Medical University of South Carolina View Institution's Website 35 articles published in JoVE Cancer Research Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models Dorea P. Jenkins1, Brittany Turner-Ivey1,2, Jody Fromm Longo1, Steven L. Carroll1,2 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Hollings Cancer Center, Medical University of South Carolina We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGFβ3 mouse model. Cancer Research Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor Brittany Turner-Ivey1, Jody Fromm Longo2, Dorea P. Jenkins2, Stephen T. Guest3, Steven L. Carroll1,2 1Hollings Cancer Center, Medical University of South Carolina, 2Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 3Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type. Neuroscience The Evoked Potential Operant Conditioning System (EPOCS): A Research Tool and an Emerging Therapy for Chronic Neuromuscular Disorders N. Jeremy Hill1,2, Disha Gupta1,2, Amir Eftekhar1, Jodi A. Brangaccio1, James J. S. Norton1,2, Michelle McLeod3, Tim Fake1, Jonathan R. Wolpaw1,2,4, Aiko K. Thompson3 1National Center for Adaptive Neurotechnologies, Stratton VA Medical Center, 2Electrical & Computer Engineering Department, State University of New York at Albany, 3College of Health Professions, Medical University of South Carolina, 4Department of Biomedical Sciences, State University of New York at Albany The Evoked Potential Operant Conditioning System aids scientific investigation of sensorimotor function and can administer targeted neurobehavioral training that can impact sensorimotor rehabilitation in neuromuscular disorders. This article describes its capabilities and illustrates its application in modifying a simple spinal reflex to achieve lasting improvement in motor function. Medicine A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents Morgan A. Hill1, Jennie H. Kwon1, Brielle Gerry1, Minoo Kavarana1, Satish N. Nadig*1, Taufiek K. Rajab*1 1Department of Surgery, Medical University of South Carolina This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves. Cancer Research Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells Jody Fromm Longo1, Stephanie N. Brosius2,3, Steven L. Carroll1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Pediatrics and Neurology, Perelman School of Medicine of the University of Pennsylvania, 3Division of Child Neurology, Children's Hospital of Philadelphia Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo. Behavior Standing Neurophysiological Assessment of Lower Extremity Muscles Post-Stroke John Harvey Kindred1,2, Christian Finetto3, Jasmine Jamilah Cash3, Mark Goodman Bowden1,2,3 1Department of Research and Development, Ralph H. Johnson Veterans Administration Medical Center, 2College of Health Professions, Department of Rehabilitation Sciences, Division of Physical Therapy, Medical University of South Carolina, 3College of Health Professions, Department of Health Sciences and Research, Medical University of South Carolina This protocol describes the process for performing a neurophysiological assessment of the lower extremity muscles, tibialis anterior and soleus, in a standing position using TMS in people post-stroke. This position provides a greater probability of eliciting a post-stroke TMS response and allows for the use of reduced stimulator power during neurophysiological assessments. Medicine Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice Wenyu Gou*1, Wanxing Cui*2, Yuki Cui2, Hongjun Wang1,3 1Department of Surgery, Medical University of South Carolina, 2Georgetown University, 3Ralph H. Johnson Veterans Affairs Medical Center Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice. Medicine A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion Wenyu Gou*1, Lindsay Swaby*1, A. Marissa Wolfe2, William P. Lancaster1,2, Katherine A. Morgan1, Hongjun Wang1,2 1Department of Surgery, Medical University of South Carolina, 2Ralph H. Johnson Veterans Affairs Medical Center Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS). Bioengineering Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye Marwa Belhaj1, Balasubramaniam Annamalai2, Nathaniel Parsons2, Andrew Shuler1, Jay Potts1, Bärbel Rohrer2,3,4 1Department of Cell Biology, University of South Carolina, 2Department of Ophthalmology, Division of Research, Medical University of South Carolina, 3Department of Neuroscience, Division of Research, Medical University of South Carolina, 4Division of Research, Ralph H. Johnson VA Medical Center Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes. Cancer Research Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad Lauren B. Shunkwiler1, Jill D. Haag2, Michael N. Gould2, Bart M. G. Smits1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin-Madison School of Medicine and Public Health This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique. Biology Preparation and Utilization of Freshly Isolated Human Detrusor Smooth Muscle Cells for Characterization of 9-Phenanthrol-Sensitive Cation Currents John Malysz1, Eric S. Rovner2, Robert Wake3, Georgi V. Petkov1,3,4 1Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, 2Department of Urology, Medical University of South Carolina, 3Department of Urology, College of Medicine, University of Tennessee Health Science Center, 4Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center We describe a method for preparation of the single freshly isolated detrusor smooth muscle cells from human urinary bladder specimens employing a two-step enzymatic procedure. The obtained viable DSM cells can be studied by various single cell techniques including the described amphotericin-B patch-clamp electrophysiology to reveal physiological and pharmacological properties. Neuroscience Cochlear Surface Preparation in the Adult Mouse Qiao-Jun Fang1,2, Fan Wu1, Renjie Chai2, Su-Hua Sha1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2MOE Key Laboratory of Developmental Genes and Human Disease, Institute of Life Sciences, Southeast University This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae. Neuroscience Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation Charalambos C. Charalambous1,2, Jing Nong Liang3,4, Steve A. Kautz2,5, Mark S. George5,6, Mark G. Bowden2,5,7 1Department of Neurology, New York University School of Medicine, 2Department of Health Sciences and Research, Medical University of South Carolina, 3Department of Physical Therapy, University of Nevada Las Vegas, 4Department of Health Professions, Medical University of South Carolina, 5Ralph H. Johnson VA Medical Center, 6Department of Psychiatry, Medical University of South Carolina, 7Division of Physical Therapy, Medical University of South Carolina The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system. Neuroscience Laboratory Administration of Transcutaneous Auricular Vagus Nerve Stimulation (taVNS): Technique, Targeting, and Considerations Bashar W. Badran*1,2,3, Alfred B. Yu*2, Devin Adair1, Georgia Mappin3, William H. DeVries3, Dorothea D. Jenkins4, Mark S. George3,5,6, Marom Bikson1 1Department of Biomedical Engineering, City College of New York, 2U.S. Army Research Laboratory, Aberdeen Proving Ground, 3Brain Stimulation Laboratory, Department of Psychiatry, Medical University of South Carolina, 4Department of Pediatrics, Medical University of South Carolina, 5Department of Neurology, Medical University of South Carolina, 6Ralph H. Johnson VA Medical Center A methodological description of the technique, potential targets, and proper administration of transcutaneous auricular vagus nerve stimulation (taVNS) on the human ear is described. Developmental Biology Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery Jui-Tung Liu1, Mary Paige Lamprecht1, Stephen A. Duncan1 1Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity. Bioengineering Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology Chase A. Arbra*1, Satish N. Nadig*1, Sarah Grace Dennis1, Sanket Pattanaik1, Heather A. Bainbridge1, J. Matthew Rhett1, Stephen A. Fann1, Carl Atkinson1, Michael J. Yost1 1Department of Surgery, Medical University of South Carolina Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids. Genetics Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays Sophia Emetu*1, Morgan Troiano*1, Jacob Goldmintz*1, Jensen Tomberlin1, Simon Grelet2, Philip H. Howe2, Christopher Korey3, Renaud Geslain1 1Laboratory of tRNA Biology, Department of Biology, College of Charleston, 2Department of Biochemistry and Molecular Biology, MUSC, 3Department of Biology, College of Charleston We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours. Medicine Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization Yogin Patel1, Ji S. Lee2, Hexin Chen1 1Department of Biological Science, Center for Colon Cancer Research, University of South Carolina, 2Department of Pathology, Chonnam National University Medical School Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization. Bioengineering Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels Veronica Rodriguez-Rivera1, John W. Weidner2, Michael J. Yost1 1Department of Surgery - Division of General Surgery, Medical University of South Carolina, 2Department of Chemical Engineering, University of South Carolina An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues. Neuroscience Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons Hailong Li*1, Marina Aksenova*1, Sarah J. Bertrand1, Charles F. Mactutus1, Rosemarie Booze1 1Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures. Medicine Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR Melissa A. Abt1, Christina L. Grek2, Gautam S. Ghatnekar2, Elizabeth S. Yeh1 1Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 2FirstString Research, Inc. This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue. Medicine The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration Shawn M. Stevens1, LaShardai N. Brown2, Paula C. Ezell3, Hainan Lang2 1Department of Otolaryngology Head and Neck Surgery, Medical University of South Carolina, 2Department of Pathology and Laboratory Sciences, Medical University of South Carolina, 3Intuitive Surgical, Inc Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible. Bioengineering Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer Sarah Grace Dennis1, Thomas Trusk2, Dylan Richards3, Jia Jia3, Yu Tan3, Ying Mei3, Stephen Fann1, Roger Markwald3, Michael Yost1 1Department of Surgery, Medical University of South Carolina, 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, 3Department of Bioengineering, Clemson University A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced. Medicine Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts Daniel J. Herr1, Sverre E. Aune1, Donald R. Menick1,2 1Department of Medicine/Cardiology, Gazes Cardiac Research Institute, Medical University of South Carolina, 2Ralph H. Johnson VA Medical Center, Medical University of South Carolina The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult. Developmental Biology Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation Khujista D. Haque1, Atul K. Pandey1, Matthew W. Kelley2, Chandrakala Puligilla1 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, College of Medicine, 2Laboratory of Cochlear Development, NIDCD, NIH We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development. Medicine A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys Liwei Huang1, An Xiao1, Andrea Wecker1, Daniel A. McBride1, Soo Young Choi2, Weibin Zhou3, Joshua H. Lipschutz2 1Department of Medicine, Eastern Virginia Medical School, 2Department of Medicine, Medical University of South Carolina, 3Department of Pediatrics, University of Michigan We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. Biology Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands Rushin Shojaii1, Stephanie Bacopulos2,3, Wenyi Yang2,3, Tigran Karavardanyan4, Demetri Spyropoulos5, Afshin Raouf6, Anne Martel1,4, Arun Seth2,3 1Department of Medical Biophysics, University of Toronto, 2Platform Biological Sciences, Sunnybrook Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto, 4Physical Sciences, Sunnybrook Research Institute, 5Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 6Manitoba Institute of Cell Biology, University of Manitoba We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands. Medicine An Orthotopic Bladder Cancer Model for Gene Delivery Studies Laura Kasman1, Christina Voelkel-Johnson1 1Department of Microbiology & Immunology, Medical University of South Carolina Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery. Medicine A Contusion Model of Severe Spinal Cord Injury in Rats Vibhor Krishna1, Hampton Andrews1, Xing Jin2, Jin Yu1, Abhay Varma1, Xuejun Wen3, Mark Kindy1 1Department of Neuroscience, Division of Neurosurgery, Medical University of South Carolina, 2Bioengineering, Clemson University, 3Clemson-MUSC Bioengineering Joint Program A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model. Medicine Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model Ningfei An1, Yubin Kang1 1Division of Hematology-oncology, Department of Medicine, Medical University of South Carolina Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions. Neuroscience Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging Philip O'Herron1, Zhiming Shen1, Zhongyang Lu1, Adrien E. Schramm1, Manuel Levy1, Prakash Kara1 1Department of Neuroscience, Medical University of South Carolina A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map. Medicine Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs Thais Federici1, Carl V. Hurtig1, Kentrell L. Burks1, Jonathan P. Riley1, Vibhor Krishna2, Brandon A. Miller1, Eric A. Sribnick1, Joseph H. Miller3, Natalia Grin1, Jason J. Lamanna1,4,5, Nicholas M. Boulis1 1Department of Neurosurgery, Emory University, 2Department of Neuroscience, Medical University of South Carolina, 3Division of Neurosurgery, University of Alabama, Birmingham, 4Department of Biomedical Engineering, Georgia Institute of Technology, 5Department of Biomedical Engineering, Emory University Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application. Neuroscience Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection Carlene S. Brandon1, Christina Voelkel-Johnson2, Lindsey A. May3, Lisa L. Cunningham3 1Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 2Department of Microbiology & Immunology, Medical University of South Carolina, 3National Institute on Deafness and Other Communication Disorders, National Institutes of Health Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles. Immunology and Infection 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens David A. Schofield1, Ian J. Molineux2, Caroline Westwater3 1BioSciences Division, Guild Associates, Inc., 2Department of Molecular Genetics and Microbiology, University of Texas at Austin, 3Department of Craniofacial Biology, Medical University of South Carolina A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis. Neuroscience Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging Nicholas P. Boyer1, Chunhe Chen1, Yiannis Koutalos1 1Storm Eye Institute, Medical University of South Carolina A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.
