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Vanderbilt University Medical Center

66 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Intracranial Orthotopic Allografting of Medulloblastoma Cells in Immunocompromised Mice
Xi Huang 1, Anuraag Sarangi 2, Tatiana Ketova 1, Ying Litingtung 1, Michael K. Cooper 2, Chin Chiang 1
1Department of Cell and Developmental Biology, Vanderbilt University, 2Department of Neurology, Vanderbilt University

This protocol describes the isolation and dissociation of mouse medulloblastoma tissue, and subsequent allografting of the tumor cells into immunocompromised recipient mice in order to initiate secondary medulloblastoma.

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Neuroscience

Operant Sensation Seeking in the Mouse
Christopher M. Olsen 1, Danny G. Winder 1
1Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Kennedy Center for Human Development, Vanderbilt University Medical Center

In this protocol we describe a method of operant learning using sensory stimuli as a reinforcer in the mouse. It requires no prior training or food restriction, and it allows the study of motivated behavior without the use of a pharmacological or natural reinforcer such as food.

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Medicine

Microvascular Decompression: Salient Surgical Principles and Technical Nuances
Jonathan Forbes 1, Calvin Cooper *2, Walter Jermakowicz *2, Joseph Neimat *1, Peter Konrad *1
1Department of Neurosurgery, Vanderbilt University Medical Center, 2Vanderbilt School of Medicine, Vanderbilt University Medical Center

There are many available options for management of the patient with trigeminal neuralgia. Microvascular decompression, while the most invasive of all options, is also the most effective at achieving long term remission of symptoms. Video instruction on how to maximize efficacy and minimize complications with this procedure is described.

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Medicine

The Polyvinyl Alcohol Sponge Model Implantation
Desirae L. Deskins 1,2, Shidrokh Ardestani 1,2, Pampee P. Young 1,2,3
1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine

A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.

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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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Medicine

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-κB Reporter Transgenic
Whitney Barham 1, Taylor Sherrill 2, Linda Connelly 3, Timothy S. Blackwell 2, Fiona E. Yull 1
1Cancer Biology Department, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Department of Pharmaceutical Sciences, University of Hawaii at Hilo College of Pharmacy

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-κB) signaling, visualized through bioluminescent imaging of an NF-κB luciferase reporter mouse.

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Neuroscience

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube
Elise R. Pfaltzgraff 1, Nathan A. Mundell 1,2, Patricia A. Labosky 3
1Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 2Department of Pharmacology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 3Vanderbilt University Medical Center

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

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Neuroscience

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Dennis P. Buehler 1, Carrie B. Wiese 1, S. B. Skelton 1, E. Michelle Southard-Smith 1
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

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Immunology and Infection

piggyBac Transposon System Modification of Primary Human T Cells
Sunandan Saha 1,2, Yozo Nakazawa 3, Leslie E. Huye 4,5, Joseph E. Doherty 2,6, Daniel L. Galvan 2, Cliona M. Rooney 4,5,7, Matthew H. Wilson 1,2,4,8
1Program in Translational Biology and Molecular Medicine, Baylor College of Medicine , 2Department of Medicine, Division of Nephrology, Baylor College of Medicine , 3Department of Immunology and Pathology, Shinshu University School of Medicine, 4Center for Cell and Gene Therapy, Baylor College of Medicine , 5Department of Pediatrics, Baylor College of Medicine , 6Program in Cell and Molecular Biology, Baylor College of Medicine , 7Department of Molecular Virology and Microbiology, Baylor College of Medicine , 8Michael E. DeBakey VA Medical Center

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

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JoVE Journal

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice
Wen-Chi Chen *1, Sung-Hyun Park *1, Carol Hoffman 1, Cecil Philip 1, Linda Robinson 2, James West 2, Gabriele Grunig 1,3
1Department of Environmental Medicine, New York University School of Medicine, Tuxedo, 2Division of Allergy, Pulmonary, & Critical Care Medicine, Department of Medicine, Vanderbilt University Medical Center, 3Division of Pulmonary Medicine, New York University School of Medicine

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

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Immunology and Infection

Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs
Brian C. Evans *1,2, Christopher E. Nelson *1,2, Shann S. Yu *1,2, Kelsey R. Beavers 2,3, Arnold J. Kim 1, Hongmei Li 1,2, Heather M. Nelson 4, Todd D. Giorgio 1,2,5,6, Craig L. Duvall 1,2
1Department of Biomedical Engineering, Vanderbilt University, 2Vanderbilt Institute for Nanoscale Science & Engineering, Vanderbilt University, 3Interdisciplinary Materials Science Program, Vanderbilt University, 4Monroe Carell Jr. Children's Hospital, Vanderbilt University Medical Center, 5Department of Chemical & Biomolecular Engineering, Vanderbilt University, 6Department of Cancer Biology, Vanderbilt University

A hemolysis assay can be used as a rapid, high-throughput screen of drug delivery systems' cytocompatibility and endosomolytic activity for intracellular cargo delivery. The assay measures the disruption of erythrocyte membranes as a function of environmental pH.

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Medicine

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice
Nataliya I. Skrypnyk 1, Raymond C. Harris 1, Mark P. de Caestecker 1
1Division of Nephrology, Vanderbilt University Medical Center

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

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Medicine

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation
J. Geraldo Valadez 1, Anuraag Sarangi 1, Christopher J. Lundberg 1, Michael K. Cooper 1,2,3
1Department of Neurology, Vanderbilt University Medical Center, 2Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, 3Neurology Service, Veteran Affairs TVHS

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

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Medicine

Echocardiographic Assessment of the Right Heart in Mice
Evan Brittain 1, Niki L. Penner 2, James West 2, Anna Hemnes 2
1Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

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Biology

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes
Poojitha Sitaram 1, Sarah Grace Hainline 1, Laura Anne Lee 1
1Department of Cell and Developmental Biology, Vanderbilt University Medical Center

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein. 

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Biology

Isolation and Physiological Analysis of Mouse Cardiomyocytes
Gretchen M. Roth 1, David M. Bader 1,2, Elise R. Pfaltzgraff 2
1Department of Medicine, Vanderbilt University, 2Department of Cell and Developmental Biology, Vanderbilt University

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

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JoVE Journal

Reconstitution Of β-catenin Degradation In Xenopus Egg Extract
Tony W. Chen *1, Matthew R. Broadus *1, Stacey S. Huppert 2, Ethan Lee 1,3
1Department of Cell and Developmental Biology and Program in Developmental Biology, Vanderbilt University Medical Center, 2Division of Gastroenterology, Hepatology & Nutrition and Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, 3Vanderbilt Ingram Cancer Center, Vanderbilt University School of Medicine

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

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Medicine

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells
Rachel J. Jerrell 1, Aron Parekh 1
1Department of Otolaryngology, Vanderbilt University Medical Center

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

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Medicine

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease
Bruce M. Damon 1,2,3,4, Ke Li 1,2, Richard D. Dortch 1,2, E. Brian Welch 1,2, Jane H. Park 1,2,4, Amanda K. W. Buck 1,2, Theodore F. Towse 1,2,5, Mark D. Does 1,2,3, Daniel F. Gochberg 1,2,6, Nathan D. Bryant 1,2
1Institute of Imaging Science, Vanderbilt University, 2Department of Radiology and Radiological Sciences, Vanderbilt University, 3Department of Biomedical Engineering, Vanderbilt University, 4Department of Molecular Physiology and Biophysics, Vanderbilt University, 5Department of Physical Medicine and Rehabilitation, Vanderbilt University, 6Department of Physics and Astronomy, Vanderbilt University

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

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Medicine

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images
Aliya Gifford 1, Theodore F. Towse 2, Ronald C. Walker 3, Malcolm J. Avison 4, E. Brian Welch 3
1Chemical and Physical Biology Program, Vanderbilt University, 2Department of Physical Medicine and Rehabilitation, Vanderbilt University School of Medicine, 3Radiology & Radiological Sciences, Vanderbilt University Medical Center, 4Department of Pharmacology, Vanderbilt University

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 °C) and cold conditions (17 °C) in order to map brown adipose tissue (BAT) in adult human subjects.

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Behavior

Testing Sensory and Multisensory Function in Children with Autism Spectrum Disorder
Sarah H. Baum 1, Ryan A. Stevenson 2, Mark T. Wallace 3
1Vanderbilt Brain Institute, Vanderbilt University Medical Center, 2Department of Psychology, University of Toronto, 3Department of Hearing and Speech Sciences, Vanderbilt University

We describe how to implement a battery of behavioral tasks to examine the processing and integration of sensory stimuli in children with ASD. The goal is to characterize individual differences in temporal processing of simple auditory and visual stimuli and relate these to higher order perceptual skills like speech perception.

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Biology

High Throughput Danio Rerio Energy Expenditure Assay
Savannah Y. Williams 1, Benjamin J. Renquist 2
1Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 2School of Animal and Comparative Biomedical Sciences, University of Arizona

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

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Biology

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix
Matthew K. Stephenson 1, Sean Lenihan 2,3, Roman Covarrubias 2,3, Ryan M. Huttinger 2,3, Richard J. Gumina 2,3, Douglas B. Sawyer 4, Cristi L. Galindo 2,3
1Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 2Department of Medicine, Vanderbilt University Medical Center, 3Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 4Cardiovascular Institute, Maine Medical Center

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

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Bioengineering

Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres
Raymond C. Pasek 1, Taylor E. Kavanaugh 2, Craig L. Duvall 2, Maureen A. Gannon 1,3
1Department of Medicine, Vanderbilt University Medical Center, 2School of Engineering, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Health Authority

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.

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Immunology and Infection

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension
Fanny Laroumanie 1, Bethany L. Dale 2, Mohamed A. Saleh 3, Meena S. Madhur 1
1Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Molecular Physiology and Biophysics, Vanderbilt University, 3Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University

This article provides detailed methodology to identify and quantify functional T lymphocyte subsets present within murine kidney, aorta and lymph nodes by intracellular staining and flow cytometry. The model of angiotensin II induced hypertension was chosen to explain, step-by-step, the procedures and fundamental principles of flow cytometry and intracellular staining.

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Biology

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney
Lauren E. Woodard 1,2,3, Richard C. Welch 2, Felisha M. Williams 2, Wentian Luo 2, Jizhong Cheng 3, Matthew H. Wilson 1,2,3
1Department of Veterans Affairs, Tennessee Valley Healthcare System, 2Departments of Medicine and Pharmacology, Vanderbilt University Medical Center, 3Department of Medicine, Baylor University College of Medicine

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

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Developmental Biology

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
David E. Maridas 1, Elizabeth Rendina-Ruedy 1, Phuong T. Le 1, Clifford J. Rosen 1
1Maine Medical Center Research Institute

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

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Medicine

Fat-Water Phantoms for Magnetic Resonance Imaging Validation: A Flexible and Scalable Protocol
Emily C. Bush 1, Aliya Gifford 2, Crystal L. Coolbaugh 1, Theodore F. Towse 1,3,4, Bruce M. Damon 1,5,6,7, E. Brian Welch 1,5
1Vanderbilt University Institute of Imaging Science (VUIIS), Vanderbilt University Medical Center, 2Department of Biomedical Informatics, Vanderbilt University Medical Center, 3Department of Physical Medicine and Rehabilitation, Vanderbilt University Medical Center, 4Department of Biomedical Sciences, Grand Valley State University, 5Department of Radiology & Radiological Sciences, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University, 7Department of Molecular Physiology and Biophysics, Vanderbilt University

The purpose of this work is to describe a protocol for creating a practical fat-water phantom that can be customized to produce phantoms with varying fat percentages and volumes.

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Neuroscience

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue
Aaron A. May-Zhang 1, Karen K Deal 1, E. Michelle Southard-Smith 1
1Vanderbilt University Medical Center

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

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Biochemistry

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis
Francisco J. Sierra-Valdez 1, Richard A. Stein 2, Phanindra Velissety 1,3, Valeria Vasquez 1, Julio F. Cordero-Morales 1
1Department of Physiology, University of Tennessee Health Science Center, 2Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, 3CuriRX, Inc.

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

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JoVE Core

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K
Hanan B. Elajaili 1, Laura Hernandez-Lagunas 1, Kalina Ranguelova 2, Sergey Dikalov 3, Eva Nozik-Grayck 1
1Cardiovascular Pulmonary Research Laboratories and Pediatric Critical Care Medicine, Department of Pediatrics, University of Colorado Anschutz Medical Campus, 2Bruker BioSpin Corp, 3Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

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JoVE Core

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT
Katherine Kim 1, Shan Huang 2, Laura A. Fletcher 1, Alana E. O'Mara 1, Ilan Tal 3, Robert J. Brychta 1, Aaron M. Cypess 1, Kong Y. Chen 1, Brooks P. Leitner 1
1Diabetes, Endocrinology, and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 2National Cancer Institute, National Institutes of Health, 3Division of Nuclear Medicine and Molecular Imaging, Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

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Medicine

Transdermal Measurement of Glomerular Filtration Rate in Mice
Lauren Scarfe 1,2, Daniel Schock-Kusch 3, Lorenzo Ressel 4, Jochen Friedemann 3, Yury Shulhevich 3, Patricia Murray 2, Bettina Wilm 2, Mark de Caestecker 1
1Division of Nephrology, Department of Medicine, Vanderbilt University Medical Center, 2Department of Cellular and Molecular Physiology, University of Liverpool, 3MediBeacon GmbH, 4Department of Veterinary Pathology and Public Health, Institute of Veterinary Science, University of Liverpool

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

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Medicine

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells
Raïko Blondonnet 1,2, Bertille Paquette 1,2, Damien Richard 3, Rémi Bourg 2,4, Géraldine Laplace 2,4, Romain Segurel 2,4, Henria Pouvelle 2,4, Corinne Belville 2, Loic Blanchon 2, Thomas Godet 1,2, Jean-Michel Constantin 1,2, Jean-Etienne Bazin 1,2, Vincent Sapin 2, Matthieu Jabaudon 1,2
1Department of Perioperative Medicine, CHU Clermont-Ferrand, 2Centre National de la Recherche Scientifique Unité Mixte de Recherche (CNRS UMR) 6293, Institut National de la Santé et de la Recherche Médicale (INSERM) U1103, Laboratoire de Génétique, Reproduction et Développement (GReD), Université Clermont Auvergne, 3Department of Pharmacology, CHU Clermont-Ferrand, 4Nurse Anesthetist School, CHU Clermont-Ferrand

We describe an easy protocol specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro in order to improve our understanding of mechanisms involved in the epithelial lung injury and to test novel therapies for acute respiratory distress syndrome.

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Neuroscience

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury
Brandon Foreman 1, Daryn Cass 2, Jonathan Forbes 2, Laura B. Ngwenya 2,3
1Department of Neurology and Rehabilitation Medicine, University of Cincinnati, 2Department of Neurosurgery, University of Cincinnati, 3Neurotrauma Center, University of Cincinnati Gardner Neuroscience Institute

A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described.

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Immunology and Infection

Isolation and Adoptive Transfer of High Salt Treated Antigen-presenting Dendritic Cells
Justin P. Van Beusecum 1, Liang Xiao 1, Natalia R. Barbaro 1, David M. Patrick 1, Annet Kirabo 1
1Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center

Here, we present a protocol to isolate dendritic cells from murine spleens by magnetic cell sorting and subsequent adoptive transfer into naïve mice. The model of high-salt activated dendritic cells was chosen to explain the step-by-step procedures of adoptive transfer and flow cytometry.

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Bioengineering

Growth and Characterization of Irradiated Organoids from Mammary Glands
Benjamin C. Hacker 1, Javier D. Gomez 1, Carlos A. Silvera Batista 1, Marjan Rafat 1,2,3
1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Radiation Oncology, Vanderbilt University Medical Center

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

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Cancer Research

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels
Steven M Alves 1, Tian Zhu 1, Anastasia Shostak 1, Ninna S. Rossen 2, Marjan Rafat 1,3,4
1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2Department of Radiation Oncology, Stanford University, 3Depattment of Biomedical Engineering, Vanderbilt University, 4Department of Radiation Oncology, Vanderbilt University Medical Center

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

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Developmental Biology

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence
Zhentao Zhang 1,2,3, Young-Jae Nam 1,2,3
1Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Vanderbilt Center for Stem Cell Biology, Vanderbilt University

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

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Neuroscience

An Implantable System For Chronic In Vivo Electromyography
David Zealear 1, Yike Li 1, Shan Huang 1
1Department of Otolaryngology, Vanderbilt University Medical Center

Presented here is a protocol for the manufacturing of an implantable system for in vivo chronological recording of evoked and spontaneous electromyographic potentials. The system is applied to the investigation of reinnervation of laryngeal muscles following nerve injury.   

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Developmental Biology

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption
Joseph M. Elsakr 1, Charles Deeter 2, Valerie Ricciardi 3, Maureen Gannon 1,4,5,6
1Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Agilent Technologies, 3Department of Biological Sciences, Vanderbilt University, 4Department of Veterans Affairs Tennessee Valley, 5Department of Medicine, Vanderbilt University Medical Center, 6Department of Cell and Developmental Biology, Vanderbilt University

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

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Biology

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts
Meiling Melzer 1, David Beier 1, Pampee P. Young 1,2, Sarika Saraswati 1
1Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 2American Red Cross, National Headquarters

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

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Medicine

A Modified Two Kidney One Clip Mouse Model of Renin Regulation in Renal Artery Stenosis
Mohammad Saleem 1, Pierina Barturen-Larrea 1, Luz Saavedra 1, Jose A. Gomez 1
1Department of Medicine/Clinical Pharmacology Division, Vanderbilt University Medical Center

A modified 2 kidney 1 clip (2K1C) Goldblatt mouse model was developed using polyurethane tubing to initiate renal artery stenosis, inducing an increase in renin expression and kidney injury. Here, we describe a detailed procedure of preparing and placing the cuff onto the renal artery to generate a reproducible and consistent 2K1C mouse model.

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Chemistry

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Albert B. Arul 1, Renã A.S. Robinson 1,2,3,4
1Department of Chemistry, Vanderbilt University, 2Vanderbilt Memory & Alzheimer's Center, Vanderbilt University Medical Center, 3Vanderbilt Brain Institute, Vanderbilt University Medical Center, 4Vanderbilt Institute of Chemical Biology, Vanderbilt University Medical Center

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

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JoVE Core

Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device
Raiko Blondonnet 1,2, Bertille Paquette 1,2, Jules Audard 1,2, Ridvan Guler 1,2, François-Xavier Roman 1,2, Ruoyang Zhai 2, Corinne Belville 2, Loïc Blanchon 2, Thomas Godet 1, Emmanuel Futier 1,2, Jean-Etienne Bazin 1, Jean-Michel Constantin 3, Vincent Sapin 2,4, Matthieu Jabaudon 1,2,5
1Department of Perioperative Medicine, CHU Clermont-Ferrand, 2GReD, CNRS, INSERM, Université Clermont Auvergne, 3GRC 29, AP-HP, DMU DREAM, Department of Anesthesiology and Critical Care, Pitié-Salpêtrière Hospital, Sorbonne University, 4Department of Biochemistry and Molecular Genetics, CHU Clermont-Ferrand, 5Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center

We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair.

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Developmental Biology

Assessing Cardiac Reprogramming using High Content Imaging Analysis
Zhentao Zhang 1,2,3, Young-Jae Nam 1,2,3
1Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Vanderbilt Center for Stem Cell Biology, Vanderbilt University

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

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Developmental Biology

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity
Yvon Woappi 1,2, Geraldine Ezeka 3, Justin Vercellino 4, Sean M. Bloos 5, Kim E. Creek 6, Lucia Pirisi 1
1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, 3Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Baltimore, 4Department of Pathology, Albert Einstein College of Medicine, 5Department of Pediatrics, Vanderbilt University Medical Center, 6Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

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Medicine

A New Model of Heart Failure Post-Myocardial Infarction in the Rat
Honglei Li 1,2, Jiansheng Huang 3, Caihong Liu 1, Zhentao Zhang 3, Kuiquan Song 2, Ke Ma 1, Connor W. Dennewitz 3, Shijun Wang 1,4
1College of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, 2Department of Intensive Care Unit, Jinan Municipal Hospital of Traditional Chinese Medicine, 3Department of Medicine, Vanderbilt University Medical Center, 4Shandong Co-Innovation Center of Classic TCM Formula, Shandong Provincial Education Department

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

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Biology

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence
Cassandra Warden *1, Alan J. Simmons *2, Lejla Pasic 3, Ashley Pitzer 4,6, Sean S. Davies 4, Justin H. Layer 5, Raymond L. Mernaugh 3, Annet Kirabo 4,6
1Vanderbilt Eye Institute, Vanderbilt University Medical Center, 2Department of Cell and Developmental Biology, Vanderbilt University, 3Department of Biochemistry, Vanderbilt University, 4Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University Medical Center, 5Division of Hematology and Oncology, Indiana University School of Medicine, 6Department of Molecular Physiology and Biophysics, Vanderbilt University

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

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Bioengineering

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep
Rei Ukita 1, John W. Stokes 1, W. Kelly Wu 1, Jennifer Talackine 1, Nancy Cardwell 1, Yatrik Patel 1, Clayne Benson 5, Caitlin T. Demarest 1, Erika B. Rosenzweig 3, Keith Cook 2, Emily J. Tsai 4, Matthew Bacchetta 1,6
1Department of Thoracic Surgery, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Carnegie Mellon University, 3Department of Pediatrics, Columbia University Medical Center, Columbia University Irving Medical Center, 4Department of Medicine, Columbia University Medical Center, Columbia University Irving Medical Center, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Biomedical Engineering, Vanderbilt University

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

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Biology

Development of a Cell Co-Culture Model to Mimic Cardiac Ischemia/Reperfusion In Vitro
Zhu Li 1, Matthew J. W. Hampton 1, Matthew B. Barajas 1, Matthias L. Riess 1,2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Anesthesiology, TVHS VA Medical Center, 3Department of Pharmacology, Vanderbilt University

Spatial distance is a key parameter in assessing hypoxia/reoxygenation injury in a co-culture model of separate endothelial and cardiomyocyte cell layers, suggesting, for the first time, that optimizing the co-culture spatial environment is necessary to provide a favorable in vitro model for testing the role of endothelial cells in cardiomyocyte protection.

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JoVE Core

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics
Shobana Jayapalan 1,2, Ananya Nandy 1,2, Elizabeth Rendina-Ruedy 1,2,3
1Vanderbilt Center for Bone Biology, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University Medical Center, 3Department of Molecular Physiology and Biophysics, Vanderbilt University

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

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Biology

Identification of the Source of Secreted Proteins in the Kidney by Brefeldin A Injection
Kensei Taguchi 1, Sho Sugahara 1, Bertha C. Elias 1, Craig R. Brooks 1
1Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center

Identifying the cell type responsible for secreting cytokines is necessary to understand the pathobiology of kidney disease. Here, we describe a method to quantitatively stain kidney tissue for cytokines produced by kidney epithelial or interstitial cells using brefeldin A, a secretion inhibitor, and cell-type-specific markers.

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Medicine

Modified Technique for the Use of Neonatal Murine Hearts in the Langendorff Preparation
Matthew B. Barajas 1, Richard J. Levy 2
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Anesthesiology, Columbia University Medical Center

The present protocol describes aortic cannulation and retrograde perfusion of the ex-vivo neonatal murine heart. A two-person strategy, using a dissecting microscope and a blunted small gauge needle, permits reliable cannulation. Quantification of longitudinal contractile tension is achieved using a force transducer connected to the apex of the left ventricle.

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Immunology and Infection

Murine Fecal Isolation and Microbiota Transplantation
Jeanne A. Ishimwe 1, Jianyong Zhong 2,3, Valentina Kon 2, Annet Kirabo 1
1Division of Clinical Pharmacology, Department of Medicine Vanderbilt University Medical Center and Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Division of Pediatric Nephrology Department of Pediatrics, Vanderbilt University Medical Center, 3Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

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Methods For Studying Osteoenergetics And Metabolism
Elizabeth Rendina-Ruedy 1, Courtney M. Karner 2
1Department of Medicine, Clinical Pharmacology, Vanderbilt University Medical Center, 2Department of Internal Medicine, Charles and Jane Pak Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center

Methods For Studying Osteoenergetics And Metabolism

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Biology

Design and Implementation of a Rat Ex Vivo Lung Perfusion Model
William J. Cleveland *1, Josephine E. Hees *1,2, Claudius Balzer 1,2, Hunter F. Douglas 1, Troy Stevens 3,4,5, Matthias L. Riess 1,6,7
1Anesthesiology, Vanderbilt University Medical Center, 2Anesthesiology, University of Greifswald, 3Department of Physiology and Cell Biology, University of South Alabama, 4Department of Medicine, University of South Alabama, 5Center for Lung Biology, University of South Alabama, 6Anesthesiology, Tennessee Valley Healthcare System Veterans Affairs Medical Center, 7Pharmacology, Vanderbilt University

Ex vivo lungs are useful for a variety of experiments to collect physiological data while excluding the confounding variables of in vivo experiments. Commercial setups are often expensive and limited in the types of data they can collect. We describe a method for building a fully modular setup, adaptable for various study designs.

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Current and Novel Experimental Methods in Ischemia/Reperfusion Research: From Cutting-Edge Models to Innovative Therapies
Matthew B. Barajas 1,2, Matthias L. Riess 1,2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Anesthesiology, TVHS VA Medical Center, 3Department of Pharmacology, Vanderbilt University

Current and Novel Experimental Methods in Ischemia/Reperfusion Research: From Cutting-Edge Models to Innovative Therapies

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Biology

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles
Tiffany M. Richardson *1, Yasminye D. Pettway *1, John T. Walker 1, Heather A. Nelson 2, Matthew Ishahak 3, Gregory Poffenberger 2, Radhika Aramandla 2, Conrad Reihsmann 2, Ashutosh Agarwal 3, Alvin C. Powers 1,2,4, Marcela Brissova 2
1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 3Department of Biomedical Engineering, University of Miami, 4VA Tennessee Valley Healthcare System

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

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Medicine

Gastric Point of Care Ultrasound in Adults: Image Acquisition and Interpretation
Eric R. Heinz 1, Omar Al-Qudsi 2, David L. Convissar 3, Marianne D. David 1, Jennifer E. Dominguez 2, Stephen Haskins 4, Christina Jelly 5, Anahi Perlas 6, Anita N. Vincent 1, Yuriy S. Bronshteyn 2
1Department of Anesthesiology and Critical Care Medicine, George Washington University, 2Department of Anesthesiology, Duke University School of Medicine, Duke University Health System, 3Department of Anesthesiology, Massachusetts General Hospital, 4Department of Anesthesiology, Critical Care & Pain Management, Hospital for Special Surgery, 5Department of Anesthesiology, Vanderbilt University Medical Center, 6Department of Anesthesia and Pain Management, Toronto Western Hospital, University Health Network

This protocol introduces two methods for image acquisition in gastric ultrasonography. Additionally, tips are provided for interpreting this information to assist in medical decision-making.

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Medicine

A Porcine Model of Acute Respiratory Failure with a Continuous Infusion of Oleic Acid
Bret D. Alvis 1,2, Colleen Brophy 3, Joyce Cheung-Flynn 3, Marisa Case 1, Kyle Hocking 2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Surgery, Vanderbilt University Medical Center

Infusing oleic acid continuously into the pulmonary artery of an anesthetized adult pig induces acute respiratory failure, enabling controlled experimentation during acute respiratory decompensation.

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Medicine

Adult and Pediatric Porcine Model of Acute Volume Overload
Bret D. Alvis 1,2, Jenna Helmer Sobey 1, Lexie Vaughn 3, Christina Jelly 1, Dawson Wervey 4, Joyce Cheung-Flynn 3, Marisa Case 1, Colleen Brophy 3, Kyle Hocking 2,3
1Department of Anesthesiology, Vanderbilt University Medical Center, 2Department of Biomedical Engineering, Vanderbilt University, 3Department of Surgery, Vanderbilt University Medical Center, 4Ohio University Heritage College of Osteopathic Medicine

The protocol here shows how continuous administration of crystalloids into the central veins of a euvolemic pig/piglet allows for the appropriate investigation of the physiological effects of acute volume overload.

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Medicine

Enhanced Communication of Tumor Margins Using 3D Scanning and Mapping
Carly Fassler 1, Alexis Miller 1, Kayvon Sharif 1, Kavita Prasad 1, Marina Aweeda 1, Jim Lewis 2, Michael C. Topf 1,3
1Department of Otolaryngology - Head and Neck Surgery, Vanderbilt University Medical Center, 2Department of Pathology, Vanderbilt University Medical Center, 3Vanderbilt University School of Engineering

A novel method for 3D scanning and virtual mapping of cancer resections is proposed with the goal of improving communication among the multidisciplinary cancer care team.

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Medicine

Identifying Frailty Using Point-of-Care Ultrasonography: Image Acquisition and Assessment
Eric R. Heinz 1, Rachel Bernardo 2, Sarah Elizabeth Birk 3, William Manson 4, David B. MacLeod 5, Jeroen Molinger 5, Anita Vincent 1, Yuriy S. Bronshteyn 5
1Department of Anesthesiology and Critical Care Medicine, George Washington University, 2The George Washington University School of Medicine and Health Sciences Washington, 3Vanderbilt University Medical Center, 4Department of Anesthesiology, UVA Health, 5Department of Anesthesiology, Duke University School of Medicine, Duke University Health System

Here, we present a protocol to assess frailty in the perioperative setting using point-of-care ultrasound to measure quadriceps thickness. This method offers a practical, non-invasive alternative to traditional assessment methods, potentially enhancing perioperative care by quickly identifying frail patients.

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Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics
Darian T. Carroll 1, Sarah R. Lindsley 2, Melissa Kirigiti 2, Alex Buko 3, Angela Jones 4, Paul Kievit 2, Maureen Gannon 1,5,6,7
1Department of Molecular Physiology and Biophysics, Vanderbilt University, 2Division of Metabolic Health and Disease, Oregon National Primate Research Center, 3Human Metabolome Technologies (HMT) America, 4Vanderbilt Technologies for Advanced Genomics, Vanderbilt University Medical Center, 5Department of Medicine, Vanderbilt University Medical Center, 6Department of Veterans Affairs Tennessee Valley, 7Department of Cell and Developmental Biology, Vanderbilt University

This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.

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Neuroscience

System for Focal, Closed-System Central Nervous System Injury
Amy N. Stahl 1, Elisabeth Artis 2, Purnima Ghose 2, Tonia S. Rex 2
1Neuroscience Graduate Program, Vanderbilt University, 2Vanderbilt Eye Institute, Vanderbilt University Medical Center

This protocol describes a custom overpressure air system designed to induce closed-system central nervous system (CNS) injuries in mice, including ocular, brain, and spinal cord traumas. The goal of this protocol is to provide a framework for researchers to easily adapt and expand the system for their unique CNS trauma studies.

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