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The Hospital for Sick Children

13 ARTICLES PUBLISHED IN JoVE

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Medicine

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model
Meaghan A. O'Reilly 1, Adam C. Waspe 1,2, Rajiv Chopra 1,2, Kullervo Hynynen 1,3
1Imaging Research, Sunnybrook Research Institute, 2Department of Medical Biophysics, University of Toronto, 3Department of Medical Biophysics, and Institute of Biomaterials & Biomedical Engineering (IBBME), University of Toronto

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

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Biology

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations
Eric J. Horstick 1, Elizabeth M. Gibbs 1, Xingli Li 1, Ann E. Davidson 1, James J. Dowling 1
1Departments of Pediatrics and Neurology, University of Michigan

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

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Medicine

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation
J. Moritz Kaths 1,2, Vinzent N. Spetzler 1, Nicolas Goldaracena 1, Juan Echeverri 1, Kristine S. Louis 1, Daniel B. Foltys 3, Mari Strempel 4, Paul Yip 5, Rohan John 5, Istvan Mucsi 1, Anand Ghanekar 1, Darius Bagli 6,7, Lisa Robinson 2, Markus Selzner 1
1Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, 2Division of Nephrology, The Hospital for Sick Children, Toronto, 3Department of General, Visceral & Transplant Surgery, University Medical Center Mainz, 4Department of Abdominal, Vascular & Transplant Surgery, Merheim Medical Center Cologne, 5Laboratory Medicine & Pathobiology, Toronto General Hospital, 6Departments of Surgery (Urology) & Physiology, The Hospital for Sick Children, Toronto, 7Developmental & Stem Cell Biology, The Hospital for Sick Children, Toronto

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to  transplantation.

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Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye
Sarah J. Smith *1,2, Eric J. Horstick *3,4, Ann E. Davidson 1,2, James Dowling 1,2,4
1Program in Genetics & Genome Biology, The Hospital for Sick Children, 2Department of Molecular Genetics, The University of Toronto, 3Program in Genomics of Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 4Departments of Pediatrics and Neurology, University of Michigan

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

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Medicine

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol
J. Moritz Kaths 1,2, Juan Echeverri 1,3, Nicolas Goldaracena 1, Kristine S. Louis 1, Paul Yip 4, Rohan John 4, Istvan Mucsi 5, Anand Ghanekar 1, Darius Bagli 6, Markus Selzner 1, Lisa A. Robinson 2
1Multi Organ Transplant Program, Department of Surgery, Toronto General Hospital, 2Division of Nephrology, The Hospital for Sick Children, 3Programa de Doctorat en Medicina, La Universitat Autónoma de Barcelona, 4Laboratory Medicine and Pathobiology, Toronto General Hospital, 5Department of Medicine, Toronto General Hospital, 6Departments of Surgery (Urology) & Physiology, Developmental & Stem Cell Biology, The Hospital for Sick Children

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

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Developmental Biology

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach
Yun-Ui Bae 1, Hoon-Ki Sung 2,3, Jae-Ryong Kim 1
1Department of Biochemistry and Molecular Biology and Smart-aging Convergence Research Center, College of Medicine, Yeungnam University, 2Physiology and Experimental Medicine Program, The Hospital for Sick Children Research Institute, 3Department of Laboratory Medicine and Pathobiology, University of Toronto

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

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Medicine

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents
Martin Sidler 1,2,3, Karen J. Aitken 1, Jia Xin Jiang 4, Darius J. Bägli 1,2,3
1Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, 2Institute of Medical Science, University of Toronto, 3Pediatric Urology, The Hospital for Sick Children, 4Molecular Structure and Function, Research Institute, The Hospital for Sick Children

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

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Cancer Research

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion
Ian Restall 1, Danielle Anne Bozek 1, Charles Chesnelong 1,2, Samuel Weiss 1, H. Artee Luchman 1
1Hotchkiss Brain Institute, Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, 2Department of Developmental and Stem Cell Biology Program, Arthur and Sonia Labatt Brain Tumor Research Centre, The Hospital for Sick Children

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

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Biology

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining
Yanqing Jiang *1, Joanna Lan-Hing Yeung *1, Ju Hee Lee 1,2, James An 1, Patrick E. Steadman 3, Jae-Ryong Kim 4, Hoon-Ki Sung 1,2,5
1Translational Medicine Program, The Hospital for Sick Children, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Neurosciences & Mental Health Program, The Hospital for Sick Children, 4Department of Biochemistry and Molecular Biology, Smart-Aging Convergence Research Center, College of Medicine, Yeungnam University, 5Banting and Best Diabetes Centre, University of Toronto

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

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Biology

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice
Ri Youn Kim *1,2, Ju Hee Lee *3,4, Yena Oh 1,2, Hoon-Ki Sung 3,4,5, Kyoung-Han Kim 1,2
1University of Ottawa Heart Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa, 3Translational Medicine Program, The Hospital for Sick Children, 4Department of Laboratory Medicine and Pathobiology, University of Toronto, 5Banting and Best Diabetes Centre, University of Toronto

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

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Medicine

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation
Datta Singh Goolaub 1,2, Davide Marini 3,4, Mike Seed 4,5, Christopher K. Macgowan 1,2
1Department of Medical Biophysics, University of Toronto, 2Division of Translational Medicine, The Hospital for Sick Children, 3Labatt Family Heart Centre, The Hospital for Sick Children, 4Department of Pediatrics, University of Toronto, 5Division of Pediatric Cardiology, The Hospital for Sick Children

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.

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Cancer Research

Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model
Claire Wunker 1,2, Karolina Piorkowska 3, Ben Keunen 3, Yael Babichev 2, Suzanne M. Wong 3,4, Maximilian Regenold 5, Michael Dunne 5, Julia Nomikos 1,2, Maryam Siddiqui 6, Samuel Pichardo 6, Warren Foltz 7, Adam C. Waspe 3,8, Justin T. Gerstle 3,9, James M. Drake 1,3,4,10, Rebecca A. Gladdy 1,2,10
1Institute of Medical Science, University of Toronto, 2Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 3The Wilfred and Joyce Posluns Centre for Image-Guided Innovation and Therapeutic Intervention, The Hospital for Sick Children, 4Institute of Biomedical Engineering, University of Toronto, 5Leslie Dan Faculty of Pharmacy, University of Toronto, 6Departments of Radiology and Clinical Neurosciences, University of Calgary, 7Department of Radiation Oncology, University of Toronto, 8Department of Medical Imaging, University of Toronto, 9Department of Pediatric Surgery, University of Toronto, 10Department of Surgery, University of Toronto

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

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Immunology and Infection

Scoring Central Nervous System Inflammation, Demyelination, and Axon Injury in Experimental Autoimmune Encephalomyelitis
Carmen C. Ucciferri 1, Annette Gower 2, Nuria Alvarez-Sanchez 1,2, Heather Whetstone 3, Valeria Ramaglia 1, Jennifer L. Gommerman 1, Koroboshka Brand-Arzamendi 2, Raphael Schneider 2,4, Shannon E Dunn 1,2,5
1Department of Immunology, University of Toronto, 2Keenan Research Centre for Biomedical Science of St. Michael’s Hospital, 3Sickkids Research Institute, The Hospital for Sick Children, 4BARLO MS Centre, St. Michael’s Hospital, 5Women’s College Research Institute, Women’s College Hospital

Experimental autoimmune encephalomyelitis (EAE) serves as an animal model of multiple sclerosis. This article describes an approach for scoring spinal cord inflammation, demyelination, and axonal injury in EAE. Additionally, a method to quantify soluble neurofilament light levels in the mice serum is presented, facilitating the assessment of axonal injury in live mice.

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