Thomas Jefferson University View Institution's Website 17 articles published in JoVE Neuroscience Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods Sean J. O'Sullivan*1,2,3, Ankita Srivastava*1, Rajanikanth Vadigepalli1, James S. Schwaber1 1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine The combination of laser capture microdissection and microfluidic RT-qPCR provides anatomic and biotechnical specificity in measuring the transcriptome in single neurons and glia. Applying creative methods with a system's biology approach to psychiatric disease may lead to breakthroughs in understanding and treatment such as the neuroinflammation antireward hypothesis in addiction. Neuroscience Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice Lianteng Zhi1, Jingyu Zhao1, David Jaffe1, Yuanxin Chen1, Ninghan Wang1,2, Qi Qin1, Erin L. Seifert3, Chenjian Li4, Hui Zhang1 1Department of Neuroscience, Thomas Jefferson University, 2School of Biomedical Engineering, Drexel University, 3Department of Pathology, MitoCare Center, Thomas Jefferson University, 4School of Life Sciences, Peking University Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods. Cancer Research An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth Lorela Ciraku1, Rebecca A. Moeller1, Emily M. Esquea1, Wiktoria A. Gocal1, Edward J. Hartsough2,4, Nicole L. Simone3,4, Joshua G. Jackson2, Mauricio J. Reginato1,4 1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 2Department of Pharmacology and Physiology, Drexel University College of Medicine, 3Department of Radiation Oncology, Thomas Jefferson University, 4Sidney Kimmel Cancer Center, Thomas Jefferson University We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface. Neuroscience Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis Karthik Krishnamurthy*1, Davide Trotti1, Piera Pasinelli1, Brigid Jensen*1 1Jefferson Weinberg ALS Center, Thomas Jefferson University Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons. Bioengineering Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension Flemming Forsberg1, Ipshita Gupta1,2, Priscilla Machado1, Colette M. Shaw1, Jonathan M. Fenkel3, Kirk Wallace4, John R. Eisenbrey1 1Department of Radiology, Thomas Jefferson University, 2School of Biomedical Engineering, Sciences and Health Systems, Drexel University, 3Department of Medicine, Division of Gastroenterology and Hepatology, Thomas Jefferson University, 4GE Global Research A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease. Genetics Combining Laser Capture Microdissection and Microfluidic qPCR to Analyze Transcriptional Profiles of Single Cells: A Systems Biology Approach to Opioid Dependence Sean J. O'Sullivan1,2, Beverly A.S. Reyes3, Rajanikanth Vadigepalli1, Elisabeth J. Van Bockstaele3, James S. Schwaber1 1Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Pharmacology & Physiology, Drexel University College of Medicine This protocol explains how to collect single neurons, microglia, and astrocytes from the central nucleus of the amygdala with high accuracy and anatomic specificity using laser capture microdissection. Additionally, we explain our use of microfluidic RT-qPCR to measure a subset of the transcriptome of these cells. Cancer Research Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice Ken Kageyama1,3, Shinji Ozaki2,3, Takami Sato3 1Department of Diagnostic and Interventional Radiology, Osaka City University Graduate School of Medicine, 2Department of Surgery, National Hospital Organization, Kure Medical Cancer Center, 3Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines. Immunology and Infection Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue Maria Steblyanko1, Nadia Anikeeva1, Marcus Buggert2,3, Michael R. Betts2, Yuri Sykulev4 1Department of Microbiology and Immunology, Thomas Jefferson University, 2Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, 3Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital Huddinge, 4Departments of Microbiology and Immunology and Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood. Genetics Using Mouse Oocytes to Assess Human Gene Function During Meiosis I Diego Marin*1,2, Alexandra L. Nguyen*3, Richard T. Scott, Jr.1,2, Karen Schindler3 1IVI-RMA New Jersey, 2Jefferson College of Biomedical Sciences, Thomas Jefferson University, 3Department of Genetics, Rutgers, The State University of New Jersey As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes. Developmental Biology Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies Junning Yang1, Chuanshen Wu2, Ioana Stefanescu1, Arie Horowitz1,3 1Department of Medicine, Cardeza Vascular Research Center, Sidney Kimmel Medical College, Thomas Jefferson University, 2Department of Molecular Cardiology, Cleveland Clinic Foundation, 3Department of Cancer Biology, Cardeza Vascular Research Center, Sidney Kimmel Medical College, Thomas Jefferson University We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting. Biology Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy Gabriele Di Sante1, Mathew C. Casimiro1, Timothy G. Pestell2, Richard G. Pestell1,2,3 1Department of Cancer Biology, Thomas Jefferson University, SKCC, 2Department of Medical Oncology, Thomas Jefferson University, SKCC, 3Kazan Federal University Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions. Medicine A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy Cynthia Cheng*1, Chadd W. Lee*2, Constantine Daskalakis3 1Department of Family and Community Medicine, Thomas Jefferson University, 2Sidney Kimmel Medical College, Thomas Jefferson University, 3Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm. Developmental Biology Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment Janice L. Walker1, Brigid M. Bleaken1, Iris M. Wolff1, A. Sue Menko1 1Pathology, Anatomy and Cell Biology, Thomas Jefferson University Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. Neuroscience Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons Melanie Martin1,2, Ke Li1, Megan C. Wright2, Angelo C. Lepore1 1Department of Neuroscience, Farber Institute for Neurosciences, Sidney Kimmel Medical College at Thomas Jefferson University, 2Department of Biology, Arcadia University Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease. Medicine Non-invasive Assessment of Microvascular and Endothelial Function Cynthia Cheng1, Constantine Daskalakis2, Bonita Falkner3 1Department of Family and Community Medicine, Thomas Jefferson University, 2Department of Pharmacology and Experimental Therapeutics, Biostatistics Division, Thomas Jefferson University, 3Department of Internal Medicine, Thomas Jefferson University Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function. Biology Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples Chen Lu1, Joshua L. Wonsidler1, Jianwei Li2, Yanming Du1, Timothy Block3, Brian Haab4, Songming Chen1 1Institute for Hepatitis and Virus Research, 2Department of Microbiology and Immunology, Thomas Jefferson University, 3Drexel University College of Medicine, 4Van Andel Research Institute In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure. Medicine Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury Angelo C. Lepore1 1Department of Neuroscience, Thomas Jefferson University Medical College Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.