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Boston Children's Hospital, Harvard Medical School

13 ARTICLES PUBLISHED IN JoVE

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Medicine

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants
Pei-Yi Lin 1, Nadege Roche-Labarbe 1,2, Mathieu Dehaes 3, Stefan Carp 1, Angela Fenoglio 3, Beniamino Barbieri 4, Katherine Hagan 1, P. Ellen Grant 3, Maria Angela Franceschini 1
1Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 2Lab. PALM, Université de Caen Basse-Normandie, 3Fetal-Neonatal Neuroimaging and Developmental Science Center, Boston Children's Hospital, Harvard Medical School, 4ISS, INC.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

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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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Biology

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland
Silva Krause 1, Amy Brock 2, Donald E. Ingber 1,2,3
1Vascular Biology Program, Department of Surgery, Boston Children's Hospital and Harvard Medical School, 2Wyss Institute for Biologically Inspired Engineering, Harvard University, 3Harvard School of Engineering and Applied Sciences

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

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Biology

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays
Laura L. Smith 1, Alan H. Beggs 1, Vandana A. Gupta 1
1Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.

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Neuroscience

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons
Dorothy P. Schafer 1, Emily K. Lehrman 1, Christopher T. Heller 1, Beth Stevens 1
1Department of Neurology, F.M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

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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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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

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device
Hyun Jung Kim 1, Jaewon Lee 1, Jin-Ha Choi 1, Anthony Bahinski 2, Donald E. Ingber 2,3,4
1Department of Biomedical Engineering, The University of Texas at Austin, 2Wyss Institute for Biologically Inspired Engineering at Harvard University, 3Vascular Biology Program, Boston Children's Hospital, Harvard Medical School, 4John A. Paulson School of Engineering and Applied Sciences, Harvard University

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

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JoVE Core

Interictal High Frequency Oscillations Detected with Simultaneous Magnetoencephalography and Electroencephalography as Biomarker of Pediatric Epilepsy
Christos Papadelis 1, Eleonora Tamilia 1, Steven Stufflebeam 2, Patricia E. Grant 1, Joseph R. Madsen 3, Phillip L. Pearl 4, Naoaki Tanaka 2
1Fetal-Neonatal Neuroimaging and Developmental Science Center, Division of Newborn Medicine, Department of Medicine, Boston Children's Hospital, Harvard Medical School, 2Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, 3Division of Epilepsy Surgery, Department of Neurosurgery, Boston Children's Hospital, Harvard Medical School, 4Division of Epilepsy and Clinical Neurophysiology, Department of Neurology, Boston Children's Hospital, Harvard Medical School

High Frequency Oscillations (HFOs) have emerged as presurgical biomarkers for the identification of the epileptogenic zone in pediatric patients with medically refractory epilepsy. A methodology for the noninvasive recording, detection, and localization of HFOs with simultaneous scalp electroencephalography (EEG) and magnetoencephalography (MEG) is presented.

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Medicine

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease
Martin Wagner 1, Claudia Baer 1, Werner Zuschratter 2, Monika Riek-Burchardt 3, Christian Deffge 1, Soenke Weinert 1, Jerry C Lee 4, Ruediger C Braun-Dullaeus 1, Joerg Herold 1
1Department of Cardiology and Angiology, University of Magdeburg, 2Leibniz Institute for Neurobiology, 3Institute of Molecular and Clinical Immunology, University of Magdeburg, 4Division of Hematology/Oncology, Boston Children's Hospital, Harvard Medical School

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

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Genetics

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes
Christoph N. Schlaffner 1,2,3, Georg J. Pirklbauer 2, Andreas Bender 3, Judith A.J. Steen 1, Jyoti S. Choudhary 2,4
1Department of Neurobiology, F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, 2Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute, Wellcome Genome Campus, 3Centre for Molecular Informatics, Department of Chemistry, University of Cambridge, 4Functional Proteomics Group, Chester Beatty Laboratories, Institute of Cancer Research

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

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Biology

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis
Yohei Tomita 1, Zhuo Shao 2, Bertan Cakir 1, Yumi Kotoda 1, Zhongjie Fu 1,3, Lois E.H. Smith 1
1Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 2Department of Clinical and Metabolic Genetics, Hospital for Sick Children, University of Toronto, 3Manton Center for Orphan Disease, Harvard Medical School, Boston Children's Hospital

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

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Medicine

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability
Kiran Bora *1, Zhongxiao Wang *1, Felix Yemanyi 1, Meenakshi Maurya 1, Alexandra K. Blomfield 1, Yohei Tomita 1, Jing Chen 1
1Department of Ophthalmology, Boston Children’s Hospital, Harvard Medical School

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

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