University of Virginia View Institution's Website 34 articles published in JoVE Medicine Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation William J. Garrison1, John P. Mugler III1,2, Jaime F. Mata2, Roselove N. Nunoo-Asare2, Y. Michael Shim3, G. Wilson Miller1,2,4 1Department of Biomedical Engineering, University of Virginia, 2Department of Radiology and Medical Imaging, University of Virginia, 3Department of Medicine, University of Virginia, 4Department of Physics, University of Virginia Hyperpolarized 129Xe magnetic resonance imaging (MRI) is a method for studying regionally resolved aspects of pulmonary function. This work presents an end-to-end standardized workflow for hyperpolarized 129Xe MRI of lung ventilation, with specific attention to pulse sequence design, 129Xe dose preparation, scan workflow, and best practices for subject safety monitoring. Neuroscience Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina Shichu Chang1, Wenjin Xu1, Weijia Fan2, John A. McDaniel1, Marta Grannonico1, David A. Miller2, Mingna Liu1, Hao F. Zhang2, Xiaorong Liu1,3,4,5 1Department of Biology, University of Virginia, 2Department of Biomedical Engineering, Northwestern University, 3Department of Ophthalmology, University of Virginia, 4Program in Fundamental Neuroscience, University of Virginia, 5Department of Psychology, University of Virginia The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images. Medicine Development of a Uterosacral Ligament Suspension Rat Model Beverly J. Miller1, Brian K. Jones2, Jonathan S. Turner3, Steven R. Caliari1,3, Monique H. Vaughan4 1Department of Chemical Engineering, University of Virginia, 2Electrophysiology Division, Abbott, 3Department of Biomedical Engineering, University of Virginia, 4Department of Obstetrics and Gynecology, Division of Pelvic Medicine and Reconstructive Surgery, University of Virginia Pelvic organ prolapse affects millions of women worldwide and yet some common surgical interventions have failure rates as high as 40%. The lack of standard animal models to investigate this condition impedes progress. We propose the following protocol as a model for uterosacral ligament suspension and in vivo tensile testing. Developmental Biology Polysome Purification from Soybean Symbiotic Nodules María Martha Sainz1, Carla Valeria Filippi1, Guillermo Eastman2,3, Mariana Sotelo-Silveira1, C. Mauro Martinez1, Omar Borsani1, José Sotelo-Silveira2,4 1Laboratorio de Bioquímica, Departamento de Biología Vegetal, Facultad de Agronomía, Universidad de la República, 2Departamento de Genómica, Instituto de Investigaciones Biológicas Clemente Estable, 3Department of Biology, University of Virginia, 4Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels. Bioengineering Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold Colleen Roosa1, Lauren Pruett1, Juliana Trujillo1, Areli Rodriguez1, Blaise Pfaff1, Nicholas Cornell1, Clare Flanagan1, Donald Richieri Griffin1,2 1Department of Biomedical Engineering, School of Engineering and Applied Sciences, University of Virginia, 2Department of Chemical Engineering, School of Engineering and Applied Sciences, University of Virginia This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications. Neuroscience Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants Cami Naomi Keliinui1, Susan E. Doyle1, Sarah E. Siegrist1 1Biology department, University of Virginia A method to reactivate quiescent neural stem cells in cultured Drosophila brain explants has been established. Using this method, the role of systemic signals can be uncoupled from tissue-intrinsic signals in the regulation of neural stem cell quiescence, entry and exit. Neuroscience Construction of Local Field Potential Microelectrodes for in vivo Recordings from Multiple Brain Structures Simultaneously Anastasia Brodovskaya*1, Shinnosuke Shiono*1, Tamal Batabyal1, John Williamson1, Jaideep Kapur1,2 1Department of Neurology, University of Virginia, 2UVA Brain Institute, University of Virginia The present protocol describes the construction of custom-made microelectrode arrays to record local field potentials in vivo from multiple brain structures simultaneously. Neuroscience Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy Justyna Straub1, Iuliia Vitko1, Ronald P. Gaykema1, Edward Perez-Reyes1,2 1Department of Pharmacology, University of Virginia, 2UVA Brain Institute, University of Virginia This report describes the methods to generate a model of temporal lobe epilepsy based on the electrical kindling of transgenic VGAT-Cre mice. Kindled VGAT-Cre mice may be useful in determining what causes epilepsy and for screening novel therapies. Developmental Biology Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells Elizabeth A. Nelson1,2, Jingyao Qiu3,4, Nicholas W. Chavkin1,2, Karen K. Hirschi1,2,3,4,5 1Department of Cell Biology, University of Virginia, 2Cardiovascular Research Center, University of Virginia, 3Department of Medicine, Yale University School of Medicine, 4Department of Genetics, Yale University School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week. Neuroscience Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry Wilson Wang*1, Yanrong Zhang*2, Matthew J. Anzivino1, Edward H. Bertram3, James Woznak1,4, Alexander Klibanov5, Erik Dumont6, Max Wintermark*2, Kevin S. Lee*1,7,8 1Department of Neuroscience, University of Virginia, 2Department of Radiology, Stanford University, 3Department of Neurology, University of Virginia, 4Global Internship Program, Focused Ultrasound Foundation, 5Department of Medicine, University of Virginia, 6Image Guided Therapy, 7Department of Neurosurgery, University of Virginia, 8Center for Brain, Immunology, and Glia, University of Virginia The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma. Immunology and Infection Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice Kanchan Bisht1,2, Kaushik Sharma1,2, Ukpong B. Eyo1,2 1Center for Brain Immunology and Glia (BIG), University of Virginia, 2Department of Neuroscience, University of Virginia This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations. Bioengineering Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes Victoria Osinski1,2, Alexander L Klibanov1,3, Coleen A McNamara1,3 1Robert M. Berne Cardiovascular Research Center, University of Virginia, 2Department of Pathology, University of Virginia, 3Department of Medicine, Division of Cardiovascular Medicine, University of Virginia The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level. Neuroscience Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice Pravin K. Wagley1,2, John Williamson2, Daria Skwarzynska3, Jaideep Kapur2,4,5, Jennifer Burnsed1,2 1Department of Pediatrics, University of Virginia, 2Department of Neurology, University of Virginia, 3Neuroscience Graduate Program, University of Virginia, 4Brain Institute, University of Virginia, 5Department of Neuroscience, University of Virginia This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia. Immunology and Infection Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production Aditi Upadhye1, Melissa Marshall2, James C. Garmey2, Timothy P. Bender3, Coleen McNamara2 1Department of Microbiology, Immunology, Cancer Biology, University of Virginia, 2Cardiovascular Research Center, University of Virginia, 3Beirne B. Carter Center for Immunology Research, University of Virginia Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer. Cancer Research Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody Gururaj Shivange*1,2, Tanmoy Mondal*1,2, Evan Lyerly1,2, Jeremy Gatesman3, Jogender Tushir-Singh1,2 1Laboratory of Novel Biologics, University of Virginia School of Medicine, 2Department of Biochemistry and Molecular Genetics, UVA Cancer Center, University of Virginia School of Medicine, 3Center for Comparative Medicine, University of Virginia Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models. Immunology and Infection Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain Fernando González Ibanez1,2, Katherine Picard1,2, Maude Bordeleau1,3, Kaushik Sharma1,2,4, Kanchan Bisht1,2,4, Marie-Ève Tremblay1,2 1Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, 2Département de Médecine Moléculaire, Faculté de Médecine, Université Laval, 3Integrated Program in Neuroscience, McGill University, 4Center for Brain Immunology and Glia (BIG), University of Virginia This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue. Biochemistry Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules Julian M Rocha1, Andreas Gahlmann1,2 1Department of Chemistry, University of Virginia, 2Department of Molecular Physiology & Biological Physics, University of Virginia School of Medicine 3D single-molecule localization microscopy is utilized to probe the spatial positions and motion trajectories of fluorescently labeled proteins in living bacterial cells. The experimental and data analysis protocol described herein determines the prevalent diffusive behaviors of cytosolic proteins based on pooled single-molecule trajectories. Medicine Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice Sidney Mahan1, Mingjun Liu1, Richard A. Baylis2,3, Delphine Gomez1,4 1Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, 2Robert M. Berne Cardiovascular Research Center, University of Virginia, 3Department of Biochemistry and Molecular Genetics, University of Virginia, 4Division of Cardiology, University of Pittsburgh School of Medicine We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice. Medicine Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections Jessica X. Yuan1, Jennifer M. Munson1 1Biomedical Engineering, University of Virginia This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ. Neuroscience Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space John Odackal*1, Robert Colbourn*2,3, Namrita Jain Odackal4, Lian Tao5, Charles Nicholson5, Sabina Hrabetova2 1Department of Medicine, University of Virginia, 2Department of Cell Biology, SUNY Downstate Medical Center, 3Neural and Behavioral Science Graduate Program, SUNY Downstate Medical Center, 4Division of Neonatology, University of Virginia, 5Department of Neuroscience and Physiology, New York University School of Medicine This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS. Bioengineering Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair Ellen L. Mintz1, Juliana A. Passipieri2, Daniel Y. Lovell2, George J. Christ2,3 1Department of Pathology, University of Virginia, 2Department of Biomedical Engineering, University of Virginia, 3Department of Orthopaedic Surgery, University of Virginia We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration. Developmental Biology Functional Cloning Using a Xenopus Oocyte Expression System Carol Zygar Plautz1, Hannah C. Williams1, Robert M. Grainger2 1Department of Biology, Shepherd University, 2Department of Biology, University of Virginia We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products. Medicine Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens Kasey Jividen*1, Mercedeh Javanbakht Movassagh*1, Amir Jazaeri2, Hui Li1 1Department of Pathology, University of Virginia, 2Department of Obstetrics & Gynecology, University of Virginia Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients. Immunology and Infection Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells M. Brittany Johnson1, Alison K. Criss1 1Department of Microbiology, Immunology, and Cancer Biology, University of Virginia Health Sciences Center Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. Neuroscience Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish Gwendolyn M. Lewis1, Sarah Kucenas1 1Department of Biology, University of Virginia Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration. Medicine Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling Alexander Michael Guendel*1, Kyle S. Martin*1, Joshua Cutts2, Patricia L. Foley3, Alexander M. Bailey1, Feilim Mac Gabhann4, Trevor R. Cardinal2, Shayn M. Peirce1 1Department of Biomedical Engineering, University of Virginia, 2Department of Biomedical Engineering, California Polytechnic State University, 3Office of Animal Welfare, University of Virginia, 4Department of Biomedical Engineering & Institute for Computational Medicine, Johns Hopkins University We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy. Chemistry Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples Jennifer Patterson1, Cameron Mura1 1Department of Chemistry, University of Virginia A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples. Immunology and Infection Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction Michael R. Davis, Jr.1, Joanna B. Goldberg1 1Department of Microbiology, Immunology, & Cancer Biology, University of Virginia Health System We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot. Biology Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis Ming Yang1, Peter C. Stapor1, Shayn M. Peirce2, Aline M. Betancourt3, Walter L. Murfee1 1Department of Biomedical Engineering, Tulane University, 2Department of Biomedical Engineering, University of Virginia, 3Center for Stem Cell Research and Regenerative Medicine, Tulane University This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level. Immunology and Infection Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging Joel R. Meyerson1,2, Tommi A. White1, Donald Bliss3, Amy Moran3, Alberto Bartesaghi1, Mario J. Borgnia1, M. Jason V. de la Cruz1, David Schauder1, Lisa M. Hartnell1, Rachna Nandwani1,4, Moez Dawood5, Brianna Kim6, Jun Hong Kim7, John Sununu8, Lisa Yang9, Siddhant Bhatia10, Carolyn Subramaniam1, Darrell E. Hurt11, Laurent Gaudreault12, Sriram Subramaniam1 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2The Medical Research Council Mitochondrial Biology Unit, University of Cambridge, 3National Library of Medicine, National Institutes of Health, 4Massachusetts Institute of Technology, 5William Fremd High School, 6University of Virginia, 7Duke University, 8Yale University, 9University of Notre Dame, 10Washington University in St. Louis, 11Bioinformatics and Computational Biosciences Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12Thomas Jefferson High School for Science and Technology The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis. Bioengineering Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation Caitlin W. Burke1, Richard J. Price1,2 1Department of Biomedical Engineering, University of Virginia, 2Neurological Surgery , University of Virginia Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas. Immunology and Infection Development of a Negative Selectable Marker for Entamoeba histolytica Mayuresh M Abhyankar1, Sarah M Haviland1, Carol A Gilchrist1, William A Petri, Jr.1 1Division of Infectious Disease and International Health, University of Virginia Health System We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine. Biology Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects Gary Balian1, Gina Beck1, Vedavathi Madhu1, Robert Sikes2, Quanjun Cui3, Haixiang Liang1, Joshua Bush1 1Orthopaedics Research, University of Virginia, 2Biological Sciences, University of Delaware, 3Orthopaedic Surgery, University of Virginia A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration. Biology Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells Lauren J. Buro1, Shaili Shah1, Melissa A. Henriksen1 1Department of Biology, University of Virginia This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.
