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Boston Children's Hospital

38 ARTICLES PUBLISHED IN JoVE

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Biology

Fabrication of Myogenic Engineered Tissue Constructs
Christina A. Pacak 1,2, Douglas B. Cowan 1,2
1Department of Anesthesiology, Children's Hospital Boston and Harvard Medical School, 2Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

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Biology

Optical Mapping of Langendorff-perfused Rat Hearts
Bjoern Sill 1, Peter E. Hammer 1,2, Douglas B. Cowan 1
1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Departments of Cardiac Surgery, Children's Hospital Boston and Harvard Medical School

This article describes a high temporal and spatial resolution technique to optically image action potential movement on the surface of Langendorff-perfused rat hearts using a potentiometric dye (di-8-ANEPPS).

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Biology

Implantation of Engineered Tissue in the Rat Heart
Bjoern Sill 1, Ivan V. Alpatov 2, Christina A. Pacak 2, Douglas B. Cowan 2
1Department of Anesthesiology, Perioperative and Pain Medicine, Children's Hospital Boston and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Children’s Hospital Boston

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

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Neuroscience

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants
Mauro W. Zappaterra 1, Anthony S. LaMantia 2, Christopher A. Walsh 3,4, Maria K. Lehtinen 5
1Department of Physical Medicine and Rehabilitation, VA Greater Los Angeles Healthcare System, 2Department of Pharmacology and Physiology, Institute for Neuroscience, The George Washington University School of Medicine and Health Sciences, 3Division of Genetics, Department of Medicine, Boston Children's Hospital, 4Howard Hughes Medical Institute, Boston Children's Hospital, 5Department of Pathology, Boston Children's Hospital, Harvard Medical School

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

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Bioengineering

An Improved Method for the Preparation of Type I Collagen From Skin
Christina A. Pacak 1,2, Allison A. MacKay 1, Douglas B. Cowan 1,2
1Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital, 2Department of Anesthesia, Harvard Medical School

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

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Neuroscience

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Amy E. Birsner *1, Ofra Benny *2, Robert J. D'Amato 1,3
1Vascular Biology Program, Boston Children's Hospital, 2Institute for Drug Research, School of Pharmacy, The Hebrew University of Jerusalem, 3Department of Ophthalmology, Harvard Medical School

The protocol describes the corneal micropocket assay as developed in mice.

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Medicine

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice
Terence W. Prendiville 1, Qing Ma 1, Zhiqiang Lin 1, Pingzhu Zhou 1, Aibin He 1, William T. Pu 1,2
1Department of Cardiology, Boston Children's Hospital, 2Harvard Stem Cell Institute, Harvard University

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

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Biology

Tissue Triage and Freezing for Models of Skeletal Muscle Disease
Hui Meng 1, Paul M.L. Janssen 2, Robert W. Grange 3, Lin Yang 4, Alan H. Beggs 5, Lindsay C. Swanson 5, Stacy A. Cossette 1,6, Alison Frase 7, Martin K. Childers 8, Henk Granzier 9, Emanuela Gussoni 5, Michael W. Lawlor 1
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

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Biology

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration
Janine M. Preble 1, Christina A. Pacak 2, Hiroshi Kondo 1, Allison A. MacKay 2, Douglas B. Cowan 2, James D. McCully 1
1Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center and Harvard Medical School, 2Department of Anesthesiology, Perioperative and Pain Medicine, Boston Children's Hospital and Department of Anesthesia, Harvard Medical School

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is <30 min compared to 60 - 100 min using alternative methods.

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JoVE Journal

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Daniel E. Bauer *1,2,3, Matthew C. Canver *1, Stuart H. Orkin 1,2,3,4
1Harvard Medical School, 2Division of Hematology/Oncology, Boston Children's Hospital, 3Department of Pediatric Oncology, Dana-Farber Cancer Institute, 4Howard Hughes Medical Institute

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

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Medicine

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects
Lukas Andereggen 3,4,5, Volker Neuschmelting 1,6, Michael von Gunten 7, Hans Rudolf Widmer 5, Jukka Takala 1, Stephan M. Jakob 1, Javier Fandino 1,2, Serge Marbacher 1,2
1Department of Intensive Care Medicine, University and Bern University Hospital (Inselspital), 2Department of Neurosurgery, Kantonsspital Aarau, 3Laboratories for Neuroscience Research in Neurosurgery, Boston Children's Hospital, 4Harvard Medical School, Boston Children's Hospital, 5Department of Neurosurgery, University and Bern University Hospital (Inselspital), 6Department of Neurosurgery, University Hospital Cologne, 7Institute of Pathology, Länggasse Bern

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures — subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

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Medicine

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
Jerome D. Robin 1, Woody E. Wright 1, Yaqun Zou 2, Stacy A. Cossette 3, Michael W. Lawlor 3, Emanuela Gussoni 4
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

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Bioengineering

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models
Dorothy R. Ahlf Wheatcraft 1,2, Xin Liu 1,2, Amanda B. Hummon 1,2
1Department of Chemistry and Biochemistry, University of Notre Dame, 2Harper Cancer Research Institute, University of Notre Dame

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

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Chemistry

Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles
Andrew R. Bader 1, Tina Li 1, Weiping Wang 2, Daniel S. Kohane 2, Joseph Loscalzo 1, Ying-Yi Zhang 1
1Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 2Laboratory for Biomaterials and Drug Delivery, Department of Anesthesiology, Division of Critical Care, Boston Children's Hospital

The objective of this protocol is to incorporate SDF-1α, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1α from the nanoparticles.

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Immunology and Infection

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions
Achille Broggi 1,2, Clara Cigni 1, Ivan Zanoni 1,2,3, Francesca Granucci 1,3
1Department of Biotechnology and Biosciences, University of Milano-Bicocca, 2Boston Children's Hospital, Division of Gastroenterology, Harvard Medical School, 3Humanitas Clinical and Research Center

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

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Medicine

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats
Lauren L. Jantzie 1,2, Jesse L. Winer 3, Jessie R. Maxwell 1, Lindsay A.S. Chan 3, Shenandoah Robinson 3,4
1Department of Pediatrics, University of New Mexico, 2Department of Neurosciences, University of New Mexico, 3Department of Neurosurgery, Boston Children's Hospital, 4Department of Neurology, Harvard Medical School

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

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Developmental Biology

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish
Christian Lawrence 1, Jason Best 1, Jason Cockington 2, Eric C. Henry 3, Shane Hurley 1, Althea James 1, Christopher Lapointe 1, Kara Maloney 1, Erik Sanders 4
1Aquatic Resources Program, Boston Children's Hospital, 2UQ Biological Resources, University of Queensland, 3Reed Mariculture, 4Aquatics Lab Services

Larval zebrafish are adapted to feed on zooplankton. It is possible to capitalize on this natural feature in the laboratory by growing first feeding fish together in the same system with live saltwater rotifers. This "polyculture" strategy promotes high growth and survival with minimal labor and disturbance to the larvae.

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Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse
Ke Yuan 1,2, Mark E. Orcholski 1,2, Ngan F. Huang 2,3, Vinicio A. de Jesus Perez 1,2
1Division of Pulmonary and Critical Care Medicine, School of Medicine, Stanford University, 2Stanford Cardiovascular Institute, School of Medicine, Stanford University, 3VA Palo Alto Health Care System, Department of Cardiothoracic Surgery, School of Medicine, Stanford University

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

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Developmental Biology

Rapid Acquisition of 3D Images Using High-resolution Episcopic Microscopy
Haochuan Zhang *1,2,3, JunGang Huang *2,3,4, Xin Liu 2,3, Ping Zhu 4, Zhongrong Li 1, Xue Li 2,3
1Department of Pediatric Surgery, The Second Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, 2Departments of Urology, Boston Children's Hospital, 3Department of Surgery, Harvard Medical School, 4Department of Cardiovascular Surgery, Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences

We describe a detailed protocol using high-resolution episcopic microscopy to acquire three-dimensional (3D) images of mouse embryos. This improved protocol utilizes a modified tissue preparation method to enhance penetration of the fluorescent dye, thereby permitting morphometric analysis of both small and large-sized specimens.

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Genetics

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts
Jian Ding 1,2, Zhi-Qiang Lin 1,2, Jian-Ming Jiang 3,4, Christine E. Seidman 3,4, Jonathan G. Seidman 3,4, William T. Pu 1,2, Da-Zhi Wang 1,2
1Department of Cardiology, Boston Children's Hospital, 2Department of Pediatrics, Harvard Medical School, 3Department of Genetics, Harvard Medical School, 4Howard Hughes Medical Institute

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

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Genetics

Single Molecule Analysis of Laser Localized Psoralen Adducts
Jing Huang 1, Himabindu Gali 1, Julia Gichimu 1, Marina A. Bellani 1, Durga Pokharel 1, Manikandan Paramasivam 1, Michael M. Seidman 1
1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

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Chemistry

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry
Xue Li *1,2, Dan D. Huang *3, Rui Du 1,2, Zhi J. Zhang 1, Chak K. Chan 3, Zheng X. Huang 1,2, Zhen Zhou 1,2
1Institute of Mass Spectrometer and Atmospheric Environment, Jinan University, 2Guangdong Provincial Engineering Research Center for On-line Source Apportionment System of Air Pollution, 3School of Energy and Environment, City University of Hong Kong

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

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Cancer Research

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells
Jing Huang *1,2, Peng Guo *1,2, Marsha A. Moses 1,2
1Vascular Biology Program, Boston Children's Hospital, 2Department of Surgery, Harvard Medical School and Boston Children's Hospital

Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method.

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Immunology and Infection

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses
William Santus 1, Francesca Mingozzi 1, Marina Vai 1, Francesca Granucci *1, Ivan Zanoni *1,2
1Department of Biotechnology and Biosciences, University of Milano-Bicocca, 2Harvard Medical School and Division of Gastroenterology, Boston Children's Hospital

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

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Immunology and Infection

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method
Thea Brennan-Krohn 1,2,3, James E Kirby 1,3
1Department of Pathology, Beth Israel Deaconess Medical Center, 2Division of Infectious Diseases, Boston Children's Hospital, 3Harvard Medical School

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

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JoVE Journal

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase
Nicholas McCaul 1,2, Hui Ying Yeoh 1, Guus van Zadelhoff 1, Naomi Lodder 1, Bertrand Kleizen 1, Ineke Braakman 1
1Cellular Protein Chemistry, Utrecht University, 2Program in Cellular and Molecular Medicine, Boston Children's Hospital

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

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Neuroscience

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth
Mary C. Whitman 1,2,3, Jessica L. Bell 1,3, Elaine H. Nguyen 1,3, Elizabeth C. Engle 1,2,3,4,5,6
1Department of Ophthalmology, Boston Children's Hospital, 2Department of Ophthalmology, Harvard Medical School, 3F.M. Kirby Neurobiology Center, Boston Children's Hospital, 4Department of Neurology, Boston Children's Hospital, 5Department of Neurology, Harvard Medical School, 6Howard Hughes Medical Institute

An ex vivo slice assay allows oculomotor nerve outgrowth to be imaged in real time. Slices are generated by embedding E10.5 IslMN:GFP embryos in agarose, slicing on a vibratome, and growing in a stage-top incubator. The role of axon guidance pathways is assessed by adding inhibitors to the culture media.

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Biology

Detecting Establishment of Shared Blood Supply in Parabiotic Mice by Caudal Vein Glucose Injection
Xin Liu 1, Xue Bai 1, Mingqi Li 1, Huimin Li 1, Yong Zhang 1,2, Baofeng Yang 1,3
1Department of Pharmacology, The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China, Key Laboratory of Cardiovascular Research, Ministry of Education, College of Pharmacy, Harbin Medical University, 2Institute of Metabolic Disease, Heilongjiang Academy of Medical Science, 3Department of Pharmacology and Therapeutics, Melbourne School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences University of Melbourne

Here we describe a new method of detecting successful establishment of shared blood circulation of two parabionts through a caudal vein injection of glucose, which causes minimal damage and is not fatal to the parabionts.

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Neuroscience

Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice
Ryosuke Fujiki 1,2,3,4,9, Joun Y. Lee 1,2,10, Julie A. Jurgens 1,2,3,7, Mary C. Whitman 2,5,6, Elizabeth C. Engle 1,2,3,4,5,6,7,8
1Department of Neurology, Boston Children's Hospital, 2FM Kirby Neurobiology Center, Boston Children's Hospital, 3Department of Neurology, Harvard Medical School, 4Medical Genetics Training Program, Harvard Medical School, 5Department of Ophthalmology, Boston Children's Hospital, 6Department of Ophthalmology, Harvard Medical School, 7Broad Institute of M.I.T. and Harvard, 8Howard Hughes Medical Institute, 9Department of Neurology, Kokura Memorial Hospital, 10Department of Genetics, Albert Einstein College of Medicine

This work presents a protocol to yield homogeneous cell cultures of primary oculomotor, trochlear, and spinal motor neurons. These cultures can be used for comparative analyses of the morphological, cellular, molecular, and electrophysiological characteristics of ocular and spinal motor neurons.

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Neuroscience

Arterial Pouch Microsurgical Bifurcation Aneurysm Model in the Rabbit
Stefan Wanderer 1,2, Claudia Waltenspuel 2, Basil E. Grüter 1,2, Fabio Strange 1,2, Sivani Sivanrupan 2, Luca Remonda 3, Hans Rudolf Widmer 4, Daniela Casoni 5, Lukas Andereggen 1,2, Javier Fandino 1,2, Serge Marbacher 1,2
1Department of Neurosurgery, Kantonsspital Aarau, 2Cerebrovascular Research Group, Department for BioMedical Research, University of Bern, 3Division of Neuroradiology, Department of Radiology, Kantonsspital Aarau, 4Department of Neurosurgery, Neurocenter and Regenerative Neuroscience Cluster, Inselspital, Bern University Hospital, University of Bern, 5Department for Biomedical Research, Faculty of Medicine, University of Bern

Developing and testing endovascular devices for intracranial aneurysm treatment is still of great importance. Most aneurysm models used today miss either the important characteristics of an arterial degenerated wall or the hemodynamics of a true bifurcation. Therefore, we aimed to design a novel arterial pouch bifurcation model in rabbits.

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Biology

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury
Hussein Atta *1,2, Ali Hashemi Gheinani *1,2, Amanda Wacker 1, Yaser Heshmati 3,4,5, Alex Bigger-Allen 1,6, George Lambrinos 1,2, Yao Gao 2,7, Diane R. Bielenberg 2,7, Rosalyn M. Adam 1,2
1Department of Urology, Boston Children's Hospital, 2Department of Surgery, Harvard Medical School, 3Division of Hematology/Oncology, Harvard Medical School Boston, 4Dana-Farber Cancer Institute, 5Broad Institute, 6Biological Biomedical Sciences Program, Division of Medical Sciences, Harvard Medical School, 7Vascular Biology Program, Boston Children's Hospital

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

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Medicine

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model
Yuan Yuan 1, Meng-hua Chen 1, Jing Huang 2, Yuan Tian 2, Ke Qin 3, Zhang Yuan 1, Wen-yan Wang 1, Zhi-jiang Wu 1, Xin-yue Tian 1, Yubin Zhang 4
1Department of Intensive Care Unit, The Second Affiliated Hospital of Guangxi Medical University, 2Department of hepatobiliary surgery, The Affiliated Ningbo Medical Center Lihuili Hospital of Medical School of Ningbo University, 3Department of transplantation, The Second Affiliated Hospital of Guangxi Medical University, 4Digital Technology and Engineering college, Ningbo University of Finance & Economics

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

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Immunology and Infection

Isolating Brown Adipocytes from Murine Interscapular Brown Adipose Tissue for Gene and Protein Expression Analysis
Steven G. Negron 1, Bing Xu 1, Zhiqiang Lin 1
1Masonic Medical Research Institute

This study describes a new method of isolating murine brown adipocytes for gene and protein expression analysis.

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Medicine

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies
Timothy Klouda 1, Hyunbum Kim 1, Jiwon Kim 1, Gary Visner 1, Ke Yuan 1
1Divisions of Pulmonary Medicine, Boston Children's Hospital

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

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Medicine

Creation of Two Saccular Elastase-Digested Aneurysms with Different Hemodynamics in One Rabbit
Gwendoline Boillat 1,2, Tim Franssen 2, Basil Grüter 1,2, Stefan Wanderer 1,2, Kristina Catalano 2, Daniela Casoni 3, Lukas Andereggen 1,2, Serge Marbacher 1,2
1Department of Neurosurgery, Kantonsspital Aarau, 2Cerebrovascular Research Group, Department for BioMedical Research, University of Bern, 3Experimental Surgery Facility, Department for Biomedical Research, Faculty of Medicine, University of Bern

This protocol describes the steps for the creation of a rabbit model with two elastase-digested aneurysms with different hemodynamics (stump and bifurcation constellation). This allows the testing of novel endovascular devices in aneurysms with different angioarchitecture and hemodynamic conditions within a single animal.

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Neuroscience

Using a Cell-Tracer Injection to Investigate the Origin of Neointima-Forming Cells in a Rat Saccular Side Wall Model
Stefan Wanderer *1,2, Basil E. Grüter *1,2, Jeannine Kümin 1,2, Gwendoline Boillat 1,2, Sivani Sivanrupan 2, Kristina Catalano 1,2, Michael von Gunten 3, Hans Rudolf Widmer 4, Serge Marbacher 1,2,5, Lukas Andereggen 1,2,5
1Department of Neurosurgery, Kantonsspital Aarau, 2Cerebrovascular Research Group, Department for BioMedical Research, University of Bern, 3Institute of Pathology Laenggasse, 4Department of Neurosurgery, Neurocenter and Regenerative Neuroscience Cluster, Inselspital, Bern University Hospital, University of Bern, 5Faculty of Medicine, University of Bern

We performed a one-point, lipophilic cell-tracer injection to track endothelial cells, followed by an arteriotomy and suturing of sidewall aneurysms on the abdominal rat aorta. Neointima formation seemed dependent on the parent artery in decellularized aneurysms and was promoted by the recruitment from aneurysm wall cells in vital cell-rich walls.

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Medicine

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging
Fan Chen *1,2, Chun Liu *3, Peng Guo 3, Weihui Zheng 1,2
1Key Laboratory of Head & Neck Cancer Translational Research of Zhejiang Province, Zhejiang Cancer Hospital, 2The Cancer Hospital of the University of Chinese Academy of Sciences (Zhejiang Cancer Hospital), 3Institute of Basic Medicine and Cancer (IBMC), Chinese Academy of Sciences

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

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Immunology and Infection

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers
Daniel S. Shin 1,2,3, Juliana Barreto de Albuquerque 1,3, James J. Moon 1,3,4
1Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, 2Division of Immunology, Boston Children's Hospital, 3Harvard Medical School, 4Division of Pulmonary and Critical Care Medicine, Massachusetts General Hospital

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

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