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University of Virginia

47 ARTICLES PUBLISHED IN JoVE

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Biology

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells
Lauren J. Buro 1, Shaili Shah 1, Melissa A. Henriksen 1
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.

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Bioengineering

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation
Caitlin W. Burke 1, Richard J. Price 1,2
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.

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Biology

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects
Gary Balian 1, Gina Beck 1, Vedavathi Madhu 1, Robert Sikes 2, Quanjun Cui 3, Haixiang Liang 1, Joshua Bush 1
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.

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Immunology and Infection

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
Wei Han 1, Hui Li 1, Brahm H. Segal 2,3, Timothy S. Blackwell 1
1Department of Medicine, Vanderbilt University School of Medicine, 2Departments of Medicine and Immunology, Roswell Park Cancer Institute, 3Department of Medicine, University at Buffalo School of Medicine

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

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Biology

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis
Ming Yang 1, Peter C. Stapor 1, Shayn M. Peirce 2, Aline M. Betancourt 3, Walter L. Murfee 1
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.

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Chemistry

Gas Chromatography (GC) with Flame-Ionization Detection
Barbara Jill Venton 1
1University of Virginia

Gas Chromatography (GC) with Flame-Ionization Detection

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Chemistry

Calibration Curves
Barbara Jill Venton 1
1University of Virginia

Calibration Curves

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Chemistry

Ultraviolet-Visible (UV-Vis) Spectroscopy
Barbara Jill Venton 1
1University of Virginia

Ultraviolet-Visible (UV-Vis) Spectroscopy

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Chemistry

Sample Preparation for Analytical Characterization
Barbara Jill Venton 1
1University of Virginia

Sample Preparation for Analytical Characterization

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Chemistry

Internal Standards
Barbara Jill Venton 1
1University of Virginia

Internal Standards

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Chemistry

Capillary Electrophoresis (CE)
Barbara Jill Venton 1
1University of Virginia

Capillary Electrophoresis (CE)

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Chemistry

Ion-Exchange Chromatography
Barbara Jill Venton 1
1University of Virginia

Ion-Exchange Chromatography

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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 Cutts 2, Patricia L. Foley 3, Alexander M. Bailey 1, Feilim Mac Gabhann 4, Trevor R. Cardinal 2, Shayn M. Peirce 1
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.

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Medicine

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens
Kasey Jividen *1, Mercedeh Javanbakht Movassagh *1, Amir Jazaeri 2, Hui Li 1
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. 

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Developmental Biology

Functional Cloning Using a Xenopus Oocyte Expression System
Carol Zygar Plautz 1, Hannah C. Williams 1, Robert M. Grainger 2
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.

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Biology

Measurement of Carbon Dioxide Production from Radiolabeled Substrates in Drosophila melanogaster
Michelle L. Bland 1
1Department of Pharmacology, University of Virginia

This paper describes a method for the measurement of fuel oxidation in Drosophila melanogaster in which trace amounts of specific radiolabeled metabolic substrates are fed to flies. The exhaled radiolabeled CO2 that is a produced from fuel oxidation is collected and measured.

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Neuroscience

Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques
Kanchan Bisht *1, Hassan El Hajj *1, Julie C. Savage 1, Maria G. Sánchez 1, Marie-Ève Tremblay 1
1Neurosciences Axis, CHU de Québec Research Center

This article describes a protocol for visualizing amyloid Aβ plaques in Alzheimer's disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

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Developmental Biology

Isolation of Murine Embryonic Hemogenic Endothelial Cells
Jennifer S. Fang *1, Emily C. Gritz *2, Kathrina L. Marcelo 3, Karen K. Hirschi 1
1Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine, 2Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine, 3Department of Molecular and Cellular Biology, Baylor College of Medicine

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

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Bioengineering

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair
Ellen L. Mintz 1, Juliana A. Passipieri 2, Daniel Y. Lovell 2, George J. Christ 2,3
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.

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Biology

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum
Anna J. Saltman *1,2, May Barakat *1, Donald M. Bryant 1, Anastasia Brodovskaya 1, Jessica L. Whited 1
1Department of Orthopedic Surgery, Harvard Medical School and Brigham and Women's Hospital, 2Division of Graduate Medical Sciences, Boston University

Using a lipophilic 1,1'-Dioctadecy-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

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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 Odackal 4, Lian Tao 5, Charles Nicholson 5, Sabina Hrabetova 2
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.

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Medicine

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections
Jessica X. Yuan 1, Jennifer M. Munson 1
1Biomedical Engineering, University of Virginia

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

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JoVE Core

Preparation of Aligned Steel Fiber Reinforced Cementitious Composite and Its Flexural Behavior
Ru Mu 1, Luansu Wei 1, Xiaowei Wang 1, Hui Li 1, Longbang Qing 1, Jian Zhou 1, Quanming Zhao 2
1School of Civil and Transportation Engineering, Hebei University of Technology, 2School of Information Engineering, Hebei University of Technology

This protocol describes an approach for manufacturing aligned steel fiber reinforced cementitious composite by applying a uniform electromagnetic field. Aligned steel fiber reinforced cementitious composite exhibits superior mechanical properties to ordinary fiber reinforced concrete.

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Medicine

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice
Sidney Mahan 1, Mingjun Liu 1, Richard A. Baylis 2,3, Delphine Gomez 1,4
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.

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Biochemistry

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
Julian M Rocha 1, Andreas Gahlmann 1,2
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.

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Immunology and Infection

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain
Fernando González Ibanez 1,2, Katherine Picard 1,2, Maude Bordeleau 1,3, Kaushik Sharma 1,2,4, Kanchan Bisht 1,2,4, Marie-Ève Tremblay 1,2
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.

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Cancer Research

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Gururaj Shivange *1,2, Tanmoy Mondal *1,2, Evan Lyerly 1,2, Jeremy Gatesman 3, Jogender Tushir-Singh 1,2
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.

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A Xenograft Mouse Model to Assess Efficacy of Therapeutic Agents for Human Acute Leukemia
Chandrika Gowda *1, Charyguly Annageldiyev *2,3, Pavan Kumar Dhanyamraju 1, Morgann Klink 1, Sinisa Dovat 1, Mark Kester 4,5, Thomas P. Loughran, Jr 4,6, David Claxton 2,3, Arati Sharma 1,2,7
1Department of Pediatrics, Pennsylvania State University College of Medicine, 2Penn State Hershey Cancer Institute, Pennsylvania State University College of Medicine, 3Departments of Medicine, Division of Hematology and Oncology, Pennsylvania State University College of Medicine, 4University of Virginia Cancer Center, 5nanoSTAR Institute, University of Virginia, 6Division of Hematology and Oncology, Department of Medicine, University of Virginia School of Medicine, 7Department of Pharmacology, Pennsylvania State University College of Medicine

Mouse (Mus Musculus) models are being widely used to develop xenografts using human leukemia cells. These models provide a comparable biological system to study drug efficacy, pharmacodynamics, and pharmacokinetics. Modeling acute myeloid leukemia in immunocompromised mice is described in detail using the U937 cell line xenograft as an example.

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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 Upadhye 1, Melissa Marshall 2, James C. Garmey 2, Timothy P. Bender 3, Coleen McNamara 2
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.

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Behavior

Using a Virtual Reality Walking Simulator to Investigate Pedestrian Behavior
Hyun Chae Chung 1, Soon Ho Kim 2, Gyoojae Choi 3, Jong Won Kim 4, Moo Young Choi 2, Hui Li 1
1Department of Sport and Exercise Sciences, Kunsan National University, 2Department of Physics and Astronomy, Seoul National University, 3School of Mechanical and Automotive Engineering, Kunsan National University, 4Department of Healthcare Information Technology, Inje University

This protocol describes use of a walking simulator that serves as a safe and ecologically valid method to study pedestrian behavior in the presence of moving traffic.

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Neuroscience

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry
Wilson Wang *1, Yanrong Zhang *2, Matthew J. Anzivino 1, Edward H. Bertram 3, James Woznak 1,4, Alexander Klibanov 5, Erik Dumont 6, 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.

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Neuroscience

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice
Pravin K. Wagley 1,2, John Williamson 2, Daria Skwarzynska 3, Jaideep Kapur 2,4,5, Jennifer Burnsed 1,2
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.

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Immunology and Infection

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice
Kanchan Bisht 1,2, Kaushik Sharma 1,2, Ukpong B. Eyo 1,2
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.

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Bioengineering

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes
Victoria Osinski 1,2, Alexander L Klibanov 1,3, Coleen A McNamara 1,3
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.

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Developmental Biology

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells
Elizabeth A. Nelson 1,2, Jingyao Qiu 3,4, Nicholas W. Chavkin 1,2, Karen K. Hirschi 1,2,3,4,5
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.

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Neuroscience

Preparation and Implantation of Electrodes for Electrically Kindling VGAT-Cre Mice to Generate a Model for Temporal Lobe Epilepsy
Justyna Straub 1, Iuliia Vitko 1, Ronald P. Gaykema 1, Edward Perez-Reyes 1,2
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.

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Developmental Biology

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing
Nicholas W. Chavkin 1,2, Shelby Cain 1, Kenneth Walsh 2,3,4, Karen K. Hirschi 1,2,4,5
1Department of Cell Biology, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Cardiology, University of Virginia School of Medicine, 4Hematovascular Biology Center, University of Virginia School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

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Neuroscience

Neural Stem Cell Reactivation in Cultured Drosophila Brain Explants
Cami Naomi Keliinui 1, Susan E. Doyle 1, Sarah E. Siegrist 1
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.

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Neuroscience

Construction of Local Field Potential Microelectrodes for in vivo Recordings from Multiple Brain Structures Simultaneously
Anastasia Brodovskaya *1, Shinnosuke Shiono *1, Tamal Batabyal 1, John Williamson 1, Jaideep Kapur 1,2
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.

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Biology

Intravital Subcellular Microscopy of the Mammary Gland
Yeap Ng 1, Andrius Masedunskas 1,2, Marco Heydecker 1, Seham Ebrahim 1,3, Roberto Weigert 1, Ian H. Mather 1
1Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 2Charles Perkins Centre, Central Clinical School, Faculty of Medicine and Health, The University of Sydney, 3Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

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Bioengineering

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold
Colleen Roosa 1, Lauren Pruett 1, Juliana Trujillo 1, Areli Rodriguez 1, Blaise Pfaff 1, Nicholas Cornell 1, Clare Flanagan 1, Donald Richieri Griffin 1,2
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.

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Developmental Biology

Polysome Purification from Soybean Symbiotic Nodules
María Martha Sainz 1, Carla Valeria Filippi 1, Guillermo Eastman 2,3, Mariana Sotelo-Silveira 1, C. Mauro Martinez 1, Omar Borsani 1, José Sotelo-Silveira 2,4
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.

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Medicine

Development of a Uterosacral Ligament Suspension Rat Model
Beverly J. Miller 1, Brian K. Jones 2, Jonathan S. Turner 3, Steven R. Caliari 1,3, Monique H. Vaughan 4
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.

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Neuroscience

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina
Shichu Chang 1, Wenjin Xu 1, Weijia Fan 2, John A. McDaniel 1, Marta Grannonico 1, David A. Miller 2, Mingna Liu 1, Hao F. Zhang 2, Xiaorong Liu 1,3,4,5
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.

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Medicine

Acquiring Hyperpolarized 129Xe Magnetic Resonance Images of Lung Ventilation
William J. Garrison 1, John P. Mugler III 1,2, Jaime F. Mata 2, Roselove N. Nunoo-Asare 2, Y. Michael Shim 3, G. Wilson Miller 1,2,4
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.

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In Vivo Functional Assessment of Rat Masseter Muscle Following Surgical Creation of a Volumetric Muscle Loss (VML) Injury
Sydney B. Shriver 1, Ramzi J. Khairallah 2, George J. Christ 1,3
1Department of Biomedical Engineering, University of Virginia, 2Myologica LLC, 3Department of Orthopedic Surgery, University of Virginia

Volumetric muscle loss (VML) injuries exceed endogenous regenerative ability, resulting in permanent functional deficits. Current VML research primarily focuses on limb and trunk muscles. To extend mechanistic studies of VML to craniofacial muscles, this article describes an in vivo method for longitudinal assessment of masseter muscle function pre- and post-VML injury.

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Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice
Magdalena Pikus 1, Elzbieta Dulko 2, Mark Beenhakker 3, Nadia Lunardi 1
1Department of Anesthesiology, University of Virginia, 2Neuroscience Graduate Program, University of Virginia, 3Department of Pharmacology, University of Virginia

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

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