Boston Children's Hospital 15 articles published in JoVE Medicine Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies Timothy Klouda1, Hyunbum Kim1, Jiwon Kim1, Gary Visner1, Ke Yuan1 1 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. 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 Wacker1, Yaser Heshmati3,4,5, Alex Bigger-Allen1,6, George Lambrinos1,2, Yao Gao2,7, Diane R. Bielenberg2,7, Rosalyn M. Adam1,2 1 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. Neuroscience Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from Prenatal Islmn:GFP Transgenic Mice Ryosuke Fujiki1,2,3,4,9, Joun Y. Lee1,2,10, Julie A. Jurgens1,2,3,7, Mary C. Whitman2,5,6, Elizabeth C. Engle1,2,3,4,5,6,7,8 1 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. Neuroscience Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth Mary C. Whitman1,2,3, Jessica L. Bell1,3, Elaine H. Nguyen1,3, Elizabeth C. Engle1,2,3,4,5,6 1 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. Immunology and Infection Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method Thea Brennan-Krohn1,2,3, James E Kirby1,3 1Department of Pathology, Beth Israel Deaconess Medical Center, 2 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. Biochemistry Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase Nicholas McCaul1,2, Hui Ying Yeoh1, Guus van Zadelhoff1, Naomi Lodder1, Bertrand Kleizen1, Ineke Braakman1 1Cellular Protein Chemistry, Utrecht University, 2 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. Biology Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption Elizabeth S. DeWitt*1, Katherine J. Black*1, John N. Kheir1 1 Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions. Medicine Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats Lauren L. Jantzie1,2, Jesse L. Winer3, Jessie R. Maxwell1, Lindsay A.S. Chan3, Shenandoah Robinson3,4 1Department of Pediatrics, University of New Mexico, 2Department of Neurosciences, University of New Mexico, 3 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. Chemistry Preparation and Characterization of SDF-1α-Chitosan-Dextran Sulfate Nanoparticles Andrew R. Bader1, Tina Li1, Weiping Wang2, Daniel S. Kohane2, Joseph Loscalzo1, Ying-Yi Zhang1 1Laboratory 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. Medicine Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling Jerome D. Robin1, Woody E. Wright1, Yaqun Zou2, Stacy A. Cossette3, Michael W. Lawlor3, Emanuela Gussoni4 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, 4 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. Biology Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 Daniel E. Bauer*1,2,3, Matthew C. Canver*1, Stuart H. Orkin1,2,3,4 1Harvard Medical School, 2 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. Medicine Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis Tadanori Mammoto1, Akiko Mammoto1 1 Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse. Medicine The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects Lukas Andereggen3,4,5, Volker Neuschmelting1,6, Michael von Gunten7, Hans Rudolf Widmer5, Jukka Takala1, Stephan M. Jakob1, Javier Fandino1,2, Serge Marbacher1,2 1Department of Intensive Care Medicine, University and Bern University Hospital (Inselspital), 2Department of Neurosurgery, Kantonsspital Aarau, 3 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. Neuroscience The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye Amy E. Birsner*1, Ofra Benny*2, Robert J. D'Amato1,3 1 The protocol describes the corneal micropocket assay as developed in mice. Bioengineering An Improved Method for the Preparation of Type I Collagen From Skin Christina A. Pacak1,2, Allison A. MacKay1, Douglas B. Cowan1,2 1 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.