of the ribs by automatically sonicating between the ribs, as demonstrated by temperature measurements using thermocouples placed at different location on the ribcage. It is also discussed how this aberration correction process could be achieved non invasively for clinical application. PMID:18475006

  10. Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

    PubMed Central

    Piotrowska-Nitsche, Karolina; Caspary, Tamara

    2013-01-01

    We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time. PMID:23666396

  11. Thermal analysis of laser interstitial thermotherapy in ex vivo fibro-fatty tissue using exponential functions

    NASA Astrophysics Data System (ADS)

    Salas, Nelson, Jr.; Manns, Fabrice; Milne, Peter J.; Denham, David B.; Minhaj, Ahmed M.; Parel, Jean-Marie; Robinson, David S.

    2004-05-01

    A therapeutic procedure to treat small, surface breast tumours up to 10 mm in radius plus a 5 mm margin of healthy, surrounding tissue using laser interstitial thermotherapy (LITT) is currently being investigated. The purpose of this study is to analyse and model the thermal and coagulative response of ex vivo fibro-fatty tissue, a model for breast tissue, during experimental laser interstitial thermotherapy at 980 nm. Laser radiation at 980 nm was delivered interstitially through a diffusing tip optical fibre inserted into a fibro-fatty tissue model to produce controlled heating at powers ranging from 3.2 to 8.0 W. Tissue temperature was measured with thermocouples placed at 15 positions around the fibre. The induced coagulation zone was measured on gross anatomical sections. Thermal analysis indicates that a finite sum of exponential functions is an approximate solution to the heat conduction equation that more accurately predicts the time-temperature dependence in tissue prior to carbonization (T < 100 °C) during LITT than the traditional model using a single exponential function. Analysis of the ellipsoid coagulation volume induced in tissue indicates that the 980 nm wavelength does not penetrate deep enough in fibro-fatty tissue to produce a desired 30 mm diameter (14.1 × 103 mm3) coagulation volume without unwanted tissue liquefaction and carbonization.

  12. Effect of penetration enhancers on gel formulation of Zidovudine: in vivo and ex vivo studies.

    PubMed

    Pokharkar, Varsha; Dhar, Sheetal; Singh, Nripendra

    2010-01-01

    To overcome many challenges associated with antiretroviral drug therapy, novel drug delivery systems present an opportunity for formulation scientists to improve the management of patients with HIV/AIDS. The purpose of this study was to prepare a transdermal delivery system for zidovudine using different penetration enhancers incorporated in carbopol 971P gel and to evaluate the same for rheology, percent drug content, drug deposition, in vitro, ex vivo, and in vivo permeation across rat skin. The rheology studies indicated that 1% w/w carbopol gel had a higher linear viscoelastic region, good creep recovery, and desirable viscosity. Among all gel formulations, gel containing cineole and menthol as penetration enhancers attained a steady-state flux of 5.9 mg/cm(2)/h and 5.4 mg/cm(2)/h of zidovudine, respectively, leading to plasma concentration in the therapeutic range. The drug deposition was also found to be highest in the case of gel containing cineole and menthol as penetration enhancers. The results indicated a linear relationship between in vitro flux and in vivo bioavailability of zidovudine transdermal gel. PMID:21502034

  13. Virulence Diversity among Bacteremic Aeromonas Isolates: Ex Vivo, Animal, and Clinical Evidences

    PubMed Central

    Chen, Po-Lin; Wu, Chi-Jung; Tsai, Pei-Jane; Tang, Hung-Jen; Chuang, Yin-Ching; Lee, Nan-Yao; Lee, Ching-Chi; Li, Chia-Wen; Li, Ming-Chi; Chen, Chi-Chung; Tsai, Hung-Wen; Ou, Chun-Chun; Chen, Chang-Shi; Ko, Wen-Chien

    2014-01-01

    Background The objective of this study was to compare virulence among different Aeromonas species causing bloodstream infections. Methodology/Principal Findings Nine of four species of Aeromonas blood isolates, including A. dhakensis, A. hydrophila, A. veronii and A. caviae were randomly selected for analysis. The species was identified by the DNA sequence matching of rpoD. Clinically, the patients with A. dhakensis bacteremia had a higher sepsis-related mortality rate than those with other species (37.5% vs. 0%, P = 0.028). Virulence of different Aeromonas species were tested in C. elegans, mouse fibroblast C2C12 cell line and BALB/c mice models. C. elegans fed with A. dhakensis and A. caviae had the lowest and highest survival rates compared with other species, respectively (all P values <0.0001). A. dhakensis isolates also exhibited more cytotoxicity in C2C12 cell line (all P values <0.0001). Fourteen-day survival rate of mice intramuscularly inoculated with A. dhakensis was lower than that of other species (all P values <0.0001). Hemolytic activity and several virulence factor genes were rarely detected in the A. caviae isolates. Conclusions/Significance Clinical data, ex vivo experiments, and animal studies suggest there is virulence variation among clinically important Aeromonas species. PMID:25375798

  14. Two-photon excited fluorescence microscopy application for ex vivo investigation of ocular fundus samples

    NASA Astrophysics Data System (ADS)

    Peters, Sven; Hammer, Martin; Schweitzer, Dietrich

    2011-07-01

    Two-photon excited fluorescence (TPEF) imaging of ocular tissue has recently become a promising tool in ophthalmology for diagnostic and research purposes. The feasibility and the advantages of TPEF imaging, namely deeper tissue penetration and improved high-resolution imaging of microstructures, have been demonstrated lately using human ocular samples. The autofluorescence properties of endogenous fluorophores in ocular fundus tissue are well known from spectrophotometric analysis. But fluorophores, especially when it comes to fluorescence lifetime, typically display a dependence of their fluorescence properties on local environmental parameters. Hence, a more detailed investigation of ocular fundus autofluorescence ideally in vivo is of utmost interest. The aim of this study is to determine space-resolved the stationary and time-resolved fluorescence properties of endogenous fluorophores in ex vivo porcine ocular fundus samples by means of two-photon excited fluorescence spectrum and lifetime imaging microscopy (FSIM/FLIM). By our first results, we characterized the autofluorescence of individual anatomical structures of porcine retina samples excited at 760 nm. The fluorescence properties of almost all investigated retinal layers are relatively homogenous. But as previously unknown, ganglion cell bodies show a significantly shorter fluorescence lifetime compared to the adjacent mueller cells. Since all retinal layers exhibit bi-exponential autofluorescence decays, we were able to achieve a more precise characterization of fluorescence properties of endogenous fluorophores compared to a present in vivo FLIM approach by confocal scanning laser ophthalmoscope (cSLO).

  15. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation

    NASA Astrophysics Data System (ADS)

    Jackson, E. J.; Coussios, C.-C.; Cleveland, R. O.

    2014-06-01

    Thermal ablation by high intensity focused ultrasound (HIFU) has a great potential for the non-invasive treatment of solid tumours. Due to the high pressure amplitudes involved, nonlinear acoustic effects must be understood and the relevant medium property is the parameter of nonlinearity B/A. Here, B/A was measured in ex vivo bovine liver, over a heating/cooling cycle replicating temperatures reached during HIFU ablation, adapting a finite amplitude insertion technique, which also allowed for measurement of sound-speed and attenuation. The method measures the nonlinear progression of a plane wave through liver and B/A was chosen so that numerical simulations matched the measured waveforms. To create plane-wave conditions, sinusoidal bursts were transmitted by a 100 mm diameter 1.125 MHz unfocused transducer and measured using a 15 mm diameter 2.25 MHz broadband transducer in the near field. Attenuation and sound-speed were calculated using a reflected pulse from the smaller transducer using the larger transducer as the reflecting interface. Results showed that attenuation initially decreased with heating then increased after denaturation, the sound-speed initially increased with temperature and then decreased, and B/A showed an increase with temperature but no significant post-heating change. The B/A data disagree with other reports that show a significant change and we suggest that any nonlinear enhancement in the received ultrasound signal post-treatment is likely due to acoustic cavitation rather than changes in tissue nonlinearity.

  16. Fluorescence Lifetime Imaging and Intravascular Ultrasound: Co-Registration Study Using Ex Vivo Human Coronaries

    PubMed Central

    Gorpas, Dimitris; Fatakdawala, Hussain; Bec, Julien; Ma, Dinglong; Yankelevich, Diego R.; Qi, Jinyi

    2015-01-01

    Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques. PMID:25163056

  17. Metabolic profiling during ex vivo machine perfusion of the human liver.

    PubMed

    Bruinsma, Bote G; Sridharan, Gautham V; Weeder, Pepijn D; Avruch, James H; Saeidi, Nima; Özer, Sinan; Geerts, Sharon; Porte, Robert J; Heger, Michal; van Gulik, Thomas M; Martins, Paulo N; Markmann, James F; Yeh, Heidi; Uygun, Korkut

    2016-01-01

    As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from 'transplantable' DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers. PMID:26935866

  18. The isolated perfused equine distal limb as an ex vivo model for pharmacokinetic studies.

    PubMed

    Friebe, M; Stahl, J; Kietzmann, M

    2013-06-01

    Even though intra-articular injections play an important role in the treatment of joint-related lameness in horses, little is known about pharmacokinetic properties of substances used. Therefore, an ex vivo model for pharmacokinetic studies was developed using distal forelimbs of slaughtered horses. The extremity was perfused with gassed Tyrode solution for up to 8 h. Tissue viability was confirmed by measurements of glucose consumption, lactate production, and lactate dehydrogenase activity in the perfusate. Standard criteria for tissue viability had been determined in preliminary experiments (n = 11), which also included histological examinations of the joint capsule. As the model's first implementation, the articular efflux rate of betamethasone (BM), administered as BM disodium phosphate intra-articularly to the fetlock joint (4 mg BM/joint), was investigated. The concentration of BM in the venous perfusate of the radial vein was measured by means of high-performance liquid chromatography. The average BM efflux rate per minute was calculated to be 5.1 μg/min with values ranging from 9 μg/min to 2.9 μg/min. 7.5 h after i.a. application, 2.3 mg BM had left the joint via the radial vein. Using this inexpensive setup, the presented model allows studying a variety of pharmacological topics without the ethical limitations of animal studies. PMID:22913456

  19. Ex vivo imaging of early dental caries within the interproximal space

    NASA Astrophysics Data System (ADS)

    Choo-Smith, Lin-P'ing; Hewko, Mark D.; Dufour, Marc L.; Fulton, Crystal; Qiu, Pingli; Gauthier, Bruno; Padioleau, Christian; Bisaillon, Charles-Etienne; Dong, Cecilia; Cleghorn, Blaine M.; Lamouche, Guy; Sowa, Michael G.

    2009-02-01

    Optical coherence tomography (OCT) is emerging as a technology that can potentially be used for the detection and monitoring of early dental enamel caries since it can provide high-resolution depth imaging of early lesions. To date, most caries detection optical technologies are well suited for examining caries at facial, lingual, incisal and occlusal surfaces. The approximal surfaces between adjacent teeth are difficult to examine due to lack of visual access and limited space for these new caries detection tools. Using a catheter-style probe developed at the NRC-Industrial Materials Institute, the probe was inserted into the interproximal space to examine the approximal surfaces with OCT imaging at 1310 nm. The probe was rotated continuously and translated axially to generate depth images in a spiral fashion. The probe was used in a mock tooth arch model consisting of extracted human teeth mounted with dental rope wax in their anatomically correct positions. With this ex vivo model, the probe provided images of the approximal surfaces revealing morphological structural details, regions of calculus, and especially regions of early dental caries (white spot lesions). Results were compared with those obtained from OCT imaging of individual samples where the approximal surfaces of extracted teeth are accessible on a lab-bench. Issues regarding access, regions of interest, and factors to be considered in an in vivo setting will be discussed. Future studies are aimed at using the probe in vivo with patient volunteers.

  20. Prion structure investigated in situ, ex vivo, and in vitro by FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Kneipp, Janina; Miller, Lisa M.; Spassov, Sashko; Sokolowski, Fabian; Lasch, Peter; Beekes, Michael; Naumann, Dieter

    2004-07-01

    Syrian hamster nervous tissue was investigated by FTIR microspectroscopy with conventional and synchrotron infrared light sources. Various tissue structures from the cerebellum and medulla oblongata of scrapie-infected and control hamsters were investigated at a spatial resolution of 50 μm. Single neurons in dorsal root ganglia of scrapie-infected hamsters were analyzed by raster scan mapping at 6 μm spatial resolution. These measurements enabled us to (i) scrutinize structural differences between infected and non-infected tissue and (ii) analyze for the first time the distribution of different protein structures in situ within single nerve cells. Single nerve cells exhibited areas of increased β-sheet content, which co-localized consistently with accumulations of the pathological prion protein (PrPSc). Spectral data were also obtained from purified, partly proteinase K digested PrPSc isolated from scrapie-infected nervous tissue of hamsters to elucidate similarities/dissimilarities between prion structure in situ and ex vivo. A further comparison is drawn to the recombinant Syrian hamster prion protein SHaPrP90-232, whose in vitro transition from the predominantly a-helical isoform to β-sheet rich oligomeric structures was also investigated by FTIR spectroscopy.

  1. Changes in gluconeogenesis and intracellular lipid accumulation characterize uremic human hepatocytes ex vivo.

    PubMed

    Li, Meng; Ellis, Ewa; Johansson, Helene; Nowak, Greg; Isaksson, Bengt; Gnocchi, Davide; Parini, Paolo; Axelsson, Jonas

    2016-06-01

    It is well known that reduced glomerular filtration rate (GFR) leads to an increased risk of dyslipidemia, insulin resistance, and cardiovascular mortality. The liver is a central organ for metabolism, but its function in the uremic setting is still poorly characterized. We used human primary hepatocytes isolated from livers of nine donors with normal renal function to investigate perturbations in key metabolic pathways following exposure to uremic (n = 8) or healthy (n = 8) sera, and to serum-free control medium. Both uremic and healthy elicited consistent responses from hepatocytes from multiple donors and compared with serum-free control. However, at physiological insulin concentrations, uremic cells accumulated 56% more intracellular lipids. Also, when comparing uremic with healthy medium after culture, it contained more very-low-density lipoprotein-triglyceride and glucose. These changes were accompanied by decreased phosphorylation of AktS473 mRNA levels of key regulators of gluconeogenesis in uremic sera-treated hepatocytes such as phosphoenolpyruvate carboxykinase 1 and glucose 6-phosphate were elevated. We also found increased expression of 11β-hydroxysteroid dehydrogenase mRNA in uremic cells, along with high phosphorylation of downstream p53 and phospholipase C-γ1Y783 Thus our ex vivo data suggest that the uremic hepatocytes rapidly develop a glycogenic and lipogenic condition accompanied by perturbations in a large number of signaling networks. PMID:27056725

  2. Fluorescence lifetime imaging and intravascular ultrasound: co-registration study using ex vivo human coronaries.

    PubMed

    Gorpas, Dimitris; Fatakdawala, Hussain; Bec, Julien; Ma, Dinglong; Yankelevich, Diego R; Qi, Jinyi; Marcu, Laura

    2015-01-01

    Fluorescence lifetime imaging (FLIM) has demonstrated potential for robust assessment of atherosclerotic plaques biochemical composition and for complementing conventional intravascular ultrasound (IVUS), which provides information on plaque morphology. The success of such a bi-modal imaging modality depends on accurate segmentation of the IVUS images and proper angular registration between these two modalities. This paper reports a novel IVUS segmentation methodology addressing this issue. The image preprocessing consisted of denoising, using the Wiener filter, followed by image smoothing, implemented through the application of the alternating sequential filter on the edge separability metric images. Extraction of the lumen/intima and media/adventitia boundaries was achieved by tracing the gray-scale peaks over the A-lines of the IVUS preprocessed images. Cubic spline interpolation, in both cross-sectional and longitudinal directions, ensured boundary smoothness and continuity. The detection of the guide-wire artifact in both modalities is used for angular registration. Intraluminal studies were conducted in 13 ex vivo segments of human coronaries. The IVUS segmentation accuracy was assessed against independent manual tracings, providing 91.82% sensitivity and 97.55% specificity. The proposed methodology makes the bi-modal FLIM and IVUS approach feasible for comprehensive intravascular diagnosis by providing co-registered biochemical and morphological information of atherosclerotic plaques. PMID:25163056

  3. Dynamic vocal fold parameters with changing adduction in ex-vivo hemilarynx experiments.

    PubMed

    Döllinger, Michael; Berry, David A; Kniesburges, Stefan

    2016-05-01

    Ex-vivo hemilarynx experiments allow the visualization and quantification of three-dimensional dynamics of the medial vocal fold surface. For three excised human male larynges, the vibrational output, the glottal flow resistance, and the sound pressure during sustained phonation were analyzed as a function of vocal fold adduction for varying subglottal pressure. Empirical eigenfunctions, displacements, and velocities were investigated along the vocal fold surface. For two larynges, an increase of adduction level resulted in an increase of the glottal flow resistance at equal subglottal pressures. This caused an increase of lateral and vertical oscillation amplitudes and velocity indicating an improved energy transfer from the airflow to the vocal folds. In contrast, the third larynx exhibited an amplitude decrease for rising adduction accompanying reduction of the flow resistance. By evaluating the empirical eigenfunctions, this reduced flow resistance was assigned to an unbalanced oscillation pattern with predominantly lateral amplitudes. The results suggest that adduction facilitates the phonatory process by increasing the glottal flow resistance and enhancing the vibrational amplitudes. However, this interrelation only holds for a maintained balanced ratio between vertical and lateral displacements. Indeed, a balanced vertical-lateral oscillation pattern may be more beneficial to phonation than strong periodicity with predominantly lateral vibrations. PMID:27250133

  4. Functional Blocking of Staphylococcus aureus Adhesins following Growth in Ex Vivo Media

    PubMed Central

    Massey, Ruth C.; Dissanayeke, Shobana R.; Cameron, Brian; Ferguson, David; Foster, Timothy J.; Peacock, Sharon J.

    2002-01-01

    Defining the role of Staphylococcus aureus adhesins in disease pathogenesis may depend on the use of bacteria grown in culture media that more closely reflect the human milieu than conventional broth. This study examined the functional effect on S. aureus adhesins following growth in an ex vivo medium containing a complex mixture of human proteins (used peritoneal dialysate) relative to growth in Todd-Hewitt broth. The adherence of S. aureus, cultured in dialysate, to fibronectin and fibrinogen was markedly reduced despite the expresion of full-length ClfA, ClfB, and fibronectin-binding proteins. Growth in dialysate resulted in the acquisition of a surface coat, as visualized by transmission electron microscopy, which was shown to contain fibronectin, fibrinogen, and immunoglobulins. Adherence of S. aureus to fibrinogen following growth in dialysate was significantly reduced by expression of protein A but was restored following growth in immunoglobulin-depleted dialysate. We conclude that bacterial adherence to solid-phase protein is critically dependent on the culture medium, that S. aureus adhesins may become saturated with target protein prior to contact with solid surfaces, and that there is an interaction between fibrinogen-binding proteins and immunoglobulin bound to protein A following contact with host proteins. These findings have important implications for future studies of S. aureus adhesins. PMID:12228257

  5. Ex vivo peripheral nerve detection of rats by spontaneous Raman spectroscopy

    PubMed Central

    Minamikawa, Takeo; Harada, Yoshinori; Takamatsu, Tetsuro

    2015-01-01

    Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future. PMID:26602842

  6. Cholesterol-lowering properties of Ganoderma lucidum in vitro, ex vivo, and in hamsters and minipigs

    PubMed Central

    Berger, A; Rein, D; Kratky, E; Monnard, I; Hajjaj, H; Meirim, I; Piguet-Welsch, C; Hauser, J; Mace, K; Niederberger, P

    2004-01-01

    Introduction There has been renewed interest in mushroom medicinal properties. We studied cholesterol lowering properties of Ganoderma lucidum (Gl), a renowned medicinal species. Results Organic fractions containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes. In hamsters, 5% Gl did not effect LDL; but decreased total cholesterol (TC) 9.8%, and HDL 11.2%. Gl (2.5 and 5%) had effects on several fecal neutral sterols and bile acids. Both Gl doses reduced hepatic microsomal ex-vivo HMG-CoA reductase activity. In minipigs, 2.5 Gl decreased TC, LDL- and HDL cholesterol 20, 27, and 18%, respectively (P < 0.05); increased fecal cholestanol and coprostanol; and decreased cholate. Conclusions Overall, Gl has potential to reduce LDL cholesterol in vivo through various mechanisms. Next steps are to: fully characterize bioactive components in lipid soluble/insoluble fractions; evaluate bioactivity of isolated fractions; and examine human cholesterol lowering properties. Innovative new cholesterol-lowering foods and medicines containing Gl are envisioned. PMID:14969592

  7. In vitro and ex vivo assays of virulence in Candida albicans.

    PubMed

    Calderone, Richard A

    2009-01-01

    The measurement of virulence using ex vivo and in vitro models is discussed in the context of the human pathogenic yeast, Candida albicans. The models described are of two types. First, reconstituted tissues of various sorts are used that are derived from human carcinomas. The tissues are grown in vitro in complex media, attain a three-dimensional tissue structure, and retain cell-surface antigens typical of the specific tissue. Both adherence and invasion of tissues can be studied following infection with strains of C. albicans (1, 2). Further, one can increase the level of complexity by providing infected tissues with host phagocytes or cytokines such that an immune contribution to protection can be followed (3-5). The second model employs Drosophila melanogaster larvae that are infected with C. albicans (6). In this model, the progression of virulence is followed after injection of strains of a pathogen of interest into the fly abdomen. Thus, in the case of human pathogenic fungi, the recognition of host tissues and invasion by the specific pathogen can be studied in vitro and correlations developed for human disease. The obvious advantage to using animal models (e.g., mice) is reduced cost, such that large numbers of C. albicans strains can be assessed for their virulence properties. Additionally, another application of these models is in drug discovery. It is clear that there are both advantages and disadvantages of the use of alternate models other than a murine model, to evaluate disease, and this is discussed below. PMID:19152042

  8. CD1c tetramers detect ex vivo T cell responses to processed phosphomycoketide antigens

    PubMed Central

    Ly, Dalam; Kasmar, Anne G.; Cheng, Tan-Yun; de Jong, Annemieke; Huang, Shouxiong; Roy, Sobhan; Bhatt, Apoorva; van Summeren, Ruben P.; Altman, John D.; Jacobs, William R.; Adams, Erin J.; Minnaard, Adriaan J.; Porcelli, Steven A.

    2013-01-01

    CD1c is expressed with high density on human dendritic cells (DCs) and B cells, yet its antigen presentation functions are the least well understood among CD1 family members. Using a CD1c-reactive T cell line (DN6) to complete an organism-wide survey of M. tuberculosis lipids, we identified C32 phosphomycoketide (PM) as a previously unknown molecule and a CD1c-presented antigen. CD1c binding and presentation of mycoketide antigens absolutely required the unusual, mycobacteria-specific lipid branching patterns introduced by polyketide synthase 12 (pks12). Unexpectedly, one TCR responded to diversely glycosylated and unglycosylated forms of mycoketide when presented by DCs and B cells. Yet cell-free systems showed that recognition was mediated only by the deglycosylated phosphoantigen. These studies identify antigen processing of a natural bacterial antigen in the human CD1c system, indicating that cells act on glycolipids to generate a highly simplified neoepitope composed of a sugar-free phosphate anion. Using knowledge of this processed antigen, we generated human CD1c tetramers, and demonstrate that CD1c–PM complexes stain T cell receptors (TCRs), providing direct evidence for a ternary interaction among CD1c-lipid-TCR. Furthermore, PM-loaded CD1c tetramers detect fresh human T cells from peripheral blood, demonstrating a polyclonal response to PM antigens in humans ex vivo. PMID:23530121

  9. 4D optical coherence tomography of aortic valve dynamics in a murine mouse model ex vivo

    NASA Astrophysics Data System (ADS)

    Schnabel, Christian; Jannasch, Anett; Faak, Saskia; Waldow, Thomas; Koch, Edmund

    2015-07-01

    The heart and its mechanical components, especially the heart valves and leaflets, are under enormous strain during lifetime. Like all highly stressed materials, also these biological components undergo fatigue and signs of wear, which impinge upon cardiac output and in the end on health and living comfort of affected patients. Thereby pathophysiological changes of the aortic valve leading to calcific aortic valve stenosis (AVS) as most frequent heart valve disease in humans are of particular interest. The knowledge about changes of the dynamic behavior during the course of this disease and the possibility of early stage diagnosis could lead to the development of new treatment strategies and drug-based options of prevention or therapy. ApoE-/- mice as established model of AVS versus wildtype mice were introduced in an ex vivo artificially stimulated heart model. 4D optical coherence tomography (OCT) in combination with high-speed video microscopy were applied to characterize dynamic behavior of the murine aortic valve and to characterize dynamic properties during artificial stimulation. OCT and high-speed video microscopy with high spatial and temporal resolution represent promising tools for the investigation of dynamic behavior and their changes in calcific aortic stenosis disease models in mice.

  10. Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry

    NASA Astrophysics Data System (ADS)

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ashraf, Sumara; Ahmad, Shakil; Ikram, Masroor

    2015-05-01

    Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT), retardance (RT), depolarization (ΔT), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT, ΔL, RT,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DT and D showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues.

  11. Nerve damage assessment following implant placement in human cadaver jaws: an ex vivo comparative study.

    PubMed

    Murat, Sema; Kamburoğlu, Kıvanç; Kılıç, Cenk; Ozen, Tuncer; Gurbuz, Ayhan

    2014-02-01

    The present study compared the use of cone beam computerized tomography (CBCT) images and intra-oral radiographs in the placement of final implant drills in terms of nerve damage to cadaver mandibles. Twelve cadaver hemimandibles obtained from 6 cadavers were used. Right hemimandibles were imaged using peri-apical radiography and left hemimandibles using CBCT, and the images obtained were used in treatment planning for the placement of implant drills (22 for each modality, for a total of 44 final drills). Specimens were dissected, and the distances between the apex of the final implant drill and the inferior alveolar neurovascular bundle and incisive nerve were measured using a digital calliper. Nerves were assessed as damaged or not damaged, and the Chi-square test was used to compare nerve damage between modalities (P < 0.05). Nerve damage occurred with 7 final drills placed based on peri-apical radiography (31.8%) and 1 final drill placed using CBCT images (4.5%). The difference in nerve damage between imaging modalities was statistically significant (P = 0.023), with CBCT outperforming intraoral film in the placement of final implant drills ex vivo. In order to prevent nerve damage, CBCT is recommended as the principal imaging modality for pre-implant assessment. PMID:22150347

  12. Ex vivo imaging of human thyroid pathology using integrated optical coherence tomography and optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Zhou, Chao; Wang, Yihong; Aguirre, Aaron D.; Tsai, Tsung-Han; Cohen, David W.; Connolly, James L.; Fujimoto, James G.

    2010-01-01

    We evaluate the feasibility of optical coherence tomography (OCT) and optical coherence microscopy (OCM) for imaging of benign and malignant thyroid lesions ex vivo using intrinsic optical contrast. 34 thyroid gland specimens are imaged from 17 patients, covering a spectrum of pathology ranging from normal thyroid to benign disease/neoplasms (multinodular colloid goiter, Hashimoto's thyroiditis, and follicular adenoma) and malignant thyroid tumors (papillary carcinoma and medullary carcinoma). Imaging is performed using an integrated OCT and OCM system, with <4 μm axial resolution (OCT and OCM), and 14 μm (OCT) and <2 μm (OCM) transverse resolution. The system allows seamless switching between low and high magnifications in a way similar to traditional microscopy. Good correspondence is observed between optical images and histological sections. Characteristic features that suggest malignant lesions, such as complex papillary architecture, microfollicules, psammomatous calcifications, or replacement of normal follicular architecture with sheets/nests of tumor cells, can be identified from OCT and OCM images and are clearly differentiable from normal or benign thyroid tissues. With further development of needle-based imaging probes, OCT and OCM could be promising techniques to use for the screening of thyroid nodules and to improve the diagnostic specificity of fine needle aspiration evaluation.

  13. CRITICAL ROLE OF ACROLEIN IN SECONDARY INJURY FOLLOWING EX VIVO SPINAL CORD TRAUMA

    PubMed Central

    Hamann, Kristin; Durkes, Abigail; Ouyang, Hui; Pond, Amber

    2008-01-01

    The pathophysiology of spinal cord injury (SCI) is characterized by the initial, primary injury followed by secondary injury processes in which oxidative stress is a critical component. Secondary injury processes not only exacerbate pathology at the site of primary injury, but also result in spreading of injuries to the adjacent, otherwise healthy tissue. The lipid peroxidation byproduct acrolein has been implicated as one potential mediator of secondary injury. In order to further and rigorously elucidate the role of acrolein in secondary injury, a unique ex vivo model is utilized to isolate the detrimental effects of mechanical injury from toxins such as acrolein that are produced endogenously following SCI. We demonstrate that: 1) acrolein-lys adducts are capable of diffusing from compressed tissue to adjacent, otherwise uninjured tissue; 2) secondary injury by itself produces significant membrane damage and increased superoxide production; and 3) these injuries are significantly attenuated by the acrolein scavenger hydralazine. Furthermore, hydralazine treatment results in significantly less membrane damage 2 hours following compression injury, but not immediately after. These findings support our hypothesis that, following SCI, acrolein is increased to pathologic concentrations, contributes significantly to secondary injury, and thus represents a novel target for scavenging to promote improved recovery. PMID:18710419

  14. In vivo and ex vivo applications of gold nanoparticles for biomedical SERS imagingi

    PubMed Central

    Yigit, Mehmet V; Medarova, Zdravka

    2012-01-01

    Surface enhanced Raman scattering (SERS) is a signal-increasing phenomenon that occurs whenever Raman scattering on a metal surface is enhanced many orders of magnitude. Recently SERS has received considerable attention due to its ultrasensitive multiplex imaging capability with strong photostability. It provides rich molecular information on any Raman molecule adsorbed to rough metal surfaces. The signal enhancement is so remarkable that identification of a single molecule is possible. SERS has become a genuine molecular imaging technique. Gold nanoparticles, encoded with Raman reporters, provide a SERS signal and have been used as imaging probes, often referred to as SERS nanoparticles. They have been used for molecular imaging in vivo, ex vivo and in vitro. Detection of picomolar concentrations of target molecules has been achieved by functionalizing the nanoparticles with target recognition ligands. This review focuses on recent achievements in utilizing SERS nanoparticles for in vivo molecular imaging. In the near future, SERS technology may allow detection of disease markers at the single cell level. PMID:23133814

  15. Elastic Cherenkov effects in transversely isotropic soft materials-II: Ex vivo and in vivo experiments

    NASA Astrophysics Data System (ADS)

    Li, Guo-Yang; He, Qiong; Qian, Lin-Xue; Geng, Huiying; Liu, Yanlin; Yang, Xue-Yi; Luo, Jianwen; Cao, Yanping

    2016-09-01

    In part I of this study, we investigated the elastic Cherenkov effect (ECE) in an incompressible transversely isotropic (TI) soft solid using a combined theoretical and computational approach, based on which an inverse method has been proposed to measure both the anisotropic and hyperelastic parameters of TI soft tissues. In this part, experiments were carried out to validate the inverse method and demonstrate its usefulness in practical measurements. We first performed ex vivo experiments on bovine skeletal muscles. Not only the shear moduli along and perpendicular to the direction of muscle fibers but also the elastic modulus EL and hyperelastic parameter c2 were determined. We next carried out tensile tests to determine EL, which was compared with the value obtained using the shear wave elastography method. Furthermore, we conducted in vivo experiments on the biceps brachii and gastrocnemius muscles of ten healthy volunteers. To the best of our knowledge, this study represents the first attempt to determine EL of human muscles using the dynamic elastography method and inverse analysis. The significance of our method and its potential for clinical use are discussed.

  16. Comparative evaluation of bond strength of three contemporary self-etch adhesives: An ex vivo study

    PubMed Central

    Nikhil, Vineeta; Singh, Vijay; Chaudhry, Suruchi

    2011-01-01

    Aim: This study evaluated the effect of 2-hydroxymethyl methacrylate (HEMA) and the type of solvent on the tensile bond strength of the following three self-etch adhesives: Adper easy one (HEMA-rich adhesive) which contained ethanol, G-Bond (HEMA-free adhesive) which contained acetone, and Xeno V (HEMA-free adhesive) which contained butanol as a solvent. Material and Methods: Intact mandibular molars were mounted in self-cured resin and the occlusal surfaces were ground with # 600 SiC paper. Adhesives were applied on the prepared dentinal surfaces and the resin composite was condensed in the split brass mold (5 × 3 mm) placed over the adhesive surface. The specimens were stored in normal saline and placed in incubator at 37°C. After 24 hours, the specimens were tested in tensile mode at a crosshead speed of 1 mm/min. Statistical analysis was done using One way ANOVA and Tukey's HSD test. Results: The mean bond strengths of Adper easy one, G-Bond, and Xeno V were 12.41 MPa, 10.09 MPa, and 8.67 MPa, respectively. Conclusions: Comparison of contemporary adhesives in this ex vivo study revealed that the ethanol-based HEMA-rich self-etch adhesive is better than HEMA-free self-etch adhesive that contained acetone and butanol as the solvents, when compared in terms of bond strength. PMID:21957383

  17. Ex vivo absorption of thymol and thymol-β-D-glucopyranoside in piglet everted jejunal segments.

    PubMed

    Petrujkić, Branko T; Sedej, Ivana; Beier, Ross C; Anderson, Robin C; Harvey, Roger B; Epps, Sharon V R; Stipanovic, Robert D; Krueger, Nathan A; Nisbet, David J

    2013-04-17

    Food-producing animals are reservoirs of Campylobacter, a leading bacterial cause of human foodborne illness. The natural product thymol can reduce the survivability of Campylobacter, but its rapid absorption in the proximal gastrointestinal tract may preclude its use as a feed additive to reduce intestinal colonization of these pathogens. This work examined the ex vivo absorption of thymol and thymol-β-d-glucopyranoside in everted porcine jejunal segments, as the latter was hypothesized to be more resistant to absorption. A modified gas chromatography and extraction method was developed to determine 1.0-500 mg/L thymol. From 1 and 3 mM solutions, 0.293 ± 0.04 and 0.898 ± 0.212 mM thymol, respectively, p = 0.0347, were absorbed, and 0.125 ± 0.041 and 0.317 ± 0.143 mM thymol-β-d-glucopyranoside, respectively, p = 0.0892, were absorbed. Results indicate that thymol-β-d-glucopyranoside was absorbed 2.3 to 2.8 times less effectively than thymol, thus providing evidence that thymol-β-d-glucopyranoside may potentially be used as a feed additive to transport thymol to the piglet lower gut. PMID:23551201

  18. Effect of surface hydrophilicity on ex vivo blood compatibility of segmented polyurethanes.

    PubMed

    Takahara, A; Okkema, A Z; Cooper, S L; Coury, A J

    1991-04-01

    The relationship between surface, bulk and ex vivo blood-contacting properties of segmented polyurethanes with various polyol soft segment was investigated. The polyols used in this study were poly(ethylene oxide), poly(tetramethylene oxide), hydrogenated poly(butadiene), poly(butadiene) and poly(dimethylsiloxane). The hard segment of these segmented polyurethanes was composed of 4,4' diphenylmethane diisocyanate and 1,4 butanediol, present at 50 wt%. An experimental polyurethane, Biostable PUR, which has shown excellent biostability, was used in this study. The segmented polyurethanes based on the hydrophobic polyols such as poly(dimethylsiloxane) and hydrogenated poly(butadiene) showed distinct microphase separation between hard and soft segments. X-ray photoelectron spectroscopy revealed the surface enrichment of the hydrophobic component at the air-solid interface. Dynamic contact angle measurements indicated that the poly(dimethylsiloxane)-based segmented polyurethane possessed a hydrophobic surface in water. The poly(dimethylsiloxane)-based segmented polyurethane had the lowest platelet adhesion among the segmented polyurethanes investigated in this study, whilst the platelet deposition on the poly(ethylene oxide)-based polymer increased with time. PMID:1854901

  19. Cardiac tissue engineering, ex-vivo: design principles in biomaterials and bioreactors.

    PubMed

    Shachar, Michal; Cohen, Smadar

    2003-07-01

    Cardiac tissue engineering has emerged as a promising approach to replace or support an infarcted cardiac tissue and thus may hold a great potential to treat and save the lives of patients with heart diseases. By its broad definition, tissue engineering involves the construction of tissue equivalents from donor cells seeded within 3-D biomaterials, then culturing and implanting the cell-seeded scaffolds to induce and direct the growth of new, healthy tissue. In this review, we present an up-to-date summary of the research in cardiac tissue engineering, with an emphasis on the design principles and selection criteria that have been used in two key technologies employed in tissue engineering, (1) biomaterials technology, for the creation of 3-D porous scaffolds which are used to support and guide the tissue formation from dissociated cells, and (2) bioreactor cultivation of the 3-D cell constructs during ex-vivo tissue engineering, which aims to duplicate the normal stresses and flows experienced by the tissues. PMID:12878836

  20. Ex vivo generation of functional immune cells by mitochondria-targeted photosensitization of cancer cells.

    PubMed

    Marrache, Sean; Tundup, Smanla; Harn, Donald A; Dhar, Shanta

    2015-01-01

    Stimulating the immune system for potent immune therapy against cancer is potentially a revolutionary method to eradicate cancer. Tumors stimulated with photosensitizers (PSs) not only kill cancer cells but also help to boost the immune system. We recently reported that tumor-associated antigens (TAAs) generated by delivery of a mitochondria-acting PS zinc phthalocyanine (ZnPc) to MCF-7 breast cancer cells followed by laser irradiation can lead to ex vivo stimulation of mouse bone marrow-derived dendritic cells (BMDCs). The antigens generated from the breast cancer cells were also found to cause significant DC maturation and the activated DCs were able to stimulate T cells to cytotoxic CD8(+) T cells. In this protocol, we describe methods to engineer a mitochondria-targeted biodegradable nanoparticle (NP) formulation, T-ZnPc-NPs for delivery of ZnPc to the mitochondria of MCF-7 cells, subsequent photodynamic therapy (PDT) using a long wavelength laser irradiation to produce TAAs, DC stimulation by the TAAs to secrete interferon-gamma (IFN-γ), and matured DC-driven T-cell activation. PMID:25634271

  1. In vitro antioxidant and ex vivo protective activities of green and roasted coffee.

    PubMed

    Daglia, M; Papetti, A; Gregotti, C; Bertè, F; Gazzani, G

    2000-05-01

    The antioxidant properties of green and roasted coffee, in relation to species (Coffea arabica and Coffea robusta) and degree of roasting (light, medium, dark), were investigated. These properties were evaluated by determining the reducing substances (RS) of coffee and its antioxidant activity (AA) in vitro (model system beta-carotene-linoleic acid) and ex vivo as protective activity (PA) against rat liver cell microsome lipid peroxidation measured as TBA-reacting substances. RS of C. robustasamples were found to be significantly higher when compared to those of C. arabica samples (p < 0.001). AA for green coffee samples were slightly higher than for the corresponding roasted samples while PA was significantly lower in green coffee compared to that of all roasted samples (p < 0.001). Extraction with three different organic solvents (ethyl acetate, ethyl ether, and dichloromethane) showed that the most protective compounds are extracted from acidified dark roasted coffee solutions with ethyl acetate. The analysis of acidic extract by gel filtration chromatography (GFC) gave five fractions. Higher molecular mass fractions were found to possess antioxidant activity while the lower molecular mass fractions showed protective activity. The small amounts of these acidic, low molecular mass protective fractions isolated indicate that they contain very strong protective agents. PMID:10820041

  2. Ex vivo absorption of promestriene from oil-in-water emulsion into infant foreskin.

    PubMed

    Salmon, D; Kassai, B; Roussel, L; Mouriquand, P; Gérard, C; Gorduza, D B; Serre, C; Falson, F; Pivot, C; Pirot, F

    2013-11-01

    Hypospadias is a birth defect in which the urinary tract opening is not at the tip of the penis. Hypospadias surgery is frequently complicated by healing deficiencies. Topical treatments with oestrogens were reported to improve healing. In the present study, ex vivo percutaneous absorption of promestriene, a synthetic oestrogen resulting of the double esterification of estradiol was conducted as a pre-requisite for further clinical trial in infants. Penetration of promestriene into infant foreskin treated with commercial oil in water emulsion (10 μg mg(-1)) for 24 h was characterized showing controlled release properties enabling epidermal concentration more than six times higher than dermal concentration (4.13±2.46 mg g(-1) versus 0.62±0.84 mg g(-1), respectively). Furthermore, apparent promestriene fluxes into and through the skin (i.e., 1.5 μg cm(-2) h(-1) and<0.89 μg cm(-2) h(-1), respectively) were calculated from (i) drug amount retained into epidermis and dermis, or (ii) the limit of detection into the receptor fluid. In conclusion, less than 2% of initial dose were absorbed within 24h which compared well with others steroids applied topically in colloidal systems. PMID:23968783

  3. Assembly and characterization of a nonlinear optical microscopy for in vivo and ex vivo tissue imaging

    NASA Astrophysics Data System (ADS)

    Pratavieira, S.; Buzzá, H. H.; Jorge, A. E.; Grecco, C.; Pires, L.; Cosci, A.; Bagnato, V. S.; Kurachi, C.

    2014-02-01

    The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. The microscope allows the adjustment for in vitro, in vivo and ex vivo imaging of biological samples. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The excitation is done using a tunable femtosecond Ti: Sapphire laser. The light is focused in tissue by an objective lens 20X, water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The detection system is composed by a cut off filter that eliminates laser light back reflections and diverse dichroic filters can be chosen to split the emitted signal for the two photomultiplier detector. The calibration and resolution of the microscope was done using a stage micrometer with 10 μm divisions and fluorescent particle slide, respectively. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue, the images have a sub-cellular spatial resolution. Further characterization and differentiation of tissue layers can be obtained by performing axial scanning. By means of the microscope it is possible to have a three dimensional reconstruction of tissues with sub-cellular resolution.

  4. Ex Vivo Assessment of a Parabolic-Tip Inflow Cannula for Pediatric Continuous-Flow VADs.

    PubMed

    Griffin, Michael T; Grzywinski, Matthew F; Voorhees, Hannah J; Kameneva, Marina V; Olia, Salim E

    2016-01-01

    To address the challenge of unloading the left ventricle during pediatric mechanical circulatory support using next-generation rotary blood pumps, a novel inflow cannula was developed. This unique inflow cannula for pediatric, continuous-flow, left ventricular assist devices (VADs) with a parabolic-shaped inlet entrance was evaluated alongside a bevel-tip and fenestrated-tip cannula via an ex vivo, isolated-heart experimental setup. Performance was characterized using two clinical scenarios of over-pumping and hypovolemia, created by varying pump speed and filling preload pressure, respectively, at ideal and off-axis cannula placement to assess ventricular unloading and positional sensitivity. Quantitative and qualitative assessments were performed on the resultant hemodynamics and intra-ventricular boroscopic images to classify conditions of nonsuction, partial, gradual or severe entrainment, and ventricular collapse. The parabolic-tip cannula was found to be significantly less sensitive to placement position (p < 0.001) than the bevel-tip cannula under all conditions, while not statistically different from the fenestrated cannula. Visual analysis of the parabolic-tip cannula showed complete resistance to entrainment, whereas the fenestrated-tip had partial entrainment in 90% and 87% of the over-pumping and hypovolemic studies, respectively. We conclude that future pediatric VAD designs may benefit from incorporating the parabolic-tip inflow cannula design to maximize unloading of the left ventricle in ideal and nonoptimal conditions. PMID:27442862

  5. Enumeration of Cytotoxic CD8 T Cells Ex Vivo during the Response to Listeria monocytogenes Infection▿

    PubMed Central

    Zaiss, Dietmar M. W.; Sijts, Alice J. A. M.; Mosmann, Tim R.

    2008-01-01

    Cytotoxicity is a key effector function of CD8 T cells. However, what proportion of antigen-specific CD8 T cells in vivo exert cytotoxic activity during a functional CD8 T-cell response to infection still remains unknown. We used the Lysispot assay to directly enumerate cytotoxic CD8 T cells from the spleen ex vivo during the immune response to infection with the intracellular bacterium Listeria monocytogenes. We demonstrate that not all antigen-responsive gamma interferon (IFN-γ)-secreting T cells display cytotoxic activity. Most CD8 T cells detected at early time points of the response were cytotoxic. This percentage continuously declined during both the expansion and contraction phases to about 50% at the peak and to <10% of IFN-γ-producing cells in the memory phase. As described for clonal expansion, this elaboration of a program of differentiation after an initial stimulus was not affected by antigen or CD4 help but, like proliferation, could be influenced by later reinfection. These data indicate that cytotoxic effector function during the response to infection is regulated independently from IFN-γ secretion or expansion or contraction of the overall CD8 T-cell response. PMID:18678661

  6. Titanium dioxide nanoparticle impact and translocation through ex vivo, in vivo and in vitro gut epithelia

    PubMed Central

    2014-01-01

    Background TiO2 particles are commonly used as dietary supplements and may contain up to 36% of nano-sized particles (TiO2-NPs). Still impact and translocation of NPs through the gut epithelium is poorly documented. Results We show that, in vivo and ex vivo, agglomerates of TiO2-NPs cross both the regular ileum epithelium and the follicle-associated epithelium (FAE) and alter the paracellular permeability of the ileum and colon epithelia. In vitro, they accumulate in M-cells and mucus-secreting cells, much less in enterocytes. They do not cause overt cytotoxicity or apoptosis. They translocate through a model of FAE only, but induce tight junctions remodeling in the regular ileum epithelium, which is a sign of integrity alteration and suggests paracellular passage of NPs. Finally we prove that TiO2-NPs do not dissolve when sequestered up to 24 h in gut cells. Conclusions Taken together these data prove that TiO2-NPs would possibly translocate through both the regular epithelium lining the ileum and through Peyer’s patches, would induce epithelium impairment, and would persist in gut cells where they would possibly induce chronic damage. PMID:24666995

  7. Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

    PubMed

    de Bekker, Charissa; Smith, Philip B; Patterson, Andrew D; Hughes, David P

    2013-01-01

    Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied. PMID:23940603

  8. Metabolomics Reveals the Heterogeneous Secretome of Two Entomopathogenic Fungi to Ex Vivo Cultured Insect Tissues

    PubMed Central

    de Bekker, Charissa; Smith, Philip B.; Patterson, Andrew D.; Hughes, David P.

    2013-01-01

    Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied. PMID:23940603

  9. Successful emergent lung transplantation after remote ex vivo perfusion optimization and transportation of donor lungs.

    PubMed

    Wigfield, C H; Cypel, M; Yeung, J; Waddell, T; Alex, C; Johnson, C; Keshavjee, S; Love, R B

    2012-10-01

    A recent clinical trial provided evidence that ex vivo lung perfusion (EVLP) results in optimized human donor lungs for transplantation. Excellent recipient outcomes were documented after 4 h of normothermic perfusion. We report a clinical case utilizing remote EVLP to assess and improve function of initially otherwise unacceptable injured donor lungs followed by transportation and subsequent bilateral lung transplantation in a patient with virally induced refractory respiratory failure supported with extracorporeal membrane oxygenation. This is the first lung transplantation with the application of remote EVLP, wherein the donor lungs were transported from the donor hospital to a center for EVLP and then transported to another hospital for transplantation. It is also the first case of lung transplantation in the United States utilizing EVLP for functional optimization leading to successful transplantation. Organ procurement data, EVLP assessment, and the pre- and postoperative course of the recipient are presented. The available evidence supporting EVLP, the humanitarian and cooperative utilization of lungs otherwise discarded, are discussed. PMID:23009140

  10. Ex vivo lung perfusion: a comprehensive review of the development and exploration of future trends.

    PubMed

    Roman, Marius A; Nair, Sukumaran; Tsui, Steven; Dunning, John; Parmar, Jasvir S

    2013-09-01

    There is a critical mismatch between the number of donor lungs available and the demand for lungs for transplantation. This has created unacceptably high waiting-list mortality for lung transplant recipients. Currently (2012) in the United Kingdom, there are 216 patients on the lung transplant waiting list and 17 on heart and lung transplant list. The waiting times for suitable lungs average 412 days, with an increasing mortality and morbidity among the patients on the lung transplant list. Ex vivo lung perfusion (EVLP) has emerged as a technique for the assessment, resuscitation, and potential repair of suboptimal donor lungs. This is a rapidly developing field with significant clinical implications. In this review article, we critically appraise the background developments that have led to our current clinical practice. In particular, we focus on the human and animal experience, the different perfusion-ventilation strategies, and the impact of different perfusates and leukocyte filters. Finally, we examine EVLP as a potential research tool. This will provide insight into EVLP and its future development in the field of clinical lung transplantation. PMID:23694953

  11. Temperature increase of ex vivo corneas from multiple 2.01-micron incident laser pulses

    NASA Astrophysics Data System (ADS)

    Kelly, Edward; Johnson, Thomas

    2011-03-01

    Current laser safety standards for multiple pulse lasers are based primarily on modeling and the results of single pulse studies. Previous thermal effects studies have focused on histological and visible endpoints, with only a few studies examining the actual temperatures achieved. The goal of this research was to probe the actual vertical temperature profile produced by 2.01 micron laser pulses in the cornea. In this study the corneal temperature rise from multiple 2.01 micron Tm:YAG laser pulses was investigated using ex-vivo rabbit eyes. A thermal-measurement data set for a different number of pulses was collected and compared. An infrared thermal camera employing microbolometer detectors captured surface temperature rises resulting from laser pulses. Single 10 ms pulses as well as two, three, and four pulse sequences were utilized while the total energy delivered was held constant. A comparison of the data to temperatures required for denaturing proteins and the current laser safety guidelines will be presented.

  12. Detection of Calcifications In Vivo and Ex Vivo After Brain Injury in Rat Using SWIFT

    PubMed Central

    Lehto, Lauri Juhani; Sierra, Alejandra; Corum, Curtis Andrew; Zhang, Jinjin; Idiyatullin, Djaudat; Pitkänen, Asla; Garwood, Michael; Gröhn, Olli

    2012-01-01

    Calcifications represent one component of pathology in many brain diseases. With MRI, they are most often detected by exploiting negative contrast in magnitude images. Calcifications are more diamagnetic than tissue, leading to a magnetic field disturbance that can be seen in phase MR images. Most phase imaging studies use gradient recalled echo based pulse sequences. Here, the phase component of SWIFT, a virtually zero acquisition delay sequence, was used to detect calcifications ex vivo and in vivo in rat models of status epilepticus and traumatic brain injury. Calcifications were detected in phase and imaginary SWIFT images based on their dipole like magnetic field disturbances. In magnitude SWIFT images, calcifications were distinguished as hypointense and hyperintense. Hypointense calcifications showed large crystallized granules with few surrounding inflammatory cells, while hyperintense calcifications contained small granules with the presence of more inflammatory cells. The size of the calcifications in SWIFT magnitude images correlated with that in Alizarin stained histological sections. Our data indicate that SWIFT is likely to better preserve signal in the proximity of a calcification or other field perturber in comparison to gradient echo due to its short acquisition delay and broad excitation bandwidth. Furthermore, a quantitative description for the phase contrast near dipole magnetic field inhomogeneities for the SWIFT pulse sequence is given. In vivo detection of calcifications provides a tool to probe the progression of pathology in neurodegenerative diseases. In particular, it appears to provide a surrogate marker for inflammatory cells around the calcifications after brain injury. PMID:22425671

  13. Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis

    PubMed Central

    Kjelgaard-Petersen, Cecilie; Siebuhr, Anne Sofie; Christiansen, Thorbjørn; Ladel, Christoph; Karsdal, Morten; Bay-Jensen, Anne-Christine

    2015-01-01

    Abstract Objective: Characterize biomarkers measuring extracellular matrix turnover of inflamed osteoarthritis synovium. Methods: Human primary fibroblast-like synoviocytes and synovial membrane explants (SMEs) treated with various cytokines and growth factors were assessed by C1M, C3M, and acMMP3 in the conditioned medium. Results: TNFα significantly increased C1M up to seven-fold (p = 0.0002), C3M up to 24-fold (p = 0.0011), and acMMP3 up to 14-fold (p < 0.0001) in SMEs. IL-1β also significantly increased C1M up to five-fold (p = 0.00094), C3M four-fold (p = 0.007), and acMMP3 18-fold (p < 0.0001) in SMEs. Conclusion: The biomarkers C1M, C3M, and acMMP-3 were synovitis biomarkers ex vivo and provide a translational tool together with the SME model. PMID:26863055

  14. Ex-vivo holographic microscopy and spectroscopic analysis of head and neck cancer

    NASA Astrophysics Data System (ADS)

    Holler, Stephen; Wurtz, Robert; Auyeung, Kelsey; Auyeung, Kris; Paspaley-Grbavac, Milan; Mulroe, Brigid; Sobrero, Maximiliano; Miles, Brett

    2015-03-01

    Optical probes to identify tumor margins in vivo would greatly reduce the time, effort and complexity in the surgical removal of malignant tissue in head and neck cancers. Current approaches involve visual microscopy of stained tissue samples to determine cancer margins, which results in the excision of excess of tissue to assure complete removal of the cancer. Such surgical procedures and follow-on chemotherapy can adversely affect the patient's recovery and subsequent quality of life. In order to reduce the complexity of the process and minimize adverse effects on the patient, we investigate ex vivo tissue samples (stained and unstained) using digital holographic microscopy in conjunction with spectroscopic analyses (reflectance and transmission spectroscopy) in order to determine label-free, optically identifiable characteristic features that may ultimately be used for in vivo processing of cancerous tissues. The tissue samples studied were squamous cell carcinomas and associated controls from patients of varying age, gender and race. Holographic microscopic imaging scans across both cancerous and non-cancerous tissue samples yielded amplitude and phase reconstructions that were correlated with spectral signatures. Though the holographic reconstructions and measured spectra indicate variations even among the same class of tissue, preliminary results indicate the existence of some discriminating features. Further analyses are presently underway to further this work and extract additional information from the imaging and spectral data that may prove useful for in vivo surgical identification.

  15. Computed Tomographic Tenography of Normal Equine Digital Flexor Tendon Sheath: An Ex Vivo Study

    PubMed Central

    Lacitignola, Luca; De Luca, Pasquale; Guarracino, Alessandro; Crovace, Antonio

    2015-01-01

    Aim of this study was to document the normal computed tomographic tenography findings of digital flexor tendon sheath. Six ex vivo normal equine forelimbs were used. An axial approach was used to inject 185 mg/mL of iopamidol in a total volume of 60 mL into the digital flexor tendon sheaths. Single-slice helical scans, with 5 mm thickness, spaced every 3 mm, for a pitch of 0.6, and with bone algorithm reconstruction, were performed before and after injections of contrast medium. To obtain better image quality for multiplanar reconstruction and 3D reformatting, postprocessing retroreconstruction was performed to reduce the images to submillimetre thickness. Computed tomographic tenography of digital flexor tendon sheaths could visualize the following main tendon structures for every forelimb in contrast-enhanced images as low densities surrounded by high densities: superficial digital flexor tendon, deep digital flexor tendon, manica flexoria, mesotendons, and synovial recess. Results of this study suggest that computed tomographic tenography can be used with accuracy and sensitivity to evaluate the common disorders of the equine digital flexor tendon sheath and the intrathecal structures. PMID:26185709

  16. Repopulating Decellularized Kidney Scaffolds: An Avenue for Ex Vivo Organ Generation

    PubMed Central

    McKee, Robert A.; Wingert, Rebecca A.

    2016-01-01

    Recent research has shown that fully developed organs can be decellularized, resulting in a complex scaffold and extracellular matrix (ECM) network capable of being populated with other cells. This work has resulted in a growing field in bioengineering focused on the isolation, characterization, and modification of organ derived acellular scaffolds and their potential to sustain and interact with new cell populations, a process termed reseeding. In this review, we cover contemporary advancements in the bioengineering of kidney scaffolds including novel work showing that reseeded donor scaffolds can be transplanted and can function in recipients using animal models. Several major areas of the field are taken into consideration, including the decellularization process, characterization of acellular and reseeded scaffolds, culture conditions, and cell sources. Finally, we discuss future avenues based on the advent of 3D bioprinting and recent developments in kidney organoid cultures as well as animal models of renal genesis. The ongoing mergers and collaborations between these fields hold the potential to produce functional kidneys that can be generated ex vivo and utilized for kidney transplantations in patients suffering with renal disease. PMID:27375844

  17. Ex vivo characterization of normal and adenocarcinoma colon samples by Mueller matrix polarimetry.

    PubMed

    Ahmad, Iftikhar; Ahmad, Manzoor; Khan, Karim; Ashraf, Sumara; Ahmad, Shakil; Ikram, Masroor

    2015-05-01

    Mueller matrix polarimetry along with polar decomposition algorithm was employed for the characterization of ex vivo normal and adenocarcinoma human colon tissues by polarized light in the visible spectral range (425-725 nm). Six derived polarization metrics [total diattenuation (DT ), retardance (RT ), depolarization(ΔT ), linear diattenuation (DL), retardance (δ), and depolarization (ΔL)] were compared for normal and adenocarcinoma colon tissue samples. The results show that all six polarimetric properties for adenocarcinoma samples were significantly higher as compared to the normal samples for all wavelengths. The Wilcoxon rank sum test illustrated that total retardance is a good candidate for the discrimination of normal and adenocarcinoma colon samples. Support vector machine classification for normal and adenocarcinoma based on the four polarization properties spectra (ΔT , ΔL, RT ,and δ) yielded 100% accuracy, sensitivity, and specificity, while both DTa nd DL showed 66.6%, 33.3%, and 83.3% accuracy, sensitivity, and specificity, respectively. The combination of polarization analysis and given classification methods provides a framework to distinguish the normal and cancerous tissues. PMID:26021717

  18. Ex vivo lymphocyte phenotyping during Plasmodium falciparum sporozoite immunization in humans.

    PubMed

    Bijker, E M; Schats, R; Visser, L G; Sauerwein, R W; Scholzen, A

    2015-11-01

    Immunization of malaria-naïve volunteers under chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) efficiently and reproducibly induces sterile protection and thus constitutes an excellent model to study protective immune responses against malaria. Here, we performed the first longitudinal assessment of lymphocyte activation and differentiation kinetics during sporozoite immunization in 15 volunteers by ex vivo lymphocyte flow cytometry analysis. Both CD4 and CD8 T cells as well as γδT cells, NK cells and CD3+ CD56+ cells showed increased activation and proliferation following immunization. Transient induction of the transcription factor T-bet and the cytotoxic molecule granzyme B indicated a role of Th1 responses and cytotoxic T cells in CPS-induced immunity. The absolute number of γδT cells as well as the proportion of granzyme B-containing γδT cells showed a significant and sustained increase. Regulatory T-cell (Treg) proliferation was significantly higher after the second immunization in subjects subsequently not protected against challenge infection. These findings indicate an important role for γδT cells, Th1 and cytotoxic responses in whole sporozoite immunization with a possibly suppressive role of Tregs. PMID:26363409

  19. Biomarkers in bladder cancer: A metabolomic approach using in vitro and ex vivo model systems.

    PubMed

    Rodrigues, Daniela; Jerónimo, Carmen; Henrique, Rui; Belo, Luís; de Lourdes Bastos, Maria; de Pinho, Paula Guedes; Carvalho, Márcia

    2016-07-15

    Metabolomics has recently proved to be useful in the area of biomarker discovery for cancers in which early diagnostic and prognostic biomarkers are urgently needed, as is the case of bladder cancer (BC). This article presents a comprehensive review of the literature on the metabolomic studies on BC, highlighting metabolic pathways perturbed in this disease and the altered metabolites as potential biomarkers for BC detection. Current disease model systems used in the study of BC metabolome include in vitro-cultured cancer cells, ex vivo neoplastic bladder tissues and biological fluids, mainly urine but also blood serum/plasma, from BC patients. The major advantages and drawbacks of each model system are discussed. Based on available data, it seems that BC metabolic signature is mainly characterized by alterations in metabolites related to energy metabolic pathways, particularly glycolysis, amino acid and fatty acid metabolism, known to be crucial for cell proliferation, as well as glutathione metabolism, known to be determinant in maintaining cellular redox balance. In addition, purine and pyrimidine metabolism as well as carnitine species were found to be altered in BC. Finally, it is emphasized that, despite the progress made in respect to novel biomarkers for BC diagnosis, there are still some challenges and limitations that should be addressed in future metabolomic studies to ensure their translatability to clinical practice. PMID:26804544

  20. Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

    PubMed Central

    Bandara, G; Mueller, G M; Galea-Lauri, J; Tindal, M H; Georgescu, H I; Suchanek, M K; Hung, G L; Glorioso, J C; Robbins, P D; Evans, C H

    1993-01-01

    Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy. Images Fig. 1 Fig. 2 PMID:8248169

  1. Ex Vivo and In Vivo Lentivirus-Mediated Transduction of Airway Epithelial Progenitor Cells.

    PubMed

    Leoni, Giulia; Wasowicz, Marguerite Y; Chan, Mario; Meng, Cuixiang; Farley, Raymond; Brody, Steven L; Inoue, Makoto; Hasegawa, Mamoru; Alton, Eric W F W; Griesenbach, Uta

    2015-01-01

    A key challenge in pulmonary gene therapy for cystic fibrosis is to provide long-term correction of the genetic defect. This may be achievable by targeting airway epithelial stem/progenitor cells with an integrating vector. Here, we evaluated the ability of a lentiviral vector, derived from the simian immunodeficiency virus and pseudotyped with F and HN envelope proteins from Sendai virus, to transduce progenitor basal cells of the mouse nasal airways. We first transduced basal cell-enriched cultures ex vivo and confirmed efficient transduction of cytokeratin-5 positive cells. We next asked whether progenitor cells could be transduced in vivo. We evaluated the transduction efficiency in mice pretreated by intranasal administration of polidocanol to expose the progenitor cell layer. Compared to control mice, polidocanol treated mice demonstrated a significant increase in the number of transduced basal cells at 3 and 14 days post vector administration. At 14 days, the epithelium of treated mice contained clusters (4 to 8 adjacent cells) of well differentiated ciliated, as well as basal cells suggesting a clonal expansion. These results indicate that our lentiviral vector can transduce progenitor basal cells in vivo, although transduction required denudation of the surface epithelium prior to vector administration. PMID:26471068

  2. Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system

    PubMed Central

    2013-01-01

    Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants. PMID:24564863

  3. Prostate Cryotherapy Monitoring Using Vibroacoustography: Preliminary Results of an Ex Vivo Study and Technical Feasibility

    PubMed Central

    Davis, Brian J.; Alizad, Azra; Greenleaf, James F.; Wilson, Torrence M.; Mynderse, Lance A.; Fatemi, Mostafa

    2009-01-01

    The objective of this research is to prospectively evaluate the feasibility of vibroacoustography (VA) imaging in monitoring prostate cryotherapy in an ex vivo model. Baseline scanning of an excised human prostate is accomplished by a VA system apparatus in a tank of degassed water. Alcohol and dry ice mixture are used to freeze two prostate tissue samples. The frozen prostates are subsequently placed within the water tank at 27°C and rescanned. VA images were acquired at prescribed time intervals to characterize the acoustic properties of the partially frozen tissue. The frozen prostate tissue appears in the images as hypoemitting signal. Once the tissue thaws, previously frozen regions show coarser texture than prior to freezing. The margin of the frozen tissue is delineated with a well-defined rim. The thawed cryolesions show a different contrast compared with normal unfrozen prostate. In conclusion, this pilot study shows that VA produces clear images of a frozen prostate at different temperature stages. The frozen tissue appears as a uniform region with well-defined borders that are readily identified. These characteristic images should allow safer and more efficient application of prostatic cryosurgery. These results provide substantial motivation to further investigate VA as a potential modality to monitor prostate cryotherapy intraoperatively. PMID:18990628

  4. A method to measure the hyperelastic parameters of ex vivo breast tissue samples

    NASA Astrophysics Data System (ADS)

    Samani, Abbas; Plewes, Donald

    2004-09-01

    Over the past decade, there has been increasing interest in modelling soft tissue deformation. This topic has several biomedical applications ranging from medical imaging to robotic assisted telesurgery. In these applications, tissue deformation can be very large due to low tissue stiffness and lack of physical constraints. As a result, deformation modelling of such organs often requires a treatment, which reflects nonlinear behaviour. While computational techniques such as nonlinear finite element methods are well developed, the required intrinsic nonlinear mechanical parameters of soft tissues that are critical to develop reliable tissue deformation models are not well known. To address this issue, we developed a system to measure the hyperelastic parameters of small ex vivo tissue samples. This measurement technique consists of indenting an unconfined small block of tissue using a computer controlled loading system while measuring the resulting indentation force. The nonlinear tissue force-displacement response is used to calculate the hyperelastic parameters via an appropriate inversion technique. This technique is based on a nonlinear least squares formulation that uses a nonlinear finite element model as the direct problem solver. The features of the system are demonstrated with two samples of breast tissue and typical hyperelastic results are presented.

  5. Development of novel polymer-stabilized diosmin nanosuspensions: in vitro appraisal and ex vivo permeation.

    PubMed

    Freag, May S; Elnaggar, Yosra S R; Abdallah, Ossama Y

    2013-09-15

    Scanty solubility and permeability of diosmin (DSN) are perpetrators for its poor oral absorption and high inter-subject variation. This article investigated the potential of novel DSN nanosuspensions to improve drug delivery characteristics. Bottom-up nanoprecipitation technique has been employed for nanosuspension development. Variables optimized encompassed polymeric stabilizer type, DSN: stabilizer ratio, excess stabilizer removal, spray drying, and mannitol incorporation. In vitro characterization included particle size (PS), infrared spectroscopy (IR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM), and dissolution profile. Ex vivo permeation was assessed in rats using non-everted sac technique and HPLC. Optimal DSN nanosuspension (DSN:hydroxypropylmethyl cellulose HPMC 2:1) was prepared with acid base neutralization technique. The formula exhibited the lowest PS (336 nm) with 99.9% drug loading and enhanced reconstitution properties after mannitol incorporation. SEM and TEM revealed discrete, oval drug nanocrystals with higher surface coverage with HPMC compared to MC. DSN nanosuspension demonstrated a significant enhancement in DSN dissolution (100% dissolved) compared to crude drug (51%). Permeation studies revealed 89% DSN permeated from the nanosuspension after 120 min compared to non-detected amounts from drug suspension. Conclusively, novel DSN nanosuspension could successful improve its dissolution and permeation characteristics with promising consequences of better drug delivery. PMID:23830765

  6. Ex vivo assessment of carbon tetrachloride (CCl(4))-induced chronic injury using polarized light spectroscopy.

    PubMed

    Ahmad, Manzoor; Ali, Safdar; Mehmood, Malik Sajjad; Ali, Hamid; Khurshid, Ahmat; Firdous, Shamaraz; Muhammad, Saleh; Ikram, Masroor

    2013-12-01

    The liver performs various functions, such as the production and detoxification of chemicals; therefore, it is susceptible to hepatotoxins such as carbon tetrachloride (CCl4), which causes chronic injury. Thus, assessment of injury and its status of severity are of prime importance. Current work reports an ex vivo study for probing the severance of hepatic injury induced by CCl4 with polarized light over the spectral range 400-800 nm. Different concentrations of CCl4 were used to induce varying severity of hepatic injury in a rat model. Linear retardance, depolarization rates, and diagonal Mueller matrix elements (m22, m33, and m44), were successfully used as the distinguishing criterion for normal and different liver injuries. Our results show that linear retardance for injured liver samples with lower doses of CCl4 tends to increase when compared with normal liver samples, while samples injured at higher doses of CCl4 offer almost no retardance. Total, linear, and circular depolarizations follow decreasing trends with increased liver injury severity over the entire investigated wavelength range. Linear polarization states were observed to be better maintained as compared to circular polarization states for all samples. Furthermore, numerical values of diagonal elements of the experimentally measured Mueller matrix also increase with increasing doses of CCl4. Liver fibroses, change in transport albedo, and the relative refractive index of the extracellular matrix caused by CCl4 are responsible for the observed differences. These results will provide a pathway to gauge the severity of injury caused by toxic chemicals. PMID:24359651

  7. An ex Vivo Model for Evaluating Blood-Brain Barrier Permeability, Efflux, and Drug Metabolism.

    PubMed

    Hellman, Karin; Aadal Nielsen, Peter; Ek, Fredrik; Olsson, Roger

    2016-05-18

    The metabolism of drugs in the brain is difficult to study in most species because of enzymatic instability in vitro and interference from peripheral metabolism in vivo. A locust ex vivo model that combines brain barrier penetration, efflux, metabolism, and analysis of the unbound fraction in intact brains was evaluated using known drugs. Clozapine was analyzed, and its major metabolites, clozapine N-oxide (CNO) and N-desmethylclozapine (NDMC), were identified and quantified. The back-transformation of CNO into clozapine observed in humans was also observed in locusts. In addition, risperidone, citalopram, fluoxetine, and haloperidol were studied, and one preselected metabolite for each drug was analyzed, identified, and quantified. Metabolite identification studies of clozapine and midazolam showed that the locust brain was highly metabolically active, and 18 and 14 metabolites, respectively, were identified. The unbound drug fraction of clozapine, NDMC, carbamazepine, and risperidone was analyzed. In addition, coadministration of drugs with verapamil or fluvoxamine was performed to evaluate drug-drug interactions in all setups. All findings correlated well with the data in the literature for mammals except for the stated fact that CNO is a highly blood-brain barrier permeant compound. Overall, the experiments indicated that invertebrates might be useful for screening of blood-brain barrier permeation, efflux, metabolism, and analysis of the unbound fraction of drugs in the brain in early drug discovery. PMID:26930271

  8. Furosemide Loaded Silica-Lipid Hybrid Microparticles: Formulation Development, in vitro and ex vivo Evaluation

    PubMed Central

    Sambaraj, Swapna; Ammula, Divya; Nagabandi, Vijaykumar

    2015-01-01

    Purpose: The main objective of the current research work was to formulate and evaluate furosemide loaded silica lipid hybrid microparticles for improved oral delivery. A novel silica-lipid hybrid microparticulate system is used for enhancing the oral absorption of low solubility and low permeability of (BCS Class IV) drugs. Silica-lipid hybrid microparticles include the drug solubilising effect of dispersed lipids and stabilizing effect of hydrophilic silica particles to increase drug solubilisation, which leads to enhanced oral bioavailability. Methods: The slica lipid hybrid (SLH) microparticles were composed of poorly soluble drug (furosemide), dispersion of oil phase (Soya bean oil and miglyol) in lecithin (Phospholipoid 90H), non-ionic surfactant (Polysorbate 80) and adsorbent (Aerosol 380). Saturation solubility studies were performed in different oils and surfactants with increased concentration of drug revealed increased solubility of furosemide. Results: In vitro dissolution studies conducted under simulated gastric medium revealed 2-4 fold increase in dissolution efficiencies for SLH microparticles compared to that of pure drug (furosemide) and marketed formulation Lasix®. Ex vivo studies showed enhanced lipid digestibility, which improved drug permeability. Solid-state characterization of SLH microparticles by X-ray powder diffraction and Fourier transform infrared spectroscopic analysis confirmed non-crystalline nature and more compatibility of furosemide in silica-lipid hybrid microparticles. Conclusion: It can be concluded that the role of lipids and hydrophilic silica based carrier highlighted in enhancing solubility and permeability, and hence the oral bioavailability of poorly soluble drugs. PMID:26504763

  9. The Use of Herpes Simplex Virus in Ex Vivo Slice Culture

    PubMed Central

    Friedman, Allyson K.

    2015-01-01

    Herpes simplex virus (HSV) can be used for a wide range of genetic manipulation in ex vivo slices of central nervous system tissue from both young and adult rodents. The fast expression of the HSV viral mediated gene transfer, which can be engineered to produce cell-type specificity, can be utilized in slice cultures for a variety of purposes over a 1-4 day period with spatial and temporal specificity. This protocol exploits the rapid expression of HSV viral vectors by utilizing slice culture for electrophysiological recordings, avoiding the need to do intra-cranial viral injections. Brain slice cultures maintain many aspects of in vivo biology, including functional local synaptic circuitry with preserved brain architecture, while allowing good experimental access and precise control of the extracellular environment, making them ideal platforms for quick access to evaluate expression effects of HSV viral mediated gene transfer on the molecular and cellular properties of specific neurons. This protocol provides an easy way to study neuronal function following expression of these HSV viruses. PMID:26131662

  10. Ex vivo estimation of cementless acetabular cup stability using an impact hammer.

    PubMed

    Michel, Adrien; Bosc, Romain; Sailhan, Frédéric; Vayron, Romain; Haiat, Guillaume

    2016-02-01

    Obtaining primary stability of acetabular cup (AC) implants is one of the main objectives of press-fit procedures used for cementless hip arthroplasty. The aim of this study is to investigate whether the AC implant primary stability can be evaluated using the signals obtained with an impact hammer. A hammer equipped with a force sensor was used to impact the AC implant in 20 bovine bone samples. For each sample, different stability conditions were obtained by changing the cavity diameter. For each configuration, the inserted AC implant was impacted four times with a maximum force comprised between 2500 and 4500 N. An indicator I was determined based on the partial impulse estimation and the pull-out force was measured. The implant stability and the value of the indicator I reached a maximum value for an interference fit equal to 1 mm for 18 out of 20 samples. When pooling all samples and all configurations, the implant stability and I were significantly correlated (R(2) = 0.83). The AC implant primary stability can be assessed through the analysis of the impact force signals obtained using an impact hammer. Based on these ex vivo results, a medical device could be developed to provide a decision support system to the orthopedic surgeons. PMID:26671784

  11. Flagellin Induces β-Defensin 2 in Human Colonic Ex vivo Infection with Enterohemorrhagic Escherichia coli

    PubMed Central

    Lewis, Steven B.; Prior, Alison; Ellis, Samuel J.; Cook, Vivienne; Chan, Simon S. M.; Gelson, William; Schüller, Stephanie

    2016-01-01

    Enterohemorrhagic E.coli (EHEC) is an important foodborne pathogen in the developed world and can cause life-threatening disease particularly in children. EHEC persists in the human gut by adhering intimately to colonic epithelium and forming characteristic attaching/effacing lesions. In this study, we investigated the innate immune response to EHEC infection with particular focus on antimicrobial peptide and protein expression by colonic epithelium. Using a novel human colonic biopsy model and polarized T84 colon carcinoma cells, we found that EHEC infection induced expression of human β-defensin 2 (hBD2), whereas hBD1, hBD3, LL-37, and lysozyme remained unchanged. Infection with specific EHEC deletion mutants demonstrated that this was dependent on flagellin, and apical exposure to purified flagellin was sufficient to stimulate hBD2 and also interleukin (IL)-8 expression ex vivo and in vitro. Flagellin-mediated hBD2 induction was significantly reduced by inhibitors of NF-κB, MAP kinase p38 and JNK but not ERK1/2. Interestingly, IL-8 secretion by polarized T84 cells was vectorial depending on the side of stimulation, and apical exposure to EHEC or flagellin resulted in apical IL-8 release. Our results demonstrate that EHEC only induces a modest immune response in human colonic epithelium characterized by flagellin-dependent induction of hBD2 and low levels of IL-8. PMID:27446815

  12. Development of an Ex Vivo, Beating Heart Model for CT Myocardial Perfusion

    PubMed Central

    Pelgrim, Gert Jan; Das, Marco; Haberland, Ulrike; Slump, Cees; Handayani, Astri; van Tuijl, Sjoerd; Stijnen, Marco; Klotz, Ernst; Oudkerk, Matthijs; Wildberger, Joachim E.; Vliegenthart, Rozemarijn

    2015-01-01

    Objective. To test the feasibility of a CT-compatible, ex vivo, perfused porcine heart model for myocardial perfusion CT imaging. Methods. One porcine heart was perfused according to Langendorff. Dynamic perfusion scanning was performed with a second-generation dual source CT scanner. Circulatory parameters like blood flow, aortic pressure, and heart rate were monitored throughout the experiment. Stenosis was induced in the circumflex artery, controlled by a fractional flow reserve (FFR) pressure wire. CT-derived myocardial perfusion parameters were analysed at FFR of 1 to 0.10/0.0. Results. CT images did not show major artefacts due to interference of the model setup. The pacemaker-induced heart rhythm was generally stable at 70 beats per minute. During most of the experiment, blood flow was 0.9–1.0 L/min, and arterial pressure varied between 80 and 95 mm/Hg. Blood flow decreased and arterial pressure increased by approximately 10% after inducing a stenosis with FFR ≤ 0.50. Dynamic perfusion scanning was possible across the range of stenosis grades. Perfusion parameters of circumflex-perfused myocardial segments were affected at increasing stenosis grades. Conclusion. An adapted Langendorff porcine heart model is feasible in a CT environment. This model provides control over physiological parameters and may allow in-depth validation of quantitative CT perfusion techniques. PMID:26185756

  13. In vivo and Ex vivo MR Imaging of Slowly Cycling Melanoma Cells

    PubMed Central

    Magnitsky, S.; Roesch, A.; Herlyn, M.; Glickson, J.D.

    2011-01-01

    Slowly cycling cells are believed to play a critical role in tumor progression and metastatic dissemination. The goal of this study was to develop a method for in vivo detection of slowly cycling cells. To distinguish these cells from more rapidly proliferating cells that constitute the vast majority of cells in tumors, we utilized the well-known effect of label dilution due to division of cells with normal cycle and retention of contrast agent in slowly dividing cells. To detect slowly cycling cells melanoma cells were labeled with iron oxide particles. After labeling, we observed dilution of contrast agent in parallel with cell proliferation in the vast majority of normally cycling cells. A small and distinct sub-population of iron-retaining cells was detected by flow cytometry after 20 days of in vitro proliferation. These iron-retaining cells exhibited high expression of a biological marker of slowly cycling cells, JARID1B. After implantation of labeled cells as xenografts into immunocompromised mice, iron-retaining cells were detected in vivo and ex vivo by MRI that was confirmed by Prussian Blue staining. MR imaging detects not only iron retaining melanoma cells but also iron positive macrophages. Proposed method opens up opportunities to image subpopulation of melanoma cells, which is critical for continuous tumor growth. PMID:21523820

  14. Ex Vivo Liver Resection and Autotransplantation for End-Stage Alveolar Echinococcosis: A Case Series.

    PubMed

    Wen, H; Dong, J-H; Zhang, J-H; Duan, W-D; Zhao, J-M; Liang, Y-R; Shao, Y-M; Ji, X-W; Tai, Q-W; Li, T; Gu, H; Tuxun, T; He, Y-B; Huang, J-F

    2016-02-01

    The role of autotransplantation in end-stage hepatic alveolar echinococcosis (AE) is unclear. We aimed to present our 15-case experience and propose selection criteria for autotransplantation. All patients were considered to have unresectable hepatic AE by conventional resection due to critical invasion to retrohepatic vena cava, hepatocaval region along with three hepatic veins, and the tertiary portal and arterial branches. All patients successfully underwent ex vivo extended right hepatectomy and autotransplantation without intraoperative mortality. The median autograft weight was 706 g (380-1000 g); operative time was 15.5 hours (11.5-20.5 hours); and anhepatic time was 283.8 minutes (180-435 min). Postoperative hospital stay was 32.3 days (12-60 days). Postoperative complication Clavien-Dindo grade IIIa or higher occurred in three patients including one death that occurred 12 days after the surgery due to acute liver failure. One patient was lost to follow-up after the sixth month. Thirteen patients were followed for a median of 21.6 months with no relapse. This is the largest reported series of patients with end-stage hepatic AE treated with liver autotransplantation. The technique requires neither organ donor nor postoperative immunosuppressant. The early postoperative mortality was low with acceptable morbidity. Preoperative precise assessment and strict patient selection are of utmost importance. PMID:26460900

  15. Invasion of Herpes Simplex Virus Type 1 into Murine Epidermis: An Ex Vivo Infection Study.

    PubMed

    Rahn, Elena; Petermann, Philipp; Thier, Katharina; Bloch, Wilhelm; Morgner, Jessica; Wickström, Sara A; Knebel-Mörsdorf, Dagmar

    2015-12-01

    Herpes simplex virus type 1 (HSV-1) invades its human host via the skin or mucosa. We aim to understand how HSV-1 overcomes the barrier function of the host epithelia, and for this reason, we established an ex vivo infection assay initially with murine skin samples. Here, we report how tissue has to be prepared to be susceptible to HSV-1 infection. Most efficient infection of the epidermis was achieved by removing the dermis. HSV-1 initially invaded the basal epidermal layer, and from there, spreading to the suprabasal layers was observed. Strikingly, in resting stage hair follicles, only the hair germ was infected, whereas the quiescent bulge stem cells (SCs) were resistant to infection. However, during the growth phase, infected cells were also detected in the activated bulge SCs. We demonstrated that cell proliferation was not a precondition for HSV-1 invasion, but SC activation was required as shown by infection of aberrantly activated bulge SCs in integrin-linked kinase (ILK)-deficient hair follicles. These results suggest that the status of the bulge SCs determines whether HSV-1 can reach its receptors, whereas the receptors on basal keratinocytes are accessible irrespective of their proliferation status. PMID:26203638

  16. Ex vivo generated natural killer cells acquire typical natural killer receptors and display a cytotoxic gene expression profile similar to peripheral blood natural killer cells.

    PubMed

    Lehmann, Dorit; Spanholtz, Jan; Osl, Markus; Tordoir, Marleen; Lipnik, Karoline; Bilban, Martin; Schlechta, Bernhard; Dolstra, Harry; Hofer, Erhard

    2012-11-01

    Ex vivo differentiation systems of natural killer (NK) cells from CD34+ hematopoietic stem cells are of potential importance for adjuvant immunotherapy of cancer. Here, we analyzed ex vivo differentiation of NK cells from cord blood-derived CD34+ stem cells by gene expression profiling, real-time RT-PCR, flow cytometry, and functional analysis. Additionally, we compared the identified characteristics to peripheral blood (PB) CD56(bright) and CD56(dim) NK cells. The data show sequential expression of CD56 and the CD94 and NKG2 receptor chains during ex vivo NK cell development, resulting finally in the expression of a range of genes with partial characteristics of CD56(bright) and CD56(dim) NK cells from PB. Expression of characteristic NK cell receptors and cytotoxic genes was mainly found within the predominant ex vivo generated population of NKG2A+ NK cells, indicating the importance of NKG2A expression during NK cell differentiation and maturation. Furthermore, despite distinct phenotypic characteristics, the detailed analysis of cytolytic genes expressed within the ex vivo differentiated NK cells revealed a pattern close to CD56(dim) NK cells. In line with this finding, ex vivo generated NK cells displayed potent cytotoxicity. This supports that the ex vivo differentiation system faithfully reproduces major steps of the differentiation of NK cells from their progenitors, constitutes an excellent model to study NK cell differentiation, and is valuable to generate large-scale NK cells appropriate for immunotherapy. PMID:22571679

  17. Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

    PubMed Central

    Yeung, Jonathan C; Wagnetz, Dirk; Cypel, Marcelo; Rubacha, Matthew; Koike, Terumoto; Chun, Yi-Min; Hu, Jim; Waddell, Thomas K; Hwang, David M; Liu, Mingyao; Keshavjee, Shaf

    2012-01-01

    Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function. PMID:22453765

  18. Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

    PubMed

    Carmelo, Joana G; Fernandes-Platzgummer, Ana; Cabral, Joaquim M S; da Silva, Cláudia Lobato

    2015-01-01

    The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations. PMID:25063496

  19. Ex vivo identification of atherosclerotic plaque calcification by a 31P solid-state magnetic resonance imaging technique.

    PubMed

    Hallock, Kevin J; Hamilton, James A

    2006-12-01

    Calcified tissue is a common component of atherosclerotic plaques, and occurs most often in mature plaques. The process of calcification is a poorly understood risk factor that may contribute to a plaque's vulnerability to sudden rupture. In this study a solid-state imaging sequence, termed single-point imaging (SPI), was used to observe calcification directly in ex vivo atherosclerotic plaques. Standards were used to validate the ability of (31)P SPI to detect and differentiate calcification from crystalline cholesterol, phospholipids, and other plaque components. After suitable experimental parameters were found, human carotid specimens obtained by endarterectomy were imaged ex vivo by (31)P solid-state imaging and standard (1)H methods. In contrast to (1)H imaging methods, (31)P imaging detected only the calcification in the plaque. PMID:17089379

  20. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility.

    PubMed

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N; Marfurt, Jutta

    2016-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. PMID:26525783

  1. Towards a PBMC “virogram assay” for precision medicine: concordance between ex vivo and in vivo viral infection transcriptomes

    PubMed Central

    Gardeux, Vincent; Bosco, Anthony; Li, Jianrong; Halonen, Marilyn J.; Jackson, Daniel; Martinez, Fernando D.; Lussier, Yves A.

    2016-01-01

    Background Understanding individual patient host-response to viruses is key to designing optimal personalized therapy. Unsurprisingly, in vivo human experimentation to understand individualized dynamic response of the transcriptome to viruses are rarely studied because of the obviously limitations stemming from ethical considerations of the clinical risk. Objective In this rhinovirus study, we first hypothesized that ex vivo human cells response to virus can serve as proxy for otherwise controversial in vivo human experimentation. We further hypothesized that the N-of-1-pathways framework, previously validated in cancer, can be effective in understanding the more subtle individual transcriptomic response to viral infection. Method N-of-1-pathways computes a significance score for a given list of gene sets at the patient level, using merely the ‘omics profiles of two paired samples as input. We extracted the peripheral blood mononuclear cells (PBMC) of four human subjects, aliquoted in two paired samples, one subjected to ex vivo rhinovirus infection. Their dysregulated genes and pathways were then compared to those of 9 human subjects prior and after intranasal inoculation in vivo with rhinovirus. Additionally, we developed the Similarity Venn Diagram, a novel visualization method that goes beyond conventional overlap to show the similarity between two sets of qualitative measures. Results We evaluated the individual N-of-1-pathways results using two established cohort-based methods: GSEA and enrichment of differentially expressed genes. Similarity Venn Diagrams and individual patient ROC curves illustrate and quantify that the in vivo dysregulation is recapitulated ex vivo both at the gene and pathway level (p-values≤0.004). Conclusion We established the first evidence that an interpretable dynamic transcriptome metric, conducted as an ex vivo assays for a single subject, has the potential to predict individualized response to infectious disease without the

  2. Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications

    PubMed Central

    Han, Shuhong; Huang, Yuju; Liang, Yin; Ho, Yuchin; Wang, Yichen; Chang, Lung-Ji

    2009-01-01

    Ex vivo activation and expansion of lymphocytes for adoptive cell therapy has demonstrated great success. To improve safety and therapeutic efficacy, increased antigen specificity and reduced non-specific response of the ex vivo generated immune cells are necessary. Here, using a complete protein-spanning pool of pentadecapeptides of the latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV), a weak viral antigen which is associated with EBV lymphoproliferative diseases, we investigated the phenotype and function of immune effector cells generated based on IFN-γ or CD137 activation marker selection and dendritic cell (DC) activation. These ex vivo prepared immune cells exhibited a donor- and antigen-dependent T cell response; the IFN-γ-selected immune cells displayed a donor-related CD4- or CD8-dominant T cell phenotype; however, the CD137-enriched cells showed an increased ratio of CD4 T cells. Importantly, the pentadecapeptide antigens accessed both class II and class I MHC antigen processing machineries and effectively activated EBV-specific CD4 and CD8 T cells. Phenotype and kinetic analyses revealed that the IFN-γ and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-γ-selected effector cells showed the highest level of antigen-specificity and effector activities. While all three approaches generated immune cells with comparable antigen-specific activities, the IFN-γ selection followed by ex vivo expansion produced high quality and quantity of antigen-specific effector cells. Our studies presented the optimal approach for generating therapeutic immune cells with potential for emergency and routine clinical applications. PMID:19660111

  3. Expression of Plasmodium vivax crt-o Is Related to Parasite Stage but Not Ex Vivo Chloroquine Susceptibility

    PubMed Central

    Pava, Zuleima; Handayuni, Irene; Wirjanata, Grennady; To, Sheren; Trianty, Leily; Noviyanti, Rintis; Poespoprodjo, Jeanne Rini; Auburn, Sarah; Price, Ric N.

    2015-01-01

    Chloroquine (CQ)-resistant Plasmodium vivax is present in most countries where P. vivax infection is endemic, but the underlying molecular mechanisms responsible remain unknown. Increased expression of P. vivax crt-o (pvcrt-o) has been correlated with in vivo CQ resistance in an area with low-grade resistance. We assessed pvcrt-o expression in isolates from Papua (Indonesia), where P. vivax is highly CQ resistant. Ex vivo drug susceptibilities to CQ, amodiaquine, piperaquine, mefloquine, and artesunate were determined using a modified schizont maturation assay. Expression levels of pvcrt-o were measured using a novel real-time quantitative reverse transcription-PCR method. Large variations in pvcrt-o expression were observed across the 51 isolates evaluated, with the fold change in expression level ranging from 0.01 to 59 relative to that seen with the P. vivax β-tubulin gene and from 0.01 to 24 relative to that seen with the P. vivax aldolase gene. Expression was significantly higher in isolates with the majority of parasites at the ring stage of development (median fold change, 1.7) compared to those at the trophozoite stage (median fold change, 0.5; P < 0.001). Twenty-nine isolates fulfilled the criteria for ex vivo drug susceptibility testing and showed high variability in CQ responses (median, 107.9 [range, 6.5 to 345.7] nM). After controlling for the parasite stage, we found that pvcrt-o expression levels did not correlate with the ex vivo response to CQ or with that to any of the other antimalarials tested. Our results highlight the importance of development-stage composition for measuring pvcrt-o expression and suggest that pvcrt-o transcription is not a primary determinant of ex vivo drug susceptibility. A comprehensive transcriptomic approach is warranted for an in-depth investigation of the role of gene expression levels and P. vivax drug resistance. PMID:26525783

  4. Recent advances in haploidentical hematopoietic stem cell transplantation using ex vivo T cell-depleted graft in children and adolescents

    PubMed Central

    Koh, Kyung-Nam; Seo, Jong Jin

    2016-01-01

    Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for children and adolescents with various malignant and non-malignant diseases. While human leukocyte antigen (HLA)-identical sibling donor is the preferred choice, matched unrelated volunteer donor is another realistic option for successful HSCT. Unfortunately, it is not always possible to find a HLA-matched donor for patients requiring HSCT, leading to a considerable number of deaths of patients without undergoing transplantation. Alternatively, allogeneic HSCT from haploidentical family members could provide donors for virtually all patients who need HSCT. Although the early attempts at allogeneic HSCT from haploidentical family donor (HFD) were disappointing, recent advances in the effective ex vivo depletion of T cells or unmanipulated in vivo regulation of T cells, better supportive care, and optimal conditioning regimens have significantly improved the outcomes of haploidentical HSCT. The ex vivo techniques used to remove T cells have evolved from the selection of CD34+ hematopoietic stem cell progenitors to the depletion of CD3+ cells, and more recently to the depletion of αβ+ T cells. The recent emerging evidence for ex vivo T cell-depleted haploidentical HSCT has provided additional therapeutic options for pediatric patients with diseases curable by HSCT but has not found a suitable related or unrelated donor. This review discusses recent advances in haploidentical HSCT, focusing on transplant using ex vivo T cell-depleted grafts. In addition, our experiences with this novel approach for the treatment of pediatric patients with malignant and non-malignant diseases are described. PMID:27104186

  5. An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine

    PubMed Central

    Schwerdtfeger, Luke A.; Ryan, Elizabeth P.

    2015-01-01

    Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation. PMID:26680736

  6. Cryopreserved Ex Vivo-Expanded Allogeneic Myeloid Progenitor Cell Product Protects Neutropenic Mice From a Lethal Fungal Infection.

    PubMed

    Domen, Jos; Christensen, Julie L; Gille, Daphne; Smith-Berdan, Stephanie; Fong, Timothy; Brown, Janice M Y; Sedello, Anna K

    2016-01-01

    Severe neutropenia induced by chemotherapy or conditioning for hematopoietic cell transplantation often results in morbidity and mortality due to infection by opportunistic pathogens. A system has been developed to generate ex vivo-expanded mouse myeloid progenitor cells (mMPCs) that produce functional neutrophils in vivo upon transplantation in a pathogen challenge model. It has previously been demonstrated that transplantation of large numbers of freshly isolated myeloid progenitors from a single donor provides survival benefit in radiation-induced neutropenic mice. In the present work, an ex vivo-expanded and cryopreserved mMPC product generated from an allogeneic donor pool retains protective activity in vivo in a lethal fungal infection model. Infusion of the allogeneic pooled mMPC product is effective in preventing death from invasive Aspergillus fumigatus in neutropenic animals, and protection is dose dependent. Cell progeny from the mMPC product is detected in the bone marrow, spleen, blood, and liver by flow cytometry 1 week postinfusion but is no longer evident in most animals 4 weeks posttransplant. In this model, the ex vivo-generated pooled allogeneic mMPC product (i) expands and differentiates in vivo; (ii) is functional and prevents death from invasive fungal infection; and (iii) does not permanently engraft or cause allosensitization. These data suggest that an analogous ex vivo-expanded human myeloid progenitor cell product may be an effective off-the-shelf bridging therapy for the infectious complications that develop during hematopoietic recovery following hematopoietic cell transplantation or intensive chemotherapy. PMID:25812169

  7. Ex vivo human trabecular bone model for biocompatibility evaluation of calcium phosphate composites modified with spray dried biodegradable microspheres.

    PubMed

    Schnieders, Julia; Gbureck, Uwe; Germershaus, Oliver; Kratz, Marita; Jones, David B; Kissel, Thomas

    2013-10-01

    Our aim was to study the suitability of the ex-vivo human trabecular bone bioreactor ZetOS to test the biocompatibility of calcium phosphate bone cement composites modified with spray dried, drug loaded microspheres. We hypothesized, that this bone bioreactor could be a promising alternative to in vivo assessment of biocompatibility in living human bone over a defined time period. Composites consisting of tetracycline loaded poly(lactic-co-glycolic acid) microspheres and calcium phosphate bone cement, were inserted into in vitro cultured human femora head trabecular bone and incubated over 30 days at 37°C in the incubation system. Different biocompatibility parameters, such as lactate dehydrogenase activity, alkaline phosphatase release and the expression of relevant cytokines, IL-1β, IL-6, and TNF-α, were measured in the incubation medium. No significant differences in alkaline phosphatase, osteocalcin, and lactate dehydrogenase activity were measured compared to control samples. Tetracycline was released from the microspheres, delivered and incorporated into newly formed bone. In this study we demonstrated that ex vivo biocompatibility testing using human trabecular bone in a bioreactor is a potential alternative to animal experiments since bone metabolism is still maintained in a physiological environment ex vivo. PMID:23568426

  8. Integrity and stability of oral liposomes containing bile salts studied in simulated and ex vivo gastrointestinal media.

    PubMed

    Hu, Shunwen; Niu, Mengmeng; Hu, Fuqiang; Lu, Yi; Qi, Jianping; Yin, Zongning; Wu, Wei

    2013-01-30

    The objective of this study was to investigate the integrtity and stability of oral liposomes containing glycocholate (SGC-Lip) in simulated gastrointestinal (GI) media and ex vivo GI media from rats in comparison with conventional liposomes (CH-Lip) composed of soybean phosphatidylcholine and cholesterol. Membrane integrity of liposomes was evaluated by monitoring calcein release, particle size and distribution in different simulated GI media. The stability of liposomes encapsulating insulin was investigated in simulated GI fluids containing pepsin or pancreatin and ex vivo GI enzyme fluids. Simulated GI media with low pH or physiological bile salts resulted in significant increase in calcein release, but dynamic laser scattering data showed that the size and distribution were generally stable. SGC-Lip retained the major amount of the initially encapsulated insulin as compared with CH-Lip in simulated GI fluids (SGF, FaSSGF, SIF and FeSSIF-V2). SGC-Lip retained respectively 17.1% and 20.5% of the initially encapsulated insulin in ex vivo GI fluid, which were also significantly more than CH-Lip. These results suggested that SGC-Lip could protect insulin from degradation to some degree during their transit through the gastrointestinal tract and contributed to enhanced oral absorption. PMID:23089580

  9. Sustained Growth of the Ex Vivo Ablation Zones' Critical Short Axis Using Gas-cooled Radiofrequency Applicators

    SciTech Connect

    Rempp, Hansjoerg; Scharpf, Marcus; Voigtlaender, Matthias; Schraml, Christina; Schmidt, Diethard; Fend, Falko; Claussen, Claus D.; Enderle, Markus D.; Pereira, Philippe L.; Clasen, Stephan

    2011-02-15

    Purpose: To evaluate the ablation zones created with a gas-cooled bipolar radiofrequency applicator performed on ex vivo bovine liver tissue. Materials and Methods: A total of 320 ablations with an internally gas-cooled bipolar radiofrequency applicator were performed on fresh ex vivo bovine liver tissue, varying the ablation time (5, 10, 15, and 20 min), power (20, 30, 40, and 50 W), and gas pressure of the CO{sub 2} used for cooling (585, 600, 615, 630, 645 psi), leading to a total of 80 different parameter combinations. Size and shape of the white coagulation zone were assessed. Results: The largest complete ablation zone was achieved after 20 min of implementing 50 W and 645 psi, resulting in a short axis of mean 46 {+-} 1 mm and a long axis of 56 {+-} 2 mm (mean {+-} standard deviation). Short-axis diameters increased between 5 and 20 min of ablation time at 585 psi (increase of the short axis was 45% at 30 W, 29% at 40 W, and 39% at 50 W). This increase was larger at 645 psi (113% at 30 W, 67% at 40 W, and 70% at 50 W). Macroscopic assessment and NADH (nicotinamide adenine dinucleotide) staining revealed incompletely ablated tissue along the needle track in 18 parameter combinations including low-power settings (20 and 30 W) and different cooling levels and ablation times. Conclusion: Gas-cooled radiofrequency applicators increase the short-axis diameter of coagulation in an ex vivo setting if appropriate parameters are selected.

  10. Precision cut intestinal slices are an appropriate ex vivo model to study NSAID-induced intestinal toxicity in rats.

    PubMed

    Niu, Xiaoyu; de Graaf, Inge A M; van der Bij, Hendrik A; Groothuis, Geny M M

    2014-10-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used therapeutic agents, however, they are associated with a high prevalence of intestinal side effects. In this investigation, rat precision cut intestinal slices (PCIS) were evaluated as an ex vivo model to study NSAID-induced intestinal toxicity. Firstly, PCIS were incubated with 0-200 μM diclofenac (DCF), one of the most intensively studied NSAIDs, to investigate whether they could correctly reflect the toxic mechanisms. DCF induced intestinal toxicity in PCIS was shown by morphological damage and ATP depletion. DCF induced endoplasmic-reticulum (ER) stress, mitochondrial injury and oxidative stress were reflected by up-regulated HSP-70 (heat shock protein 70) and BiP (binding immunoglobulin protein) gene expression, caspase 9 activation, GSH (glutathione) depletion and HO-1 (heme oxygenase 1) gene up-regulation respectively. Furthermore, DCF intestinal metabolites, which gave rise to protein adduct but not toxicity, were detected in PCIS. Secondly, PCIS were incubated with various concentrations of five NSAIDs. Typical NSAID-induced morphological changes were observed in PCIS. The ex vivo toxicity ranking (diflunisal> diclofenac = indomethacin > naproxen ≫ aspirin) showed good correlation with published in vitro and in vivo data, with diflunisal being the only exception. In conclusion, PCIS correctly reflect the various mechanisms of DCF-induced intestinal toxicity, and can serve as an ex vivo model for the prediction of NSAID-induced intestinal toxicity. PMID:25014874

  11. Ex-vivo-examination of ultrastructural changes in organotypic retina culture using near-infrared imaging and optical coherence tomography.

    PubMed

    Schnichels, Sven; Dorfi, Tanja; Schultheiss, Maximilian; Arango-Gonzalez, Blanca; Bartz-Schmidt, Karl-Ulrich; Januschowski, Kai; Spitzer, Martin S; Ziemssen, Focke

    2016-06-01

    Optical coherence tomography (OCT) dramatically changed the way of diagnostic assessment in retinal diseases during the last years. Using this technique in-vivo in-depth analysis of the retina and its layers is possible. Since animal research is changing by intrinsic and extrinsic pressure to animal-(in-vivo)-free methods, we adapted OCT-measurements to organotypic cultures. An easy to use protocol was generated to assess standardized OCT assessments in organotypic culture. First, two custom-made devices need to be made to change any commercially available OCT for examinations in humans into a device allowing ex-vivo analyses of organotypic culture. The modification is feasible within seconds. After OCT measurement of the ex-vivo tissues, quantitative evaluation of the retinas were performed via ImageJ software. OCT pictures of ex-vivo retinas were obtained for time periods of seven days and the thickness of retinal tissue was evaluated. The reproducibility of the pictures and measurements was very high (SD < 15%). In conclusion, an easy to use protocol for the investigation of different effects on retinal cultures with commercially available OCT devices was successfully established. PMID:27109031

  12. Comparative analysis of human ex vivo-generated platelets vs megakaryocyte-generated platelets in mice: a cautionary tale.

    PubMed

    Wang, Yuhuan; Hayes, Vincent; Jarocha, Danuta; Sim, Xiuli; Harper, Dawn C; Fuentes, Rudy; Sullivan, Spencer K; Gadue, Paul; Chou, Stella T; Torok-Storb, Beverly J; Marks, Michael S; French, Deborah L; Poncz, Mortimer

    2015-06-01

    Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application. PMID:25852052

  13. Numerical and ex vivo studies of a bioprobe developed for laser-induced thermotherapy (LITT) in contact with liver tissue.

    PubMed

    Chartier, T; Carpentier, O; Genestie, B; Hornez, J-C; Monchau, F

    2016-08-01

    This work is based on the production of a bioprobe that is compatible with magnetic resonance imaging (MRI) for laser-induced thermotherapy (LITT) in liver cancer laser therapy. This probe is made of an alumina tube (3-mm diameter) in which an optical fibre is centred and fixed. A shooting window (20mm) is created using a mechanical rectifier. The device is then consolidated by the injection of a transparent and heat-resistant resin. Through numerical modelling, the thermal power damping of the laser source is evaluated as well as the propagation of the heat in the ex vivo liver tissue according to different heating scenarios. These analyses allow for an estimation of the irradiated volume. Ex vivo tests were performed on bovine liver to confirm the adequacy of the bioprobe for LITT and of the irradiated volumes predicted by the numerical model. There was a difference of 8% between the simulations and ex vivo experiments. The pulsed mode heating scenario was the most effective under the experimental conditions. PMID:27212211

  14. Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model

    PubMed Central

    Weidensdorfer, Marko; Chae, Ju Ik; Makobe, Celestine; Stahl, Julia; Averhoff, Beate; Müller, Volker; Schürmann, Christoph; Brandes, Ralf P.; Wilharm, Gottfried; Ballhorn, Wibke; Christ, Sara; Linke, Dirk; Fischer, Doris; Göttig, Stephan

    2015-01-01

    Bacterial adherence determines the virulence of many human-pathogenic bacteria. Experimental approaches elucidating this early infection event in greater detail have been performed using mainly methods of cellular microbiology. However, in vitro infections of cell monolayers reflect the in vivo situation only partially, and animal infection models are not available for many human-pathogenic bacteria. Therefore, ex vivo infection of human organs might represent an attractive method to overcome these limitations. We infected whole human umbilical cords ex vivo with Bartonella henselae or Acinetobacter baumannii under dynamic flow conditions mimicking the in vivo infection situation of human endothelium. For this purpose, methods for quantifying endothelium-adherent wild-type and trimeric autotransporter adhesin (TAA)-deficient bacteria were set up. Data revealed that (i) A. baumannii binds in a TAA-dependent manner to endothelial cells, (ii) this organ infection model led to highly reproducible adherence rates, and furthermore, (iii) this model allowed to dissect the biological function of TAAs in the natural course of human infections. These findings indicate that infection models using ex vivo human tissue samples (“organ microbiology”) might be a valuable tool in analyzing bacterial pathogenicity with the capacity to replace animal infection models at least partially. PMID:26712205

  15. In vitro, ex vivo and in vivo characterization of PLGA nanoparticles loading pranoprofen for ocular administration.

    PubMed

    Cañadas, Cristina; Alvarado, Helen; Calpena, Ana C; Silva, Amélia M; Souto, Eliana B; García, Maria L; Abrego, Guadalupe

    2016-09-25

    Pranoprofen (PF) is a NSAID considered as a safe anti-inflammatory treatment for strabismus and/or cataract surgery. The drug has been formulated in poly (lactic/glycolic) acid (PLGA) nanoparticles (PF-F1NPs with cPF 1.5mg/mL, PF-F2NPs with cPF 1mg/mL) produced by solvent displacement technique and tested the in vitro cytotoxicity, ex vivo corneal permeation, in vivo ocular tolerance and in vivo anti-inflammatory efficacy of PF-F1NPs, PF-F2NPs, in comparison to eye drops conventional dosage form (Oftalar(®), PF 1mg/mL) and free drug solution (PF dissolved in PBS, 1.5mg/mL). The mean particle size of both formulations was around 350nm, with polydispersity index below 0.1, and a net negative charge of -7.41mV and -8.5mV for PF-F1NPs and PF-F2NPs, respectively. Y-79 human retinoblastoma cell line was used to evaluate the cytotoxicity of PF-F1NPs and PF-F2NPs, which were compared to blank NPs and free drug solution (PF dissolved in PBS, 1.5mg/mL). Concentrations up to 75μg/mL exhibited no toxicity to Y-79 cells, whereas at 150μg/mL a decrease of about 80% on the cell viability was observed after exposing the cells to PF-F1NPs. When treating the Y-79 cells with concentrations of PF-F2NPs between 1μg/mL to 100μg/mL, the cell viability was similar to control values after 24h and 48h of exposure. An ex vivo corneal permeation study was carried out in New Zealand rabbits. A very similar profile has been observed for the permeation of PF through the cornea when administered as eye drops and as free drug solution, which was kept much lower in comparison to PF-NPs formulations. The permeated amount of PF from the PF-F1NPs was slightly smaller than from PF-F2NPs, attributed to the increase of viscosity of the formulations with the increase of cPVA concentration. New Zealand white rabbits were also used to evaluate the irritancy of PF-F1NPs and PF-F2NPs, which demonstrated to be well-tolerated to the eye (i.e. the mean total score (MTS) was 0). PF-F2NPs exhibited the

  16. Ex vivo and in vivo coherent Raman imaging of the peripheral and central nervous system

    NASA Astrophysics Data System (ADS)

    Huff, Terry Brandon

    A hallmark of nervous system disorders is damage or degradation of the myelin sheath. Unraveling the mechanisms underlying myelin degeneration and repair represent one of the great challenges in medicine. This thesis work details the development and utilization of advanced optical imaging methods to gain insight into the structure and function of myelin in both healthy and diseased states in the in vivo environment. This first part of this thesis discusses ex vivo studies of the effects of high-frequency stimulation of spinal tissues on the structure of the node of Ranvier as investigated by coherent anti-Stokes Raman scattering (CARS) imaging (manuscript submitted to Journal of Neurosciece). Reversible paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation, beginning minutes after the onset and continuing for up to 10 min after stimulation was ceased. A mechanistic study revealed a Ca2+ dependent pathway: high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down. Also, the construction of dual-scanning CARS microscope for large area mapping of CNS tissues is detailed (Optics Express, 2008, 16:19396-193409). A confocal scanning head equipped with a rotating polygon mirror provides high speed, high resolution imaging and is coupled with a motorized sample stage to generate high-resolution large-area images of mouse brain coronal section and guinea pig spinal cord cross section. The polygon mirror decreases the mosaic acquisition time significantly without reducing the resolution of individual images. The ex vivo studies are then extended to in vivo imaging of mouse sciatic nerve tissue by CARS and second harmonic generation (SHG) imaging (Journal of Microscopy, 2007, 225: 175-182). Following a minimally invasive surgery to open the skin, CARS imaging of myelinated axons and SHG imaging of the

  17. An ex vivo gene therapy approach in X-linked retinoschisis

    PubMed Central

    Bashar, Abu E.; Metcalfe, Andrew L.; Viringipurampeer, Ishaq A.; Yanai, Anat; Gregory-Evans, Cheryl Y.

    2016-01-01

    Purpose X-linked retinoschisis (XLRS) is juvenile-onset macular degeneration caused by haploinsufficiency of the extracellular cell adhesion protein retinoschisin (RS1). RS1 mutations can lead to either a non-functional protein or the absence of protein secretion, and it has been established that extracellular deficiency of RS1 is the underlying cause of the phenotype. Therefore, we hypothesized that an ex vivo gene therapy strategy could be used to deliver sufficient extracellular RS1 to reverse the phenotype seen in XLRS. Here, we used adipose-derived, syngeneic mesenchymal stem cells (MSCs) that were genetically modified to secrete human RS1 and then delivered these cells by intravitreal injection to the retina of the Rs1h knockout mouse model of XLRS. Methods MSCs were electroporated with two transgene expression systems (cytomegalovirus (CMV)-controlled constitutive and doxycycline-induced Tet-On controlled inducible), both driving expression of human RS1 cDNA. The stably transfected cells, using either constitutive mesenchymal stem cell (MSC) or inducible MSC cassettes, were assayed for their RS1 secretion profile. For single injection studies, 100,000 genetically modified MSCs were injected into the vitreous cavity of the Rs1h knockout mouse eye at P21, and data were recorded at 2, 4, and 8 weeks post-injection. The control groups received either unmodified MSCs or vehicle injection. For the multiple injection studies, the mice received intravitreal MSC injections at P21, P60, and P90 with data collection at P120. For the single- and multiple-injection studies, the outcomes were measured with electroretinography, optokinetic tracking responses (OKT), histology, and immunohistochemistry. Results Two lines of genetically modified MSCs were established and found to secrete RS1 at a rate of 8 ng/million cells/day. Following intravitreal injection, RS1-expressing MSCs were found mainly in the inner retinal layers. Two weeks after a single injection of MSCs, the

  18. Prevalidation of the ex-vivo model PCLS for prediction of respiratory toxicity.

    PubMed

    Hess, A; Wang-Lauenstein, L; Braun, A; Kolle, S N; Landsiedel, R; Liebsch, M; Ma-Hock, L; Pirow, R; Schneider, X; Steinfath, M; Vogel, S; Martin, C; Sewald, K

    2016-04-01

    In acute inhalation toxicity studies, animals inhale substances at given concentrations. Without additional information, however, appropriate starting concentrations for in-vivo inhalation studies are difficult to estimate. The goal of this project was the prevalidation of precision-cut lung slices (PCLS) as an ex-vivo alternative to reduce the number of animals used in inhalation toxicity studies. According to internationally agreed principles for Prevalidation Studies, the project was conducted in three independent laboratories. The German BfR provided consultancy in validation principles and independent support with biostatistics. In all laboratories, rat PCLS were prepared and exposed to 5 concentrations of 20 industrial chemicals under submerged culture conditions for 1h. After 23h post-incubation, toxicity was assessed by measurement of released lactate dehydrogenase and mitochondrial activity. In addition, protein content and pro-inflammatory cytokine IL-1α were measured. For all endpoints IC50 values were calculated if feasible. For each endpoint test acceptance criteria were established. This report provides the final results for all 20 chemicals. More than 900 concentration-response curves were analyzed. Log10[IC50 (μM)], obtained for all assay endpoints, showed best intra- and inter-laboratory consistency for the data obtained by WST-1 and BCA assays. While WST-1 and LDH indicated toxic effects for the majority of substances, only some of the substances induced an increase in extracellular IL-1α. Two prediction models (two-group classification model, prediction of LC50 by IC50) were developed and showed promising results. PMID:26778741

  19. Air leak seal for lung dissection plane with diode laser irradiation: an ex vivo study

    NASA Astrophysics Data System (ADS)

    Gotoh, Maya; Tokunaga, Hisako; Kaneko, Kenji; Arai, Tsunenori

    2007-02-01

    In order to seal air leak from lung dissection plane in thoracotomy, we studied diode laser irradiation (wavelength: 810nm) with surface stain of indocyanine green (ICG, peak absorption wavelength: 805nm) ex vivo. In general, this air leak is sealed by suturing with weak tension and large margin of parenchyma. This suturing requires surgeon's skill and takes long time. Moreover, lung ventilatory performance is significantly impaired. Since laser tissue welding is novel method to adhere living tissue with thin thermally denatured attachment layer, we propose to seal the lung dissection plane with laser irradiation. Our aim of this study is to investigate the sealing mechanism as well as optimum condition to develop reliable laser sealing method for dissected lung plane in surgery, using practical laser-dye combination. Compartment of extracted porcine lung was prepared as a lung model, which volume was approximately 60cm^3. ICG solution (2.5mg/ml) was applied to the dissection plane of this lung model with 1minute. The diode laser (power density: 8-40W/cm^2) irradiated to the plane, moving the laser spot with constant speed (v=1mm/s). The heat degeneration depth and smoothness of the laser irradiated surface were observed by a microscope. When power density was over 24W/cm^2, heat degeneration depth was over 1.5E-4 m. There were no pin holes on the surface and the air leak seemed to be sealed completely. We also evaluated the air leak by endotracheal pressure. In the case of above condition, the heat degeneration depth was the same that of previous reported result with CO2 laser.

  20. Ethosomes for skin delivery of ropivacaine: preparation, characterization and ex vivo penetration properties.

    PubMed

    Zhai, Yingjie; Xu, Rui; Wang, Yi; Liu, Jiyong; Wang, Zimin; Zhai, Guangxi

    2015-01-01

    Ropivacaine, a novel long-acting local anesthetic, has been proved to own superior advantage. However, Naropin® Injection, the applied form in clinic, can cause patient non-convenience. The purpose of this study was to formulate ropivacaine (RPV) in ethosomes and evaluate the potential of ethosome formulation in delivering RPV transdermally. The RPV-loaded ethosomes were prepared with thin-film dispersion technique and the formulation was characterized in terms of size, zeta potential, differential scanning calorimetry (DSC) analysis and X-ray diffraction (XRD) study. The results showed that the optimized RPV-ethosomes displayed a typical lipid bilayer structure with a narrow size distribution of 73.86 ± 2.40 nm and drug loading of 8.27 ± 0.37%, EE of 68.92 ± 0.29%. The results of DSC and XRD study indicated that RPV was in amorphous state when encapsulated into ethosomes. Furthermore, the results of ex vivo permeation study proved that RPV-ethosomes could promote the permeability in a high-efficient, rapid way (349.0 ± 11.5 μg cm(-2) at 12 h and 178.8 ± 7.1 μg cm(-2) at 0.5 h). The outcomes of histopathology study forecasted that the interaction between ethosomes and skin could loosen the tight conjugation of corneocyte layers and weaken the permeation barrier. In conclusion, RPV-ethosomes could be a promising delivery system to encapsulate RPV and deliver RPV for transdermal administration. PMID:25625544

  1. Rapid infrared laser sealing and cutting of porcine renal vessels, ex vivo

    NASA Astrophysics Data System (ADS)

    Giglio, Nicholas C.; Hutchens, Thomas C.; Perkins, William C.; Latimer, Cassandra; Ward, Arlen; Nau, William H.; Fried, Nathaniel M.

    2014-03-01

    Suture ligation with subsequent cutting of blood vessels to maintain hemostasis during surgery is time consuming and skill intensive. Energy-based, electrosurgical and ultrasonic devices are often used to replace sutures and mechanical clips to provide rapid hemostasis, and decrease surgical time. Some of these devices may create undesirably large collateral zones of thermal damage and tissue necrosis, or require separate mechanical blades for cutting. Infrared lasers are currently being explored as alternative energy sources for vessel sealing applications. In a previous study, a 1470-nm laser was used to seal vessels of 1-6 mm in diameter in 5 s, yielding burst pressures of ~ 500 mmHg. The purpose of this study was to provide faster sealing, incorporate transection of the sealed vessels, and increase the burst pressure. A 110-Watt, 1470-nm laser beam was transmitted through a fiber and beam shaping optics, producing a linear beam 3.0 mm by 9.5 mm for sealing, and 1.1 mm by 9.6 mm for cutting (FWHM). A twostep process sealed then transected ex vivo porcine renal vessels (1-8.5 mm diameter) in a bench top setup. Seal and cut times were 1.0 s each. A standard burst pressure system measured resulting seal strength, and gross and histologic thermal damage measurements were also recorded. All blood vessels tested (n = 30) were sealed and cut, with total irradiation times of 2.0 s, mean burst pressures > 1000 mmHg (compared to normal systolic blood pressure of 120 mmHg), and combined seal/collateral thermal coagulation zones of 2-3 mm. The results of this study demonstrated that an optical-based system is capable of precisely sealing and cutting a wide range of porcine renal vessel sizes, and with further development, may provide an alternative to radiofrequency and ultrasound-based vessel sealing devices.

  2. Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

    PubMed Central

    Kendig, Derek M.; Hurst, Norm R.; Grider, John R.

    2016-01-01

    Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues

  3. Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

    PubMed

    Sidgwick, G P; McGeorge, D; Bayat, A

    2016-07-01

    A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-β1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p < 0.05). Histological analysis indicated a reduction in mast cell tryptase and chymase positive cell numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p < 0.05). Determination of transdermal uptake indicated that GTCs were detected in the biopsies. This model could be adapted to study a range of different topical formulations in both normal and diseased skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment. PMID:27086034

  4. Radial Diffusivity Predicts Demyelination in ex-vivo Multiple Sclerosis Spinal Cords

    PubMed Central

    Klawiter, Eric C.; Schmidt, Robert E.; Trinkaus, Kathryn; Liang, Hsiao-Fang; Budde, Matthew D.; Naismith, Robert T.; Song, Sheng-Kwei; Cross, Anne H.; Benzinger, Tammie L.

    2011-01-01

    Objective Correlation of diffusion tensor imaging (DTI) with histochemical staining for demyelination and axonal damage in multiple sclerosis (MS) ex vivo human cervical spinal cords. Background In MS, demyelination, axonal degeneration, and inflammation contribute to disease pathogenesis to variable degrees. Based upon in vivo animal studies with acute injury and histopathologic correlation, we hypothesized that DTI can differentiate between axonal and myelin pathologies within humans. Methods DTI was performed at 4.7 Tesla on 9 MS and 5 normal control fixed cervical spinal cord blocks following autopsy. Sections were then stained for Luxol fast blue (LFB), Bielschowsky silver, and hematoxylin and eosin (H&E). Regions of interest (ROIs) were graded semi-quantitatively as normal myelination, mild (<50%) demyelination, or moderate-severe (>50%) demyelination. Corresponding axonal counts were manually determined on Bielschowsky silver. ROIs were mapped to co-registered DTI parameter slices. DTI parameters evaluated included standard quantitative assessments of apparent diffusion coefficient (ADC), relative anisotropy (RA), axial diffusivity and radial diffusivity. Statistical correlations were made between histochemical gradings and DTI parameters using linear mixed models. Results: Within ROIs in MS subjects, increased radial diffusivity distinguished worsening severities of demyelination. Relative anisotropy was decreased in the setting of moderate-severe demyelination compared to normal areas and areas of mild demyelination. Radial diffusivity, ADC, and RA became increasingly altered within quartiles of worsening axonal counts. Axial diffusivity did not correlate with axonal density (p=0.091). Conclusions Increased radial diffusivity can serve as a surrogate for demyelination. However, radial diffusivity was also altered with axon injury, suggesting that this measure is not pathologically specific within chronic human MS tissue. We propose that radial diffusivity

  5. Ex Vivo Modeling of Multidomain Peptide Hydrogels with Intact Dental Pulp.

    PubMed

    Moore, A N; Perez, S C; Hartgerink, J D; D'Souza, R N; Colombo, J S

    2015-12-01

    Preservation of a vital dental pulp is a central goal of restorative dentistry. Currently, there is significant interest in the development of tissue engineering scaffolds that can serve as biocompatible and bioactive pulp-capping materials, driving dentin bridge formation without causing cytotoxic effects. Our earlier in vitro studies described the biocompatibility of multidomain peptide (MDP) hydrogel scaffolds with dental pulp-derived cells but were limited in their ability to model contact with intact 3-dimensional pulp tissues. Here, we utilize an established ex vivo mandible organ culture model to model these complex interactions. MDP hydrogel scaffolds were injected either at the interface of the odontoblasts and the dentin or into the pulp core of mandible slices and subsequently cultured for up to 10 d. Histology reveals minimal disruption of tissue architecture adjacent to MDP scaffolds injected into the pulp core or odontoblast space. Additionally, the odontoblast layer is structurally preserved in apposition to the MDP scaffold, despite being separated from the dentin. Alizarin red staining suggests mineralization at the periphery of MDP scaffolds injected into the odontoblast space. Immunohistochemistry reveals deposition of dentin sialophosphoprotein by odontoblasts into the adjacent MDP hydrogel, indicating continued functionality. In contrast, no mineralization or dentin sialophosphoprotein deposition is evident around MDP scaffolds injected into the pulp core. Collagen III expression is seen in apposition to gels at all experimental time points. Matrix metalloproteinase 2 expression is observed associated with centrally injected MDP scaffolds at early time points, indicating proteolytic digestion of scaffolds. Thus, MDP scaffolds delivered centrally and peripherally within whole dental pulp tissue are shown to be biocompatible, preserving local tissue architecture. Additionally, odontoblast function and pulp vitality are sustained when MDP

  6. Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation In Vivo and Osteoprogenitor Differentiation Ex Vivo.

    PubMed

    Frara, Nagat; Abdelmagid, Samir M; Sondag, Gregory R; Moussa, Fouad M; Yingling, Vanessa R; Owen, Thomas A; Popoff, Steven N; Barbe, Mary F; Safadi, Fayez F

    2016-01-01

    Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased threefold in mutant bones, compared to normal. OA mRNA and protein expression increase during active bone regeneration post-fracture, and primary rat osteoblasts show increased OA expression during differentiation in vitro. To further examine OA/gpnmb as an osteoinductive agent, we characterized the skeletal phenotype of transgenic mouse overexpressing OA/gpnmb under the CMV-promoter (OA-Tg). Western blot analysis showed increased OA/gpnmb in OA-Tg osteoblasts, compared to wild-type (WT). In OA-Tg mouse femurs versus WT littermates, micro-CT analysis showed increased trabecular bone volume and thickness, and cortical bone thickness; histomorphometry showed increased osteoblast numbers, bone formation and mineral apposition rates in OA-Tg mice; and biomechanical testing showed higher peak moment and stiffness. Given that OA/gpnmb is also over-expressed in osteoclasts in OA-Tg mice, we evaluated bone resorption by ELISA and histomorphometry, and observed decreased serum CTX-1 and RANK-L, and decreased osteoclast numbers in OA-Tg, compared to WT mice, indicating decreased bone remodeling in OA-Tg mice. The proliferation rate of OA-Tg osteoblasts in vitro was higher, compared to WT, as was alkaline phosphatase staining and activity, the latter indicating enhanced differentiation of OA-Tg osteoprogenitors. Quantitative RT-PCR analysis showed increased TGF-β1 and TGF-β receptors I and II expression in OA-Tg osteoblasts, compared to WT. Together, these data suggest that OA overexpression has an osteoinductive effect on bone mass in vivo and stimulates osteoprogenitor differentiation ex vivo. PMID:25899717

  7. Mean scatterer spacing estimation in normal and thermally coagulated ex vivo bovine liver.

    PubMed

    Rubert, Nicholas; Varghese, Tomy

    2014-04-01

    The liver has been hypothesized to have a unique arrangement of microvasculature that presents as an arrangement of quasiperiodic scatterers to an interrogating ultrasound pulse. The mean scatterer spacing (MSS) of these quasiperiodic scatterers has been proposed as a useful quantitative ultrasound biomarker for characterizing liver tissue. Thermal ablation is an increasingly popular method for treating hepatic tumors, and ultrasonic imaging approaches for delineating the extent of thermal ablation are in high demand. In this work, we examine the distribution of estimated MSS in thermally coagulated bovine liver and normal untreated bovine liver ex vivo. We estimate MSS by detecting local maxima in the spectral coherence function of radio frequency echoes from a clinical transducer, the Siemens VFX 9L4 transducer operating on an S2000 scanner. We find that normal untreated bovine liver was characterized by an MSS of approximately 1.3 mm. We examined regions of interest 12 mm wide laterally, and ranging from 12 mm to 18 mm axially, in 2 mm increments. Over these parameters, the mode of the MSS estimates was between 1.25 and 1.37 mm. On the other hand, estimation of MSS in thermally coagulated liver tissue yields a distribution of MSS estimates whose mode varied between 0.45 and 1.0 mm when examining regions of interest over the same sizes. We demonstrate that the estimated MSS in thermally coagulated liver favors small spacings because the randomly positioned scatterers in this tissue are better modeled as aperiodic scatterers. The submillimeter spacings result from the fact that this was the most probable spacing to be estimated if the discretely sampled spectral coherence function was a uniformly random two-dimensional function. PMID:24554290

  8. Improving donor lung suitability: from protective strategies to ex-vivo reconditioning.

    PubMed

    Solidoro, Paolo; Schreiber, Annia; Boffini, Massimo; Braido, Fulvio; DI Marco, Fabiano

    2016-06-01

    Lung transplant is a therapeutic option for end stage lung diseases, but only a limited number of lung grafts is considered suitable for transplantation. It has been recently suggested an approach to improve and maximize donor lung suitability, namely ventilation strategies to prevent lung damage and preserve function before transplantation. In potential lung donor patients, the use of lung-protective ventilatory strategies may protect against and at least partially reverse some conditions, such as ventilator-induced lung injury, atelectasis and neurogenic pulmonary edema, resulting in improved donor organ procurement. The novelty recently proposed lies in the integration of ventilatory strategies of previous studies, using an algorithmic approach for the management of potential donors, based on their clinical response and PaO2/FiO2 ratio. This approach could be further improved by using lung ultrasound (LUS) which demonstrated to be more accurate than bedside chest radiography in detecting and distinguishing different degrees of aeration loss, and could be useful in the evaluation of alveolar recruitment following the application of PEEP or after performing any recruitment maneuver. Finally, the close future is the exploration of ex-vivo reconditioning which introduces the exciting concept of both a protective ventilation and a protective perfusion, reducing the risk of ventilation associated damage, and, on the other hand, washing out potential inflammatory cytokines by low volume high oncotic pressure perfusion, managing the risk of edema by capillary leakage. Addressing these challenges will allow more patients with end-stage lung disease access to a lung transplant. PMID:27424500

  9. Matrix-type transdermal drug delivery system of trandolapril: in vitro and ex vivo characterization.

    PubMed

    Tirunagari, Mamatha; Rao Jangala, Venkateswara; Khagga, Mukkanti; Gannu, Ramesh

    2010-01-01

    The purpose of the investigation was to develop and evaluate matrix-type transdermal drug delivery systems (TDDSs) of trandolapril. Matrix-type TDDSs of trandolapril were prepared by solvent evaporation technique. Eight formulations (composed of Eudragit RL 100 and Hydroxypropyl methyl cellulose 15 cps at a ratios of 2:8, 4:6, 6:4, 8:2 in formulations A1, A2, A3, A4; and Eudragit RS 100 and Hydroxypropyl methyl cellulose 15 cps in the same ratios in formulations B1, B2, B3, B4, respectively) were prepared. All formulations contained 5% w/w menthol as penetration enhancer and 15% w/w propylene glycol as plasticizer in ethanol as solvent. The prepared TDDSs were evaluated for physicochemical characteristics, in vitro release and ex vivo permeation. The physicochemical interactions between trandolapril and polymers were investigated by Fourier transform infrared spectroscopy. The results suggested that there is no physicochemical interaction between drug and polymers. The maximum drug release in 24 h for A series formulations was 95.45% (A1), 95.82% (A2), and it was 95.26% (B1), 95.69% (B2) for B series formulations, which are significantly (P < 0.05) different than the lowest values 78.79% (A3), 66.9% (A4) and 82.64% (B3), 71.67% (B4). The formulations A1 (flux 25.03 ± 0.98 μg/cm(2)/h) and B1 (flux 24.62 ± 0.63 μg/cm(2)/h) showed maximum skin permeation in the respective series. The flux obtained with formulations A1 and B1 meets the required flux (37.04 μg/h/cm(2)) with a minimum patch area (3.9 cm(2)). Matrix-type transdermal therapeutic systems of trandolapril could be prepared with the required flux using menthol as penetration enhancer. PMID:21502003

  10. An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

    PubMed Central

    Bansal, Devendra; Ave, Patrick; Kerneis, Sophie; Frileux, Pascal; Boché, Olivier; Baglin, Anne Catherine; Dubost, Geneviève; Leguern, Anne-Sophie; Prevost, Marie-Christine; Bracha, Rivka; Mirelman, David; Guillén, Nancy; Labruyère, Elisabeth

    2009-01-01

    Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion. PMID:19936071

  11. Laser-induced cartilage damage: an ex-vivo model using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Frenz, Martin; Zueger, Benno J.; Monin, D.; Weiler, C.; Mainil-Varlet, P. M.; Weber, Heinz P.; Schaffner, Thomas

    1999-06-01

    Although there is an increasing popularity of lasers in orthopedic surgery, there is a growing concern about negative side effects of this therapy e.g. prolonged restitution time, radiation damage to adjacent cartilage or depth effects like bone necrosis. Despite case reports and experimental investigations over the last few years little is known about the extent of acute cartilage damage induced by different lasers types and energies. Histological examination offers only limited insights in cell viability and metabolism. Ho:YAG and Er:YAG lasers emitting at 2.1 micrometer and 2.94 micrometer, respectively, are ideally suited for tissue treatment because these wavelengths are strongly absorbed in water. The Purpose of the present study is to evaluate the effect of laser type and energy on chondrocyte viability in an ex vivo model. Free running Er:YAG (E equals 100 and 150 mJ) and Ho:YAG (E equals 500 and 800 mJ) lasers were used at different energy levels using a fixed pulse length of 400 microseconds. The energy was delivered at 8 Hz through optical fibers. Fresh bovine hyaline cartilage samples were mounted in a water bath at room temperature and the fiber was positioned at 30 degree and 180 degree angles relative to the tissue surface. After laser irradiation the samples were assessed by a life-dead cell viability test using a confocal microscope and by standard histology. Thermal damage was much deeper with Ho:YAG (up to 1800 micrometer) than with the Er:YAG laser (up to 70 micrometer). The cell viability test revealed a damage zone about twice the one determined by standard histology. Confocal microscopy is a powerful tool for assessing changes in tissue structure after laser treatment. In addition this technique allows to quantify these alterations without necessitating time consuming and expensive animal experiments.

  12. An ex vivo swine tracheal organ culture for the study of influenza infection

    PubMed Central

    Nunes, Sandro F.; Murcia, Pablo R.; Tiley, Laurence S.; Brown, Ian H.; Tucker, Alexander W.; Maskell, Duncan J.; Wood, James Lionel N.

    2009-01-01

    Background  The threat posed by swine influenza viruses with potential to transmit from pig populations to other hosts, including humans, requires the development of new experimental systems to study different aspects of influenza infection. Ex vivo organ culture (EVOC) systems have been successfully used in the study of both human and animal respiratory pathogens. Objectives  We aimed to develop an air interface EVOC using pig tracheas in the study of influenza infection demonstrating that tracheal explants can be effectively maintained in organ culture and support productive influenza infection. Methods  Tracheal explants were maintained in the air interface EVOC system for 7 days. Histological characteristics were analysed with different staining protocols and co‐ordinated ciliary movement on the epithelial surface was evaluated through a bead clearance assay. Explants were infected with a swine H1N1 influenza virus. Influenza infection of epithelial cells was confirmed by immunohistochemistry and viral replication was quantified by plaque assays and real‐time RT‐PCR. Results  Histological analysis and bead clearance assay showed that the tissue architecture of the explants was maintained for up to 7 days, while ciliary movement exhibited a gradual decrease after 4 days. Challenge with swine H1N1 influenza virus showed that the EVOC tracheal system shows histological changes consistent with in vivo influenza infection and supported productive viral replication over multiple cycles of infection. Conclusion  The air interface EVOC system using pig trachea described here constitutes a useful biological tool with a wide range of applications in the study of influenza infection. PMID:20021502

  13. Ex vivo perfusion of the isolated rat small intestine as a novel model of Salmonella enteritis.

    PubMed

    Boyle, Erin C; Dombrowsky, Heike; Sarau, Jürgen; Braun, Janin; Aepfelbacher, Martin; Lautenschläger, Ingmar; Grassl, Guntram A

    2016-01-15

    Using an ex vivo perfused rat small intestinal model, we examined pathological changes to the tissue, inflammation induction, as well as dynamic changes to smooth muscle activity, metabolic competence, and luminal fluid accumulation during short-term infection with the enteropathogenic bacteria Salmonella enterica serovar Typhimurium and Yersinia enterocolitica. Although few effects were seen upon Yersinia infection, this system accurately modeled key aspects associated with Salmonella enteritis. Our results confirmed the importance of the Salmonella Pathogenicity Island 1 (SPI1)-encoded type 3 secretion system (T3SS) in pathology, tissue invasion, inflammation induction, and fluid secretion. Novel physiological consequences of Salmonella infection of the small intestine were also identified, namely, SPI-1-dependent vasoconstriction and SPI-1-independent reduction in the digestive and absorptive functions of the epithelium. Importantly, this is the first small animal model that allows for the study of Salmonella-induced fluid secretion. Another major advantage of this model is that one can specifically determine the contribution of resident cell populations. Accordingly, we can conclude that recruited cell populations were not involved in the pathological damage, inflammation induction, fluid accumulation, nutrient absorption deficiency, and vasoconstriction observed. Although fluid loss induced by Salmonella infection is hypothesized to be due to damage caused by recruited neutrophils, our data suggest that bacterial invasion and inflammation induction in resident cell populations are sufficient for fluid loss into the lumen. In summary, this model is a novel and useful tool that allows for detailed examination of the early physiopathological effects of Salmonella infection on the small intestine. PMID:26564721

  14. ULTRASOUND-ENHANCED rt-PA THROMBOLYSIS IN AN EX VIVO PORCINE CAROTID ARTERY MODEL

    PubMed Central

    Hitchcock, Kathryn E.; Ivancevich, Nikolas M.; Haworth, Kevin J.; Caudell Stamper, Danielle N.; Vela, Deborah C.; Sutton, Jonathan T.; Pyne-Geithman, Gail J.; Holland, Christy K.

    2014-01-01

    Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model. PMID:21723448

  15. Amniotic fluid for ex vivo skin preservation: a comparative study of tissue preservation solutions.

    PubMed

    Buseman, Jason; Rinker, Alexander B; Rinker, Brian

    2013-12-01

    Ex vivo skin preservation is important for skin banks, burn centers, and in research; however, the optimal preservation solution is not known. Human amniotic fluid (HAF), in addition to its role in fetal wound healing, has promise as an effective and readily available preservation solution. The purpose of this study was to compare the efficacy of several solutions, including HAF, in full-thickness skin preservation. Human amniotic fluid was obtained from patients undergoing amniocentesis. Full-thickness skin obtained during abdominoplasty was divided into 1-cm(2) samples. These specimens were preserved in either saline, HAF from a single patient, pooled HAF, University of Wisconsin solution, or custodial histidine-tryptophan-ketoglutarate solution at 4°C. There were 5 samples in each group. Specimens were examined for keratinocyte survival at 7, 14, 21, 28, and 35 days using the trypan blue assay. The first 200 cells identified were counted to calculate the degree of cell death. Comparisons were made between the groups, and a multivariable repeated-measures analysis was performed to determine statistical significance, which was defined as P < 0.05. The individual and pooled HAF showed greater keratinocyte survival than the other solutions, at each time point. The difference was statistically significant (P < 0.05) when compared to saline and custodial solution but not statistically significant when compared to University of Wisconsin solution. When used as a tissue preservation solution, HAF produces superior keratinocyte survival than several commercially available solutions. Amniotic fluid, nature's tissue preservation solution, may be an inexpensive, readily available alternative for use in skin banks, burn centers, and research. PMID:24231574

  16. EX VIVO STUDY OF QUANTITATIVE ULTRASOUND PARAMETERS IN FATTY RABBIT LIVERS

    PubMed Central

    Ghoshal, Goutam; Lavarello, Roberto J.; Kemmerer, Jeremy P.; Miller, Rita J.; Oelze, Michael L.

    2012-01-01

    Nonalcoholic fatty liver disease (NAFLD) affects more than 30% of Americans, and with increasing problems of obesity in the United States, NAFLD is poised to become an even more serious medical concern. At present, accurate classification of steatosis (fatty liver) represents a significant challenge. In this study, the use of high-frequency (8 to 25 MHz) quantitative ultrasound (QUS) imaging to quantify fatty liver was explored. QUS is an imaging technique that can be used to quantify properties of tissue giving rise to scattered ultrasound. The changes in the ultrasound properties of livers in rabbits undergoing atherogenic diets of varying durations were investigated using QUS. Rabbits were placed on a special fatty diet for 0, 3, or 6 weeks. The fattiness of the livers was quantified by estimating the total lipid content of the livers. Ultrasonic properties, such as speed of sound, attenuation, and backscatter coefficients, were estimated in ex vivo rabbit liver samples from animals that had been on the diet for varying periods. Two QUS parameters were estimated based on the backscatter coefficient: effective scatterer diameter (ESD) and effective acoustic concentration (EAC), using a spherical Gaussian scattering model. Two parameters were estimated based on the backscattered envelope statistics (the k parameter and the μ parameter) according to the homodyned K distribution. The speed of sound decreased from 1574 to 1565 m/s and the attenuation coefficient increased from 0.71 to 1.27 dB/cm/MHz, respectively, with increasing fat content in the liver. The ESD decreased from 31 to 17 μm and the EAC increased from 38 to 63 dB/cm3 with increasing fat content in the liver. A significant increase in the μ parameter from 0.18 to 0.93 scatterers/mm3 was observed with increasing fat content in the liver samples. The results of this study indicate that QUS parameters are sensitive to fat content in the liver. PMID:23062376

  17. Novel Techniques for Ex Vivo Expansion of Cord Blood: Clinical Trials

    PubMed Central

    Mehta, Rohtesh S.; Rezvani, Katayoun; Olson, Amanda; Oran, Betul; Hosing, Chitra; Shah, Nina; Parmar, Simrit; Armitage, Sue; Shpall, Elizabeth J.

    2015-01-01

    Cord blood (CB) provides an excellent alternative source of hematopoietic progenitor cells (HPC) for patients lacking human leukocyte antigen-matched peripheral blood or bone marrow graft for transplantation. However, due to the limited cell dose in CB graft, it is associated with prolonged time to engraftment, risk of graft rejection, infections, and treatment-related mortality. To increase the cell dose, a variety of ex vivo expansion techniques have been developed. Results of traditional methods of CB expansion using cytokines alone were disappointing. Expanding CB cells with mesenchymal progenitor cells led to sizeable increase in graft content and improved engraftment. Other methods used HPC-differentiation blockers, such as nicotinamide analogs, copper chelators, inducing constitutive Notch signaling, or an aryl hydrocarbon receptor antagonist (StemReginin1). Many of these methods lead to substantial expansions of total nucleated cells and CD34+ cells, and significantly improved time to neutrophil or platelet engraftment in patients transplanted with the expanded products compared to the recipients of unmanipulated CBT. These studies differ not only in the expansion method but also with regards to the cytokines used, patient population, conditioning regimens, and transplantation practices, to name a few. Some of these methods employed expansion of a portion of CB unit in the setting of single CBT, while others in the setting of double CBT. Here, we review various procedures used for CB expansion and highlight some of the key differences. Novel methods of improving engraftment that aim at improving bone marrow homing potential of CB cells are not reviewed. PMID:26697430

  18. Measurement of the hyperelastic properties of 44 pathological ex vivo breast tissue samples

    NASA Astrophysics Data System (ADS)

    O'Hagan, Joseph J.; Samani, Abbas

    2009-04-01

    The elastic and hyperelastic properties of biological soft tissues have been of interest to the medical community. There are several biomedical applications where parameters characterizing such properties are critical for a reliable clinical outcome. These applications include surgery planning, needle biopsy and brachtherapy where tissue biomechanical modeling is involved. Another important application is interpreting nonlinear elastography images. While there has been considerable research on the measurement of the linear elastic modulus of small tissue samples, little research has been conducted for measuring parameters that characterize the nonlinear elasticity of tissues included in tissue slice specimens. This work presents hyperelastic measurement results of 44 pathological ex vivo breast tissue samples. For each sample, five hyperelastic models have been used, including the Yeoh, N = 2 polynomial, N = 1 Ogden, Arruda-Boyce, and Veronda-Westmann models. Results show that the Yeoh, polynomial and Ogden models are the most accurate in terms of fitting experimental data. The results indicate that almost all of the parameters corresponding to the pathological tissues are between two times to over two orders of magnitude larger than those of normal tissues, with C11 showing the most significant difference. Furthermore, statistical analysis indicates that C02 of the Yeoh model, and C11 and C20 of the polynomial model have very good potential for cancer classification as they show statistically significant differences for various cancer types, especially for invasive lobular carcinoma. In addition to the potential for use in cancer classification, the presented data are very important for applications such as surgery planning and virtual reality based clinician training systems where accurate nonlinear tissue response modeling is required.

  19. Ex vivo 3D osteocyte network construction with primary murine bone cells

    PubMed Central

    Sun, Qiaoling; Gu, Yexin; Zhang, Wenting; Dziopa, Leah; Zilberberg, Jenny; Lee, Woo

    2015-01-01

    Osteocytes reside as three-dimensionally (3D) networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because: (1) current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and (2) primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to: (1) construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and (2) reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to: (1) distribute and entrap cells within the interstitial spaces between the microbeads and (2) maintain average cell-to-cell distance to be about 19 µm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions (SOST and FGF23) and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of: (1) studying flow-induced shear stress on the mechanotransduction function of primary osteocytes, (2) studying physiological functions of 3D-networked osteocytes with in vitro convenience, and (3) developing clinically relevant human bone disease models. PMID:26421212

  20. Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation

    PubMed Central

    Freag, May S; Elnaggar, Yosra SR; Abdallah, Ossama Y

    2013-01-01

    Diosmin (DSN) is an outstanding phlebotonic flavonoid with a tolerable potential for the treatment of colon and hepatocellular carcinoma. Being highly insoluble, DSN bioavailability suffers from high inter-subject variation due to variable degrees of permeation. This work endeavored to develop novel DSN loaded phytosomes in order to improve drug dissolution and intestinal permeability. Three preparation methods (solvent evaporation, salting out, and lyophilization) were compared. Nanocarrier optimization encompassed different soybean phospholipid (SPC) types, different solvents, and different DSN:SPC molar ratios (1:1, 1:2, and 1:4). In vitro appraisal encompassed differential scanning calorimetry, infrared spectroscopy, particle size, zeta potential, polydispersity index, transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed under sink versus non-sink conditions. Ex vivo intestinal permeation studies were performed on rats utilizing noneverted sac technique and high-performance liquid chromatography analysis. The results revealed lyophilization as the optimum preparation technique using SPC and solvent mixture (Dimethyl sulphoxide:t-butylalchol) in a 1:2 ratio. Complex formation was contended by differential scanning calorimetry and infrared data. Optimal lyophilized phytosomal nanocarriers (LPNs) exhibited the lowest particle size (316 nm), adequate zeta-potential (−27 mV), and good in vitro stability. Well formed, discrete vesicles were revealed by transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed. LPNs demonstrated significant enhancement in DSN dissolution compared to crude drug, physical mixture, and generic and brand DSN products. Permeation studies revealed 80% DSN permeated from LPNs via oxygenated rat intestine compared to non-detectable amounts from suspension. In this study, LPNs (99% drug loading) could be successfully

  1. Use of formalin-fixed tissues for ex-vivo imaging with optical coherence tomography (OCT)

    NASA Astrophysics Data System (ADS)

    Shortkroff, Sonya; Goodwin, Alicia; Giattina, Susanne; Liu, Bin; Brezinski, Mark E.

    2006-02-01

    Structural and compositional analysis of normal and pathological tissues by OCT often is performed ex vivo and subsequently compared to the histology. Many of the tissues of interest require immediate fixation to prevent degradation of the sample. Frequently, samples are obtained up to a week prior to procuring images by OCT. We investigated whether fixation affects OCT image analysis by acquiring images of freshly isolated bovine ligament samples and repeating OCT imaging of the same area after fixation at 24 hours and at one week. Samples were divided into two groups: group one was fixed in 10% neutral buffered formalin for 24 hours and placed in normal saline while group two remained in formalin for one week. Tissue samples were processed for paraffin embedment and stained with Masson's trichrome or with picrosirius red. The banding pattern contrast ratio of the OCT images before and after fixation for both groups was measured and compared for possible differences. Histology was evaluated for tissue integrity and compared to the OCT images. The mean contrast ratio at time 0 was 5.41 +/- 1.1 and 5.31 +/- 0.6 for groups 1 and 2, respectively. Results at 1 week were slightly lower with 5.11 +/- 0.3 and 5.20 +/- 0.7, respectively. Statistical analysis of the data by ANOVA showed no difference in the contrast ratios with time or with treatment. This data indicates that 24 hours in formalin is sufficient to fix these small ligament samples with little effect on imaging up to one week after fixation.

  2. Detection of human brain cancer infiltration ex vivo and in vivo using quantitative optical coherence tomography.

    PubMed

    Kut, Carmen; Chaichana, Kaisorn L; Xi, Jiefeng; Raza, Shaan M; Ye, Xiaobu; McVeigh, Elliot R; Rodriguez, Fausto J; Quiñones-Hinojosa, Alfredo; Li, Xingde

    2015-06-17

    More complete brain cancer resection can prolong survival and delay recurrence. However, it is challenging to distinguish cancer from noncancer tissues intraoperatively, especially at the transitional, infiltrative zones. This is especially critical in eloquent regions (for example, speech and motor areas). This study tested the feasibility of label-free, quantitative optical coherence tomography (OCT) for differentiating cancer from noncancer in human brain tissues. Fresh ex vivo human brain tissues were obtained from 32 patients with grade II to IV brain cancer and 5 patients with noncancer brain pathologies. On the basis of volumetric OCT imaging data, pathologically confirmed brain cancer tissues (both high- and low-grade) had significantly lower optical attenuation values at both cancer core and infiltrated zones when compared with noncancer white matter, and OCT achieved high sensitivity and specificity at an attenuation threshold of 5.5 mm(-1) for brain cancer patients. We also used this attenuation threshold to confirm the intraoperative feasibility of performing in vivo OCT-guided surgery using a murine model harboring human brain cancer. Our OCT system was capable of processing and displaying a color-coded optical property map in real time at a rate of 110 to 215 frames per second, or 1.2 to 2.4 s for an 8- to 16-mm(3) tissue volume, thus providing direct visual cues for cancer versus noncancer areas. Our study demonstrates the translational and practical potential of OCT in differentiating cancer from noncancer tissue. Its intraoperative use may facilitate safe and extensive resection of infiltrative brain cancers and consequently lead to improved outcomes when compared with current clinical standards. PMID:26084803

  3. Optimization of Direct Current-Enhanced Radiofrequency Ablation: An Ex Vivo Study

    SciTech Connect

    Tanaka, Toshihiro Isfort, Peter; Bruners, Philipp; Penzkofer, Tobias; Kichikawa, Kimihiko; Schmitz-Rode, Thomas; Mahnken, Andreas H.

    2010-10-15

    The purpose of this study was to investigate the optimal setting for radiofrequency (RF) ablation combined with direct electrical current (DC) ablation in ex vivo bovine liver. An electrical circuit combining a commercially available RF ablation system with DC was developed. The negative electrode of a rectifier that provides DC was connected to a 3-cm multitined expandable RF probe. A 100-mH inductor was used to prevent electrical leakage from the RF generator. DC was applied for 15 min and followed by RF ablation in freshly excised bovine livers. Electric current was measured by an ammeter. Coagulation volume, ablation duration, and mean amperage were assessed for various DC voltages (no DC, 2.2, 4.5, and 9.0 V) and different RF ablation protocols (stepwise increase from 40 to 80 W, 40 W fixed, and 80 W fixed). Results were compared using Kruskal-Wallis and Mann-Whitney U test. Applying DC with 4.5 or 9.0 V, in combination with 40 W fixed or a stepwise increase of RF energy, resulted in significantly increased zone of ablation size compared with 2.2 V or no DC (P = 0.009). At 4.5 V DC, the stepwise increase of RF energy resulted in the same necrosis size as a 40 W fixed protocol (26.6 {+-} 3.9 vs. 26.5 {+-} 4.0 ml), but ablation duration was significantly decreased (296 {+-} 85 s vs. 423 {+-} 104 s; P = 0.028). Mean amperage was significantly lower at 4.5 V compared with 9.0 V (P = 0.028). Combining a stepwise increase of RF energy with a DC voltage of 4.5 V is most appropriate to increase coagulation volume and to minimize procedure time.

  4. Ex-vivo tissue classification of cell surface receptor concentrations using kinetic modeling

    NASA Astrophysics Data System (ADS)

    Sinha, Lagnojita; Wang, Yu; Yang, Cynthia; Khan, Altaz; Liu, Jonathan T.; Tichauer, Kenneth M.

    2015-03-01

    One of the major challenges in the complete resection of cancer is the difficulty of distinctly classifying tumor and healthy tissue. This paper investigates the capability of competing kinetic modeling approaches for identifying different tissue types based on differential cell-surface receptor expressions. These approaches require fresh resected tissues to be stained with a mixture of two probes: one targeted to a cancer specific cell-surface receptor, and another left "untargeted" to account for nonspecific retention of the targeted agent, with subsequent repeated rinsing and imaging of the probe concentrations. Analysis of the results were carried out in simulations and in animal experiments for the cancer target, epidermal growth factor receptor (EGFR), a cell surface receptor overexpressed by many cancers. In the animal experiments, subcutaneous xenografts of human glioma (U251; moderate EGFR) and human epidermoid (A431; high EGFR) tumors, grown in six athymic mice, were excised and stained with an EGFR targeted surface-enhanced Raman scattering nanoparticle (SERS NP) and untargeted SERS NP pair. The salient finding in this study was that significant non-specific retention was observed for the EGFR targeted probe [anti-EGFR antibody labeled with a surface-enhanced Raman scattering (SERS) nanoparticle], but could be corrected for by the equivalent non-specific retention of the untargeted probe (isotype control antibody labeled with a different SERS nanoparticle). Once this non-specific binding was accounted for, the kinetic model was able to predict the expected differences in EGFR concentration among different tissue types: healthy, U251, and A431 in accordance with an ex vivo flow cytometry analysis, successfully classifying different tissue types.

  5. In Vitro, Ex Vivo, and In Vivo Determination of Thyroid Hormone Modulating Activity of Benzothiazoles.

    PubMed

    Hornung, Michael W; Kosian, Patricia A; Haselman, Jonathan T; Korte, Joseph J; Challis, Katie; Macherla, Chitralekha; Nevalainen, Erica; Degitz, Sigmund J

    2015-08-01

    As in vitro assays are increasingly used to screen chemicals for their potential to produce endocrine disrupting adverse effects, it is important to understand their predictive capacity. The potential for a set of 6 benzothiazoles to affect endpoints related to thyroid hormone synthesis inhibition were assessed using in vitro, ex vivo, and in vivo assays. Inhibition of thyroid peroxidase (TPO) derived from pig thyroid glands was determined for benzothiazole (BTZ), 2-mercaptobenzothiazole (MBT), 5-chloro-2-mercaptobenzothiazole (CMBT), 2-aminobenzothiazole (ABT), 2-hydroxybenzothiazole (HBT), and 2-methylthiobenzothiazole (MTBT). Their rank order potency for TPO inhibition was MBT=CMBT>ABT>BTZ, whereas HBT and MTBT exhibited no inhibitory activity. The benzothiazoles were tested further in a Xenopus laevis thyroid gland explant culture assay in which inhibition of thyroxine (T4) release was the measured endpoint. In this assay all 6 benzothiazoles inhibited T4 release. The activity of the benzothiazoles for disrupting thyroid hormone activity was verified in vivo using X. laevis tadpoles in a 7-day assay. The 2 most potent chemicals for TPO inhibition, MBT and CMBT, produced responses in vivo indicative of T4 synthesis inhibition including induction of sodium iodide symporter mRNA and decreases in glandular and circulating thyroid hormones. The capability to measure thyroid hormone levels in the glands and blood by ultrahigh performance LC-MS/MS methods optimized for small tissue samples was critical for effects interpretation. These results indicate that inhibition of TPO activity in vitro was a good indicator of a chemical's potential for thyroid hormone disruption in vivo and may be useful for prioritizing chemicals for further investigation. PMID:25953703

  6. Ex-vivo Assessment of Coronary Artery Atherosclerosis by Magnetic Resonance Imaging: Correlation with Histopathology

    PubMed Central

    Gomes, Everli P. S. Gonçalves; Rochitte, Carlos Eduardo; Azevedo, Clerio F.; Lemos, Pedro A.; Gutierrez, Paulo Sampaio; César, Luiz Antonio M.

    2014-01-01

    Introduction: In recent years, high-resolution magnetic resonance imaging (MRI) has emerged as a very promising technique for studying atherosclerotic disease in humans. Aim: In the present study we sought to determine whether MRI allowed for the morphological characterization of the coronary vessel wall and atherosclerotic plaques using histopathological assessment as the reference standard. Methods: The study population consisted of 13 patients who died of acute myocardial infarction and underwent autopsy. The proximal portions of the coronary arteries were excised and were evaluated both by MRI and by histopathology. For each arterial segment, the following parameters were calculated through manual planimetry: 1. total vessel area (TVA); 2. luminal area (LA) and 3. plaque area (PA). Results: A total of 207 coronary artery cross-sections were found to be suitable for analysis by both MRI and histopathology and were included in the final analyses. Both methods demonstrated moderate to good agreement for the quantification of TVA (mean difference = 2.4±2.4 mm2, 95‰ limits of agreement from -2.4 to +7.2 mm2; CCC = 0.69, 95‰ CI from 0.63 to 0.75), LA (mean difference = 0.0±1.7 mm2, 95‰ limits of agreement from -3.3 to + 3.3 mm2; CCC = 0.84, 95‰ CI from 0.80 to 0.88) and PA (mean difference = 2.4±2.4 mm2, 95‰ limits of agreement from -2.3 to + 7.1 mm2; CCC = 0.64, 95‰ CI from 0.58 to 0.71). Conclusion: In this ex vivo experimental model we demonstrated good agreement between coronary artery morphometrical measurements obtained by high-resolution MRI and by histopathology. PMID:24847387

  7. An Ex Vivo Study of the Correlation between Acoustic Emission and Microvascular Damage

    PubMed Central

    Samuel, Stanley; Cooper, Michol A.; Bull, Joseph L.; Fowlkes, J. Brian; Miller, Douglas L.

    2009-01-01

    The objective of this study was to conduct an ex vivo examination of correlation between acoustic emission and tissue damage. Intravital microscopy was employed in conjunction with ultrasound exposure in cremaster muscle of male Wistar rats. Definity® microbubbles were administered intravenously through the tail vein (80 μL.kg-1.min-1infusion rate) with the aid of a syringe pump. For the pulse repetition frequency (PRF) study, exposures were performed at four locations of the cremaster at a PRF of 1000, 500, 100 and 10 Hz (one location per PRF per rat). The 100-pulse exposures were implemented at a peak rarefactional pressure (Pr) of 2 MPa, frequency of 2.25 MHz with 46 cycle pulses. For the pressure amplitude threshold study, 100-pulse exposures (46 cycle pulses) were conducted at various peak rarefactional pressures from 0.5 MPa to 2 MPa at a frequency of 2.25 MHz and PRF of 100 Hz. Photomicrographs were captured before and 2-minutes post exposure. On a pulse-to-pulse basis, the 10 Hz acoustic emission was considerably higher and more sustained than those at other PRFs (1000, 500, and 100 Hz) (p < 0.05). Damage, measured as area of extravasation of red blood cells (RBC's), was also significantly higher at 10 Hz PRF than at 1000, 500, and 100 Hz (p < 0.01). The correlation of acoustic emission to tissue damage showed a trend of increasing damage with increasing cumulative function of the relative integrated power spectrum (CRIPS; R2 = 0.75). No visible damage was present at Pr ≤ 0.85 MPa. Damage, however, was observed at Pr ≥ 1.0 MPa, and it increased with increasing acoustic pressure. PMID:19560856

  8. Assessing leukocyte-endothelial interactions under flow conditions in an ex vivo autoperfused microflow chamber assay.

    PubMed

    Mulki, Lama; Sweigard, J Harry; Connor, Kip M

    2014-01-01

    Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation. PMID:25590688

  9. Bridging the gap: functional healing of embryonic small intestine ex vivo

    PubMed Central

    Coletta, Riccardo; Roberts, Neil A.; Oltrabella, Francesca; Khalil, Basem A.; Morabito, Antonino

    2015-01-01

    Abstract The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2–3 mm tubes, which were placed in pairs, separated by a small gap, on semi‐permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum‐free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin+ neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies. ©2015. The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd PMID:26234729

  10. Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets.

    PubMed

    Mohan, K V K; Rao, S Sainath; Gao, Y; Atreya, C D

    2014-01-01

    Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 μM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria. PMID:23926880

  11. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    PubMed

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage. PMID:21401652

  12. Mucociliary clearance and submucosal gland secretion in the ex vivo ferret trachea.

    PubMed

    Jeong, Jin Hyeok; Joo, Nam Soo; Hwang, Peter H; Wine, Jeffrey J

    2014-07-01

    In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends on gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho HJ, Joo NS, Wine JJ. Am J Physiol Lung Cell Mol Physiol 299: L124-L136, 2010). Stimulated MCC velocities (mm/min, means ± SE for 10- to 35-min period poststimulation) were as follows: 1 μM carbachol: 19.1 ± 3.3 > 10 μM phenylephrine: 15.3 ± 2.4 ≈ 10 μM isoproterenol: 15.0 ± 1.9 ≈ 10 μM forskolin: 14.6 ± 3.1 > 1 μM vasoactive intestinal peptide (VIP): 10.2 ± 2.2 > basal (t15): 1.8 ± 0.3; n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70% and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3 (-)-free solutions did not significantly inhibit MCC to either intracellular Ca(2+) concentration or intracellular cAMP concentration ([cAMP]i)-elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion, and bumetanide almost completely inhibited [cAMP]i-stimulated MCC while it had a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this. PMID:24793168

  13. Ex vivo effects of naphthoquinones on allergen-sensitized mononuclear cells in mice.

    PubMed

    Tanaka, M; Inoue, K; Shimada, A; Takano, H

    2016-09-01

    Naphthoquinone (NQ), one of the extractable chemical compounds of diesel exhaust particles, enhances allergic asthma traits in mice. However, it remains unknown whether: (1) several types of NQs have the same potential to facilitate allergies; and (2) NQs synergistically disrupt the functional phenotypes of immune cells. The aim of the present study was to investigate the effects of two types (1,2- and 1,4-) of NQs on sensitized mononuclear cells using an ex vivo assay. Male BALB/c mice were repeatedly and intraperitoneally administered ovalbumin (OVA: 20 µg) plus alum with or without two different doses of each NQ. After the final administration, splenocytes (mononuclear cells) were isolated from these mice and cultured in the presence of OVA. Helper T-related cytokines in the culture supernatants and downstream molecules were then evaluated. Protein levels of interferon-γ were higher in the supernatants from 1,2-NQ and 1,4-NQ at low dose + OVA-exposed mononuclear cells following the OVA stimulation than in those from OVA-exposed mononuclear cells. Interleukin (IL)-13 levels were higher in the supernatants from low dose NQs + OVA-exposed mononuclear cells. IL-17 levels were significantly higher in the supernatants from low dose 1,2-NQ + OVA-exposed mononuclear cells. The quantity of phosphorylated STAT6 in the nuclei of these cells was significantly greater in the low dose NQ + OVA groups than in the OVA group. These findings suggest NQs differently enhance allergen sensitization in the context of the Th response against mononuclear cells such as lymphocytes. PMID:26884456

  14. Ex-Vivo Uterine Environment (EVE) Therapy Induced Limited Fetal Inflammation in a Premature Lamb Model

    PubMed Central

    Miura, Yuichiro; Saito, Masatoshi; Usuda, Haruo; Woodward, Eleanor; Rittenschober-Böhm, Judith; Kannan, Paranthaman S.; Musk, Gabrielle C.; Matsuda, Tadashi; Newnham, John P.; Kemp, Matthew W.

    2015-01-01

    Introduction Ex-vivo uterine environment (EVE) therapy uses an artificial placenta to provide gas exchange and nutrient delivery to a fetus submerged in an amniotic fluid bath. Development of EVE may allow us to treat very premature neonates without mechanical ventilation. Meanwhile, elevations in fetal inflammation are associated with adverse neonatal outcomes. In the present study, we analysed fetal survival, inflammation and pulmonary maturation in preterm lambs maintained on EVE therapy using a parallelised umbilical circuit system with a low priming volume. Methods Ewes underwent surgical delivery at 115 days of gestation (term is 150 days), and fetuses were transferred to EVE therapy (EVE group; n = 5). Physiological parameters were continuously monitored; fetal blood samples were intermittently obtained to assess wellbeing and targeted to reference range values for 2 days. Age-matched animals (Control group; n = 6) were surgically delivered at 117 days of gestation. Fetal blood and tissue samples were analysed and compared between the two groups. Results Fetal survival time in the EVE group was 27.0 ± 15.5 (group mean ± SD) hours. Only one fetus completed the pre-determined study period with optimal physiological parameters, while the other 4 animals demonstrated physiological deterioration or death prior to the pre-determined study end point. Significant elevations (p<0.05) in: i) inflammatory proteins in fetal plasma; ii) selected cytokine/chemokine mRNA expression levels in fetal tissues; and iii) histological inflammatory score in fetal lung, were observed in the EVE group compared to the Control group. There was no significant difference (p>0.05) in surfactant protein mRNA expression level between the two groups. Conclusion In this study, we achieved limited fetal survival using EVE therapy. Despite this, EVE therapy only induced a modest fetal inflammatory response and did not promote lung maturation. These data provide additional insight into

  15. Detection of Human Brain Cancer Infiltration ex vivo and in vivo Using Quantitative Optical Coherence Tomography*

    PubMed Central

    Kut, Carmen; Chaichana, Kaisorn L.; Xi, Jiefeng; Raza, Shaan M.; Ye, Xiaobu; McVeigh, Elliot R.; Rodriguez, Fausto J.; Quinones-Hinojosa, Alfredo; Li, Xingde

    2015-01-01

    More complete brain cancer resection can prolong survival and delay recurrence. However, it is challenging to distinguish cancer from non-cancer tissues intraoperatively, especially at the transitional, infiltrative zones. This is especially critical in eloquent regions (e.g. speech and motor areas). This study tested the feasibility of label-free, quantitative optical coherence tomography (OCT) for differentiating cancer from non-cancer in human brain tissues. Fresh ex vivo human brain tissues were obtained from 32 patients with grades II-IV brain cancer and 5 patients with non-cancer brain pathologies. Based on volumetric OCT imaging data, pathologically confirmed brain cancer tissues (both high-grade and low-grade) had significantly lower optical attenuation values at both cancer core and infiltrated zones when compared with non-cancer white matter, and OCT achieved high sensitivity and specificity at an attenuation threshold of 5.5 mm-1 for brain cancer patients. We also used this attenuation threshold to confirm the intraoperative feasibility of performing in vivo OCT-guided surgery using a murine model harboring human brain cancer. Our OCT system was capable of processing and displaying a color-coded optical property map in real time at a rate of 110-215 frames per second, or 1.2-2.4 seconds for an 8-16 mm3 tissue volume, thus providing direct visual cues for cancer versus non-cancer areas. Our study demonstrates the translational and practical potential of OCT in differentiating cancer from non-cancer tissue. Its intraoperative use may facilitate safe and extensive resection of infiltrative brain cancers and consequently lead to improved outcomes when compared with current clinical standards. PMID:26084803

  16. Ex vivo vs. in vivo antibacterial activity of two antiseptics on oral biofilm

    PubMed Central

    Prada-López, Isabel; Quintas, Víctor; Casares-De-Cal, Maria A.; Suárez-Quintanilla, Juan A.; Suárez-Quintanilla, David; Tomás, Inmaculada

    2015-01-01

    Aim: To compare the immediate antibacterial effect of two application methods (passive immersion and active mouthwash) of two antiseptic solutions on the in situ oral biofilm. Material and Methods: A randomized observer-masked crossover study was conducted. Fifteen healthy volunteers wore a specific intraoral device for 48 h to form a biofilm in three glass disks. One of these disks was used as a baseline; another one was immersed in a solution of 0.2% Chlorhexidine (0.2% CHX), remaining the third in the device, placed in the oral cavity, during the 0.2% CHX mouthwash application. After a 2-weeks washout period, the protocol was repeated using a solution of Essential Oils (EO). Samples were analyzed for bacterial viability with the confocal laser scanning microscope after previous staining with LIVE/DEAD® BacLight™. Results: The EO showed a better antibacterial effect compared to the 0.2% CHX after the mouthwash application (% of bacterial viability = 1.16 ± 1.00% vs. 5.08 ± 5.79%, respectively), and was more effective in all layers (p < 0.05). In the immersion, both antiseptics were significantly less effective (% of bacterial viability = 26.93 ± 13.11%, EO vs. 15.17 ± 6.14%, 0.2% CHX); in the case of EO immersion, there were no significant changes in the bacterial viability of the deepest layer in comparison with the baseline. Conclusions: The method of application conditioned the antibacterial activity of the 0.2% CHX and EO solutions on the in situ oral biofilm. The in vivo active mouthwash was more effective than the ex vivo passive immersion in both antiseptic solutions. There was more penetration of the antiseptic inside the biofilm with an active mouthwash, especially with the EO. Trial registered in clinicaltrials.gov with the number NCT02267239. URL: https://clinicaltrials.gov/ct2/show/NCT02267239. PMID:26191050

  17. Infrared laser thermal fusion of blood vessels: preliminary ex vivo tissue studies

    NASA Astrophysics Data System (ADS)

    Cilip, Christopher M.; Rosenbury, Sarah B.; Giglio, Nicholas; Hutchens, Thomas C.; Schweinsberger, Gino R.; Kerr, Duane; Latimer, Cassandra; Nau, William H.; Fried, Nathaniel M.

    2013-05-01

    Suture ligation of blood vessels during surgery can be time-consuming and skill-intensive. Energy-based, electrosurgical, and ultrasonic devices have recently replaced the use of sutures and mechanical clips (which leave foreign objects in the body) for many surgical procedures, providing rapid hemostasis during surgery. However, these devices have the potential to create an undesirably large collateral zone of thermal damage and tissue necrosis. We explore an alternative energy-based technology, infrared lasers, for rapid and precise thermal coagulation and fusion of the blood vessel walls. Seven near-infrared lasers (808, 980, 1075, 1470, 1550, 1850 to 1880, and 1908 nm) were tested during preliminary tissue studies. Studies were performed using fresh porcine renal vessels, ex vivo, with native diameters of 1 to 6 mm, and vessel walls flattened to a total thickness of 0.4 mm. A linear beam profile was applied normal to the vessel for narrow, full-width thermal coagulation. The laser irradiation time was 5 s. Vessel burst pressure measurements were used to determine seal strength. The 1470 nm laser wavelength demonstrated the capability of sealing a wide range of blood vessels from 1 to 6 mm diameter with burst strengths of 578±154, 530±171, and 426±174 mmHg for small, medium, and large vessel diameters, respectively. Lateral thermal coagulation zones (including the seal) measured 1.0±0.4 mm on vessels sealed at this wavelength. Other laser wavelengths (1550, 1850 to 1880, and 1908 nm) were also capable of sealing vessels, but were limited by lower vessel seal pressures, excessive charring, and/or limited power output preventing treatment of large vessels (>4 mm outer diameter).

  18. Adenosine A2A Agonist Improves Lung Function During Ex-vivo Lung Perfusion

    PubMed Central

    Emaminia, Abbas; LaPar, Damien J.; Zhao, Yunge; Steidle, John F.; Harris, David A.; Linden, Joel; Kron, Irving L.; Lau, Christine L.

    2012-01-01

    Background Ex-vivo lung perfusion (EVLP) is a novel technique to assess, and potentially repair marginal lungs that may otherwise be rejected for transplantation. Adenosine has been shown to protect against lung ischemia-reperfusion injury through its A2A receptor. We hypothesized that combining EVLP with adenosine A2A receptor agonist treatment would enhance lung functional quality and increase donor lung usage. Methods Eight bilateral pig lungs were harvested and flushed with cold Perfadex. After 14 hours storage at 4°C, EVLP was performed for 5 hours on two explanted lung groups: 1) Control group lungs (n=4), were perfused with Steen Solution and Dimethyl sulfoxide (DMSO), and 2) treated group lungs (n=4) received 10μM CGS21680, a selective A2A receptor agonist, in a Steen Solution-primed circuit. Lung histology, tissue cytokines, gas analysis and pulmonary function were compared between groups. Results Treated lungs demonstrated significantly less edema as reflected by wet-dry weight ratio (6.6 vs. 5.2, p<0.03) and confirmed by histology. In addition, treated lung demonstrated significantly lower levels of interferon gamma (45.1 vs. 88.5, p<0.05). Other measured tissue cytokines (interleukin (IL) 1 beta, IL-6, and IL-8) were lower in treatment group, but values failed to reach statistical significance. Oxygenation index was improved in the treated group (1.5 vs. 2.3, p<0.01) as well as mean airway pressure (10.3 vs. 13 p<0.009). Conclusions EVLP is a novel and efficient way to assess and optimize lung function and oxygen exchange within donor lungs, and the use of adenosine A2A agonist potentiates its potential. EVLP with the concomitant administration of A2A agonist may enhance donor lung quality and could increase the donor lung pool for transplantation. PMID:22051279

  19. Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii.

    PubMed

    Graham, Joseph G; Winchell, Caylin G; Kurten, Richard C; Voth, Daniel E

    2016-05-01

    Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells, C. burnetii generates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes. C. burnetii requires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand how C. burnetii evades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection of ex vivo human lung tissue, defining a valuable approach for characterizing C. burnetii interactions with a human host. Within whole lung tissue, C. burnetii preferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response to C. burnetii. PMID:26902725

  20. Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

    PubMed Central

    Phillips, Ryan M.; Dailey, Lisa A.; Bair, Eric; Samet, James M.; Allbritton, Nancy L.

    2014-01-01

    We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min−1 mg−1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min−1 mg−1, and was also effectively inhibited by pre-incubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2- naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min−1 mg−1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34–36 pmol min−1 mg−1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. PMID:24380370

  1. Inhibition of human platelet function in vitro and ex vivo by acetaminophen.

    PubMed

    Lages, B; Weiss, H J

    1989-03-15

    The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, 14C-5HT secretion, and thromboxane B2 (TxB2) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB2 formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB2 formation was inhibited by 40-99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB2 formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion in 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB2 formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with 3H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD2 and PGE2, as well as TxB2. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro. PMID:2499947

  2. Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

    NASA Astrophysics Data System (ADS)

    Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

    2016-02-01

    Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

  3. Mean Scatterer Spacing Estimation in Normal and Thermally Coagulated Ex Vivo Bovine Liver

    PubMed Central

    Rubert, Nicholas; Varghese, Tomy

    2014-01-01

    The liver has been hypothesized to have a unique arrangement of microvasculature that presents as an arrangement of quasiperiodic scatterers to an interrogating ultrasound pulse. The mean scatterer spacing (MSS) of these quasiperiodic scatterers has been proposed as a useful quantitative ultrasound biomarker for characterizing liver tissue. Thermal ablation is an increasingly popular method for treating hepatic tumors, and ultrasonic imaging approaches for delineating the extent of thermal ablation are in high demand. In this work, we examine the distribution of estimated MSS in thermally coagulated bovine liver and normal untreated bovine liver ex vivo. We estimate MSS by detecting local maxima in the spectral coherence function of radio frequency echoes from a clinical transducer, the Siemens VFX 9L4 transducer operating on an S2000 scanner. We find that normal untreated bovine liver was characterized by an MSS of approximately 1.3 mm. We examined regions of interest 12 mm wide laterally, and ranging from 12 mm to 18 mm axially, in 2 mm increments. Over these parameters, the mode of the MSS estimates was between 1.25 and 1.37 mm. On the other hand, estimation of MSS in thermally coagulated liver tissue yields a distribution of MSS estimates whose mode varied between 0.45 and 1.0 mm when examining regions of interest over the same sizes. We demonstrate that the estimated MSS in thermally coagulated liver favors small spacings because the randomly positioned scatterers in this tissue are better modeled as aperiodic scatterers. The submillimeter spacings result from the fact that this was the most probable spacing to be estimated if the discretely sampled spectral coherence function was a uniformly random two-dimensional function. PMID:24554290

  4. Ex vivo effects of lysine clonixinate on cyclooxygenases in rat lung and stomach preparations.

    PubMed

    Franchi, A M; Girolamo, G D; De los Santos, A R; Marti, M L; Gimeno, M A

    1999-01-01

    Lysine clonixinate (LC) is an anti-inflammatory, anti-pyretic and analgesic drug with minor digestive side effects, which might suggest a weak COX-1 inhibitor. The aim of this study focused on ex vivo effects of LC 40 mg/kg ip and indomethacin (INDO) 10 mg/kg ip in lung and stomach preparations of control rats and LPS-treated rats (5 mg/kg ip). The non-steroidal antiinflammatory drugs were administered concomitantly, following three hours and before one, two or three hours of LPS treatment. Tissues were weighed and incubated in 2 ml of Kress Ringer Bicarbonate buffer containing glucose (11 mM) under an atmosphere of 95% oxygen and 5% CO(2). Approximately 200 mg of tissue were used for each determination; 0.25 microCi of (14)C-arachidonic acid was added to each tube and the tissues were incubated for 60 min. Prostanoids were extracted from the incubation medium and separated by TLC. Results were expressed as a percentage of the total radioactivity of the plates (% of cpm on plate/100 mg ww). It was found that LC animals that were not given LPS did not modify the synthesis of PGE(2); in lung and stomach tissues showing that did not inhibit COX-1 activity. However, LC inhibited clearly the synthesis of PGE(2) in both preparations obtained from LPS-treated animals. The inhibition was shown when the rats were treated concomitantly, 3 h after or 1 or 2 h before the injection of LPS. PMID:17657442

  5. Ex vivo Raman spectroscopic study of breast metastatic lesions in lungs in animal models.

    PubMed

    Bhattacharjee, Tanmoy; Tawde, Sneha; Hudlikar, Rasika; Mahimkar, Manoj; Maru, Girish; Ingle, Arvind; Murali Krishna, C

    2015-08-01

    The lung is one of the most common sites of metastases, with approximately 50% of patients with extrathoracic cancer exhibiting pulmonary metastases. Correct identification of the metastatic status of a lung lesion is vital to therapeutic planning and better prognosis. However, currently available diagnostic techniques, such as conventional radiography and low dose computed tomography (LDCT), may fail to identify metastatic lesions. Alternative techniques such as Raman spectroscopy (RS) are hence being extensively explored for correct diagnosis of metastasis. The current ex vivo study aims to evaluate the ability of a fiber optic-based Raman system to distinguish breast cancer metastasis in lung from primary breast and lung tumor in animal models. In this study, spectra were acquired from normal breast, primary breast tumor, normal lung, primary lung tumor, and breast cancer metastasis in lung tissues and analyzed using principal component analysis and principal component-linear discriminant analysis. Breast cancer metastasis in lung could be classified with 71% classification efficiency. Approximately 6% breast metastasis spectra were misclassified with breast tumor, probably due to the presence of breast cancer cells in metastasized lungs. Test prediction results show 64% correct prediction of breast metastasis, while 13% breast metastasis spectra were wrongly predicted as breast tumor, suggesting the possible influence of breast cancer cells. Thus, findings of this study, the first of such explorations, demonstrate the potential of RS in classifying breast metastasis in lungs from primary lung and primary breast tumor. Prospective evaluation on a larger cohort with better multivariate analysis, combined with LDCT and recently developed real-time in vivo probes, RS can play a significant role in nonsurgical screening of lesions, which can lead to individualized therapeutic regimes and improved prognoses. PMID:26295177

  6. A Comparison of Direct Heating During Radiofrequency and Microwave Ablation in Ex Vivo Liver

    SciTech Connect

    Andreano, Anita; Brace, Christopher L.

    2013-04-15

    This study was designed to determine the magnitude and spatial distribution of temperature elevations when using 480 kHz RF and 2.45 GHz microwave energy in ex vivo liver models. A total of 60 heating cycles (20 s at 90 W) were performed in normal, RF-ablated, and microwave-ablated liver tissues (n = 10 RF and n = 10 microwave in each tissue type). Heating cycles were performed using a 480-kHz generator and 3-cm cooled-tip electrode (RF) or a 2.45-GHz generator and 14-gauge monopole (microwave) and were designed to isolate direct heating from each energy type. Tissue temperatures were measured by using fiberoptic thermosensors 5, 10, and 15 mm radially from the ablation applicator at the depth of maximal heating. Power delivered, sensor location, heating rates, and maximal temperatures were compared using mixed effects regression models. No significant differences were noted in mean power delivered or thermosensor locations between RF and microwave heating groups (P > 0.05). Microwaves produced significantly more rapid heating than RF at 5, 10, and 15 mm in normal tissue (3.0 vs. 0.73, 0.85 vs. 0.21, and 0.17 vs. 0.09 Degree-Sign C/s; P < 0.05); and at 5 and 10 mm in ablated tissues (2.3 {+-} 1.4 vs. 0.7 {+-} 0.3, 0.5 {+-} 0.3 vs. 0.2 {+-} 0 Degree-Sign C/s, P < 0.05). The radial depth of heating was {approx}5 mm greater for microwaves than RF. Direct heating obtained with 2.45-GHz microwave energy using a single needle-like applicator is faster and covers a larger volume of tissue than 480-kHz RF energy.

  7. Fabrication of Large Size Ex Vivo-Produced Oral Mucosal Equivalents for Clinical Application.

    PubMed

    Kato, Hiroko; Marcelo, Cynthia L; Washington, James B; Bingham, Eve L; Feinberg, Stephen E

    2015-09-01

    The soft tissue reconstruction of significant avulsed and/or surgically created tissue defects requires the ability to manufacture substantial soft tissue constructs for repair of the resulting wounds. In this study, we detail the issues that need to be addressed in upsizing the manufacture of larger tissue-engineered devices (ex vivo-produced oral mucosa equivalent [EVPOME]) in vitro from a methodology previously used for smaller constructs. The larger-sized EVPOME, consisting of autologous human oral keratinocytes and a dermal substitute, AlloDerm(®), was fabricated for the purpose of reconstructing large clinical defects. Regulated as an autologous somatic cell therapy product, the fabrication process abided by current Good Manufacturing Practices and current Good Tissue Practices as required by the Center for Biologics Evaluation and Research (CBER) of the United States Food and Drug Administration (FDA). Successful fabrication of large EVPOMEs utilized a higher cell seeding density (5.3×10(5) cells/cm(2)) with a relatively thinner AlloDerm, ranging from 356.6 to 508.0 μm in thickness. During the air-liquid interface culture, the thickness of the scaffold affected the medium diffusion rate, which, in turn, resulted in changes of epithelial stratification. Histologically, keratinocyte progenitor (p63), proliferation (Ki-67), and late differentiation marker (filaggrin) expression showed differences correlating with the expression of glucose transporter-1 (GLUT1) in the EVPOMEs from the thickest (550-1020 μm) to the thinnest (228.6-330.2 μm) AlloDerm scaffold. Glucose consumption and 2-deoxyglucose (2DG) uptake showed direct correlation with scaffold thickness. The scaffold size and thickness have an impact on the cellular phenotype and epithelial maturation in the manufacturing process of the EVPOME due to the glucose accessibility influenced by the diffusion rate. These outcomes provide basic strategies to manufacture a large-sized, healthy EVPOME

  8. Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo

    PubMed Central

    Beans, Elizabeth J.; Fournogerakis, Dennis; Gauntlett, Carolyn; Heumann, Lars V.; Kramer, Rainer; Marsden, Matthew D.; Murray, Danielle; Zack, Jerome A.; Wender, Paul A.

    2013-01-01

    Highly active antiretroviral therapy (HAART) decreases plasma viremia below the limits of detection in the majority of HIV-infected individuals, thus serving to slow disease progression. However, HAART targets only actively replicating virus and is unable to eliminate latently infected, resting CD4+ T cells. Such infected cells are potentially capable of reinitiating virus replication upon cessation of HAART, thus leading to viral rebound. Agents that would eliminate these reservoirs, when used in combination with HAART, could thus provide a strategy for the eradication of HIV. Prostratin is a preclinical candidate that induces HIV expression from latently infected CD4+ T cells, potentially leading to their elimination through a virus-induced cytopathic effect or host anti-HIV immunity. Here, we report the synthesis of a series of designed prostratin analogs and report in vitro and ex vivo studies of their activity relevant to induction of HIV expression. Members of this series are up to 100-fold more potent than the preclinical lead (prostratin) in binding to cell-free PKC, and in inducing HIV expression in a latently infected cell line and prostratin-like modulation of cell surface receptor expression in primary cells from HIV-negative donors. Significantly, selected members were also tested for HIV induction in resting CD4+ T cells isolated from infected individuals receiving HAART and were found to exhibit potent induction activity. These more potent agents and by extension related tunable analogs now accessible through the studies described herein should facilitate research and preclinical advancement of this strategy for HIV/AIDS eradication. PMID:23812750

  9. Correlation of Subchondral Bone Density and Structure from Plain Radiographs with Micro Computed Tomography Ex Vivo.

    PubMed

    Hirvasniemi, Jukka; Thevenot, Jérôme; Kokkonen, Harri T; Finnilä, Mikko A; Venäläinen, Mikko S; Jämsä, Timo; Korhonen, Rami K; Töyräs, Juha; Saarakkala, Simo

    2016-05-01

    Osteoarthritis causes changes in the subchondral bone structure and composition. Plain radiography is a cheap, fast, and widely available imaging method. Bone tissue can be well seen from plain radiograph, which however is only a 2D projection of the actual 3D structure. Therefore, the aim was to investigate the relationship between bone density- and structure-related parameters from 2D plain radiograph and 3D bone parameters assessed from micro computed tomography (µCT) ex vivo. Right tibiae from eleven cadavers without any diagnosed joint disease were imaged using radiography and with µCT. Bone density- and structure-related parameters were calculated from four different locations from the radiographs of proximal tibia and compared with the volumetric bone microarchitecture from the corresponding regions. Bone density from the plain radiograph was significantly related with the bone volume fraction (r = 0.86; n = 44; p < 0.01). Mean homogeneity index for orientation of local binary patterns (HIangle,mean) and fractal dimension of vertical structures (FDVer) were related (p < 0.01) with connectivity density (HIangle,mean: r = -0.73, FDVer: r = 0.69) and trabecular separation (HIangle,mean: r = 0.73, FDVer: r = -0.70) when all ROIs were pooled together (n = 44). Bone density and structure in tibia from standard clinically available 2D radiographs are significantly correlated with true 3D microstructure of bone. PMID:26369637

  10. Lyophilized phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation.

    PubMed

    Freag, May S; Elnaggar, Yosra S R; Abdallah, Ossama Y

    2013-01-01

    Diosmin (DSN) is an outstanding phlebotonic flavonoid with a tolerable potential for the treatment of colon and hepatocellular carcinoma. Being highly insoluble, DSN bioavailability suffers from high inter-subject variation due to variable degrees of permeation. This work endeavored to develop novel DSN loaded phytosomes in order to improve drug dissolution and intestinal permeability. Three preparation methods (solvent evaporation, salting out, and lyophilization) were compared. Nanocarrier optimization encompassed different soybean phospholipid (SPC) types, different solvents, and different DSN:SPC molar ratios (1:1, 1:2, and 1:4). In vitro appraisal encompassed differential scanning calorimetry, infrared spectroscopy, particle size, zeta potential, polydispersity index, transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed under sink versus non-sink conditions. Ex vivo intestinal permeation studies were performed on rats utilizing noneverted sac technique and high-performance liquid chromatography analysis. The results revealed lyophilization as the optimum preparation technique using SPC and solvent mixture (Dimethyl sulphoxide:t-butylalchol) in a 1:2 ratio. Complex formation was contended by differential scanning calorimetry and infrared data. Optimal lyophilized phytosomal nanocarriers (LPNs) exhibited the lowest particle size (316 nm), adequate zeta-potential (-27 mV), and good in vitro stability. Well formed, discrete vesicles were revealed by transmission electron microscopy, drug content, and in vitro stability. Comparative dissolution studies were performed. LPNs demonstrated significant enhancement in DSN dissolution compared to crude drug, physical mixture, and generic and brand DSN products. Permeation studies revealed 80% DSN permeated from LPNs via oxygenated rat intestine compared to non-detectable amounts from suspension. In this study, LPNs (99% drug loading) could be successfully

  11. Manganese ferrite-based nanoparticles induce ex vivo, but not in vivo, cardiovascular effects

    PubMed Central

    Nunes, Allancer DC; Ramalho, Laylla S; Souza, Álvaro PS; Mendes, Elizabeth P; Colugnati, Diego B; Zufelato, Nícholas; Sousa, Marcelo H; Bakuzis, Andris F; Castro, Carlos H

    2014-01-01

    Magnetic nanoparticles (MNPs) have been used for various biomedical applications. Importantly, manganese ferrite-based nanoparticles have useful magnetic resonance imaging characteristics and potential for hyperthermia treatment, but their effects in the cardiovascular system are poorly reported. Thus, the objectives of this study were to determine the cardiovascular effects of three different types of manganese ferrite-based magnetic nanoparticles: citrate-coated (CiMNPs); tripolyphosphate-coated (PhMNPs); and bare magnetic nanoparticles (BaMNPs). The samples were characterized by vibrating sample magnetometer, X-ray diffraction, dynamic light scattering, and transmission electron microscopy. The direct effects of the MNPs on cardiac contractility were evaluated in isolated perfused rat hearts. The CiMNPs, but not PhMNPs and BaMNPs, induced a transient decrease in the left ventricular end-systolic pressure. The PhMNPs and BaMNPs, but not CiMNPs, induced an increase in left ventricular end-diastolic pressure, which resulted in a decrease in a left ventricular end developed pressure. Indeed, PhMNPs and BaMNPs also caused a decrease in the maximal rate of left ventricular pressure rise (+dP/dt) and maximal rate of left ventricular pressure decline (−dP/dt). The three MNPs studied induced an increase in the perfusion pressure of isolated hearts. BaMNPs, but not PhMNPs or CiMNPs, induced a slight vasorelaxant effect in the isolated aortic rings. None of the MNPs were able to change heart rate or arterial blood pressure in conscious rats. In summary, although the MNPs were able to induce effects ex vivo, no significant changes were observed in vivo. Thus, given the proper dosages, these MNPs should be considered for possible therapeutic applications. PMID:25031535

  12. Development of an Ex Vivo Protocol to Model Bone Fracture in Laying Hens Resulting from Collisions

    PubMed Central

    Toscano, Michael J.; Wilkins, Lindsay J.; Millburn, Georgina; Thorpe, Katherine; Tarlton, John F.

    2013-01-01

    Fractures of the keel bone, a bone extending ventrally from the sternum, are a serious health and welfare problem in free range laying hens. Recent findings suggest that a major cause of keel damage within extensive systems is collisions with internal housing structures, though investigative efforts have been hindered by difficulties in examining mechanisms and likely influencing factors at the moment of fracture. The objectives of this study were to develop an ex vivo impact protocol to model bone fracture in hens caused by collision, to assess impact and bird-related factors influencing fracture occurrence and severity, and to identify correlations of mechanical and structural properties between different skeletal sites. We induced keel bone fractures in euthanized hens using a drop-weight impact tester able to generate a range of impact energies, producing fractures that replicate those commonly found in commercial settings. The results demonstrated that impact energies of a similar order to those expected in normal housing were able to produce fractures, and that greater collision energies resulted in an increased likelihood of fractures and of greater severity. Relationships were also seen with keel’s lateral surface bone mineral density, and the peak reactive force (strength) at the base of the manubrial spine. Correlations were also identified between the keel and long bones with respect to both strength and bone mineral density. This is the first study able to relate impact and bone characteristics with keel bone fracture at the moment of collision. Greater understanding of these relationships will provide means to reduce levels of breakage and severity in commercial systems. PMID:23785487

  13. Isolation and characterization of ex vivo expanded mesenchymal stem cells obtained from a surgical patient

    PubMed Central

    HUANG, JIA; SHA, HUIFAN; WANG, GUAN; BAO, GUOLIANG; LU, SHUN; LUO, QINGQUAN; TAN, QIANG

    2015-01-01

    The aim of the present study was to investigate the morphological characteristics and pluripotent differentiation potential of human bone marrow mesenchymal stem cells (hBMMSCs) in vitro and in vivo. Bone marrow cells were isolated from a rib fragment of an adult surgical patient, hBMMSCs were isolated based on plastic adherence and expanded ex vivo and phenotyping was performed. Pluripotent differentiation assays for adipogenesis, myogenesis and osteogenesis were conducted. Hematopoietic reconstruction of sublethally irradiated nude mice was performed by infusion of hBMMSCs. The gene expression profiles of early and late hBMMSCs were examined. The rate of CD31-positive cells was 31.1% in passage (P)4 hBMMSCs and 18.6% in P10 hBMMSCs. CD105 and CD106 were expressed in 99 and 95% of P25 hBMMSCs, respectively. Lipid droplets appeared at day 18 post induction. For osteogenesis, palpable masses were grossly observed from day 35 post inoculation of hBMMSCs. Hematoxylin and eosin staining further revealed chondrocytes and bone tissues. For myogenesis, at day six post subcutaneous inoculation, hBMMSCs differentiated into myocytes and were positive for myoglobin and MyoD1. In irradiated nude mice reconstituted by hBMMSCs, the white blood cell count briefly decreased following irradiation; however, it gradually recovered. In the irradiated nude mice reconstituted with hBMMSCs, CD45- and CD34-positive cells were detected 72 h post induction. Gene microarray analysis of P7 and P57 hBMMSCs demonstrated that 20 genes were upregulated >2 fold and 40 genes were downregulated >2 fold in P57 hBMMSCs. In conclusion, the isolated HBMMSCs possessed pluripotent differentiation potential and it was feasible and safe to use hBMMSCs within 30 passages. PMID:25376882

  14. LANTCET: laser nanotechnology for screening and treating tumors ex vivo and in vivo

    NASA Astrophysics Data System (ADS)

    Lapotko, Dmitri O.; Lukianova-Hleb, Ekaterina Y.; Zhdanok, Sergei A.; Hafner, Jason H.; Rostro, Betty C.; Scully, Peter; Konopleva, Marina; Andreeff, Michael; Li, Chun; Hanna, Ehab Y.; Myers, Jeffrey N.; Oraevsky, Alexander A.

    2007-06-01

    LANTCET (laser-activated nano-thermolysis as cell elimination technology) was developed for selective detection and destruction of individual tumor cells through generation of photothermal bubbles around clusters of light absorbing gold nanoparticles (nanorods and nanoshells) that are selectively formed in target tumor cells. We have applied bare nanoparticles and their conjugates with cell-specific vectors such as monoclonal antibodies CD33 (specific for Acute Myeloid Leukemia) and C225 (specific for carcinoma cells that express epidermal growth factor -EGF). Clusters were formed by using vector-receptor interactions with further clusterization of nanoparticles due to endocytosis. Formation of clusters was verified directly with optical resonance scattering microscopy and microspectroscopy. LANTCET method was tested in vitro for living cell samples with: (1) model myeloid K562 cells (CD33 positive), (2) primary human bone marrow CD33-positive blast cells from patients with the diagnosis of acute myeloid leukemia, (3) monolayers of living EGF-positive carcinoma cells (Hep-2C), (4) human lymphocytes and red blood cells as normal cells. The LANTCET method was also tested in vivo using rats with experimental polymorphic sarcoma. Photothermal bubbles were generated and detected in vitro with a photothermal microscope equipped with a tunable Ti-Sa pulsed laser. We have found that cluster formation caused an almost 100-fold decrease in the bubble generation threshold of laser pulse fluence in tumor cells compared to the bubble generation threshold for normal cells. The animal tumor that was treated with a single laser pulse showed a necrotic area of diameter close to the pump laser beam diameter and a depth of 1-2 mm. Cell level selectivity of tumor damage with single laser pulse was demonstrated. Combining lightscattering imaging with bubble imaging, we introduced a new image-guided mode of the LANTCET operation for screening and treatment of tumors ex vivo and in vivo.

  15. A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain.

    PubMed

    Ghoochani, Ali; Yakubov, Eduard; Sehm, Tina; Fan, Zheng; Hock, Stefan; Buchfelder, Michael; Eyüpoglu, Ilker Y; Savaskan, Nicolai E

    2016-01-12

    Primary brain tumors are hallmarked for their destructive activity on the microenvironment and vasculature. However, solely few experimental techniques exist to access the tumor microenvironment under anatomical intact conditions with remaining cellular and extracellular composition. Here, we detail an ex vivo vascular glioma impact method (VOGIM) to investigate the influence of gliomas and chemotherapeutics on the tumor microenvironment and angiogenesis under conditions that closely resemble the in vivo situation. We generated organotypic brain slice cultures from rats and transgenic mice and implanted glioma cells expressing fluorescent reporter proteins. In the VOGIM, tumor-induced vessels presented the whole range of vascular pathologies and tumor zones as found in human primary brain tumor specimens. In contrast, non-transformed cells such as primary astrocytes do not alter the vessel architecture. Vascular characteristics with vessel branching, junctions and vessel meshes are quantitatively assessable as well as the peritumoral zone. In particular, the VOGIM resembles the brain tumor microenvironment with alterations of neurons, microglia and cell survival. Hence, this method allows live cell monitoring of virtually any fluorescence-reporter expressing cell. We further analyzed the vasculature and microglia under the influence of tumor cells and chemotherapeutics such as Temozolamide (Temodal/Temcad®). Noteworthy, temozolomide normalized vasculare junctions and branches as well as microglial distribution in tumor-implanted brains. Moreover, VOGIM can be facilitated for implementing the 3Rs in experimentations. In summary, the VOGIM represents a versatile and robust technique which allows the assessment of the brain tumor microenvironment with parameters such as angiogenesis, neuronal cell death and microglial activity at the morphological and quantitative level. PMID:26673818

  16. Assessment of human herpesvirus-6 infection in mesenchymal stromal cells ex vivo expanded for clinical use.

    PubMed

    Pessina, A; Bonomi, A; Coccè, V; Bernardo, M E; Cometa, A M; Ferrari, M; Sisto, F; Cavicchini, L; Locatelli, F

    2009-12-01

    Infection or reactivation of human herpesvirus (HHV)-6 represents a potentially serious complication (often involving the central nervous system) in patients receiving either solid organ or hematopoietic stem cell transplantation. The objective of this study was to assess the risk of HHV-6 infection/reactivation in mesenchymal stromal cells (MSCs). MSCs are multipotent cells displaying immunomodulatory properties that have been already successfully used in the clinical setting to enhance hematopoietic stem cell engraftment and to treat steroid-refractory acute graft-versus-host disease. We analyzed 20 samples of ex vivo expanded MSCs, at different passages of culture, isolated both from bone marrow and from umbilical cord blood. Through Western blotting and immunocytochemistry techniques, we investigated the presence of the HHV-6 receptor (CD46) on cell surface, whereas the presence of HHV-6 DNA was evaluated by nested polymerase chain reaction assay. All of the MSC samples tested were positive for the virus receptor (CD46), suggesting their potential susceptibility to HHV-6. However, none of the MSC samples derived from cultures, performed in the perspective of clinical use, was found to harbor HHV-6. This preliminary observation on a consistent number of MSC samples, some of them tested at late in vitro passages, indicates a good safety profile of the product in terms of HHV-6 contamination. Nevertheless, it remains important to set up in vitro experimental models to study MSCs' susceptibility to HHV-6 (and HHV-7) infection, to verify their capacity to integrate the virus into cellular DNA, and to investigate which experimental conditions are able to induce virus reactivation. PMID:19664021

  17. Removing Biofilms from Microstructured Titanium Ex Vivo: A Novel Approach Using Atmospheric Plasma Technology

    PubMed Central

    Rupf, Stefan; Idlibi, Ahmad Nour; Marrawi, Fuad Al; Hannig, Matthias; Schubert, Andreas; von Mueller, Lutz; Spitzer, Wolfgang; Holtmann, Henrik; Lehmann, Antje; Rueppell, Andre; Schindler, Axel

    2011-01-01

    The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 µm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h) to 91 µm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease. PMID:22016784

  18. A versatile ex vivo technique for assaying tumor angiogenesis and microglia in the brain

    PubMed Central

    Ghoochani, Ali; Yakubov, Eduard; Sehm, Tina; Fan, Zheng; Hock, Stefan; Buchfelder, Michael

    2016-01-01

    Primary brain tumors are hallmarked for their destructive activity on the microenvironment and vasculature. However, solely few experimental techniques exist to access the tumor microenvironment under anatomical intact conditions with remaining cellular and extracellular composition. Here, we detail an ex vivo vascular glioma impact method (VOGIM) to investigate the influence of gliomas and chemotherapeutics on the tumor microenvironment and angiogenesis under conditions that closely resemble the in vivo situation. We generated organotypic brain slice cultures from rats and transgenic mice and implanted glioma cells expressing fluorescent reporter proteins. In the VOGIM, tumor-induced vessels presented the whole range of vascular pathologies and tumor zones as found in human primary brain tumor specimens. In contrast, non-transformed cells such as primary astrocytes do not alter the vessel architecture. Vascular characteristics with vessel branching, junctions and vessel length are quantitatively assessable as well as the peritumoral zone. In particular, the VOGIM resembles the brain tumor microenvironment with alterations of neurons, microglia and cell survival. Hence, this method allows live cell monitoring of virtually any flu