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Yale University School of Medicine

52 ARTICLES PUBLISHED IN JoVE

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Biology

Fabrication of Amperometric Electrodes
Carolyn M. Pike 1, Chad P. Grabner 2, Amy B. Harkins 1
1Department of Pharmacological and Physiological Science, Saint Louis University School of Medicine, 2Yale University School of Medicine

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

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Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.
Shiaulou Yuan 1, Zhaoxia Sun 1
1Department of Genetics, Yale University School of Medicine

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

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Biology

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy
Christina Joselevitch 1, David Zenisek 1
1Cellular and Molecular Physiology, Yale University School of Medicine

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

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Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes
Silvio Sacchetti 1, Kambiz N. Alavian 1, Emma Lazrove 1, Elizabeth A. Jonas 1
1Department of Internal Medicine, Yale University

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

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Medicine

Mouse Models for Graft Arteriosclerosis
Lingfeng Qin 1, Luyang Yu 2, Wang Min 2
1Department of Surgery, Yale University School of Medicine , 2Department of Pathology, Yale University School of Medicine

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

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Neuroscience

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells
Erin M. Boisvert 1,2, Kyle Denton 1, Ling Lei 1, Xue-Jun Li 1,3
1Department of Neuroscience, The University of Connecticut Health Center, 2Department of Genetics and Developmental Biology, The University of Connecticut Health Center, 3Stem Cell Institute, The University of Connecticut Health Center

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

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Medicine

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence
Natalia J. Sumi 1, Eydis Lima 1, John Pizzonia 2, Sean P. Orton 3, Vinicius Craveiro 1, Wonduk Joo 1, Jennie C. Holmberg 1, Marta Gurrea 1, Yang Yang-Hartwich 1, Ayesha Alvero 1, Gil Mor 1
1Department of Obstetrics, Gynecology and Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, 2NatureMost Laboratories, 3Bruker Preclinical Imaging

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

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Neuroscience

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination
Robert A. Hill 1,3, Jelena Medved 1, Kiran D. Patel 1, Akiko Nishiyama 1,2
1Department of Physiology and Neurobiology, University of Connecticut, 2Stem Cell Institute, University of Connecticut, 3Department of Neurology, Yale University School of Medicine

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

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Biology

Generation of Transgenic Hydra by Embryo Microinjection
Celina E. Juliano 1, Haifan Lin 1, Robert E. Steele 2
1Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, 2Department of Biological Chemistry and the Developmental Biology Center, University of California, Irvine

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.

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Neuroscience

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus
Tharakeswari Selvakumar 1, Kambiz N. Alavian 2, Travis Tierney 1
1Department of Neurosurgery, Brigham & Women's Hospital, Harvard Medical School, 2Division of Brain Sciences, Department of Medicine, Imperial College London

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

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Neuroscience

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains
Maria Weinert 1, Tharakeswari Selvakumar 2, Travis S. Tierney 2, Kambiz N. Alavian 1,3
1Division of Brain Sciences, Department of Medicine, Imperial College London, 2Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 3Department of Internal Medicine, Endocrinology, Yale University School of Medicine

The causes of degeneration of midbrain dopaminergic neurons during Parkinson’s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

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Neuroscience

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System
Andrew Zayachkivsky 1, Mark J. Lehmkuhle 2, F. Edward Dudek 2
1Department of Neurosurgery, Yale University School of Medicine, 2Department of Neurosurgery, University of Utah

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

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Medicine

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation
Samir Tivari 1, Reju Korah 1, Michael Lindy 1, Robert Wieder 1
1Department of Medicine and New Jersey Medical School Cancer Center, Rutgers New Jersey Medical School

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

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Bioengineering

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography
Dustin R. Morado 1, Bo Hu 1, Jun Liu 1
1Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at Houston

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

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Biology

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments
Feinan Wu 1, Brennan G. Olson 1, Jie Yao 1
1Department of Cell Biology, Yale University School of Medicine

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

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JoVE Core

Knowledge Based Cloud FE Simulation of Sheet Metal Forming Processes
Du Zhou 1, Xi Yuan 1, Haoxiang Gao 1, Ailing Wang 1, Jun Liu 1, Omer El Fakir 1, Denis J. Politis 1, Liliang Wang 1, Jianguo Lin 1
1Department of Mechanical Engineering, Imperial College London

The following paper presents a novel FE simulation technique (KBC-FE), which reduces computational cost by performing simulations on a cloud computing environment, through the application of individual modules. Moreover, it establishes a seamless collaborative network between world leading scientists, enabling the integration of cutting edge knowledge modules into FE simulations.

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Immunology and Infection

Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice
Yi Yao 1,2, Yun Liu 1,3, Akira Takashima 1
1Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, 2Department of Internal Medicine, Yale University School of Medicine, 3Department of Pathophysiology, Southern Medical University (China)

This current protocol employs fluorescent reporters, in vivo labeling, and intravital imaging techniques to enable monitoring of the dynamic process of neutrophil priming in living animals.

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Medicine

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging
Victoria L. Patterson *1, Brian S. Thompson *1, Catherine Cherry *1, Shao-bin Wang 2, Bo Chen 2, Josephine Hoh 1,2
1Department of Environmental Health Sciences, Yale University School of Medicine, 2Department of Ophthalmology, Yale University School of Medicine

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

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Developmental Biology

Isolation of Murine Embryonic Hemogenic Endothelial Cells
Jennifer S. Fang *1, Emily C. Gritz *2, Kathrina L. Marcelo 3, Karen K. Hirschi 1
1Departments of Medicine, Genetics and Biomedical Engineering, Yale Cardiovascular Research Center, Vascular Biology and Therapeutics Program, Yale Stem Cell Center, Yale University School of Medicine, 2Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Yale University School of Medicine, 3Department of Molecular and Cellular Biology, Baylor College of Medicine

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

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Neuroscience

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy
Joerg Nikolaus 1,2, Erdem Karatekin 1,2,3,4
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 2Nanobiology Institute, Yale University, 3Department of Molecular Biophysics and Biochemistry, Yale University, 4Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS)

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

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Use of Decellularized Mammalian Lung Tissues to Study Cell: Matrix Interactions
Christina N. Blaul 1,2, Jenna Balestrini 3,4, Hongyi Pan 5, Julia Winkler 5, Erica L. Herzog 5, Huanxing Sun 5
1NSF Funded Biomedical MD-PhD REU in affiliation with Yale School of Medicine, Yale Pathology Department, Section of Pulmonary, Critical Care, and Sleep Medicine, Yale University School of Medicine, 2School of Natural Sciences: Molecular and Cellular Biology, University of California, Merced, 3Department of Anesthesiology, Yale School of Medicine, 4Cell Bioprocessing, Draper, 5Department of Internal Medicine, Section of Pulmonary, Critical Care, and Sleep Medicine, Yale School of Medicine

This study presents an ex vivo platform based on decellularized lung tissue to study cell: matrix interactions in the healthy and diseased adult lung.

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JoVE Core

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos
Lorena Benedetti 1,2,3, Anna Ghilardi 4, Laura Prosperi 4, Maura Francolini *1, Luca Del Giacco *4
1Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, 2Department of Neuroscience; Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, 3Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, 4Department of BioSciences, Università degli Studi di Milano

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

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Developmental Biology

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity
Shen Zhang 1, Yonggen Wu 1, Yimin Weng 2, Zhihui Xu 1, Wenmin Chen 3, Dahan Zheng 4, Wei Lin 5, Jun Liu 6, Ying Zhou 1,7
1Reproductive Medicine Center, The First Affiliated Hospital of Wenzhou Medical University, 2Department of Orthopaedics, The Second Affiliated Hospital of Wenzhou Medical University, 3Department of Obstetrics, The First Affiliated Hospital of Wenzhou Medical University, 4School of Laboratory Medicine and Life Science, Wenzhou Medical University, 5School of Pharmaceutical Science, Wenzhou Medical University, 6Stem Cells and Genetic Engineering Group, AgriBioscience Research Centre, Department of Economic Development, Jobs, Transport and Resources, 7Department of Histology and Embryology, Wenzhou Medical University

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

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Developmental Biology

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta
Ashish Misra 1, Zhonghui Feng 1, Jiasheng Zhang 1, Zhi-Yin Lou 2,3, Daniel M. Greif 1
1Yale Cardiovascular Research Center, Section of Cardiovascular Medicine, Department of Internal Medicine, Yale University School of Medicine, 2Department of Neurology, Yale University School of Medicine, 3Department of Neurology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

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Bioengineering

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement
Jun Liu 1, John Wilson 2, Claire Davis 1, Anthony Peyton 2
1Advanced Steel Research Centre, Warwick Manufacturing Group, University of Warwick, 2School of Electrical and Electronic Engineering, University of Manchester

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

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Cancer Research

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics
Taylor C. Brown 1, Norman G. Nicolson 2, Joyce Cheng 1, Reju Korah 1, Tobias Carling 1
1Department of Surgery & Yale Endocrine Neoplasia Laboratory, Yale University School of Medicine, 2Yale New Haven Hospital

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in vitro.

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Medicine

Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment
Laura E. Stevens *1, Anna Arnal-Estapé *1, Don X. Nguyen 1,2
1Department of Pathology, Yale University School of Medicine, 2Department of Medical Oncology, Yale University School of Medicine

We describe a method to significantly enhance orthotopic engraftment of lung cancer cells into the murine lungs by pre-conditioning the airways with injury. This approach may also be applied to study stromal interactions within the lung microenvironment, metastatic dissemination, lung cancer co-morbidities, and to more efficiently generate patient derived xenografts.

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JoVE Core

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)
Matthew Zimmerman 1, Landry Blanc 1, Pei-Yu Chen 1, Véronique Dartois 1, Brendan Prideaux 1
1Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

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Genetics

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization
Caitlin E. Moss 1, Andrew Robson 2, Erol Fikrig 3, Sukanya Narasimhan 3
1Department of Microbial Pathogenesis, Yale University School of Medicine, 2Program in Vertebrate Developmental Biology, Departments of Pediatrics and Genetics, Yale University School of Medicine, 3Department of Internal Medicine, Yale University School of Medicine

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

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Immunology and Infection

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection
Ana Maria Ortega-Prieto 1, Jessica Katy Skelton 1, Catherine Cherry 1, Marco Antonio Briones-Orta 1, Charlotte Alexandra Hateley 1, Marcus Dorner 1
1Section of Virology, Department of Medicine, Imperial College

The goal of this protocol is to provide a step-by-step guide to perform 3-D "liver-on-a-chip" infection experiments with the hepatitis B virus.

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Immunology and Infection

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System
Irfan Ullah 1,2, Kunho Chung 1, Jagadish Beloor 2, Sang-Kyung Lee 1, Priti Kumar 2
1Department of Bioengineering and Institute of Nanoscience and Technology, Hanyang University, 2Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in the central nervous system.

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Immunology and Infection

Novel Protocol for Generating Physiologic Immunogenic Dendritic Cells
Alessandra Ventura *1,2, Aaron Vassall *1, Alp Yurter 1, Eve Robinson 1, Renata Filler 1, Douglas Hanlon 1, Katrina Meeth 1, Harib Ezaldein 1, Michael Girardi 1, Olga Sobolev 1, Marcus W. Bosenberg 1,3, Richard L. Edelson 1
1Department of Dermatology, Yale University School of Medicine, 2Dermatology Department, University of Rome Tor Vergata, 3Department of Pathology, Yale University School of Medicine

The transimmunization (TI) device or plate and related protocols have been developed to replicate the key features of extracorporeal photochemotherapy (ECP), in an experimental setting, allowing for production of physiologically activated, tunable dendritic cells (DCs) for cancer immunotherapy.

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Neuroscience

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons
Brent Aulston 1, Qing Liu 1, Patrick Reilly 1, Shauna H. Yuan 1
1Department of Neurosciences, University of California, San Diego

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

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Developmental Biology

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution
Leighton H. Duncan 1,5, Mark W. Moyle 1,5, Lin Shao 1,5, Titas Sengupta 1,5, Richard Ikegami 1,5, Abhishek Kumar 4,5, Min Guo 4,5, Ryan Christensen 4,5, Anthony Santella 2,5, Zhirong Bao 2,5, Hari Shroff 4,5, William Mohler 3,5, Daniel A. Colón-Ramos 1,5,6
1Department of Neuroscience and Department of Cell Biology, Yale University School of Medicine, 2Developmental Biology Program, Sloan Kettering Institute, 3Department of Genetics and Genome Sciences and Center for Cell Analysis and Modeling, University of Connecticut Health Center, 4Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, 5WormGUIDES.org, 6Instituto de Neurobiología, Recinto de Ciencias Médicas, Universidad de Puerto Rico

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

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Immunology and Infection

Measurement of Chitinase Activity in Biological Samples
Alyssa K. Amick 1, Qing Liu 1, Samir Gautam 1, Geoffrey Chupp 1, Charles S. Dela Cruz *1, Lokesh Sharma *1
1Pulmonary, Critical Care and Sleep Medicine, Yale University School of Medicine

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

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Developmental Biology

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells
Erin M. Boisvert 1, Robert E. Means 1, Michael Michaud 1, Jason J. Thomson 2, Joseph A. Madri 1, Samuel G. Katz 1
1Department of Pathology, Yale University School of Medicine, 2Stem Cell Center, Yale University School of Medicine

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

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Immunology and Infection

Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System
David Chetrit 1, Donghyun Park 1,2, Bo Hu 3, Jun Liu 1,2, Craig R. Roy 1
1Department of Microbial Pathogenesis, Boyer Center for Molecular Medicine, Yale University School of Medicine, 2Microbial Sciences Institute, Yale University, 3Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston

Imaging of bacterial cells is an emerging systems biology approach focused on defining static and dynamic processes that dictate the function of large macromolecular machines. Here, integration of quantitative live cell imaging and cryo-electron tomography is used to study Legionella pneumophila type IV secretion system architecture and functions.

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Medicine

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube
Dmitry Frank *1, Shiri Savir *1, Benjamin F. Gruenbaum 2, Israel Melamed 3, Julia Grinshpun 1, Ruslan Kuts 1, Boris Knyazer 4, Alexander Zlotnik 1, Max Vinokur 1, Matthew Boyko 1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

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Neuroscience

Laser-Induced Brain Injury in the Motor Cortex of Rats
Ruslan Kuts *1, Israel Melamed *2, Honore N. Shiyntum 3, Benjamin F. Gruenbaum 4, Dmitry Frank 1, Boris Knyazer 5, Dmitry Natanel 1, Olena Severynovska 3, Max Vinokur 1, Matthew Boyko 1
1Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Biochemistry and Physiology, Faculty of Biology, Ecology, and Medicine, Oles Honchar Dnipro National University, 4Department of Anesthesiology, Yale University School of Medicine, 5Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

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Immunology and Infection

Three-Dimensional Imaging of the Vertebral Lymphatic Vasculature and Drainage using iDISCO+ and Light Sheet Fluorescence Microscopy
Laurent Jacob *1, Jose Brito *1,2, Jean-Leon Thomas 1,3
1Université Pierre et Marie Curie Paris, Sorbonne Université, Institut du Cerveau et de la Moelle Epinière, 2Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 3Department of Neurology, Yale University School of Medicine

A protocol is presented combining tissue clearing with light sheet fluorescence microscopy (LSFM) to obtain three-dimensional and cellular resolution images of the lymphatic vessels and lymph nodes (LNs) collecting the cerebrospinal fluid (CSF) and spinal epidural fluid.

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Neuroscience

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration
Dmitry Frank *1, Israel Melamed *2, Benjamin F. Gruenbaum 3, Julia Grinshpun 1, Ruslan Kuts 1, Rachel Shvartsur 4, Abed N. Azab 4, Mohamad H. Assadi 5, Max Vinokur 1, Matthew Boyko 1
1Division of Anesthesia and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Anesthesiology, Yale University School of Medicine, 4Recanati School for Community Health Professions, Faculty of Health Sciences, Ben-Gurion University of the Negev, 5Department of microbiology and immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

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JoVE Core

Imaging and Analysis of Oil Red O-Stained Whole Aorta Lesions in an Aneurysm Hyperlipidemia Mouse Model
Pei-Yu Chen 1, Lingfeng Qin 2, Michael Simons 1,3
1Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 2Department of Surgery, Yale University School of Medicine, 3Department of Cell Biology, Yale University School of Medicine

This protocol provides a step-by-step procedure to analyze atherosclerotic burden in mice. Investigators can use this protocol to compare the abundance, location, and size of atherosclerotic lesions in different animals.

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Neuroscience

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Julia Grinshpun 1, Israel Melamed 3, Olena Severynovska 4, Ruslan Kuts 1, Michael Semyonov 1, Evgeni Brotfain 1, Alexander Zlotnik 1, Matthew Boyko 1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology, Yale University School of Medicine, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

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Behavior

A Complex Diving-For-Food Task to Investigate Social Organization and Interactions in Rats
Benjamin F. Gruenbaum *1, Dmitry Frank *2, Shiri Savir 2, Honore N. Shiyntum 3, Ruslan Kuts 2, Max Vinokur 2, Israel Melamed 4, Michael Dubilet 2, Alexander Zlotnik 2, Matthew Boyko 2
1Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 2Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 3Department of Biophysics and Biochemistry, Oles Honchar Dnipro National University, 4Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

This protocol describes a method of examining social hierarchy in a rat model. Rats perform a complex diving-for-food task in which they form a distinct hierarchy according to their willingness to dive underwater and swim to obtain a food pellet. This method is used to understand decision making and social relationships among highly social animals in small groups.

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Neuroscience

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Israel Melamed 3, Julia Grinshpun 1, Yair Benjamin 1, Ievgeni Vzhetson 1, Nadia Kravchenko 4, Michael Dubilet 1, Matthew Boyko *1, Alexander Zlotnik *1
1Department of Anesthesiology and Critical Care, Soroka Medical Center, Ben-Gurion University of the Negev, 2Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 3Department of Neurosurgery, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 4Department of Physiology, Faculty of Biology, Ecology and Medicine, Dnepropetrovsk State University

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

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Developmental Biology

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells
Elizabeth A. Nelson 1,2, Jingyao Qiu 3,4, Nicholas W. Chavkin 1,2, Karen K. Hirschi 1,2,3,4,5
1Department of Cell Biology, University of Virginia, 2Cardiovascular Research Center, University of Virginia, 3Department of Medicine, Yale University School of Medicine, 4Department of Genetics, Yale University School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

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Biology

Visualization and Quantification of Brown and Beige Adipose Tissues in Mice using [18F]FDG Micro-PET/MR Imaging
Qing Liu *1,2, Kel Vin Tan *3, Hing-Chiu Chang 3, Pek-Lan Khong 3, Xiaoyan Hui 1,2,4
1State Key Laboratory of Pharmaceutical Biotechnology, The University of Hong Kong, 2Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 3Department of Diagnostic Radiology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 4School of Biomedical Sciences, Institute of Vascular Medicine, Chinese University of Hong Kong

Functional imaging and quantitation of thermogenic adipose depots in mice using a micro-PET/MR imaging-based approach.

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Developmental Biology

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing
Nicholas W. Chavkin 1,2, Shelby Cain 1, Kenneth Walsh 2,3,4, Karen K. Hirschi 1,2,4,5
1Department of Cell Biology, University of Virginia School of Medicine, 2Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 3Department of Cardiology, University of Virginia School of Medicine, 4Hematovascular Biology Center, University of Virginia School of Medicine, 5Yale Cardiovascular Research Center, Yale University School of Medicine

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

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Medicine

Multiple Intravenous Bolus Dosing and Invasive Hemodynamic Assessment in a Hypoxia-Induced Mouse Pulmonary Artery Hypertension Model
Lingfeng Qin 1, Bo Jiang 2, Krisztina Zsebo 3, Henricus J. Duckers 3, Michael Simons 4,5, Pei-Yu Chen 4
1Department of Surgery, Yale University School of Medicine, 2Department of Vascular Surgery, The First Hospital of China Medical University, Key Laboratory of Pathogenesis, Prevention, and Therapeutics of Aortic Aneurysm, 3VasoRx Inc., 4Yale Cardiovascular Research Center, Department of Internal Medicine, Yale University School of Medicine, 5Department of Cell Biology, Yale University School of Medicine

This protocol provides a step-by-step procedure for executing multiple intravenous bolus dose administration and invasive hemodynamic monitoring in mice. Investigators can use this protocol for future therapeutic compound screening for pulmonary artery hypertension.

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Behavior

Assessing Dominant-Submissive Behavior in Adult Rats Following Traumatic Brain Injury
Dmitry Frank *1, Benjamin F. Gruenbaum *2, Michael Semyonov 1, Yair Binyamin 1, Olena Severynovska 3, Ron Gal 1, Amit Frenkel 1, Boris Knazer 4, Matthew Boyko 1, Alexander Zlotnik 1
1Division of Anesthesiology and Critical Care, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev, 2Department of Anesthesiology and Perioperative Medicine, Mayo Clinic, 3Department of Biochemistry and Physiology of the Faculty of Biology and Ecology, Oles Gonchar of the Dnipro National University, 4Department of Ophthalmology, Soroka University Medical Center and the Faculty of Health Sciences, Ben-Gurion University of the Negev

The present protocol describes a rat model of fluid percussion-induced traumatic brain injury followed by a series of behavioral tests to understand the development of dominant and submissive behavior. Using this model of traumatic brain injury in conjunction with specific behavioral tests enables the study of social impairments following brain injury.

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Neuroscience

Simultaneous Data Collection of fMRI and fNIRS Measurements Using a Whole-Head Optode Array and Short-Distance Channels
Sara Sanchez-Alonso 1, Rebecca R. Canale 2, Isabel F. Nichoson 1, Richard N. Aslin 1,2,3
1Child Study Center, Yale University School of Medicine, 2Department of Psychology, University of Connecticut, 3Department of Psychology, Yale University

We present a method for simultaneously collecting fMRI and fNIRS signals from the same subjects with whole-head fNIRS coverage. The protocol has been tested with three young adults and can be adapted for data collection for developmental studies and clinical populations.

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Neuroscience

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery
Xiaona Lu 1, Yong-hui Jiang 1,2,3
1Department of Genetics, Yale University School of Medicine, 2Department of Neuroscience, Yale University School of Medicine, 3Department of Pediatrics, Yale University School of Medicine

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

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