Duke University Medical Center 29 articles published in JoVE Neuroscience Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice Simone Degan1, Yu Feng1, Ulrike Hoffmann2, Dennis A. Turner1,3,4,5 1Department of Neurosurgery, Duke University Medical Center, 2Department of Anesthesiology and Pain Management, University Texas Southwestern Medical School, 3Department of Neurobiology, Duke University Medical Center, 4Department of Biomedical Engineering, Duke University, 5Research and Surgery Services, Durham VA Medical Center We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations. Biology Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells Mohammad Fazli1, Chantell S. Evans1 1Department of Cell Biology, Duke University Medical Center This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging. Neuroscience A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice Huaxin Sheng1, Lihong Dang1, Xuan Li1, Zhong Yang1, Wei Yang1 1Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University Medical Center This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes. Neuroscience Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation Ran Li1, Weina Duan1, Dong Zhang2, Ulrike Hoffmann3, Junjie Yao2, Wei Yang1, Huaxin Sheng1 1Multidisciplinary Brain Protection Program, Department of Anesthesiology, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University, 3Department of Anesthesiology and Pain Management, UT Southwestern University Medical Center This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation. Immunology and Infection Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus) Chelsea D. Landon1,2, Gabriella Dancourt1, Vivian Shing2, Herman F. Staats2,3,4 1Division of Laboratory Animal Resources, Duke University Medical Center, 2Department of Pathology, Duke University Medical Center, 3Department of Immunology, Duke University Medical Center, 4Duke Human Vaccine Institute, Duke University Medical Center This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization. Neuroscience Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development Urann Chan1, Diwas Gautam1, Anne E. West1 1Department of Neurobiology, Duke University Medical Center Here we describe a method to visualize synaptogenesis of granule neurons in the mouse cerebellum over the time course of postnatal brain development when these cells refine their synaptic structures and form synapses to integrate themselves into the overall brain circuit. Neuroscience Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase Claire A. de March1, Yosuke Fukutani1,2, Aashutosh Vihani1,3, Hitoshi Kida1,4, Hiroaki Matsunami1,3,5,6 1Department of Molecular Genetics and Microbiology, Duke University Medical Center, 2Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 3Department of Neurobiology, Duke University Medical Center, 4Department of Mechanical Systems, Engineering, Tokyo University of Agriculture and Technology, 5Institute of Global Innovation Research, Tokyo University of Agriculture and Technology, 6Duke Institute for Brain Sciences, Duke University Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase. Genetics Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models Lidia Tagliafierro1,2, Ekaterina Ilich3, Malik Moncalvo3, Jeffrey Gu1, Ahila Sriskanda1,2, Carole Grenier4, Susan K. Murphy4, Ornit Chiba-Falek1,2, Boris Kantor3 1Department of Neurology, Duke University Medical Center, 2Center for Genomic and Computational Biology, Duke University Medical Center, 3Viral Vector Core, Duke University Medical Center, 4Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Duke University Medical Center Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons. Bioengineering Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography Alex Y. Lin1,2, Yifu Ding1,2,3, Daniel J. Vanselow1,2, Spencer R. Katz1,2,3, Maksim A. Yakovlev1,2, Darin P. Clark4, David Mandrell5, Jean E. Copper1,2, Damian B. van Rossum1,2, Keith C. Cheng1,2 1The Jake Gittlen Laboratories for Cancer Research, Penn State College of Medicine, 2Division of Experimental Pathology, Department of Pathology, Penn State College of Medicine, 3Medical Scientist Training Program, Penn State College of Medicine, 4Center for In Vivo Microscopy, Duke University Medical Center, 5KTM Research We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT. Medicine A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration Chao Xiong1, Zhiquan Zhang1, Gurpreet S. Baht2,3, Niccolo Terrando1,4 1Department of Anesthesiology, Duke University Medical Center, 2Department of Orthopaedic Surgery, Duke University Medical Center, 3Duke Molecular Physiology Institute, 4Center for Translational Pain Medicine, Duke University Medical Center This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging. Behavior Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates Leah C Acker1,2,3, Erica N. Pino1,4,5, Edward S. Boyden1,6,7, Robert Desimone1,7 1McGovern Institute, Massachusetts Institute of Technology, 2Harvard-MIT Division of Heath Sciences and Technology, 3Department of Anesthesiology, Duke University Medical Center, 4Department of Biology, Massachusetts Institute of Technology, 5Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 6Media Lab and Department of Biological Engineering, Massachusetts Institute of Technology, 7Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology A protocol to build a tissue penetrating illuminator for delivering light over large volumes with minimal diameter is presented. Neuroscience Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay Yuetian Zhang*1, Yi Pan*1, Hiroaki Matsunami2,3, Hanyi Zhuang1,4 1Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Shanghai Jiao Tong University School of Medicine, 2Department of Molecular Genetics and Microbiology, Duke University Medical Center, 3Department of Neurobiology, Duke Institute for Brain Sciences, Duke University Medical Center, 4Institute of Health Science, Chinese Academy of Science/Shanghai Jiao Tong University School of Medicine Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay. Developmental Biology Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells Kathryn E. Ware1, Shivee Gilja1, Shenghan Xu1, Samantha Shetler1, Mohit K. Jolly2, Xueyang Wang3, Suzanne Bartholf Dewitt4, Alexander J. Hish1, Sarah Jordan1, William Eward5, Herbert Levine2, Andrew J. Armstrong4, Jason A. Somarelli1 1Department of Medicine, Duke University, 2Department of Bioengineering, Rice University, 3Department of Molecular Genetics and Microbiology, Duke University, 4Solid Tumor Program and the Duke Prostate Center, Duke University Medical Center, 5Duke University Medical Center We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments. Medicine Methods for Acute and Subacute Murine Hindlimb Ischemia Michael E. Padgett1, Timothy J. McCord1, Joseph M. McClung1, Christopher D. Kontos1 1Division of Cardiology, Department of Medicine, Duke University Medical Center Surgical induction of hindlimb ischemia in the mouse is useful to examine angiogenesis, however this is compromised in certain inbred mouse strains that display marked ischemia-induced tissue necrosis. Methods are described to induce subacute limb ischemia using ameroid constrictors to circumvent this problem through the induction of gradual arterial occlusion. Neuroscience A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice Melanie Maya Kaelberer1,2, Sven-Eric Jordt2 1Department of Cellular & Molecular Physiology, Yale University, 2Department of Anesthesiology, Duke University Medical Center Organ specific sensory neurons are difficult to identify. Fast Blue tracing is used to identify nodose neurons innervating the airways for cell sorting. Sorted nodose neurons are used to extract high quality ribonucleic acid (RNA) for sequencing. Using this protocol, gene expression of airway specific neurons is determined. Medicine Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension Zhiyuan Ma1, Lan Mao1, Sudarshan Rajagopal1 1Department of Medicine, Duke University Medical Center Pulmonary arterial hypertension (PAH) is a disease of pulmonary arterioles that leads to their obliteration and the development of right ventricular failure. Rodent models of PAH are critical in understanding the pathophysiology of PAH. Here we demonstrate hemodynamic characterization, with right heart catheterization and echocardiography, in the mouse and rat. Chemistry Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos Sarah C. Rothschild1, Ludmila Francescatto2, Robert M. Tombes1 1Department of Biology, Virginia Commonwealth University, 2Center for Human Disease Modeling, Duke University Medical Center Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins. Behavior Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia Sarah J. Hart1,2, Joseph J. Shaffer1,3, Joshua Bizzell1,2, Mariko Weber1,3, Mary A. McMahon2, Hongbin Gu1, Diana O. Perkins1, Aysenil Belger1,2 1Department of Psychiatry, University of North Carolina at Chapel Hill School of Medicine, 2Duke-UNC Brain Imaging and Analysis Center, Duke University Medical Center, 3Curriculum in Neurobiology, University of North Carolina at Chapel Hill This paper describes how to use the emotional oddball task and fMRI to measure brain activation in children and adolescents at familial high risk for schizophrenia (FHR). FMRI was used to measure differences in fronto-striato-limbic regions during an emotional oddball task. Children with FHR exhibited abnormal functional activation during adolescence. Medicine In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose Venkatesh Krishnan1, Robert Clark1, Marina Chekmareva2, Amy Johnson1, Sophia George3, Patricia Shaw4, Victoria Seewaldt4,5, Carrie Rinker-Schaeffer1 1Section of Urology, Department of Surgery, The University of Chicago, 2Department of Pathology, Robert Wood Johnson Medical School, 3Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, 4Department of Laboratory Medicine and Pathobiology, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University of Toronto, University Health Network, 5Departments of Medicine, Pharmacology, and Cancer Biology, Duke University Medical Center We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose. Biology Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice Dennis Abraham1, Lan Mao1 1Department of Medicine, Duke University Medical Center Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. Neuroscience Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling Gustavo Arriaga1, Joshua J. Macopson1, Erich D. Jarvis1,2 1Department of Neurobiology, Duke University Medical Center, 2Howard Hughes Medical Institute Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons. Biology Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell Diego Bohórquez1, Fariha Haque2, Satish Medicetty3, Rodger A. Liddle1 1Department of Medicine, Duke University Medical Center, 2Department of Chemistry, University of North Carolina - Chapel Hill, 3Renovo Neural Incorporated Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy. Immunology and Infection Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation Victoria R. Rendell1, Charles Giamberardino2, Jie Li2, M. Louise Markert2,3, Todd V. Brennan1 1Department of Surgery, Duke University Medical Center, 2Department of Pediatrics, Duke University Medical Center, 3Department of Immunology, Duke University Medical Center Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. Medicine Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats Gert Blueschke*1, Gabi Hanna*2, Andrew N. Fontanella2, Gregory M. Palmer2, Alina Boico2, Hooney Min2, Mark W. Dewhirst2, David C. Irwin3, Yulin Zhao2, Thies Schroeder4 1Division of Plastic, Maxillofacial, and Oral Surgery, Duke University Medical Center, 2Department of Radiation Oncology, Duke University Medical Center, 3Department of Cardiology, University of Colorado Denver, 4Department of Physical Chemistry, University of Mainz A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents. Medicine An Isolated Working Heart System for Large Animal Models Matthew A. Schechter1, Kevin W. Southerland1, Bryan J. Feger1, Dean Linder Jr.1, Ayyaz A. Ali2, Linda Njoroge1, Carmelo A. Milano1, Dawn E. Bowles1 1Department of Surgery, Duke University Medical Center, 2Department of Cardiothoracic Surgery, University Hospital of South Manchester Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition. Neuroscience Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex Louis-Jan Pilaz1, Debra L. Silver1,2 1Department of Molecular Genetics and Microbiology, Duke University Medical Center, 2Departments of Neurobiology and Cell Biology, Duke Institute for Brain Sciences, Duke University Medical Center Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment. Behavior Development of a Virtual Reality Assessment of Everyday Living Skills Stacy A. Ruse1, Vicki G. Davis1, Alexandra S. Atkins1, K. Ranga R. Krishnan2,3, Kolleen H. Fox4, Philip D. Harvey5, Richard S.E. Keefe1,3 1NeuroCog Trials, Inc., 2Duke-NUS Graduate Medical Center, 3Duke University Medical Center, 4Fox Evaluation and Consulting, PLLC, 5University of Miami Miller School of Medicine A challenge for proving treatment efficacy for cognitive impairments in schizophrenia is finding the optimizing measurement of skills related to everyday functioning. The Virtual Reality Functional Capacity Assessment Tool (VRFCAT) is an interactive gaming based computerized measure aimed at skills associated with everyday functioning, including baseline impairments and treatment related changes. Neuroscience Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons Nissa L. Carrodus*1, Kathleen Sue-Lyn Teng*1, Kathryn M. Munro1, Matthew J. Kennedy2, Jenny M. Gunnersen1,3 1Department of Anatomy & Neuroscience, The University of Melbourne, 2Department of Neurobiology, Duke University Medical Center, 3Florey Institute of Neuroscience & Mental Health, The University of Melbourne We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation. Immunology and Infection Forward Genetic Approaches in Chlamydia trachomatis Bidong D. Nguyen1, Raphael H. Valdivia1 1Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.