University of Maryland School of Medicine 15 articles published in JoVE Biology Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability Hugo Bibollet1, Daniel F. Bennett1, Martin F. Schneider1, Erick O. Hernández-Ochoa1 1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers. Cancer Research Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection Safwan Alomari1, Jayanidhi Kedda1, Adarsha P. Malla2,3, Victor Pacis1, Pavlos Anastasiadis2,3, Su Xu4, Emylee McFarland2,3, Lilia Sukhon1, Bruno Gallo5, Jordina Rincon-Torroella1, Netanel Ben-Shalom1, Heather M. Ames3,6, Henry Brem1,7, Graeme F. Woodworth2,3, Betty Tyler1 1Department of Neurosurgery, Johns Hopkins University School of Medicine, 2Department of Neurosurgery, University of Maryland School of Medicine, 3Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, 4Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, 5Pontifical Catholic University of Parana, 6Department of Pathology, University of Maryland School of Medicine, 7Departments of Ophthalmology, Oncology and Biomedical Engineering, Johns Hopkins University School of Medicine The present protocol describes a standardized resection of brain tumors in rodents through a minimally invasive approach with an integrated tissue preservation system. This technique has implications for accurately mirroring the standard of care in rodent and other animal models. 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. Developmental Biology Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity Yvon Woappi1,2, Geraldine Ezeka3, Justin Vercellino4, Sean M. Bloos5, Kim E. Creek6, Lucia Pirisi1 1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2 Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions. Medicine Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart Guiling Zhao1, Humberto C. Joca1, W. Jonathan Lederer1 1Laboratory of Molecular Cardiology, Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized. Medicine Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies Adeel Kaiser1, John G. Eley1, Nasarachi E. Onyeuku1, Stephanie R. Rice1, Carleen C. Wright2, Nathan E. McGovern2, Megan Sank2, Mingyao Zhu1, Zeljko Vujaskovic1, Charles B. Simone 2nd1, Arif Hussain3 1Department of Radiation Oncology, University of Maryland School of Medicine, 2Department of Radiation Oncology, University of Maryland Medical Center, 3Department of Medicine, University of Maryland School of Medicine, University of Maryland Greenebaum Comprehensive Cancer Center The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients. Behavior A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats Annalisa M. Baratta1, Shaun S. Viechweg2, Jessica A. Mong2, Ana Pocivavsek1 1Maryland Psychiatric Research Center, Department of Psychiatry, University of Maryland School of Medicine, 2Department of Pharmacology, University of Maryland School of Medicine Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats. Medicine Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection Brittany O. Aicher1, Subhradip Mukhopadhyay1, Xin Lu2, Selen C. Muratoglu1, Dudley K. Strickland1, Areck A. Ucuzian1,3 1Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 2Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, 3Division of Vascular Surgery, University of Maryland School of Medicine This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection. Genetics Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish Timothy L. Hostelley*1, Jessica E. Nesmith*1, Norann A. Zaghloul1 1Division of Endocrinology, Diabetes and Nutrition, Department of Medicine, University of Maryland School of Medicine This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes. Biology Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function Dan Yu1,4, George Makkar2, Rajabrata Sarkar2,3,4, Dudley K. Strickland2,3,4, Thomas S. Monahan1,2,4 1Department of Surgery, Baltimore Veterans Affairs Medical Center, 2Department of Surgery, University of Maryland School of Medicine, 3Department of Physiology, University of Maryland School of Medicine, 4Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo. Medicine Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport Jessica Felton1, Kunrong Cheng2, Anan Said2, Aaron C. Shang2, Su Xu3, Diana Vivian4, Melissa Metry5, James E. Polli5, Jean-Pierre Raufman2,6 1Department of Surgery, University of Maryland School of Medicine, 2Department of Medicine, University of Maryland School of Medicine, 3Department of Radiology, University of Maryland School of Medicine, 4Food and Drug Administration, 5Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 6VA Maryland Health Care System Tools to diagnose bile acid malabsorption and measure bile acid transport in vivo are limited. An innovative approach in live animals is described that utilizes combined proton (1H) plus fluorine (19F) magnetic resonance imaging; this novel methodology has translational potential to screen for bile acid malabsorption in clinical practice. Medicine The Rodent Model of Nonarteritic Anterior Ischemic Optic Neuropathy (rNAION) Yan Guo1, Zara Mehrabian1, Steven L. Bernstein1,2 1Department of Ophthalmology and Visual Sciences, University of Maryland-Baltimore School of Medicine, 2Anatomy and Neurobiology, University of Maryland-Baltimore School of Medicine The following report describes how to replicate the rodent model of nonarteritic anterior ischemic optic neuropathy (rNAION), using the appropriate dye, contact lens and laser parameters. We also reveal the appropriate steps for evaluating the rNAION lesion in vivo. Bioengineering Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity Rosangela Salerno-Goncalves1, Alessio Fasano2, Marcelo B. Sztein1 1Center for Vaccine Development, Department of Pediatrics, University of Maryland School of Medicine, 2Mucosal Immunology and Biology Research Center, Massachusetts General Hospital for Children Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors. Biology Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1 Karthika Natarajan1,2, Yi Xie1,3, Takeo Nakanishi4, Rebecca S. Moreci5,6, Pancharatnam Jeyasuria7, Arif Hussain1,3,8,9, Douglas D. Ross1,3,8,9,10,11 1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Pharmaceutical Sciences, University of Maryland School of Pharmacy, 3Baltimore VA Medical Center, 4Membrane Transport and Biopharmaceutics, School of Pharmaceutical Sciences, Kanazawa University, 5Obstetrics, Gynecology and Reproductive Science, University of Pittsburgh, 6 With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays. Bioengineering Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles Eva Wagner*1,2,3, Sören Brandenburg*1,2, Tobias Kohl1,2, Stephan E. Lehnart1,2,3,4 1Heart Research Center Goettingen, 2Clinic of Cardiology & Pulmonology, University Medical Center Goettingen, 3German Center for Cardiovascular Research (DZHK) partner site Goettingen, 4BioMET, Center for Biomedical Engineering & Technology, University of Maryland School of Medicine In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.
