U.S. Department of Veterans Affairs View Institution's Website 30 articles published in JoVE Medicine Novel Percutaneous Approach for Deployment of 3D Printed Coronary Stenosis Implants in Swine Models of Ischemic Heart Disease John J. Hollowed1,2, Caroline M. Colbert3,4, Jesse W. Currier1,2, Kim-Lien Nguyen1,2,3,4 1Division of Cardiology, David Geffen School of Medicine, University of California Los Angeles, 2VA Greater Los Angeles Healthcare System, U.S. Department of Veterans Affairs, 3Physics and Biology in Medicine Graduate Program, University of California Los Angeles, 4Diagnostic Cardiovascular Imaging Laboratory, Department of Radiological Sciences, David Geffen School of Medicine, University of California Los Angeles We describe a novel, cost-effective, and efficient technique for percutaneous delivery of three-dimensionally printed coronary implants to create closed-chest swine models of ischemic heart disease. The implants were fixed in place using a mother-and-child extension catheter with high success rate. Genetics Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury Zhihong Yuan1,2, Brahmchetna Bedi1, Ruxana T. Sadikot1,2 1Department of Veterans Affairs, Atlanta VAMC, 2Division of Pulmonary and Critical Care Medicine, Department of Medicine, Emory University Mice exposed to intraperitoneal LPS secrete exosomes in Broncho-alveolar lavage (BAL) fluid that are packaged with miRNAs. Using a co-culture system, we show that exosomes released in the BAL fluid disrupt expression of tight junction proteins in bronchial epithelial cells and increase expression of pro-inflammatory cytokines that accentuate lung injury. Biochemistry Expression, Purification, and Antimicrobial Activity of S100A12 Emmanuel Jackson1, Saffron Little1, Dana S. Franklin1, Jennifer A. Gaddy2,3, Steven M. Damo1,4 1Department of Life and Physical Sciences, Fisk University, 2Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs, 3Department of Medicine - Division of Infectious Diseases, Vanderbilt University Medical School, 4Department of Biochemistry, Vanderbilt University Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori. Medicine Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats Daniel R. Machin1, Miriam E. Leary2, Yuxia He1,3, Yan-Ting Shiu1,3, Hirofumi Tanaka2, Anthony J. Donato1,4,5,6 1Department of Internal Medicine, University of Utah, 2Department of Kinesiology and Health Education, University of Texas at Austin, 3Division of Nephrology and Hypertension, University of Utah, 4Department of Biochemistry, University of Utah, 5Department of Exercise and Sport Science, University of Utah, 6Geriatric Research Education and Clinical Center, Department of Veterans Affairs Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats. Neuroscience Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications Yazan M. Dweiri1,3, Matthew A. Stone2, Dustin J. Tyler1, Grant A. McCallum1, Dominique M. Durand1 1Neural Engineering Center, Case Western Reserve University, 2Advanced Platform Technology Center, U.S. Department of Veterans Affairs, 3Jordan University of Science and Technology This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves. Immunology and Infection High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability Kathryn Patricia Haley1, Eric Joshua Blanz1, Jennifer Angeline Gaddy1,2 1Department of Medicine - Division of Infectious Diseases, Vanderbilt University School of Medicine, 2Tennessee Valley Healthcare Systems, U. S. Dept. of Veterans Affairs Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability. Biology A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice Timo Rieg1,2 1Division of Nephrology-Hypertension, Department of Medicine, University of California, San Diego, 2San Diego VA Healthcare System Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model. Bioengineering Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering Bahar Bilgen1,2, Danielle Chu3, Robert Stefani1, Roy K. Aaron1,2 1Department of Orthopaedics, The Warren Alpert Brown Medical School of Brown University and the Rhode Island Hospital, 2Center for Restorative and Regenerative Medicine, VA Medical Center, Providence, RI, 3University of Texas Southwestern Medical Center We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect. Bioengineering Bacterial Detection & Identification Using Electrochemical Sensors Colin Halford1,2, Vincent Gau3, Bernard M. Churchill2, David A. Haake2,4,5 1Research Service, Veterans Affairs Greater Los Angeles Healthcare System, 2Department of Urology, The David Geffen School of Medicine, University of California, Los Angeles, 3GeneFluidics, 4Division of Infectious Diseases, Veterans Affairs Greater Los Angeles Healthcare System, 5Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current. Medicine Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR Lik Chuan Lee1, Zhang Zhihong1, Andrew Hinson2, Julius M. Guccione1 1Department of Surgery, UCSF/VA Medical Center, 2Clinical & Regulatory, LoneStar Heart, Inc. This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans. Neuroscience Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants Mauro W. Zappaterra1, Anthony S. LaMantia2, Christopher A. Walsh3,4, Maria K. Lehtinen5 1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF. Medicine Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique Timothy C. Chang1,2, Jen-Jane Liu1,2, Joseph C. Liao1,2 1Department of Urology, Stanford University School of Medicine, 2Veterans Affairs Palo Alto Health Care System Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection. Immunology and Infection piggyBac Transposon System Modification of Primary Human T Cells Sunandan Saha1,2, Yozo Nakazawa3, Leslie E. Huye4,5, Joseph E. Doherty2,6, Daniel L. Galvan2, Cliona M. Rooney4,5,7, Matthew H. Wilson1,2,4,8 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. Bioengineering Skin Tattooing As A Novel Approach For DNA Vaccine Delivery Yung-Nung Chiu1, Jared M. Sampson1, Xunqing Jiang1, Susan B. Zolla-Pazner2,3, Xiang-Peng Kong1 1Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 2Department of Pathology, New York University School of Medicine, 3Healthcare System, Veterans Affairs New York Harbor Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy. Neuroscience Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis Joshua N. Douglas1,2,3, Lidia A. Gardner1,2, Sangmin Lee1,2, Yoojin Shin1,2, Chassidy J. Groover1,2, Michael C. Levin1,2,3 1Research Service, Veterans Administration Medical Center, Memphis, TN, 2Department of Neurology, University of Tennessee Health Science Center, Memphis, TN, 3Department of Anatomy/Neurobiology, University of Tennessee Health Science Center, Memphis, TN A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells. Medicine Models of Bone Metastasis J. Preston Campbell1,2, Alyssa R. Merkel2,3,4, S. Kathryn Masood-Campbell2,4, Florent Elefteriou1,2,4,5, Julie A. Sterling2,3,4,5 1Department of Pharmacology, Vanderbilt University, 2Vanderbilt Center for Bone Biology, Vanderbilt University, 3Department of Veterans Affairs, Tennessee Valley Healthcare System (VISN 9), 4Department of Medicine, Division of Clinical Pharmacology, Vanderbilt University, 5Department of Cancer Biology, Vanderbilt University Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models. Medicine Generation of Alginate Microspheres for Biomedical Applications Omaditya Khanna1, Jeffery C. Larson2, Monica L. Moya3, Emmanuel C. Opara4, Eric M. Brey2,5 1Department of Chemical and Biological Engineering, Illinois Institute of Technology, 2Department of Biomedical Engineering, Illinois Institute of Technology, 3Department of Biomedical Engineering, University of California at Irvine, 4Wake Forest Institute for Regenerative Medicine and Department of Biomedical Engineering, Wake Forest University Health Sciences, 5Research Service, Hines Veterans Administration Hospital In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes. Medicine Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry Wenhong Zhu1, Richard L. Gallo2,3, Chun-Ming Huang2,3,4 1Sanford-Burnham Medical Research Institute, 2Division of Dermatology, University of California, San Diego, 3VA San Diego Healthcare Center, 4Moores Cancer Center, University of California, San Diego Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable. Biology Synthesis of an In vivo MRI-detectable Apoptosis Probe Justin Lam1, Paul C. Simpson2,3, Phillip C. Yang1, Rajesh Dash1 1Division of Cardiovascular Medicine, Department of Medicine, Stanford University Medical Center, 2Division of Cardiology, Department of Medicine, University of California, San Francisco, 3San Francisco VAMC Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes. Medicine The Polyvinyl Alcohol Sponge Model Implantation Desirae L. Deskins1,2, Shidrokh Ardestani1,2, Pampee P. Young1,2,3 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. Immunology and Infection Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers Kathleen C. Prins*1,2, Gaia Vasiliver-Shamis*2,3, Michael Cammer1,2, David Depoil1,2, Michael L. Dustin2, Catarina E. Hioe1,4 1Department of Pathology, New York University Langone School of Medicine, 2Program in Molecular Pathogenesis, Marty and Helen Kimmel Center for Biology and Medicine and Skirball Institute for Biomolecular Medicine, 3Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 4Veteran Affairs New York Harbor Healthcare System This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation. Neuroscience Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition Gloria Wong1,2, Sanda Dolcos1,3, Ekaterina Denkova1, Rajendra Morey4,5,6, Lihong Wang4,5, Gregory McCarthy6,7, Florin Dolcos1,2,3,8,9 1Department of Psychiatry, University of Alberta, 2Centre for Neuroscience, University of Alberta, 3Department of Psychology, University of Illinois, 4Brain Imaging and Analysis Center, Duke University, 5Department of Psychiatry and Behavioral Sciences, Duke University, 6Mid-Atlantic Mental Illness Research Education and Clinical Center, VA Medical Center, 7Department of Psychology, Yale University, 8Neuroscience Program, University of Illinois, 9Beckman Institute for Advanced Science & Technology, University of Illinois We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants. Neuroscience Live Imaging of Dorsal Root Axons after Rhizotomy Andrew Skuba1, B. Timothy Himes2,3, Young-Jin Son4 1Temple University, Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, 2Medical Research Service, Department of Veterans Affairs Hospital, 3Department of Neurobiology and Anatomy, Drexel University College of Medicine, 4Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS. Immunology and Infection Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins Marija Pinne1,2, David Haake3,4 1Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, 2Research service, 151, Veterans Affairs Greater Los Angeles Healthcare System, 3Departments of Medicine, Urology at David Geffen School of Medicine and Department of Microbiology, Immunology and Molecular Gentics, University of California Los Angeles (UCLA), 4Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Health Care System An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction. Biology Modified Mouse Embryonic Stem Cell based Assay for Quantifying Cardiogenic Induction Efficiency Ada Ao1, Charles H. Williams1, Jijun Hao1, Charles C. Hong1,2,3,4 1Division of Cardiovascular Medicine, Department of Medicine, Vanderbilt University School of Medicine, 2Department of Pharmacology, Vanderbilt University School of Medicine, 3Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, 4Research Medicine, Veterans Administration TVHS We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/β-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages. Neuroscience The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds Michelle K. Leach1, Zhang-Qi Feng2, Caitlyn C. Gertz3, Samuel J. Tuck3, Tara M. Regan3, Youssef Naim3, Andrea M. Vincent3, Joseph M. Corey1,3,4 1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatric Research, Education and Clinical Center, Veterans Affairs Ann Arbor Health System Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included. Bioengineering Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters Michelle K. Leach1, Zhang-Qi Feng1,2, Samuel J. Tuck3, Joseph M. Corey1,3,4 1Department of Biomedical Engineering, University of Michigan, 2State Key Laboratory of Bioelectronics, Southeast University, 3Department of Neurology, University of Michigan, 4Geriatrics Research, Education and Clinical Center, Veterans Affairs Ann Arbor Healthcare Center Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented. Medicine Technique to Collect Fungiform (Taste) Papillae from Human Tongue Andrew I. Spielman*1, M. Yanina Pepino2, Roy Feldman3,4,5, Joseph G. Brand*4,6 1Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University, 2Department of Internal Medicine and Department of Psychiatry, School of Medicine, Washington University in St. Louis, 3Veterans Affairs Medical Center, 4School of Dental Medicine, Department of Biochemistry, University of Pennsylvania-School of Medicine, 5Monell Chemical Senses Center, 6Monell Chemical Senses Center Knowledge of molecular mechanisms underlying gustatory transduction has recently enjoyed significant advances, largely due to using animal models. However, the wide diversity in taste sensitivity and specificity among mammals warrants studies in human tissue. We describe a biopsy technique to collect living taste cells from the papillae on human tongue. Immunology and Infection In vivo Imaging of Transgenic Leishmania Parasites in a Live Host Colin J. Thalhofer1, Joel W. Graff2, Laurie Love-Homan3, Suzanne M. Hickerson4, Noah Craft5, Stephen M. Beverley4, Mary E. Wilson6,7 1Interdisciplinary Immunology Program, University of Iowa, and the VA Medical Center, 2Department of Biochemistry, University of Iowa, and the VA Medical Center, 3Department of Internal Medicine, University of Iowa, 4Department of Molecular Microbiology, Washington University School of Medicine, 5Division of Dermatology, Harbor-UCLA Medical Center, Hanley-Hardison Research Center, 6Interdisciplinary Immunology Program, Iowa City VA Medical Center, 7Departments of Internal Medicine, Microbiology and Epidemiology, University of Iowa An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection. Biology Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees Brad M. Isaacson1,2, Jeroen G. Stinstra3, Rob S. MacLeod2,3, Joseph B. Webster1,4, James P. Beck1,5, Roy D. Bloebaum1,2,5 1Department of Veteran Affairs, 2Department of Bioengineering, University of Utah, 3Scientific Computing and Imaging Institute , University of Utah, 4Department of Physical Medicine and Rehabilitation, University of Utah, 5Department of Orthopaedics, University of Utah There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.