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University of North Carolina at Chapel Hill

53 ARTICLES PUBLISHED IN JoVE

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Biology

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays
Pauline Chugh *1, Kristen Tamburro *1, Dirk P Dittmer 1
1Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, Center for AIDS Research, University of North Carolina at Chapel Hill

We will demonstrate the setup and analysis of pre-microRNA 96-well arrays for QPCR using a robot as well as by hand with a Thermo Scientific Matrix multichannel pipette.

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Medicine

Eye Tracking Young Children with Autism
Noah J. Sasson 1, Jed T. Elison 2
1School of Behavioral and Brain Sciences, University of Texas at Dallas, 2Carolina Institute for Developmental Disabilities, School of Medicine, University of North Carolina at Chapel Hill

Eye tracking has long been used to study gaze patterns in typically-developing individuals, but recent technological advancements have made its use with clinical populations, including autism, more feasible. While eye-tracking young children with autism can offer insight into early symptom manifestations, it involves methodological challenges. Suggestions for best practices are provided.

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Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart
Laura A. Dyer 1, Cam Patterson 1
1McAllister Heart Institute, University of North Carolina at Chapel Hill

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

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Biology

Isolation of Embryonic Ventricular Endothelial Cells
Laura A. Dyer 1, Cam Patterson 1
1McAllister Heart Institute, University of North Carolina at Chapel Hill

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

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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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Biology

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region
Laura A. Dyer 1, Yaxu Wu 1, Cam Patterson 2
1McAllister Heart Institute, University of North Carolina at Chapel Hill, 2New York-Presbyterian Hospital/Weill-Cornell Medical Center

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

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Biology

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum
David M. Linz *1, Courtney M. Clark-Hachtel *1, Ferran Borràs-Castells *1, Yoshinori Tomoyasu 1
1Department of Biology, Miami University

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

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Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice
Erin C. Steinbach 1,2, Gregory R. Gipson 1, Shehzad Z. Sheikh 1,3,4
1Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, 2Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 3Department of Genetics, University of North Carolina at Chapel Hill, 4Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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Medicine

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye
Larry Kagemann 1,2, Bo Wang 2, Gadi Wollstein 1, Hiroshi Ishikawa 1,2, Brandon Mentley 1, Ian Sigal 1,2,3, Richard A Bilonick 1,4, Joel S Schuman 1,2,3
1Department of Ophthalmology, UPMC Eye Center, Eye and Ear Institute, Ophthalmology and Visual Science Research Center, University of Pittsburgh School of Medicine, 2Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh, 3The McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, 4Deptartment of Biostatistics, Graduate School of Public Health, University of Pittsburgh

Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

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Neuroscience

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae
Jared D. Chrispell 1, Tatiana I. Rebrik 2, Ellen R. Weiss 1
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Ophthalmology, Duke University

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

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Immunology and Infection

Isolation and Transplantation of Different Aged Murine Thymic Grafts.
Y. Maurice Morillon II *1, Fatima Manzoor *1, Bo Wang 1, Roland Tisch 1
1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

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Medicine

Surgical Models of Gastroesophageal Reflux with Mice
Jinxi He 1,2, Yu Fang 2,3, Xiaoxin Chen 2,4
1Department of Thoracic Surgery, Ningxia Medical University General Hospital, 2Cancer Research Program, North Carolina Central University, 3Department of Thoracic & Cardiovascular Surgery, The First Affiliated Hospital of Chongqing Medical University, 4Department of Medicine, Center for Esophageal Disease and Swallowing, Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett’s esophagus and esophageal adenocarcinoma.

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Medicine

Isolation and Culture Expansion of Tumor-specific Endothelial Cells
Lin Xiao 1, James V. McCann 1, Andrew C. Dudley 1,2,3
1Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 3McAllister Heart Institute, University of North Carolina at Chapel Hill

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

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Chemistry

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions
Marlon Lawrence 1, Anze Testen 1, Tilen Koklic 2, Oliver Smithies 1
1Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, 2Condensed Matter Physics Department, Laboratory of Biophysics, Jozef Stefan Institute

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

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Developmental Biology

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5
Li Wang 1, Ziqing Liu 1, Chaoying Yin 1, Yang Zhou 1, Jiandong Liu 1, Li Qian 1
1Department of Pathology and Laboratory Medicine, McAllister Heart Institute, University of North Carolina, Chapel Hill

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

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Neuroscience

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis
Cortney C. Winkle 1, Christopher C. Hanlin 2, Stephanie L. Gupton 1,2
1Curriculum in Neurobiology, The University of North Carolina at Chapel Hill, 2Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

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Developmental Biology

Isolation and Characterization of Single Cells from Zebrafish Embryos
Leigh Ann Samsa 1,2, Nicole Fleming 2,3, Scott Magness 1, Li Qian 2,3, Jiandong Liu 2,3
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2McAllister Heart Institute, University of North Carolina at Chapel Hill, 3Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

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Biology

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
Kathryn A. Ramsey 1,2, Zachary L. Rushton 1, Camille Ehre 1,3
1Marsico Lung Institute/CF Center, University of North Carolina at Chapel Hill, 2Telethon Kids Institute, University of Western Australia, 3Department of Pediatrics, University of North Carolina at Chapel Hill

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

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Medicine

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals
Phillip G. Durham 1, Shumaila N. Hanif 2, Lucia Garcia Contreras 2, Ellen F. Young 3, Miriam S. Braunstein 3, Anthony J. Hickey 1
1RTI International, 2Oklahoma University Health Science Center, 3University of North Carolina at Chapel Hill

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

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Biochemistry

Measuring Protein Binding to F-actin by Co-sedimentation
Jonathon A. Heier 1, Daniel J. Dickinson 2, Adam V. Kwiatkowski 1
1Department of Cell Biology, University of Pittsburgh School of Medicine, 2Department of Biology, University of North Carolina at Chapel Hill

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

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Biochemistry

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications
Rachel A. Battaglia 1, Parijat Kabiraj 1, Helen H. Willcockson 1, Melinda Lian 1, Natasha T. Snider 1
1Department of Cell Biology and Physiology, School of Medicine, University of North Carolina at Chapel Hill

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

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Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification
Deirdre K Tucker 1,2, Julie F Foley 3, Schantel A Bouknight 4, Suzanne E Fenton 2
1Curriculum in Toxicology, University of North Carolina at Chapel Hill, 2National Toxicology Program Laboratory (NTPL), DNTP, National Institute of Environmental Health Sciences, 3Cellular and Molecular Pathology Branch, DNTP, National Institute of Environmental Health Sciences, 4Charles River Laboratories Inc.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

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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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Environment

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory
Ann G. Matthysse 1
1Department of Biology, University of North Carolina at Chapel Hill

An easy method for measuring and characterizing bacterial adhesion to plants, particularly roots and sprouts, is described in this article.

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Cancer Research

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors
Elizabeth M. Corteselli 1, James M. Samet 2, Eugene A. Gibbs-Flournoy 2,3
1Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, 2Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 3Oak Ridge Institute for Science and Education

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

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Cancer Research

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer
Milica Momcilovic 1, Sean T. Bailey 2, Jason T. Lee 3, Charles Zamilpa 3, Anthony Jones 3, Gihad Abdelhady 1, James Mansfield 4, Kevin P. Francis 5, David B. Shackelford 1
1Division of Pulmonary and Critical Care Medicine, University of California Los Angeles David Geffen School of Medicine, 2University of North Carolina at Chapel Hill, 3Department of Molecular and Medical Pharmacology, University of California Los Angeles, 4Andor Technology, 5Division of Orthopaedic Surgery, University of California Los Angeles David Geffen School of Medicine

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

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Education

The Diffusion of Passive Tracers in Laminar Shear Flow
Manuchehr Aminian 1,2, Francesca Bernardi 1, Roberto Camassa 1, Daniel M. Harris 1,3, Richard M. McLaughlin 1
1Department of Mathematics, University of North Carolina at Chapel Hill, 2Department of Mathematics, Colorado State University, 3School of Engineering, Brown University

A protocol for the study of the diffusion of passive tracers in laminar pressure-driven flow is presented. The procedure is applicable to various capillary pipe geometries.

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Developmental Biology

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay
Salma H. Azam 1, Mitchell Smith 2, Vivek Somasundaram 2, Chad V. Pecot 2,3
1Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 3Department of Medicine, University of North Carolina at Chapel Hill

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

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Biology

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining
Wilber Escorcia 1, Dana L. Ruter 1,2,3, James Nhan 1,2, Sean P. Curran 1,2
1Leonard Davis School of Gerontology, University of Southern California, 2Molecular & Computational Biology Section, University of Southern California, 3Integrative Program for Biological & Genome Sciences, University of North Carolina at Chapel Hill

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

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Medicine

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds
Kevin T. Sheets 1, Juli R. Bagó 1, Ivory L. Paulk 1, Shawn D. Hingtgen 1,2
1Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

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Medicine

Electromagnetic Navigation Transthoracic Nodule Localization for Minimally Invasive Thoracic Surgery
Sohini Ghosh 1, David Chambers 1, Adam R. Belanger 1, Allen Cole Burks 1, Christina MacRosty 1, Anna Conterato 1, Jason Long 2, Benjamin Haithcock 2, M. Patricia Rivera 1, Jason A. Akulian 1
1Section of Interventional Pulmonology, Division of Pulmonary and Critical Care Medicine, University of North Carolina at Chapel Hill, 2Section of Thoracic Surgery, Division of Cardiothoracic Surgery, University of North Carolina at Chapel Hill

Presented here is a protocol for lung nodule localization using dye marking via electromagnetically navigated transthoracic needle access. The technique described here can be accomplished in the peri-operative period to optimize nodule localization and to successful resection when performing minimally invasive thoracic surgery.

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Biochemistry

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker
Aleksandra Skrajna 1, Xiao-Cui Yang 1, Zbigniew Dominski 1,2
1Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, 2Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

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Developmental Biology

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo
Trevor Henley 1, Kandace Thomas 1, Michael Bressan 1
1Department of Cell Biology and Physiology, McAllister Heart Institute, University of North Carolina at Chapel Hill

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

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Immunology and Infection

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System
Susanna L. Harris 1, Cesar A. Pelaez 2, Elizabeth A. Shank 1,2
1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, 2Department of Biology, University of North Carolina at Chapel Hill

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

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Biochemistry

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress
Caitlin E Tolbert 1, Lindsey Palmquist 2, Hannah Lee Dixon 2, Melissa C. Srougi 2,3
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, 2Department of Chemistry, High Point University, 3Biotechnology Program and the Department of Molecular Biomedical Sciences, North Carolina State University

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

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Neuroscience

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes
Blake A. Creighton *1, Theodore W. Ruffins *1, Damaris N. Lorenzo 1
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

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Cancer Research

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports
Kelly Pittman 1, Shelton Earp 1, Eric Ubil 2
1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 2O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham

Here, we present a method using permeable membrane supports to facilitate the study of non-contact paracrine signaling used by tumor cells to suppress the immune response. The system is amenable to studying the role of tumor-secreted factors in dampening macrophage activation.

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Medicine

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application
Nicholas E. Buglak 1,2,3,5, Edward S. M. Bahnson 1,2,3,4,5
1Department of Surgery, Division of Vascular Surgery, University of North Carolina at Chapel Hill, 2Center for Nanotechnology in Drug Delivery, University of North Carolina at Chapel Hill, 3Curriculum in Toxicology & Environmental Medicine, University of North Carolina at Chapel Hill, 4Department of Cell Biology & Physiology, University of North Carolina at Chapel Hill, 5McAllister Heart Institute, University of North Carolina at Chapel Hill

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

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Bioengineering

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy
Ying Yin *1, Siyu Wang *1, Danni Hu 1, Jingyao Cai 1, Fubin Chen 1, Bo Wang 1, Yu Gao 1
1Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

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Developmental Biology

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging
Russell H. Knutsen 1, Leah M. Gober 1, Joseph R. Sukinik 1, Danielle R. Donahue 2, Elise K. Kronquist 1, Mark D. Levin 1, Sean E. McLean 3, Beth A. Kozel 1
1Translational Vascular Medicine Branch, National Heart Lung and Blood Institute, National Institutes of Health, 2Mouse Imaging Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 3Division of Pediatric Surgery, Department of Surgery, University of North Carolina at Chapel Hill

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

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JoVE Journal

RNA Interference in Aquatic Beetles as a Powerful Tool for Manipulating Gene Expression at Specific Developmental Time Points
Shubham Rathore 1, Jenni Hassert 1, Courtney M. Clark-Hachtel 2,3, Aaron Stahl 1,4, Yoshinori Tomoyasu 2, Elke K. Bushbeck 1
1Department of Biological Sciences, University of Cincinnati, 2Department of Biology, Miami University, 3Department of Biology, University of North Carolina at Chapel Hill, 4Department of Neuroscience, The Scripps Research Institute

RNA interference is a widely applicable, powerful technique for manipulating gene expression at specific developmental stages. Here, we describe the necessary steps for implementing this technique in the aquatic diving beetle Thermonectus marmoratus, from the acquisition of gene sequences to the knockdown of genes that affect structure or behavior.

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Biology

Automated Detection and Analysis of Exocytosis
Fabio Urbina 1, Stephanie L. Gupton 1
1UNC Neuroscience Center, Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill

We developed automated computer vision software to detect exocytic events marked by pH-sensitive fluorescent probes. Here, we demonstrate the use of a graphical user interface and RStudio to detect fusion events, analyze and display spatiotemporal parameters of fusion, and classify events into distinct fusion modes.

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Cancer Research

Sentinel Lymph Node Mapping and Biopsy for Endometrial Cancer at Early Stage with Laparoscopy
Bo Wang 1, Yu Xue 1, Qian Wang 1, Yan Xu 1, Xiaojun Chen 1, Chao Wang 1
1Obstetrics and Gynecology Hospital of Fudan University

This protocol describes the identification and resection of sentinel lymph nodes to make the operation as easy and minimally invasive as possible.

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Medicine

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models
Jacquelyn J. Bower 1,3, Zhenwei Song 2, Liujiang Song 1,2
1Department of Ophthalmology, University of North Carolina at Chapel Hill, 2Gene Therapy Center, University of North Carolina at Chapel Hill, 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

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Neuroscience

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study
Sara Oberrauch 1, Jing Lu 1, Linda Cornthwaite-Duncan 1, Maytham Hussein 1, Jian Li 2, Gauri Rao 3, Tony Velkov 1
1Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 2Department of Microbiology, Biomedicine Discovery Institute, Monash University, 3UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill

Delivery of therapeutics directly into the central nervous system is one way of circumventing the blood-brain barrier. The present protocol demonstrates intracerebroventricular injection for subsequent collection of cerebrospinal fluid and bodily organs. This facilitates the investigation of drug pharmacokinetics and pharmacodynamics in animal models for developing new treatments.

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Medicine

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer
Jason A. Wykoff 1, Kendall M. Shaffer 1, Kenza C. Araba 1, Matthew R. Markovetz 1, Jérémy Patarin 2, Matthieu Robert de Saint Vincent 2, Scott H. Donaldson 1,3, Camille Ehre 1,4
1Marsico Lung Institute / CF Center, The University of North Carolina at Chapel Hill, 2Rheonova, 3Department of Pulmonary and Critical Care Medicine, The University of North Carolina at Chapel Hill, 4Department of Pediatric, Pediatric Pulmonology Division, The University of North Carolina at Chapel Hill

The viscoelastic properties of mucus play a critical role in mucociliary clearance. However, traditional mucus rheological techniques require complex and time-consuming approaches. This study provides a detailed protocol for the use of a benchtop rheometer that can rapidly and reliably perform viscoelastic measurements.

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Bioengineering

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells
Madelyn VanBlunk 1, Pritha Agarwalla 1,2, Sharda Pandit 1,2, Yevgeny Brudno 1,2,3
1Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, 2Comparative Medicine Institute, North Carolina State University, 3Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill

Herein is a protocol for creating dry macroporous alginate scaffolds that mediate efficient viral gene transfer for use in genetic engineering of T cells, including T cells for CAR-T cell therapy. The scaffolds were shown to transduce activated primary T cells with >85% transduction.

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Biochemistry

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions
Brandy N. Curtis *1, Ellysa J. D. Vogt *2, Kevin S. Cannon 1,3, Amy S. Gladfelter 1,2,3
1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, 2Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 3Department of Biology, University of North Carolina at Chapel Hill

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

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Immunology and Infection

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection
Rajnikant Sharma 1, Shekhar Yeshwante 1, Quentin Vallé 1, Maytham Hussein 2, Varsha Thombare 2, Sean Michael McCann 1, Robert Maile 3,4,5, Jian Li 6, Tony Velkov 2, Gauri Rao 1
1UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, 2Department of Biochemistry & Pharmacology, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 3Department of Microbiology & Immunology, University of North Carolina School of Medicine, 4Department of Surgery, University of North Carolina at Chapel Hill, 5Curriculum in Toxicology and Environmental Medicine, University of North Carolina at Chapel Hill, 6Department of Microbiology, Monash Biomedicine Discovery Institute, Monash University

A model mimicking the clinical scenario of burn injury and infection is necessary for furthering burn research. The present protocol demonstrates a simple and reproducible rat burn infection model comparable to that in humans. This facilitates the study of burn and infections following burn for developing new topical antibiotic treatments.

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Medicine

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome
Lauren C. Frazer 1, Yukihiro Yamaguchi 1, Corey M. Jania 1, Wyatt E. Lanik 2, Qingqing Gong 3, Dhirendra K. Singh 1, Stephen Mackay 1, Natalia S. Akopyants 1, Misty Good 1
1Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of North Carolina at Chapel Hill, 2University of Nebraska College of Medicine, 3Department of Surgery, Washington University School of Medicine

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

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Neuroscience

Clinical Indications for Rapid Sequence MRI in Pediatric Neurosurgical Patients and the Limitations and Barriers to Implementation
Deveney Franklin 1, Candace Barr 1, Diana Nguyen 1, Declan O’Shaughnessy 2, Olivia E. Gilbert 1,3, Carolyn Quinsey 1,3
1School of Medicine, University of North Carolina, Chapel Hill, 2Department of Radiology, University of North Carolina at Chapel Hill, 3Department of Neurosurgery, University of North Carolina at Chapel Hill

Here, we present a protocol to increase the use of rapid sequence magnetic resonance imaging (RS-MRI) for pediatric patients for spine, traumatic brain injury (TBI), and hydrocephalus while documenting limitations and barriers to universal implementation.

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Medicine

Extracellular Vesicle Tissue Factor Activity Assay
Yohei Hisada 1, Nigel Mackman 1
1Division of Hematology, UNC Blood Research Center, University of North Carolina at Chapel Hill

Here we describe an in-house extracellular vesicle tissue factor activity assay. Activity-based assays and antigen-based assays have been used to measure tissue factor in extracellular vesicles from human plasma samples. Activity-based assays have higher sensitivity and specificity than antigen-based assays.

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Behavior

Behavioral Training Procedures for Head-fixed Virtual Reality in Mice
Jared Karl Glorius 1, Leslie Wilson 1, Ayland Letsinger 2, Parth Shirolkar 1,3, Jesse Cushman 1
1Neurobehavioral Core, National Institute of Environmental Health Sciences, 2Ion Channel Physiology Group, National Institute of Environmental Health Sciences, 3University of North Carolina at Chapel Hill

The article describes the experimental procedures for the commonly used linear track virtual reality (VR) paradigm in mice as well as determining the feasibility of running complex VR tasks by testing a Y-shaped signal discrimination task.

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