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Duke University Medical Center

43 ARTICLES PUBLISHED IN JoVE

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Biology

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging
Zhong Yang 1, Qing Zeng 2, Zhiyuan Ma 1, Yaming Wang 1, Xiaoyin Xu 2
1Department of Anesthesia, Brigham and Woman's Hospital, 2Department of Radiology, Brigham and Woman's Hospital

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

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Neuroscience

Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells
Sandeepa Dey 1, Senmiao Zhan 2, Hiroaki Matsunami 1
1Department of Molecular Genetics and Molecular Biology , Duke University, 2Department of Chemistry, Duke University

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

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Bioengineering

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model
Alexandra E. Jantzen 1, Whitney O. Lane 2, Shawn M. Gage 3, Justin M. Haseltine 1, Lauren J. Galinat 1, Ryan M. Jamiolkowski 4, Fu-Hsiung Lin 3, George A. Truskey 1, Hardean E. Achneck 3
1Department of Biomedical Engineering, Duke University , 2School of Medicine, Duke University , 3Department of Surgery, Duke University Medical Center, 4School of Medicine, University of Pennsylvania

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

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Bioengineering

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress
Whitney O. Lane 1, Alexandra E. Jantzen 2, Tim A. Carlon 2, Ryan M. Jamiolkowski 3, Justin E. Grenet 1, Melissa M. Ley 1, Justin M. Haseltine 2, Lauren J. Galinat 2, Fu-Hsiung Lin 1, Jason D. Allen 4, George A. Truskey 2, Hardean E. Achneck 1
1Department of Surgery, Duke University Medical Center, 2Department of Biomedical Engineering, Duke University , 3School of Medicine, University of Pennsylvania , 4Department of Medicine, Division of Cardiology, Duke University Medical Center

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

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Biology

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Chun-Chun Chen 1, Kazuhiro Wada 2, Erich D. Jarvis 1
1Howard Hughes Medical Institute, Department of Neurobiology, Duke University , 2Department of Biological Sciences, Hokkaido University

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

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Neuroscience

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI
Gabriel P. Howles 1, Yi Qi 2, Stephen J. Rosenzweig 3, Kathryn R. Nightingale 3, G. Allan Johnson 2
1Department of Radiology, Stanford University , 2Center for In Vivo Microscopy, Duke University Medical Center, 3Department of Biomedical Engineering, Duke University

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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Biology

In Vivo Modeling of the Morbid Human Genome using Danio rerio
Adrienne R. Niederriter 1,2, Erica E. Davis 1,3, Christelle Golzio 1, Edwin C. Oh 1, I-Chun Tsai 1, Nicholas Katsanis 1
1Center for Human Disease Modeling, Department of Cell Biology, Duke University Medical Center, 2Department of Evolutionary Anthropology, Duke University, 3Department of Pediatrics, Duke University Medical Center

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

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Immunology and Infection

Forward Genetic Approaches in Chlamydia trachomatis
Bidong D. Nguyen 1, Raphael H. Valdivia 1
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.

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Neuroscience

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling
Gustavo Arriaga 1, Joshua J. Macopson 1, Erich D. Jarvis 1,2
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.

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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. Munro 1, Matthew J. Kennedy 2, Jenny M. Gunnersen 1,3
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.

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Neuroscience

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex
Louis-Jan Pilaz 1, Debra L. Silver 1,2
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.

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Behavior

Development of a Virtual Reality Assessment of Everyday Living Skills
Stacy A. Ruse 1, Vicki G. Davis 1, Alexandra S. Atkins 1, K. Ranga R. Krishnan 2,3, Kolleen H. Fox 4, Philip D. Harvey 5, Richard S.E. Keefe 1,3
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.

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Behavior

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia
Sarah J. Hart 1,2, Joseph J. Shaffer 1,3, Joshua Bizzell 1,2, Mariko Weber 1,3, Mary A. McMahon 2, Hongbin Gu 1, Diana O. Perkins 1, Aysenil Belger 1,2
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.

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Medicine

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats
Gert Blueschke *1, Gabi Hanna *2, Andrew N. Fontanella 2, Gregory M. Palmer 2, Alina Boico 2, Hooney Min 2, Mark W. Dewhirst 2, David C. Irwin 3, Yulin Zhao 2, Thies Schroeder 4
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.

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Medicine

An Isolated Working Heart System for Large Animal Models
Matthew A. Schechter 1, Kevin W. Southerland 1, Bryan J. Feger 1, Dean Linder Jr. 1, Ayyaz A. Ali 2, Linda Njoroge 1, Carmelo A. Milano 1, Dawn E. Bowles 1
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.

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Immunology and Infection

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation
Victoria R. Rendell 1, Charles Giamberardino 2, Jie Li 2, M. Louise Markert 2,3, Todd V. Brennan 1
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.

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JoVE Core

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose
Venkatesh Krishnan 1, Robert Clark 1, Marina Chekmareva 2, Amy Johnson 1, Sophia George 3, Patricia Shaw 4, Victoria Seewaldt 4,5, Carrie Rinker-Schaeffer 1
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.

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Biology

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell
Diego Bohórquez 1, Fariha Haque 2, Satish Medicetty 3, Rodger A. Liddle 1
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.

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Biology

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice
Dennis Abraham 1, Lan Mao 1
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.

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Medicine

Hemodynamic Characterization of Rodent Models of Pulmonary Arterial Hypertension
Zhiyuan Ma 1, Lan Mao 1, Sudarshan Rajagopal 1
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.

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Chemistry

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos
Sarah C. Rothschild 1, Ludmila Francescatto 2, Robert M. Tombes 1
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.

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Neuroscience

A Method to Target and Isolate Airway-innervating Sensory Neurons in Mice
Melanie Maya Kaelberer 1,2, Sven-Eric Jordt 2
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.

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Behavior

Eliciting and Analyzing Male Mouse Ultrasonic Vocalization (USV) Songs
Jonathan Chabout 1,2, Joshua Jones-Macopson 1, Erich D. Jarvis 1,2,3
1Department of Neurobiology, Duke University, 2Howard Hughes Medical Institute, 3The Rockefeller University

Mice produce a complex multisyllabic repertoire of ultrasonic vocalizations (USVs). These USVs are widely used as readouts for neuropsychiatric disorders. This protocol describes some of the practices we learned and developed to consistently induce, collect, and analyze the acoustic features and syntax of mouse songs.

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Medicine

Methods for Acute and Subacute Murine Hindlimb Ischemia
Michael E. Padgett 1, Timothy J. McCord 1, Joseph M. McClung 1, Christopher D. Kontos 1
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.

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Developmental Biology

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells
Kathryn E. Ware 1, Shivee Gilja 1, Shenghan Xu 1, Samantha Shetler 1, Mohit K. Jolly 2, Xueyang Wang 3, Suzanne Bartholf Dewitt 4, Alexander J. Hish 1, Sarah Jordan 1, William Eward 5, Herbert Levine 2, Andrew J. Armstrong 4, Jason A. Somarelli 1
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.

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Medicine

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy
Xiaoxi Yang 1, Taylor D. Osborne 1, Martha Delahunty 2, Marilyn J. Telen 2, Jason S. Kim 1
1BioMedomics Inc., 2Division of Hematology, Department of Medicine, Duke University Medical Center

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

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Neuroscience

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay
Yuetian Zhang *1, Yi Pan *1, Hiroaki Matsunami 2,3, Hanyi Zhuang 1,4
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.

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Medicine

Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart
Muath Bishawi 1, Jun-Neng Roan 1,2, Jordan Richards 1, Zachary Brown 1, Laura Blue 1, Mani A. Daneshmand 1, Jacob N. Schroder 1, Dawn E. Bowles 3, Carmelo A. Milano 1
1Division of Cardiothoracic Surgery, Department of Surgery, Duke University, 2Division of Cardiovascular Surgery, Department of Surgery, National Cheng Kung University Hospital and College of Medicine, 3Division of Surgical Sciences, Department of Surgery, Duke University

Here, we present a protocol using two centrifugal pumps as a total artificial heart replacement.

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Behavior

Large Volume, Behaviorally-relevant Illumination for Optogenetics in Non-human Primates
Leah C Acker 1,2,3, Erica N. Pino 1,4,5, Edward S. Boyden 1,6,7, Robert Desimone 1,7
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.

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Medicine

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration
Chao Xiong 1, Zhiquan Zhang 1, Gurpreet S. Baht 2,3, Niccolo Terrando 1,4
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.

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Genetics

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
Sriram Vijayraghavan 1, Boris Kantor 1
1Duke Viral Vector Core, Department of Neurobiology, Duke University School of Medicine

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

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Neuroscience

A Rat Model of Central Fatigue Using a Modified Multiple Platform Method
Weiyue Zhang *1, Wei Zhang *1, Ning Dai *1, Chenxia Han 2, Fengzhi Wu 1, Xu Wang 1, Libo Tan 1, Jie Li 1, Feng Li 1, Qingjia Ren 3
1School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, 2Department of Integrated Traditional Chinese and Western Medicine, West China Hospital, Sichuan University, 3Institute of Tibetan Medicine, Tibetan Traditional Medical College

Here, we present a protocol to introduce a rat model of central fatigue using the modified multiple platform method (MMPM).

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Bioengineering

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography
Alex Y. Lin 1,2, Yifu Ding 1,2,3, Daniel J. Vanselow 1,2, Spencer R. Katz 1,2,3, Maksim A. Yakovlev 1,2, Darin P. Clark 4, David Mandrell 5, Jean E. Copper 1,2, Damian B. van Rossum 1,2, Keith C. Cheng 1,2
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.

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Genetics

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models
Lidia Tagliafierro 1,2, Ekaterina Ilich 3, Malik Moncalvo 3, Jeffrey Gu 1, Ahila Sriskanda 1,2, Carole Grenier 4, Susan K. Murphy 4, Ornit Chiba-Falek 1,2, Boris Kantor 3
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.

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Neuroscience

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase
Claire A. de March 1, Yosuke Fukutani 1,2, Aashutosh Vihani 1,3, Hitoshi Kida 1,4, Hiroaki Matsunami 1,3,5,6
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.

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Immunology and Infection

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)
Chelsea D. Landon 1,2, Gabriella Dancourt 1, Vivian Shing 2, Herman F. Staats 2,3,4
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.

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Neuroscience

Utilizing In Vivo Postnatal Electroporation to Study Cerebellar Granule Neuron Morphology and Synapse Development
Urann Chan 1, Diwas Gautam 1, Anne E. West 1
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.

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Medicine

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft
Michelle Mendiola Pla 1, Amy Evans 2, Franklin H. Lee 3, Yuting Chiang 1, Muath Bishawi 1, Andrew Vekstein 1, Lillian Kang 1, Diego Zapata 4, Ryan Gross 3, Alexis Carnes 2, Lynden E. Gault 5, Julie A. Balko 6, Desiree Bonadonna 2, Sam Ho 5, Paul Lezberg 7, Benjamin S. Bryner 1, Jacob N. Schroder 1, Carmelo A. Milano 1, Dawn E. Bowles 3
1Division of Cardiothoracic Surgery, Department of Surgery, Duke University, 2Perfusion Services, Duke University, 3Division of Surgical Sciences, Department of Surgery, Duke University, 4Division of Laboratory Animal Resources, Duke University, 5Gift of Hope Organ and Tissue Donor Network, 6College of Veterinary Medicine, North Carolina State University, 7TransMedics, Inc

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

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Neuroscience

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice
Simone Degan 1, Yu Feng 1, Ulrike Hoffmann 2, Dennis A. Turner 1,3,4,5
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.

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Biology

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells
Mohammad Fazli 1, Chantell S. Evans 1
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.

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Neuroscience

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation
Ran Li 1, Weina Duan 1, Dong Zhang 2, Ulrike Hoffmann 3, Junjie Yao 2, Wei Yang 1, Huaxin Sheng 1
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.

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Neuroscience

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice
Huaxin Sheng 1, Lihong Dang 1, Xuan Li 1, Zhong Yang 1, Wei Yang 1
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.

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Neuroscience

Isolating Immune Cells from Mouse Brain and Skull
Ran Zhang 1, Jin Zhang 1, Ata Ur Rehman 1, Lihong Dang 1, Xinyuan Yu 1, Wei Yang 1
1Multidisciplinary Brain Protection Program (MBPP), Department of Anesthesiology, Duke University Medical Center

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

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