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University of Maryland School of Medicine

23 ARTICLES PUBLISHED IN JoVE

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Bioengineering

Patterned Photostimulation with Digital Micromirror Devices to Investigate Dendritic Integration Across Branch Points
Conrad W. Liang *1, Michael Mohammadi *1, M. Daniel Santos 1, Cha-Min Tang 1
1Department of Neurology, Baltimore VA Medical Center, University of Maryland School of Medicine

Digital micromirror devices (DMD) can generate complex patterns in time and space with which to control neuronal excitability. Issues relevant to the design, construction, and operation of DMD systems are discussed. Such a system enabled the demonstration of non-linear integration across distal dendritic branch points.

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Medicine

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery
Richard M. Lovering 1,2, Joseph A. Roche 1, Mariah H. Goodall 2, Brett B. Clark 2, Alan McMillan 3
1Department of Physiology, University of Maryland School of Medicine, 2Department of Orthopaedics, University of Maryland School of Medicine, 3Department of Diagnostic Radiology, University of Maryland School of Medicine

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

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Neuroscience

The Mouse Forced Swim Test
Adem Can 1, David T. Dao 2, Michal Arad 1, Chantelle E. Terrillion 3, Sean C. Piantadosi 1, Todd D. Gould 1,3,4
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 4The Program in Neuroscience, University of Maryland

The forced swim test is validated as an experimental approach to assess potential antidepressant efficacy in rodents. Experimental animals are placed in a tank of water and escape-related mobility behavior is quantified. The common procedures for the mouse version of this test are described.

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Neuroscience

The Tail Suspension Test
Adem Can *1, David T. Dao *1,2, Chantelle E. Terrillion 3, Sean C. Piantadosi 1, Shambhu Bhat 1, Todd D. Gould 1,3,4
1Department of Psychiatry, University of Maryland School of Medicine, 2Tulane University School of Medicine, 3The Program in Neuroscience, University of Maryland , 4Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine

The tail-suspension test is validated as an experimental procedure to assess antidepressant efficacy of drug treatments in mice. Mice are suspended by their tails for six minutes and escape-related behaviors are assessed. We describe procedures used in conducting the tail suspension test.

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Advanced Biology

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts
Dominique R. Bollino 1, Eric A. Legenzov 2, Tonya J. Webb 1
1Department of Microbiology and Immunology, University of Maryland School of Medicine and the Marlene and Stewart Greenebaum Comprehensive Cancer Center, 2Center for Biomedical Engineering and Technology, University of Maryland School of Medicine

Confocal Fluorescence Microscopy: A Technique to Determine the Localization of Proteins in Mouse Fibroblasts

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Biology

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents
Karen F. Underwood 1, Maria T. Mochin 1, Jessica L. Brusgard 2, Moran Choe 3, Avi Gnatt 4, Antonino Passaniti 3,5
1Greenebaum Cancer Center, University of Maryland School of Medicine, 2Program in Molecular Medicine, University of Maryland School of Medicine, 3Department of Biochemistry & Molecular Biology, University of Maryland School of Medicine, 4Department of Pharmacology & Experimental Therapeutics, University of Maryland School of Medicine, 5Department of Pathology and Biochemistry & Molecular Biology, University of Maryland School of Medicine

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

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Bioengineering

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Eva Wagner *1,2,3, Sören Brandenburg *1,2, Tobias Kohl 1,2, Stephan E. Lehnart 1,2,3,4
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.

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Biology

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
Karthika Natarajan 1,2, Yi Xie 1,3, Takeo Nakanishi 4, Rebecca S. Moreci 5,6, Pancharatnam Jeyasuria 7, Arif Hussain 1,3,8,9, Douglas D. Ross 1,3,8,9,10,11
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, 6Magee Women's Research Institute, 7Obstetrics, Gynecology, Perinatal Research Branch (NICHD), Wayne State University School of Medicine, 8Medicine, University of Maryland School of Medicine, 9Pathology, University of Maryland School of Medicine, 10Pharmacology, University of Maryland School of Medicine, 11Experimental Therapeutics, University of Maryland School of Medicine

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.

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Bioengineering

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity
Rosangela Salerno-Goncalves 1, Alessio Fasano 2, Marcelo B. Sztein 1
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.

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Medicine

Using Multi-fluorinated Bile Acids and In Vivo Magnetic Resonance Imaging to Measure Bile Acid Transport
Jessica Felton 1, Kunrong Cheng 2, Anan Said 2, Aaron C. Shang 2, Su Xu 3, Diana Vivian 4, Melissa Metry 5, James E. Polli 5, Jean-Pierre Raufman 2,6
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.

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Biology

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function
Dan Yu 1,4, George Makkar 2, Rajabrata Sarkar 2,3,4, Dudley K. Strickland 2,3,4, Thomas S. Monahan 1,2,4
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.

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Genetics

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish
Timothy L. Hostelley *1, Jessica E. Nesmith *1, Norann A. Zaghloul 1
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.

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Medicine

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection
Brittany O. Aicher 1, Subhradip Mukhopadhyay 1, Xin Lu 2, Selen C. Muratoglu 1, Dudley K. Strickland 1, Areck A. Ucuzian 1,3
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.

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Behavior

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats
Annalisa M. Baratta 1, Shaun S. Viechweg 2, Jessica A. Mong 2, Ana Pocivavsek 1
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.

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Medicine

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies
Adeel Kaiser 1, John G. Eley 1, Nasarachi E. Onyeuku 1, Stephanie R. Rice 1, Carleen C. Wright 2, Nathan E. McGovern 2, Megan Sank 2, Mingyao Zhu 1, Zeljko Vujaskovic 1, Charles B. Simone 2nd 1, Arif Hussain 3
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.

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Medicine

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice
Frederico Gerlinger-Romero 1, Alex B. Addinsall 2, Richard M. Lovering 3, Victoria C. Foletta 4, Chris van der Poel 5, Paul A. Della-Gatta 4, Aaron P. Russell 4
1School of Exercise and Nutrition Sciences, Deakin University, 2Centre for Molecular and Medical Research, School of Medicine, Deakin University, 3Department of Orthopaedics, School of Medicine, University of Maryland, 4Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, 5Department of Physiology, Anatomy and Microbiology, La Trobe University

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

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Biochemistry

Nitropeptide Profiling and Identification Illustrated by Angiotensin II
Shan Feng 1,2, Xiaofei Wen 3, Xin Lu 2,4,5,6
1Mass Spectrometry Core Facility, School of Life Sciences, Westlake University, 2Department of Biological Sciences, University of Notre Dame, 3Department of Urology, Shanghai East Hospital, Tongji University School of Medicine, 4Boler-Parseghian Center for Rare and Neglected Diseases, University of Notre Dame, 5Harper Cancer Research Institute, University of Notre Dame, 6Tumor Microenvironment and Metastasis Program, Indiana University Melvin and Bren Simon Cancer Center

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

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Biology

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes
Libet Garber 1,2, Humberto C. Joca 1, Andrew K. Coleman 1, Liron Boyman 1, W. Jonathan Lederer 1, Maura Greiser 1
1Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, 2Fischell Department of Bioengineering, University of Maryland

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

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Behavior

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice
Yi Xie 1, Sai-Yue Deng 1, Si-Miao Chen 1, Xue-Jiao Chen 1, Wen-Wen Lai 1, Li-Fang Huang 1, Li Ba 1, Wei Wang 1,2, Feng-Fei Ding 1,3
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000, China, 2Key Laboratory of Neurological Diseases of Chinese Ministry of Education, The School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430000,China, 3Department of Pharmacology, School of Basic Medical Science; State Key Laboratory of Medical Neurobiology, Institutes of Brain Science and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

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Medicine

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart
Guiling Zhao 1, Humberto C. Joca 1, W. Jonathan Lederer 1
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.

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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 He 1, Liqin Xiao 1, Huimin Yue 1, Shulin Wang 1, Yuting Sun 1, Yajun Wu 1, Liyang Wang 1,4, George Ghartey-Kwansah 1, Odell D. Jones 5, Joseph L. Bryant 6, MengMeng Xu 7, Jianjie Ma 3, Xuehon Xu 1
1National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China/CGDB, Shaanxi Normal University College of Life Sciences, 2Shaanxi Key Laboratory of Ischemic Cardiovascular Disease, Shaanxi Key Laboratory of Brain disorders, Institute of Basic & Translational Medicine, Xi'an Medical University, 3Ohio State University College of Medicine, 4Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 5University of Pennsylvania ULAR, 6University of Maryland School of Medicine, 7Columbia University Medical Center

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.

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Cancer Research

Implementation of Minimally Invasive Brain Tumor Resection in Rodents for High Viability Tissue Collection
Safwan Alomari 1, Jayanidhi Kedda 1, Adarsha P. Malla 2,3, Victor Pacis 1, Pavlos Anastasiadis 2,3, Su Xu 4, Emylee McFarland 2,3, Lilia Sukhon 1, Bruno Gallo 5, Jordina Rincon-Torroella 1, Netanel Ben-Shalom 1, Heather M. Ames 3,6, Henry Brem 1,7, Graeme F. Woodworth 2,3, Betty Tyler 1
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.

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Biology

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability
Hugo Bibollet 1, Daniel F. Bennett 1, Martin F. Schneider 1, Erick O. Hernández-Ochoa 1
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.

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