University Health Network (UHN) View Institution's Website 31 articles published in JoVE Medicine Murine Intrapulmonary Tracheal Transplantation: A Model for Investigating Obliterative Airway Disease After Lung Transplantation Yamato Suzuki1,2, Stephen Juvet1,2, Mingyao Liu1,2, Shaf Keshavjee1,2 1Latner Thoracic Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Temerty Faculty of Medicine, University of Toronto The murine intrapulmonary tracheal transplantation (IPTT) model is valuable for studying obliterative airway disease (OAD) after lung transplantation. It offers insights into lung-specific immunological and angiogenic behavior in airway obliteration after allotransplantation with high reproducibility. Here, we describe the IPTT procedure and its expected results. Medicine Measuring Diaphragm Thickness and Function Using Point-of-Care Ultrasound Catherine A. Bellissimo1, Idunn S. Morris2,3,4, Jenna Wong1, Ewan C. Goligher1,2,3,5 1Toronto General Hospital Research Institute, 2Interdepartmental Division of Critical Care Medicine, University of Toronto, 3Department of Physiology, Faculty of Medicine, University of Toronto, 4Deparatment of Intensive Care Medicine, Nepean Hospital, 5Division of Respirology, Department of Medicine, University Health Network Diaphragm thickness and function can be assessed in healthy individuals and critically ill patients using point-of-care ultrasound. This technique offers an accurate, reproducible, feasible, and well-tolerated method for evaluating diaphragm structure and function. Immunology and Infection Culturing Lymphocytes in Simulated Microgravity Using a Rotary Cell Culture System Marieke de Korte1,2, Armand Keating1,2,4, Chen Wang1,3 1Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, 2Krembil Research Institute, Toronto Western Hospital, University Health Network, 3Pathology and Lab Medicine, Hematopathology, Mount Sinai Hospital, Sinai Health Systems, 4Medical Oncology and Hematology, Princess Margaret Cancer Centre This is a step-by-step guide for using a commercially available rotary cell culture system to culture lymphocytes in simulated microgravity using specialized disposable culture vessels. This culturing method may be applied to any suspension-type cell culture. Bioengineering Procurement and Perfusion-Decellularization of Porcine Vascularized Flaps in a Customized Perfusion Bioreactor Michael S. Xu1, Golnaz Karoubi1,2, Thomas K. Waddell1,3, Siba Haykal1,4 1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Institute of Laboratory Medicine and Pathobiology, University of Toronto, 3Division of Thoracic Surgery, Department of Surgery, University of Toronto, 4Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Toronto The protocol describes the surgical procurement and subsequent decellularization of vascularized porcine flaps by the perfusion of sodium dodecyl sulfate detergent through the flap vasculature in a customized perfusion bioreactor. Neuroscience Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis Darren M. O'Hara1,2, Minesh Kapadia1,2, Susan Ping1,2, Suneil K. Kalia1,2,3, Lorraine V. Kalia1,2,4,5,6 1Krembil Research Institute, Toronto Western Hospital, University Health Network, 2Department of Laboratory Medicine and Pathobiology, University of Toronto, 3Department of Surgery, Division of Neurosurgery, University of Toronto, 4Department of Medicine, Division of Neurology, University of Toronto, 5Department of Medicine, Division of Neurology, Edmond J. Safra Program in Parkinson's Disease and the Morton and Gloria Shulman Movement Disorders Clinic, Toronto Western Hospital, University Health Network, 6Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta. Bioengineering Capturing Representative Hand Use at Home Using Egocentric Video in Individuals with Upper Limb Impairment Meng-Fen Tsai1,3, Andrea Bandini3, Rosalie H. Wang2,3,5, José Zariffa1,3,4,5 1Institute of Biomedical Engineering, University of Toronto, 2Department of Occupational Science and Occupational Therapy, University of Toronto, 3KITE, Toronto Rehabilitation Institute, University Health Network, 4Edward S. Rogers Sr. Department of Electrical and Computer Engineering, University of Toronto, 5Rehabilitation Sciences Institute, University of Toronto A protocol is proposed to capture natural hand function of individuals with hand impairments during their daily routines using an egocentric camera. The goal of the protocol is to ensure that the recordings are representative of an individual's typical hand use during activities of daily living at home. Medicine Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation Xu-Chun Chen*1, Manmeet Sekhon*1,2, Xue-Zhong Ma*1, Justin Manuel1, Sai Chung1,2, Eddie He1, Agata Bartczak1, Sandra Fischer3, Cornelia Thoeni3, Graziano Oldani1, Catia T. Perciani1, Sonya MacParland1,2,3, Ian McGilvray1 1Multi-Organ Transplant Program, University Health Network, 2Department of Immunology, University of Toronto, 3Department of Laboratory Medicine and Pathobiology, University of Toronto The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver's common hepatic artery to the recipient liver's proper hepatic artery. Immunology and Infection Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils Jeffrey W. Chadwick1,2, Michael Glogauer1,2 1Department of Dental Oncology and Maxillofacial Prosthetics, Princess Margaret Cancer Centre, University Health Network, 2Faculty of Dentistry, University of Toronto This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described. Cancer Research An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells Lauren Philp1,2, Harley Chan3, Marjan Rouzbahman4, Ariana Rostami5, Lili Ding6, Scott V. Bratman5,6, Margarete K. Akens5,7,8, Jonathan C. Irish7,9,11, Marcus Q. Bernardini10, Gang Zheng5,11 1Institute of Medical Science, University of Toronto, 2Department of Obstetrics and Gynecology, University of Toronto, 3Guided Therapeutics Laboratory, TECHNA Institute, University Health Network, 4Department of Pathology, Princess Margaret Cancer Center, University Health Network, 5Department of Medical Biophysics, University of Toronto, 6Princess Margaret Cancer Center, University Health Network, 7Techna Institute, University Health Network, 8Department of Surgery, University of Toronto, 9Department of Otolaryngology, Head and Neck Surgery, University of Toronto, 10Division of Gynecologic Oncology, University of Toronto /Princess Margaret Cancer Center, University Health Network, 11Princess Margaret Cancer Center, University Health Network This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases. Medicine Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch Yaguang Li1, Junhong Liu2,3, Gaixia Liu1, Zhenhong Pan2,3, Mingxia Zhang1, Yao Ma4, Qingxia Wei1,5, Hongping Xia1,6, Rui Xue Zhang1,2, Junjun She1 1 The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease. Medicine Pre-clinical Model of Cardiac Donation after Circulatory Death Henry Aceros*1, Leyla Joulali*2, Mélanie Borie1, Roberto Vanin Pinto Ribeiro3, Mitesh Vallabh Badiwala3, Shant Der Sarkissian1,4, Nicolas Noiseux1,4 1 This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death. Medicine A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation Roberto V. P. Ribeiro1,2, Juglans S. Alvarez1, Frank Yu1, Mitchell B. Adamson1,2, Naoto Fukunaga1, Cyril Serrick3, Ved Bissoondath1, Massimiliano Meineri4,5, Mitesh V. Badiwala1,6, Vivek Rao1,2,6 1Division of Cardiovascular Surgery, Peter Munk Cardiac Center, Toronto General Hospital, University Health Network, 2Institute of Medical Science, University of Toronto, 3Perfusion and Anesthesia Services, Toronto General Hospital, University Health Network, 4Department of Anesthesia and Pain Management, Toronto General Hospital, University Health Network, 5Department of Anesthesia, University of Toronto, 6Department of Surgery, Faculty of Medicine, University of Toronto Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies. Bioengineering Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles Hisato Konoeda1, Hong Yang2, Chengliang Yang1, Annette Gower1, Chun Xu1, Wei Zhang1, Mingyao Liu1,3 1Latner Thoracic Surgery Research Laboratories, Toronto General Hospital Research Institute, University Health Network, 2Respiratory Medicine Research Laboratory, Institute of Translation Medicine, Shanghai General Hospital, Shanghai Jiaotong University, 3Institute of Medical Science, Faculty of Medicine, University of Toronto We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly. Immunology and Infection Isolation and Identification of Extravascular Immune Cells of the Heart Laura Aronoff1,2, Slava Epelman1,2,3,4,5, Xavier Clemente-Casares1,2 1Toronto General Hospital Research Institute, University Health Network (UHN), 2Dept of Laboratory Medicine and Pathobiology, University of Toronto, 3Dept of Immunology, University of Toronto, 4Peter Munk Cardiac Centre, 5Ted Rogers Centre for Heart Research This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry. Cancer Research Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice Rui Xue Zhang1, Tian Zhang1, King Chen1, Ji Cheng1, Paris Lai1, Andrew M. Rauth2, K. Sandy Pang1, Xiao Yu Wu1 1Department of Pharmaceutical Sciences, University of Toronto, 2Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute, University Health Network This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination. Medicine Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid Shaun Pacheco1, Shan-Yu Fung2, Mingyao Liu1,3 1Latner Thoracic Surgery Research Laboratories, University Health Network, 2 This protocol describes a clinically-applicable means of dissolving hydrophobic compounds in an aqueous environment using combinations of self-assembling peptide and amino acid solutions. Our method resolves a major limitation of hydrophobic therapeutics, which lack safe, efficient means of solubility and delivery methods into clinical settings. Developmental Biology Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation Xuetao Sun1, Sara S. Nunes1,2,3,4 1Toronto General Research Institute, University Health Network, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto, 4Laboratory of Medicine and Pathobiology, University of Toronto The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform. Immunology and Infection Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry Stephen C. Juvet1, Sajad Moshkelgosha2, Sharon Sanderson3, Joanna Hester4, Kathryn J. Wood4, Andrew Bushell4 1Division of Respirology, Departments of Medicine and Immunology, Toronto Lung Transplant Program, Multiorgan Transplant Program, Toronto General Research Institute, University of Toronto and University Health Network, 2Latner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, 3National Institutes of Health Research, Oxford Biomedical Research Centre, Translational Immunology Laboratory, NDORMS, Kennedy Institute of Rheumatology, University of Oxford, 4Transplantation Research Immunology Group, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry. Developmental Biology Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors Emily C. McGaugh1,2,3, M. Cristina Nostro1,2,3 1Toronto General Research Institute, University Health Network, 2McEwen Centre for Regenerative Medicine, University Health Network, 3Department of Physiology, University of Toronto Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines. Immunology and Infection Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-γ Responses Jeeyoon Jennifer Ahn*1, Thirumahal Selvanantham*1, Monan Angela Zhang*1, Thierry Mallevaey1, Shannon E. Dunn1,2,3 1Department of Immunology, University of Toronto, 2Toronto General Research Institute, University Health Network, 3 This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen. Immunology and Infection Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain Tingxi Guo1,2, Toshiki Ochi*2, Munehide Nakatsugawa*2, Yuki Kagoya2, Mark Anczurowski1,2, Chung-Hsi Wang1,2, Muhammed A. Rahman2, Kayoko Saso2, Marcus O. Butler1,2, Naoto Hirano1,2 1Department of Immunology, University of Toronto, 2Princess Margaret Cancer Centre, University Health Network Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCRα or TCRβ of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity. Immunology and Infection Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies Andrzej Chruscinski1, Flora Y. Y. Huang1, Antigona Ulndreaj2, Conan Chua1, Michael Fehlings3, Vivek Rao4, Heather J. Ross1, Gary A. Levy1 1Multi-Organ Transplant Program, University Health Network, 2Institute of Medical Science, University of Toronto, 3Divison of Neurosurgery, University Health Network, 4Division of Cardiac Surgery, University Health Network We describe here a method to generate customizable antigen microarrays that can be used for the simultaneous detection of serum IgG and IgM autoantibodies from humans and mice. These arrays allow for high-throughput and quantitative detection of antibodies against any antigens or epitopes of interest. Medicine Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors Shawn Stapleton1,2,3, Daniel Mirmilshteyn2, Jinzi Zheng3,4, Christine Allen2,4,5, David A. Jaffray1,2,3,4,5,6 1Department of Medical Biophysics, University of Toronto, 2Leslie Dan Faculty of Pharmacy, University of Toronto, 3STTARR Innovation Centre, Princess Margaret Cancer Centre, 4Institute of Biomaterials and Biomedical Engineering, University of Toronto, 5Techna Institute, University Health Network, 6Radiation Medicine Program, Princess Margaret Cancer Centre The heterogeneous intra-tumoral accumulation of liposomes has been linked to an abnormal tumor microenvironment. Herein methods are presented to measure tumor microcirculation by perfusion imaging and elevated interstitial fluid pressure (IFP) using an image-guided robotic system. Measurements are compared to the intra-tumoral accumulation of liposomes, determined using volumetric micro-CT imaging. Immunology and Infection Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow Yonghong Chen*1, Azusa Maeda*1,2, Jiachuan Bu1, Ralph DaCosta1,2,3 1Princess Margaret Cancer Centre, 2Department of Medical Biophysics, University of Toronto, 3Techna Institute, University Health Network The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time. Bioengineering Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance Yannan N. Dou1, Robert A. Weersink2,5, Warren D. Foltz5,6, Jinzi Zheng5,6, Naz Chaudary7, David A. Jaffray2,3,4,5,6, Christine Allen1 1Leslie Dan Faculty of Pharmacy, University of Toronto, 2Department of Radiation Oncology, University of Toronto, 3Medical Biophysics, University of Toronto, 4Institute of Biomaterials & Biomedical Engineering, University of Toronto, 5Techna Institute and Radiation Medicine Program, Princess Margaret Cancer Center, University Health Network, 6STTARR Innovation Center, University Health Network, 7Ontario Cancer Institute, University Health Network AN MRI-compatible custom-designed laser-based heating apparatus has been developed to provide local heating of subcutaneous tumors in order to activate release of agents from thermosensitive liposomes specifically at the tumor region. Medicine In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose Venkatesh Krishnan1, Robert Clark1, Marina Chekmareva2, Amy Johnson1, Sophia George3, Patricia Shaw4, Victoria Seewaldt4,5, Carrie Rinker-Schaeffer1 1Section of Urology, Department of Surgery, The University of Chicago, 2Department of Pathology, Robert Wood Johnson Medical School, 3Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, 4Department of Laboratory Medicine and Pathobiology, Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University of Toronto, University Health Network, 5Departments of Medicine, Pharmacology, and Cancer Biology, Duke University Medical Center We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose. Immunology and Infection An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection Constance Finney1, Lena Serghides2 1Department of Biological Sciences, University of Calgary, 2Toronto General Research Institute, University Health Network Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection. Medicine MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder Katharine Dunlop1, Pauline Gaprielian6, Daniel Blumberger5,7, Zafiris J. Daskalakis5,7, Sidney H. Kennedy2,3,5, Peter Giacobbe2,3,5, Jonathan Downar2,3,4,5 1Institute of Medical Sciences, University of Toronto, 2MRI-Guided rTMS Clinic, University Health Network, 3Department of Psychiatry, University Health Network, 4Toronto Western Research Institute, University Health Network, 5Department of Psychiatry, University of Toronto, 6Faculty of Arts and Science, University of Toronto, 7Temerty Centre for Therapeutic Brain Intervention, Centre for Addiction and Mental Health Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder. Medicine Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury Klaus Zweckberger1, Yang Liu1, Jian Wang1, Nicole Forgione1, Michael G. Fehlings1,2,3 1Department of Genetics and Development, Toronto Western Research Institute and Spinal Program, University Health Network, Krembil Neuroscience Centre, 2Department of Surgery, University of Toronto, 3Institute of Medical Sciences, University of Toronto Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established. Medicine Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice Sharif U. Ahmed1, Murtuza Zair1, Kui Chen1, Matthew Iu1, Feng He1, Oyedele Adeyi2, Sean P. Cleary3, Anand Ghanekar1,3 1Toronto General Research Institute, University Health Network, 2Department of Pathology, University Health Network, 3Division of General Surgery, University Health Network Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment. Immunology and Infection Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry Christina Farr1, Stuart Berger1,2 1Immunology, University of Toronto, 2Arthritis and Immune Disorder Research Centre, University Health Network (UHN) This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.
