The Ohio State University College of Medicine 17 articles published in JoVE Medicine Successful Orthotopic Liver Transplantation in Mice Utilizing Microcomputed Tomography Angiography Qiang Zeng1, Doug A. Gouchoe2,3, Mahboubeh Nabavinia4, Yong Gyu Lee2, Xi Wang2, Terri A. Shaffer5, Mitchel R. Stacy4,5,6, Blake R. Peterson7,8, Bryan A. Whitson2,9, Christopher Breuer1,4, Sylvester M. Black1,10 1Center for Regenerative Medicine, The Research Institute at Nationwide Children’s Hospital, 5Division of Vascular Diseases and Surgery, The Ohio State University College of Medicine, 6Interdisciplinary Biophysics Graduate Program, The Ohio State University, 7Division of Medicinal Chemistry and Pharmacognosy, The Ohio State University College of Pharmacy, 8The Ohio State University Comprehensive Cancer Center, 9Division of Cardiac Surgery, Department of Surgery, The Ohio State Wexner Medical Center, 10Division of Transplantation, Department of Surgery, The Ohio State Wexner Medical Center In this protocol, we discuss the implementation of a model of successful orthotopic liver transplantation (OLT) in mice. Additionally, adjuvants to further analyze allograft patency after successful OLT in a mouse are discussed as well, specifically utilizing microcomputed tomography (microCT) scans. Biochemistry Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay Pavan S. Krishnan1,2, Fernando T. Zamuner1,3, Carolyn M. Jenks1, Johnny Y. Xie4, Lisa Zhang5, Mohammed Lehar1, Neal S. Fedarko6, Mariana Brait1,3, John P. Carey1 1Department of Otolaryngology-Head & Neck Surgery, Johns Hopkins University School of Medicine, 2Virginia Commonwealth University School of Medicine, 3Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, 4Department of Otolaryngology-Head and Neck Surgery, University of Michigan Medical School, 5Department of Otolaryngology-Head and Neck Surgery, The Ohio State University College of Medicine, 6ICTR Clinical Research Core Laboratory, Johns Hopkins University School of Medicine Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma. Developmental Biology A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo Wei Zhang*1, Xinjuan Lei*1, Xin Zhou*2,3, Boling He1, Liqin Xiao1, Huimin Yue1, Shulin Wang1, Yuting Sun1, Yajun Wu1, Liyang Wang1,4, George Ghartey-Kwansah1, Odell D. Jones5, Joseph L. Bryant6, MengMeng Xu7, Jianjie Ma3, Xuehon Xu1 1National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB, Shaanxi Normal University College of Life Sciences, 2 Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization. Neuroscience In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes Cassandra N. Dennys1, Julieth A. Sierra-Delgado1, Shrestha Sinha Ray1, Annalisa M. Hartlaub1, Florence S. Roussel1, Yacidzohara Rodriguez1, Kathrin Meyer1,2 1 We describe a protocol to reprogram human skin-derived fibroblasts into induced Neuronal Progenitor Cells (iNPCs), and their subsequent differentiation into induced Astrocytes (iAs). This method leads to fast and reproducible generation of iNPCs and iAs in large quantities. Developmental Biology Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells Shiqiao Ye1,2, Xiaoping Wan3, Juan Su1,2, Akshar Patel1,2,4, Blake Justis1,2,5, Isabelle Deschênes3, Ming-Tao Zhao1,2,5,6 1The Heart Center, Nationwide Children’s Hospital, 3Department of Physiology and Cell Biology, The Ohio State University College of Medicine, 4Department of Anatomy, The Ohio State University College of Medicine, 5MCDB Graduate Program, The Ohio State University, 6Department of Pediatrics, The Ohio State University College of Medicine Here, we present a protocol to robustly generate and expand human cardiomyocytes from patient peripheral blood mononuclear cells. Developmental Biology Analysis of Hematopoietic Stem Progenitor Cell Metabolism Giorgia Scapin1,2,3, Marie C. Goulard1,2,3, Priyanka R. Dharampuriya1,2,3, Jennifer L. Cillis1,2,3, Dhvanit I. Shah1,2,3 1 Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs. Bioengineering Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model Matthew G. Wiet*1,2, Sayali Dharmadhikari*1,3, Audrey White1,2, Susan D. Reynolds4, Jed Johnson5, Christopher K. Breuer3,6, Tendy Chiang1,3 1 Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation. Immunology and Infection Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase Miguel A. Lopez Jr.1, Randi M. Mackler1, Matthew P. Altman1, Kristine E. Yoder1 1Department of Cancer Biology and Genetics, The Ohio State University College of Medicine Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme. Biology Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations Dorrelyn P. Martin1, Jharna Miya1, Julie W. Reeser1, Sameek Roychowdhury1,2 1Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University, 2Department of Pharmacology, The Ohio State University We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer. Biology Isolation of Murine Coronary Vascular Smooth Muscle Cells Kathryn E. Husarek1,2, Xiaojin Zhang2, Patricia E. McCallinhart2, Pamela A. Lucchesi2,3, Aaron J. Trask2,3 1School of Biomedical Science, The Ohio State University College of Medicine, 2 The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques. Immunology and Infection Nasal Wipes for Influenza A Virus Detection and Isolation from Swine Jacqueline M. Nolting1, Christine M. Szablewski1, Jody L. Edwards1, Sarah W. Nelson1, Andrew S. Bowman1 1Department of Veterinary Preventive Medicine, The Ohio State University The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. Engineering Experiments on Ultrasonic Lubrication Using a Piezoelectrically-assisted Tribometer and Optical Profilometer Sheng Dong1, Marcelo Dapino1 1Smart Vehicle Concepts Center, Department of Mechanical and Aerospace Engineering, The Ohio State University We present a protocol for using a piezoelectrically-assisted tribometer and optical profilometer to investigate the dependence of ultrasonic wear and friction reduction on linear velocity, contact pressure, and surface properties. Bioengineering Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect Daniel S. Oh1, Alia Koch1, Sidney Eisig1, Sahng Gyoon Kim2, Yoon Hyuk Kim3, Do-Gyoon Kim4, Jae Hyuck Shim5 1Oral and Maxillofacial Surgery, Columbia University, 2Endodontics, Columbia University, 3Mechanical Engineering, Kyung Hee University, South Korea, 4Orthodontics, The Ohio State University, 5Pathology, Weill Cornell Medical College A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels. Medicine Design, Fabrication, and Administration of the Hand Active Sensation Test (HASTe) Alexandra Borstad1, Alex Altenburger1, Aaron Hannigan1, Joshua LaPorte1, Rachael Mott1, Deborah S. Nichols-Larsen2 1Division of Physical Therapy, The Ohio State University, 2School of Health and Rehabilitation Sciences, The Ohio State University The Hand Active Sensation Test (HASTe) is a valid and reliable measure of haptic performance, which has been used successfully to identify impaired haptic touch in individuals with stroke. The purpose of this paper is to describe the design, fabrication and administration of the HASTe. Behavior Mindfulness in Motion (MIM): An Onsite Mindfulness Based Intervention (MBI) for Chronically High Stress Work Environments to Increase Resiliency and Work Engagement Maryanna Klatt1, Beth Steinberg2, Anne-Marie Duchemin3 1Department of Family Medicine, The Ohio State University College of Medicine, 2Critical Care Nursing, Wexner Medical Center, 3Department of Psychiatry, Stress, Trauma, and Resilience (STAR) Program, The Ohio State University College of Medicine The Mindfulness in Motion (MIM) protocol offers a pragmatic Mindfulness Based Intervention (MBI) on-site, for persons working in chronically high-stress work environments that significantly increases resiliency and work engagement. The protocol has proven feasible, beneficial, and is easily adaptable to other high-stress workplaces. Medicine Diagnostic Ultrasound Imaging of Mouse Diaphragm Function Li Zuo1,2, William J. Roberts1,2, Kevin D. Evans1 1Radiologic Sciences & Respiratory Therapy Division, School of Health and Rehabilitation Sciences, Davis Heart and Lung Research Institute, The Ohio State University College of Medicine, 2Department of Biological Sciences, Oakland University Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function. Medicine A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery Zhaobin Xu1, Jenna Alloush1, Eric Beck1, Noah Weisleder1 1Davis Heart & Lung Research Institute, Department of Physiology and Cell Biology, The Ohio State University We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.
