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Sample records for interventions phantom ex-vivo

  1. 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.

  2. 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

  3. WE-EF-210-06: Ultrasound 2D Strain Measurement of Radiation-Induced Toxicity: Phantom and Ex Vivo Experiments

    SciTech Connect

    Liu, T; Torres, M; Rossi, P; Jani, A; Curran, W; Yang, X

    2015-06-15

    Purpose: Radiation-induced fibrosis is a common long-term complication affecting many patients following cancer radiotherapy. Standard clinical assessment of subcutaneous fibrosis is subjective and often limited to visual inspection and palpation. Ultrasound strain imaging describes the compressibility (elasticity) of biological tissues. This study’s purpose is to develop a quantitative ultrasound strain imaging that can consistently and accurately characterize radiation-induce fibrosis. Methods: In this study, we propose a 2D strain imaging method based on deformable image registration. A combined affine and B-spline transformation model is used to calculate the displacement of tissue between pre-stress and post-stress B-mode image sequences. The 2D displacement is estimated through a hybrid image similarity measure metric, which is a combination of the normalized mutual information (NMI) and normalized sum-of-squared-differences (NSSD). And 2D strain is obtained from the gradient of the local displacement. We conducted phantom experiments under various compressions and compared the performance of our proposed method with the standard cross-correlation (CC)- based method using the signal-to-noise (SNR) and contrast-to-noise (CNS) ratios. In addition, we conducted ex-vivo beef muscle experiment to further validate the proposed method. Results: For phantom study, the SNR and CNS values of the proposed method were significantly higher than those calculated from the CC-based method under different strains. The SNR and CNR increased by a factor of 1.9 and 2.7 comparing to the CC-based method. For the ex-vivo experiment, the CC-based method failed to work due to large deformation (6.7%), while our proposed method could accurately detect the stiffness change. Conclusion: We have developed a 2D strain imaging technique based on the deformable image registration, validated its accuracy and feasibility with phantom and ex-vivo data. This 2D ultrasound strain imaging

  4. Adaptive Thermal Therapy using Planar Ultrasound Transducers with Real-time MR Temperature Feedback: Demonstration in Gel Phantoms and Ex-vivo Tissues

    NASA Astrophysics Data System (ADS)

    Tang, Kee; Choy, Vanessa; Chopra, Rajiv; Bronskill, Michael

    2007-05-01

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. The main goal of this study was to evaluate active temperature feedback on a clinical 1.5T MR imager to control conformal thermal therapy. MR thermometry was performed during heating in both thermal gel phantoms and ex-vivo tissue with a single-element transurethral heating applicator. The applicator rotation rate and power were controlled based on MRI-temperature measurements. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) was also investigated in gel phantoms. The 55°C isotherm generated during heating closely matched the targeted prostate shape, with an average distance error of 0.9 mm ± 0.4 mm in turkey breasts, 1.3 mm ± 0.5 mm in gel phantoms without rectal cooling and 1.4 mm ± 0.6 mm in gel phantoms with rectal cooling. Accurate, MRI-guided, active feedback has been successfully demonstrated experimentally and has the capability to adjust for unpredictable and varying tissue properties during the treatment.

  5. 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.

  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.

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

    PubMed

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

    2014-03-01

    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.

  8. 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

  9. Combined chirp coded tissue harmonic and fundamental ultrasound imaging for intravascular ultrasound: 20–60 MHz phantom and ex vivo results

    PubMed Central

    Park, Jinhyoung; Li, Xiang; Zhou, Qifa; Shung, K. Kirk

    2013-01-01

    The application of chirp coded excitation to pulse inversion tissue harmonic imaging can increase signal to noise ratio. On the other hand, the elevation of range side lobe level, caused by leakages of the fundamental signal, has been problematic in mechanical scanners which are still the most prevalent in high frequency intravascular ultrasound imaging. Fundamental chirp coded excitation imaging can achieve range side lobe levels lower than –60 dB with Hanning window, but it yields higher side lobes level than pulse inversion chirp coded tissue harmonic imaging (PI-CTHI). Therefore, in this paper a combined pulse inversion chirp coded tissue harmonic and fundamental imaging mode (CPI-CTHI) is proposed to retain the advantages of both chirp coded harmonic and fundamental imaging modes by demonstrating 20–60 MHz phantom and ex vivo results. A simulation study shows that the range side lobe level of CPI-CTHI is 16 dB lower than PI-CTHI, assuming that the transducer translates incident positions by 50 μm when two beamlines of pulse inversion pair are acquired. CPI-CTHI is implemented for a proto-typed intravascular ultrasound scanner capable of combined data acquisition in real-time. A wire phantom study shows that CPI-CTHI has a 12 dB lower range side lobe level and a 7 dB higher echo signal to noise ratio than PI-CTHI, while the lateral resolution and side lobe level are 50 μm finer and –3 dB less than fundamental chirp coded excitation imaging respectively. Ex vivo scanning of a rabbit trachea demonstrates that CPI-CTHI is capable of visualizing blood vessels as small as 200 μm in diameter with 6 dB better tissue contrast than either PI-CTHI or fundamental chirp coded excitation imaging. These results clearly indicate that CPI-CTHI may enhance tissue contrast with less range side lobe level than PI-CTHI. PMID:22871273

  10. Combined chirp coded tissue harmonic and fundamental ultrasound imaging for intravascular ultrasound: 20-60 MHz phantom and ex vivo results.

    PubMed

    Park, Jinhyoung; Li, Xiang; Zhou, Qifa; Shung, K Kirk

    2013-02-01

    The application of chirp coded excitation to pulse inversion tissue harmonic imaging can increase signal to noise ratio. On the other hand, the elevation of range side lobe level, caused by leakages of the fundamental signal, has been problematic in mechanical scanners which are still the most prevalent in high frequency intravascular ultrasound imaging. Fundamental chirp coded excitation imaging can achieve range side lobe levels lower than -60dB with Hanning window, but it yields higher side lobes level than pulse inversion chirp coded tissue harmonic imaging (PI-CTHI). Therefore, in this paper a combined pulse inversion chirp coded tissue harmonic and fundamental imaging mode (CPI-CTHI) is proposed to retain the advantages of both chirp coded harmonic and fundamental imaging modes by demonstrating 20-60MHz phantom and ex vivo results. A simulation study shows that the range side lobe level of CPI-CTHI is 16dB lower than PI-CTHI, assuming that the transducer translates incident positions by 50μm when two beamlines of pulse inversion pair are acquired. CPI-CTHI is implemented for a proto-typed intravascular ultrasound scanner capable of combined data acquisition in real-time. A wire phantom study shows that CPI-CTHI has a 12dB lower range side lobe level and a 7dB higher echo signal to noise ratio than PI-CTHI, while the lateral resolution and side lobe level are 50μm finer and -3dB less than fundamental chirp coded excitation imaging respectively. Ex vivo scanning of a rabbit trachea demonstrates that CPI-CTHI is capable of visualizing blood vessels as small as 200μm in diameter with 6dB better tissue contrast than either PI-CTHI or fundamental chirp coded excitation imaging. These results clearly indicate that CPI-CTHI may enhance tissue contrast with less range side lobe level than PI-CTHI.

  11. Combined ultrasound and photoacoustic imaging to detect and stage deep vein thrombosis: phantom and ex vivo studies.

    PubMed

    Karpiouk, Andrei B; Aglyamov, Salavat R; Mallidi, Srivalleesha; Shah, Jignesh; Scott, W Guy; Rubin, Jonathan M; Emelianov, Stanislav Y

    2008-01-01

    Treatment of deep venous thrombosis (DVT)--a primary cause of potentially fatal pulmonary embolism (PE)--depends on the age of the thrombus. The existing clinical imaging methods are capable of visualizing a thrombus but cannot determine the age of the blood clot. Therefore, there is a need for an imaging technique to reliably diagnose and adequately stage DVT. To stage DVT (i.e., to determine the age of the thrombus, and therefore, to differentiate acute from chronic DVT), we explored photoacoustic imaging, a technique capable of noninvasive measurements of the optical absorption in tissue. Indeed, optical absorption of the blood clot changes with age, since maturation of DVT is associated with significant cellular and molecular reorganization. The ultrasound and photoacoustic imaging studies were performed using DVT-mimicking phantoms and phantoms with embedded acute and chronic thrombi obtained from an animal model of DVT. The location and structure of the clots were visualized using ultrasound imaging, while the composition, and therefore age, of thrombi were related to the magnitude and spatiotemporal characteristics of the photoacoustic signal. Overall, the results of our study suggest that combined ultrasound and photoacoustic imaging of thrombi may be capable of simultaneous detection and staging of DVT.

  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.

  13. 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

  14. 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

  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.

  16. 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

  17. 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

  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.

  19. Conformal thermal therapy using planar ultrasound transducers and adaptive closed-loop MR temperature control: demonstration in gel phantoms and ex vivo tissues

    NASA Astrophysics Data System (ADS)

    Tang, K.; Choy, V.; Chopra, R.; Bronskill, M. J.

    2007-05-01

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. Integrating a multi-element planar transducer with active MR temperature feedback can enable three-dimensional conformal thermal therapy of a target region within the prostate gland while sparing surrounding normal tissues. Continuous measurement of the temperature distribution in tissue enables dynamic compensation for unknown changes in blood flow and tissue properties during treatment. The main goal of this study was to evaluate the feasibility of using active temperature feedback on a clinical 1.5 T MR imager for conformal thermal therapy. MR thermometry was performed during heating in both gel phantoms and excised tissue with a transurethral heating applicator, and the rotation rate and power were varied based on the thermal measurements. The capability to produce a region of thermal damage that matched a target boundary was evaluated. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) on the desired pattern of thermal damage was also investigated in gel phantoms. Results showed high correlation between the desired target boundary and the 55 °C isotherm generated during heating with an average distance error of 0.9 mm ± 0.4 mm (n = 6) in turkey breasts, 1.4 mm ± 0.6 mm (n = 4) in gel phantoms without rectal cooling and 1.4 mm ± 0.6 mm (n = 3) in gel phantoms with rectal cooling. The results were obtained using a temporal update rate of 5 s, a spatial resolution of 3 × 3 × 10 mm for the control point, and a temperature uncertainty of approximately 1 °C. The performance of the control algorithm under these conditions was comparable to that of simulations conducted previously by our group. Overall, the feasibility of generating targeted regions of thermal damage with a transurethral heating applicator and active MR temperature feedback has been demonstrated experimentally. This method of treatment

  20. Conformal thermal therapy using planar ultrasound transducers and adaptive closed-loop MR temperature control: demonstration in gel phantoms and ex vivo tissues.

    PubMed

    Tang, K; Choy, V; Chopra, R; Bronskill, M J

    2007-05-21

    MRI-guided transurethral ultrasound therapy offers a minimally invasive approach for the treatment of localized prostate cancer. Integrating a multi-element planar transducer with active MR temperature feedback can enable three-dimensional conformal thermal therapy of a target region within the prostate gland while sparing surrounding normal tissues. Continuous measurement of the temperature distribution in tissue enables dynamic compensation for unknown changes in blood flow and tissue properties during treatment. The main goal of this study was to evaluate the feasibility of using active temperature feedback on a clinical 1.5 T MR imager for conformal thermal therapy. MR thermometry was performed during heating in both gel phantoms and excised tissue with a transurethral heating applicator, and the rotation rate and power were varied based on the thermal measurements. The capability to produce a region of thermal damage that matched a target boundary was evaluated. The influence of a cooling gradient (to simulate cooling of the rectum or urethra) on the desired pattern of thermal damage was also investigated in gel phantoms. Results showed high correlation between the desired target boundary and the 55 degrees C isotherm generated during heating with an average distance error of 0.9 mm +/- 0.4 mm (n = 6) in turkey breasts, 1.4 mm +/- 0.6 mm (n = 4) in gel phantoms without rectal cooling and 1.4 mm +/- 0.6 mm (n = 3) in gel phantoms with rectal cooling. The results were obtained using a temporal update rate of 5 s, a spatial resolution of 3 x 3 x 10 mm for the control point, and a temperature uncertainty of approximately 1 degrees C. The performance of the control algorithm under these conditions was comparable to that of simulations conducted previously by our group. Overall, the feasibility of generating targeted regions of thermal damage with a transurethral heating applicator and active MR temperature feedback has been demonstrated experimentally. This method

  1. Gnotobiotic Human Colon Ex Vivo

    PubMed Central

    McDermott, Frank D.; Folan, David M. A.; Winter, Des C.; Folan, Michael A.; Baird, Alan W.

    2015-01-01

    Background A novel emulsion with efficacy as an agent for eliminating biofilms was selected. The aim of this study was to examine efficacy and effect of a formulation of ML:8 against commensal bacteria harvested from ex vivo human colonic tissues. Methods Mucosal sheets, obtained at the time of surgery, were exposed for 2 minutes to one of four solutions: Krebs-Hensleit (KH) solution, saline (NaCl; 0.9%), povidone iodine (1%), or ML:8 (2%); n = 4. Lumenal surfaces were swabbed for culture under aerobic or anaerobic conditions. Following treatment, each sheet was mounted in Ussing chambers and voltage clamped. Tissues were challenged with carbachol. Permeability coefficient (Papp) was determined using mannitol fluxes. At the end of each experiment, tissues were examined histologically. Results Similar colony forming units grew in aerobic and anaerobic conditions in both control and NaCl treated tissues. Iodine reduced and ML:8 virtually abolished viable bacteria. Basal electrophysiological parameters were not different between treatments. Transepithelial electrical resistance values did not differ between groups. All tissues responded to carbachol, although this was attenuated in iodine treated tissue. Papp values were slightly elevated in all treated tissues but this did not reach significance. Histopathological assessment revealed no overt damage to tissues. Conclusion Brief exposure to ML:8 reduced culturable bacterial burden from human intestinal tissues harvested at the time of surgical resection. Such gnotobiotic tissues retain structural and functional integrity. This is a novel approach to reduce bacterial burden. PMID:27785304

  2. 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

  3. Ebola Virus Persistence in Semen Ex Vivo.

    PubMed

    Fischer, Robert J; Judson, Seth; Miazgowicz, Kerri; Bushmaker, Trent; Munster, Vincent J

    2016-02-01

    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.

  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. Ex vivo expansion of hematopoietic stem cells.

    PubMed

    Xie, JingJing; Zhang, ChengCheng

    2015-09-01

    Ex vivo expansion of hematopoietic stem cells (HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

  6. Human lung ex vivo infection models.

    PubMed

    Hocke, Andreas C; Suttorp, Norbert; Hippenstiel, Stefan

    2017-03-01

    Pneumonia is counted among the leading causes of death worldwide. Viruses, bacteria and pathogen-related molecules interact with cells present in the human alveolus by numerous, yet poorly understood ways. Traditional cell culture models little reflect the cellular composition, matrix complexity and three-dimensional architecture of the human lung. Integrative animal models suffer from species differences, which are of particular importance for the investigation of zoonotic lung diseases. The use of cultured ex vivo infected human lung tissue may overcome some of these limitations and complement traditional models. The present review gives an overview of common bacterial lung infections, such as pneumococcal infection and of widely neglected pathogens modeled in ex vivo infected lung tissue. The role of ex vivo infected lung tissue for the investigation of emerging viral zoonosis including influenza A virus and Middle East respiratory syndrome coronavirus is discussed. Finally, further directions for the elaboration of such models are revealed. Overall, the introduced models represent meaningful and robust methods to investigate principles of pathogen-host interaction in original human lung tissue.

  7. Comprehensive phantom for interventional fluorescence molecular imaging

    NASA Astrophysics Data System (ADS)

    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.

  8. Transplantation of ex vivo expanded cord blood.

    PubMed

    Shpall, Elizabeth J; Quinones, Ralph; Giller, Roger; Zeng, Chan; Baron, Anna E; Jones, Roy B; Bearman, Scott I; Nieto, Yago; Freed, Brian; Madinger, Nancy; Hogan, Christopher J; Slat-Vasquez, Vicki; Russell, Peggy; Blunk, Betsy; Schissel, Deborah; Hild, Elaine; Malcolm, Janet; Ward, William; McNiece, Ian K

    2002-01-01

    Umbilical cord blood (CB) from unrelated donors is increasingly used to restore hematopoiesis after myeloablative therapy. CB transplants are associated with higher rates of delayed and failed engraftment than are bone marrow transplants, particularly for adult patients. We studied the ex vivo expansion of CB in an attempt to improve time to engraftment and reduce the graft failure rate in the recipients. In this feasibility study, 37 patients (25 adults, 12 children) with hematologic malignancies (n = 34) or breast cancer (n = 3) received high-dose therapy followed by unrelated allogeneic CB transplantation. A fraction of each patient's CB allograft was CD34-selected and cultured ex vivo for 10 days prior to transplantation in defined media with stem cell factor, granulocyte colony-stimulating factor, and megakaryocyte growth and differentiation factor. The remainder of the CB graft was infused without further manipulation. Two sequential cohorts of patients were accrued to the study. The first cohort had 40% and the second cohort had 60% of their CB graft expanded. Patients received a median of 0.99 x 10(7) total nucleated cells (expanded plus unexpanded) per kilogram. The median time to engraftment of neutrophils was 28 days (range, 15-49 days) and of platelets was 106 days (range, 38-345 days). All evaluable patients who were followed for 28 days or longer achieved engraftment of neutrophils. Grade III/IV acute GVHD was documented in 40% and extensive chronic GVHD in 63% of patients. At a median follow-up of 30 months, 13 (35%) of 37 of patients survived. This study demonstrates that the CD34 selection and ex vivo expansion of CB prior to transplantation of CB is feasible. Additional accrual will be required to assess the clinical efficacy of expanded CB progenitors.

  9. 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

  10. 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.

  11. 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

  12. Ex vivo expansion of human hematopoietic stem and progenitor cells

    PubMed Central

    Dahlberg, Ann; Delaney, Colleen

    2011-01-01

    Despite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field. PMID:21436068

  13. Novel chemical attempts at ex vivo hematopoietic stem cell expansion.

    PubMed

    Zhang, Yu; Gao, Yingdai

    2016-05-01

    Hematopoietic stem cells (HSCs) are the most extensively studied stem cell type in adults, and the only stem cell type with proof of clinical utility. However, the greatest challenge for the broader use of HSCs remains the true expansion of the stem cells ex vivo. The development of researches on small-molecule compounds that support the safe and efficient ex vivo expansion of HSCs would help to promote the clinical application of HSCs. In recent years, several novel small-molecule compounds have been reported to improve ex vivo HSC expansion by promoting self-renewal, delaying differentiation, increasing homing, and inhibiting apoptosis. Here, we review recent chemical developments in stem cell research and the mechanisms underlying these compounds' effects.

  14. In vitro and ex vivo strategies for intracellular delivery

    NASA Astrophysics Data System (ADS)

    Stewart, Martin P.; Sharei, Armon; Ding, Xiaoyun; Sahay, Gaurav; Langer, Robert; Jensen, Klavs F.

    2016-10-01

    Intracellular delivery of materials has become a critical component of genome-editing approaches, ex vivo cell-based therapies, and a diversity of fundamental research applications. Limitations of current technologies motivate development of next-generation systems that can deliver a broad variety of cargo to diverse cell types. Here we review in vitro and ex vivo intracellular delivery approaches with a focus on mechanisms, challenges and opportunities. In particular, we emphasize membrane-disruption-based delivery methods and the transformative role of nanotechnology, microfluidics and laboratory-on-chip technology in advancing the field.

  15. 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

  16. Analytical Advances in the Ex Vivo Challenge Efficacy Assay.

    PubMed

    Richardson-Harman, Nicola; Parody, Robert; Anton, Peter; McGowan, Ian; Doncel, Gustavo; Thurman, Andrea Ries; Herrera, Carolina; Kordy, Kattayoun; Fox, Julie; Tanner, Karen; Swartz, Glenn; Dezzutti, Charlene S

    2017-04-01

    The ex vivo challenge assay is being increasingly used as an efficacy endpoint during early human clinical trials of HIV prevention treatments. There is no standard methodology for the ex vivo challenge assay, although the use of different data collection methods and analytical parameters may impact results and reduce the comparability of findings between trials. In this analysis, we describe the impact of data imputation methods, kit type, testing schedule and tissue type on variability, statistical power, and ex vivo HIV growth kinetics. Data were p24 antigen (pg/ml) measurements collected from clinical trials of candidate microbicides where rectal (n = 502), cervical (n = 88), and vaginal (n = 110) tissues were challenged with HIV-1BaL ex vivo. Imputation of missing data using a nonlinear mixed effect model was found to provide an improved fit compared to imputation using half the limit of detection. The rectal virus growth period was found to be earlier and of a relatively shorter duration than the growth period for cervical and vaginal tissue types. On average, only four rectal tissue challenge assays in each treatment and control group would be needed to find a one log difference in p24 to be significant (alpha = 0.05), but a larger sample size was predicted to be needed for either cervical (n = 21) or vaginal (n = 10) tissue comparisons. Overall, the results indicated that improvements could be made in the design and analysis of the ex vivo challenge assay to provide a more standardized and powerful assay to compare efficacy of microbicide products.

  17. In vitro and ex vivo retina angiogenesis assays.

    PubMed

    Rezzola, Sara; Belleri, Mirella; Gariano, Giuseppina; Ribatti, Domenico; Costagliola, Ciro; Semeraro, Francesco; Presta, Marco

    2014-07-01

    Pathological angiogenesis of the retina is a key component of irreversible causes of blindness, as observed in proliferative diabetic retinopathy, age-related macular degeneration, and retinopathy of prematurity. Seminal studies in the early 1980 s about the angiogenic activity exerted by mammalian retinal tissue extracts on the chick embryo chorioallantoic membrane and the later discovery of vascular endothelial growth factor (VEGF) accumulation in eyes of patients with diabetic retinopathy paved the way for the development of anti-angiogenic VEGF blockers for the treatment of retinal neovascularization. Since then, numerous preclinical and clinical studies about diabetic retinopathy and other retinal disorders have opened new lines of angiogenesis inquiry, indicating that limitations to anti-VEGF therapies may exist. Moreover, the production of growth factors other than VEGF may affect the response to anti-VEGF approaches. Thus, experimental models of retinal angiogenesis remain crucial for investigating novel anti-angiogenic therapies and bringing them to patients. To this aim, in vitro and ex vivo angiogenesis assays may be suitable for a rapid screening of potential anti-angiogenic molecules before in vivo validation of the putative lead compounds. This review focuses on the different in vitro and ex vivo angiogenesis assays that have been developed over the years based on the isolation of endothelial cells from the retina of various animal species and ex vivo cultures of neonatal and adult retina explants. Also, recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryos, an in vivo animal platform experimentally analogous to in vitro/ex vivo models, may represent a novel target for the identification of angiogenesis inhibitors. When compared to in vivo assays, in vitro and ex vivo models of retina neovascularization, including zebrafish embryo, may represent cost-effective and rapid tools for the screening of novel anti

  18. 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.

  19. Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

    PubMed Central

    Rajendran, Simon; Salwa, Slawomir; Gao, Xuefeng; Tabirca, Sabin; O'Hanlon, Deirdre; O'Sullivan, Gerald C.; Tangney, Mark

    2010-01-01

    This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial. PMID:21326169

  20. 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

  1. Ex Vivo Metrics, a preclinical tool in new drug development.

    PubMed

    Curtis, C Gerald; Bilyard, Kevin; Stephenson, Hugo

    2008-01-23

    Among the challenges facing translational medicine today is the need for greater productivity and safety during the drug development process. To meet this need, practitioners of translational medicine are developing new technologies that can facilitate decision making during the early stages of drug discovery and clinical development. Ex Vivo Metrics is an emerging technology that addresses this need by using intact human organs ethically donated for research. After hypothermic storage, the organs are reanimated by blood perfusion, providing physiologically and biochemically stable preparations. In terms of emulating human exposure to drugs, Ex Vivo Metrics is the closest biological system available for clinical trials. Early application of this tool for evaluating drug targeting, efficacy, and toxicity could result in better selection among promising drug candidates, greater drug productivity, and increased safety.

  2. 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

  3. Passive cavitation detection during pulsed HIFU exposures of ex vivo tissues and in vivo mouse pancreatic tumors.

    PubMed

    Li, Tong; Chen, Hong; Khokhlova, Tatiana; Wang, Yak-Nam; Kreider, Wayne; He, Xuemei; Hwang, Joo Ha

    2014-07-01

    Pulsed high-intensity focused ultrasound (pHIFU) has been shown to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rare factional focal pressures (1-12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms and pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KrasLSL.G12 D/+; p53 R172 H/+; PdxCretg/+ (KPC) mice and closely re-capitulate human disease in their morphology. The cavitation threshold, defined at 50% cavitation probability, was found to vary broadly among the investigated tissues (within 2.5-10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but it decreased rapidly and stopped

  4. Ex Vivo and In Silico Feasibility Study of Monitoring Electric Field Distribution in Tissue during Electroporation Based Treatments

    PubMed Central

    Kranjc, Matej; Bajd, Franci; Sersa, Igor; Woo, Eung Je; Miklavcic, Damijan

    2012-01-01

    Magnetic resonance electrical impedance tomography (MREIT) was recently proposed for determining electric field distribution during electroporation in which cell membrane permeability is temporary increased by application of an external high electric field. The method was already successfully applied for reconstruction of electric field distribution in agar phantoms. Before the next step towards in vivo experiments is taken, monitoring of electric field distribution during electroporation of ex vivo tissue ex vivo and feasibility for its use in electroporation based treatments needed to be evaluated. Sequences of high voltage pulses were applied to chicken liver tissue in order to expose it to electric field which was measured by means of MREIT. MREIT was also evaluated for its use in electroporation based treatments by calculating electric field distribution for two regions, the tumor and the tumor-liver region, in a numerical model based on data obtained from clinical study on electrochemotherapy treatment of deep-seated tumors. Electric field distribution inside tissue was successfully measured ex vivo using MREIT and significant changes of tissue electrical conductivity were observed in the region of the highest electric field. A good agreement was obtained between the electric field distribution obtained by MREIT and the actual electric field distribution in evaluated regions of a numerical model, suggesting that implementation of MREIT could thus enable efficient detection of areas with insufficient electric field coverage during electroporation based treatments, thus assuring the effectiveness of the treatment. PMID:23029212

  5. 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.

  6. 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.

  7. 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.

  8. Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

    PubMed

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

    2015-05-01

    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.

  9. Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices.

    PubMed

    Bosch, Daniel; Asede, Douglas; Ehrlich, Ingrid

    2016-04-05

    Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

  10. Ex vivo gene therapy for HIV-1 treatment.

    PubMed

    Scherer, Lisa J; Rossi, John J

    2011-04-15

    Until recently, progress in ex vivo gene therapy (GT) for human immunodeficiency virus-1 (HIV-1) treatment has been incremental. Long-term HIV-1 remission in a patient who received a heterologous stem cell transplant for acquired immunodeficiency syndrome-related lymphoma from a CCR5(-/-) donor, even after discontinuation of conventional therapy, has energized the field. We review the status of current approaches as well as future directions in the areas of therapeutic targets, combinatorial strategies, vector design, introduction of therapeutics into stem cells and enrichment/expansion of gene-modified cells. Finally, we discuss recent advances towards clinical application of HIV-1 GT.

  11. Ex vivo gene therapy cures a blistering skin disease.

    PubMed

    Featherstone, Carol; Uitto, Jouni

    2007-06-01

    A recent publication that describes gene therapy treatment of a patient with an inherited blistering skin disease, epidermolysis bullosa, demonstrates for the first time that gene therapy can cure a disease of solid tissue. The treatment relies on ex vivo transduction of autologous epidermal stem cells with a normal copy of the defective gene, followed by reconstitution of the patient's skin with epithelial sheets that are grown from these genetically corrected cells. This approach holds promise for treatment not only of inherited disorders of the skin but also of other solid tissues that are becoming amenable to tissue engineering.

  12. 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

  13. Ex vivo culture of the intestinal epithelium: strategies and applications.

    PubMed

    Leushacke, Marc; Barker, Nick

    2014-08-01

    Limited pools of resident adult stem cells are critical effectors of epithelial renewal in the intestine throughout life. Recently, significant progress has been made regarding the isolation and in vitro propagation of fetal and adult intestinal stem cells in mammals. It is now possible to generate ever-expanding, three-dimensional epithelial structures in culture that closely parallel the in vivo epithelium of the intestine. Growing such organotypic epithelium ex vivo facilitates a detailed description of endogenous niche factors or stem-cell characteristics, as they can be monitored in real time. Accordingly, this technology has already greatly contributed to our understanding of intestinal adult stem-cell renewal and differentiation. Transplanted organoids have also been proven to readily integrate into, and effect the long-term repair of, mouse colonic epithelia in vivo, establishing the organoid culture as a promising tool for adult stem cell/gene therapy. In another exciting development, novel genome-editing techniques have been successfully employed to functionally repair disease loci in cultured intestinal stem cells from human patients with a hereditary defect. It is anticipated that this technology will be instrumental in exploiting the regenerative medicine potential of human intestinal stem cells for treating human disorders in the intestinal tract and for creating near-physiological ex vivo models of human gastrointestinal disease.

  14. 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.

  15. 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

  16. [Ex vivo dermoscopy: synchronic evaluation between dermatologist and dermatopathologist of melanocytic lesions].

    PubMed

    Maia, Marcus; Lellis, Rute Facchini; Marta, Alessandra Cristine

    2009-01-01

    Clinicopathologic correlation is essential for diagnostic accuracy. Even though interdependent, dermatology and dermatopathology have become apart. In order to minimize this distance, we have performed ex vivo dermoscopic examinations. We performed comparative in vivo and ex vivo dermoscopy study followed by histopathological mapping. We observed that ex vivo dermoscopy identified the same structures visualized by the in vivo one, but with significant change of colors.

  17. Cellular senescence: ex vivo p53-dependent asymmetric cell kinetics

    PubMed Central

    2001-01-01

    Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture. PMID:12488624

  18. Mesenchymal Stem Cells in ex vivo Cord Blood Expansion

    PubMed Central

    Robinson, Simon N.; Simmons, Paul J.; Yang, Hong; Alousi, Amin M; de Lima, Marcos J.

    2013-01-01

    Umbilical cord blood (CB) is becoming an important source of haematopoietic support for transplant patients lacking human leukocyte antigen matched donors. The ethnic diversity, relative ease of collection, ready availability as cryopreserved units from CB banks, reduced incidence and severity of graft versus host disease and tolerance of higher degrees of HLA disparity between donor and recipient, are positive attributes when compared to bone marrow or cytokine-mobilized peripheral blood. However, CB transplantation is associated with significantly delayed neutrophil and platelet engraftment and an elevated risk of graft failure. These hurdles are thought to be due, at least in part, to low total nucleated cell and CD34+ cell doses transplanted. Here, current strategies directed at improving TNC and CD34+ cell doses at transplant are discussed, with particular attention paid to the use of a mesenchymal stem cell (MSC)/CB mononuclear cell ex vivo co-culture expansion system. PMID:21396596

  19. 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.

  20. In vivo versus ex vivo CRISPR therapies for retinal dystrophy

    PubMed Central

    Bakondi, Benjamin

    2017-01-01

    SUMMARY Two therapeutic paths have been proposed to treat inherited retinal dystrophy using clustered regularly interspaced short palindromic repeats (CRISPR). One strategy is to genetically correct patient cells ex vivo for autologous transplant, whereas the second is to modify cells in vivo by delivering CRISPR effectors to the retina. The feasibility of both editing strategies has been demonstrated within three years of CRISPR’s adaptation to mammalian systems. However, the functional integration of transplanted cells into host retinae has been a long-standing challenge that currently represents the 2025 moonshot of the National Eye Institute’s Audacious Goals Initiative. The clinical translatability of each path is discussed with regard to current investigations and whether cell replacement can be circumvented by in vivo editing. PMID:28163772

  1. Robot Assisted Stapedotomy ex vivo with an Active Handheld Instrument*

    PubMed Central

    Vendrametto, Tobia; McAfee, Jacob S.; Hirsch, Barry E.; Riviere, Cameron N.; Ferrigno, Giancarlo; De Momi, Elena

    2015-01-01

    Micron is a fully handheld active micromanipulator that helps to improve position accuracy and precision in microsurgery by cancelling hand tremor. This work describes adaptation, tuning, and testing of the Micron system for stapedotomy, a microsurgical procedure performed in the middle ear to restore hearing that requires accurate manipulation in narrow spaces. Two end-effectors, a handle, and a brace (or rest) were designed and prototyped. The control system was adapted for the new hardware. The system was tested ex vivo in stapedotomy procedure comparing manually-performed and Micron-assisted surgical tasks. Tremor amplitude was found to be reduced significantly. Further testing is needed in order to obtain statistically significant results regarding other parameters dealing with regularity of the fenestra shape. PMID:26737386

  2. Energy dissipation in Ex-Vivo Porcine Liver during Electrosurgery.

    PubMed

    Karaki, Wafaa; Akyildiz, Ali; De, Suvranu; Borca Tasciuc, Diana-Andra

    2016-07-27

    This paper explores energy dissipation in ex-vivo liver tissue during radiofrequency current excitation with application in electrosurgery. Tissue surface temperature for monopolar electrode configuration is measured using infrared thermometry. The experimental results are fitted to a finite element model for transient heat transfer taking into account energy storage and conduction in order to extract information about "apparent" specific heat, which encompasses storage and phase change. The average apparent specific heat determined for low temperatures is in agreement with published data. However, at temperatures approaching the boiling point of water, apparent specific heat increased by a factor of five, indicating that vaporization plays an important role in the energy dissipation through latent heat loss.

  3. Assessment of donor heart viability during ex vivo heart perfusion.

    PubMed

    White, Christopher W; Ambrose, Emma; Müller, Alison; Li, Yun; Le, Hoa; Hiebert, Brett; Arora, Rakesh; Lee, Trevor W; Dixon, Ian; Tian, Ganghong; Nagendran, Jayan; Hryshko, Larry; Freed, Darren

    2015-10-01

    Ex vivo heart perfusion (EVHP) may facilitate resuscitation of discarded donor hearts and expand the donor pool; however, a reliable means of demonstrating organ viability prior to transplantation is required. Therefore, we sought to identify metabolic and functional parameters that predict myocardial performance during EVHP. To evaluate the parameters over a broad spectrum of organ function, we obtained hearts from 9 normal pigs and 37 donation after circulatory death pigs and perfused them ex vivo. Functional parameters obtained from a left ventricular conductance catheter, oxygen consumption, coronary vascular resistance, and lactate concentration were measured, and linear regression analyses were performed to identify which parameters best correlated with myocardial performance (cardiac index: mL·min(-1)·g(-1)). Functional parameters exhibited excellent correlation with myocardial performance and demonstrated high sensitivity and specificity for identifying hearts at risk of poor post-transplant function (ejection fraction: R(2) = 0.80, sensitivity = 1.00, specificity = 0.85; stroke work: R(2) = 0.76, sensitivity = 1.00, specificity = 0.77; minimum dP/dt: R(2) = 0.74, sensitivity = 1.00, specificity = 0.54; tau: R(2) = 0.51, sensitivity = 1.00, specificity = 0.92), whereas metabolic parameters were limited in their ability to predict myocardial performance (oxygen consumption: R(2) = 0.28; coronary vascular resistance: R(2) = 0.20; lactate concentration: R(2) = 0.02). We concluded that evaluation of functional parameters provides the best assessment of myocardial performance during EVHP, which highlights the need for an EVHP device capable of assessing the donor heart in a physiologic working mode.

  4. 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.

  5. Surface-enhanced Raman spectroscopy for the in-vitro and ex-vivo detection of excitatory amino acids

    NASA Astrophysics Data System (ADS)

    O'Neal, D. P.; Motamedi, Massoud; Chen, Jefferson; Cote, Gerard L.

    1999-04-01

    Traditionally methods for the detection of excitatory amino acids, which have been linked to secondary injury following head trauma, can be excessively time consuming clinically. A near real-time measurement system could provide clinical information in anticipation of pharmaceutical intervention for head injured patients. Our studies have shown that surface-enhanced Raman spectroscopy (SERS) with silver colloids has the ability to measure physiological concentrations of in vitro excitatory amino acids using short scan times. Employing a damage model for ischemia, preliminary ex vivo rat extracellular grain fluid analysis shows an intriguing correlation between SERS spectral features and expected Glutamate concentration fluctuations following head injuries.

  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. Evaluation of Robotic Needle Steering in ex vivo Tissue

    PubMed Central

    Majewicz, Ann; Wedlick, Thomas R.; Reed, Kyle B.; Okamura, Allison M.

    2010-01-01

    Insertion velocity, tip asymmetry, and shaft diameter may influence steerable needle insertion paths in soft tissue. In this paper we examine the effects of these variables on needle paths in ex vivo goat liver, and demonstrate practical applications of robotic needle steering for ablation, biopsy, and brachytherapy. All experiments were performed using a new portable needle steering robot that steers asymmetric-tip needles under fluoroscopic imaging. For bevel-tip needles, we found that larger diameter needles resulted in less curvature, i.e. less steerability, confirming previous experiments in artificial tissue. The needles steered with radii of curvature ranging from 3:4 cm (for the most steerable pre-bent needle) to 2:97m (for the least steerable bevel needle). Pre-bend angle significantly affected needle curvature, but bevel angle did not. We hypothesize that biological tissue characteristics such as inhomogeneity and viscoelasticity significantly increase path variability. These results underscore the need for closed-loop image guidance for needle steering in biological tissues with complex internal structure. PMID:21339851

  8. Improved ex vivo method for microbiocidal activity across vertebrate species

    PubMed Central

    French, Susannah S.; Neuman-Lee, Lorin A.

    2012-01-01

    Summary The field of ecoimmunology is currently undergoing rapid expansion, whereby biologists from a wide range of ecological disciplines are increasingly interested in assessing immunocompetence in their study organisms. One of the key challenges to researchers is determining what eco-immune measures to use in a given experiment. Moreover, there are limitations depending on study species, requirements for specific antibodies, and relevance of the methodology to the study organism. Here we introduce an improved ex vivo method for microbiocidal activity across vertebrate species. The utility of this assay is that it determines the ability of an organism to remove a pathogen that could be encountered in the wild, lending ecological relevancy to the technique. The applications of this microbiocidal assay are broad, as it is readily adaptable to different types of microbes as well as a wide variety of study species. We describe a method of microbiocidal analysis that will enable researchers across disciplines to effectively employ this method to accurately quantify microbial killing ability, using readily available microplate absorbance readers. PMID:23213440

  9. Molecular convergence in ex vivo models of Diamond Blackfan anemia.

    PubMed

    O'Brien, Kelly A; Farrar, Jason E; Vlachos, Adrianna; Anderson, Stacie M; Tsujiura, Crystiana A; Lichtenberg, Jens; Blanc, Lionel; Atsidaftos, Eva; Elkahloun, Abdel; An, Xiuli; Ellis, Steven R; Lipton, Jeffery M; Bodine, David M

    2017-04-04

    Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor GATA1 Erythroid cells from patients with DBA have not been well characterized and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an ex vivo culture system to expand peripheral blood CD34(+) progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation compared to controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translation is a common feature of DBA caused by both RP and GATA1 mutations.

  10. 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

  11. Safe ex vivo coronary angiography with isosmotic contrast agent.

    PubMed

    Schmit, D B; Kern, J A; Mauney, M C; Kron, I L; Tribble, C G

    1996-08-01

    Plain-film coronary angiography of the cardiac explant on the operating table should be considered when conventional cardiac catheterization is desired but unavailable. We compared the effects of three contrast solutions on cold-preserved, isolated guinea pig hearts. Hearts were excised, perfused for 30 minutes, and arrested with Plegisol solution at 7 degree C. Twenty minutes after arrest, experimental hearts were perfused with one of three solutions: hyperosmolar Hexabrix solution (n = 6), hyperosmolar Renografin-76 solution (n = 6), or diluted, isosmotic Omnipaque solution (n = 8). The hearts were flushed with cold Plegisol solution 5 minutes later. Control hearts received no contrast during arrest (n = 9). The hearts were reperfused after 1 hour of arrest, and coronary blood flow (in millimeters per minute), left ventricular developed pressure (in millimeters of mercury), and rate of developed pressure (in millimeters of mercury per second) were measured. Endothelium-dependent smooth muscle relaxation to bradykinin administration and endothelium-independent relaxation to sodium nitroprusside administration were also assessed. No significant difference in myocardial or endothelial function was noted between control hearts and hearts perfused with Omnipaque solution. Hearts perfused with Renografin solution or Hexabrix solution, however, were found to have significantly impaired endothelial and myocardial function. We conclude that an isosmotic contrast solution should be used for ex vivo coronary angiography in cold-preserved hearts to avoid impairment of endothelial and myocardial function.

  12. 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.

  13. Ex vivo investigation of magnetically targeted drug delivery system

    NASA Astrophysics Data System (ADS)

    Yoshida, Y.; Fukui, S.; Fujimoto, S.; Mishima, F.; Takeda, S.; Izumi, Y.; Ohtani, S.; Fujitani, Y.; Nishijima, S.

    2007-03-01

    In conventional systemic drug delivery the drug is administered by intravenous injection; it then travels to the heart from where it is pumped to all regions of the body. When the drug is aimed at a small target region, this method is extremely inefficient and leads to require much larger doses than those being necessary. In order to overcome this problem a number of targeted drug delivery methods are developed. One of these, magnetically targeted drug delivery system (MT-DDS) will be a promising way, which involves binding a drug to small biocompatible magnetic particles, injecting these into the blood stream and using a high gradient magnetic field to pull them out of suspension in the target region. In the present paper, we describe an ex vivo experimental work. It is also reported that navigation and accumulation test of the magnetic particles in the Y-shaped glass tube was performed in order to examine the threshold of the magnetic force for accumulation. It is found that accumulation of the magnetic particles was succeeded in the blood vessel when a permanent magnet was placed at the vicinity of the blood vessel. This result indicates the feasibility of the magnetically drug targeting in the blood vessel.

  14. Terahertz pulse imaging of ex vivo basal cell carcinoma.

    PubMed

    Woodward, Ruth M; Wallace, Vincent P; Pye, Richard J; Cole, Bryan E; Arnone, Donald D; Linfield, Edmund H; Pepper, Michael

    2003-01-01

    Terahertz pulse imaging has been used for the first time to study basal cell carcinoma ex vivo, the most common form of skin cancer. This noninvasive technique uses part of the electromagnetic spectrum in the frequency range 0.1-2.7 THz. A total of 21 samples were imaged; the study was performed blind and results were compared to histology. Each image consisted of possible diseased tissue and normal tissue from the same patient. The diseased tissue showed an increase in absorption compared to normal tissue, which is attributed to either an increase in the interstitial water within the diseased tissue or a change in the vibrational modes of water molecules with other functional groups. Seventeen of the images showed a significant difference between the normal and the diseased tissue. These were confirmed by histology to be basal cell carcinomas. Of the remaining four cases, three showed no contrast and were confirmed as blind controls of normal tissue; the fourth case was a suspected basal cell carcinoma but showed no contrast, and histology showed no tumor. Cross-sections of the terahertz images, showing the terahertz absorption, were compared to histology. Regions of increased terahertz absorption agreed well with the location of the tumor sites. Resolutions at 1 THz of 350 microm laterally and 40 microm axially in skin were attainable with our system. These results demonstrate the ability of terahertz pulse imaging to distinguish basal cell carcinoma from normal tissue, and this macroscopic technique may, in the future, help plan surgery.

  15. Effects of Ex Vivo y-Tocopherol on Airway Macrophage ...

    EPA Pesticide Factsheets

    Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate y-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6)and mild house dust mite-sensitive allergic asthmatics (n =6) were treated ex vivo with GT (300 uM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206,CD36 and CD86 in allergic asthmatics but not in corntrols. Overall, GT caused down regulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor. Recent studies on the effects of the fat-soluble steriod hormone vitamins D and E suggest that dietary suplementation with these vitamins may be helpful for the prevention or in the treatment of inflammatory and immune-mediated diseases, including atopic asthma.

  16. In vitro and ex vivo antiangiogenic activity of Salvia officinalis.

    PubMed

    Keshavarz, Maryam; Mostafaie, Ali; Mansouri, Kamran; Bidmeshkipour, Ali; Motlagh, Hamid Reza Mohammadi; Parvaneh, Shahram

    2010-10-01

    Angiogenesis is a key process in the promotion of cancer and its metastasis. Herein, the antiangiogenic activity of Salvia officinalis extract and its fractions was investigated. S. officinalis aerial parts were extracted with ethanol and its successive hexane, ethyl acetate, n-butanol and aqueous fractions were evaluated for their antiangiogenic activities using human umbilical vein endothelial cells (HUVEC) capillary tube formation and rat aorta models in a three-dimensional collagen matrix. Furthermore, antimigrative effects of the fractions were assessed using a wound healing model. The ethanol extract of S. officinalis (ESO) potently inhibited capillary tube formation in HUVEC and rat aorta models of angiogenesis, and its hexane fraction (HSO) exerted the highest inhibitory effect. In addition, the ethanol extract of S. officinalis and its hexane fraction showed a dose-dependent inhibitory activity on the migration of the endothelial cells in the wound healing model. Furthermore, ESO inhibited endothelial cell proliferation at 50-200 μg/mL in a dose-dependent manner. These findings indicated some new pharmacological activities of S. officinalis such as antiangiogenic in vitro and ex vivo, and antimigrative activity in vitro. Therefore, S. officinalis could be a candidate as a useful herb with therapeutic or preventive activity against angiogenesis related disorders.

  17. 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.

  18. 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

  19. Liver metastases: Microenvironments and ex-vivo models

    PubMed Central

    Clark, Amanda M; Ma, Bo; Taylor, D Lansing; Griffith, Linda

    2016-01-01

    The liver is a highly metastasis-permissive organ, tumor seeding of which usually portends mortality. Its unique and diverse architectural and cellular composition enable the liver to undertake numerous specialized functions, however, this distinctive biology, notably its hemodynamic features and unique microenvironment, renders the liver intrinsically hospitable to disseminated tumor cells. The particular focus for this perspective is the bidirectional interactions between the disseminated tumor cells and the unique resident cell populations of the liver; notably, parenchymal hepatocytes and non-parenchymal liver sinusoidal endothelial, Kupffer, and hepatic stellate cells. Understanding the early steps in the metastatic seeding, including the decision to undergo dormancy versus outgrowth, has been difficult to study in 2D culture systems and animals due to numerous limitations. In response, tissue-engineered biomimetic systems have emerged. At the cutting-edge of these developments are ex vivo ‘microphysiological systems’ (MPS) which are cellular constructs designed to faithfully recapitulate the structure and function of a human organ or organ regions on a milli- to micro-scale level and can be made all human to maintain species-specific interactions. Hepatic MPSs are particularly attractive for studying metastases as in addition to the liver being a main site of metastatic seeding, it is also the principal site of drug metabolism and therapy-limiting toxicities. Thus, using these hepatic MPSs will enable not only an enhanced understanding of the fundamental aspects of metastasis but also allow for therapeutic agents to be fully studied for efficacy while also monitoring pharmacologic aspects and predicting toxicities. The review discusses some of the hepatic MPS models currently available and although only one MPS has been validated to relevantly modeling metastasis, it is anticipated that the adaptation of the other hepatic models to include tumors will not

  20. An ex vivo culture system to study thyroid development.

    PubMed

    Delmarcelle, Anne-Sophie; Villacorte, Mylah; Hick, Anne-Christine; Pierreux, Christophe E

    2014-06-06

    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.

  1. 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.

  2. Evaluation of the in vivo and ex vivo optical properties in a mouse ear model

    NASA Astrophysics Data System (ADS)

    Salomatina, E.; Yaroslavsky, A. N.

    2008-06-01

    Determination of in vivo optical properties is a challenging problem. Absorption and scattering measured ex vivo are often used for in vivo applications. To investigate the validity of this approach, we have obtained and compared the optical properties of mouse ears in vivo and ex vivo in the spectral range from 370 to 1650 nm. Integrating sphere spectrophotometry in combination with the inverse Monte Carlo technique was employed to determine absorption coefficients, μa, scattering coefficients, μs, and anisotropy factors, g. Two groups of mice were used for the study. The first group was measured in vivo and ex vivo within 5-10 min post mortem. The second group was measured in vivo and ex vivo every 24 h for up to 72 h after sacrifice. Between the measurements the tissues were kept at 4 °C wrapped in a gauze moistened with saline solution. Then the specimens were frozen at -25 °C for 40 min, thawed and measured again. The results indicate that the absorption coefficients determined in vivo and ex vivo within 5-10 min post mortem differed considerably only in the spectral range dominated by hemoglobin. These changes can be attributed to rapid deoxygenation of tissue and blood post mortem. Absorption coefficients determined ex vivo up to 72 h post mortem decreased gradually with time in the spectral regions dominated by hemoglobin and water, which can be explained by the continuing loss of blood. Absorption properties of the frozen-thawed ex vivo tissues showed increase in oxygenation, which is likely caused by the release of hemoglobin from hemolyzed erythrocytes. Scattering of the ex vivo tissues decreased gradually with time in the entire spectral range due to the continuing loss of blood and partial cell damage. Anisotropy factors did not change considerably.

  3. 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

  4. A Method for Whole Brain Ex Vivo Magnetic Resonance Imaging with Minimal Susceptibility Artifacts

    PubMed Central

    Shatil, Anwar S.; Matsuda, Kant M.; Figley, Chase R.

    2016-01-01

    Magnetic resonance imaging (MRI) is a non-destructive technique that is capable of localizing pathologies and assessing other anatomical features (e.g., tissue volume, microstructure, and white matter connectivity) in postmortem, ex vivo human brains. However, when brains are removed from the skull and cerebrospinal fluid (i.e., their normal in vivo magnetic environment), air bubbles and air–tissue interfaces typically cause magnetic susceptibility artifacts that severely degrade the quality of ex vivo MRI data. In this report, we describe a relatively simple and cost-effective experimental setup for acquiring artifact-free ex vivo brain images using a clinical MRI system with standard hardware. In particular, we outline the necessary steps, from collecting an ex vivo human brain to the MRI scanner setup, and have also described changing the formalin (as might be necessary in longitudinal postmortem studies). Finally, we share some representative ex vivo MRI images that have been acquired using the proposed setup in order to demonstrate the efficacy of this approach. We hope that this protocol will provide both clinicians and researchers with a straight-forward and cost-effective solution for acquiring ex vivo MRI data from whole postmortem human brains. PMID:27965620

  5. Culture materials affect ex vivo expansion of hematopoietic progenitor cells.

    PubMed

    LaIuppa, J A; McAdams, T A; Papoutsakis, E T; Miller, W M

    1997-09-05

    Ex vivo expansion of hematopoietic cells is important for applications such as cancer treatment, gene therapy, and transfusion medicine. While cell culture systems are widely used to evaluate the biocompatibility of materials for implantation, the ability of materials to support proliferation of primary human cells in cultures for reinfusion into patients has not been addressed. We screened a variety of commercially available polymer (15 types), metal (four types), and glass substrates for their ability to support expansion of hematopoietic cells when cultured under conditions that would be encountered in a clinical setting. Cultures of peripheral blood (PB) CD34+ cells and mononuclear cells (MNC) were evaluated for expansion of total cells and colony-forming unit-granulocyte monocyte (CFU-GM; progenitors committed to the granulocyte and/or monocyte lineage). Human hematopoietic cultures in serum-free medium were found to be extremely sensitive to the substrate material. The only materials tested that supported expansion at or near the levels of polystyrene were tissue culture polystyrene, Teflon perfluoroalkoxy, Teflon fluorinated ethylene propylene, cellulose acetate, titanium, new polycarbonate, and new polymethylpentene. MNC were less sensitive to the substrate materials than the primitive CD34+ progenitors, although similar trends were seen for expansion of the two cell populations on the substrates tested. CFU-GM expansion was more sensitive to substrate materials than was total cell expansion. The detrimental effects of a number of the materials on hematopoietic cultures appear to be caused by protein adsorption and/or leaching of toxins. Factors such as cleaning, sterilization, and reuse significantly affected the performance of some materials as culture substrates. We also used PB CD34+ cell cultures to examine the biocompatibility of gas-permeable cell culture and blood storage bags and several types of tubing commonly used with biomedical equipment

  6. 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

  7. Dual instrument for in vivo and ex vivo OCT imaging in an ENT department

    PubMed Central

    Cernat, Ramona; Tatla, Taran S.; Pang, Jingyin; Tadrous, Paul J.; Bradu, Adrian; Dobre, George; Gelikonov, Grigory; Gelikonov, Valentin; Podoleanu, Adrian Gh.

    2012-01-01

    A dual instrument is assembled to investigate the usefulness of optical coherence tomography (OCT) imaging in an ear, nose and throat (ENT) department. Instrument 1 is dedicated to in vivo laryngeal investigation, based on an endoscope probe head assembled by compounding a miniature transversal flying spot scanning probe with a commercial fiber bundle endoscope. This dual probe head is used to implement a dual channel nasolaryngeal endoscopy-OCT system. The two probe heads are used to provide simultaneously OCT cross section images and en face fiber bundle endoscopic images. Instrument 2 is dedicated to either in vivo imaging of accessible surface skin and mucosal lesions of the scalp, face, neck and oral cavity or ex vivo imaging of the same excised tissues, based on a single OCT channel. This uses a better interface optics in a hand held probe. The two instruments share sequentially, the swept source at 1300 nm, the photo-detector unit and the imaging PC. An aiming red laser is permanently connected to the two instruments. This projects visible light collinearly with the 1300 nm beam and allows pixel correspondence between the en face endoscopy image and the cross section OCT image in Instrument 1, as well as surface guidance in Instrument 2 for the operator. The dual channel instrument was initially tested on phantom models and then on patients with suspect laryngeal lesions in a busy ENT practice. This feasibility study demonstrates the OCT potential of the dual imaging instrument as a useful tool in the testing and translation of OCT technology from the lab to the clinic. Instrument 1 is under investigation as a possible endoscopic screening tool for early laryngeal cancer. Larger size and better quality cross-section OCT images produced by Instrument 2 provide a reference base for comparison and continuing research on imaging freshly excised tissue, as well as in vivo interrogation of more superficial skin and mucosal lesions in the head and neck patient

  8. Dual instrument for in vivo and ex vivo OCT imaging in an ENT department.

    PubMed

    Cernat, Ramona; Tatla, Taran S; Pang, Jingyin; Tadrous, Paul J; Bradu, Adrian; Dobre, George; Gelikonov, Grigory; Gelikonov, Valentin; Podoleanu, Adrian Gh

    2012-12-01

    A dual instrument is assembled to investigate the usefulness of optical coherence tomography (OCT) imaging in an ear, nose and throat (ENT) department. Instrument 1 is dedicated to in vivo laryngeal investigation, based on an endoscope probe head assembled by compounding a miniature transversal flying spot scanning probe with a commercial fiber bundle endoscope. This dual probe head is used to implement a dual channel nasolaryngeal endoscopy-OCT system. The two probe heads are used to provide simultaneously OCT cross section images and en face fiber bundle endoscopic images. Instrument 2 is dedicated to either in vivo imaging of accessible surface skin and mucosal lesions of the scalp, face, neck and oral cavity or ex vivo imaging of the same excised tissues, based on a single OCT channel. This uses a better interface optics in a hand held probe. The two instruments share sequentially, the swept source at 1300 nm, the photo-detector unit and the imaging PC. An aiming red laser is permanently connected to the two instruments. This projects visible light collinearly with the 1300 nm beam and allows pixel correspondence between the en face endoscopy image and the cross section OCT image in Instrument 1, as well as surface guidance in Instrument 2 for the operator. The dual channel instrument was initially tested on phantom models and then on patients with suspect laryngeal lesions in a busy ENT practice. This feasibility study demonstrates the OCT potential of the dual imaging instrument as a useful tool in the testing and translation of OCT technology from the lab to the clinic. Instrument 1 is under investigation as a possible endoscopic screening tool for early laryngeal cancer. Larger size and better quality cross-section OCT images produced by Instrument 2 provide a reference base for comparison and continuing research on imaging freshly excised tissue, as well as in vivo interrogation of more superficial skin and mucosal lesions in the head and neck patient.

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

    PubMed

    Scott, Yanille M; Park, Seo Young; Dezzutti, Charlene S

    2016-02-01

    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.

  10. Ex vivo normothermic machine perfusion and viability testing of discarded human donor livers.

    PubMed

    op den Dries, S; Karimian, N; Sutton, M E; Westerkamp, A C; Nijsten, M W N; Gouw, A S H; Wiersema-Buist, J; Lisman, T; Leuvenink, H G D; Porte, R J

    2013-05-01

    In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion may reduce preservation injury, improve graft viability and potentially allows ex vivo assessment of graft viability before transplantation. We have studied the feasibility of normothermic machine perfusion in four discarded human donor livers. Normothermic machine perfusion consisted of pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion for 6 h. Two hollow fiber membrane oxygenators provided oxygenation of the perfusion fluid. Biochemical markers in the perfusion fluid reflected minimal hepatic injury and improving function. Lactate levels decreased to normal values, reflecting active metabolism by the liver (mean lactate 10.0 ± 2.3 mmol/L at 30 min to 2.3 ± 1.2 mmol/L at 6 h). Bile production was observed throughout the 6 h perfusion period (mean rate 8.16 ± 0.65 g/h after the first hour). Histological examination before and after 6 h of perfusion showed well-preserved liver morphology without signs of additional hepatocellular ischemia, biliary injury or sinusoidal damage. In conclusion, this study shows that normothermic machine perfusion of human donor livers is technically feasible. It allows assessment of graft viability before transplantation, which opens new avenues for organ selection, therapeutic interventions and preconditioning.

  11. Assessment of Ex Vivo Transport Function in Isolated Rodent Brain Capillaries.

    PubMed

    Chan, Gary N Y; Cannon, Ronald E

    2017-03-17

    The blood-brain barrier plays an important role in neuroprotection; however, it can be a major obstacle for drug delivery to the brain. This barrier primarily resides in the brain capillaries and functions as an interface between the brain and peripheral blood circulation. Several anatomical and biochemical elements of the blood-brain barrier are essential to regulate the permeability of nutrients, ions, hormones, toxic metabolites, and xenobiotics into and out of the brain. In particular, high expression of ATP-driven efflux transporters at the blood-brain barrier is a major obstacle in the delivery of CNS pharmacotherapeutics to the brain. The complete understanding of these elements can offer insights on how to modulate barrier functions for neuroprotection against CNS drug toxicity and to enhance drug delivery to the brain. In the literature, preclinical models of the blood-brain barrier are widely utilized to predict drug pharmacokinetics and pharmacodynamics properties in the brain. In addition, these models are essential tools to investigate cellular mechanisms and novel interventions that alter barrier function and permeability. This unit presents procedures to isolate fresh and viable rodent brain capillaries for the assessment of ex vivo transport activity at the blood-brain barrier. © 2017 by John Wiley & Sons, Inc.

  12. 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

  13. Helicobacter pylori-induced gastric pathology: insights from in vivo and ex vivo models

    PubMed Central

    Williams, Jonathan M.

    2017-01-01

    ABSTRACT Gastric colonization with Helicobacter pylori induces diverse human pathological conditions, including superficial gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma and its precursors. The treatment of these conditions often relies on the eradication of H. pylori, an intervention that is increasingly difficult to achieve and that does not prevent disease progression in some contexts. There is, therefore, a pressing need to develop new experimental models of H. pylori-associated gastric pathology to support novel drug development in this field. Here, we review the current status of in vivo and ex vivo models of gastric H. pylori colonization, and of Helicobacter-induced gastric pathology, focusing on models of gastric pathology induced by H. pylori, Helicobacter felis and Helicobacter suis in rodents and large animals. We also discuss the more recent development of gastric organoid cultures from murine and human gastric tissue, as well as from human pluripotent stem cells, and the outcomes of H. pylori infection in these systems. PMID:28151409

  14. Ex Vivo Lung Perfusion in the Rat: Detailed Procedure and Videos

    PubMed Central

    Lonati, Caterina; Brambilla, Daniela; Rapido, Francesca; Valenza, Franco; Gatti, Stefano

    2016-01-01

    Ex vivo lung perfusion (EVLP) is a promising procedure for evaluation, reconditioning, and treatment of marginal lungs before transplantation. Small animal models can contribute to improve clinical development of this technique and represent a substantial platform for bio-molecular investigations. However, to accomplish this purpose, EVLP models must sustain a prolonged reperfusion without pharmacological interventions. Currently available protocols only partly satisfy this need. The aim of the present research was accomplishment and optimization of a reproducible model for a protracted rat EVLP in the absence of anti-inflammatory treatment. A 180 min, uninjured and untreated perfusion was achieved through a stepwise implementation of the protocol. Flow rate, temperature, and tidal volume were gradually increased during the initial reperfusion phase to reduce hemodynamic and oxidative stress. Low flow rate combined with open atrium and protective ventilation strategy were applied to prevent lung damage. The videos enclosed show management of the most critical technical steps. The stability and reproducibility of the present procedure were confirmed by lung function evaluation and edema assessment. The meticulous description of the protocol provided in this paper can enable other laboratories to reproduce it effortlessly, supporting research in the EVLP field. PMID:27936178

  15. Behavior of tip-steerable needles in ex vivo and in vivo tissue.

    PubMed

    Majewicz, Ann; Marra, Steven P; van Vledder, Mark G; Lin, MingDe; Choti, Michael A; Song, Danny Y; Okamura, Allison M

    2012-10-01

    Robotic needle steering is a promising technique to improve the effectiveness of needle-based clinical procedures, such as biopsies and ablation, by computer-controlled, curved insertions of needles within solid organs. In this paper, we explore the capabilities, challenges, and clinical relevance of asymmetric-tip needle steering through experiments in ex vivo and in vivo tissue. We evaluate the repeatability of needle insertion in inhomogeneous biological tissue and compare ex vivo and in vivo needle curvature and insertion forces. Steerable needles curved more in kidney than in liver and prostate, likely due to differences in tissue properties. Pre-bent needles produced higher insertion forces in liver and more curvature in vivo than ex vivo. When compared to straight stainless steel needles, steerable needles did not cause a measurable increase in tissue damage and did not exert more force during insertion. The minimum radius of curvature achieved by prebent needles was 5.23 cm in ex vivo tissue, and 10.4 cm in in vivo tissue. The curvatures achieved by bevel tip needles were negligible for in vivo tissue. The minimum radius of curvature for bevel tip needles in ex vivo tissue was 16.4 cm; however, about half of the bevel tip needles had negligible curvatures. We also demonstrate a potential clinical application of needle steering by targeting and ablating overlapping regions of cadaveric canine liver.

  16. Behavior of Tip-Steerable Needles in ex vivo and in vivo Tissue

    PubMed Central

    Majewicz, Ann; Marra, Steven P.; van Vledder, Mark G.; Lin, MingDe; Choti, Michael A.; Song, Danny Y.; Okamura, Allison M.

    2012-01-01

    Robotic needle steering is a promising technique to improve the effectiveness of needle-based clinical procedures, such as biopsies and ablation, by computer-controlled, curved insertions of needles within solid organs. In this paper, we explore the capabilities, challenges, and clinical relevance of asymmetric-tip needle steering though experiments in ex vivo and in vivo tissue. We evaluate the repeatability of needle insertion in inhomogeneous biological tissue and compare ex vivo and in vivo needle curvature and insertion forces. Steerable needles curved more in kidney than in liver and prostate, likely due to differences in tissue properties. Pre-bent needles produced higher insertion forces in liver and more curvature in vivo than ex vivo. When compared to straight stainless steel needles, steerable needles did not cause a measurable increase in tissue damage and did not exert more force during insertion. The minimum radius of curvature achieved by pre-bent needles was 5.23 cm in ex vivo tissue, and 10.4 cm in in vivo tissue. The curvatures achieved by bevel tip needles were negligible for in vivo tissue. The minimum radius of curvature for bevel tip needles in ex vivo tissue was 16.4 cm; however, about half of the bevel tip needles had negligible curvatures. We also demonstrate a potential clinical application of needle steering by targeting and ablating overlapping regions of cadaveric canine liver. PMID:22711767

  17. 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.

  18. The ex vivo pig eye as a replacement model for laser safety testing.

    PubMed

    Fyffe, James G; Neal, Thomas A; Butler, William P; Johnson, Thomas E

    2005-12-01

    The purpose of this study was to evaluate the viability of ex vivo pig eyes as a replacement model for in vivo testing in the establishment of laser eye safety standards. Previous studies of pulsed energy absorption at 3.8 microm were performed using rhesus monkey cornea at pulse durations two orders of magnitude shorter than the 8-micros pulses used in the current study. Ex vivo pig eyes were exposed to laser pulses of various energies and then evaluated to establish the statistical threshold for corneal damage. Tissue analysis (histologic evaluation) was used to determine the extent of damage to the cornea. These results can be used in the establishment of safety standards for laser use; our findings also suggest that ex vivo pig eyes are suitable models for this purpose.

  19. Biomonitoring and hormone-disrupting effect biomarkers of persistent organic pollutants in vitro and ex vivo.

    PubMed

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

    2014-07-01

    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.

  20. 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

  1. 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.

  2. 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

  3. Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells from Umbilical Cord Blood

    PubMed Central

    Sotnezova, E.V.; Andreeva, E.R.; Grigoriev, A.I.; Buravkova, L.B.

    2016-01-01

    Transplantation of umbilical cord blood cells is currently widely used in modern cell therapy. However, the limited number of hematopoietic stem and progenitor cells (HSPCs) and prolonged time of recovery after the transplantation are significant limitations in the use of cord blood. Ex vivo expansion with various cytokine combinations is one of the most common approaches for increasing the number of HSPCs from one cord blood unit. In addition, there are protocols that enable ex vivo amplification of cord blood cells based on native hematopoietic microenvironmental cues, including stromal components and the tissue-relevant oxygen level. The newest techniques for ex vivo expansion of HSPCs are based on data from the elucidation of the molecular mechanisms governing the hematopoietic niche function. Application of these methods has provided an improvement of several important clinical outcomes. Alternative methods of cord blood transplantation enhancement based on optimization of HPSC homing and engraftment in patient tissues have also been successful. The goal of the present review is to analyze recent methodological approaches to cord blood HSPC ex vivo amplification. PMID:27795840

  4. Functional and Molecular Characterization of Ex Vivo Cultured Epiretinal Membrane Cells from Human Proliferative Diabetic Retinopathy

    PubMed Central

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNFα activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFRβ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNFα activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo. PMID:24195074

  5. Functional and molecular characterization of ex vivo cultured epiretinal membrane cells from human proliferative diabetic retinopathy.

    PubMed

    Veréb, Zoltán; Lumi, Xhevat; Andjelic, Sofija; Globocnik-Petrovic, Mojca; Urbancic, Mojca; Hawlina, Marko; Facskó, Andrea; Petrovski, Goran

    2013-01-01

    Characterization of the cell surface marker phenotype of ex vivo cultured cells growing out of human fibrovascular epiretinal membranes (fvERMs) from proliferative diabetic retinopathy (PDR) can give insight into their function in immunity, angiogenesis, and retinal detachment. FvERMs from uneventful vitrectomies due to PDR were cultured adherently ex vivo. Surface marker analysis, release of immunity- and angiogenesis-pathway-related factors upon TNF α activation and measurement of the intracellular calcium dynamics upon mechano-stimulation using fluorescent dye Fura-2 were all performed. FvERMs formed proliferating cell monolayers when cultured ex vivo, which were negative for endothelial cell markers (CD31, VEGFR2), partially positive for hematopoietic- (CD34, CD47) and mesenchymal stem cell markers (CD73, CD90/Thy-1, and PDGFR β ), and negative for CD105. CD146/MCAM and CD166/ALCAM, previously unreported in cells from fvERMs, were also expressed. Secretion of 11 angiogenesis-related factors (DPPIV/CD26, EG-VEGF/PK1, ET-1, IGFBP-2 and 3, IL-8/CXCL8, MCP-1/CCL2, MMP-9, PTX3/TSG-14, Serpin E1/PAI-1, Serpin F1/PEDF, TIMP-1, and TSP-1) were detected upon TNF α activation of fvERM cells. Mechano-stimulation of these cells induced intracellular calcium propagation representing functional viability and role of these cells in tractional retinal detachment, thus serving as a model for studying tractional forces present in fvERMs in PDR ex vivo.

  6. A simple method for ex vivo evaluation of biomaterial interaction with blood platelets.

    PubMed

    Mantovani, F; Marconi, W; Caprino, L; Goglia, G; Togna, G

    1980-09-01

    The process of thrombus formation, as a consequence of the interaction of artificial surfaces with blood, is related to the activation of blood platelets. A simple ex vivo method, which is suitable for the evaluation of the platelet-surface interaction is described. This method has been used to compare the haemocompatibility of several artificial materials, including nylon-6, Silastic and pyrolytic carbon.

  7. Antimicrobial efficacy of biocides tested on skin using an ex-vivo test.

    PubMed

    Maillard, J Y; Messager, S; Veillon, R

    1998-12-01

    An ex-vivo test was used to evaluate the activity of antimicrobials against three microorganisms, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The ex-vivo test is a carrier test using freshly excised animal skin samples maintained in viable conditions for a short period of time. Skin samples came from a veterinary practice and were excised from either dogs or cats. The antimicrobial activity of povidone iodine, chlorhexidine diacetate, cetrimide and benzalkonium chloride was also evaluated with suspension and glass-carrier tests. Generally, the activity of the antimicrobials tested was reduced when applied to the skin surface. Apart from povidone iodine (2%) against S. aureus, the biocides investigated failed to achieve a 5 log10 reduction in bacterial titre when tested with the ex-vivo method. There was no significant difference in reduction of bacterial titres after treatment with antimicrobials between the glass-carrier and the suspension tests. Furthermore, the drying process of bacterial inoculum was less detrimental on skin than on glass surfaces. This study confirmed that the activity of a biocide tested in suspension or on an inanimate surface did not reflect its activity when tested on skin. Further development of the ex-vivo test may be useful, especially for testing the antimicrobial activity of formulations with antiseptic properties.

  8. In vitro and ex vivo studies of antioxidant activity of carrageenans, sulfated polysaccharides from red algae.

    PubMed

    Sokolova, E V; Barabanova, A O; Homenko, V A; Solov'eva, T F; Bogdanovich, R N; Yermak, I M

    2011-02-01

    Antioxidant properties of structurally different sulfated polysaccharides (carrageenans) were studied in vitro and ex vivo. Ferric reducing antioxidant activity of carrageenans and their inhibitory effects on hydroxyl radicals and superoxide anion radicals were demonstrated in vitro. Activity of carrageenans depends on the polysaccharide structure. Carrageenans stimulate catalytic activity of SOD from donor erythrocyte.

  9. 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.

  10. 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...

  11. 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...

  12. 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...

  13. 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,...

  14. Simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system.

    PubMed

    Vinberg, Frans; Kefalov, Vladimir

    2015-05-06

    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.

  15. 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

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and 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...

  16. 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

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Tissue culture media for human ex vivo tissue and 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...

  17. 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

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Tissue culture media for human ex vivo tissue and 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...

  18. 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

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Tissue culture media for human ex vivo tissue and 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...

  19. 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

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Tissue culture media for human ex vivo tissue and 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...

  20. Soluble complement receptor 1 inhibits both complement and granulocyte activation during ex vivo hemodialysis.

    PubMed

    Himmelfarb, J; McMonagle, E; Holbrook, D; Toth, C

    1995-10-01

    Hemodialysis with cellulosic membranes results in both complement and granulocyte activation. We investigated the effects of soluble complement receptor 1 (sCR1), a potent complement inhibitor, on both complement and granulocyte activation in an ex vivo model of dialysis. Measurements were made of complement activation (radioimmunoassay for C3a desArg) as well as granulocyte activation (flow cytometric measurements of reactive oxygen species production, granulocyte CD11b/CD18 (MAC-1) expression and CD62L (L-selectin) expression). sCR1 completely abolished the generation of plasma C3a desArg during ex vivo hemodialysis. Without sCR1, C3a desArg levels rose from 968 +/- 373 ng/ml to 4961 +/- 40 ng/ml by the end of the ex vivo procedure (p < 0.001). sCR1 also completely inhibited MAC-1 upregulation and L-selectin shedding from granulocytes during ex vivo hemodialysis. With sCR1 there was still a statistically significant increase in granulocyte reactive oxygen species production (from 2.42 +/- 0.1 fluorescence channels to 6.47 +/- 0.7 fluorescence channels, p < 0.01) but a 50% inhibition when compared with experiments without sCR1 (3.15 +/- 0.5 to 11.2 +/- 1.9, p < 0.01). We conclude that sCR1 completely abolishes complement activation and changes in granulocyte cell adhesion molecules during ex vivo hemodialysis with cellulosic membranes. sCR1 partially inhibits granulocyte reactive oxygen species formation.

  1. 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.

  2. 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

  3. Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.

    PubMed

    Sutton, Michael E; op den Dries, Sanna; Karimian, Negin; Weeder, Pepijn D; de Boer, Marieke T; Wiersema-Buist, Janneke; Gouw, Annette S H; Leuvenink, Henri G D; Lisman, Ton; Porte, Robert J

    2014-01-01

    Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1) steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group), and (2) a cumulative bile production <20 g in 6 h (low bile output group). Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

  4. Regional T1 relaxation time constants in Ex vivo human brain: Longitudinal effects of formalin exposure

    PubMed Central

    Raman, Mekala R.; Shu, Yunhong; Lesnick, Timothy G.; Jack, Clifford R.

    2016-01-01

    Purpose Relaxation time constants are useful as markers of tissue properties. Imaging ex vivo tissue is done for research purposes; however, T1 relaxation time constants are altered by tissue fixation in a time‐dependent manner. This study investigates regional changes in T1 relaxation time constants in ex vivo brain tissue over 6 months of fixation. Methods Five ex vivo human brain hemispheres in 10% formalin were scanned over 6 months. Mean T1 relaxation time constants were measured in regions of interest (ROIs) representing gray matter (GM) and white matter (WM) regions and analyzed as a function of fixation time. Results Cortical GM ROIs had longer T1 relaxation time constants than WM ROIs; the thalamus had T1 relaxation time constants similar to those of WM ROIs. T1 relaxation time constants showed rapid shortening within the first 6 weeks after fixation followed by a slower rate of decline. Conclusion Both GM and WM T1 relaxation time constants of fixed brain tissue show rapid decline within the first 6 weeks after autopsy and slow by 6 months. This information is useful for optimizing MR imaging acquisition parameters according to fixation time for ex vivo brain imaging studies. Magn Reson Med 77:774–778, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMID:26888162

  5. Multiple effects of IL-21 on human NK cells in ex vivo expansion.

    PubMed

    Li, Qi; Ye, Lin-Jie; Ren, Hai-Long; Huyan, Ting; Li, Jing; Shi, Jun-Ling; Huang, Qing-Sheng

    2015-07-01

    Natural killer (NK) cells (CD56(+)CD3(-)) are large, granular immunocytes that play a very pivotal role in the anti-inflammatory response and tumor surveillance. As an ideal cytotoxic lymphocyte (CTL), NK cells have attracted much attention in clinical trials. However, an insufficient number and their limited life span are bottlenecks that limit the application of NK cells in adoptive immunotherapy. Interleukins such as IL-2, IL-15 and IL-18 are recognized as factors that stimulate NK cells and have been used in NK cells ex vivo expansion. Similar to IL-2 and IL-15, IL-21 is a common γ-chain cytokine that is important in NK cell activation, maturation and proliferation. The present study aims to assess the effects of membrane-bound and soluble IL-21 on primary human NK cells during ex vivo expansion. IL-21 was found to have multiple effects on NK cells, increasing their cytotoxicity in a concentration-dependent manner by up-regulating IFN-γ and Granzyme-B expression. Nevertheless, at a high concentration (50 ng/mL), IL-21 curtailed the life span of NK cells by significantly inducing apoptosis. Moreover, when treated with IL-21, the number of NKT (CD56(+)CD3(+)) cells increased among peripheral blood mononuclear cells (PBMCs) during ex vivo expansion in a concentration-dependent manner. IL-21 also promoted expanded cells to enter into S phase of the cell cycle during the first to second weeks of culture. All these results suggest that IL-21 has multiple effects on NK cell development and functions. More attention should be given to the dosage and multiple effects of IL-21 when it was applied to NK cells in ex vivo expansion.

  6. Antimicrobial Blue Light Therapy for Infectious Keratitis: Ex Vivo and In Vivo Studies

    PubMed Central

    Zhu, Hong; Kochevar, Irene E.; Behlau, Irmgard; Zhao, Jie; Wang, Fenghua; Wang, Yucheng; Sun, Xiaodong; Hamblin, Michael R.; Dai, Tianhong

    2017-01-01

    Purpose To investigate the effectiveness of antimicrobial blue light (aBL) as an alternative or adjunctive therapeutic for infectious keratitis. Methods We developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis. A bioluminescent strain of Pseudomonas aeruginosa was used as the causative pathogen, allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging. Quantitation of bacterial luminescence was correlated to colony-forming units (CFU). Using the ex vivo and in vivo models, the effectiveness of aBL (415 nm) for the treatment of keratitis was evaluated as a function of radiant exposure when aBL was delivered at 6 or 24 hours after bacterial inoculation. The aBL exposures calculated to reach the retina were compared to the American National Standards Institute standards to estimate aBL retinal safety. Results Pseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation. Bacterial luminescence in the infected corneas correlated linearly to CFU (R2 = 0.921). Bacterial burden in the infected corneas was rapidly and significantly reduced (>2-log10) both ex vivo and in vivo after a single exposure of aBL. Recurrence of infection was observed in the aBL-treated mice at 24 hours after aBL exposure. The aBL toxicity to the retina is largely dependent on the aBL transmission of the cornea. Conclusions Antimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis. Further studies of corneal and retinal safety using large animal models, in which the ocular anatomies are similar to that of humans, are warranted. PMID:28129422

  7. Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

    PubMed Central

    Kerkhofs, P; Adam, E; Droogmans, L; Portetelle, D; Mammerickx, M; Burny, A; Kettmann, R; Willems, L

    1996-01-01

    Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression. PMID:8642639

  8. 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.

  9. 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

  10. 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.

  11. Parsley extract inhibits in vitro and ex vivo platelet aggregation and prolongs bleeding time in rats.

    PubMed

    Gadi, Dounia; Bnouham, Mohamed; Aziz, Mohammed; Ziyyat, Abderrahim; Legssyer, Abdelkhaleq; Legrand, Chantal; Lafeve, Françoise Fauvel; Mekhfi, Hassane

    2009-08-17

    Many cardiovascular diseases are associated with an increase in blood platelet activity. In Morocco, parsley (Petroselinum crispum, Apiaceae) is one of the medicinal herbs used to treat cardiovascular diseases such as arterial hypertension. In this study, crude aqueous extract (CAE) of parsley was evaluated for its anti-platelet activity in experimental animals on platelet aggregation in vitro and ex vivo; and on bleeding time in vivo. The in vitro aggregation was monitored after pre-incubation of platelets with CAE. The bleeding time and ex vivo aggregation were performed after oral treatment. CAE inhibited dose dependently platelet aggregation in vitro induced by thrombin, ADP, collagen and epinephrine. The oral administration of CAE (3g/kg) inhibited significantly (p<0.001) platelet aggregation ex vivo and prolonged bleeding time (p<0.001) without changes in the platelet amount. The prolongation of bleeding time by CAE may be attributed to the observed inhibition of platelet aggregation. These effects could be related in part to the polyphenolic compounds present in the extract. These results support the hypothesis that the dietary intake of parsley may be benefit in the normalization of platelet hyperactivation, in the nutritional prevention of cardiovascular diseases and are potentially interesting in the development of new prevention strategies.

  12. In vivo visualization and ex vivo quantification of experimental myocardial infarction by indocyanine green fluorescence imaging

    PubMed Central

    Sonin, Dmitry; Papayan, Garry; Pochkaeva, Evgeniia; Chefu, Svetlana; Minasian, Sarkis; Kurapeev, Dmitry; Vaage, Jarle; Petrishchev, Nickolay; Galagudza, Michael

    2016-01-01

    The fluorophore indocyanine green accumulates in areas of ischemia-reperfusion injury due to an increase in vascular permeability and extravasation of the dye. The aim of the study was to validate an indocyanine green-based technique of in vivo visualization of myocardial infarction. A further aim was to quantify infarct size ex vivo and compare this technique with the standard triphenyltetrazolium chloride staining. Wistar rats were subjected to regional myocardial ischemia (30 minutes) followed by reperfusion (n = 7). Indocyanine green (0.25 mg/mL in 1 mL of normal saline) was infused intravenously for 10 minutes starting from the 25th minute of ischemia. Video registration in the near-infrared fluorescence was performed. Epicardial fluorescence of indocyanine green corresponded to the injured area after 30 minutes of reperfusion. Infarct size was similar when determined ex vivo using traditional triphenyltetrazolium chloride assay and indocyanine green fluorescent labeling. Intravital visualization of irreversible injury can be done directly by fluorescence on the surface of the heart. This technique may also be an alternative for ex vivo measurements of infarct size. PMID:28101408

  13. Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

    PubMed

    Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

    2010-10-01

    Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration.

  14. 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.

  15. 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.

  16. 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

  17. Development of an ex vivo human-porcine respiratory model for preclinical studies

    PubMed Central

    Perinel, Sophie; Pourchez, Jérémie; Leclerc, Lara; Avet, John; Durand, Marc; Prévôt, Nathalie; Cottier, Michèle; Vergnon, Jean M.

    2017-01-01

    Anatomical models to study aerosol delivery impose huge limitations and extrapolation to humans remains controversial. This study aimed to develop and validate an ex vivo human-like respiratory tract model easy to use and relevant to compare to in vivo human data. A human plastinated head is connected to an ex vivo porcine pulmonary tract ventilated artificially by passive expansion. A physiological study measures “pleural” depressions, tidal volumes, and minute ventilation for the respiratory rates chosen (10, 15, and 20 per minute) with three inspiratory/expiratory ratios (1/1, 1/2, and 1/3). Scintigraphy with 81mKrypton assesses the homogeneity of the ventilation. Forty different experiments were set for validation, with 36 (90%) ventilating successfully. At a respiratory rate of 15/minute with inspiratory/expiratory ratio of 1/2, the tidal volume average was 824 mL (standard deviation, 207 mL). The scintigraphy performed on 16 ex vivo models (44.4%), showed homogenous ventilation with great similarity to human physiological studies. Ratio of the peripheral to central count rates were equally correlated with human data published in the literature. This new model, combining research feasibility and human physiology likeness, provides a realistic approach to human inhalation and therefore can be an interesting tool in aerosol regional deposition studies. PMID:28233793

  18. 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

  19. Robust methods to create ex vivo minimum deformation atlases for brain mapping.

    PubMed

    Janke, Andrew L; Ullmann, Jeremy F P

    2015-02-01

    Highly detailed ex vivo 3D atlases of average structure are of critical importance to neuroscience and its current push to understanding the global microstructure of the brain. Multiple single slice histology sections can no longer provide sufficient detail of inter-slice microstructure and lack out of plane resolution. Two ex vivo methods have emerged that can create such detailed models. High-field micro MRI with the addition of contrast media has allowed intact whole brain microstructure imaging with an isotropic resolution of 15 μm in mouse. Blockface imaging has similarly evolved to a point where it is now possible to image an entire brain in a rigorous fashion with an out of plane resolution of 10 μm. Despite the destruction of the tissue as part of this process it allows a reconstructed model that is free from cutting artifacts. Both of these methods have been utilised to create minimum deformation atlases that are representative of the respective populations. The MDA atlases allow us unprecedented insight into the commonality and differences in microstructure in cortical structures in specific taxa. In this paper we provide an overview of how to create such MDA models from ex vivo data.

  20. X-ray cell tracking: from ex-vivo to in-vivo experiments

    NASA Astrophysics Data System (ADS)

    Astolfo, A.; Schültke, E.; Menk, R.-H.; Hall, C.; Juurlink, B.; Arfelli, F.

    2013-06-01

    The capacity to track cells (cell tracking) using x-rays on ex-vivo specimens of both malignant and non-malignant cell lines on small animals has been demonstrated recently. Gold nanoparticles have been used as a cellular contrast agent to render cells visible in x-ray microCT acquisitions. The limits of the technique proposed are basically driven by the imaging system used. Single cell resolution can be achieved using synchrotron radiation in-vitro or ex-vivo samples. Micro-focus x-ray tubes can be used to obtain high resolution cell tracking but with some limitations. However, the translation from ex-vivo to in-vivo experiments is not straightforward. The dose restrictions required for in-vivo longitudinal experiments set severe limitations on the technique. Here we present a detailed investigation showing a significant reduction of x-ray dose for the tracking of brain tumour cells. Monte Carlo simulations have been performed considering different spatial resolutions, photon fluence, number of projections, lesion dimension and cell contrast dilution. The findings are compared with real samples imaged using the same parameters. A pioneering in-vivo experiment conducted at the SYRMEP beamline (Elettra, Basovizza, Italy) is presented here as proof of principle of in-vivo longitudinal x-ray cell tracking experiments on small animals at low x-ray doses.

  1. Development of an ex vivo human-porcine respiratory model for preclinical studies.

    PubMed

    Perinel, Sophie; Pourchez, Jérémie; Leclerc, Lara; Avet, John; Durand, Marc; Prévôt, Nathalie; Cottier, Michèle; Vergnon, Jean M

    2017-02-24

    Anatomical models to study aerosol delivery impose huge limitations and extrapolation to humans remains controversial. This study aimed to develop and validate an ex vivo human-like respiratory tract model easy to use and relevant to compare to in vivo human data. A human plastinated head is connected to an ex vivo porcine pulmonary tract ventilated artificially by passive expansion. A physiological study measures "pleural" depressions, tidal volumes, and minute ventilation for the respiratory rates chosen (10, 15, and 20 per minute) with three inspiratory/expiratory ratios (1/1, 1/2, and 1/3). Scintigraphy with (81m)Krypton assesses the homogeneity of the ventilation. Forty different experiments were set for validation, with 36 (90%) ventilating successfully. At a respiratory rate of 15/minute with inspiratory/expiratory ratio of 1/2, the tidal volume average was 824 mL (standard deviation, 207 mL). The scintigraphy performed on 16 ex vivo models (44.4%), showed homogenous ventilation with great similarity to human physiological studies. Ratio of the peripheral to central count rates were equally correlated with human data published in the literature. This new model, combining research feasibility and human physiology likeness, provides a realistic approach to human inhalation and therefore can be an interesting tool in aerosol regional deposition studies.

  2. 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

  3. Ex Vivo Virotherapy With Myxoma Virus Does Not Impair Hematopoietic Stem and Progenitor Cells

    PubMed Central

    Villa, Nancy Y.; Bais, Swarna; Meacham, Amy M.; Wise, Elizabeth; Rahman, Masmudur M.; Moreb, Jan S; Rosenau, Emma H.; Wingard, John R.; McFadden, Grant; Cogle, Christopher R.

    2016-01-01

    Background Relapsing disease is a major challenge after hematopoietic cell transplant for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells, and define the optimal treatment conditions for ex vivo virotherapy. Methods Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematological malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated by flow cytometry for MYXV infection, LTC-IC assay, and CFC assay. Results MYXV initiated infection in up to 45% of antigen presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in > 95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens but all cases, less than 10% of CD34+ cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared to mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change percentage of infected cells, LTC-IC colony formation or CFC colony formation. Conclusions Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment. PMID:26857235

  4. [Use of stem cells cultured ex vivo for ocular surface reconstruction].

    PubMed

    Ricardo, José Reinaldo da Silva; Gomes, José Alvaro Pereira

    2010-01-01

    Lesions on the ocular surface can destroy the stem cells from the limbus and cause limbal stem cell deficiency. The limbal stem cell deficiency is marked by conjunctivalization, which can be defined as the invasion of conjunctival epithelium over the cornea. This process is accompanied by varying degrees of corneal changes such as neovascularization, inflammation, recurrent erosions, persistent epithelial defects, destruction of basement membrane of epithelium and stromal healing. Often, these changes are associated with poor visual acuity, photophobia and ocular discomfort. The best treatment for this disease is not known and varies in unilateral or bilateral cases. Among the treatments available, transplantation of limbal autograft or allograft is one of the most used. To improve the outcome of allotransplantation, some researchers use the transplantation of corneal epithelium cultured in the laboratory by ex vivo expansion of limbal stem cells, but due to limited availability of autologous tissue from the limbus and the risk of complications associated with immunosuppression in allogeneic tissue transplantation, researches of others options of stem cell cultured ex vivo have been described in experimental and clinical stage. This review describes the new types of stem cells cultured ex vivo, their current results and future potential.

  5. Synaptic signal streams generated by ex vivo neuronal networks contain non-random, complex patterns.

    PubMed

    Lee, Sangmook; Zemianek, Jill M; Shultz, Abraham; Vo, Anh; Maron, Ben Y; Therrien, Mikaela; Courtright, Christina; Guaraldi, Mary; Yanco, Holly A; Shea, Thomas B

    2014-11-01

    Cultured embryonic neurons develop functional networks that transmit synaptic signals over multiple sequentially connected neurons as revealed by multi-electrode arrays (MEAs) embedded within the culture dish. Signal streams of ex vivo networks contain spikes and bursts of varying amplitude and duration. Despite the random interactions inherent in dissociated cultures, neurons are capable of establishing functional ex vivo networks that transmit signals among synaptically connected neurons, undergo developmental maturation, and respond to exogenous stimulation by alterations in signal patterns. These characteristics indicate that a considerable degree of organization is an inherent property of neurons. We demonstrate herein that (1) certain signal types occur more frequently than others, (2) the predominant signal types change during and following maturation, (3) signal predominance is dependent upon inhibitory activity, and (4) certain signals preferentially follow others in a non-reciprocal manner. These findings indicate that the elaboration of complex signal streams comprised of a non-random distribution of signal patterns is an emergent property of ex vivo neuronal networks.

  6. Piscine orthoreovirus (PRV) replicates in Atlantic salmon (Salmo salar L.) erythrocytes ex vivo.

    PubMed

    Wessel, Øystein; Olsen, Christel Moræus; Rimstad, Espen; Dahle, Maria Krudtaa

    2015-03-06

    Piscine orthoreovirus (PRV) is a reovirus that has predominantly been detected in Atlantic salmon (Salmo salar L.). PRV is associated with heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon, and recently erythrocytes were identified as major target cells. The study of PRV replication and pathogenesis of the infection has been impeded by the inability to propagate PRV in vitro. In this study we developed an ex vivo cultivation system for PRV in Atlantic salmon erythrocytes. PRV was successfully passaged to naïve erythrocytes using lysates of blood cells from infected salmon. During cultivation a significant increase in viral load was observed by RT-qPCR and flow cytometry, which coincided with the formation of cytoplasmic inclusions. The inclusions resembled viral factories and contained both PRV protein and dsRNA. In addition, the erythrocytes generated an antiviral immune gene activation after PRV infection, with significant up-regulation of IFN-α, RIG-I, Mx and PKR transcripts. Supernatants from the first passage successfully transmitted virus to naïve erythrocytes. This study demonstrates that PRV replicates in Atlantic salmon erythrocytes ex vivo. The ex vivo infection model closely reflects the situation in vivo and can be used to study the infection and replication mechanisms of PRV, as well as the antiviral immune responses of salmonid erythrocytes.

  7. An observational study of Donor Ex Vivo Lung Perfusion in UK lung transplantation: DEVELOP-UK.

    PubMed Central

    Fisher, Andrew; Andreasson, Anders; Chrysos, Alexandros; Lally, Joanne; Mamasoula, Chrysovalanto; Exley, Catherine; Wilkinson, Jennifer; Qian, Jessica; Watson, Gillian; Lewington, Oli; Chadwick, Thomas; McColl, Elaine; Pearce, Mark; Mann, Kay; McMeekin, Nicola; Vale, Luke; Tsui, Steven; Yonan, Nizar; Simon, Andre; Marczin, Nandor; Mascaro, Jorge; Dark, John

    2016-01-01

    BACKGROUND Many patients awaiting lung transplantation die before a donor organ becomes available. Ex vivo lung perfusion (EVLP) allows initially unusable donor lungs to be assessed and reconditioned for clinical use. OBJECTIVE The objective of the Donor Ex Vivo Lung Perfusion in UK lung transplantation study was to evaluate the clinical effectiveness and cost-effectiveness of EVLP in increasing UK lung transplant activity. DESIGN A multicentre, unblinded, non-randomised, non-inferiority observational study to compare transplant outcomes between EVLP-assessed and standard donor lungs. SETTING Multicentre study involving all five UK officially designated NHS adult lung transplant centres. PARTICIPANTS Patients aged ≥ 18 years with advanced lung disease accepted onto the lung transplant waiting list. INTERVENTION The study intervention was EVLP assessment of donor lungs before determining suitability for transplantation. MAIN OUTCOME MEASURES The primary outcome measure was survival during the first 12 months following lung transplantation. Secondary outcome measures were patient-centred outcomes that are influenced by the effectiveness of lung transplantation and that contribute to the health-care costs. RESULTS Lungs from 53 donors unsuitable for standard transplant were assessed with EVLP, of which 18 (34%) were subsequently transplanted. A total of 184 participants received standard donor lungs. Owing to the early closure of the study, a non-inferiority analysis was not conducted. The Kaplan-Meier estimate of survival at 12 months was 0.67 [95% confidence interval (CI) 0.40 to 0.83] for the EVLP arm and 0.80 (95% CI 0.74 to 0.85) for the standard arm. The hazard ratio for overall 12-month survival in the EVLP arm relative to the standard arm was 1.96 (95% CI 0.83 to 4.67). Patients in the EVLP arm required ventilation for a longer period and stayed longer in an intensive therapy unit (ITU) than patients in the standard arm, but duration of overall hospital

  8. 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

  9. Comparison of two in vivo and two ex vivo tests to assess the antibacterial activity of several antiseptics.

    PubMed

    Messager, S; Goddard, P A; Dettmar, P W; Maillard, J-Y

    2004-10-01

    An ex vivo test was adapted to mimic the in vivo conditions of testing antiseptic activity on human forearms and in the European Standard Hygienic Handwash Test (BSEN 1499). The study was to validate the ex vivo protocols using 4.8% (w/v) para-chloro-meta-xylenol (PCMX, neat Dettol), 0.5% (w/v) triclosan in 70% (v/v) isopropanol, and 2% (v/v) povidone-iodine against a high bacterial inoculum (>10(8) cfu/mL) of Escherichia coli NCTC 10538. Two ex vivo tests using human skin samples, including one introducing a mechanical rubbing effect, were compared with two corresponding in vivo tests (the forearm test and the BSEN handwashing test). All antiseptics assessed in vivo (forearm and handwash tests) produced reductions in bacterial counts that were significantly greater than those for the non-medicated soft soap control. When assessed ex vivo without rubbing, only PCMX and povidone-iodine achieved reductions significantly greater than soft soap. When assessed ex vivo with mechanical rubbing, only PCMX and triclosan achieved reductions significantly greater than soft soap. Overall, the antiseptics at the concentrations tested were more active when tested in vivo than ex vivo. The addition of a mechanical effect, either in vivo by the volunteers washing their hands or ex vivo by a drill rubbing two skin samples against each other, produced a significantly greater reduction in bacterial concentrations. The ex vivo tests were easily adapted to mimic in vivo protocols. The value of such tests, particularly the one that includes a rubbing effect, may be significant as they avoid the need for human volunteers.

  10. 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

  11. Assessment of Anti-Scarring Therapies in Ex Vivo Organ Cultured Rabbit Corneas

    PubMed Central

    Sriram, Sriniwas; Gibson, Daniel; Robinson, Paulette; Pi, Liya; Tuli, Sonal; Lewin, Alfred S.; Schultz, Gregory

    2014-01-01

    The effects of a triple combination of siRNAs targeting key scarring genes was assessed using an ex vivo organ culture model of excimer ablated rabbit corneas. The central 6 mm diameter region of fresh rabbit globes was ablated to a depth of 155 microns with an excimer laser. Corneas were excised, cultured at the air-liquid interface in defined culture medium supplemented with transforming growth factor beta 1 (TGFB1), and treated with either 1% prednisolone acetate or with 22.5 μM cationic nanoparticles complexed with a triple combination of siRNAs (NP-siRNA) targeting TGFB1, TGFB Receptor (TGFBR2) and connective tissue growth factor (CTGF). Scar formation was measured using image analysis of digital images and levels of smooth muscle actin (SMA) were assessed in ablated region of corneas using qRT-PCR and immunostaining. Ex vivo cultured corneas developed intense haze-like scar in the wounded areas and levels of mRNAs for pro-fibrotic genes were significantly elevated 3 to 8 fold in wounded tissue compared to unablated corneas. Treatment with NP-siRNA or steroid significantly reduced quantitative haze levels by 55% and 68%, respectively, and reduced SMA mRNA and immunohistostaining. This ex vivo corneal culture system reproduced key molecular patterns of corneal scarring and haze formation generated in rabbits. Treatment with NP-siRNAs targeting key scarring genes or an anti-inflammatory steroid reduced corneal haze and SMA mRNA and protein. PMID:24971495

  12. Rapid ex vivo imaging of PAIII prostate to bone tumor with SWIFT-MRI

    PubMed Central

    Luhach, Ihor; Idiyatullin, Djaudat; Lynch, Conor C.; Corum, Curt; Martinez, Gary V.; Garwood, Michael; Gillies, Robert J.

    2013-01-01

    Introduction The limiting factor for MRI of skeletal/mineralized tissue is fast transverse relaxation. A recent advancement in MRI technology, SWIFT (Sweep Imaging with Fourier Transform), is emerging as a new approach to overcome this difficulty. Among other techniques like UTE, ZTE and WASPI, the application of SWIFT technology has the strong potential to impact preclinical and clinical imaging, particularly in the context of primary or metastatic bone cancers since it has the added advantage of imaging water in mineralized tissues of bone allowing MRI images to be obtained of tissues previously visible only with modalities such as CT. The goal of the current study is to examine the feasibility of SWIFT for the assessment of the prostate cancer induced changes in bone formation (osteogenesis) and destruction (osteolysis) in ex vivo specimens. Methods A luciferase expressing prostate cancer cell line (PAIII) or saline control was inoculated directly into the tibia of 6-week old immunocompromised male mice. Tumor growth was assessed weekly for three weeks prior to euthanasia and dissection of the tumor bearing and sham tibias. The ex vivo mouse tibia specimens were imaged with a 9.4T and 7T MRI systems. SWIFT images are compared with traditional gradient-echo and spin-echo MRI images as well as CT and histological sections. Results SWIFT images with nominal resolution of 78 μm are obtained with the tumor and different bone structures identified. Prostate cancer induced changes in the bone microstructure are visible in SWIFT images, which is supported by spin-echo, high resolution CT and histological analysis. Conclusions SWIFT MRI is capable of high-quality high-resolution ex vivo imaging of bone tumor and surrounding bone and soft tissues. Furthermore, SWIFT MRI shows promise for in vivo bone tumor imaging, with the added benefits of non-exposure to ionizing radiation, quietness and speed. PMID:24155275

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

    PubMed

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

    2007-03-07

    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.

  14. The atheroma plaque secretome stimulates the mobilization of endothelial progenitor cells ex vivo.

    PubMed

    Vega, Francisco M; Gautier, Violette; Fernandez-Ponce, Cecilia M; Extremera, M J; Altelaar, A F M; Millan, Jaime; Tellez, Juan C; Hernandez-Campos, Jose A; Conejero, Rosario; Bolivar, Jorge; Pardal, Ricardo; Garcia-Cózar, Francisco J; Aguado, Enrique; Heck, Albert J R; Duran-Ruiz, Mª Carmen

    2017-04-01

    Endothelial progenitor cells (EPCs) constitute a promising alternative in cardiovascular regenerative medicine due to their assigned role in angiogenesis and vascular repair. In response to injury, EPCs promote vascular remodeling by replacement of damaged endothelial cells and/or by secreting angiogenic factors over the damaged tissue. Nevertheless, such mechanisms need to be further characterized. In the current approach we have evaluated the initial response of early EPCs (eEPCs) from healthy individuals after direct contact with the factors released by carotid arteries complicated with atherosclerotic plaques (AP), in order to understand the mechanisms underlying the neovascularization and remodeling properties assigned to these cells. Herein, we found that the AP secretome stimulated eEPCs proliferation and mobilization ex vivo, and such increase was accompanied by augmented permeability, cell contraction and also an increase of cell-cell adhesion in association with raised vinculin levels. Furthermore, a comparative mass spectrometry analysis of control versus stimulated eEPCs revealed a differential expression of proteins in the AP treated cells, mostly involved in cell migration, proliferation and vascular remodeling. Some of these protein changes were also detected in the eEPCs isolated from atherosclerotic patients compared to eEPCs from healthy donors. We have shown, for the first time, that the AP released factors activate eEPCs ex vivo by inducing their mobilization together with the expression of vasculogenic related markers. The present approach could be taken as a ex vivo model to study the initial activation of vascular cells in atherosclerosis and also to evaluate strategies looking to potentiate the mobilization of EPCs prior to clinical applications.

  15. 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.

  16. Ex-Vivo Magnetic Resonance Imaging in South African Manganese Mine Workers

    PubMed Central

    Criswell, Susan R; Nelson, Gill; Gonzalez-Cuyar, Luis F; Huang, John; Shimony, Joshua S; Checkoway, Harvey; Simpson, Christopher D; Dills, Russell; Seixas, Noah S.; Racette, Brad A

    2015-01-01

    Background Manganese (Mn) exposure is associated with increased T1-weighted Magnetic Resonance Imaging (MRI) signal in the basal ganglia. T1 signal intensity has been correlated with occupational Mn exposure but not with clinical symptomatology or neuropathology. Objectives This study investigated predictors of ex-vivo T1 MRI basal ganglia signal intensity in neuropathologic tissue obtained from deceased South African mine workers. Methods A 3.0T MRI was performed on ex-vivo brain tissue obtained from 19 Mn mine workers and 10 race- and sex-matched mine workers of other commodities. Basal ganglia regions of interest were identified for each subject with T1-weighted intensity indices generated for each region. In a pathology subset, regional T1 indices were compared to neuronal and glial cell density and tissue metal concentrations. Results Intensity indices were higher in Mn mine workers than non-Mn mine workers for the globus pallidus, caudate, anterior putamen, and posterior putamen with the highest values in subjects with the longest cumulative Mn exposure. Intensity indices were inversely correlated with the neuronal cell density in the caudate (p = 0.040) and putamen (p = 0.050). Tissue Mn concentrations were similar in Mn and non-Mn mine workers. Tissue iron (Fe) concentration trended lower across all regions in Mn mine workers. Conclusions Mn mine workers demonstrated elevated basal ganglia T1 indices when compared to non-Mn mine workers. Predictors of ex-vivo T1 MRI signal intensity in Mn mine workers include duration of Mn exposure and neuronal density. PMID:25912463

  17. 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.

  18. Genomic landscape of human, bat, and ex vivo DNA transposon integrations.

    PubMed

    Campos-Sánchez, Rebeca; Kapusta, Aurélie; Feschotte, Cédric; Chiaromonte, Francesca; Makova, Kateryna D

    2014-07-01

    The integration and fixation preferences of DNA transposons, one of the major classes of eukaryotic transposable elements, have never been evaluated comprehensively on a genome-wide scale. Here, we present a detailed study of the distribution of DNA transposons in the human and bat genomes. We studied three groups of DNA transposons that integrated at different evolutionary times: 1) ancient (>40 My) and currently inactive human elements, 2) younger (<40 My) bat elements, and 3) ex vivo integrations of piggyBat and Sleeping Beauty elements in HeLa cells. Although the distribution of ex vivo elements reflected integration preferences, the distribution of human and (to a lesser extent) bat elements was also affected by selection. We used regression techniques (linear, negative binomial, and logistic regression models with multiple predictors) applied to 20-kb and 1-Mb windows to investigate how the genomic landscape in the vicinity of DNA transposons contributes to their integration and fixation. Our models indicate that genomic landscape explains 16-79% of variability in DNA transposon genome-wide distribution. Importantly, we not only confirmed previously identified predictors (e.g., DNA conformation and recombination hotspots) but also identified several novel predictors (e.g., signatures of double-strand breaks and telomere hexamer). Ex vivo integrations showed a bias toward actively transcribed regions. Older DNA transposons were located in genomic regions scarce in most conserved elements-likely reflecting purifying selection. Our study highlights how DNA transposons are integral to the evolution of bat and human genomes, and has implications for the development of DNA transposon assays for gene therapy and mutagenesis applications.

  19. 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

  20. Dual-modality optical biopsy of glioblastomas multiforme with diffuse reflectance and fluorescence: ex vivo retrieval of optical properties

    NASA Astrophysics Data System (ADS)

    Du Le, Vinh Nguyen; Provias, John; Murty, Naresh; Patterson, Michael S.; Nie, Zhaojun; Hayward, Joseph E.; Farrell, Thomas J.; McMillan, William; Zhang, Wenbin; Fang, Qiyin

    2017-02-01

    Glioma itself accounts for 80% of all malignant primary brain tumors, and glioblastoma multiforme (GBM) accounts for 55% of such tumors. Diffuse reflectance and fluorescence spectroscopy have the potential to discriminate healthy tissues from abnormal tissues and therefore are promising noninvasive methods for improving the accuracy of brain tissue resection. Optical properties were retrieved using an experimentally evaluated inverse solution. On average, the scattering coefficient is 2.4 times higher in GBM than in low grade glioma (LGG), and the absorption coefficient is 48% higher. In addition, the ratio of fluorescence to diffuse reflectance at the emission peak of 460 nm is 2.6 times higher for LGG while reflectance at 650 nm is 2.7 times higher for GBM. The results reported also show that the combination of diffuse reflectance and fluorescence spectroscopy could achieve sensitivity of 100% and specificity of 90% in discriminating GBM from LGG during ex vivo measurements of 22 sites from seven glioma specimens. Therefore, the current technique might be a promising tool for aiding neurosurgeons in determining the extent of surgical resection of glioma and, thus, improving intraoperative tumor identification for guiding surgical intervention.

  1. Solitary kidney with renal artery aneurysm repaired by ex vivo reconstruction.

    PubMed

    Palcau, Laura; Gouicem, Djelloul; Joguet, Etienne; Cameliere, Lucie; Berger, Ludovic

    2014-01-01

    A 22-year-old pregnant female with pyelonephritis was found to have a 26-mm left renal artery aneurysm with unknown right kidney agenesis diagnosed by magnetic resonance imaging. Computed tomographic angiography with 3-dimensional reconstructions confirmed a saccular aneurysm localized at the bifurcation of the left posterior segmental artery. The patient ultimately underwent successful ex vivo left renal artery aneurysm repair with autotransplantation. Pathologic evaluation of the resected aneurysm confirmed the diagnosis of fibromuscular dysplasia. Fibromuscular dysplasia is the most common cause of renal artery stenosis and renovascular hypertension and can, in rare cases, be associated with the development of renal artery aneurysms.

  2. Ex vivo expansion of hematopoietic stem cell by fusion protein TAT-Zfx

    SciTech Connect

    Xu Chong; Zhang Yanbing; Jiang Hua

    2009-02-13

    The relative inability of hemopoietic stem cells (HSCs) to reproduce themselves (self-renew) ex vivo imposes substantial limitations on the current use of HSC transplantation. Recently, the transcription factor Zfx has been demonstrated that played an important in controlling the self-renewal of hematopoietic stem cells. Here, we reported that Zfx could enable high-level expansion of HSCs in vitro, by combination of protein transduction domain, TAT. Furthermore, we also demonstrated that expanded HSCs population retains their normal in vivo potential of pluripotency. It is thus that TAT-Zfx has the potential to expand HSCs significantly in vitro, and will have enormous clinical potentials.

  3. Human ex-vivo oral tissue imaging using spectral domain polarization sensitive optical coherence tomography.

    PubMed

    Sharma, Priyanka; Verma, Yogesh; Sahu, Khageswar; Kumar, Sudhir; Varma, Amit V; Kumawat, Jyoti; Gupta, Pradeep Kumar

    2017-01-01

    We report the use of spectral domain polarization sensitive optical coherence tomography for ex-vivo imaging of human oral mandibular tissue samples. Our results show that compared to the changes observed in the epithelium thickness and the decay constant of A-scan intensity profile, a much larger degree of change was observed in the phase retardation for tissue sites progressing from normal to the malignant state. These results suggest that monitoring of tissue retardance can help in better differentiation of normal and cancerous oral tissue sites.

  4. 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

  5. Effect of implanted brachytherapy seeds on optical fluence distribution: preliminary ex vivo study

    NASA Astrophysics Data System (ADS)

    Hetzel, Fred W.; Chen, Qun; Ding, Meisong; Newman, Francis; Dole, Kenneth C.; Huang, Zheng; Blanc, Dominique

    2007-02-01

    Photodynamic therapy (PDT) has gradually found its place in the treatment of malignant and non-malignant human diseases. Currently, interstitial PDT is being explored as an alternative modality for newly diagnosed and recurrent organ-confined prostate cancer. The interstitial PDT for the treatment of prostate cancer might be considered to treat prostates with permanent radioactive seeds implantation. However, the effect of implanted brachytherapy seeds on the optical fluence distribution of PDT light has not been studied before. This study investigated, for the first time, the effect of brachytherapy seed on the optical fluence distribution of 760 nm light in ex vivo models (meat and canine prostate).

  6. Ultrasound-enhanced drug delivery in a perfused ex vivo artery model

    NASA Astrophysics Data System (ADS)

    Hitchcock, Kathryn E.

    Acoustically driven stable cavitation may improve treatments of diseases in which passive penetration of drug into the target tissue is poor. Examples include atherosclerosis, in which the endothelium can prevent penetration of therapeutics into the plaque, and ischemic stroke, in which pathologically low flow of blood impedes the delivery of intravenous drugs to the clot. Understanding the way in which ultrasound cavitation agents nucleate cavitation in flowing blood-mimicking solutions is an important step in optimizing ultrasound-enhanced drug delivery. The use of a perfused, living ex vivo artery model permitted study of this phenomenon while still providing information on arterial bioeffects. Cavitation-enhanced delivery of anti-ICAM-1-targeted echogenic liposomes into and beyond the ex vivo murine aortic endothelium was demonstrated using 1-MHz continuous wave ultrasound. Acoustic cavitation had no apparent effect on the health of the murine arterial tissue. A method of maximizing the energy of stable cavitation through the use of intermittent 120-kHz ultrasound with quiescent periods to allow contrast agent inflow was developed. Using this insonificaiton method, sonothrombolysis was studied in ex vivo porcine carotid arteries using a 120-kHz center frequency and 0.44 MPa peak-to-peak pressure amplitude. Clot mass loss was used as a metric of thrombolytic efficacy. Clots exposed to recombinant tissue plasminogen activator and the ultrasound contrast agent, DefinityRTM in flowing porcine plasma without ultrasound experienced 34% mass loss. When robust stable cavitation was induced via 120-kHz insonation, the mean clot mass loss rose to 83%, which constituted a significant improvement (n = 6, p<0.0001). Without DefinityRTM there was no thrombolytic enhancement by ultrasound exposure alone at the same insonation pressure (n = 6, p<0.0001). Significant loss of endothelium occurred in 64% of the porcine carotid arteries, possibly due to poor oxygen delivery by the

  7. 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

  8. Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy

    PubMed Central

    Holmes, Holly E.; Powell, Nick M.; Ma, Da; Ismail, Ozama; Harrison, Ian F.; Wells, Jack A.; Colgan, Niall; O'Callaghan, James M.; Johnson, Ross A.; Murray, Tracey K.; Ahmed, Zeshan; Heggenes, Morten; Fisher, Alice; Cardoso, M. Jorge; Modat, Marc; O'Neill, Michael J.; Collins, Emily C.; Fisher, Elizabeth M. C.; Ourselin, Sébastien; Lythgoe, Mark F.

    2017-01-01

    With increasingly large numbers of mouse models of human disease dedicated to MRI studies, compromises between in vivo and ex vivo MRI must be fully understood in order to inform the choice of imaging methodology. We investigate the application of high resolution in vivo and ex vivo MRI, in combination with tensor-based morphometry (TBM), to uncover morphological differences in the rTg4510 mouse model of tauopathy. The rTg4510 mouse also offers a novel paradigm by which the overexpression of mutant tau can be regulated by the administration of doxycycline, providing us with a platform on which to investigate more subtle alterations in morphology with morphometry. Both in vivo and ex vivo MRI allowed the detection of widespread bilateral patterns of atrophy in the rTg4510 mouse brain relative to wild-type controls. Regions of volume loss aligned with neuronal loss and pathological tau accumulation demonstrated by immunohistochemistry. When we sought to investigate more subtle structural alterations in the rTg4510 mice relative to a subset of doxycycline-treated rTg4510 mice, ex vivo imaging enabled the detection of more regions of morphological brain changes. The disadvantages of ex vivo MRI may however mitigate this increase in sensitivity: we observed a 10% global shrinkage in brain volume of the post-mortem tissues due to formalin fixation, which was most notable in the cerebellum and olfactory bulbs. However, many central brain regions were not adversely affected by the fixation protocol, perhaps due to our “in-skull” preparation. The disparity between our TBM findings from in vivo and ex vivo MRI underlines the importance of appropriate study design, given the trade-off between these two imaging approaches. We support the utility of in vivo MRI for morphological phenotyping of mouse models of disease; however, for subtler phenotypes, ex vivo offers enhanced sensitivity to discrete morphological changes.

  9. Cocoa polyphenols and their influence on parameters involved in ex vivo skin restructuring.

    PubMed

    Gasser, P; Lati, E; Peno-Mazzarino, L; Bouzoud, D; Allegaert, L; Bernaert, H

    2008-10-01

    Polyphenols in general are compounds that are known to promote health and have a preventive effect against various chronic diseases. The influence of cocoa polyphenols on skin, however, has scarcely been studied from a histological point of view. The aim of this study is to assess the influence of cocoa polyphenols on several indicators of skin elasticity and skin tonus, namely, glycosaminoglycans and collagen I, III and IV. This was carried out by using a model of ex vivo human skin explants maintained in survival, on which a cocoa polyphenol extract was applied. After processing by standard histological techniques (fixation, paraffin embedding, sectioning, staining, immunostaining and microscopical observation), the influence of cocoa polyphenols on the evaluated parameters was quantified by image analysis. The results obtained show that cocoa polyphenols exhibit a positive action on the parameters assessed, and the dose at which they improve the most parameters associated with skin tonus and elasticity was determined. Their activity was compared with a commercially available product, and the results obtained show that their efficacy is equivalent. Moreover, an enhancing effect of cocoa butter on activity of cocoa polyphenol was highlighted. Now that the properties of cocoa polyphenols on ex vivo skin restructuring parameters have been assessed, the next step could include their evaluation in vivo.

  10. High embryonic recovery rates with in vivo and ex vivo techniques in the bitch.

    PubMed

    Luz, M R; de Holanda, C C; Pereira, J J; Freitas, P M C; Salgado, A E P; Giannotti, J Di Giorgio; de Oliveira, S B; Teixeira, N S; Guaitolini, C R de Freitas

    2011-08-01

    The embryonic collection techniques in dogs present a vast methodological variation and low recovery rates. The objectives were to compare and describe two techniques as to the recovery of canine embryos, on the 12th day after the first mating or artificial insemination. Embryos were recovered through uterine horn flushing in vivo, before performing the ovariohysterectomy (OHE) (Group 1; n = 9) or ex vivo, immediately after the OHE (Group 2; n = 9). In total, 43 and 47 embryonic structures were recovered in Groups 1 and 2, respectively. There was no significant difference (p > 0.05) between groups on recovery rates (72.8% and 81.0%, respectively). We inferred that both in vivo and ex vivo techniques allow a high rate of embryonic recovery; in the collection technique prior to the OHE, it is essential to carefully handle the reproductive system during the trans-surgical period and that the 12th day (D12) after the first mating/artificial insemination is an efficient option for the high recovery rate of morulae and blastocysts.

  11. 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.

  12. 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.

  13. Pushing the Envelope in Tissue Engineering: Ex Vivo Production of Thick Vascularized Cardiac Extracellular Matrix Constructs

    PubMed Central

    Sarig, Udi; Nguyen, Evelyne Bao-Vi; Wang, Yao; Ting, Sherwin; Bronshtein, Tomer; Sarig, Hadar; Dahan, Nitsan; Gvirtz, Maskit; Reuveny, Shaul; Oh, Steve K.W.; Scheper, Thomas; Boey, Yin Chiang Freddy; Venkatraman, Subbu S.

    2015-01-01

    Functional vascularization is a prerequisite for cardiac tissue engineering of constructs with physiological thicknesses. We previously reported the successful preservation of main vascular conduits in isolated thick acellular porcine cardiac ventricular ECM (pcECM). We now unveil this scaffold's potential in supporting human cardiomyocytes and promoting new blood vessel development ex vivo, providing long-term cell support in the construct bulk. A custom-designed perfusion bioreactor was developed to remodel such vascularization ex vivo, demonstrating, for the first time, functional angiogenesis in vitro with various stages of vessel maturation supporting up to 1.7 mm thick constructs. A robust methodology was developed to assess the pcECM maximal cell capacity, which resembled the human heart cell density. Taken together these results demonstrate feasibility of producing physiological-like constructs such as the thick pcECM suggested here as a prospective treatment for end-stage heart failure. Methodologies reported herein may also benefit other tissues, offering a valuable in vitro setting for “thick-tissue” engineering strategies toward large animal in vivo studies. PMID:25602926

  14. Dopamine D2High receptors measured ex vivo are elevated in amphetamine-sensitized animals.

    PubMed

    Seeman, Philip

    2009-03-01

    Although dopamine supersensitivity is a fundamental aspect of diseases such as schizophrenia and Parkinson's disease, the molecular basis of dopamine supersensitivity is not known. Because behavioral dopamine supersensitivity is associated with a marked elevation of striatal dopamine D2(High) receptors in vitro, it is important to develop methods to measure D2(High) receptors in vivo. The present ex vivo study found that the dopamine agonist NPA ([-]-N-propyl-norapomorphine) inhibited the binding of the agonist [(3)H](+)PHNO to rat striatal D2 receptors significantly more than the D2 antagonist [(3)H]raclopride, when NPA was coinjected i.v. with each radioligand. These results suggest that the greater sensitivity of [(3)H](+)PHNO to inhibition by the coinjected NPA reflects in vivo competition at D2(High) receptors. Using rats that had been sensitized to amphetamine, this ex vivo method found that the specific binding of [(3)H](+)PHNO that was displaced by 10 microg/kg of NPA was 2.4-fold higher than that for control rats. These data agree with in vitro data showing a marked increase in D2(High) sites after amphetamine sensitization. Therefore, it is recommended that this method of co-injecting the D2 radioligand and the dopamine agonist displacer be used in human positron tomography to detect D2(High) receptors in health and disease.

  15. Selective ex-vivo photothermal ablation of human pancreatic cancer with albumin functionalized multiwalled carbon nanotubes

    PubMed Central

    Mocan, Lucian; Tabaran, Flaviu A; Mocan, Teodora; Bele, Constantin; Orza, Anamaria Ioana; Lucan, Ciprian; Stiufiuc, Rares; Manaila, Ioana; Iulia, Ferencz; Dana, Iancu; Zaharie, Florin; Osian, Gelu; Vlad, Liviu; Iancu, Cornel

    2011-01-01

    The process of laser-mediated ablation of cancer cells marked with biofunctionalized carbon nanotubes is frequently called “nanophotothermolysis”. We herein present a method of selective nanophotothermolisys of pancreatic cancer (PC) using multiwalled carbon nanotubes (MWCNTs) functionalized with human serum albumin (HSA). With the purpose of testing the therapeutic value of these nanobioconjugates, we have developed an ex-vivo experimental platform. Surgically resected specimens from patients with PC were preserved in a cold medium and kept alive via intra-arterial perfusion. Additionally, the HSA-MWCNTs have been intra-arterially administered in the greater pancreatic artery under ultrasound guidance. Confocal and transmission electron microscopy combined with immunohistochemical staining have confirmed the selective accumulation of HSA-MWCNTs inside the human PC tissue. The external laser irradiation of the specimen has significantly produced extensive necrosis of the malign tissue after the intra-arterial administration of HSA-MWCNTs, without any harmful effects on the surrounding healthy parenchyma. We have obtained a selective photothermal ablation of the malign tissue based on the selective internalization of MWCNTs with HSA cargo inside the pancreatic adenocarcinoma after the ex-vivo intra-arterial perfusion. PMID:21720504

  16. Diet-induced obesity skin changes monitored by in vivo SHG and ex vivo CARS microscopy

    PubMed Central

    Haluszka, Dóra; Lőrincz, Kende; Kiss, Norbert; Szipőcs, Róbert; Kuroli, Enikő; Gyöngyösi, Nóra; Wikonkál, Norbert M.

    2016-01-01

    Obesity related metabolic syndrome and type 2 diabetes have severe consequences on our skin. Latest developments in nonlinear microscopy allow the use of noninvasive, label free imaging methods, such as second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS), for early diagnosis of metabolic syndrome-related skin complications by 3D imaging of the skin and the connective tissue. Our aim was to study effects of various types of diet-induced obesity in mice using these methods. We examined mice on different diets for 32 weeks. The collagen morphology was evaluated four times in vivo by SHG microscopy, and adipocytes were examined once at the end of experiment by ex vivo CARS method. A strong correlation was found between the body weight and the adipocyte size, while we found that the SHG intensity of dermal collagen reduces considerably with increasing body weight. Obese mice on high-fat diet showed worse results than those on high-fat - high-fructose diet. Animals on high-fructose diet did not gain more weight than those on ordinary diet despite of the increased calorie intake, but their collagen damage was nonetheless significant. Obesity and high sugar intake damages the skin, mainly the dermal connective tissue and subcutaneous adipose tissue, which efficiently can be monitored by in vivo SHG and ex vivo CARS microscopy. PMID:27895989

  17. Instrumented urethral catheter and its ex vivo validation in a sheep urethra

    NASA Astrophysics Data System (ADS)

    Ahmadi, Mahdi; Rajamani, Rajesh; Timm, Gerald; Sezen, Serdar

    2017-03-01

    This paper designs and fabricates an instrumented catheter for instantaneous measurement of distributed urethral pressure profiles. Since the catheter enables a new type of urological measurement, a process for accurate ex vivo validation of the catheter is developed. A flexible sensor strip is first fabricated with nine pressure sensors and integrated electronic pads for an associated sensor IC chip. The flexible sensor strip and associated IC chip are assembled on a 7 Fr Foley catheter. A sheep bladder and urethra are extracted and used in an ex vivo set up for verification of the developed instrumented catheter. The bladder-urethra are suspended in a test rig and pressure cuffs placed to apply known static and dynamic pressures around the urethra. A significant challenge in the performance of the sensor system is the presence of parasitics that introduce large bias and drift errors in the capacitive sensor signals. An algorithm based on use of reference parasitic transducers is used to compensate for the parasitics. Extensive experimental results verify that the developed compensation method works effectively. Results on pressure variation profiles circumferentially around the urethra and longitudinally along the urethra are presented. The developed instrumented catheter will be useful in improved urodynamics to more accurately diagnose the source of urinary incontinence in patients.

  18. 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

  19. In vivo prevention of transplant arteriosclerosis by ex vivo-expanded human regulatory T cells.

    PubMed

    Nadig, Satish N; Wieckiewicz, Joanna; Wu, Douglas C; Warnecke, Gregor; Zhang, Wei; Luo, Shiqiao; Schiopu, Alexandru; Taggart, David P; Wood, Kathryn J

    2010-07-01

    Transplant arteriosclerosis is the hallmark of chronic allograft dysfunction (CAD) affecting transplanted organs in the long term. These fibroproliferative lesions lead to neointimal thickening of arteries in all transplanted allografts. Luminal narrowing then leads to graft ischemia and organ demise. To date, there are no known tolerance induction strategies that prevent transplant arteriosclerosis. Therefore, we designed this study to test the hypothesis that human regulatory T cells (T(reg) cells) expanded ex vivo can prevent transplant arteriosclerosis. Here we show the comparative capacity of T(reg) cells, sorted via two separate strategies, to prevent transplant arteriosclerosis in a clinically relevant chimeric humanized mouse system. We found that the in vivo development of transplant arteriosclerosis in human arteries was prevented by treatment of ex vivo-expanded human T(reg) cells. Additionally, we show that T(reg) cells sorted on the basis of low expression of CD127 provide a more potent therapy to conventional T(reg) cells. Our results demonstrate that human T(reg) cells can inhibit transplant arteriosclerosis by impairing effector function and graft infiltration. We anticipate our findings to serve as a foundation for the clinical development of therapeutics targeting transplant arteriosclerosis in both allograft transplantation and other immune-mediated causes of vasculopathy.

  20. Consequences of Mrp2 deficiency for diclofenac toxicity in the rat intestine ex vivo.

    PubMed

    Niu, Xiaoyu; de Graaf, Inge A M; van de Vegte, Dennis; Langelaar-Makkinje, Miriam; Sekine, Shuichi; Groothuis, Geny M M

    2015-02-01

    The non-steroidal anti-inflammatory drug diclofenac (DCF) has a high prevalence of intestinal side effects in humans and rats. It has been reported that Mrp2 transporter deficient rats (Mrp2) are more resistant to DCF induced intestinal toxicity. This was explained in vivo by impaired Mrp2-dependent biliary transport of DCF-acylglucuronide (DAG), leading to decreased intestinal exposure to DAG and DCF. However, it is not known to what extent adaptive changes in the Mrp2 intestine itself influence its sensitivity to DCF toxicity without the influence of liver metabolites. To investigate this, DCF toxicity and disposition were studied ex vivo by precision-cut intestinal slices and Ussing chamber using intestines from wild type(WT) and Mrp2 rats. The results show that adaptive changes due to Mrp2 deficiency concerning Mrp2, Mrp3 and BCRP gene expression, GSH content and DAG formation were different between liver and intestine. Furthermore, Mrp2 intestine was intrinsically more resistant to DCF toxicity than its WT counterpart ex vivo. This can at least partly be explained by a reduced DCF uptake by the Mrp2 intestine, but isnot related to the other adaptive changes in the intestine. The extrapolation of this data to humans with MRP2 deficiency is uncertain due to species differences in activity and regulation of transporters.

  1. Ex Vivo Produced Oral Mucosa Equivalent by Using the Direct Explant Cell Culture Technique

    PubMed Central

    Bayar, Gürkan Raşit; Aydıntuğ, Yavuz Sinan; Günhan, Ömer; Öztürk, Kamile; Gülses, Aydın

    2012-01-01

    Objective: The aim of this study is the histological and immunohistochemical evaluation of ex vivo produced oral mucosal equivalents using keratinocytes cultured by direct explant technique. Material and Methods: Oral mucosa tissue samples were obtained from the keratinized gingival tissues of 14 healthy human subjects. Human oral mucosa keratinocytes from an oral mucosa biopsy specimen were dissociated by the explant technique. Once a sufficient population of keratinocytes was reached, they were seeded onto the type IV collagen coated “AlloDerm” and taken for histological and immunohistochemical examinations at 11 days postseeding of the keratinocytes on the cadaveric human dermal matrix. Results: Histopathologically and immunohistochemically, 12 out of 14 successful ex vivo produced oral mucosa equivalents (EVPOME) that consisted of a stratified epidermis on a dermal matrix have been developed with keratinocytes cultured by the explant technique. Conclusion: The technical handling involved in the direct explant method at the beginning of the process has fewer steps than the enzymatic method and use of the direct explant technique protocol for culturing of human oral mucosa keratinocyte may be more adequate for EVPOME production. PMID:25207018

  2. Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats.

    PubMed

    Benetti, Camillo; Flammini, Lisa; Vivo, Valentina; Colombo, Paolo; Colombo, Gaia; Elviri, Lisa; Scarpignato, Carmelo; Buttini, Francesca; Bettini, Ruggero; Barocelli, Elisabetta; Rossi, Alessandra

    2016-10-10

    The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

  3. 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.

  4. 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.

  5. A Leukocyte Filter Does Not Provide Further Benefit During Ex Vivo Lung Perfusion.

    PubMed

    Luc, Jessica G Y; Aboelnazar, Nader S; Himmat, Sayed; Hatami, Sanaz; Haromy, Alois; Matsumura, Nobutoshi; Vasanthan, Vishnu; White, Christopher W; Mengel, Michael; Freed, Darren H; Nagendran, Jayan

    2017-02-20

    Normothermic ex vivo lung perfusion (EVLP) allows for assessment and reconditioning of donor lungs. Though a leukocyte filter (LF) is routinely incorporated into the EVLP circuit, its efficacy remains to be determined. Twelve pig lungs were perfused and ventilated ex vivo in a normothermic state for 12 hours. Lungs (n=3) were allocated to 4 groups according to perfusate composition and the presence or absence of a LF in the circuit (acellular ± LF, cellular ± LF). Acceptable physiologic lung parameters were achieved during EVLP; however, increased amounts of pro-inflammatory cytokines (TNF-α, IL-6) and leukocytes in the perfusate were observed despite the presence or absence of a LF. Analysis of cells washed off the LF demonstrates that it trapped leukocytes though was ineffective throughout perfusion as it became saturated over 12 hours. We conclude that there is no objective evidence to support the routine incorporation of a LF during EVLP as it does not provide further benefit and its removal does not appear to cause harm. The lack of hypothesized benefit to a LF may be due to the saturation of the LF with donor leukocytes, leading to similar amounts of circulating leukocytes still present in the perfusate with and without a LF.

  6. 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.

  7. Short term ex-vivo expansion of circulating head and neck tumour cells

    PubMed Central

    Kulasinghe, Arutha; Perry, Chris; Warkiani, Majid E.; Blick, Tony; Davies, Anthony; O'Byrne, Ken; Thompson, Erik W.; Nelson, Colleen C.; Vela, Ian; Punyadeera, Chamindie

    2016-01-01

    Minimally invasive techniques are required for the identification of head and neck cancer (HNC) patients who are at an increased risk of metastasis, or are not responding to therapy. An approach utilised in other solid cancers is the identification and enumeration of circulating tumour cells (CTCs) in the peripheral blood of patients. Low numbers of CTCs has been a limiting factor in the HNC field to date. Here we present a methodology to expand HNC patient derived CTCs ex-vivo. As a proof of principle study, 25 advanced stage HNC patient bloods were enriched for circulating tumour cells through negative selection and cultured in 2D and 3D culture environments under hypoxic conditions (2% O2, 5% CO2). CTCs were detected in 14/25 (56%) of patients (ranging from 1–15 CTCs/5 mL blood). Short term CTC cultures were successfully generated in 7/25 advanced stage HNC patients (5/7 of these cultures were from HPV+ patients). Blood samples from which CTC culture was successful had higher CTC counts (p = 0.0002), and were predominantly from HPV+ patients (p = 0.007). This is, to our knowledge, the first pilot study to culture HNC CTCs ex-vivo. Further studies are warranted to determine the use of short term expansion in HNC and the role of HPV in promoting culture success. PMID:27517751

  8. Subnormothermic machine perfusion for ex vivo preservation and recovery of the human liver for transplantation.

    PubMed

    Bruinsma, B G; Yeh, H; Ozer, 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 J; Yarmush, M L; Uygun, K; Izamis, M-L

    2014-06-01

    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 pretransplantation. To determine the feasibility in human livers, we assessed the effect of 3 h 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 [3.70-5.99] to 2.29 [1.20-3.43] mmol/L) and adenosine triphosphate content (45.0 [70.6-87.5] pmol/mg preperfusion 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 postischemia.

  9. In vitro and ex vivo activity of Melaleuca alternifolia against protoscoleces of Echinococcus ortleppi.

    PubMed

    Monteiro, Danieli Urach; Azevedo, Maria Isabel; Weiblen, Carla; DE Avila Botton, Sônia; Funk, Nadine Lysyk; DE Bona DA Silva, Cristiane; Zanette, Régis Adriel; Schwanz, Thiago Guilherme; DE LA Rue, Mário Luiz

    2017-02-01

    Cystic echinococcosis is a zoonotic disease of difficult diagnosis and treatment. The use of protoscolicidal agents in procedures is of utmost importance for treatment success. This study was aimed at analysing the in vitro and ex vivo activity of Melaleuca alternifolia oil (tea tree oil - TTO), its nanoemulsion formulation (NE-TTO) and its major component (terpinen-4-ol) against Echinococcus ortleppi protoscoleces obtained from cattle. Concentrations of 2·5, 5 and 10 mg mL-1 of TTO, 10 mg mL-1 of NE-TTO and 1, 1·5 and 2 mg mL-1 of terpinen-4-ol were evaluated in vitro against protoscoleces at 5, 10, 15 and 30 min. TTO was also injected directly into hydatid cysts (ex vivo analysis, n = 20) and the viability of protoscoleces was evaluated at 5, 15 and 30 min. The results indicated protoscolicidal effect at all tested formulations and concentrations. Terpinen-4-ol (2 mg mL-1) activity was superior when compared with the highest concentration of TTO. NE-TTO reached a gradual protoscolicidal effect. TTO at 20 mg mL-1 showed 90% protoscolicidal action in hydatid cysts at 5 min. The results showed that TTO affects the viability of E. ortleppi protoscoleces, suggesting a new protoscolicidal option to the treatment of cystic equinococcosis.

  10. 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).

  11. 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.

  12. 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.

  13. 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

  14. 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

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

    PubMed

    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.

  16. 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.

  17. Preoperative CT-Angiography Predicts Ex Vivo Vein Length for Right Kidneys After Laparoscopic Donor Nephrectomy.

    PubMed

    Özdemir-van Brunschot, Denise M D; Rottier, Simone J; den Ouden, Judith E; van der Jagt, Michel F; d'Ancona, Frank C; Kloke, Heinrich; van der Vliet, Daan J A; Schultze Kool, Leo J; Warlé, Michiel C

    2015-09-10

    BACKGROUND Implantation of a kidney with a short renal vein is technically more challenging and therefore prone for technique-related complications. It remains unclear whether pre-operative computed tomography angiography (CTA), to assess vascular anatomy of the donor kidney, can be used to predict renal vein length. MATERIAL AND METHODS Right and left renal vein lengths of 100 consecutive kidney donors were measured in an oblique-coronal plane multiplanar reconstruction image of 100 consecutive kidney donors in whom ex vivo vein length was measured after recovery. In a second retrospective cohort of 100 consecutive kidney donors donating a right kidney, preoperative CTA vein length measurements were correlated to anastomosis time and early graft outcome. RESULTS Left and right renal vein lengths, measured on CTA, were 43.2 mm and 30.0 mm, respectively. No correlation was found between CTA and ex vivo measurements for the left renal vein (p=.610), whereas a significant correlation was found for the right renal vein (p=.021). In the retrospective cohort, right renal vein length was significantly correlated with the anastomosis time but not with early graft outcome. CONCLUSIONS The length of the right, but not the left, renal vein can be predicted by preoperative CTA, but this does not hold true for the left renal vein.

  18. Cochlear implants and ex vivo BDNF gene therapy protect spiral ganglion neurons.

    PubMed

    Rejali, Darius; Lee, Valerie A; Abrashkin, Karen A; Humayun, Nousheen; Swiderski, Donald L; Raphael, Yehoash

    2007-06-01

    Spiral ganglion neurons often degenerate in the deaf ear, compromising the function of cochlear implants. Cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant. Brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Providing enhanced levels of BDNF in human ears may be accomplished by one of several different methods. The goal of these experiments was to test a modified design of the cochlear implant electrode that includes a coating of fibroblast cells transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, we transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF. We then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. We determined that the BDNF expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes. This protective effect decreased in the higher cochlear turns. The data demonstrate the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival.

  19. In vitro and ex vivo microbial leakage assessment in endodontics: A literature review

    PubMed Central

    Savadkouhi, Sohrab Tour; Bakhtiar, Hengameh; Ardestani, Safoura Emami

    2016-01-01

    The aim of this study was to perform a literature review of published in-vitro and ex-vivo studies, which evaluated microbial leakage in endodontics in the past 10 years. A comprehensive electronic literature search was carried out in PubMed database for English articles published from 2005 to 2016 using the keywords “endodontics,” “in vitro,” “ex vivo,” “microbial leakage,” “microbial penetration,” “saliva,” “Enterococcus faecalis,” “E. faecalis,” “endodontic sealers,” “temporary filling material,” “apical plug,” “mineral trioxide aggregate,” and “MTA.” The keywords were combined using Boolean operators AND/OR. Based on our search strategy, 33 relevant articles were included in the study. There are three main methods for assessment of bacterial microleakage, namely, (A) the dual-chamber leakage model, (B) detection of bacteria using a scanning electron microscope (SEM), and (C) polymerase chain reaction. All bacterial leakage models have some limitations and may yield different results compared to other microleakage evaluation techniques (i.e., dye penetration, fluid filtration, or electrochemical tests). The results of SEM correlated with those of microbial leakage test in most studies. Microbial leakage test using saliva better simulates the clinical setting for assessment of the leakage of single or mixed bacterial species. PMID:28032041

  20. The use of rats and mice as animal models in ex vivo bone growth and development studies.

    PubMed

    Abubakar, A A; Noordin, M M; Azmi, T I; Kaka, U; Loqman, M Y

    2016-12-01

    In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610-618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

  1. Radical protection in the visible and infrared by a hyperforin-rich cream--in vivo versus ex vivo methods.

    PubMed

    Arndt, Sophia; Haag, Stefan F; Kleemann, Anke; Lademann, Juergen; Meinke, Martina C

    2013-05-01

    The formation of radicals plays an important role in the development of atopic eczema or barrier-disrupted skin. We evaluated the radical scavenging effect of a cream containing a Hypericum perforatum extract rich in hyperforin in a double-blind placebo-controlled study on 11 healthy volunteers. Electron paramagnetic resonance spectroscopy was applied to determine radical formation during VIS/NIR irradiation of the inner forearm. The results were compared to ex vivo investigations on excised porcine ear skin after a single application of the creams. The non-treated skin was measured as control. The absolute values and the kinetics are not comparable for ex vivo and in vivo radical formation. Whereas in vivo, the radical production decreases with time, it remains stable ex vivo over the investigated timescale. Nevertheless, ex vivo methods could be developed to estimate the protection efficiency of creams. In vivo as well as ex vivo, the radical formation could be reduced by almost 80% when applying the hyperforin-rich cream onto the skin, whereas placebo resulted in about 60%. In vivo, a daylong protection effect could be validated after a 4-week application time of the cream indicating that a regular application is necessary to obtain the full effect.

  2. The use of rats and mice as animal models in ex vivo bone growth and development studies

    PubMed Central

    Abubakar, A. A.; Noordin, M. M.; Azmi, T. I.; Kaka, U.

    2016-01-01

    In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine. Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610–618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2. PMID:27965220

  3. Investigation of biphasic tumor oxygen dynamics induced by hyperoxic gas intervention: the dynamic phantom approach

    NASA Astrophysics Data System (ADS)

    Kim, Jae G.; Liu, Hanli

    2008-01-01

    We have developed dynamic tumor vascular phantoms and utilized them to investigate the biphasic behavior of increases in light absorption, which is directly associated with oxygenated hemoglobin concentration that was observed in vivo from rat breast tumor experiments during carbogen/oxygen inhalation. The experimental setup for the phantom study included a continuous-wave, multichannel, near-infrared spectroscopy (NIRS) system and syringe pumps to drive the simulated blood through the dynamic vascular phantoms. The results from such phantom experiments clearly show that the two time constants observed in tumor oxygenation dynamics in vivo can result from two different perfusion rates or two different blood flow velocities. We provide experimental support for our previous hypothesis: the biphasic tumor hemodynamic feature stems from a well-perfused and poorly perfused region that could be detected with the two time constants of the NIRS signals. With a multichannel approach, noninvasive NIRS measurements may have useful and prognostic values to quantify the therapeutic effects of cancer treatments.

  4. 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

  5. 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

  6. 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

  7. Use of ex vivo and in vitro cultures of the human respiratory tract to study the tropism and host responses of highly pathogenic avian influenza A (H5N1) and other influenza viruses.

    PubMed

    Chan, Renee W Y; Chan, Michael C W; Nicholls, John M; Malik Peiris, J S

    2013-12-05

    The tropism of influenza viruses for the human respiratory tract is a key determinant of host-range, and consequently, of pathogenesis and transmission. Insights can be obtained from clinical and autopsy studies of human disease and relevant animal models. Ex vivo cultures of the human respiratory tract and in vitro cultures of primary human cells can provide complementary information provided they are physiologically comparable in relevant characteristics to human tissues in vivo, e.g. virus receptor distribution, state of differentiation. We review different experimental models for their physiological relevance and summarize available data using these cultures in relation to highly pathogenic avian influenza H5N1, in comparison where relevant, with other influenza viruses. Transformed continuous cell-lines often differ in important ways to the corresponding tissues in vivo. The state of differentiation of primary human cells (respiratory epithelium, macrophages) can markedly affect virus tropism and host responses. Ex vivo cultures of human respiratory tissues provide a close resemblance to tissues in vivo and may be used to risk assess animal viruses for pandemic threat. Physiological factors (age, inflammation) can markedly affect virus receptor expression and virus tropism. Taken together with data from clinical studies on infected humans and relevant animal models, data from ex vivo and in vitro cultures of human tissues and cells can provide insights into virus transmission and pathogenesis and may provide understanding that leads to novel therapeutic interventions.

  8. Apparatus for Histological Validation of In Vivo and Ex Vivo Magnetic Resonance Imaging of the Human Prostate

    PubMed Central

    Bourne, Roger M.; Bailey, Colleen; Johnston, Edward William; Pye, Hayley; Heavey, Susan; Whitaker, Hayley; Siow, Bernard; Freeman, Alex; Shaw, Greg L.; Sridhar, Ashwin; Mertzanidou, Thomy; Hawkes, David J.; Alexander, Daniel C.; Punwani, Shonit; Panagiotaki, Eleftheria

    2017-01-01

    This article describes apparatus to aid histological validation of magnetic resonance imaging studies of the human prostate. The apparatus includes a 3D-printed patient-specific mold that facilitates aligned in vivo and ex vivo imaging, in situ tissue fixation, and tissue sectioning with minimal organ deformation. The mold and a dedicated container include MRI-visible landmarks to enable consistent tissue positioning and minimize image registration complexity. The inclusion of high spatial resolution ex vivo imaging aids in registration of in vivo MRI and histopathology data. PMID:28393049

  9. Experimental in vivo and ex vivo models for the study of human aortic dissection: promises and challenges

    PubMed Central

    Jiang, Ding-Sheng; Yi, Xin; Zhu, Xue-Hai; Wei, Xiang

    2016-01-01

    Aortic dissection (AD) is a life-threatening aortopathy with high mortality. To mimic spontaneous AD, investigate the pathogenesis of AD and develop novel therapeutic targets and measures, multiple AD experimental models have been generated, including drugs or chemicals induced experimental models, genetically modified experimental models, surgically or invasively induced experimental models, and ex vivo models. However, the perfect model of AD that replicates every aspect of the natural disease has not be generated yet. This review provides an overview of the experimental models used in AD preclinical research. The value and challenges of each in vivo and ex vivo model are discussed. PMID:28077990

  10. 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.

  11. Enhanced ex vivo intestinal absorption of olmesartan medoxomil nanosuspension: Preparation by combinative technology.

    PubMed

    Attari, Zenab; Bhandari, Amita; Jagadish, P C; Lewis, Shaila

    2016-01-01

    The purpose of this study was to develop nanosuspension based on combinative technology to enhance the intestinal absorption of Olmesartan medoxomil (OLM), a potent antihypertensive agent with limited oral bioavailability. Two combinative approaches were employed and then characterized. In vitro intestinal absorption of OLM nanosuspension and plain OLM was studied using non-everted rat intestinal sac model. Optimal OLM nanosuspension was prepared by a combination of ball milling and probe sonication using stabilizer, Poloxamer 407. The formula exhibited particle size of 469.9 nm and zeta potential of -19.1 mV, which was subjected to ex vivo studies. The flux and apparent permeability coefficient in intestine from OLM nanosuspension was higher than the plain drug, thereby suggesting better drug delivery.

  12. [Advances in ex vivo expansion and immunotherapy application of regulatory T cells].

    PubMed

    Yan, Li; Shao, Zong-Hong

    2015-04-01

    CD4+ CD25+ regulatory T cells (Treg) play a fundamental role in the establishment and maintenance of immune tolerance. In a some of experimental models, it was found that Tregs can quench autoimmune diseases, maintain allogeneic transplants, and prevent allergic diseases. A major obstacle to their clinical application is related to their definitive phenotype and very limited number of these cells in peripheral circulation, no more than 5%-10% of total CD4+ T cells. Recent progress of technologies for Treg sorting with multicolor flow cytometry and immuno-absorbing columns has overcome these obstacles, and opened the doors to the clinical application of Treg. This review highlight the characteristics of Treg, describe the current information of cell sorting and ex vivo expansion techniques, and outline the adoptive transfer experiments and clinical trials of immunotherapy that have been developed in recent years. It is foreseeable that Treg adoptive transfusion will be a promising immunosuppressive therapy.

  13. Ex-vivo partial nephrectomy after living donor nephrectomy: Surgical technique for expanding kidney donor pool

    PubMed Central

    Nyame, Yaw A.; Babbar, Paurush; Aboumohamed, Ahmed A.; Mori, Ryan L.; Flechner, Stuart M.; Modlin, Charles S.

    2017-01-01

    Renal transplantation has profound improvements in mortality, morbidity, and overall quality of life compared to renal replacement therapy. This report aims to illustrate the use of ex-vivo partial nephrectomy in a patient with a renal angiomyolipoma prior to living donor transplantation. The surgical outcomes of the donor nephrectomy and recipient transplantation are reported with 2 years of follow-up. Both the donor and recipient are healthy and without any significant comorbidities. In conclusion, urologic techniques such as partial nephrectomy can be used to expand the living donor pool in carefully selected and well informed transplant recipients. Our experience demonstrated a safe and positive outcome for both the recipient and donor, and is consistent with other reported outcomes in the literature. PMID:28216945

  14. Safety and efficient ex vivo expansion of stem cells using platelet-rich plasma technology.

    PubMed

    Anitua, Eduardo; Prado, Roberto; Orive, Gorka

    2013-09-01

    The goal of this Review is to provide an overview of the cell culture media supplements used in the ex vivo expansion of stem cells intended for cell therapy. Currently, the gold standard is the culture supplemented with fetal bovine serum, however, their use in cell therapy raises many concerns. The alternatives to its use are presented, ranging from the use of human serum to platelet-rich plasma (PRP), to serum-free media or extracellular matrix components. Finally, various growth factors present in PRP are described, which make it a safe and effective stem cell expansion supplement. These growth factors could be responsible for their efficiency, as they increase both stem cell proliferation and survival. The different PRP formulations are also discussed, as well as the need for protocol standardization.

  15. Whole-brain ex-vivo quantitative MRI of the cuprizone mouse model

    PubMed Central

    Hurley, Samuel A.; Vernon, Anthony C.; Torres, Joel; Dell’Acqua, Flavio; Williams, Steve C.R.; Cash, Diana

    2016-01-01

    Myelin is a critical component of the nervous system and a major contributor to contrast in Magnetic Resonance (MR) images. However, the precise contribution of myelination to multiple MR modalities is still under debate. The cuprizone mouse is a well-established model of demyelination that has been used in several MR studies, but these have often imaged only a single slice and analysed a small region of interest in the corpus callosum. We imaged and analyzed the whole brain of the cuprizone mouse ex-vivo using high-resolution quantitative MR methods (multi-component relaxometry, Diffusion Tensor Imaging (DTI) and morphometry) and found changes in multiple regions, including the corpus callosum, cerebellum, thalamus and hippocampus. The presence of inflammation, confirmed with histology, presents difficulties in isolating the sensitivity and specificity of these MR methods to demyelination using this model. PMID:27833805

  16. 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

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

    PubMed

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

    2015-01-01

    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.

  18. Optical coherence tomography and Raman spectroscopy of the ex-vivo retina

    PubMed Central

    Evans, Julia W.; Zawadzki, Robert J.; Liu, Rui; Chan, James W.; Lane, Stephen M.; Werner, John S.

    2009-01-01

    Imaging the structure and correlating it with the biochemical content of the retina holds promise for fundamental research and for clinical applications. Optical coherence tomography (OCT) is commonly used to image the 3D structure of the retina and while the added functionality of biochemical analysis afforded by Raman scattering could provide critical molecular signatures for clinicians and researchers, there are many technical challenges to combine these imaging modalities. We describe an OCT microscope for ex-vivo imaging combined with Raman spectroscopy capable of collecting morphological and molecular information about a sample simultaneously. We present our first results and discuss the challenges to further development of this dual-mode instrument and limitations for future in-vivo retinal imaging. PMID:19569116

  19. Effect of NSAIDs and diuretics on nephrogenesis in an ex vivo embryogenic kidney model.

    PubMed

    Bueters, Ruud Rg; Klaasen, Annelies; van den Heuvel, Lambertus P; Schreuder, Michiel F

    2013-12-01

    The kidney is one of the key organs in clearing foreign compounds. The effects of drugs on the developing kidney are relatively unknown. We studied the direct effect of furosemide, hydrochlorothiazide, ibuprofen, and indomethacin on kidney development in an ex vivo embryonic kidney model. At embryonic day 13, metanephroi were dissected from mice and cultured in control media or media supplemented with various clinically relevant concentrations of drugs. The ureteric tree was visualized by whole-mount staining and branching was evaluated by counting. Additionally, gene expression levels of Wt1, Sox9, Bmp7, Fgf8, and Gdnf were investigated. No distinct differences were noted on either ureteric tip development or gene expression analysis for each drug after 24 hr of exposure. Even though short-term exposure to clinically relevant concentrations seems not to disturb renal development, future research is needed to study prolonged or repeated exposures.

  20. Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.

    PubMed

    Yu, Min; Bardia, Aditya; Aceto, Nicola; Bersani, Francesca; Madden, Marissa W; Donaldson, Maria C; Desai, Rushil; Zhu, Huili; Comaills, Valentine; Zheng, Zongli; Wittner, Ben S; Stojanov, Petar; Brachtel, Elena; Sgroi, Dennis; Kapur, Ravi; Shioda, Toshihiro; Ting, David T; Ramaswamy, Sridhar; Getz, Gad; Iafrate, A John; Benes, Cyril; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A

    2014-07-11

    Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor-positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

  1. Differentiation of ex vivo human breast tissue using polarization-sensitive optical coherence tomography

    PubMed Central

    South, Fredrick A.; Chaney, Eric J.; Marjanovic, Marina; Adie, Steven G.; Boppart, Stephen A.

    2014-01-01

    Successful treatment of breast cancer typically requires surgical removal of the tumor. Optical coherence tomography (OCT) has been previously developed for real-time imaging of the surgical margin. However, it can be difficult to distinguish between normal stromal tissue and cancer tissue based on scattering intensity and structure alone. Polarization-sensitive optical coherence tomography (PS-OCT) is sensitive to form birefringence of biological tissue. We report on the development of a high-speed PS-OCT system and imaging of ex vivo human breast tissue, showing enhanced contrast between healthy and cancerous tissues based upon collagen content confirmed with corresponding histology. These results demonstrate the feasibility of using PS-OCT to supplement structural OCT as a possible method for intraoperative tumor margin evaluation. PMID:25360360

  2. Ex vivo T-cell depletion in allogeneic hematopoietic stem cell transplant: past, present and future.

    PubMed

    Saad, A; Lamb, L S

    2017-03-20

    The most common cause of post-transplant mortality in patients with hematological malignancy is relapse, followed by GvHD, infections, organ toxicity and second malignancy. Immune-mediated complications such as GvHD continue to be challenging, yet amenable to control through manipulation of the T-cell compartment of the donor graft with subsequent immunomodulation after transplant. However, risk of both relapse and infection increase concomitantly with T-cell depletion (TCD) strategies that impair immune recovery. In this review, we discuss the clinical outcome of current and emerging strategies of TCD in allogeneic hematopoietic stem cell transplant that have developed during the modern transplantation era, focusing specifically on ex vivo strategies that target selected T-cell subsets.Bone Marrow Transplantation advance online publication, 20 March 2017; doi:10.1038/bmt.2017.22.

  3. Steerable intravitreal inserts for drug delivery: in vitro and ex vivo mobility experiments.

    PubMed

    Bergeles, Christos; Kummer, Michael P; Kratochvil, Bradley E; Framme, Carsten; Nelson, Bradley J

    2011-01-01

    The progress of wet age-related macular degeneration can now be controlled by intravitreal drug injection. This approach requires repeated injections, which could be avoided by delivering the drug to the retina. Intraocular implants are a promising solution for drug delivery near the retina. Currently, their accurate placement is challenging, and they can only be removed after a vitrectomy. In this paper, we introduce an approach for minimally invasive retinal drug delivery using magnetic intraocular inserts. We briefly discuss the electromagnetic-control system for magnetic implants and then focus on evaluating their ability to move in the vitreous humor. The mobility of magnetic intraocular implants is estimated in vitro with synthesized vitreous humors, and ex vivo with experiments on cadaver porcine eyes. Preliminary results show that with such magnetic implants a vitrectomy can be avoided.

  4. 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.

  5. Temperature profile of ex-vivo organs during radio frequency thermal ablation by fiber Bragg gratings

    NASA Astrophysics Data System (ADS)

    Palumbo, Giovanna; Iadicicco, Agostino; Tosi, Daniele; Verze, Paolo; Carlomagno, Nicola; Tammaro, Vincenzo; Ippolito, Juliet; Campopiano, Stefania

    2016-11-01

    We report on the integration of fiber optic sensors with commercial medical instrumentation for temperature monitoring during radio frequency ablation for tumor treatment. A suitable configuration with five fiber Bragg grating sensors bonded to a bipolar radio frequency (RF) probe has been developed to monitor the area under treatment. A series of experiments were conducted on ex-vivo animal kidney and liver and the results confirm that we were able to make a multipoint measurement and to develop a real-time temperature profile of the area, with a temperature resolution of 0.1°C and a spatial resolution of 5 mm during a series of different and consecutive RF discharges.

  6. 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.

  7. 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 = 0.871, 0.948 and 0.966) compared to integrated backscatter imaging (AUROC = 0.865, 0.877 and 0.832).

  8. 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.

  9. Ex vivo photometric and polarimetric multilayer characterization of human healthy colon by multispectral Mueller imaging.

    PubMed

    Pierangelo, Angelo; Manhas, Sandeep; Benali, Abdelali; Fallet, Clément; Antonelli, Maria-Rosaria; Novikova, Tatiana; Gayet, Brice; Validire, Pierre; De Martino, Antonello

    2012-06-01

    Healthy human colon samples were analyzed ex vivo with a multispectral imaging Mueller polarimeter operating from 500 to 700 nm in a backscattering configuration with diffuse light illumination impinging on the innermost tissue layer, the mucosa. The intensity and polarimetric responses were taken on whole tissues first and after progressive exfoliation of the outer layers afterwards. Moreover, these measurements were carried out with two different substrates (one bright and the other dark) successively placed beneath each sample, allowing a reasonably accurate evaluation of the contributions to the overall backscattered light by the various layers. For the shorter investigated wavelengths (500 to 550 nm) the major contribution comes from mucosa and submucosa, while for the longer wavelengths (650 to 700 nm) muscular tissue and fat also contribute significantly. The depolarization has also been studied and is found to be stronger in the red part of the spectrum, mainly due to the highly depolarizing power of the muscular and fat layers.

  10. Matrix type transdermal therapeutic system containing captopril: formulation optimization, in vitro and ex vivo characterization.

    PubMed

    Kerimoğlu, Oya; Keskin, Ebru; Dortunç, Betül; Anah, Sela

    2013-01-01

    Transdermal therapeutic systems (TTS) containing captopril were developed by using synthetic and pH independent polymers, Eudragit RL 100 and RS 100. The formulations were characterized in terms of their appearance, thickness, captopril content, in vitro release rate and diffusion profiles. In vitro release studies demonstrated controlled release for each formulation developed. In viro and ex vivo diffusion rate studies were performed through various synthetic membranes with different thickness, pore size and type (hydrophilic and hydrophobic) and through human skin by using Franz diffusion cells. Type of membrane and composition of the formulation affected the diffusion profiles of captopril from the transdermal therapeutic systems. Transdermal therapeutic systems containing captopril were successfully prepared and especially two of the formulations (F15 and F16) are considered to be suitable to administer captopril via skin.

  11. [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.

  12. CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo

    PubMed Central

    Mladenov, Radoslav; Hristodorov, Dmitrij; Cremer, Christian; Gresch, Gerrit; Grieger, Elena; Schenke, Lea; Klose, Diana; Amoury, Manal; Woitok, Mira; Jost, Edgar; Brümmendorf, Tim H.; Fendel, Rolf; Fischer, Rainer; Stein, Christoph; Thepen, Theo; Barth, Stefan

    2016-01-01

    Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15–50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia. PMID:27564103

  13. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

    PubMed

    Mustata, Roxana C; Van Loy, Tom; Lefort, Anne; Libert, Frédérick; Strollo, Sandra; Vassart, Gilbert; Garcia, Marie-Isabelle

    2011-06-01

    Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

  14. CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo.

    PubMed

    Mladenov, Radoslav; Hristodorov, Dmitrij; Cremer, Christian; Gresch, Gerrit; Grieger, Elena; Schenke, Lea; Klose, Diana; Amoury, Manal; Woitok, Mira; Jost, Edgar; Brümmendorf, Tim H; Fendel, Rolf; Fischer, Rainer; Stein, Christoph; Thepen, Theo; Barth, Stefan

    2016-10-11

    Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

  15. Assessment of stiffness changes in the ex vivo porcine aortic wall using magnetic resonance elastography

    PubMed Central

    Xu, Lei; Chen, Jun; Yin, Meng; Glaser, Kevin J.; Chen, Qingshan; Woodrum, David A.; Ehman, Richard L.

    2011-01-01

    Magnetic resonance elastography (MRE) is a noninvasive phase-contrast technique for estimating the mechanical properties of tissues by imaging propagating mechanical waves within the tissue. In this study, we hypothesize that changes in arterial wall stiffness, experimentally induced by formalin fixation, can be measured using MRE in ex vivo porcine aortas. In agreement with our hypothesis, the significant stiffness increase after sample fixation were clearly demonstrated by MRE and confirmed by mechanical testing. The results indicate that MRE can be used to examine the stiffness changes of the aorta. This study has provided evidence of the effectiveness of using MRE to directly assess the stiffness change in aortic wall. The results offer motivation to pursue MRE as a noninvasive method for the evaluation of arterial wall mechanical properties. PMID:22055848

  16. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells.

    PubMed

    Zhang, Cheng Cheng; Kaba, Megan; Ge, Guangtao; Xie, Kathleen; Tong, Wei; Hug, Christopher; Lodish, Harvey F

    2006-02-01

    Successful ex vivo expansion of hematopoietic stem cells (HSCs) would greatly benefit the treatment of disease and the understanding of crucial questions of stem cell biology. Here we show, using microarray studies, that the HSC-supportive mouse fetal liver CD3(+) cells specifically express the proteins angiopoietin-like 2 (Angptl2) and angiopoietin-like 3 (Angptl3). We observed a 24- or 30-fold net expansion of long-term HSCs by reconstitution analysis when we cultured highly enriched HSCs for 10 days in the presence of Angptl2 or Angptl3 together with saturating levels of other growth factors. The coiled-coil domain of Angptl2 was capable of stimulating expansion of HSCs. Furthermore, angiopoietin-like 5, angiopoietin-like 7 and microfibril-associated glycoprotein 4 also supported expansion of HSCs in culture.

  17. 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.

  18. 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.

  19. Ex Vivo Lung Perfusion: A Key Tool for Translational Science in the Lungs.

    PubMed

    Tane, Shinya; Noda, Kentaro; Shigemura, Norihisa

    2017-02-23

    Ex vivo lung perfusion (EVLP) promises to be a comprehensive platform for assessment, re-conditioning, and preservation for donor lungs and has been dramatically changing the face of clinical lung transplantation. Besides its increasing role in lung transplantation, EVLP has also been recognized as a useful tool for translational research involving the lungs. Based on recent remarkable evidence and experience using EVLP in lung transplantation, there is growing interest in and expectations for the use of EVLP beyond the field of lung transplantation. By combining EVLP with advances in regenerative medicine, stem cell biology, and oncology, the evolving technology of EVLP has a tremendous potential to advance pulmonary medicine and science. In this review, we revisit recent advances in EVLP technology and research and discuss the future translation of EVLP applications into life-changing medicine.

  20. Microultrasound characterisation of ex vivo porcine tissue for ultrasound capsule endoscopy

    NASA Astrophysics Data System (ADS)

    Lay, H. S.; Cox, B. F.; Sunoqrot, M.; Démoré, C. E. M.; Näthke, I.; Gomez, T.; Cochran, S.

    2017-01-01

    Gastrointestinal (GI) disease development and progression is often characterised by cellular and tissue architectural changes within the mucosa and sub-mucosa layers. Current clinical capsule endoscopy and other approaches are heavily reliant on optical techniques which cannot detect disease progression below the surface layer of the tissue. To enhance the ability of clinicians to detect cellular changes earlier and more confidently, both quantitative and qualitative microultrasound (μUS) techniques are investigated in healthy ex vivo porcine GI tissue. This work is based on the use of single-element, focussed μUS transducers made with micromoulded piezocomposite operating at around 48 MHz. To explore the possibility that μUS can detect Crohn’s disease and other inflammatory bowel diseases, ex vivo porcine small bowel tissue samples were cannulised and perfused with phosphate-buffered saline followed by various dilutions of polystyrene microspheres. Comparison with fluorescent imaging showed that the microspheres had infiltrated the microvasculature of the samples and that μUS was able to successfully detect this as a mimic of inflammation. Samples without microspheres were analysed using quantitative ultrasound to assess mechanical properties. Attenuation coefficients of 1.78 ± 0.66 dB/mm and 1.92 ± 0.77 dB/mm were obtained from reference samples which were surgically separated from the muscle layer. Six intact samples were segmented using a software algorithm and the acoustic impedance, Z, for varying tissue thicknesses, and backscattering coefficient, BSC, were calculated using the reference attenuation values and tabulated.

  1. Light-induced disulfide dimerization of recoverin under ex vivo and in vivo conditions.

    PubMed

    Zernii, Evgeni Yu; Nazipova, Aliya A; Gancharova, Olga S; Kazakov, Alexey S; Serebryakova, Marina V; Zinchenko, Dmitry V; Tikhomirova, Natalya K; Senin, Ivan I; Philippov, Pavel P; Permyakov, Eugene A; Permyakov, Sergei E

    2015-06-01

    Despite vast knowledge of the molecular mechanisms underlying photochemical damage of photoreceptors, linked to progression of age-related macular degeneration, information on specific protein targets of the light-induced oxidative stress is scarce. Here, we demonstrate that prolonged intense illumination (halogen bulb, 1500 lx, 1-5 h) of mammalian eyes under ex vivo (cow) or in vivo (rabbit) conditions induces disulfide dimerization of recoverin, a Ca(2+)-dependent inhibitor of rhodopsin kinase. Western blotting and mass spectrometry analysis of retinal extracts reveals illumination time-dependent accumulation of disulfide homodimers of recoverin and its higher order disulfide cross-linked species, including a minor fraction of mixed disulfides with intracellular proteins (tubulins, etc.). Meanwhile, monomeric bovine recoverin remains mostly reduced. These effects are accompanied by accumulation of disulfide homodimers of visual arrestin. Histological studies demonstrate that the light-induced oxidation of recoverin and arrestin occurs in intact retina (illumination for 2 h), while illumination for 5 h is associated with damage of the photoreceptor layer. A comparison of ex vivo levels of disulfide homodimers of bovine recoverin with redox dependence of its in vitro thiol-disulfide equilibrium (glutathione redox pair) gives the lowest estimate of redox potential in rod outer segments under illumination from -160 to -155 mV. Chemical crosslinking and dynamic light scattering data demonstrate an increased propensity of disulfide dimer of bovine recoverin to multimerization/aggregation. Overall, the oxidative stress caused by the prolonged intense illumination of retina might affect rhodopsin desensitization via concerted disulfide dimerization of recoverin and arrestin. The developed herein models of eye illumination are useful for studies of the light-induced thiol oxidation of visual proteins.

  2. P-gp activity and inhibition in the different regions of human intestine ex vivo.

    PubMed

    Li, Ming; de Graaf, Inge A M; de Jager, Marina H; Groothuis, Geny M M

    2017-03-01

    Although intestinal P-glycoprotein (P-gp) has been extensively studied in vitro and in animals, its activity and the consequences of P-gp inhibition for drug disposition and toxicity in humans are still difficult to accurately extrapolate from these studies. Moreover, existing in vitro models do not take into consideration that the intestine is heterogeneous with respect to P-gp expression. Recently, we reported rat precision-cut intestinal slices (PCIS) as a physiological ex vivo model to study the regional gradient of P-gp activity and inhibition. Here we extended the application of PCIS to the human intestine. For this purpose rhodamine 123 (R123) accumulation in the presence or absence of the P-gp inhibitors verapamil, cyclosporine A, quinidine, ketoconazole, PSC833 and CP100356 was measured in PCIS of human duodenum, jejunum, ileum and colon. R123 accumulation in the presence of the P-gp inhibitors appeared to be most enhanced in the ileum compared to the other regions. Moreover, the regional differences in accumulation are in line with published differences in abundance of P-gp. The rank order of the potency of the P-gp inhibitors, reflected by their IC50 , was comparable to that in rat PCIS. However, the increase in accumulation of the P-gp substrate R123 by the inhibitors was larger in human ileum PCIS than in rat PCIS, indicating species difference in P-gp abundance. These data show that human PCIS are an appropriate ex vivo model to study the activity of intestinal P-gp and predict the inhibitory effect of drugs and of transporter-mediated drug-drug interactions in the human intestine. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase

    PubMed Central

    Nishino, Taito; Wang, Changshan; Mochizuki-Kashio, Makiko; Osawa, Mitsujiro; Nakauchi, Hiromitsu; Iwama, Atsushi

    2011-01-01

    Background Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. Methodology/Principal Findings In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34+CD38– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34+CD38– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34+ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. Conclusions/Significance Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs. PMID:21931675

  4. 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

  5. An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

    PubMed

    Di Lullo, A M; Scorza, M; Amato, F; Comegna, M; Raia, V; Maiuri, L; Ilardi, G; Cantone, E; Castaldo, G; Iengo, M

    2016-11-29

    Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed the sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicenter studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%.

  6. 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.

  7. 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.

  8. Flexor tenorrhaphy tensile strength: reduction by cyclic loading: in vitro and ex vivo porcine study.

    PubMed

    Gibbons, C E R; Thompson, D; Sandow, M J

    2009-06-01

    The integrity of the repair is critical to maintain coaptation of the severed flexor tendon end until healing has advanced sufficiently. In our hospital, we use a modified Savage repair (four-strand Adelaide technique) using 3-0 Ethibond (Ethicon, Somerville, NJ, USA) for acute flexor tenorrhaphy and an active postrepair mobilization protocol. To explain the apparent differences between the theoretical and actual repair strength of a multistrand repair in a single tension test and the reduced strength of a repair subjected to cyclic loading, we compared single and cyclical tensile loading with different suture in vitro configurations of 3-0 Ethibond (Ethicon, Somerville, NJ, USA; one, two, and four strands) and an ex vivo four-strand repair of freshly divided porcine tendon to calculate the ultimate tensile strength (UTS). Mechanical testing was repeated 15 times with both single tensile and cyclical loading for each suture configuration and porcine repair. In the in vitro model, the presence of a knot in a single strand reduced the UTS by 50%. The stiffness of a knotted strand was substantially less than the unknotted strand but became identical after cyclical loading. There was no statistical significance of the UTS between single and cyclical loading with different numbers of strands in this model. In the ex vivo four-strand porcine repair model, there was a significant reduction in UTS with cyclical loading, which equated to the number of strands times the strength of the knotted strand. This discrepancy can be explained by the change in stiffness of the knotted strand after cyclical loading and has important implications for previous studies of suture tendon repair using single tensile loading where the UTS may have been overestimated. We believe that cyclical loading is more representative of physiological loading after acute flexor tendon repair and should be the testing model of choice in suture tenorrhaphy studies.

  9. Histological evaluation of vertical laser channels from ablative fractional resurfacing: an ex vivo pig skin model.

    PubMed

    Skovbølling Haak, Christina; Illes, Monica; Paasch, Uwe; Hædersdal, Merete

    2011-07-01

    Ablative fractional resurfacing (AFR) represents a new treatment potential for various skin conditions and new laser devices are being introduced. It is important to gain information about the impact of laser settings on the dimensions of the created laser channels for obtaining a safe and efficient treatment outcome. The aim of this study was to establish a standard model to document the histological tissue damage profiles after AFR and to test a new laser device at diverse settings. Ex vivo abdominal pig skin was treated with a MedArt 620, prototype fractional carbon dioxide (CO(2)) laser (Medart, Hvidovre, Denmark) delivering single microbeams (MB) with a spot size of 165 μm. By using a constant pulse duration of 2 ms, intensities of 1-18 W, single and 2-4 stacked pulses, energies were delivered in a range from 2-144 mJ/MB. Histological evaluations included 3-4 high-quality histological measurements for each laser setting (n = 28). AFR created cone-shaped laser channels. Ablation depths varied from reaching the superficial dermis (2 mJ, median 41 μm) to approaching the subcutaneous fat (144 mJ, median 1,943 μm) and correlated to the applied energy levels in an approximate linear relation (r(2) = 0.84, p < 0.001). The dermal ablation width increased slightly within the energy range of 4-144 mJ (median 163 μm). The thickness of the coagulation zone reached a plateau around 65 μm at energies levels above 16 mJ. The calculated volumes of ablated tissue increased with increasing energies. We suggest this ex vivo pig skin model to characterize AFR laser channels histologically.

  10. Cytokine combinations on the potential for ex vivo expansion of murine hematopoietic stem cells.

    PubMed

    Lui, Wing Chi; Chan, Yuen Fan; Chan, Li Chong; Ng, Ray Kit

    2014-08-01

    Hematopoietic stem cell (HSC) is a rare cell population, which is capable of self-renewal and differentiation to all blood lineages. The clinical potential of HSCs for treating hematological disorders has led to the use of cytokine stimulation for ex vivo expansion. However, little is known about the molecular features of the HSC populations expanded under different cytokine combinations. We studied the expansion of murine HSCs cultured with six different cytokine combinations under serum-containing or serum-free conditions for 14days. We found that all the cytokine combinations promoted expansion of murine HSCs. Although SCF/IL-3/IL-6 induced the highest expansion of the immunophenotypic Lineage(-)Sca-1(+)c-Kit(+) (LSK) cells at day 14, over 90% of them were FcεRIα(+) mast cells. In contrast, the serum-free medium with SCF/Flt3-L/IL-11 effectively promoted the expansion of LSK/FcεRIα(-) HSCs by over 50-fold. HSCs expanded by SCF/Flt3-L/IL-11 combination formed compact hematopoietic colonies and demonstrated a higher degree of multipotency compared to the HSCs cultured with other cytokine combinations. Surprisingly, despite the same LSK/FcεRIα(-) immunophenotype, HSCs cultured with different cytokine combinations demonstrated differential patterns of hematopoietic gene expression. HSCs cultured with SCF/Flt3-L/IL-11 maintained a transcription profile resembling that of freshly isolated HSCs. We propose that serum-free medium supplemented with SCF/Flt3-L/IL-11 is the optimal culture condition to maintain the stemness of ex vivo expanded HSCs. This study used molecular characterization of cytokine-expanded murine HSCs to facilitate the selection of cytokine combinations that could induce fully competent HSC for clinical applications.

  11. Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

    PubMed

    Furuya, Kishio; Tan, Ju Jing; Boudreault, Francis; Sokabe, Masahiro; Berthiaume, Yves; Grygorczyk, Ryszard

    2016-11-01

    Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (<30 s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ∼10(-6) M, relevant for autocrine/paracrine signaling, but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus dependent: prolonged (100 s) inflation evoked long-lasting ATP release that terminated upon alveoli deflation/derecruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patchlike areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP release imaging in ex vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.

  12. Tolerance of human placental tissue to severe hypoxia and its relevance for dual ex vivo perfusion.

    PubMed

    Schneider, H

    2009-03-01

    In the dual ex vivo perfusion of an isolated human placental cotyledon it takes on average 20-30 min to set up stable perfusion circuits for the maternal and fetal vascular compartments. In vivo placental tissue of all species maintains a highly active metabolism and it continues to puzzle investigators how this tissue can survive 30 min of ischemia with more or less complete anoxia following expulsion of the organ from the uterus and do so without severe damage. There seem to be parallels between "depressed metabolism" seen in the fetus and the immature neonate in the peripartum period and survival strategies described in mammals with increased tolerance of severe hypoxia like hibernators in the state of torpor or deep sea diving turtles. Increased tolerance of hypoxia in both is explained by "partial metabolic arrest" in the sense of a temporary suspension of Kleiber's rule. Furthermore the fetus can react to major changes in surrounding oxygen tension by decreasing or increasing the rate of specific basal metabolism, providing protection against severe hypoxia as well as oxidative stress. There is some evidence that adaptive mechanisms allowing increased tolerance of severe hypoxia in the fetus or immature neonate can also be found in placental tissue, of which at least the villous portion is of fetal origin. A better understanding of the molecular details of reprogramming of fetal and placental tissues in late pregnancy may be of clinical relevance for an improved risk assessment of the individual fetus during the critical transition from intrauterine life to the outside and for the development of potential prophylactic measures against severe ante- or intrapartum hypoxia. Responses of the tissue to reperfusion deserve intensive study, since they may provide a rational basis for preventive measures against reperfusion injury and related oxidative stress. Modification of the handling of placental tissue during postpartum ischemia, and adaptation of the

  13. Full Mimicking of Coronary Hemodynamics for Ex-Vivo Stimulation of Human Saphenous Veins.

    PubMed

    Piola, Marco; Ruiter, Matthijs; Vismara, Riccardo; Mastrullo, Valeria; Agrifoglio, Marco; Zanobini, Marco; Pesce, Maurizio; Soncini, Monica; Fiore, Gianfranco Beniamino

    2017-04-01

    After coronary artery bypass grafting, structural modifications of the saphenous vein wall lead to lumen narrowing in response to the altered hemodynamic conditions. Here we present the design of a novel ex vivo culture system conceived for mimicking central coronary artery hemodynamics, and we report the results of biomechanical stimulation experiments using human saphenous vein samples. The novel pulsatile system used an aortic-like pressure for forcing a time-dependent coronary-like resistance to obtain the corresponding coronary-like flow rate. The obtained pulsatile pressures and flow rates (diastolic/systolic: 80/120 mmHg and 200/100 mL/min, respectively) showed a reliable mimicking of the complex coronary hemodynamic environment. Saphenous vein segments from patients undergoing coronary artery bypass grafting (n = 12) were subjected to stimulation in our bioreactor with coronary pulsatile pressure/flow patterns or with venous-like perfusion. After 7-day stimulation, SVs were fixed and stained for morphometric evaluation and immunofluorescence. Results were compared with untreated segments of the same veins. Morphometric and immunofluorescence analysis revealed that 7 days of pulsatile stimulation: (i) did not affect integrity of the vessel wall and lumen perimeter, (ii) significantly decreased both intima and media thickness, (iii) led to partial endothelial denudation, and (iv) induced apoptosis in the vessel wall. These data are consistent with the early vessel remodeling events involved in venous bypass adaptation to arterial flow/pressure patterns. The pulsatile system proved to be a suitable device to identify ex vivo mechanical cues leading to graft adaptation.

  14. 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.

  15. Viability of human chondrocytes in an ex vivo model in relation to temperature and cartilage depth.

    PubMed

    Drobnic, M; Mars, T; Alibegović, A; Bole, V; Balazic, J; Grubic, Z; Brecelj, J

    2005-01-01

    Chondrocytes in human articular cartilage remain viable post-mortem. It has however not been established yet how the storage temperature affects their survival, which is essential information when post-mortem cartilage is used for toxicologic studies. Our aim was to construct a simple model of explanted knee cartilage and to test the influences of time and temperature on the viability of chondrocytes in the ex vivo conditions. Osteochondral cylinders were procured from the cadaveric femoral condyles. The cylinders were embedded in water-tight rubber tubes, which formed separate chondral and osteal compartments. Tubes were filled with normal saline, without additives, to keep chondrocytes under close-to-normal conditions. The samples were divided into two groups stored at 4 degrees C and 35 degrees C, respectively. Three samples of each of these two groups were analysed at the time of removal, and then three and nine days later. Images of Live-Dead staining were scanned by a confocal laser microscope. Count of viable chondrocytes in four regions, from surface to bone, was obtained using image analysis software. The regression model revealed that the number of viable chondrocytes decreased every day by 19% and that an increase in temperature by 1 degree C decreased their viability by 5.8%. The temperature effect fell by 0.2 percentage points for every 100 microm from the surface to the bone. Herein we demonstrate that chondrocytes remain viable in the ex vivo model of human knee cartilage long enough to be able to serve as a model for toxicologic studies. Their viability is, however, significantly influenced by time and temperature.

  16. Impact of immunosuppressive drugs on the therapeutic efficacy of ex vivo expanded human regulatory T cells

    PubMed Central

    Scottà, Cristiano; Fanelli, Giorgia; Hoong, Sec Julie; Romano, Marco; Lamperti, Estefania Nova; Sukthankar, Mitalee; Guggino, Giuliana; Fazekasova, Henrieta; Ratnasothy, Kulachelvy; Becker, Pablo D.; Afzali, Behdad; Lechler, Robert I.; Lombardi, Giovanna

    2016-01-01

    Immunosuppressive drugs in clinical transplantation are necessary to inhibit the immune response to donor antigens. Although they are effective in controlling acute rejection, they do not prevent long-term transplant loss from chronic rejection. In addition, immunosuppressive drugs have adverse side effects, including increased rate of infections and malignancies. Adoptive cell therapy with human Tregs represents a promising strategy for the induction of transplantation tolerance. Phase I/II clinical trials in transplanted patients are already underway, involving the infusion of Tregs alongside concurrent immunosuppressive drugs. However, it remains to be determined whether the presence of immunosuppressive drugs negatively impacts Treg function and stability. We tested in vitro and in vivo the effects of tacrolimus, mycophenolate and methylprednisolone (major ISDs used in transplantation) on ex vivo expanded, rapamycin-treated human Tregs. The in vitro results showed that these drugs had no effect on phenotype, function and stability of Tregs, although tacrolimus affected the expression of chemokine receptors and IL-10 production. However, viability and proliferative capacity were reduced in a dose-dependent manner by all the three drugs. The in vivo experiments using a humanized mouse model confirmed the in vitro results. However, treatment of mice with only rapamycin maintained the viability, function and proliferative ability of adoptively transferred Tregs. Taken together, our results suggest that the key functions of ex vivo expanded Tregs are not affected by a concurrent immunosuppressive therapy. However, the choice of the drug combination and their timing and dosing should be considered as an essential component to induce and maintain tolerance by Treg. PMID:26471483

  17. 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.

  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. Hepatic ablation with multiple interstitial ultrasound applicators: initial ex vivo and computational studies

    NASA Astrophysics Data System (ADS)

    Prakash, Punit; Salgaonkar, Vasant A.; Burdette, E. Clif; Diederich, Chris J.

    2011-03-01

    Radiofrequency (RF) ablation has emerged as an effective method for treating liver tumors under 3 cm in diameter. Multiple applicator devices and techniques - using RF, microwave and other modalities - are under development for thermal ablation of large and irregularly-shaped liver tumors. Interstitial ultrasound (IUS) applicators, comprised of linear arrays of independently powered tubular transducers, enable 3D control of the spatial power deposition profile and simultaneous ablation with multiple applicators. We evaluated IUS applicator configurations (parallel, converging and diverging implants) suitable for percutaneous and laparascopic placement with experiments in ex vivo bovine tissue and computational models. Ex vivo ablation zones measured 4.6+/-0.5 x 4.2+/-0.5 × 3.3+/-0.5 cm3 and 5.6+/-0.5 × 4.9+/-0.5 x 2.8+/-0.3 cm3 using three parallel applicators spaced 2 and 3 cm apart, respectively, and 4.0+/-0.3 × 3.2+/-0.4 × 2.9+/-0.2 cm3 using two parallel applicators spaced 2 cm apart. Computational models indicate in vivo ablation zones up to 4.5 × 4.4 × 5.5 cm3 and 5.7 × 4.8 × 5.2 cm3, using three applicators spaced 2 and 3 cm apart, respectively. Converging and diverging implant patterns can also be employed for conformal ablation of irregularly-shaped tumor margins by tailoring power levels along each device. Simultaneously powered interstitial ultrasound devices can create tailored ablation zones comparable to currently available RF devices and similarly sized microwave antennas.

  20. 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.

  1. Ex Vivo Electromechanical Reshaping of Costal Cartilage in the New Zealand White Rabbit Model

    PubMed Central

    Badran, Karam; Manuel, Cyrus; Waki, Curtis; Protsenko, Dmitry; Wong, Brian J. F.

    2014-01-01

    Objectives/Hypothesis Determine the effective electromechanical reshaping (EMR) parameters for shape change and cell viability in the ex vivo rabbit costal cartilage model. Study Design Ex vivo animal study combined with computer modeling to guide electrode placement and polarity selection. Methods Rabbit costal cartilages were secured in a jig that approximated the shape of the rabbit auricle framework. Finite element modeling was used to select the initial electrode geometry, polarity, spacing, and estimate dosimetry parameters. Porcine costal cartilage was utilized to refine the selection of dosing parameters. Parametric analysis was performed to determine the effect of voltage and application time on tissue shape change. Next, rabbit rib cartilage was reshaped, varying voltage and application time to identify the lowest parameters to produce acceptable shape change mimicking native auricular cartilage. Acceptable qualitative shape change was determined on a five-point Likert scale analyzed using one-way general linear analysis of variance. Confocal microscopy with live/dead cell viability analysis determined the degree of injury and the distribution of live and dead cells. Results The minimum acceptable deformation of rabbit costal cartilage was found at 4 V–3 minutes. Viability analysis of cartilage reshaped at 4 V–3 minutes demonstrates cell injury extending 2 mm away from each electrode with viable cells found between the electrodes. Conclusions The EMR parameters of 4 V–3 minutes demonstrates appropriate shape change producing grafts that resemble the native auricle and contains the viable cells adequate for clinical evaluation. The rabbit auricular reconstruction model using EMR is a feasible one. PMID:23553270

  2. 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

  3. 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

  4. Equivalent metabolic acidosis with four colloids and saline on ex vivo haemodilution.

    PubMed

    Morgan, T J; Vellaichamy, M; Cowley, D M; Weier, S L; Venkatesh, B; Jones, M A

    2009-05-01

    Colloid infusions can cause metabolic acidosis. Mechanisms and relative severity with different colloids are incompletely understood. We compared haemodilution acid-base effects of 4% albumin, 3.5% polygeline, 4% succinylated gelatin (all weak acid colloids, strong ion difference 12 mEq/l, 17.6 mEq/l and 34 mEq/l respectively), 6% hetastarch (non-weak acid colloid, strong ion difference zero) and 0.9% saline (crystalloid, strong ion difference zero). Gelatin weak acid properties were tracked via the strong ion gap. Four-step ex vivo dilutions of pre-oxygenated human venous blood were performed to a final [Hb] near 50% baseline. With each fluid, base excess fell to approximately -13 mEq/l. Base excess/[Hb] relationships across dilution were linear and direct (R2 > or = 0.96), slopes and intercepts closely resembling saline. Baseline strong ion gap was -0.3 (2.1) mEq/l. Post-dilution increases occurred in three groups: small with saline, hetastarch and albumin (to 3.5 (02) mEq/l, 4.3 (0.3) mEq/l, 3.3 (1.4) mEq/l respectively), intermediate with polygeline (to 12.2 (0.9) mEq/l) and greatest with succinylated gelatin (to 20.8 (1.4) mEq/l). We conclude that, despite colloid weak acid activity ranging from zero (hydroxyethyl starch) to greater than that of albumin with both gelatin preparations, ex vivo dilution causes a metabolic acidosis of identical severity to saline in each case. This uniformity reflects modifications to the albumin and gelatin saline vehicles, in part aimed at pH correction. By proportionally increasing the strong ion difference, these modifications counter deviations from pure saline effects caused by colloid weak acid activity. Extrapolation in vivo requires further investigation.

  5. 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

  6. Mapping of the internal structure of human habenula with ex vivo MRI at 7T

    PubMed Central

    Strotmann, Barbara; Kögler, Carsten; Bazin, Pierre-Louis; Weiss, Marcel; Villringer, Arno; Turner, Robert

    2013-01-01

    The habenula is a small but important nucleus located next to the third ventricle in front of the pineal body. It helps to control the human reward system and is considered to play a key role in emotion, showing increased activation in major depressive disorders. Its dysfunction may underlie several neurological and psychiatric disorders. It is now possible to visualize the habenula and its anatomical subdivisions—medial habenula (MHB) and lateral habenula (LHB)—using MR techniques. The aim of this study was to further differentiate substructures within human lateral habenula (LHB) using ex vivo ultra-high field MR structural imaging, distinguishing between a medial part (m-LHB) and a lateral part (l-LHB). High resolution T1w images with 0.3-mm isotropic resolution and T2*w images with 60-micrometer isotropic resolution were acquired on a 7T MR scanner and quantitative maps of T1 and T2* were calculated. Cluster analysis of image intensity was performed using the Fuzzy and Noise Tolerant Adaptive Segmentation Method (FANTASM) tool. Ultra-high resolution structural MRI of ex vivo brain tissue at 7T provided sufficient SNR and contrast to discriminate the medial and lateral habenular nuclei. Heterogeneity was observed in the lateral habenula (LHB) nuclei, with clear distinctions between lateral and medial parts (m-LHB, l-LHB) and with the neighboring medial habenula (MHB). Clustering analysis based on the T1 and T2* maps strongly showed 4–6 clusters as subcomponents of lateral and medial habenula. PMID:24391571

  7. Respiratory-Gated Helical Computed Tomography of Lung: Reproducibility of Small Volumes in an Ex Vivo Model

    SciTech Connect

    Biederer, Juergen Dinkel, Julien; Bolte, Hendrik; Welzel, Thomas; Hoffmann, Beata M.Sc.; Thierfelder, Carsten; Mende, Ulrich; Debus, Juergen; Heller, Martin; Kauczor, Hans-Ulrich

    2007-12-01

    Purpose: Motion-adapted radiotherapy with gated irradiation or tracking of tumor positions requires dedicated imaging techniques such as four-dimensional (4D) helical computed tomography (CT) for patient selection and treatment planning. The objective was to evaluate the reproducibility of spatial information for small objects on respiratory-gated 4D helical CT using computer-assisted volumetry of lung nodules in a ventilated ex vivo system. Methods and Materials: Five porcine lungs were inflated inside a chest phantom and prepared with 55 artificial nodules (mean diameter, 8.4 mm {+-} 1.8). The lungs were respirated by a flexible diaphragm and scanned with 40-row detector CT (collimation, 24 x 1.2 mm; pitch, 0.1; rotation time, 1 s; slice thickness, 1.5 mm; increment, 0.8 mm). The 4D-CT scans acquired during respiration (eight per minute) and reconstructed at 0-100% inspiration and equivalent static scans were scored for motion-related artifacts (0 or absent to 3 or relevant). The reproducibility of nodule volumetry (three readers) was assessed using the variation coefficient (VC). Results: The mean volumes from the static and dynamic inspiratory scans were equal (364.9 and 360.8 mm{sup 3}, respectively, p = 0.24). The static and dynamic end-expiratory volumes were slightly greater (371.9 and 369.7 mm{sup 3}, respectively, p = 0.019). The VC for volumetry (static) was 3.1%, with no significant difference between 20 apical and 20 caudal nodules (2.6% and 3.5%, p = 0.25). In dynamic scans, the VC was greater (3.9%, p = 0.004; apical and caudal, 2.6% and 4.9%; p = 0.004), with a significant difference between static and dynamic in the 20 caudal nodules (3.5% and 4.9%, p = 0.015). This was consistent with greater motion-related artifacts and image noise at the diaphragm (p <0.05). The VC for interobserver variability was 0.6%. Conclusion: Residual motion-related artifacts had only minimal influence on volumetry of small solid lesions. This indicates a high

  8. “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

  9. An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine.

    PubMed

    Schwerdtfeger, Luke A; Ryan, Elizabeth P; Tobet, Stuart A

    2016-02-15

    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.

  10. Clinical Studies of Ex Vivo Expansion to Accelerate Engraftment After Umbilical Cord Blood Transplantation: A Systematic Review.

    PubMed

    Kiernan, Jeffrey; Damien, Pauline; Monaghan, Madeline; Shorr, Risa; McIntyre, Lauralyn; Fergusson, Dean; Tinmouth, Alan; Allan, David

    2016-12-23

    Cell dose limits greater use of umbilical cord blood (UCB) in hematopoietic cell transplantation. The clinical benefits of ex vivo expansion need clarity to understand its potential impact. A systematic search of studies addressing UCB ex vivo expansion was conducted. Fifteen clinical studies (349 transplanted patients) and 13 registered trials were identified. The co-infusion of an expanded unit and a second unmanipulated unit (8 studies), the fractional expansion of 12% to 60% of a single unit (5 studies), and the infusion of a single expanded unit (2 studies) were reported. More recently, published studies and 12 of 13 ongoing trials involve the use of novel small molecules in addition to traditional cytokine cocktails. Higher total cell number was closely associated with faster neutrophil engraftment. Compared with historical controls, neutrophil engraftment was significantly accelerated in more recent studies using small molecules or mesenchymal stromal cells (MSC) co-culture, and in some cases, platelet recovery was also statistically improved. Recent studies using nicotinamide and StemRegenin-1 reported long-term chimerism of the expanded unit. No significant improvement in survival or other transplant-related outcomes was demonstrated for any of the strategies. Ex vivo expansion of UCB can accelerate initial neutrophil engraftment after transplant. More recent studies suggest that long-term engraftment of ex vivo expanded cord blood units is achievable. Results of larger randomized controlled trials are needed to understand the impact on patient outcomes and health care costs.

  11. 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...

  12. 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

    2015-09-09

    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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Mouse lung slices: An ex vivo model for the evaluation of antiviral and anti-inflammatory agents against influenza viruses.

    PubMed

    Liu, Rui; An, Liwei; Liu, Ge; Li, Xiaoyu; Tang, Wei; Chen, Xulin

    2015-08-01

    The influenza A virus is notoriously known for its ability to cause recurrent epidemics and global pandemics. Antiviral therapy is effective when treatment is initiated within 48h of symptom onset, and delaying treatment beyond this time frame is associated with decreased efficacy. Research on anti-inflammatory therapy to ameliorate influenza-induced inflammation is currently underway and seems important to the impact on the clinical outcome. Both antiviral and anti-inflammatory drugs with novel mechanisms of action are urgently needed. Current methods for evaluating the efficacy of anti-influenza drugs rely mostly on transformed cells and animals. Transformed cell models are distantly related to physiological and pathological conditions. Although animals are the best choices for preclinical drug testing, they are not time- or cost-efficient. In this study, we established an ex vivo model using mouse lung slices to evaluate both antiviral and anti-inflammatory agents against influenza virus infection. Both influenza virus PR8 (H1N1) and A/Human/Hubei/3/2005 (H3N2) can replicate efficiently in mouse lung slices and trigger significant cytokine and chemokine responses. The induction of selected cytokines and chemokines were found to have a positive correlation between ex vivo and in vivo experiments, suggesting that the ex vivo cultured lung slices may closely resemble the lung functionally in an in vivo configuration when challenged by influenza virus. Furthermore, a set of agents with known antiviral and/or anti-inflammatory activities were tested to validate the ex vivo model. Our results suggested that mouse lung slices provide a robust, convenient and cost-efficient model for the assessment of both antiviral and anti-inflammatory agents against influenza virus infection in one assay. This ex vivo model may predict the efficacy of drug candidates' antiviral and anti-inflammatory activities in vivo.

  18. A small molecule modulator of prion protein increases human mesenchymal stem cell lifespan, ex vivo expansion, and engraftment to bone marrow in NOD/SCID mice.

    PubMed

    Mohanty, Sindhu T; Cairney, Claire J; Chantry, Andrew D; Madan, Sanjeev; Fernandes, James A; Howe, Steven J; Moore, Harry D; Thompson, Mark J; Chen, Beining; Thrasher, Adrian; Keith, W Nicol; Bellantuono, Ilaria

    2012-06-01

    Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine.

  19. Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

    PubMed

    Meneghini, Vasco; Frati, Giacomo; Sala, Davide; De Cicco, Silvia; Luciani, Marco; Cavazzin, Chiara; Paulis, Marianna; Mentzen, Wieslawa; Morena, Francesco; Giannelli, Serena; Sanvito, Francesca; Villa, Anna; Bulfone, Alessandro; Broccoli, Vania; Martino, Sabata; Gritti, Angela

    2016-09-16

    : Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains.

  20. Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

    PubMed

    Meneghini, Vasco; Frati, Giacomo; Sala, Davide; De Cicco, Silvia; Luciani, Marco; Cavazzin, Chiara; Paulis, Marianna; Mentzen, Wieslawa; Morena, Francesco; Giannelli, Serena; Sanvito, Francesca; Villa, Anna; Bulfone, Alessandro; Broccoli, Vania; Martino, Sabata; Gritti, Angela

    2017-02-01

    Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells

  1. Evaluation of three-dimensional temperature distributions produced by a low-frequency transcranial focused ultrasound system within ex vivo human skulls.

    PubMed

    McDannold, Nathan; Park, Eun-Joo; Mei, Chang-Sheng; Zadicario, Eyal; Jolesz, Ferenc

    2010-09-01

    Transcranial MR-guided focused ultrasound (TcMRgFUS) provides a potential noninvasive alternative to surgical resection and for other treatments for brain disorders. Use of low-frequency ultrasound provides several advantages for TcMRgFUS, but is potentially limited by reflection and standing wave effects that may cause secondary hotspots within the skull cavity. The purpose of this work was to use volumetric magnetic resonance temperature imaging (MRTI) and ex vivo human skulls filled with tissue-mimicking phantom material to search for heating distant from the focal point that may occur during sonication with a TcMRgFUS system as a result of reflections or standing wave effects. Heating during 120-s sonications was monitored within the entire skull volume for 12 different locations in two different skulls. The setup used a hemispheric array operating at 220 kHz. Multiple sonications were delivered at each location while varying the MRTI slice positions to provide full coverage of the skull cavity. An automated routine was used evaluate the MRTI to detect voxel regions that appeared to be heated by ultrasound. No secondary hotspots with a temperature rise of 15% or more of the focal heating were found. The MRTI noise level prevented the identification of possible hotspots with a lower temperature rise. These results suggest that significant secondary heating by this TcMRgFUS system at points distant from the focal point are not common.

  2. 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.

  3. 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.

  4. Development and validation of an ex vivo electron paramagnetic resonance fingernail biodosimetric method.

    PubMed

    He, Xiaoming; Swarts, Steven G; Demidenko, Eugene; Flood, Ann B; Grinberg, Oleg; Gui, Jiang; Mariani, Michael; Marsh, Stephen D; Ruuge, Andres E; Sidabras, Jason W; Tipikin, Dmitry; Wilcox, Dean E; Swartz, Harold M

    2014-06-01

    There is an imperative need to develop methods that can rapidly and accurately determine individual exposure to radiation for screening (triage) populations and guiding medical treatment in an emergency response to a large-scale radiological/nuclear event. To this end, a number of methods that rely on dose-dependent chemical and/or physical alterations in biomaterials or biological responses are in various stages of development. One such method, ex vivo electron paramagnetic resonance (EPR) nail dosimetry using human nail clippings, is a physical biodosimetry technique that takes advantage of a stable radiation-induced signal (RIS) in the keratin matrix of fingernails and toenails. This dosimetry method has the advantages of ubiquitous availability of the dosimetric material, easy and non-invasive sampling, and the potential for immediate and rapid dose assessment. The major challenge for ex vivo EPR nail dosimetry is the overlap of mechanically induced signals and the RIS. The difficulties of analysing the mixed EPR spectra of a clipped irradiated nail were addressed in the work described here. The following key factors lead to successful spectral analysis and dose assessment in ex vivo EPR nail dosimetry: (1) obtaining a thorough understanding of the chemical nature, the decay behaviour, and the microwave power dependence of the EPR signals, as well as the influence of variation in temperature, humidity, water content, and O₂ level; (2) control of the variability among individual samples to achieve consistent shape and kinetics of the EPR spectra; (3) use of correlations between the multiple spectral components; and (4) use of optimised modelling and fitting of the EPR spectra to improve the accuracy and precision of the dose estimates derived from the nail spectra. In the work described here, two large clipped nail datasets were used to test the procedures and the spectral fitting model of the results obtained with it. A 15-donor nail set with 90 nail samples

  5. Cord-Blood Engraftment with Ex Vivo Mesenchymal-Cell Coculture

    PubMed Central

    de Lima, Marcos; McNiece, Ian; Robinson, Simon N.; Munsell, Mark; Eapen, Mary; Horowitz, Mary; Alousi, Amin; Saliba, Rima; McMannis, John D.; Kaur, Indreshpal; Kebriaei, Partow; Parmar, Simrit; Popat, Uday; Hosing, Chitra; Champlin, Richard; Bollard, Catherine; Molldrem, Jeffrey J.; Jones, Roy B.; Nieto, Yago; Andersson, Borje S.; Shah, Nina; Oran, Betul; Cooper, Laurence J.N.; Worth, Laura; Qazilbash, Muzaffar H.; Korbling, Martin; Rondon, Gabriela; Ciurea, Stefan; Bosque, Doyle; Maewal, Ila; Simmons, Paul J.; Shpall, Elizabeth J.

    2013-01-01

    BACKGROUND Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×107 total nucleated cells per kilogram of body weight and 1.81×106 CD34+ cells per kilogram — doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P = 0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT

  6. Quantitative MR Imaging of Hepatic Steatosis: Validation in Ex Vivo Human Livers

    PubMed Central

    Bannas, Peter; Kramer, Harald; Hernando, Diego; Agni, Rashmi; Cunningham, Ashley M.; Mandal, Rakesh; Motosugi, Utaroh; Sharma, Samir D.; del Rio, Alejandro Munoz; Fernandez, Luis; Reeder, Scott B.

    2015-01-01

    Emerging magnetic resonance imaging (MRI) biomarkers of hepatic steatosis have demonstrated tremendous promise for accurate quantification of hepatic triglyceride concentration. These methods quantify the “proton density fat-fraction” (PDFF), which reflects the concentration of triglycerides in tissue. Previous in vivo studies have compared MRI-PDFF with histologic steatosis grading for assessment of hepatic steatosis. However, the correlation of MRI-PDFF with the underlying hepatic triglyceride content remained unknown. The aim of this ex vivo study was to validate the accuracy of MRI-PDFF as an imaging biomarker of hepatic steatosis. Using ex vivo human livers, we compared MRI-PDFF with magnetic resonance spectroscopy-PDFF (MRS-PDFF), biochemical triglyceride extraction and histology as three independent reference standards. A secondary aim was to compare the precision of MRI-PDFF relative to biopsy for the quantification of hepatic steatosis. MRI-PDFF was prospectively performed at 1.5T in 13 explanted human livers. We performed co-localized paired evaluation of liver fat content in all nine Couinaud segments using single-voxel MRS-PDFF (n=117), tissue wedges for biochemical triglyceride extraction (n=117), and five core biopsies performed in each segment for histologic grading (n=585). Accuracy of MRI-PDFF was assessed through linear regression with MRS-PDFF, triglyceride extraction and histology. Intra-observer agreement, inter-observer agreement and repeatability of MRI-PDFF and histologic grading were assessed through Bland-Altman analyses. MRI-PDFF showed an excellent correlation with MRS-PDFF (r=0.984; CI: 0.978–0.989) and strong correlation with histology (r=0.850; CI: 0.791–0.894) and triglyceride extraction (r=0.871; CI: 0.818–0.909). Intra-observer agreement, inter-observer agreement and repeatability showed a significantly smaller variance for MRI-PDFF than for histologic steatosis grading (all p<0.001). Conclusion MRI-PDFF is an accurate

  7. Control of circumferential wall stress and luminal shear stress within intact vascular segments perfused ex vivo.

    PubMed

    El-Kurdi, Mohammed S; Vipperman, Jeffrey S; Vorp, David A

    2008-10-01

    Proportional, integral, and derivative (PID) controllers have proven to be robust in controlling many applications, and remain the most widely used control system architecture. The purpose of this work was to use this architecture for designing and tuning two PID controllers. The first was used to control the physiologic arterial circumferential wall stress (CWS) and the second to control the physiologic arterial shear stress (SS) imposed on intact vascular segments that were implanted into an ex vivo vascular perfusion system (EVPS). In order to most accurately control the stresses imposed onto vascular segments perfused ex vivo, analytical models were derived to calculate the CWS and SS. The mid-vein-wall CWS was calculated using the classical Lame solution for thick-walled cylinders in combination with the intraluminal pressure and outer diameter measurements. Similarly, the SS was calculated using the Hagen-Poiseuille equation in combination with the flow rate and outer diameter measurements. Performance of each controller was assessed by calculating the root mean square of the error (RMSE) between the desired and measured process variables. The performance experiments were repeated ten times (N=10) and an average RMSE was reported for each controller. RMSE standard deviations were calculated to demonstrate the reproducibility of the results. Sterile methods were utilized for making blood gas and temperature measurements in order to maintain physiologic levels within the EVPS. Physiologic blood gases (pH, pO(2), and pCO(2)) and temperature within the EVPS were very stable and controlled manually. Blood gas and temperature levels were recorded hourly for several (N=9) 24 h perfusion experiments. RMSE values for CWS control (0.427+/-0.027 KPa) indicated that the system was able to generate a physiologic CWS wave form within 0.5% error of the peak desired CWS over each cardiac cycle. RMSE values for SS control (0.005+/-0.0007 dynescm(2)) indicated that the system

  8. 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

  9. Temperature-dependent thermal properties of ex vivo liver undergoing thermal ablation.

    PubMed

    Guntur, Sitaramanjaneya Reddy; Lee, Kang Il; Paeng, Dong-Guk; Coleman, Andrew John; Choi, Min Joo

    2013-10-01

    Thermotherapy uses a heat source that raises temperatures in the target tissue, and the temperature rise depends on the thermal properties of the tissue. Little is known about the temperature-dependent thermal properties of tissue, which prevents us from accurately predicting the temperature distribution of the target tissue undergoing thermotherapy. The present study reports the key thermal parameters (specific heat capacity, thermal conductivity and heat diffusivity) measured in ex vivo porcine liver while being heated from 20 ° C to 90 ° C and then naturally cooled down to 20 ° C. The study indicates that as the tissue was heated, all the thermal parameters resulted in plots with asymmetric quasi-parabolic curves with temperature, being convex downward with their minima at the turning temperature of 35-40 ° C. The largest change was observed for thermal conductivity, which decreased by 9.6% from its initial value (at 20 ° C) at the turning temperature (35 ° C) and rose by 45% at 90 ° C from its minimum (at 35 ° C). The minima were 3.567 mJ/(m(3) ∙ K) for specific heat capacity, 0.520 W/(m.K) for thermal conductivity and 0.141 mm(2)/s for thermal diffusivity. The minimum at the turning temperature was unique, and it is suggested that it be taken as a characteristic value of the thermal parameter of the tissue. On the other hand, the thermal parameters were insensitive to temperature and remained almost unchanged when the tissue cooled down, indicating that their variations with temperature were irreversible. The rate of the irreversible rise at 35 ° C was 18% in specific heat capacity, 40% in thermal conductivity and 38.3% in thermal diffusivity. The study indicates that the key thermal parameters of ex vivo porcine liver vary largely with temperature when heated, as described by asymmetric quasi-parabolic curves of the thermal parameters with temperature, and therefore, substantial influence on the temperature distribution of the tissue undergoing

  10. Ex vivo response to mucosal bacteria and muramyl dipeptide in inflammatory bowel disease

    PubMed Central

    Loganes, Claudia; Valencic, Erica; Pin, Alessia; Marini, Elisa; Martelossi, Stefano; Naviglio, Samuele; De Leo, Luigina; Not, Tarcisio; Monasta, Lorenzo; Tommasini, Alberto; Marcuzzi, Annalisa

    2016-01-01

    AIM To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn’s disease (CD) and ulcerative colitis (UC). METHODS Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Two tissue samples were taken from inflamed mucosal segments (in patients with active disease) or from non-inflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo. For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS We demonstrated that subjects with CD display a spontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in

  11. 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

  12. Synthesis, characterization and ex vivo evaluation of polydimethylsiloxane polyurea-urethanes.

    PubMed

    Lim, F; Yang, C Z; Cooper, S L

    1994-05-01

    A series of segmented polydimethylsiloxane (PDMS) polyurea-urethanes was synthesized based on hexane diisocyanate modified polyether-PDMS soft segments. The hard segments consisted of 4,4'-methylene diphenylene diisocyanate, which was chain extended with 1,4-butanediol. The effect of chemical composition of the polyether-PDMS soft segments on the extent of phase separation, physical properties and surface properties was studied using a variety of techniques including dynamic mechanical analysis, tensile testing, X-ray photoelectron spectroscopy (XPS) and contact angle analysis. The polymers were also evaluated for their blood-contacting properties in a canine ex vivo model. The PDMS polyurea-urethanes containing polyether-PDMS soft segments showed three distinct phases: a PDMS-rich phase, a polyol soft segment-rich phase and a hard segment-rich phase. The tensile strength and modulus of these materials were not significantly lower compared to a polymer without PDMS in the soft segment. XPS revealed the surface enrichment of the hydrophobic PDMS component at the air-solid interface. Dynamic contact angle measurements indicated that the PDMS-based polyurea-urethanes possessed a hydrophobic surface in water. The PDMS polyurea-urethanes showed lower adherent platelet and fibrinogen deposition compared to a polymer without PDMS in the soft segment. Varying the amount of PDMS in the soft segment of these polymers did not reveal significant differences in their blood-contacting properties.

  13. 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.

  14. Air trapping ability of the Spiral Gold membrane oxygenator: an ex vivo study.

    PubMed

    Mueller, X M; Tevaearai, H T; van Ness, K; Horisberger, J; Augstburger, M; Burki, M; von Segesser, L K

    1998-01-01

    Despite an overall improvement in cardiopulmonary bypass (CPB) technology and materials, air emboli still occur. The latest generation membrane oxygenator from Bentley Laboratories, the SpiralGold, was tested ex vivo for its air handling ability. The study was conducted on four calves. Bolus amounts of air of 10, 15 and 20 cm3 were each injected three times, upstream of the oxygenator and a bubble detector located directly downstream. The amount of bubbles was measured semiquantitatively on a 10 unit scale (U one semiquantitative unit). The animals were killed 10 days after the CPB. When 10 cm3 of air was injected, no bubbles were detected. With 15 and 20 cm3, respectively, 1 +/- 1.5 and 5 +/- 3.3 U of bubbles were detected. Despite a total of 135 cm3 of air injected as large bolus amounts, all the animals survived without any obvious neurological deficit secondary to air bubble manipulation. In conclusion, the SpiralGold oxygenator per se can reliably trap an air bolus of up to 10 cm3. This feature should be taken into account when choosing an oxygenator, as it offers an additional barrier to air bubbles in the CPB circuit.

  15. Confocal raman microspectral imaging of ex vivo human spinal cord tissue.

    PubMed

    Wang, Shuang; Liang, Zhuowen; Gong, Yuzhe; Yin, Yaning; Wang, Kaige; He, Qingli; Wang, Zhe; Bai, Jintao

    2016-10-01

    Confocal Raman microspectral imaging (CRMI) provides a versatile tool to illustrate the biochemical nature and structure of biological tissue without introducing any external labels. In this work, a precise correlation was established between the biochemical profile and histological architecture of ex vivo human spinal cord tissue by using CRMI with 633nm excitation. After precisely linking the spectral features to the chemical constituents, much information about the molecular composition of both gray and white matter were revealed. Two-dimensional Raman images were generated by integrating the intensities of the characteristic Raman bands in the area of the intermediate column and ventral horn. K-mean cluster analysis was further applied to visualize the underlying morphological basis of spinal cord tissue by chemical component types and their distribution pattern. Lipid-rich white matter could be visually distinguished from gray matter considering a CH2 bending/scissoring band at 1445cm(-1) and an amide III band at 1250cm(-1). Meanwhile, the formation and distribution pattern of perineuronal nets (PNNs) in the scanning area was validated by the integration of saccharides (617cm(-1)) and amide III bands. Moreover, the heme profile indicated a higher degree of vascularization in gray matter. All of the results obtained testified to the possibility that gray matter could be more susceptible to spinal cord injury (SCI) because of capillary network distribution and glycosaminoglycans (GAGs) aggregation. These findings are important for interpreting the morphological specificity of human spinal cord tissue, and also for studying the molecular basis of SCI.

  16. Amelogenin-assisted ex vivo remineralization of human enamel: Effects of supersaturation degree and fluoride concentration.

    PubMed

    Fan, Yuwei; Nelson, James R; Alvarez, Jason R; Hagan, Joseph; Berrier, Allison; Xu, Xiaoming

    2011-05-01

    The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions, and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by field emission-scanning electron microscopy, attenuated total reflection-Fourier transformed infrared and X-ray diffraction. The concentration of fluoride and the supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP)=10.2±2.0 with 1.5±0.5 mg l(-1) fluoride and 40±10 μg ml(-1) amelogenin, pH 6.8±0.4. A phase diagram summarizes the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management.

  17. Hybrid System for Ex Vivo Hemorheological and Hemodynamic Analysis: A Feasibility Study.

    PubMed

    Yeom, Eunseop; Jun Kang, Yang; Lee, Sang Joon

    2015-06-19

    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.

  18. Amelogenin-assisted ex vivo remineralization of human enamel: effects of supersaturation degree and fluoride concentration

    PubMed Central

    Fan, Yuwei; Nelson, James R.; Alvarez, Jason R.; Hagan, Joseph; Berrier, Allison; Xu, Xiaoming

    2011-01-01

    The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by FE-SEM, ATR-FTIR and XRD. The concentration of fluoride and supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP) = 10.2±2.0 with fluoride 1.5±0.5 mg/L and amelogenin 40±10 µg/mL, pH 6.8±0.4. A phase diagram summarized the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management. PMID:21256987

  19. 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

  20. Blind source separation of ex-vivo aorta tissue multispectral images

    PubMed Central

    Galeano, July; Perez, Sandra; Montoya, Yonatan; Botina, Deivid; Garzón, Johnson

    2015-01-01

    Blind Source Separation methods (BSS) aim for the decomposition of a given signal in its main components or source signals. Those techniques have been widely used in the literature for the analysis of biomedical images, in order to extract the main components of an organ or tissue under study. The analysis of skin images for the extraction of melanin and hemoglobin is an example of the use of BSS. This paper presents a proof of concept of the use of source separation of ex-vivo aorta tissue multispectral Images. The images are acquired with an interference filter-based imaging system. The images are processed by means of two algorithms: Independent Components analysis and Non-negative Matrix Factorization. In both cases, it is possible to obtain maps that quantify the concentration of the main chromophores present in aortic tissue. Also, the algorithms allow for spectral absorbance of the main tissue components. Those spectral signatures were compared against the theoretical ones by using correlation coefficients. Those coefficients report values close to 0.9, which is a good estimator of the method’s performance. Also, correlation coefficients lead to the identification of the concentration maps according to the evaluated chromophore. The results suggest that Multi/hyper-spectral systems together with image processing techniques is a potential tool for the analysis of cardiovascular tissue. PMID:26137366

  1. Blind source separation of ex-vivo aorta tissue multispectral images.

    PubMed

    Galeano, July; Perez, Sandra; Montoya, Yonatan; Botina, Deivid; Garzón, Johnson

    2015-05-01

    Blind Source Separation methods (BSS) aim for the decomposition of a given signal in its main components or source signals. Those techniques have been widely used in the literature for the analysis of biomedical images, in order to extract the main components of an organ or tissue under study. The analysis of skin images for the extraction of melanin and hemoglobin is an example of the use of BSS. This paper presents a proof of concept of the use of source separation of ex-vivo aorta tissue multispectral Images. The images are acquired with an interference filter-based imaging system. The images are processed by means of two algorithms: Independent Components analysis and Non-negative Matrix Factorization. In both cases, it is possible to obtain maps that quantify the concentration of the main chromophores present in aortic tissue. Also, the algorithms allow for spectral absorbance of the main tissue components. Those spectral signatures were compared against the theoretical ones by using correlation coefficients. Those coefficients report values close to 0.9, which is a good estimator of the method's performance. Also, correlation coefficients lead to the identification of the concentration maps according to the evaluated chromophore. The results suggest that Multi/hyper-spectral systems together with image processing techniques is a potential tool for the analysis of cardiovascular tissue.

  2. Dermal absorption and skin damage following hydrofluoric acid exposure in an ex vivo human skin model.

    PubMed

    Dennerlein, Kathrin; Kiesewetter, Franklin; Kilo, Sonja; Jäger, Thomas; Göen, Thomas; Korinth, Gintautas; Drexler, Hans

    2016-04-25

    The wide industrial use of hydrofluoric acid (HF) poses a high risk for accidental dermal exposure. Despite local and systemic hazards associated with HF, information on percutaneous penetration and tissue damage is rare. In the present ex vivo study, the dermal absorption of HF (detected in terms of fluoride ions) was quantified and the skin damaging potential as a function of concentration and exposure duration was assessed. Percutaneous penetration of HF (c=5, 30, and 50%) at 3 exposure durations (3, 5, and 10 min) was investigated in a static diffusion cell model using freshly excised human skin. Alterations of skin were histologically evaluated. HF rapidly penetrated through skin under formation of a considerable intradermal reservoir (∼ 13-67% of total absorbed fluoride). Histologically, epidermal alterations were detected already after exposure to 5% HF for 3 min. The degree of skin damage increased with rising concentration and exposure duration leading to coagulation necrosis. For HF concentrations of ≥ 30%, skin damage progressed into deeper skin layers. Topically applied HF concentration was the principal parameter determining HF induced skin effects. The intradermal HF retention capacity associated with progression and prolongation of HF induced skin effects must be considered in the review of skin decontamination procedures.

  3. Ex vivo production of autologous whole inactivated HIV-1 for clinical use in therapeutic vaccines.

    PubMed

    Gil, Cristina; Climent, Núria; García, Felipe; Hurtado, Carmen; Nieto-Márquez, Sara; León, Agathe; García, M Teresa; Rovira, Cristina; Miralles, Laia; Dalmau, Judith; Pumarola, Tomás; Almela, Manel; Martinez-Picado, Javier; Lifson, Jeffrey D; Zamora, Laura; Miró, José M; Brander, Christian; Clotet, Bonaventura; Gallart, Teresa; Gatell, José M

    2011-08-05

    This study provides a detailed description and characterization of the preparation of individualized lots of autologous heat inactivated HIV-1 virions used as immunogen in a clinical trial designed to test an autologous dendritic-cell-based therapeutic HIV-1 vaccine (Clinical Trial DCV-2, NCT00402142). For each participant, ex vivo isolation and expansion of primary virus were performed by co-culturing CD4-enriched PBMCs from the HIV-1-infected patient with PBMC from HIV-seronegative unrelated healthy volunteer donors. The viral supernatants were heat-inactivated and concentrated to obtain 1 mL of autologous immunogen, which was used to load autologous dendritic cells of each patient. High sequence homology was found between the inactivated virus immunogen and the HIV-1 circulating in plasma at the time of HIV-1 isolation. Immunogens contained up to 10⁹ HIV-1 RNA copies/mL showed considerably reduced infectivity after heat inactivation (median of 5.6 log₁₀), and were free of specified adventitious agents. The production of individualized lots of immunogen based on autologous inactivated HIV-1 virus fulfilling clinical-grade good manufacturing practice proved to be feasible, consistent with predetermined specifications, and safe for use in a clinical trial designed to test autologous dendritic cell-based therapeutic HIV-1 vaccine.

  4. 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

  5. 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

  6. An ex vivo feasibility experimental study on targeted cell surgery by high intensity focused ultrasound

    NASA Astrophysics Data System (ADS)

    Wang, Zhi Biao; Wu, Junru; Fang, Liao Qiong; Wang, Hua; Li, Fa Qi; Tian, Yun Bo; Gong, Xiao Bo; Zhang, Hong; Zhang, Lian; Feng, Ruo

    2012-10-01

    High intensity focused ultrasound (HIFU) has become a new noninvasive surgical modality in medicine. A portion of tissue seated inside a patient's body may experience coagulative necrosis after a few seconds of insonification by high intensity focused ultrasound (US) generated by an extracorporeal focusing US transducer. The region of tissue affected by coagulative necrosis (CN) usually has an ellipsoidal shape when the thermal effect due to US absorption plays the dominant role. Its long and short axes are parallel and perpendicular to the US propagation direction respectively. It was shown by ex vivo experiments that the dimension of the short and long axes of the tissue which experiences CN can be as small as 50 μm and 250 μm respectively after one second exposure of US pulse (the spatial and pulse average acoustic power is on the order of tens of Watts and the local acoustic spatial and temporal pulse averaged intensity is on the order of 3 × 104 W/cm2) generated by a 1.6 MHz HIFU transducer of 12 cm diameter and 11 cm geometric focal length (f-number = 0.92). The numbers of cells which suffered CN were estimated to be on the order of 40. This result suggests that HIFU is able to interact with tens of cells at/near its focal zone while keeping the neighboring cells minimally affected, and thus the targeted cell surgery may be achievable.

  7. 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.

  8. 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

  9. 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

  10. Detection of calcifications in vivo and ex vivo after brain injury in rat using SWIFT.

    PubMed

    Lehto, Lauri Juhani; Sierra, Alejandra; Corum, Curtis Andrew; Zhang, Jinjin; Idiyatullin, Djaudat; Pitkänen, Asla; Garwood, Michael; Gröhn, Olli

    2012-07-16

    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.

  11. XTT assay of ex vivo saliva biofilms to test antimicrobial influences

    PubMed Central

    Koban, Ina; Matthes, Rutger; Hübner, Nils-Olaf; Welk, Alexander; Sietmann, Rabea; Lademann, Jürgen; Kramer, Axel; Kocher, Thomas

    2012-01-01

    Objective: Many dental diseases are attributable to biofilms. The screening of antimicrobial substances, in particular, requires a high sample throughput and a realistic model, the evaluation must be as quick and as simple as possible. For this purpose, a colorimetric assay of the tetrazolium salt XTT (sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate) converted by saliva biofilms is recommended. Cleavage of XTT by dehydrogenase enzymes of metabolically active cells in biofilms yields a highly colored formazan product which is measured photometrically. Materials and method: The suitability of the XTT assay for detecting the vitality of ex vivo saliva biofilms was tested to determine the efficacy of chlorhexidine and ozone versus saliva biofilms grown on titanium discs. Results: The XTT method lends itself to testing the vitality of microorganisms in saliva biofilms. The sensitivity of the arrays requires a specific minimum number of pathogens, this number being different for planktonic bacteria and those occurring in biofilms. The antibacterial effect after treatment with chlorhexidine or ozone was measured by XTT conversion that was significantly reduced. The antimicrobial efficacy of 60 s 0.5% and 0.1% chlorhexidine treatment was equal and comparable with 60 s ozone treatment. Conclusion: The XTT assay is a suitable method to determine the vitality in saliva biofilms, permitting assessment of the efficacy of antimicrobial substances. Its quick and easy applicability renders it especially suitable for screening. PMID:22558040

  12. 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.

  13. Plaque characterization in ex vivo MRI evaluated by dense 3D correspondence with histology

    NASA Astrophysics Data System (ADS)

    van Engelen, Arna; de Bruijne, Marleen; Klein, Stefan; Verhagen, Hence; Groen, Harald; Wentzel, Jolanda; van der Lugt, Aad; Niessen, Wiro

    2011-03-01

    Automatic quantification of carotid artery plaque composition is important in the development of methods that distinguish vulnerable from stable plaques. MRI has shown to be capable of imaging different components noninvasively. We present a new plaque classification method which uses 3D registration of histology data with ex vivo MRI data, using non-rigid registration, both for training and evaluation. This is more objective than previously presented methods, as it eliminates selection bias that is introduced when 2D MRI slices are manually matched to histological slices before evaluation. Histological slices of human atherosclerotic plaques were manually segmented into necrotic core, fibrous tissue and calcification. Classification of these three components was voxelwise evaluated. As features the intensity, gradient magnitude and Laplacian in four MRI sequences after different degrees of Gaussian smoothing, and the distances to the lumen and the outer vessel wall, were used. Performance of linear and quadratic discriminant classifiers for different combinations of features was evaluated. Best accuracy (72.5 +/- 7.7%) was reached with the linear classifier when all features were used. Although this was only a minor improvement to the accuracy of a classifier that only included the intensities and distance features (71.6 +/- 7.9%), the difference was statistically significant (paired t-test, p<0.05). Good sensitivity and specificity for calcification was reached (83% and 95% respectively), however, differentiation between fibrous (sensitivity 85%, specificity 60%) and necrotic tissue (sensitivity 49%, specificity 89%) was more difficult.

  14. Spectral and temporal near-infrared imaging of ex vivo cancerous and normal human breast tissues.

    PubMed

    Alrubaiee, M; Gayen, S K; Alfano, R R; Koutcher, J A

    2005-10-01

    Cancerous and normal ex vivo human breast tissues were investigated using spectroscopic and time-sliced two-dimensional (2-D) transillumination imaging methods in order to demonstrate the importance and potential of spectral and temporal measurements in breast cancer detection and diagnosis. The experimental arrangement for time-sliced optical imaging used 120 fs, 1 kHz repetition-rate, 800 nm light pulses from a Ti:sapphire laser system for sample illumination, and a 80 ps resolution ultrafast gated intensified camera system for recording 2-D time-sliced images. The spectroscopic imaging arrangement used 1225-1300 nm tunable output of a Cr: forsterite laser for sample illumination, a Fourier space gate to discriminate against multiple-scattered light, and a near-infrared area camera to record 2-D images. Images recorded with earlier temporal slices of transmitted light highlighted tumors, while those recorded with later slices accentuated normal tissues. When light was tuned closer to the 1203 nm absorption resonance of adipose tissues, a marked enhancement in contrast between the images of adipose and fibrous tissues was observed. A similar wavelength-dependent difference between normal and cancerous tissues was observed. These results correlate well with pathology and nuclear magnetic resonance based analyses of the samples.

  15. 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

  16. 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

  17. 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.

  18. Long Term Ex Vivo Culture and Live Imaging of Drosophila Larval Imaginal Discs

    PubMed Central

    Lee, Yuan-Ming; Huang, Yu-Fen

    2016-01-01

    Continuous imaging of live tissues provides clear temporal sequence of biological events. The Drosophila imaginal discs have been popular experimental subjects for the study of a wide variety of biological phenomena, but long term culture that allows normal development has not been satisfactory. Here we report a culture method that can sustain normal development for 18 hours and allows live imaging. The method is validated in multiple discs and for cell proliferation, differentiation and migration. However, it does not support disc growth and cannot support cell proliferation for more than 7 to 12 hr. We monitored the cellular behavior of retinal basal glia in the developing eye disc and found that distinct glia type has distinct properties of proliferation and migration. The live imaging provided direct proof that wrapping glia differentiated from existing glia after migrating to the anterior front, and unexpectedly found that they undergo endoreplication before wrapping axons, and their nuclei migrate up and down along the axons. UV-induced specific labeling of a single carpet glia also showed that the two carpet glia membrane do not overlap and suggests a tiling or repulsion mechanism between the two cells. These findings demonstrated the usefulness of an ex vivo culture method and live imaging. PMID:27685172

  19. Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation

    PubMed Central

    Hsueh, Yi-Jen; Huang, Shiang-Fu; Lai, Jui-Yang; Ma, Shih-Chieh; Chen, Hung-Chi; Wu, Sung-En; Wang, Tze-Kai; Sun, Chi-Chin; Ma, Kevin Sheng-Kai; Chen, Jan-Kan; Lai, Chyong-Huey; Ma, David Hui-Kang

    2016-01-01

    To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75NTR immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in β-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27KIP1. Furthermore, ILK silencing led to the inhibition of nuclear β-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27KIP1; these observations suggest that ILK/β-catenin pathway may be involved in cell proliferation regulation during the ex vivo expansion of OMECs for transplantation purposes. PMID:27824126

  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. 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".

  2. The measurement of ultrasound backscattering from cell pellet biophantoms and tumors ex vivo

    PubMed Central

    Han, Aiguo; Abuhabsah, Rami; Miller, Rita J.; Sarwate, Sandhya; O'Brien, William D.

    2013-01-01

    Simple scattering media fit scattering model theories much better than more complex scattering media. Tissue is much more complex as an acoustic scattering media and to date there has not been an adequate scattering model that fits it well. Previous studies evaluated the scattering characteristics of simple media (grouping of cells at various number densities) and fit them to the concentric spheres scattering model theory. This study is to increase the complexity of the media to provide insight into the acoustic scattering characteristics of tissue, and specifically two tumor types. Complementing the data from the tumors is 100% volume fraction cell pellets of the same cell lines. Cell pellets and ex vivo tumors are scanned using high-frequency single-element transducers (9–105 MHz), and the attenuation and backscatter coefficient (BSC) are estimated. BSC comparisons are made between cell pellets and tumors. The results show that the 4T1 (ATCC #CRL-2539) cell pellets and tumors have similar BSC characteristics, whereas the MAT (ATCC #CRL-1666) cell pellets and tumors have significantly different BSC characteristics. Factors that yield such differences are explored. Also, the fluid-filled sphere and the concentric spheres models are evaluated against the BSC characteristics, demonstrating that further work is required. PMID:23862841

  3. Lentiviral vector-mediated genetic modification of human neural progenitor cells for ex vivo gene therapy.

    PubMed

    Capowski, Elizabeth E; Schneider, Bernard L; Ebert, Allison D; Seehus, Corey R; Szulc, Jolanta; Zufferey, Romain; Aebischer, Patrick; Svendsen, Clive N

    2007-07-30

    Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

  4. Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies.

    PubMed

    Akkaya, Billur; Holstein, Amanda H; Isaac, Christopher; Maz, Mitra P; Glass, Deborah D; Shevach, Ethan M; Akkaya, Munir

    2017-02-01

    Ex-vivo differentiation of regulatory T cells (Tregs) from naïve CD4(+) T-cells has been widely used in immunological research. Isolation of a highly pure naïve T cell population is the key factor that determines the efficiency of subsequent Treg differentiation. Currently, this step relies mostly on FACS sorting, which is often costly, time consuming, and inconvenient. Alternatively, magnetic separation of T-cells can be performed; yet, available protocols fail to reach sort level purity and consequently result in low Treg differentiation efficiency. Here, we present the results of a comprehensive side-by-side comparison of various magnetic separation strategies and FACS sorting in multiple levels. Additionally, we propose a novel optimized custom made magnetic separation protocol, which not only yields sort level purity and Treg differentiation but also lowers the reagent costs up to 75% compared to the commercially available purification kits. The highly pure naïve CD4(+) T-cell population obtained by this versatile method can also be used for differentiation of other T-cell subsets; therefore this protocol may have broad applications in T-cell research.

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Thiolated chitosan nanoparticles for enhancing oral absorption of docetaxel: preparation, in vitro and ex vivo evaluation.

    PubMed

    Saremi, Shahrooz; Atyabi, Fatemeh; Akhlaghi, Seyedeh Parinaz; Ostad, Seyed Nasser; Dinarvand, Rassoul

    2011-01-12

    The aim of this study was to prepare and evaluate mucoadhesive core-shell nanoparticles based on copolymerization of thiolated chitosan coated on poly methyl methacrylate cores as a carrier for oral delivery of docetaxel. Docetaxel-loaded nanoparticles with various concentrations were prepared via a radical emulsion polymerization method using cerium ammonium nitrate as an initiator. The physicochemical properties of the obtained nanoparticles were characterized by: dynamic light-scattering analysis for their mean size, size distribution, and zeta potential; scanning electron microscopy and transmission electron microscopy for surface morphology; and differential scanning calorimetry analysis for confirmation of molecular dispersity of docetaxel in the nanoparticles. Nanoparticles were spherical with mean diameter below 200 nm, polydispersity of below 0.15, and positive zeta potential values. The entrapment efficiency of the nanoparticles was approximately 90%. In vitro release studies showed a sustained release characteristic for 10 days after a burst release at the beginning. Ex vivo studies showed a significant increase in the transportation of docetaxel from intestinal membrane of rat when formulated as nanoparticles. Cellular uptake of nanoparticles was investigated using fluoresceinamine-loaded nanoparticles. Docetaxel nanoparticles showed a high cytotoxicity effect in the Caco-2 and MCF-7 cell lines after 72 hours. It can be concluded that by combining the advantages of both thiolated polymers and colloidal particles, these nanoparticles can be proposed as a drug carrier system for mucosal delivery of hydrophobic drugs.

  10. Characteristics of non-carious cervical lesions--an ex vivo study using micro computed tomography.

    PubMed

    Hur, B; Kim, H-C; Park, J-K; Versluis, A

    2011-06-01

    The aetiology of non-carious cervical lesions (NCCLs) is not well understood and still controversial. The aim of this ex-vivo study was to examine the morphological characteristics of NCCLs for clinical evidence of enamel loss above the cemento-enamel junction (CEJ) as suggested by the abfraction theory. Fifty extracted human teeth with various types of NCCLs were collected and scanned by micro computed tomography. The reconstructed three-dimensional models were evaluated from multiple aspects, including longitudinal cross-section series. The location of internal line angle and proximal exits of the lesions were evaluated in relation to the level of CEJ. The coronal margins of the lesions were inspected for evidence of enamel loss above the CEJ using the bucco-lingual longitudinal sectional images. Coronal margins of the lesions were located along and/or under the CEJ for all of the 50 samples. In most of the lesions, regardless of lesion type, the proximal exits and internal line angles were located below the CEJ. This study did not detect clinical evidence of enamel loss above the occlusal margin of NCCLs as would have been expected according to the general abfraction mechanism.

  11. Optical assessment of tissue anisotropy in ex vivo distended rat bladders.

    PubMed

    Alali, Sanaz; Aitken, Karen J; Schröder, Annette; Shröder, Annette; Bagli, Darius J; Alex Vitkin, I

    2012-08-01

    Microstructural remodelling in epithelial layers of various hollow organs, including changes in tissue anisotropy, are known to occur under mechanical distension and during disease processes. In this paper, we analyze how bladder distension alters wall anisotropy using polarized light imaging (followed by Mueller matrix decomposition). Optical retardance values of different regions of normal rat bladders under different distension pressures are derived. Then optical coherence tomography is used to measure local bladder wall thicknesses, enabling the calculation of the tissue birefringence maps as a measure of the tissue anisotropy. Selected two-photon microscopy is also performed to better understand the compositional origins of the obtained anisotropy results. The dome region of the bladder shows maximum birefringence when the bladder is distended to high pressures, whereas the ventral remains roughly isotropic during distension. In addition, the average anisotropy direction is longitudinal, along the urethra to dome. The derived wall anisotropy trends are based on birefringence as an intrinsic property of the tissue organization independent of its thickness, to aid in understanding the structure-functions relation in healthy bladders. These new insights into the wall microstructure of ex vivo distending bladders may help improve the functionality of the artificially engineered bladder tissues.

  12. Biosafety in Ex Vivo Gene Therapy and Conditional Ablation of Lentivirally Transduced Hepatocytes in Nonhuman Primates

    PubMed Central

    Menzel, Olivier; Birraux, Jacques; Wildhaber, Barbara E; Jond, Caty; Lasne, Françoise; Habre, Walid; Trono, Didier; Nguyen, Tuan H; Chardot, Christophe

    2009-01-01

    Ex vivo gene therapy is an interesting alternative to orthotopic liver transplantation (OLT) for treating metabolic liver diseases. In this study, we investigated its efficacy and biosafety in nonhuman primates. Hepatocytes isolated from liver lobectomy were transduced in suspension with a bicistronic liver-specific lentiviral vector and immediately autotransplanted (SLIT) into three cynomolgus monkeys. The vector encoded cynomolgus erythropoietin (EPO) and the conditional suicide gene herpes simplex virus-thymidine kinase (HSV-TK). Survival of transduced hepatocytes and vector dissemination were evaluated by detecting transgene expression and vector DNA. SLIT was safely performed within a day in all three subjects. Serum EPO and hematocrit rapidly increased post-SLIT and their values returned to baseline within about 1 month. Isoforms of EPO detected in monkeys' sera differed from the physiological renal EPO. In liver biopsies at months 8 and 15, we detected EPO protein, vector mRNA and DNA, demonstrating long-term survival and functionality of transplanted lentivirally transduced hepatocytes. Valganciclovir administration resulted in complete ablation of the transduced hepatocytes. We demonstrated the feasibility and biosafety of SLIT, and the long term (>1 year) functionality of lentivirally transduced hepatocytes in nonhuman primates. The HSV-TK/valganciclovir suicide strategy can increase the biosafety of liver gene therapy protocols by safely and completely ablating transduced hepatocytes on demand. PMID:19568222

  13. Improved haematopoietic recovery following transplantation with ex vivo-expanded mobilized blood cells.

    PubMed

    Prince, H Miles; Simmons, Paul J; Whitty, Genevieve; Wall, Dominic P; Barber, Lesley; Toner, Guy C; Seymour, John F; Richardson, Gary; Mrongovius, Robert; Haylock, David N

    2004-08-01

    Infusions of ex vivo-expanded (EXE) mobilized blood cells have been explored to enhance haematopoietic recovery following high dose chemotherapy (HDT). However, prior studies have not consistently demonstrated improvements in trilineage haematopoietic recovery. Three cohorts of three patients with breast cancer received three cycles of repetitive HDT supported by either unmanipulated (UM) and/or EXE cells. Efficacy was assessed by an internal comparison of each patient's consecutive HDT cycles, and to 106 historical UM infusions. Twenty-one cycles were supported by EXE cells and six by UM cells alone. Infusions of EXE cells resulted in fewer days with an absolute neutrophil count (ANC) <0.1 x 10(9)/l (median 2 vs. 4 d, P = 0.002) and 3 d faster ANC recovery to >0.1 x 10(9)/l (median 5 vs. 8 d, P = 0.0002). This resulted in a major reduction in the incidence of febrile neutropenia compared with UM cycles (0% vs. 83%; P = 0.008) and in 66% of historical UM cycles (P = 0.01) and a marked reduction in hospital re-admission. There were also fewer platelet transfusions required (43% vs. 100%; P = 0.009). We conclude that EXE cells enhance both neutrophil and platelet recovery and reduce febrile neutropenia, platelet transfusion and hospital re-admission.

  14. Effects of zinc ex vivo on taurine uptake in goldfish retinal cells

    PubMed Central

    2010-01-01

    Background Taurine and zinc exert neurotrophic effects in the central nervous system. Current studies demonstrate that Na+/Cl- dependent neurotransmitter transporters, similar to that of taurine, are modulated by micromolar concentrations of zinc. This study examined the effect of zinc sulfate ex vivo on [3H]taurine transport in goldfish retina. Methods Isolated cells were incubated in Ringer with zinc (0.1–100 µM). Taurine transport was done with 50 nM [3H]taurine or by isotopic dilution with taurine (0.001–1 mM) and 50 nM [3H]taurine. Results Zinc reduced the capacity of taurine transport without changes in affinity, and caused a noncompetitive inhibition of high affinity taurine transport, with an EC50= 0.072 µM. The mechanism by which zinc affects taurine transport is unknown at the present. Conclusions There may be a binding site of zinc in the transporter that affects union or translocation of taurine, or possibly the formation of taurine-zinc complexes, rather than free zinc, could affect the operation of the transporter. PMID:20804587

  15. 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.

  16. Patterns of histological changes following hepatic electrolytic ablation in an ex-vivo perfused model.

    PubMed

    Gravante, Gianpiero; Ong, Seok Ling; West, Kevin; McGregor, Angus; Maddern, Guy J; Metcalfe, Matthew S; Lloyd, David M; Dennison, Ashley R

    2012-10-01

    Electrolytic ablation (EA) destroys the liver by releasing toxic radicles and producing modifications in the local pH without increasing the tissue temperature. We assessed the histological changes produced by EA using an ex-vivo perfused model. Five porcine livers were harvested, preserved in ice and reperfused for six hours in an extracorporeal circuit using autologous normothermic blood. One hour after reperfusion EA was performed and liver biopsies collected at the end of the experiments. The main necrotic zone consisted of coagulative necrosis, sinusoidal dilatation and haemorrhage with an unusual morphological pattern. The coagulative necrosis and haemorrhage affected mainly the peripheral area of the lobule with relative sparing of the area surrounding the centrilobular vein. Contrasting with this sinusoidal dilatation appeared to be more prominent in the centrilobular area. EA produces patterns of tissue destruction that have not been observed with the more commonly used thermal techniques. Further studies should obtain more information about the influence of adjacent biliary and vascular structures so that appropriate clinical trials can be designed.

  17. Ex vivo correlation of the permeability of metoprolol across human and porcine buccal mucosa.

    PubMed

    Meng-Lund, Emil; Marxen, Eva; Pedersen, Anne Marie L; Müllertz, Anette; Hyrup, Birgitte; Holm, Rene; Jacobsen, Jette

    2014-07-01

    The pH partition theory proposes a correlation between fraction of unionized drug substance and permeability. The aim of this study was to compare the permeability of metoprolol and mannitol in ex vivo human and porcine buccal mucosa models at varying pH to validate whether the porcine permeability model is predictive for human buccal absorption. Human (n = 9-10) and porcine (n = 6-7) buccal mucosa were mounted in a modified Ussing chamber, and the kinetics of metoprolol and mannitol transport was assessed for a period of 5.5 h with the pH values of donor medium set at 7.4, 8.5, and 9.0. In addition, hematoxylin-eosin and Alcian blue-van Gieson were used as tissue stains to evaluate the histology and the presence of acidic polysaccharides (e.g., mucins), respectively. The permeability of metoprolol was decreased in human buccal mucosa by almost twofold when compared with porcine buccal mucosa with a positive correlation (r(2) = 0.96) between the permeability assessed in porcine and human buccal mucosa. There was no change in the degree of either epithelial swelling or desquamation when treating with the pH 9.0 donor medium for 5.5 h. These data suggest that buccal mucosa from pigs can be used to predict human buccal absorption.

  18. In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies.

    PubMed

    Tang, Xu-Dong; Guo, Shu-Liang; Wang, Guo-Zhen; Li, Ning; Wu, Yu-Yun; Fang, Dian-Chun; Fan, Ya-Han; Yang, Shi-Ming

    2014-01-01

    Previous studies have indicated that heparanase (Hpa) might represent a candidate universal tumor-associated antigen. However, vaccine therapy targeting only one cytotoxic T lymphocyte (CTL) epitope is suboptimal in preventing cancer. In the present study, we designed heparanase multi-epitope vaccines to increase the immune response to standard single heparanase epitopes. The results showed that multi-epitope vaccines Hpa525 + 277 + 405 + 16 and Hpa8 + 310 + 315 + 363 induced higher Hpa-specific lysis of various cancer cells from different tissues in a HLA-A2-restricted and heparanase-specific manner compared with the single epitope vaccines Hpa525, Hpa277, Hpa405, Hpa16, Hpa8, Hpa310, Hpa315 and Hpa363, both in vitro and ex vivo. Heparanase multi-epitope vaccines not only induced the heparanase-specific CTL to lyse tumor cells but also increased CTL secretion of interferon-γ. However, these heparanase-specific CTL did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirms the safety of these multi-epitope vaccines. Therefore, the present study provides theoretical evidence for the use of heparanase multi-epitope vaccines for clinical application.

  19. The Transcriptome of Exophiala dermatitidis during Ex-vivo Skin Model Infection

    PubMed Central

    Poyntner, Caroline; Blasi, Barbara; Arcalis, Elsa; Mirastschijski, Ursula; Sterflinger, Katja; Tafer, Hakim

    2016-01-01

    The black yeast Exophiala dermatitidis is a widespread polyextremophile and human pathogen, that is found in extreme natural habitats and man-made environments such as dishwashers. It can cause various diseases ranging from phaeohyphomycosis and systemic infections, with fatality rates reaching 40%. While the number of cases in immunocompromised patients are increasing, knowledge of the infections, virulence factors and host response is still scarce. In this study, for the first time, an artificial infection of an ex-vivo skin model with Exophiala dermatitidis was monitored microscopically and transcriptomically. Results show that Exophiala dermatitidis is able to actively grow and penetrate the skin. The analysis of the genomic and RNA-sequencing data delivers a rich and complex transcriptome where circular RNAs, fusion transcripts, long non-coding RNAs and antisense transcripts are found. Changes in transcription strongly affect pathways related to nutrients acquisition, energy metabolism, cell wall, morphological switch, and known virulence factors. The L-Tyrosine melanin pathway is specifically upregulated during infection. Moreover the production of secondary metabolites, especially alkaloids, is increased. Our study is the first that gives an insight into the complexity of the transcriptome of Exophiala dermatitidis during artificial skin infections and reveals new virulence factors. PMID:27822460

  20. Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

    PubMed Central

    Clusel, C; Ugarte, E; Enjolras, N; Vasseur, M; Blumenfeld, M

    1993-01-01

    Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes. Images PMID:7688452

  1. 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

  2. Ex vivo encapsulation of dexamethasone sodium phosphate into human autologous erythrocytes using fully automated biomedical equipment.

    PubMed

    Mambrini, Giovanni; Mandolini, Marco; Rossi, Luigia; Pierigè, Francesca; Capogrossi, Giovanni; Salvati, Patricia; Serafini, Sonja; Benatti, Luca; Magnani, Mauro

    2017-01-30

    Erythrocyte-based drug delivery systems are emerging as potential new solutions for the release of drugs into the bloodstream. The aim of the present work was to assess the performance of a fully automated process (EDS) for the ex-vivo encapsulation of the pro-drug dexamethasone sodium phosphate (DSP) into autologous erythrocytes in compliance with regulatory requirements. The loading method was based on reversible hypotonic hemolysis, which allows the opening of transient pores in the cell membrane to be crossed by DSP. The efficiency of encapsulation and the biochemical and physiological characteristics of the processed erythrocytes were investigated in blood samples from 34 healthy donors. It was found that the processed erythrocytes maintained their fundamental properties and the encapsulation process was reproducible. The EDS under study showed greater loading efficiency and reduced variability compared to previous EDS versions. Notably, these results were confirmed using blood samples from Ataxia Telangiectasia (AT) patients, 9.33±1.40 and 19.41±2.10mg of DSP (mean±SD, n=134) by using 62.5 and 125mg DSP loading quantities, respectively. These results support the use of the new EDS version 3.2.0 to investigate the effect of erythrocyte-delivered dexamethasone in regulatory trials in patients with AT.

  3. 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.

  4. 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.

  5. Ex Vivo Confocal Spectroscopy of Autofluorescence in Age-Related Macular Degeneration

    PubMed Central

    Kaluzny, Joel; Purta, Patryk; Poskin, Zach; Rogers, Jeremy D.; Fawzi, Amani A.

    2016-01-01

    Purpose We investigated the autofluorescence (AF) signature of the microscopic features of retina with age-related macular degeneration (AMD) using 488 nm excitation. Methods The globes of four donors with AMD and four age-matched controls were embedded in paraffin and sectioned through the macula. Sections were excited using a 488 nm argon laser, and the AF emission was captured using a laser scanning confocal microscope (496–610 nm, 6 nm resolution). The data cubes were then analyzed to compare peak emission spectra between the AMD and the controls. Microscopic features, including individual lipofuscin and melanolipofuscin granules, Bruch’s Membrane, as well macroscopic features, were considered. Results Overall, the AMD eyes showed a trend of blue-shifted emission peaks compared with the controls. These differences were statistically significant when considering the emission of the combined RPE/Bruch’s Membrane across all the tissue cross-sections (p = 0.02). Conclusions The AF signatures of ex vivo AMD RPE/BrM show blue-shifted emission spectra (488 nm excitation) compared with the control tissue. The magnitude of these differences is small (~4 nm) and highlights the potential challenges of detecting these subtle spectral differences in vivo. PMID:27631087

  6. Cell population kinetics of collagen scaffolds in ex vivo oral wound repair.

    PubMed

    Agis, Hermann; Collins, Amy; Taut, Andrei D; Jin, Qiming; Kruger, Laura; Görlach, Christoph; Giannobile, William V

    2014-01-01

    Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.

  7. The effect of ex vivo anticoagulants on whole blood platelet aggregation.

    PubMed

    Kalb, Madeleine L; Potura, Lukasz; Scharbert, Gisela; Kozek-Langenecker, Sibylle A

    2009-02-01

    Pre- and intraoperative platelet function monitoring is increasingly recommended in order to detect risk factors for bleeding and to target coagulation management. The ideal anticoagulant for accurate platelet aggregometry remains controversial. The aim of this experimental trial was to compare platelet aggregability in whole blood stored in citrate, heparin and direct thrombin inhibitors. Whole blood was drawn from 11 healthy adult volunteers who had not taken any medication in the previous 14 days. Blood was stored in trisodium citrate, unfractionated heparin, melagatran, lepirudin and argatroban. Platelet aggregation was performed using the impedance aggregometer Multiplate (Dynabyte, Munich, Germany) with adenosine diphosphate (ADP), thrombin receptor activating peptide (TRAP), collagen, arachidonic acid and ristocetin as agonists. Samples were analysed immediately after blood sampling (baseline), as well as 30 and 120 min afterwards. At baseline there were no significant differences in aggregability between samples containing direct thrombin inhibitors and heparin. In contrast, aggregation in response to all agonists except for ristocetin was significantly impaired in citrated blood. During storage the response to arachidonic acid and collagen was maintained by direct thrombin inhibitors and heparin, whereas ADP-, TRAP- and ristocetin-induced aggregation varied considerably over time in all ex vivo anticoagulants tested. Pre-analytical procedures should be standardized because storage duration and anticoagulants significantly affect platelet aggregability in whole blood. For point-of-care monitoring with immediate analysis after blood withdrawal all tested direct thrombin inhibitors as well as unfractionated heparin can be used as anticoagulants whereas citrate is not recommended.

  8. Retinal electrophysiology for toxicology studies: applications and limits of ERG in animals and ex vivo recordings.

    PubMed

    Rosolen, Serge Georges; Kolomiets, Bogdan; Varela, Oscar; Picaud, Serge

    2008-06-01

    Assessing retinal drug toxicity is becoming increasingly important as different molecules are now developed for the treatment of neurodegenerative diseases and vascular disorders. In pharmacology and toxicology, the electroretinogram (ERG) and the multielectrode array (MEA) recording techniques can be used to quantify the possible side effects of retino-active xenobiotics. Toxicity testing requires the use of rodent as well as non-rodent models for extrapolation to the human model when determining risk and safety. Animal species differ in their retinal anatomo-physiology: most rodents used in toxicology studies are essentially nocturnal species, whereas the non-rodent laboratory species normally used (e.g. dogs, pigs and monkeys) are diurnal. The ratio between the photoreceptor populations which varies from species to species, should be considered when designing the experiment protocol and the interpretation. The described ERG procedures are designed to comply with all applicable good laboratory practice standards. Use of these procedures should yield an acceptable level of intra- and inter-subject variability for compiling a historical database, and for detecting possible retinal toxicity in animal studies. They could therefore be used as specific and standardized tools for screening of potential retinotoxic molecules in drug discovery and development in order to compare methods and results with those obtained in human electrophysiological assessments. Recording of ganglion cell light responses on ex vivo retina with the MEA technique can further demonstrate how retino-active xenobiotics affect retinal visual information processing by eliminating potential obstacles related to bioavailability and blood barrier permeability.

  9. 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

  10. Assessment of glucocorticoid lung targeting by ex-vivo receptor binding studies in rats.

    PubMed

    Hochhaus, G; Gonzalez-Rothi, R J; Lukyanov, A; Derendorf, H; Schreier, H; Dalla Costa, T

    1995-01-01

    Triamcinolone acetonide (TA, 22 micrograms) was given to rats by intravenous (i.v.) injection or intratracheal (IT) instillation. Free glucocorticoid receptors were monitored over time in liver and lung using an ex-vivo receptor binding technique. After i.v. administration of a TA solution, the reduction of free receptors over time was very similar in lung and liver (AUCLung = 280 +/- 47% h; AUCLiver = 320 +/- 76% h). Intratracheal instillation of the same solution produced time profiles which mirrored those of i.v. injection (AUCLung = 260 +/- 41% h; AUCLiver = 330 +/- 50% h). The lack of lung targeting was also reflected in the failure to show any significant difference in the pulmonary targeting factor T (AUCLung/AUCLiver) between i.v. (T = 0.84 +/- 0.18) and IT (T = 0.78 +/- 0.03) administration. In contrast, a certain degree of lung specificity was observed after IT instillation of a glucocorticoid suspension (22 micrograms; AUCLung = 160 +/- 135% h; AUCLiver = 65 +/- 91% h, T = 2.3 +/- 0.5) as indicated by significant differences in T between i.v. injection and IT instillation (p = 0.038). The method presented provides a means of simultaneously assessing pulmonary and systemic effects after different forms and routes of administration and might be of value in further studying multiple aspects of inhalation glucocorticoid therapy.

  11. 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.

  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. 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.

  14. A comparative study of ex vivo skin optical clearing using two-photon microscopy.

    PubMed

    Sdobnov, Anton; Darvin, Maxim E; Lademann, Juergen; Tuchin, Valery

    2017-01-30

    Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (Omnipaque(TM) ) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or Omnipaque(TM) solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% Omnipaque(TM) it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than Omnipaque(TM) . However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. Omnipaque(TM) , on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover Omnipaque(TM) allows for robust multimodal optical/X-ray imaging with automatically matched optically cleared and X-ray contrasted tissue volumes. These findings make Omnipaque(TM) more prospective than glycerol for some particular application.

  15. 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.

  16. Effect of cord blood serum on ex vivo human limbal epithelial cell culture.

    PubMed

    Chakraborty, Anindita; Dutta, Jayanta; Das, Sumantra; Datta, Himadri

    2012-12-01

    Limbal cell transplantation is an efficacious procedure for rehabilitation of visual acuity in patients with severe ocular surface disorders. Cultivation of limbal epithelial stem cell with fetal bovine serum for transplantation has been a promising treatment for reconstructing the ocular surface in severe limbal stem cell deficiency caused by Steven Johnson syndrome, chemical or thermal injury. This technique of "cell therapy" has been accepted worldwide but the cost of cultivating the cells for transplantation is high. The objective of this study was to investigate the effect of cord blood serum in place of fetal bovine serum on the growth of human limbal epithelial cell culture. Our group has experimented with human cord blood serum which was obtained free of cost from willing donors. The use of human cord blood serum in place of fetal bovine serum for ex vivo culture of limbal stem cell has helped us in reducing the cost of culture. Fresh human limbal tissues from donor cadavers were cultured on intact and denuded amniotic membrane. Cells were proliferated in vitro with cell culture media containing human cord blood serum. Reverse transcription-polymerase chain reaction and immunofluorescence cytochemistry of cultured human limbal epithelial stem cell was done for characterization of the cells.

  17. 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.

  18. 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.

  19. Classical and adaptive control of ex vivo skeletal muscle contractions using Functional Electrical Stimulation (FES)

    PubMed Central

    Shoemaker, Adam; Grange, Robert W.; Abaid, Nicole; Leonessa, Alexander

    2017-01-01

    Functional Electrical Stimulation is a promising approach to treat patients by stimulating the peripheral nerves and their corresponding motor neurons using electrical current. This technique helps maintain muscle mass and promote blood flow in the absence of a functioning nervous system. The goal of this work is to control muscle contractions from FES via three different algorithms and assess the most appropriate controller providing effective stimulation of the muscle. An open-loop system and a closed-loop system with three types of model-free feedback controllers were assessed for tracking control of skeletal muscle contractions: a Proportional-Integral (PI) controller, a Model Reference Adaptive Control algorithm, and an Adaptive Augmented PI system. Furthermore, a mathematical model of a muscle-mass-spring system was implemented in simulation to test the open-loop case and closed-loop controllers. These simulations were carried out and then validated through experiments ex vivo. The experiments included muscle contractions following four distinct trajectories: a step, sine, ramp, and square wave. Overall, the closed-loop controllers followed the stimulation trajectories set for all the simulated and tested muscles. When comparing the experimental outcomes of each controller, we concluded that the Adaptive Augmented PI algorithm provided the best closed-loop performance for speed of convergence and disturbance rejection. PMID:28273101

  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. Measurements of fluorine in contemporary urban Canadians: a comparison of the levels found in human bone using in vivo and ex vivo neutron activation analysis.

    PubMed

    Mostafaei, F; McNeill, F E; Chettle, D R; Wainman, B C; Pidruczny, A E; Prestwich, W V

    2015-03-01

    content of the tea drinkers and the non-tea drinkers were found to be 0.127 (± 0.029) and 0.050 (± 0.009) mg F/g Ca per year respectively. Finally, we also obtained twelve bone samples from cadavers' skulls. Neutron activation analysis was used to determine the fluorine levels in these ex vivo samples. The rate of increase of fluorine content versus age for in vivo and ex vivo measurements were found to be 0.078  ±  0.014 and 0.078  ±  0.050 mg F/g Ca per year respectively. Excellent agreement was found between the fluorine levels determined in vivo and ex vivo using the two separate systems, providing confidence in the fluorine concentration data being measured in vivo.

  2. 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 Central

    Lehmann, Dorit; Spanholtz, Jan; Osl, Markus; Tordoir, Marleen; Lipnik, Karoline; Bilban, Martin; Schlechta, Bernhard; Dolstra, Harry

    2012-01-01

    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) CD56bright and CD56dim 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 CD56bright and CD56dim 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 CD56dim 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

  3. 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.

  4. Ex vivo adenoviral vector gene delivery results in decreased vector-associated inflammation pre- and post-lung transplantation in the pig.

    PubMed

    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

    2012-06-01

    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.

  5. Hydroxytyrosol targets extracellular matrix remodeling by endothelial cells and inhibits both ex vivo and in vivo angiogenesis.

    PubMed

    García-Vilas, Javier A; Quesada, Ana R; Medina, Miguel Ángel

    2017-04-15

    The health benefits of olive oil are attributed to their bioactive compounds, such as hydroxytyrosol. Previously, we demonstrated that hydroxytyrosol inhibits angiogenesis in vitro. The present study aimed to: i) get further insight into the effects of hydroxytyrosol on extracellular matrix remodeling; and ii) test whether hydroxytyrosol is able to inhibit angiogenesis ex vivo and in vivo. Hydroxytyrosol induced a shift toward inhibition of proteolysis in endothelial cells, with decreased expression of extracellular matrix remodeling-enzyme coding genes and increased levels of some of their inhibitors. Furthermore, this work demonstrated that hydroxytyrosol, at concentrations within the range of its content in virgin olive oil that can be absorbed from moderate and sustained virgin olive oil consumption, is a strong inhibitor of angiogenesis ex vivo and in vivo. These results suggest the need for translational studies to evaluate the potential use of hydroxytyrosol for angio-prevention and angiogenesis inhibition in clinical setting.

  6. Ex vivo uranium decontamination efficiency on wounded skin and in vitro skin toxicity of a calixarene-loaded nanoemulsion.

    PubMed

    Grives, Sophie; Phan, Guillaume; Morat, Guillaume; Suhard, David; Rebiere, Francois; Fattal, Elias

    2015-06-01

    The present work aims at studying the decontamination efficacy of a calixarene-loaded nanoemulsion on two ex vivo wounded skin models mimicking superficial stings or cuts contaminated with uranium, and on a third model using excoriation. The decontaminating formulation was compared with the currently used radio-decontaminating soapy water (Trait rouge®) treatment. Moreover, to assess skin damage potentially induced by the undiluted nanoemulsion, in vitro toxicity studies were conducted on an in vitro reconstructed human epidermis, coupled with three different toxicity tests [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, lactate dehydrogenase, and interleukin-1-α]. This work demonstrated not only a significant decontamination activity of the calixarene nanoemulsion on wounded skin, ranging from 92% to 94% of the applied uranium solution according to the ex vivo model used, but also the absence of side effects of this promising treatment.

  7. Anti-inflammatory activity of Punica granatum L. (Pomegranate) rind extracts applied topically to ex vivo skin.

    PubMed

    Houston, David M J; Bugert, Joachim; Denyer, Stephen P; Heard, Charles M

    2017-03-01

    Coadministered pomegranate rind extract (PRE) and zinc (II) produces a potent virucidal activity against Herpes simplex virus (HSV); however, HSV infections are also associated with localised inflammation and pain. Here, the objective was to determine the anti-inflammatory activity and relative depth penetration of PRE, total pomegranate tannins (TPT) and zinc (II) in skin, ex vivo. PRE, TPT and ZnSO4 were dosed onto freshly excised ex vivo porcine skin mounted in Franz diffusion cells and analysed for COX-2, as a marker for modulation of the arachidonic acid inflammation pathway, by Western blotting and immunohistochemistry. Tape stripping was carried out to construct relative depth profiles. Topical application of PRE to ex vivo skin downregulated expression of COX-2, which was significant after just 6h, and maintained for up to 24h. This was achieved with intact stratum corneum, proving that punicalagin penetrated skin, further supported by the depth profiling data. When PRE and ZnSO4 were applied together, statistically equal downregulation of COX-2 was observed when compared to the application of PRE alone; no effect followed the application of ZnSO4 alone. TPT downregulated COX-2 less than PRE, indicating that tannins alone may not be entirely responsible for the anti-inflammatory activity of PRE. Punicalagin was found throughout the skin, in particular the lower regions, indicating appendageal delivery as a significant route to the viable epidermis. Topical application of TPT and PRE had significant anti-inflammatory effects in ex vivo skin, confirming that PRE penetrates the skin and modulates COX-2 regulation in the viable epidermis. Pomegranates have potential as a novel approach in ameliorating the inflammation and pain associated with a range of skin conditions, including cold sores and herpetic stromal keratitis.

  8. Pharmacokinetics and Ex Vivo Pharmacodynamic Antimalarial Activity of Dihydroartemisinin-Piperaquine in Patients with Uncomplicated Falciparum Malaria in Vietnam ▿

    PubMed Central

    Nguyen, Dao Van Hoang; Nguyen, Quoc Phuc; Nguyen, Ngoa Dang; Le, Thuy Thi Thanh; Nguyen, The Duy; Dinh, Duy Ngoc; Nguyen, Thanh Xuan; Bui, Dai; Chavchich, Marina; Edstein, Michael D.

    2009-01-01

    Compared to healthy subjects, malaria patients show a reduction in the mean oral clearance (1.19 versus 5.87 liters/h/kg of body weight) and apparent volume of distribution (1.47 versus 8.02 liters/kg) of dihydroartemisinin in Vietnamese patients following treatment with dihydroartemisinin-piperaquine (Artekin) for uncomplicated Plasmodium falciparum. Dihydroartemisinin is responsible for most of the ex vivo antimalarial activity of dihydroartemisinin-piperaquine. PMID:19528277

  9. Phenotype and functional evaluation of ex vivo generated antigen-specific immune effector cells with potential for therapeutic applications.

    PubMed

    Han, Shuhong; Huang, Yuju; Liang, Yin; Ho, Yuchin; Wang, Yichen; Chang, Lung-Ji

    2009-08-06

    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-gamma 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-gamma-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-gamma and the CD137 selections enriched more central memory T (Tcm) cells than did the DC-activation approach, and after expansion, the IFN-gamma-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-gamma 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.

  10. In vitro-ex vivo correlations between a cell-laden hydrogel and mucosal tissue for screening composite delivery systems.

    PubMed

    Blakney, Anna K; Little, Adam B; Jiang, Yonghou; Woodrow, Kim A

    2016-11-01

    Composite delivery systems where drugs are electrospun in different layers and vary the drug stacking-order are posited to affect bioavailability. We evaluated how the formulation characteristics of both burst- and sustained-release electrospun fibers containing three physicochemically diverse drugs: dapivirine (DPV), maraviroc (MVC) and tenofovir (TFV) affect in vitro and ex vivo release. We developed a poly(hydroxyethyl methacrylate) (pHEMA) hydrogel release platform for the rapid, inexpensive in vitro evaluation of burst- and sustained-release topical or dermal drug delivery systems with varying microarchitecture. We investigated properties of the hydrogel that could recapitulate ex vivo release into nonhuman primate vaginal tissue. Using a dimethyl sulfoxide extraction protocol and high-performance liquid chromatography analysis, we achieved >93% recovery from the hydrogels and >88% recovery from tissue explants for all three drugs. We found that DPV loading, but not stacking order (layers of fiber containing a single drug) or microarchitecture (layers with isolated drug compared to all drugs in the same layer) impacted the burst release in vitro and ex vivo. Our burst-release formulations showed a correlation for DPV accumulation between the hydrogel and tissue (R(2)=( )0.80), but the correlation was not significant for MVC or TFV. For the sustained-release formulations, the PLGA/PCL content did not affect TFV release in vitro or ex vivo. Incorporation of cells into the hydrogel matrix improved the correlation between hydrogel and tissue explant release for TFV. We expect that this hydrogel-tissue mimic may be a promising preclinical model to evaluate topical or transdermal drug delivery systems with complex microarchitectures.

  11. 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

  12. Fast isolation and ex vivo culture of circulating tumor cells from the peripheral blood of lung cancer patients.

    PubMed

    Wenjun, Wu; Zhihua, Wang; Zhuo, Wang; Yuliang, Deng; Qihui, Shi

    2017-01-20

    Circulating tumor cells (CTCs) are free tumor cells shed from tumor site and enter into blood circulation. CTCs represent a reliable source of tumor cells for the molecular characteristics of the original tumor. However, the extraordinary rarity of CTCs makes the subsequent molecular and functional analysis technically challenging. Here, we describe a one-step microfludics-based immunomagnetic isolation method to isolate CTCs directly from the whole blood of lung adenocarcinoma patients. This method avoids harsh sample preparation and enrichment steps, and therefore preserves the viability (>90%) of CTCs during the in vitro isolation. The isolated CTCs are enriched in small volume (80 μL) and cultured ex vivo that leads to successful ex vivo expansion. The expanded CTCs can be frozen and thawed, which shows cell line property. Genetic sequencing on EGFR、KRAS、PIK3CA、TP53 and BRAF and metabolic assay (2-NBDG) are utilized to characterize the expanded CTCs. Our results demostrated that this method is suitable for ex vivo expansion of CTCs facilitates. The genomic, proteomic and metabolic analyses of CTCs have guiding significance in tumor precise treatment.

  13. Denopamine, a beta(1)-adrenergic agonist, increases alveolar fluid clearance in ex vivo rat and guinea pig lungs.

    PubMed

    Sakuma, T; Tuchihara, C; Ishigaki, M; Osanai, K; Nambu, Y; Toga, H; Takahashi, K; Ohya, N; Kurihara, T; Matthay, M A

    2001-01-01

    The effect of denopamine, a selective beta(1)-adrenergic agonist, on alveolar fluid clearance was determined in both ex vivo rat and guinea pig lungs. Alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue-labeled albumin over 1 h at 37 degrees C. Denopamine (10(-6) to 10(-3) M) increased alveolar fluid clearance in a dose-dependent manner in ex vivo rat lungs. Denopamine also stimulated alveolar fluid clearance in guinea pig lungs. Atenolol, a selective beta(1)-adrenergic antagonist, and amiloride, a sodium channel inhibitor, inhibited denopamine-stimulated alveolar fluid clearance. The potency of denopamine was similar to that of similar doses of isoproterenol or terbutaline. Short-term hypoxia (100% nitrogen for 1-2 h) did not alter the stimulatory effect of denopamine. Denopamine (10(-4), 10(-3) M) increased intracellular adenosine 3',5'-cyclic monophosphate levels in cultured rat alveolar type II cells. In summary, denopamine, a selective beta(1)-adrenergic agonist, stimulates alveolar fluid clearance in both ex vivo rat and guinea pig lungs.

  14. Ex vivo culture of circulating tumor cells using magnetic force-based coculture on a fibroblast feeder layer.

    PubMed

    Yamamoto, Shuhei; Shimizu, Kazunori; Fei, Jiahui; Iwata, Hiroji; Okochi, Mina; Nakanishi, Hayao; Honda, Hiroyuki

    2016-11-01

    Phenotype-based analysis of circulating tumor cells (CTCs) is a promising approach to identification of new therapeutic targets and to elucidation of the biological properties. Nonetheless, ex vivo culturing of CTCs is still a technical challenge. Here, we develop a novel ex vivo culture method for CTCs using a fibroblast feeder layer and a magnetic coculture protocol. CTCs in the blood of a mouse metastasis model are labeled magnetically with magnetite nanoparticles. The labeled CTCs are isolated by a magnetic capture column and a size-selective capture filter. The isolated CTCs are positioned on a fibroblast feeder layer by the magnetic force. As a result, we observe adhesion and proliferation of the CTCs under the conditions of the fibroblast feeder layer and the magnetic force, whereas no adhesion or proliferation is observed without the feeder layer. After that, we culture the CTCs and obtain three CTC-derived cell lines. Using these cell lines, we perform phenotype-based analyses of invasiveness and drug resistance and find that the CTC-derived cell lines are more malignant than the original cells. Thus, the proposed method would be a promising approach to ex vivo culture of CTCs for phenotype-based analysis, and possibly used in cancer treatment.

  15. In vitro and ex vivo evaluation of cyclic aminoalkyl benzilates as potential emission tomography ligands for the muscarinic receptor.

    PubMed

    Otto, C A; Mulholland, G K; Perry, S E; Combs, R; Sherman, P S; Fisher, S J

    1989-01-01

    A series of muscarinic antagonists were screened as potential receptor imaging agents. (+)2 alpha-tropanyl benzilate (TRB), N-methyl-4-piperidyl benzilate (NMPB) and several analogs amenable to labeling with positron emitting isotopes were evaluated for muscarinic binding to mouse brain tissue in vitro and ex vivo using [3H]quinuclidinyl benzilate as the probe. The in vitro assay directly compared the innate binding affinities of the compounds. The rank order of binding (IC50) was TRB (0.7 nm), QNB (0.8 nm), scopolamine (1.3 nm) and NMPB (1.6 nm). The ex vivo assay was used to gain information regarding the pharmacokinetics and brain penetration of the compounds in live animals. Ex vivo results demonstrated that TRB was rapidly taken up into the brain and was equipotent with QNB in occupying muscarinic binding sites at early time points, but TRB binding decreased twice as fast over time as QNB binding. The results suggest TRB would be a good candidate for radiolabeling and further study.

  16. Uptake of phenothiazines by the harvested chylomicrons ex vivo model: influence of self-nanoemulsifying formulation design.

    PubMed

    Shahnaz, Gul; Hartl, Markus; Barthelmes, Jan; Leithner, Katharina; Sarti, Federica; Hintzen, Fabian; Rahmat, Deni; Salvenmoser, Willi; Bernkop-Schnürch, Andreas

    2011-09-01

    The aim of this study was to examine the potential of self-nanoemulsifying drug delivery systems (SNEDDS) on the uptake of the lipophilic and poorly water soluble phenothiazines thioridazine and chlorpromazine with the isolated plasma derived chylomicron (CM) ex vivo model. The multi-component delivery systems were optimized by evaluating their ability to self-emulsify when introduced to an aqueous medium under gentle agitation. The uptake of phenothiazines by isolated plasma derived chylomicrons was investigated with short chain triglyceride (SCT) SNEDDS, medium chain triglyceride (MCT) SNEDDS, and long chain triglyceride (LCT) SNEDDS. SNEDDS were also evaluated for their stabilities, dispersibilities, percentage transmittances and by particle size analyses. For thioridazine a 5.6-fold and for chlorpromazine a 3.7-fold higher CM uptake could be observed using a LCT-SNEDDS formulation compared to the drugs without formulation. In contrast, ex vivo uptake by isolated CM was not significantly increased by SNEDDS formulations based on MCT and SCT. Compared with isolated CM, the CM sizes were increased 2.5-fold in LCT-SNEDDS, whereas in MCT-SNEDDS or SCT-SNEDDS only a small, non-significant (P<0.05) increase in CM size was observed. These results show that distinct SNEDDS formulations containing phenothiazines are efficiently uptaken by plasma derived chylomicrons ex vivo.

  17. Protandim attenuates intimal hyperplasia in human saphenous veins cultured ex vivo via a catalase-dependent pathway.

    PubMed

    Joddar, Binata; Reen, Rashmeet K; Firstenberg, Michael S; Varadharaj, Saradhadevi; McCord, Joe M; Zweier, Jay L; Gooch, Keith J

    2011-03-15

    Human saphenous veins (HSVs) are widely used for bypass grafts despite their relatively low long-term patency. To evaluate the role of reactive oxygen species (ROS) signaling in intima hyperplasia (IH), an early stage pathology of vein-graft disease, and to explore the potential therapeutic effects of up-regulating endogenous antioxidant enzymes, we studied segments of HSV cultured ex vivo in an established ex vivo model of HSV IH. Results showed that HSV cultured ex vivo exhibit an ~3-fold increase in proliferation and ~3.6-fold increase in intimal area relative to freshly isolated HSV. Treatment of HSV during culture with Protandim, a nutritional supplement known to activate Nrf2 and increase the expression of antioxidant enzymes in several in vitro and in vivo models, blocks IH and reduces cellular proliferation to that of freshly isolated HSV. Protandim treatment increased the activity of SOD, HO-1, and catalase 3-, 7-, and 12-fold, respectively, and decreased the levels of superoxide (O(2)(•-)) and the lipid peroxidation product 4-HNE. Blocking catalase activity by cotreating with 3-amino-1,2,4-triazole abrogated the protective effect of Protandim on IH and proliferation. In conclusion, these results suggest that ROS-sensitive signaling mediates the observed IH in cultured HSV and that up-regulation of endogenous antioxidant enzymes can have a protective effect.

  18. Ex vivo nanofiber expansion and genetic modification of human cord blood-derived progenitor/stem cells enhances vasculogenesis.

    PubMed

    Das, Hiranmoy; Abdulhameed, Nasreen; Joseph, Matthew; Sakthivel, Ramasamy; Mao, Hai-Quan; Pompili, Vincent J

    2009-01-01

    The stem cell therapy for treating ischemic diseases is promising; however, the limited availability and compromised quality of progenitor cells in aged and diseased patients limit its therapeutic use. Here we report a nanofiber-based ex vivo stem cell expansion technology and proangiogenic growth factors overexpression of human umbilical cord blood (UCB)-derived progenitor cells to enhance angiogenic potential of therapeutic stem cells. The progenitor cells were expanded approximately 225-fold on nanofiber-based serum-free ex vivo expansion culture technique without inducing differentiation. The expanded cells express high levels of stem cell homing receptor, CXCR4, and adhesion molecule, LFA-1. The nanofiber-expanded stem cells uptake AcLDL effectively, and migrate efficiently in an in vitro transmigration assay. These expanded cells can also differentiate into endothelial and smooth muscle cells in vitro. In a NOD/SCID mouse hind limb vascular injury model, nanofiber-expanded cells were more effective in blood flow restoration and this effect was further augmented by VEGF(164) and PDGF-BB, growth factor overexpression. The data indicate that nanofiber-based ex vivo expansion technology can provide an essential number of therapeutic stem cells. Additionally, proangiogenic growth factors overexpression in progenitor cells can potentially improve autologous or allogeneic stem cell therapy for ischemic diseases.

  19. Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function.

    PubMed

    Tsubota, Yoshiaki; Frey, Jeremy M; Raines, Elaine W

    2014-01-01

    Monocyte recruitment to inflammatory sites and their transendothelial migration into tissues are critical to homeostasis and pathogenesis of chronic inflammatory diseases. However, even short-term suspension culture of primary human monocytes leads to phenotypic changes. In this study, we characterize the functional effects of ex vivo monocyte culture on the steps involved in monocyte transendothelial migration. Our data demonstrate that monocyte diapedesis is impaired by as little as 4 h culture, and the locomotion step is subsequently compromised. After 16 h in culture, monocyte diapedesis is irreversibly reduced by ∼90%. However, maintenance of monocytes under conditions mimicking physiological flow (5-7.5 dyn/cm²) is sufficient to reduce diapedesis impairment significantly. Thus, through the application of shear during ex vivo culture of monocytes, our study establishes a novel protocol, allowing functional analyses of monocytes not currently possible under static culture conditions. These data further suggest that monocyte-based therapeutic applications may be measurably improved by alteration of ex vivo conditions before their use in patients.

  20. How to Recondition Ex Vivo Initially Rejected Donor Lungs for Clinical Transplantation: Clinical Experience from Lund University Hospital

    PubMed Central

    Lindstedt, Sandra; Eyjolfsson, Atli; Koul, Bansi; Wierup, Per; Pierre, Leif; Gustafsson, Ronny; Ingemansson, Richard

    2011-01-01

    A major problem in clinical lung transplantation is the shortage of donor lungs. Only about 20% of donor lungs are accepted for transplantation. We have recently reported the results of the first six double lung transplantations performed with donor lungs reconditioned ex vivo that had been deemed unsuitable for transplantation by the Scandiatransplant, Eurotransplant, and UK Transplant organizations because the arterial oxygen pressure was less than 40 kPa. The three-month survival of patients undergoing transplant with these lungs was 100%. One patient died due to sepsis after 95 days, and one due to rejection after 9 months. Four recipients are still alive and well 24 months after transplantation, with no signs of bronchiolitis obliterans syndrome. The donor lungs were reconditioned ex vivo in an extracorporeal membrane oxygenation circuit using STEEN solution mixed with erythrocytes, to dehydrate edematous lung tissue. Functional evaluation was performed with deoxygenated perfusate at different inspired fractions of oxygen. The arterial oxygen pressure was significantly improved in this model. This ex vivo evaluation model is thus a valuable addition to the armamentarium in increasing the number of acceptable lungs in a donor population with inferior arterial oxygen pressure values, thereby, increasing the lung donor pool for transplantation. In the following paper we present our clinical experience from the first six patients in the world. We also present the technique we used in detail with flowchart. PMID:21876780

  1. The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.

    PubMed

    Blanco, Teresa Mortera; Mantalaris, Athanasios; Bismarck, Alexander; Panoskaltsis, Nicki

    2010-03-01

    Acute myeloid leukaemia (AML) is a cancer of haematopoietic cells that develops in three-dimensional (3-D) bone marrow niches in vivo. The study of AML has been hampered by lack of appropriate ex vivo models that mimic this microenvironment. We hypothesised that fabrication and optimisation of suitable biomimetic scaffolds for culturing leukaemic cells ex vivo might facilitate the study of AML in its native 3-D niche. We evaluated the growth of three leukaemia subtype-specific cell lines, K-562, HL60 and Kasumi-6, on highly porous scaffolds fabricated from biodegradable and non-biodegradable polymeric materials, such as poly (L-lactic-co-glycolic acid) (PLGA), polyurethane (PU), poly (methyl-methacrylate), poly (D, L-lactade), poly (caprolactone), and polystyrene. Our results show that PLGA and PU supported the best seeding efficiency and leukaemic growth. Furthermore, the PLGA and PU scaffolds were coated with extracellular matrix (ECM) proteins, collagen type I (62.5 or 125 microg/ml) and fibronectin (25 or 50 microg/ml) to provide biorecognition signals. The 3 leukaemia subtype-specific lines grew best on PU scaffolds coated with 62.5 microg/ml collagen type I over 6 weeks in the absence of exogenous growth factors. In conclusion, PU-collagen scaffolds may provide a practical model to study the biology and treatment of primary AML in an ex vivo mimicry.

  2. Analysis of Endothelial Adherence of Bartonella henselae and Acinetobacter baumannii Using a Dynamic Human Ex Vivo Infection Model.

    PubMed

    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; Kempf, Volkhard A J

    2015-12-28

    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.

  3. Ex vivo digestion of carp muscle tissue--ACE inhibitory and antioxidant activities of the obtained hydrolysates.

    PubMed

    Borawska, J; Darewicz, M; Vegarud, G E; Iwaniak, A; Minkiewicz, P

    2015-01-01

    In the digestive tract of humans, bioactive peptides, i.e. protein fragments impacting the physiological activity of the body, may be released during the digestion of food proteins, including those of fish. The aim of the study was to establish the method of human ex vivo digestion of carp muscle tissue and evaluate the angiotensin I-converting enzyme inhibitory and antioxidant activities of hydrolysates obtained after digestion. It was found that the hydrolysates of carp muscle tissue obtained with the three-stage method of simulated ex vivo digestion showed ACE inhibitory as well as antioxidative activities. It was demonstrated that the degree of hydrolysis depended on the duration of individual stages and the degree of comminution of the examined material. Although the applied gastric juices initiated the process of hydrolysis of carp muscle tissue, the duodenal juices caused a rapid increase in the amount of hydrolysed polypeptide bonds. The antihypertensive and antioxidative activities of the hydrolysates of carp muscle tissue increased together with progressive protein degradation. However, the high degree of protein hydrolysis does not favour an increase in the activity of free radical scavenging. The presented results are an example of the first preliminary screening of the potential health-promoting biological activity of carp muscle tissue in an ex vivo study.

  4. 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.

  5. Biomimetic phantom for cardiac diffusion MRI

    PubMed Central

    Teh, Irvin; Zhou, Feng‐Lei; Hubbard Cristinacce, Penny L.; Parker, Geoffrey J.M.

    2015-01-01

    Purpose Diffusion magnetic resonance imaging (MRI) is increasingly used to characterize cardiac tissue microstructure, necessitating the use of physiologically relevant phantoms for methods development. Existing phantoms are generally simplistic and mostly simulate diffusion in the brain. Thus, there is a need for phantoms mimicking diffusion in cardiac tissue. Materials and Methods A biomimetic phantom composed of hollow microfibers generated using co‐electrospinning was developed to mimic myocardial diffusion properties and fiber and sheet orientations. Diffusion tensor imaging was carried out at monthly intervals over 4 months at 9.4T. 3D fiber tracking was performed using the phantom and compared with fiber tracking in an ex vivo rat heart. Results The mean apparent diffusion coefficient and fractional anisotropy of the phantom remained stable over the 4‐month period, with mean values of 7.53 ± 0.16 × 10‐4 mm2/s and 0.388 ± 0.007, respectively. Fiber tracking of the 1st and 3rd eigenvectors generated analogous results to the fiber and sheet‐normal direction respectively, found in the left ventricular myocardium. Conclusion A biomimetic phantom simulating diffusion in the heart was designed and built. This could aid development and validation of novel diffusion MRI methods for investigating cardiac microstructure, decrease the number of animals and patients needed for methods development, and improve quality control in longitudinal and multicenter cardiac diffusion MRI studies. J. MAGN. RESON. IMAGING 2016;43:594–600. PMID:26213152

  6. 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

  7. 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

  8. Ex vivo reversal of effects of rivaroxaban evaluated using thromboelastometry and thrombin generation assay

    PubMed Central

    Schenk, B.; Würtinger, P.; Streif, W.; Sturm, W.; Fries, D.; Bachler, M.

    2016-01-01

    Background In major bleeding events, the new direct oral anticoagulants pose a great challenge for physicians. The aim of the study was to test for ex vivo reversal of the direct oral anticoagulant rivaroxaban with various non-specific reversal agents: prothrombin complex concentrate (PCC), activated prothrombin complex concentrate (aPCC), recombinant activated factor VII (rFVIIa), and fibrinogen concentrate (FI). Methods Blood was obtained from healthy volunteers and from patients treated with rivaroxaban. Blood samples from healthy volunteers were spiked with rivaroxaban to test the correlation between rivaroxaban concentration and coagulation tests. Patient blood samples were spiked with various concentrations of the above-mentioned agents and analysed using thromboelastometry and thrombin generation. Results When added in vitro, rivaroxaban was significantly (P<0.05) correlated with ROTEM® thromboelastometry EXTEM (extrinsic coagulation pathway) clotting time (CT), time to maximal velocity (MaxV−t), and with all measured thrombin generation parameters. In vivo, CT, MaxV−t, lag time, and peak thrombin generation (Cmax) were significantly correlated with rivaroxaban concentrations. Regarding reversal of rivaroxaban, all tested agents significantly (P<0.05) reduced EXTEM CT, but to different extents: rFVIIa by 68%, aPCC by 47%, PCC by 17%, and FI by 9%. Only rFVIIa reversed EXTEM CT to baseline values. Both PCC (+102%) and aPCC (+232%) altered overall thrombin generation (area under the curve) and increased Cmax (+461% for PCC, +87.5% for aPCC). Conclusions Thromboelastometry and thrombin generation assays do not favour the same reversal agents for rivaroxaban anticoagulation. Controlled clinical trials are urgently needed to establish doses and clinical efficacy of potential reversal agents. Clinical trial registration EudracCT trial no. 213-00474-30. PMID:27623677

  9. 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

  10. Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM.

    PubMed

    Ng, Chee Ping; Sharif, Abdul Rahim Mohamed; Heath, Daniel E; Chow, John W; Zhang, Claire B Y; Chan-Park, Mary B; Hammond, Paula T; Chan, Jerry K Y; Griffith, Linda G

    2014-04-01

    Large-scale expansion of highly functional adult human mesenchymal stem cells (aMSCs) remains technologically challenging as aMSCs lose self renewal capacity and multipotency during traditional long-term culture and their quality/quantity declines with donor age and disease. Identification of culture conditions enabling prolonged expansion and rejuvenation would have dramatic impact in regenerative medicine. aMSC-derived decellularized extracellular matrix (ECM) has been shown to provide such microenvironment which promotes MSC self renewal and "stemness". Since previous studies have demonstrated superior proliferation and osteogenic potential of human fetal MSCs (fMSCs), we hypothesize that their ECM may promote expansion of clinically relevant aMSCs. We demonstrated that aMSCs were more proliferative (∼ 1.6 ×) on fMSC-derived ECM than aMSC-derived ECMs and traditional tissue culture wares (TCPS). These aMSCs were smaller and more uniform in size (median ± interquartile range: 15.5 ± 4.1 μm versus 17.2 ± 5.0 μm and 15.5 ± 4.1 μm for aMSC ECM and TCPS respectively), exhibited the necessary biomarker signatures, and stained positive for osteogenic, adipogenic and chondrogenic expressions; indications that they maintained multipotency during culture. Furthermore, fMSC ECM improved the proliferation (∼ 2.2 ×), size (19.6 ± 11.9 μm vs 30.2 ± 14.5 μm) and differentiation potential in late-passaged aMSCs compared to TCPS. In conclusion, we have established fMSC ECM as a promising cell culture platform for ex vivo expansion of aMSCs.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Hydrogen sulfide activates the carotid body chemoreceptors in cat, rabbit and rat ex vivo preparations.

    PubMed

    Jiao, Yingfu; Li, Qian; Sun, Biying; Zhang, Guohua; Rong, Weifang

    2015-03-01

    We and others previously reported experimental evidence suggesting an important role for hydrogen sulfide (H2S) in oxygen sensing in murine carotid body chemoreceptors. More recent data implicated abnormal H2S-mediated chemoreceptor signaling in pathological conditions such as chronic heart failure and hypertension. However, the idea of H2S as a mediator of oxygen-sensing in chemoreceptors has been challenged. In particular, it was shown that exogenous H2S inhibited the release of neurotransmitters (ACh and ATP) from the cat carotid body, raising the possibility that there exists significant species difference in H2S-mediated signaling in chemoreceptors. This study was designed specifically to determine the effect of H2S on chemoreceptors in different species. We conducted multiunit extracellular recordings of the sinus nerve in the ex vivo carotid body preparation taken from the rat, the cat and the rabbit. As observed in the mouse carotid body, H2S donors (NaHS or Na2S) evoked qualitatively similar excitatory responses of the afferent sinus nerves of the species studied here. The excitatory effects of the H2S donors were concentration-dependent and reversible. The sinus nerve responses to H2S donors were prevented by blockade of the transmission between type I cells and the afferent terminals, as was the response to hypoxia. These results demonstrate that exogenous H2S exerts qualitatively similar excitatory effects on chemoreceptor afferents of different species. The role of endogenous H2S-mediated signaling in carotid body function in different species awaits further investigation.

  18. Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo.

    PubMed

    Beans, Elizabeth J; Fournogerakis, Dennis; Gauntlett, Carolyn; Heumann, Lars V; Kramer, Rainer; Marsden, Matthew D; Murray, Danielle; Chun, Tae-Wook; Zack, Jerome A; Wender, Paul A

    2013-07-16

    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.

  19. Defective eosinophil hematopoiesis ex vivo in inbred Rocky Mountain White (IRW) mice.

    PubMed

    Dyer, Kimberly D; Garcia-Crespo, Katia E; Percopo, Caroline M; Bowen, Aaron B; Ito, Tomonobu; Peterson, Karin E; Gilfillan, Alasdair M; Rosenberg, Helene F

    2011-12-01

    We examine the proliferation and differentiation of bone marrow (BM) progenitors from inbred Rocky Mountain White (IRW) mice, a strain used primarily for retrovirus infection studies. In contrast to findings with BALB/c and C57BL/6 strains, IRW BM cells cannot proliferate or generate pure eosinophil cultures ex vivo in response to a defined cytokine regimen. Analysis of IRW BM at baseline was unremarkable, including 0.08 ± 0.03% Lin(-)Sca-1(+)c-kit(+) (LSK) hematopoietic stem cells and 5.2 ± 0.3% eosinophils; the percentage of eosinophil progenitors (EoPs; Lin(-)Sca-1(-)c-kit(+)CD34(+)IL-5Rα(+)) was similar in all three mouse strains. Transcripts encoding GM-CSFRα and the IL-3/IL-5/GM-CSF common β chain were detected at equivalent levels in IRW and BALB/c BM, whereas expression of transcripts encoding IL-5Rα, IL-3Rα, and GATA-2 was diminished in IRW BM compared with BALB/c. Expression of membrane-bound IL-5Rα and intracellular STAT5 proteins was also diminished in IRW BM cells. Diminished expression of transcripts encoding IL-5Rα and GATA-2 and immunoreactive STAT5 in IRW BM persisted after 4 days in culture, along with diminished expression of GATA-1. Western blot revealed that cells from IRW BM overexpress nonsignaling soluble IL-5Rα protein. Interestingly, OVA sensitization and challenge resulted in BM and airway eosinophilia in IRW mice; however, the responses were significantly blunted. These results suggest that IRW mice have diminished capacity to generate eosinophils in culture and in vivo, likely as a result of diminished signaling via IL-5Rα.

  20. Structural layers of ex vivo rat hippocampus at 7T MRI.

    PubMed

    Kamsu, Jeanine Manuella; Constans, Jean-Marc; Lamberton, Franck; Courtheoux, Patrick; Denise, Pierre; Philoxene, Bruno; Coquemont, Maelle; Besnard, Stephane

    2013-01-01

    Magnetic resonance imaging (MRI) applied to the hippocampus is challenging in studies of the neurophysiology of memory and the physiopathology of numerous diseases such as epilepsy, Alzheimer's disease, ischemia, and depression. The hippocampus is a well-delineated cerebral structure with a multi-layered organization. Imaging of hippocampus layers is limited to a few studies and requires high magnetic field and gradient strength. We performed one conventional MRI sequence on a 7T MRI in order to visualize and to delineate the multi-layered hippocampal structure ex vivo in rat brains. We optimized a volumic three-dimensional T2 Rapid Acquisition Relaxation Enhancement (RARE) sequence and quantified the volume of the hippocampus and one of its thinnest layers, the stratum granulare of the dentate gyrus. Additionally, we tested passive staining by gadolinium with the aim of decreasing the acquisition time and increasing image contrast. Using appropriated settings, six discrete layers were differentiated within the hippocampus in rats. In the hippocampus proper or Ammon's Horn (AH): the stratum oriens, the stratum pyramidale of, the stratum radiatum, and the stratum lacunosum moleculare of the CA1 were differentiated. In the dentate gyrus: the stratum moleculare and the stratum granulare layer were seen distinctly. Passive staining of one brain with gadolinium decreased the acquisition time by four and improved the differentiation between the layers. A conventional sequence optimized on a 7T MRI with a standard receiver surface coil will allow us to study structural layers (signal and volume) of hippocampus in various rat models of neuropathology (anxiety, epilepsia, neurodegeneration).

  1. Transgenic Expression of Osteoactivin/gpnmb Enhances Bone Formation in Vivo and Osteoprogenitor Differentiation ex Vivo

    PubMed Central

    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.

    2015-01-01

    Initial identification of osteoactivin (OA)/glycoprotein non-melanoma clone B (gpnmb) was demonstrated in an osteopetrotic rat model, where OA expression was increased 3-fold 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

  2. Detrimental arterial inflammatory effect of microparticles circulating in preeclamptic women: ex vivo evaluation in human arteries.

    PubMed

    Boisramé-Helms, Julie; Meziani, Ferhat; Sananès, Nicolas; Boisramé, Thomas; Langer, Bruno; Schneider, Francis; Ragot, Thierry; Andriantsitohaina, Ramaroson; Tesse, Angela

    2015-10-01

    Elevated plasmatic levels of lympho-monocyte and platelet microparticles (MPs) have been reported in preeclampsia. Previous studies suggest that MPs could participate in preeclampsia vascular impairment. In this study, we investigated the ex vivo vascular effects of MPs from preeclamptic women on arteries from normotensive pregnant women. Omental arteries were collected from normal pregnant women undergoing cesarean section and incubated during 24 h with MPs from normal pregnant or preeclamptic women. Vascular contraction to serotonin and phenylephrine was studied on a wire myograph with or without pharmacological selective inhibitors of inducible nitric oxide synthase (iNOS) and/or cyclo-oxygenase-2 (COX-2). Expression of iNOS, COX-2, and NF-κB and production of superoxide anion and 8-isoprostane were also assessed by immunohistological or biochemical staining and/or Western blot or ELISA assay, respectively. Microparticles from preeclamptic women, but not those from normal pregnant women, induced hyporeactivity to vasocontracturant agonists in omental arteries. Selective inhibitor of iNOS partially restored this arterial contraction, suggesting that nitric oxide (NO) is involved in vascular contractility alteration. Conversely, COX-2 induced 8-isoprostane release, a vasoconstricting metabolite modulating the agonist-induced contraction. COX-2 selective inhibitor almost abolished the arterial contraction in the same vessels. Interestingly, the association of iNOS and COX-2 selective inhibitors restored the contraction to control levels. Moreover, iNOS, COX-2, and NF-κB expressions are upregulated and superoxide anion levels increased in vessels incubated with MPs from preeclamptic women. In conclusion, circulating MPs from preeclamptic women induce vascular inflammation and enhance oxidative stress. These results suggest a possible role of MPs during preeclampsia-induced arterial dysfunction.

  3. Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology.

    PubMed

    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.

  4. 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

  5. Sealing Ability of Nano-ionomer in Primary Teeth: An ex vivo Study

    PubMed Central

    Karkare, Swati

    2016-01-01

    Introduction Microleakage is an important consideration in primary dentition because the floor of the cavity preparation may be close to the pulp. The added insult to the pulp caused by seepage of irritants around the restoration and through the thin dentin may produce irreversible pulp damage. Aim The objective of this study was to evaluate and compare the sealing ability of three light cured (LC) resin-modified glass-ionomer cements (RMGICs) in primary anterior teeth. Materials and methods Class V cavity was prepared on the labial surface of extracted primary anterior teeth which were then grouped and restored with Ketac N100, Fuji II LC, or Vitremer. Dye penetration test with methylene blue stain was used to record the microleakage. Depth of dye penetration was recorded in millimeters at the incisal and gingival margin using computer software. Results The depth of dye penetration at the incisal margin in the three groups was comparable, but at the gingival margin, Vitremer showed the least dye penetration, followed by Fuji II LC, and Ketac N100. The depth of dye penetration at the gingival margin was higher than the incisal margins in all the three groups. Conclusion Among the three RMGICs, Vitremer can be considered as the material of choice for restoring class V cavities in primary anterior teeth. Periodic recall and recare is necessary when any of the three materials are used in clinical practice. How to cite this article Siddiqui F, Karkare S. Sealing Ability of Nano-ionomer in Primary Teeth: An ex vivo Study. Int J Clin Pediatr Dent 2016;9(3):209-213. PMID:27843251

  6. 24(S)-Hydroxycholesterol protects the ex vivo rat retina from injury by elevated hydrostatic pressure

    PubMed Central

    Ishikawa, Makoto; Yoshitomi, Takeshi; Zorumski, Charles F.; Izumi, Yukitoshi

    2016-01-01

    In the central nervous system, 24(S)-hydroxycholesterol (24(S)-HC) is an oxysterol synthesized from cholesterol by cholesterol 24-hydroxylase (CYP46A1) encoded by the cyp46a1 gene. In the present study using a rat ex vivo glaucoma model, we found that retinal 24(S)-HC synthesis is facilitated by pressure elevation. Moreover, we found that 24(S)-HC is neuroprotective against pressure mediated retinal degeneration. Quantitative real-time RT-PCR, ELISA, and immunohistochemistry revealed that elevated pressure facilitated the expression of cyp46a1 and CYP46A1. Immunohistochemically, the enhanced expression of CYP46A1 was mainly observed in retinal ganglion cells (RGC). LC-MS/MS revealed that 24(S)-HC levels increased in a pressure-dependent manner. Axonal injury and apoptotic RGC death induced by 75 mmHg high pressure was ameliorated by exogenously administered 1 μM 24(S)-HC. In contrast, voriconazole, a CYP46A1 inhibitor, was severely toxic even at normobaric pressure. Under normobaric conditions, 30 μM 24(S)-HC was required to prevent the voriconazole-mediated retinal damage. Taken together, our findings indicate that 24(S)-HC is facilitated by elevated pressure and plays a neuroprotective role under glaucomatous conditions, while voriconazole, an antifungal drug, is retinotoxic. 24(S)-HC and related compounds may serve as potential therapeutic targets for protecting glaucomatous eyes from pressure-induced injuries. PMID:27653972

  7. A single epidermal stem cell strategy for safe ex vivo gene therapy

    PubMed Central

    Droz-Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz-Dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

    2015-01-01

    There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy. PMID

  8. 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.

  9. 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

  10. An ex vivo study of the correlation between acoustic emission and microvascular damage.

    PubMed

    Samuel, Stanley; Cooper, Michol A; Bull, Joseph L; Fowlkes, J Brian; Miller, Douglas L

    2009-09-01

    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 (80microL.kg(-1).min(-1)infusion 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 10Hz (one location per PRF per rat). The 100-pulse exposures were implemented at a peak rarefactional pressure (P(r)) of 2MPa, frequency of 2.25MHz with 46 cycle pulses. For the pressure amplitude threshold study, 100-pulse exposures (46 cycle pulses) were conducted at various peak rarefactional pressures from 0.5MPa to 2MPa at a frequency of 2.25MHz and PRF of 100Hz. Photomicrographs were captured before and 2-min postexposure. On a pulse-to-pulse basis, the 10Hz acoustic emission was considerably higher and more sustained than those at other PRFs (1000, 500, and 100Hz) (p<0.05). Damage, measured as area of extravasation of red blood cells (RBCs), was also significantly higher at 10Hz PRF than at 1000, 500 and 100Hz (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; R(2)=0.75). No visible damage was present at P(r)< or =0.85MPa. Damage, however, was observed at P(r)> or =1.0MPa and it increased with increasing acoustic pressure.

  11. Short communication: Glutamine modulates inflammatory responses to lipopolysaccharide in ex vivo bovine endometrium.

    PubMed

    Noleto, Pablo G; Saut, João Paulo E; Sheldon, I Martin

    2017-03-01

    Bacteria infect the endometrium lining the uterus of cattle after parturition, and clearance of these microbes depends on a robust innate immune response to bacterial molecules, such as the endotoxin lipopolysaccharide (LPS). Endometrial inflammation is characterized by secretion of the cytokines IL-1β and IL-6 and the chemokine IL-8. However, animals often fail to clear invading bacteria and develop uterine disease if they are in negative energy balance, with reduced abundance of glucose and glutamine, which are substrates for energy in tissues. Depletion of glucose blunts inflammatory responses in the endometrium, but the role of glutamine is not clear. The present study tested the hypothesis that depletion of glutamine compromises inflammatory responses to LPS in endometrial tissue. Ex vivo organ cultures of endometrium were challenged with LPS, and culture supernatants accumulated IL-1β, IL-6, and IL-8, as expected. However, reducing the availability of glutamine in culture medium containing glucose reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%. Surprisingly, in the absence of glucose, supplying increasing amounts of glutamine was not sufficient to augment inflammatory responses to LPS, whereas, in the absence of glutamine, supplying more glucose increased inflammation. Furthermore, inhibiting glycolysis reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%, even when glutamine and glucose were abundant. In conclusion, depletion of glutamine reduces inflammatory responses to LPS in the endometrium, and the activity of glutamine depends on glucose and glycolysis. These data provide mechanistic insights into how negative energy balance may be linked to postpartum uterine disease.

  12. Human whole-blood culture system for ex vivo characterization of designer-cell function.

    PubMed

    Schukur, Lina; Geering, Barbara; Fussenegger, Martin

    2016-03-01

    Encapsulated designer cells implanted into mice are currently used to validate the efficacy of therapeutic gene networks for the diagnosis and treatment of various human diseases in preclinical research. Because many human conditions cannot be adequately replicated by animal models, complementary and alternative procedures to test future treatment strategies are required. Here we describe a novel approach utilizing an ex vivo human whole-blood culture system to validate synthetic biology-inspired designer cell-based treatment strategies. The viability and functionality of transgenic mammalian designer cells co-cultured with primary human immune cells were characterized. We demonstrated that transgenic mammalian designer cells required adequate insulation from the human blood microenvironment to maintain viability and functionality. The biomaterial alginate-(poly-l-lysine)-alginate used to encapsulate the transgenic designer cells did neither affect the viability of primary granulocytes and lymphocytes nor the functionality of lymphocytes. Additionally, alginate-encapsulated transgenic designer cells remained responsive to the release of the pro-inflammatory cytokine tumor necrosis factor (TNF) from the whole-blood culture upon exposure to bacterial lipopolysaccharide (LPS). TNF diffused into the alginate capsules, bound to the specific TNF receptors on the transgenic designer cells' surface and triggered the expression of the reporter gene SEAP (human placental secreted alkaline phosphatase) that was rewired to the TNF-specific signaling cascade. Human whole-blood culture systems can therefore be considered as valuable complementary assays to animal models for the validation of synthetic circuits in genetically modified mammalian cells and may speed up preclinical research in a world of personalized medicine.

  13. 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

  14. 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.

  15. Ex vivo evaluation of new 2D and 3D dental radiographic technology for detecting caries

    PubMed Central

    Tyndall, Donald; Mol, André; Everett, Eric T; Bangdiwala, Ananta

    2016-01-01

    Objectives: Proximal dental caries remains a prevalent disease with only modest detection rates by current diagnostic systems. Many new systems are available without controlled validation of diagnostic efficacy. The objective of this study was to evaluate the diagnostic efficacy of three potentially promising new imaging systems. Methods: This study evaluated the caries detection efficacy of Schick 33 (Sirona Dental, Salzburg, Austria) intraoral digital detector images employing an advanced sharpening filter, Planmeca ProMax® (Planmeca Inc., Helsinki, Finland) extraoral “panoramic bitewing” images and Sirona Orthophos XG3D (Sirona Dental) CBCT images with advanced artefact reduction. Conventional photostimulable phosphor images served as the control modality. An ex vivo study design using extracted human teeth, ten expert observers and micro-CT ground truth was employed. Results: Receiver operating characteristic analysis indicated similar diagnostic efficacy of all systems (ANOVA p > 0.05). The sensitivity of the Schick 33 images (0.48) was significantly lower than the other modalities (0.53–0.62). The specificity of the Planmeca images (0.86) was significantly lower than Schick 33 (0.96) and XG3D (0.97). The XG3D showed significantly better cavitation detection sensitivity (0.62) than the other modalities (0.48–0.57). Conclusions: The Schick 33 images demonstrated reduced caries sensitivity, whereas the Planmeca panoramic bitewing images demonstrated reduced specificity. XG3D with artefact reduction demonstrated elevated sensitivity and specificity for caries detection, improved depth accuracy and substantially improved cavitation detection. Care must be taken to recognize potential false-positive caries lesions with Planmeca panoramic bitewing images. Use of CBCT for caries detection must be carefully balanced with the presence of metal artefacts, time commitment, financial cost and radiation dose. PMID:26670605

  16. 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.

  17. 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

  18. Design and subspace system identification of an ex vivo vascular perfusion system.

    PubMed

    El-Kurdi, Mohammed S; Vipperman, Jeffrey S; Vorp, David A

    2009-04-01

    Numerical algorithms for subspace system identification (N4SID) are a powerful tool for generating the state space (SS) representation of any system. The purpose of this work was to use N4SID to generate SS models of the flowrate and pressure generation within an ex vivo vascular perfusion system (EVPS). Accurate SS models were generated and converted to transfer functions (TFs) to be used for proportional integral and derivative (PID) controller design. By prescribing the pressure and flowrate inputs to the pumping components within the EVPS and measuring the resulting pressure and flowrate in the system,_four TFs were estimated;_two for a flowrate controller (H(RP,f) and H(RPP,f)) and two for a pressure controller (H(RP,p) and H(RPP,p)). In each controller,_one TF represents a roller pump (H(RP,f) and H(RP,p)),_and the other represents a roller pump and piston in series (H(RPP,f) and H(RPP,p)). Experiments to generate the four TFs were repeated five times (N=5) from which average TFs were calculated. The average model fits, computed as the percentage of the output variation (to_the_prescribed_inputs) reproduced by the model, were 94.93+/-1.05% for H(RP,p), 81.29+/-0.20% for H(RPP,p), 94.45+/-0.73% for H(RP,f), and 77.12+/-0.36% for H(RPP,f). The simulated step, impulse, and frequency responses indicate that the EVPS is a stable system and can respond to signals containing power of up to 70_Hz.

  19. A single epidermal stem cell strategy for safe ex vivo gene therapy.

    PubMed

    Droz-Georget Lathion, Stéphanie; Rochat, Ariane; Knott, Graham; Recchia, Alessandra; Martinet, Danielle; Benmohammed, Sara; Grasset, Nicolas; Zaffalon, Andrea; Besuchet Schmutz, Nathalie; Savioz-Dayer, Emmanuelle; Beckmann, Jacques Samuel; Rougemont, Jacques; Mavilio, Fulvio; Barrandon, Yann

    2015-04-01

    There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.

  20. Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue

    PubMed Central

    Vierkotten, Sarah; Lindner, Michael; Königshoff, Melanie; Eickelberg, Oliver

    2015-01-01

    During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment. PMID:26092995

  1. 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

  2. Ex Vivo Modeling of Multidomain Peptide Hydrogels with Intact Dental Pulp

    PubMed Central

    Moore, A.N.; Perez, S.C.; Hartgerink, J.D.; D’Souza, R.N.; Colombo, J.S.

    2015-01-01

    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

  3. Controlled ex-vivo plasma hydrolysis of valaciclovir to acyclovir demonstration using tandem mass spectrometry.

    PubMed

    Goswami, Dipanjan; Khuroo, Arshad; Gurule, Sanjay; Modhave, Yogesh; Monif, Tausif

    2011-11-01

    Plasma estimation of valaciclovir, an antiviral drug, is challenging due to both in-vivo and ex-vivo hydrolysis to active metabolite acyclovir. A simultaneous method is described involving the solid-phase ion-exchange extraction procedure requiring 100 μL of plasma volume, a reverse-phase Lichrosphere RP Select B (125 × 6 mm, 5 μm) column and isocratic mobile phase to achieve the desired chromatographic separation. ESI-MS/MS multiple reaction monitoring in positive polarity, detected mass pairs for valaciclovir (m/z 325.5 → 152.2), acyclovir (m/z 226.3 → 152.2) and respective internal standards valganciclovir (m/z 307.1 → 220.3) and acyclovir-d4 (m/z 230.2 → 152.0). Fully fledged method validation was evaluated as per current regulatory requirements and results were deemed acceptable. The plasma samples showed extensive hydrolysis of valaciclovir when collected or processed at room temperature, without buffer stabilization prior to storage at -15°C. Our results showed that using prechilled K3 EDTA vacutainers immersed in an iced-water bath during blood sample collection, and addition of 50% orthophosphoric acid solution to plasma samples prior to storage at -50°C for at least 120 days controlled the hydrolysis of valaciclovir to acyclovir. While monitoring drug absorption into systematic circulation, the valaciclovir to acyclovir formation ratio was improved to 1:20 in healthy volunteers for the first time.

  4. 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.

  5. The novel drug delivery to vascular wall using laser driven thermal balloon: basic study ex vivo

    NASA Astrophysics Data System (ADS)

    Suganuma, Kao; Homma, Rie; Shimazaki, Natsumi; Ogawa, Emiyu; Arai, Tsunenori

    2016-10-01

    To enhance drug delivery performance of popular drug eluting balloon against re-stenosis after angioplasty, we have an idea regarding to adjacent use of our unique laser driven thermal balloon of which characteristics could realize short term and uniform temperature elevation to modify drug delivery characteristics. We have already reported a delivery enhancement effect using this idea, however, detailed characteristics have not been studied yet. We studied balloon dilatation in terms of vascular circumferential tension on the heating drug delivery performance using porcine carotid artery wall ex vivo. The extracted carotid artery was used and circumferential tension of 0-30 mN/mm2 was added. Heating drug delivery was performed on this carotid artery with the heated solution of hydrophobic fluorescent Rhodamine B with 3 μg/ml in concentration at 37 and 70°C. We obtained a defined drug delivery quantity as well as delivery depth by a microscopic fluorescence measurement on a cross section of the drug delivered vessel wall. In the cases of 70°C, we found the drug penetration increase against 3°C case. We predict that the collagen thermal denaturation of the vessel wall may play important role to this penetration. In the case of 3°C, we found the drug concentration on the intimal surface with 7 mN/mm2 was increased as 10-30 times as other tension values. We found surface grooves in this case using an electron micrography. Therefore, we think that the drug delivery enhancement might be related to the groove formations of the vessel wall.

  6. Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii

    PubMed Central

    Graham, Joseph G.; Winchell, Caylin G.; Kurten, Richard C.

    2016-01-01

    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

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

    PubMed

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

    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.

  8. Terbinafine Hydrochloride Trans-ungual Delivery via Nanovesicular Systems: In Vitro Characterization and Ex Vivo Evaluation.

    PubMed

    Elsherif, Noha Ibrahim; Shamma, Rehab Nabil; Abdelbary, Ghada

    2017-02-01

    Treating a nail infection like onychomycosis is challenging as the human nail plate acts as a formidable barrier against all drug permeation. Available oral and topical treatments have several setbacks. Terbinafine hydrochloride (TBH), belonging to the allylamine class, is mainly used for treatment of onychomycosis. This study aims to formulate TBH in a nanobased spanlastic vesicular carrier that enables and enhances the drug delivery through the nail. The nanovesicles were formulated by ethanol injection method, using either Span® 60 or Span® 65, together with Tween 80 or sodium deoxycholate as an edge activator. A full factorial design was implemented to study the effect of different formulation and process variables on the prepared TBH-loaded spanlastic nanovesicles. TBH entrapment efficiency percentages, particle size diameter, percentage drug released after 2 h and 8 h were selected as dependent variables. Optimization was performed using Design-Expert® software to obtain an optimized formulation with high entrapment efficiency (62.35 ± 8.91%), average particle size of 438.45 ± 70.5 nm, and 29.57 ± 0.93 and 59.53 ± 1.73% TBH released after 2 and 8 h, respectively. The optimized formula was evaluated using differential scanning calorimetry and X-ray diffraction and was also morphologically examined using transmission electron microscopy. An ex vivo study was conducted to determine the permeation and retainment of the optimized formulation in a human cadaver nail plate, and confocal laser scanning microscope was used to show the extent of formulation permeation. In conclusion, the results confirmed that spanlastics exhibit promising results for the trans-ungual delivery of TBH.

  9. Search for PET probes for imaging the globus pallidus studied with rat brain ex vivo autoradiography.

    PubMed

    Ishiwata, K; Ogi, N; Shimada, J; Wang, W; Ishii, K; Tanaka, A; Suzuki, F; Senda, M

    2000-12-01

    We have evaluated the feasibility of using four positron emission tomography (PET) tracers for imaging the globus pallidus by ex vivo autoradiography in rats. The tracers investigated were [11C]KF18446, [11C]SCH 23390 and [11C]raclopride for mapping adenosine A2A, dopamine D1 and dopamine D2 receptors, respectively, and [18F]FDG. The highest uptake by the globus pallidus was found for [11C]SCH 23390, followed by [18F]FDG, [11C]KF18446 and [11C]raclopride. The receptor-specific uptake by the globus pallidus was observed in [11C]KF18446 and [11C]SCH 23390, but not in [11C]raclopride. Uptake ratios of globus pallidus to the striatum for [18F]FDG and [11C]KF18446 were approximately 0.6, which was twice as large as that for [11C]SCH 23390. In a rat model of degeneration of striatopallidal gamma-aminobutyric acid-ergic-enkephalin neurons induced by intrastriatal injection of quinolinic acid, the uptake of [11C]KF18446 by the striatum and globus pallidus was remarkably reduced. To prove the visualization of the globus pallidus by PET with [18F]FDG and [11C]KF18446, PET-MRI registration technique and advances in PET technologies providing high-resolution PET scanner will be required. The metabolic activity of the globus pallidus could then be measured by PET with [18F]FDG, and [11C]KF18446 may be a candidate tracer for imaging the pallidal terminals projecting from the striatum.

  10. Ozone therapy as an adjuvant for endondontic protocols: microbiological – ex vivo study and citotoxicity analyses

    PubMed Central

    NOGALES, Carlos Goes; FERREIRA, Marina Beloti; MONTEMOR, Antonio Fernando; RODRIGUES, Maria Filomena de Andrade; Lage-MARQUES, José Luiz; ANTONIAZZI, João Humberto

    2016-01-01

    ABSTRACT Objectives This study evaluated the antimicrobial efficacy of ozone therapy in teeth contaminated with Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus using a mono-species biofilm model. Parallel to this, the study aimed to evaluate the cytotoxicity of ozone for human gingival fibroblasts. Material and Methods: One hundred and eighty single-root teeth were contaminated with a mono-species biofilm of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus. Groups were formed: Group I – control; Group II – standard protocol; Group III – standard protocol + ozone gas at 40 µg/mL; and Group IV – standard protocol + aqueous ozone at 8 µg/mL. In parallel, human gingival fibroblasts were submitted to the MTT test. Cells were plated, then ozone was applied as follows: Group I (control) – broth medium; Group II – aqueous ozone at 2 µg/mL; Group III – aqueous ozone at 5 µg/mL; and Group IV – aqueous ozone at 8 µg/mL. Data were submitted to the Kruskal Wallis test and Bonferroni post hoc analyses to assess microbiology and cytotoxicity, respectively (p<0.05%). Results The results revealed antimicrobial efficacy by Group IV with no CFU count. The cytotoxicity assay showed Groups III and IV to be the most aggressive, providing a decrease in cell viability at hour 0 from 100% to 77.3% and 68.6%, respectively. Such a decrease in cell viability was reverted, and after 72 hours Groups III and IV provided the greatest increase in cell viability, being statistically different from Groups I and II. Conclusion According to the applied methodology and the limitations of this study, it was possible to conclude that ozone therapy improved the decontamination of the root canal ex vivo. Ozone was toxic to the cells on first contact, but cell viability was recovered. Thus, these findings suggest that ozone might be useful to improve root canal results. PMID:28076466

  11. Nonlinear acoustic properties of ex vivo bovine liver and the effects of temperature and denaturation.

    PubMed

    Jackson, E J; Coussios, C-C; Cleveland, R O

    2014-06-21

    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.

  12. Echo decorrelation imaging of ex vivo HIFU and bulk ultrasound ablation using image-treat arrays

    NASA Astrophysics Data System (ADS)

    Fosnight, Tyler R.; Hooi, Fong Ming; Colbert, Sadie B.; Keil, Ryan D.; Barthe, Peter G.; Mast, T. Douglas

    2017-03-01

    In this study, the ability of ultrasound echo decorrelation imaging to map and predict heat-induced cell death was tested using bulk ultrasound thermal ablation, high intensity focused ultrasound (HIFU) thermal ablation, and pulse-echo imaging of ex vivo liver tissue by a custom image-treat array. Tissue was sonicated at 5.0 MHz using either pulses of unfocused ultrasound (N=12) (7.5 s, 50.9-101.8 W/cm2 in situ spatial-peak, temporal-peak intensity) for bulk ablation or focused ultrasound (N=21) (1 s, 284-769 W/cm2 in situ spatial-peak, temporal-peak intensity and focus depth of 10 mm) for HIFU ablation. Echo decorrelation and integrated backscatter (IBS) maps were formed from radiofrequency pulse-echo images captured at 118 frames per second during 5.0 s rest periods, beginning 1.1 s after each sonication pulse. Tissue samples were frozen at -80˚C, sectioned, vitally stained, imaged, and semi-automatically segmented for receiver operating characteristic (ROC) analysis. ROC curves were constructed to assess prediction performance for echo decorrelation and IBS. Logarithmically scaled mean echo decorrelation in non-ablated and ablated tissue regions before and after electronic noise and motion correction were compared. Ablation prediction by echo decorrelation and IBS was significant for both focused and bulk ultrasound ablation. The log10-scaled mean echo decorrelation was significantly greater in regions of ablation for both HIFU and bulk ultrasound ablation. Echo decorrelation due to electronic noise and motion was significantly reduced by correction. These results suggest that ultrasound echo decorrelation imaging is a promising approach for real-time prediction of heat-induced cell death for guidance and monitoring of clinical thermal ablation, including radiofrequency ablation and HIFU.

  13. Fetal sulcation and gyrification in common marmosets (Callithrix jacchus) obtained by ex vivo magnetic resonance imaging.

    PubMed

    Sawada, K; Hikishima, K; Murayama, A Y; Okano, H J; Sasaki, E; Okano, H

    2014-01-17

    The present study characterized fetal sulcation patterns and gyrification in the cerebrum of the New World monkey group, common marmosets, using a 3D T2-weighted high-resolution anatomical magnetic resonance imaging (MRI) sequence from the fixed brain at 7-tesla ex vivo. Fetal sulcation in the marmoset cerebrum began to indent the lateral fissure and hippocampal sulcus in gestational week (GW) 12, and then the following sulci emerged: the callosal and calcarine sulci on GW 15; the superior temporal sulcus on GW 17; and the circular and occipitotemporal sulci on GW 18. The degree of cortical convolution was evaluated quantitatively based on 2D MRI slices by the gyrification index (GI) and based on 3D MRI data by sulcation index (SI). Both the mean GI and SI increased from GW 16, and were closely correlated with the cortical volume and the cortical surface area during fetal periods (their correlation coefficients marked more than 0.95). After birth, both the mean GI and SI decreased slightly by 2years of age, whereas the cortical volume and surface area continuously increased. Notably, histological analysis showed that the outer subventricular zone (oSVZ) in non-sulcal regions was thicker than that in the presumptive calcarine sulcal region on GW 13, preceding the infolding of the calcarine sulcus. The present results showed definite sulcal infolding on the cerebral cortical surface of the marmosets, with similar pattern and sequence of their emergences to other higher-order primates such as macaques and humans. Differential expansion of the oSVZ may be involved in gyral convolution and sulcal infolding in the developing cerebrum.

  14. Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy.

    PubMed

    Krishna, C Murali; Sockalingum, G D; Venteo, L; Bhat, Rani A; Kushtagi, Pralhad; Pluot, M; Manfait, M

    2005-12-05

    A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.

  15. 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.

  16. Patient and staff doses in paediatric interventional cardiology derived from experimental measurements with phantoms.

    PubMed

    Ubeda, Carlos; Vano, Eliseo; Miranda, Patricia; Aguirre, Daniel; Riquelme, Nemorino; Dalmazzo, Dandaro; Galaz, Sergio

    2016-01-01

    The aim of this paper was to determine experimentally the entrance surface air kerma (ESAK) and kerma-area product (KAP) levels to patients and scatter doses at the cardiologist's eyes during paediatric interventional cardiology (IC) procedures for Chile, on the basis of measurements taken from X-ray systems characterization for different thicknesses of polymethyl methacrylate, together with the average values of fluoroscopy time and number of cine frames for ten paediatric IC procedures. The range of cumulative ESAK values when the different clinical procedures were simulated was from 2 to 1100 mGy. KAP values ranged from 0.30 to 150 Gy cm(2). Scatter doses at cardiologist's eyes for the simulated procedures ranged from 0.20 to 116 µSv per procedure. Large differences between the X-ray systems were found in our study. Standardized guidelines in terms of X-ray system setting and protocols should be developed for hospitals that perform paediatric IC procedures in Chile.

  17. Preserved ex vivo inflammatory status and cytokine responses in naturally long-lived mice

    PubMed Central

    Arranz, Lorena; Lord, Janet M.

    2010-01-01

    Preserved immune cell function has been reported in mice that achieve extreme longevity. Since cytokines are major modulators of immune responses, we aimed to determine the levels of 21 cytokines secreted ex vivo by peritoneal leukocytes cultured under basal- and mitogen- (conconavalin A (ConA) and LPS) stimulated conditions in middle-aged (44 ± 4 weeks), old (69 ± 4 weeks), very old (92 ± 4 weeks), and extreme long-lived (125 ± 4 weeks) ICR (CD1) female mice. The secretion of cytokines was measured by multiplex luminometry. Increased basal levels of proinflammatory IL-1β, IL-6, IL-12 (p70), IFN-γ, and TNF-α were seen in the old and very old animals, accompanied by decreased IL-10. In contrast, the extreme long-lived mice maintained the overall cytokine profile of middle-aged mice, though the basal secretion of IL-2, IL-9, IL-10, IL-13, and IL-12 (p40) was raised. Under LPS- and/or ConA-stimulated conditions, leukocytes from old and very old animals showed a significantly impaired response with respect to secretion of Th1 cytokines IL-3, IL-12p70, IFN-γ, and TNF-α; Th2 cytokines IL-6, IL-4, IL-10, and IL-13; and the regulatory cytokines IL-2, IL-5, and IL-17. Extreme long-lived mice preserved the middle-aged-like cytokine profile, with the most striking effect seen for the IL-2 response to ConA, which was minimal in the old and very old mice but increased with respect to the middle-aged level in extreme long-lived mice. Chemokine responses in regard to KC, MCP-1, MIP1β, and RANTES were more variable, though similar secretion of LPS-induced KC and MCP-1 and ConA-induced MCP-1, MIP-1β, and RANTES was found in long-lived and middle-aged mice. Thus, extreme long-lived animals showed only a minimal inflammatory profile, much lower than the old and very old groups and also lower than the middle-aged, which is likely mediated by the increase of anti-inflammatory cytokines such as IL-10. This was coupled to a robust response to immune stimuli

  18. Prediction of intraocular antibody drug stability using ex-vivo ocular model.

    PubMed

    Patel, Sulabh; Stracke, Jan Olaf; Altenburger, Ulrike; Mahler, Hanns-Christian; Metzger, Philipp; Shende, Pankaj; Jere, Dhananjay

    2017-03-01

    Following intravitreal (IVT) injection, therapeutic proteins get exposed to physiological pH, temperature and components in the vitreous humor (VH) for a significantly long time. Therefore, it is of interest to study the stability of the proteins in the VH. However, the challenge posed by the isolated VH (such as pH shift upon isolation and incubation due to the formation of smaller molecular weight (MW) degradation products) can result in artefacts when investigating protein stability in relevance for the actual in vivo situation. In this current study, an ex-vivo intravitreal horizontal stability model (ExVit-HS) has been successfully developed and an assessment of long-term stability of a bi-specific monoclonal antibody (mAb) drug in the isolated VH for 3months at physiological conditions has been conducted. The stability assessment was performed using various analytical techniques such as microscopy, UV visible for protein content, target binding ELISA, Differential Scanning Calorimetry (DSC), Capillary-electrophoresis-SDS, Size Exclusion (SEC) and Ion-exchange chromatography (IEC) and SPR-Biacore. The results show that the ExVit-HS model was successful in maintaining the VH at physiological conditions and retained a majority of protein in the VH-compartment throughout the study period. The mAb exhibited significantly less fragmentation in the VH relative to the PBS control; however, chemical stability of the mAb was equally compromised in VH and PBS. Interestingly, in the PBS control, mAb showed a rapid linear loss in the binding affinity. The loss in binding was almost 20% higher compared to that in VH after 3months. The results clearly suggest that the mAb has different degradation kinetics in the VH compared to PBS. These results suggest that it is beneficial to investigate the stability in the VH for drugs intended for IVT injection and that are expected longer residence times in the VH. The studies show that the ExVit-HS model may become a valuable tool

  19. Biomimetic molecules lower catabolic expression and prevent chondroitin sulfate degradation in an osteoarthritic ex vivo model.

    PubMed

    Sharma, Shaili; Vazquez-Portalatin, Nelda; Calve, Sarah; Panitch, Alyssa

    2016-02-08

    Aggrecan, the major proteoglycan in cartilage, serves to protect cartilage tissue from damage and degradation during the progression of osteoarthritis (OA). In cartilage extracellular matrix (ECM) aggrecan exists in an aggregate composed of several aggrecan molecules that bind to a single filament of hyaluronan. Each molecule of aggrecan is composed of a protein core and glycosaminoglycan sides chains, the latter of which provides cartilage with the ability to retain water and resist compressive loads. During the progression of OA, loss of aggrecan is considered to occur first, after which other cartilage matrix components become extremely susceptible to degradation. Proteolytic cleavage of the protein core of aggrecan by enzymes such as aggrecanases, prevent its binding to HA and lower cartilage mechanical strength. Here we present the use of HA-binding or collagen type II-binding molecules that functionally mimic aggrecan but lack known cleavage sites, protecting the molecule from proteolytic degradation. These molecules synthesized with chondroitin sulfate backbones conjugated to hyaluronan- or collagen type II- binding peptides, are capable of diffusing through a cartilage explant and adhering to the ECM of this tissue. The objective of this study was to test the functional efficacy of these molecules in an ex vivo osteoarthritic model to discern the optimal molecule for further studies. Different variations of chondroitin sulfate conjugated to the binding peptides were diffused through aggrecan depleted explants and assessed for their ability to enhance compressive stiffness, prevent CS degradation, and modulate catabolic (MMP-13 and ADAMTS-5) and anabolic (aggrecan and collagen type II) gene expression. A pilot in vivo study assessed the ability to retain the molecule within the joint space of an osteoarthritic guinea pig model. The results indicate chondroitin sulfate conjugated to hyaluronan-binding peptides is able to significantly restore equilibrium

  20. Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

    PubMed Central

    Hess, Allison E.; Potter, Kelsey A.; Tyler, Dustin J.; Zorman, Christian A.; Capadona, Jeffrey R.

    2013-01-01

    Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation. The overall goal of this method is to quantitatively assess

  1. Detecting hepatic steatosis using ultrasound-induced thermal strain imaging: an ex vivo animal study

    NASA Astrophysics Data System (ADS)

    Mahmoud, Ahmed M.; Ding, Xuan; Dutta, Debaditya; Singh, Vijay P.; Kim, Kang

    2014-02-01

    .037%). Using histology as a gold standard to classify mouse livers, US-TSI had a sensitivity and specificity of 70% and 90%, respectively. The area under the receiver operating characteristic curve was 0.775. This ex vivo study demonstrates the feasibility of using US-TSI to detect fatty livers and warrants further investigation of US-TSI as a diagnostic tool for hepatic steatosis.

  2. Accuracy of five electronic foramen locators with different operating systems: an ex vivo study

    PubMed Central

    de VASCONCELOS, Bruno Carvalho; BUENO, Michelli de Medeiros; LUNA-CRUZ, Suyane Maria; DUARTE, Marco Antonio Hungaro; FERNANDES, Carlos Augusto de Oliveira

    2013-01-01

    Objective: The aim of this study was to evaluate, ex vivo, the precision of five electronic root canal length measurement devices (ERCLMDs) with different operating systems: the Root ZX, Mini Apex Locator, Propex II, iPex, and RomiApex A-15, and the possible influence of the positioning of the instrument tips short of the apical foramen. Material and Methods: Forty-two mandibular bicuspids had their real canal lengths (RL) previously determined. Electronic measurements were performed 1.0 mm short of the apical foramen (-1.0), followed by measurements at the apical foramen (0.0). The data resulting from the comparison of the ERCLMD measurements and the RL were evaluated by the Wilcoxon and Friedman tests at a significance level of 5%. Results: Considering the measurements performed at 0.0 and -1.0, the precision rates for the ERCLMDs were: 73.5% and 47.1% (Root ZX), 73.5% and 55.9% (Mini Apex Locator), 67.6% and 41.1% (Propex II), 61.7% and 44.1% (iPex), and 79.4% and 44.1