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Brigham and Women's Hospital, Harvard Medical School

19 ARTICLES PUBLISHED IN JoVE

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Biology

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium
Georg Wiese 1, Steven R. Barthel 2, Charles J. Dimitroff 2
1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

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Biology

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure
Kenneth K. Kwong 1,2, Lenuta Kloetzer 1,2,3,4, Kelvin K. Wong 5,6, Jia-Qian Ren 1,2, Braden Kuo 1,2,3,4, Yan Jiang 7, Y. Iris Chen 1,2, Suk-Tak Chan 1,2,8, Geoffrey S. Young 9, Stephen T.C. Wong 5,6
1Department of Radiology, Massachusetts General Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 4Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 5Center for biotechnology and Informatics, The Methodist Hospital Research Institute, 6Department of Radiology, The Methodist Hospital, Weill Cornell Medical College, 7Bejing University of Chinese Medicine, 8Department of Health Technology and Informatics, The Hong Kong Polytechnic University, 9Department of Radiology, Brigham and Women's Hospital, Harvard Medical School

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

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Immunology and Infection

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation
Tatiana Veremeyko 1, Sarah-Christine Starossom 1, Howard L. Weiner 1, Eugene D. Ponomarev 1
1Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

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Medicine

Ultrasonic Assessment of Myocardial Microstructure
Pranoti Hiremath 1, Michael Bauer 2, Hui-Wen Cheng 2, Kazumasa Unno 2, Ronglih Liao 2, Susan Cheng 2
1Harvard Medical School, 2Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

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Bioengineering

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow
Monica M. Burdick 1,2, Nathan M. Reynolds 1,2, Eric W. Martin 2, Jacquelyn V. Hawes 1, Grady E. Carlson 1, Chaz M. Cuckler 1, Michael C. Bates 1, Steven R. Barthel 3, Charles J. Dimitroff 3
1Department of Chemical and Biomolecular Engineering, Ohio University, 2Biomedical Engineering Program, Russ College of Engineering and Technology, Ohio University, 3Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins. Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

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Medicine

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography
Hui-Wen Cheng *1,2, Sudeshna Fisch *1, Susan Cheng 1, Michael Bauer 1, Soeun Ngoy 1, Yiling Qiu 1, Jian Guan 1, Shikha Mishra 1, Christopher Mbah 1, Ronglih Liao 1
1Cardiac Muscle Research Labratory, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, 2Cardiovascular Department, Chang Gung Memorial Hospital

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

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Immunology and Infection

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice
Ilias I. Siempos 1,3, Hilaire C. Lam 1, Yan Ding 2, Mary E. Choi 2, Augustine M. K. Choi 1, Stefan W. Ryter 1
1Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 2Department of Medicine, Renal Division, Brigham and Women's Hospital, Harvard Medical School, 3First Department of Critical Care Medicine and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

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Medicine

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats
Sudeshna Fisch 1, Ronglih Liao 1, Li-Li Hsiao 2, Tzongshi Lu 2
1Cardiac Muscle Research Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 2Renal Division, Brigham and Women's Hospital, Harvard Medical School

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

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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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Medicine

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice
Wei-Ting Chang *1,2, Sudeshna Fisch *1, Michael Chen 1, Yiling Qiu 1, Susan Cheng 1, Ronglih Liao 1
1Cardiac Muscle Research Laboratory, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 2Division of Cardiovascular Medicine, Chi-Mei Medical Center, Tainan

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

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Medicine

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis
Venkatesh Mani 1, Nadia Alie 1, Sarayu Ramachandran 1, Philip M. Robson 1, Cecilia Besa 1, Gregory Piazza 2, Michele Mercuri 3, Michael Grosso 3, Bachir Taouli 1, Samuel Z. Goldhaber 2, Zahi A. Fayad 1
1Department of Radiology, Translational and Molecular Imaging Institute, Icahn School of Medicine at Mount Sinai, 2Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 3Daiichi Sankyo Pharma Development

The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

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Medicine

Surgical Training for the Implantation of Neocortical Microelectrode Arrays Using a Formaldehyde-fixed Human Cadaver Model
Pierre Mégevand 1,2, Alain Woodtli 1, Aude Yulzari 1, G. Rees Cosgrove 3, Shahan Momjian 4, Bojan V. Stimec 5, Marco V. Corniola 4, Jean H. D. Fasel 5
1Wyss Center for Bio and Neuroengineering, Geneva, 2Division of Neurology, Department of Clinical Neuroscience, Geneva University Hospitals, 3Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, 4Division of Neurosurgery, Department of Clinical Neuroscience, Geneva University Hospitals, 5Clinical Anatomy Research Group, Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva

We designed a procedure in which a formaldehyde-fixed human cadaver is used to assist neurosurgeons in training for the implantation of microelectrode arrays into the neocortex of the human brain.

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Cancer Research

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells
Nouran S. Abdelfattah 1, Ann Mullally 1,2,3
1Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 2Broad Institute, 3Dana-Farber Cancer Institute, Harvard Medical School

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

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Biology

Visualizing Lymph Node Structure and Cellular Localization using Ex-Vivo Confocal Microscopy
Rafael M. Rezende *1, Mateus E. Lopes *2, Gustavo B Menezes 2, Howard L. Weiner 1
1Ann Romney Center for Neurologic Diseases, Brigham and Women's Hospital, Harvard Medical School, 2Center for Gastrointestinal Biology, Federal University of Minas Gerais

This protocol describes a technique to image different cell populations in draining lymph nodes without alterations in the organ structure.

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Immunology and Infection

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation
Alexander H. Tavares 1, Jennifer K. Colby 1, Bruce D. Levy 1, Raja-Elie Edward Abdulnour 1
1Pulmonary and Critical Care Medicine, Department of Internal Medicine, Brigham and Women's Hospital, Harvard Medical School

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

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Developmental Biology

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function
Alessandro Evangelisti 1, Katharina Schimmel 1, Shaurya Joshi 2, Kavya Shah 1, Sudeshna Fisch 2, Kevin M. Alexander 1, Ronglih Liao 1, Isabel Morgado 1
1Stanford Cardiovascular Institute, Stanford University, 2Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

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Immunology and Infection

Fecal (micro) RNA Isolation
Fyonn H. Dhang 1,2, Howard L. Weiner 1,2, Shirong Liu 1,2
1Ann Romney Center for Neurologic Diseases, Department of Neurology, Partners Multiple Sclerosis Center, Brigham and Women's Hospital and Harvard Medical School, 2Evergrande Center for Immunologic Diseases, Harvard Medical School and Brigham and Women's Hospital

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

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Developmental Biology

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity
Yvon Woappi 1,2, Geraldine Ezeka 3, Justin Vercellino 4, Sean M. Bloos 5, Kim E. Creek 6, Lucia Pirisi 1
1Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, 2Department of Dermatology, Brigham and Women's Hospital, Harvard Medical School, 3Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Baltimore, 4Department of Pathology, Albert Einstein College of Medicine, 5Department of Pediatrics, Vanderbilt University Medical Center, 6Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, University of South Carolina

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

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