Medical College of Wisconsin View Institution's Website 27 articles published in JoVE Medicine Long-Term Continuous Measurement of Renal Blood Flow in Conscious Rats Satoshi Shimada1, Allen W. Cowley, Jr.1 1Department of Physiology, Medical College of Wisconsin The present protocol describes a long-term continuous measurement of renal blood flow in conscious rats and simultaneously recording blood pressure with implanted catheters (fluid-filled or by telemetry). Medicine Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry Linda A. Allen*1, Maia Terashvili*1, Alison Gifford*1, Julian H. Lombard1 1Department of Physiology, Medical College of Wisconsin This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure. Medicine Inverse Probability of Treatment Weighting (Propensity Score) using the Military Health System Data Repository and National Death Index Joshua D. Mitchell1, Brian F. Gage2, Nicole Fergestrom3, Eric Novak1, Todd C. Villines4 1Cardiovascular Division, Washington University School of Medicine, 2General Medical Sciences, Washington University School of Medicine, 3Center for Advancing Population Science, Medical College of Wisconsin, 4Cardiology Service, Department of Medicine, Walter Reed National Military Medical Center When randomized controlled trials are not feasible, a comprehensive health care data source like the Military Health System Data Repository provides an attractive alternative for retrospective analyses. Incorporating mortality data from the national death index and balancing differences between groups using propensity weighting helps reduce biases inherent in retrospective designs. Biochemistry Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo Patrick Moran1,2, Yichen Guo3,4, Rong Yuan1,3, Nicholas Barnekow1, Jordan Palmer2, Adam Beck3, Bin Ren3,4,5 1Blood Research Institute, Blood Center of Wisconsin, 2Department of Medicine, Medical College of Wisconsin, 3Department of Surgery, The University of Alabama at Birmingham, 4Department of Biomedical Engineering, The University of Alabama at Birmingham, 5GBS Program, Graduate School, The University of Alabama at Birmingham We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification. Medicine Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery Paul S. Pagel1, Lonnie Dye III2, Graham E.D. Hill2, Juan L. Vega2, Justin N. Tawil2, Derek J. De Vry2, Kiran Chandrashekarappa1, Zafar Iqbal1, Brent T. Boettcher1, Julie K. Freed2 1Anesthesia Service, Clement J. Zablocki Veterans Affairs Medical Center, 2Department of Anesthesiology, Medical College of Wisconsin We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies. Neuroscience Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats Anne M. Fink1, Caron Dean2 1Department of Biobehavioral Health Science, College of Nursing, University of Illinois at Chicago, 2Department of Anesthesiology, Medical College of Wisconsin and Zablocki VA Medical Center Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival). Immunology and Infection Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells Arash Nanbakhsh1, Brad Best2, Matthew Riese3, Sridhar Rao4, Li Wang5, Jeffrey Medin6, Monica S. Thakar6, Subramaniam Malarkannan1,5,6,7 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, The Blood Center of Wisconsin, 2Vector Core Lab, Blood Research Institute, The Blood Center of Wisconsin, 3Laboratory of Lymphocyte Biology, Blood Research Institute, The Blood Center of Wisconsin, 4Laboratory of Stem Cell Transcriptional Regulation, Blood Research Institute, The Blood Center of Wisconsin, 5Department of Microbiology and Immunology, The Medical College of Wisconsin, 6Department of Pediatrics, The Medical College of Wisconsin, 7Department of Medicine, The Medical College of Wisconsin The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation. Medicine Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats Kathleen M. Lukaszewicz1, Matthew J. Durand2, Jessica R.C. Priestley3, James R. Schmidt4, L. Adrienne Allen5, Aron M. Geurts3, Julian H. Lombard3 1Department of Physical Therapy, Marquette University, 2Medical College of Wisconsin, 3Department of Physiology, Medical College of Wisconsin, 4Graduate Programs of Nurse Anesthesia, Texas Wesleyan University, 5Office of Research, Medical College of Wisconsin This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry. Immunology and Infection Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice Dorothee Weihrauch1, John G. Krolikowski1,2, Deron W. Jones3, Tahniyath Zaman3, Omoshalewa Bamkole1, Janine Struve4, Paul S. Pagel5, Nicole L. Lohr6, Kirkwood A. Pritchard, Jr.3 1Department of Anesthesiology, Medical College of Wisconsin, 2Clement J. Zablocki Veterans Affairs Medical Center, 3 We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide. Biology Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney Oleg Palygin1, Vladislav Levchenko1, Louise C. Evans1, Gregory Blass1, Allen W. Cowley Jr.1, Alexander Staruschenko1 1Department of Physiology, Medical College of Wisconsin Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney. Biology Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium Tengis S. Pavlov1, Daria V. Ilatovskaya1, Oleg Palygin1, Vladislav Levchenko1, Oleh Pochynyuk2, Alexander Staruschenko1 1Department of Physiology, Medical College of Wisconsin, 2Department of Integrative Biology & Pharmacology, University of Texas Health Science Center at Houston Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys. Biology Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli Daria V. Ilatovskaya1, Oleg Palygin1, Vladislav Levchenko1, Alexander Staruschenko1 1Department of Physiology, Medical College of Wisconsin Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli. Biology Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta Bradley T. Endres1,2, Alexander Staruschenko1, Marie Schulte4, Aron M. Geurts1,2,3, Oleg Palygin1 1Departments of Physiology, Medical College of Wisconsin, 2Human and Molecular Genetics Center, Medical College of Wisconsin, 3Cardiovascular Center, Medical College of Wisconsin, 4Blood Research Institute of Wisconsin Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta. Neuroscience Diffusion Imaging in the Rat Cervical Spinal Cord Elizabeth Zakszewski1, Brian Schmit2, Shekar Kurpad1, Matthew D. Budde1 1Department of Neurosurgery, Medical College of Wisconsin, 2Department of Biomedical Engineering, Marquette University The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts. Medicine Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells Muhammad Z. Afzal1, Jennifer L. Strande1 1Department of Medicine, Medical College of Wisconsin This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction. Medicine Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling Jerome D. Robin1, Woody E. Wright1, Yaqun Zou2, Stacy A. Cossette3, Michael W. Lawlor3, Emanuela Gussoni4 1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4 This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications. Biology High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry Subarna Bhattacharya*1, Paul W. Burridge*2, Erin M. Kropp1, Sandra L. Chuppa1, Wai-Meng Kwok3, Joseph C. Wu2, Kenneth R. Boheler4,5, Rebekah L. Gundry1,6 1Department of Biochemistry, Medical College of Wisconsin, 2Stanford Cardiovascular Institute, Stanford University School of Medicine, 3Department of Anesthesiology, Medical College of Wisconsin, 4Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, Hong Kong University, 5Division of Cardiology, Johns Hopkins University School of Medicine, 6Cardiovascular Research Center, Biotechnology and Bioengineering Center, Medical College of Wisconsin The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population. Biology Tissue Triage and Freezing for Models of Skeletal Muscle Disease Hui Meng1, Paul M.L. Janssen2, Robert W. Grange3, Lin Yang4, Alan H. Beggs5, Lindsay C. Swanson5, Stacy A. Cossette1,6, Alison Frase7, Martin K. Childers8, Henk Granzier9, Emanuela Gussoni5, Michael W. Lawlor1 1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5 The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies. Biology MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues Alison J. Kriegel1, Mingyu Liang1 1Department of Physiology, Medical College of Wisconsin This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections. Behavior How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging Nicholas L. Balderston1, Douglas H. Schultz1, Sylvain Baillet2,3, Fred J. Helmstetter1,3 1Department of Psychology, University of Wisconsin-Milwaukee, 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, 3Department of Neurology, Medical College of Wisconsin This article describes how to record amygdala activity with magnetoencephalography (MEG). In addition this article will describe how to conduct trace fear conditioning without awareness, a task that activates the amygdala. It will cover 3 topics: 1) Designing a trace conditioning paradigm using backward masking to manipulate awareness. 2) Recording brain activity during the task using magnetoencephalography. 3) Using source imaging to recover signal from subcortical structures. Neuroscience Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures Ling Zhong1, Joshua C. Brown1, Clive Wells2, Nashaat Z. Gerges1 1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 2Department of Microbiology and Molecular Genetics, Medical College of Wisconsin The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons. Immunology and Infection High-throughput Detection Method for Influenza Virus Pawan Kumar1, Allison E. Bartoszek1, Thomas M. Moran2, Jack Gorski3, Sanjib Bhattacharyya4, Jose F. Navidad4, Monica S. Thakar1,5, Subramaniam Malarkannan1,6 1Laboratory of Molecular Immunology and Immunotherapy, Blood Research Institute, 2Department of Microbiology, Mount Sinai School of Medicine, 3Laboratory of Molecular Genetics, Blood Research Institute, 4City of Milwaukee Health Department Laboratory, 5Division of Hematology-Oncology/BMT, Children's Hospital of Wisconsin, Medical College of Wisconsin, 6Division of Hematology and Oncology, Dept Medicine, Medical College of Wisconsin This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires <10 μl volume of test material and has the potential for concurrent screening of multiple pathogens. Biology Pull-down of Calmodulin-binding Proteins Kanwardeep S. Kaleka1, Amber N. Petersen1, Matthew A. Florence1, Nashaat Z. Gerges1 1Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function. Immunology and Infection Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance Jill Waukau1, Jeffrey Woodliff2, Sanja Glisic3 1Department of Pediatrics/Allergy, Medical College of Wisconsin, 2Flow Cytometry Core Facility, Medical College of Wisconsin, 3Max McGee National Research Center for Juvenile Diabetes and Human Molecular Genetics Center, Medical College of Wisconsin Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D. Neuroscience Vibratome Sectioning for Enhanced Preservation of the Cytoarchitecture of the Mammalian Organ of Corti Katherine Shim1 1Department of Pediatrics, Children’s Research Institute, Medical College of Wisconsin A simple procedure of vibratome sectioning the organ of Corti, followed by immunohistochemistry and confocal microscopy is described. This procedure allows for improved preservation of the fine cytoarchitecture of the mammalian organ of Corti, and consequently allows for accurate quantification of cell types. Medicine Performing and Processing FNA of Anterior Fat Pad for Amyloid Vinod B. Shidham1,2, Bryan Hunt1, Safwan S. Jaradeh3, Alexandru C. Barboi3, Sumana Devata4, Parameswaran Hari5 1Department of Pathology, Medical College of Wisconsin, 2Current Address: Department of Pathology, Wayne State University School of Medicine Detroit Medical Center, 3Department of Neurology, Medical College of Wisconsin, 4Department of Medicine, Medical College of Wisconsin, 5Division of Neoplastic Diseases and Related Disorders, Medical College of Wisconsin Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome. Biology Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures Maya Sieber-Blum1,2, Yaofei Hu2 1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.