University of Calgary View Institution's Website 50 articles published in JoVE Medicine Erosion Identification in Metacarpophalangeal Joints in Rheumatoid Arthritis using High-Resolution Peripheral Quantitative Computed Tomography Yousif Al-Khoury1,2, Stephanie Finzel3, Camille Figueiredo4, Andrew J. Burghardt5, Kathryn S. Stok6, Lai-Shan Tam7, Isaac Cheng7, Justin J. Tse1, Sarah L. Manske1 1Department of Radiology, McCaig Institute for Bone and Joint Health, University of Calgary, 2Department of Biomedical Engineering, University of Calgary, 3University Medical Center Freiburg, 4Department of Rheumatology, University of São Paulo, 5Department of Radiology and Biomedical Imaging, University of California, San Francisco, 6Department of Biomedical Engineering, University of Melbourne, 7Department of Medicine and Therapeutics, The Chinese University of Hong Kong Bone erosions are an important pathological feature of rheumatoid arthritis. The purpose of this work is to introduce a training tool to provide users with guidance on identifying pathological cortical breaks on high resolution peripheral quantitative computed tomography images for erosion analysis. Medicine Biobanking of Human Aqueous and Vitreous Liquid Biopsies for Molecular Analyses Julian Wolf1,2, Teja Chemudupati1,2, Aarushi Kumar1,2, Ditte K. Rasmussen1,2, Karen M. Wai2, Robert T. Chang2, Artis A. Montague2, Peter H. Tang3,4, Alexander G. Bassuk5,6,7, Antoine Dufour8,9, Prithvi Mruthrunjaya2, Vinit B. Mahajan1,2,10 1Molecular Surgery Laboratory, Stanford University, 2Department of Ophthalmology, Byers Eye Institute, Stanford University, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, 4Retina Consultants of Minnesota, 5Department of Pediatrics, University of Iowa, 6Department of Neurology, University of Iowa, 7The Iowa Neuroscience Institute (INI), University of Iowa, 8Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary, 9Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 10Veterans Affairs Palo Alto Health Care System This protocol presents an integrated biorepository platform for the standardized collection, annotation, and biobanking of high-quality human aqueous humor and vitreous liquid biopsies for molecular downstream analyses, including proteomics, metabolomics, and glycomics. Neuroscience Micro-CT Imaging and Morphometric Analysis of Mouse Neonatal Brains Jimena Barbeito-Andrés1, Laura Andrini2, Mariana Vallejo-Azar1, Sara Seguel3, Jay Devine4, Benedikt Hallgrímsson4, Paula Gonzalez1 1Unidad Ejecutora de Estudios en Neurociencias y Sistemas Complejos (ENyS, CONICET-UNAJ-HEC), 2Facultad de Ciencias Médicas, Universidad Nacional de La Plata, 3Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, 4Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary This study describes the steps for obtaining high-resolution images of neonatal mouse brains by combining micro-computed tomography (micro-CT) and a contrast agent in ex vivo samples. We describe basic morphometric analyses to quantify brain size and shape in these images. Cancer Research Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model Claire Wunker1,2, Karolina Piorkowska3, Ben Keunen3, Yael Babichev2, Suzanne M. Wong3,4, Maximilian Regenold5, Michael Dunne5, Julia Nomikos1,2, Maryam Siddiqui6, Samuel Pichardo6, Warren Foltz7, Adam C. Waspe3,8, Justin T. Gerstle3,9, James M. Drake1,3,4,10, Rebecca A. Gladdy1,2,10 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. Medicine Neuronavigation and Laparoscopy Guided Ventriculoperitoneal Shunt Insertion for the Treatment of Hydrocephalus Albert M. Isaacs1, Chad G. Ball2, Mark G. Hamilton1,3 1Division of Neurosurgery, University of Calgary, 2Department of Surgery, University of Calgary, 3Adult Hydrocephalus Program, Department of Clinical Neuroscience, University of Calgary Patient outcomes of ventriculoperitoneal (VP) shunt surgery, the mainstay treatment for hydrocephalus in adults, are poor due to high shunt failure rates. We present intraoperative footage of VP shunt insertion using neuronavigation and laparoscopy guidance, with the goal to reduce the risks of proximal and distal shunt catheter failures, respectively. Medicine Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses Kittiwoot To-on1, Usana Chatturong1, Teerapap Panklai2, Iyapa Palang1, Anjaree Inchan1, Sutthinee Wisutthathum3, Tamkeen Urooj Paracha4, Phapada Apaikawee1, Krongkarn Chootip1 1Department of Physiology, Faculty of Medical Science and Center of Excellence for Innovation in Chemistry, Naresuan University, 2Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Naresuan University, 3Cosmetics and Natural Products Research Center, Faculty of Pharmaceutical Sciences, Naresuan University, 4Snyder Institute for Chronic Diseases, Airways Inflammation Research Group, Department of Physiology and Pharmacology, University of Calgary Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli. Immunology and Infection Intravital Widefield Fluorescence Microscopy of Pulmonary Microcirculation in Experimental Acute Lung Injury Using a Vacuum-Stabilized Imaging System Stefan Hall1, Sufyan Faridi2, Irene Euodia2,3, Sophie Tanner4, Andrew Krzysztof Chojnacki5, Kamala D. Patel6,7, Juan Zhou2,4, Christian Lehmann1,2,4,8 1Department of Physiology and Biophysics, Dalhousie University, 2Department of Microbiology and Immunology, Dalhousie University, 3Department of Neuroscience, Dalhousie University, 4Department of Anesthesia, Pain Management, and Perioperative Medicine, Dalhousie University, 5Live Cell Imaging Center, University of Calgary, 6Department of Physiology and Pharmacology, University of Calgary, 7Department of Biochemistry and Molecular Biology, University of Calgary, 8Department of Pharmacology, Dalhousie University Intravital fluorescence microscopy can be utilized to study leukocyte-endothelial interactions and capillary perfusion in real-time. This protocol describes methods to image and quantify these parameters in the pulmonary microcirculation using a vacuum-stabilized lung imaging system. Biology Operation of Laboratory Photobioreactors with Online Growth Measurements and Customizable Light Regimes Marianne Haines1, Marc Strous1 1Department of Geoscience, University of Calgary This publication describes the design of laboratory photobioreactors (PBRs) with customizable light regimes. The growth of cyanobacteria or microalgae, using bicarbonate as their carbon source, is monitored continuously by measuring volumetric oxygen production. These PBRs facilitate rapid, replicated laboratory growth comparisons with little user intervention during experiments. Medicine An Intact Pericardium Ischemic Rodent Model Ali Fatehi Hassanabad1, Darrell D. Belke1, Jeannine Turnbull1, Jameson A. Dundas1, Vishnu Vasanthan1, Guoqi Teng1, Paul W. M. Fedak1, Justin F. Deniset1,2 1Section of Cardiac Surgery, Department of Cardiac Sciences, Libin Cardiovascular Institute, Cumming School of Medicine, 2Department of Pharmacology and Physiology, University of Calgary This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents. Immunology and Infection Bacteriophage Effectiveness for Biocontrol of Foodborne Pathogens Evaluated via High-Throughput Settings Yan D. Niu1, Jae Eun Hyun1, Nghi Nguyen1 1Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary This protocol describes a robust method for using high-throughput settings to screen the antibacterial efficacy of bacteriophage cocktails. Immunology and Infection Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy Liam Wilkinson1,2, Johnathan Canton2,3 1Department of Biochemistry & Microbiology, Faculty of Science, University of Victoria, 2Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Science, University of Calgary, 3Calvin, Joan and Phoebe Snyder Institute for Chronic Diseases, University of Calgary We describe protocols for measuring pH, oxidative events, and protein digestion in individual macropinosomes in live cells. An emphasis is placed on dual-fluorophore ratiometric microscopy and the advantages it offers over population-based techniques. Neuroscience Analysis of Translation in the Developing Mouse Brain using Polysome Profiling Shreeya Kedia1,3, Sarah L. Erickson2,3, Guang Yang1,2,3 1Department of Biochemistry and Molecular Biology; Cumming School of Medicine, University of Calgary, 2Department of Medical Genetics, Cumming School of Medicine, University of Calgary, 3Alberta Children's Hospital Research Institute, University of Calgary The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain. Cancer Research Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model Konstantin Stoletov1, Lian Willetts2, Perrin H. Beatty1, John D. Lewis1 1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Department of Cell Biology & Anatomy, Cumming School of Medicine, University of Calgary This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing. Medicine A Saline/Bipolar Radiofrequency Energy Device As an Adjunct for Hemostasis in Solid Organ Injury/Trauma Thomas W. Clements1, Chad G. Ball1 1Department of Surgery, University of Calgary The goal of this publication is to demonstrate the potential application of a novel device using simulated solid organ injuries in a porcine model. Immunology and Infection Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice Yujia Lin1,2, Jingna Xue2, Shan Liao2 1Department of General Surgery, The Second Affiliated Hospital of Harbin Medical University, Harbin Medical University, 2Inflammation Research Network, Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary A protocol to block lymph flow by surgical suturing of afferent lymphatic vessels is presented. Neuroscience Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments Leonardo A. Molina1,2, Victorita E. Ivan1, Aaron J. Gruber*1, Artur Luczak*1 1Canadian Center for Behavioural Neuroscience, Department of Neuroscience, University of Lethbridge, 2Clinical Neurosciences, Cumming School of Medicine, University of Calgary This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided. Medicine Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture Sadman Sakib1, Yang Yu3,4, Anna Voigt2, Mark Ungrin2,3,4, Ina Dobrinski1,2 1Biochemistry and Molecular Biology, University of Calgary, 2Comparative Biology and Experimental Medicine, University of Calgary, 3Biomedical Engineering Graduate Program, University of Calgary, 4Alberta Diabetes Institute, University of Alberta Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system. Medicine Isolation of Atrial Myocytes from Adult Mice Hailey J. Jansen1, Robert A. Rose1 1Libin Cardiovascular Institute of Alberta, Department of Cardiac Sciences, Department of Physiology and Pharmacology, Cumming School of Medicine, University of Calgary This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies. Neuroscience Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain Adrianna Giuffre1,2, Lauran Cole1,2,3, Hsing-Ching Kuo4,5, Helen L. Carlson2,4,5, Jeff Grab6, Adam Kirton2,3,4,5,7, Ephrem Zewdie3,4,5 1Department of Neurosciences, University of Calgary, 2Hotchkiss Brain Institute, University of Calgary, 3Cumming School of Medicine, University of Calgary, 4Department of Pediatrics, University of Calgary, 5Alberta Children's Hospital Research Institute, University of Calgary, 6Faculty of Medicine and Dentistry, University of Alberta, 7Department of Clinical Neurosciences, University of Calgary We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children. Medicine Protocol and Guidelines for Point-of-Care Lung Ultrasound in Diagnosing Neonatal Pulmonary Diseases Based on International Expert Consensus Jing Liu1,2, Roberto Copetti3, Erich Sorantin4, Jovan Lovrenski5, Javier Rodriguez-Fanjul6, Dalibor Kurepa7, Xing Feng8, Luigi Cattaross9, Huayan Zhang10,11, Misun Hwang12, Tsu F. Yeh13,14, Yisrael Lipener7, Abhay Lodha15, Jia-Qin Wang16, Hai-Ying Cao2,17, Cai-Bao Hu2,18, Guo-Rong Lyu19, Xin-Ru Qiu1,2, Li-Qun Jia20, Xiao-Man Wang20, Xiao-Ling Ren1,2, Jiu-Ye Guo1,2, Yue-Qiao Gao1,2, Jian-Jun Li1,2, Ying Liu1,2, Wei Fu1,2, Yan Wang21, Zu-Lin Lu1,2, Hua-Wei Wang8, Li-Li Shang22 1Department of Neonatology and NICU, Beijing Chaoyang District Maternal and Child Healthcare Hospital, 2The Neonatal Lung Ultrasound Training Base, Chinese College of Critical Ultrasound, 3Emergency Department, Cattinara University Hospital, 4Division of Pediatric Radiology, Department of Radiology, Medical University Graz, 5Faculty of Medicine, University of Novi Sad, Radiology Department, Institute for Children and Adolescents Health Care of Vojvodina, 6Pediatric Intensive Care Unit, Pediatric Service Hospital Joan XXIII Tarragona, University Rovira i Virgil, 7Department of Neonatology, Children's Hospital of Soochow University, 9Department of Neonatology, Udine University Hospital, 10Division of Neonatology, Children's Hospital of Philadelphia, 12Intensive Care Unit, Zhejiang Hospital, 19Collaborative Innovation Center for Maternal and Infant Health Service Application Technology, Quanzhou Medical College, 20Department of Neonatology and NICU, Tai'an City Central Hospital of Shandong Province, 22Department of Intensive Care Unit, The Second Affiliated Hospital of Heilongjiang University of Chinese Medicine Lung ultrasound is a noninvasive and valuable tool for bedside evaluation of neonatal lung diseases. However, a relative lack of reference standards, protocols and guidelines may limit its application. Here, we aim to develop a standardized neonatal lung ultrasound diagnostic protocol to be used in clinical decision-making. Biology Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues Jo Anne Stratton*1,2,3, Sarthak Sinha*2, Wisoo Shin2, Elodie Labit2, Tak-Ho Chu1,4, Prajay T. Shah2, Rajiv Midha1,4, Jeff Biernaskie1,2,3 1Hotchkiss Brain Institute, University of Calgary, 2Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, 3Alberta Children's Hospital Research Institute, University of Calgary, 4Department of Clinical Neurosciences, Cumming School of Medicine, University of Calgary This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided. Behavior Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies Stefanie Gierszewski1, Derek Baker1,2, Klaus Müller3, Jan-Marco Hütwohl3, Klaus-Dieter Kuhnert3, Klaudia Witte1 1Research Group of Ecology and Behavioral Biology, Institute of Biology, University of Siegen, 2University of Calgary, 3Institute of Real-Time Learning Systems, University of Siegen Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish. Biochemistry Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering Tyler Mrozowich1, Steffane McLennan2, Michael Overduin2,4, Trushar R. Patel1,3,4 1Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 2Department of Biochemistry, University of Alberta, 3Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, 4DiscoveryLab, Faculty of Medicine & Dentistry, University of Alberta Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution. Cancer Research Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion Ian Restall1, Danielle Anne Bozek1, Charles Chesnelong1,2, Samuel Weiss1, H. Artee Luchman1 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. Biochemistry Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs Joseph A. Ross1, Nehal Thakor1,2,3 1Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 2Department of Neuroscience and the Canadian Centre for Behavioral Neuroscience (CCBN), University of Lethbridge, 3Arnie Charbonneau Cancer Institute, University of Calgary Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation. Bioengineering Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System Candice C. Poon1, Vincent Ebacher2, Katherine Liu1, Voon Wee Yong*1,3, John James Patrick Kelly*1 1Department of Clinical Neurosciences, Arnie Charbonneau Cancer Institute, University of Calgary, 2Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, 3Department of Oncology, Hotchkiss Brain Institute, University of Calgary Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences. Cancer Research Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy Kristina J. Allan1,2, Douglas J. Mahoney1,4, Stephen D. Baird1, Charles A. Lefebvre1, David F. Stojdl1,2,3 1 Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally. Immunology and Infection Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material Yo Ching Cheng*1, Samia Hannaoui*1, Theodore Ralph John2, Sandor Dudas3, Stefanie Czub3, Sabine Gilch1 1Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine, University of Calgary, 2Dept. of Molecular Biology, University of Wyoming, 3Canadian Food Inspection Agency, Lethbridge Laboratories Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method. Developmental Biology Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells Celine L. Bauwens*1, Derek Toms*2, Mark Ungrin2 1Institute of Biomaterials and Biomedical Engineering, University of Toronto, 2Department of Comparative Biology and Experimental Medicine, University of Calgary Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions. Biology Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit Heather A. Paul1, Megan C. Hallam2, Raylene A. Reimer1,2 1Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis. 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. Behavior Design and Implementation of an fMRI Study Examining Thought Suppression in Young Women with, and At-risk, for Depression Caitlin L. Carew1, Erica L. Tatham2, Andrea M. Milne1, Glenda M. MacQueen3, Geoffrey B.C. Hall4 1Department of Psychiatry and Behavioural Neurosciences, McMaster University, 2McMaster Integrative Neuroscience Discovery and Study, McMaster University, 3Department of Psychiatry, University of Calgary, 4Department of Psychology, Neuroscience & Behaviour, McMaster University We aim to identify the neural correlates underlying sustained and transient thought suppression, and thought re-emergence in controls, at-risk and depressed individuals. Activation was greatest for controls compared to the at-risk and the depressed group in the dorsolateral prefrontal cortex during thought suppression and anterior cingulate cortex during thought re-emergence. Medicine A Novel Model of Mild Traumatic Brain Injury for Juvenile Rats Richelle Mychasiuk1, Allyson Farran1, Mariana Angoa-Perez2, Denise Briggs2, Donald Kuhn2, Michael J. Esser1 1Alberta Children's Hospital Research Institute, University of Calgary, 2Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine & John D. Dingell VA Medical Center The modified weight-drop technique is an easy, cost-effective procedure used for the induction of mild traumatic brain injury in juvenile rats. This novel technique produces clinically relevant symptomology that will advance the study of mild traumatic brain injury (mTBI) and concussion. Neuroscience An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time Starlyn L. M. Okada1, Nicole S. Stivers1, Peter K. Stys2, David P. Stirling1 1KY Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 2Hotchkiss Brain Institute, Department of Clinical Neurosciences, University of Calgary We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability. Behavior Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism Jacklyn D. Smith1, Jong M. Rho1, Susan A. Masino2, Richelle Mychasiuk1 1Departments of Paediatrics and Clinical Neurosciences, Alberta Children's Hospital Research Institute, University of Calgary Faculty of Medicine, 2Department of Psychology, Neuroscience Program, Trinity College Inchworming is a highly repetitive synchronous digging motion displayed by BTBR T+ Itpr3tf/J (BTBR) mice when placed in a testing cage with sufficient sawdust bedding. The procedure is a modification of the juvenile social interaction protocol and is used here to assess repetitive motor stereotypies relevant to Autism Spectrum Disorder. Bioengineering A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology Dominique Tremblay1, Charles M. Cuerrier1,2, Lukasz Andrzejewski1, Edward R. O'Brien3, Andrew E. Pelling1,4,5 1Centre for Interdisciplinary NanoPhysics, Department of Physics, University of Ottawa, 2University of Ottawa Heart Institue, University of Ottawa, 3Libin Cardiovascular Institute of Alberta, University of Calgary, 4Department of Biology, University of Ottawa, 5Institute for Science, Society and Policy, University of Ottawa We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues. Chemistry A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence Zachary J. Storms1, Elliot Cameron1, Hector de la Hoz Siegler1,2, William C. McCaffrey1 1Department of Chemical and Materials Engineering, University of Alberta, 2Department of Chemical and Petroleum Engineering, University of Calgary A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance. Bioengineering Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates Golsa Razian1, Yang Yu1, Mark Ungrin1 1Department of Comparative Biology & Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates. Immunology and Infection Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus Jo-Ann McClure-Warnier1, John M. Conly1,2,3,4,5, Kunyan Zhang1,2,3,4,5 1Centre for Antimicrobial Resistance, Alberta Health Services / Calgary Laboratory Services / University of Calgary, 2Department of Pathology & Laboratory Medicine, University of Calgary, 3Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 4Department of Medicine, University of Calgary, 5The Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories. Medicine Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns Rangaraj M. Rangayyan1,2, Shantanu Banik1, J.E. Leo Desautels1 1Department of Electrical and Computer Engineering, Schulich School of Engineering, University of Calgary, 2Department of Radiology, University of Calgary We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer. Immunology and Infection Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions Anutosh Ganguly1, Hong Zhang1, Ritu Sharma1, Sean Parsons1, Kamala D. Patel1 1Department of Physiology and Pharmacology, University of Calgary This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible. Neuroscience Wholemount Immunohistochemistry for Revealing Complex Brain Topography Joshua J. White1, Stacey L. Reeber1, Richard Hawkes2, Roy V. Sillitoe1 1Department of Neuroscience, Albert Einstein College of Medicine, Yeshiva University, 2Department of Cell Biology and Anatomy and the Hotchkiss Brain Institute, Faculty of Medicine, University of Calgary Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum. Neuroscience Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips Christophe Py1, Marzia Martina2, Robert Monette2, Tanya Comas2, Mike W. Denhoff1, Collin Luk3, Naweed I. Syed3, Geoff Mealing2 1Institute for Microstructural Sciences, National Research Council of Canada, 2Institute for Biological Sciences, National Research Council of Canada, 3Hotchkiss Brain Institute, University of Calgary We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings. Biology Germ Cell Transplantation and Testis Tissue Xenografting in Mice Lin Tang*1, Jose Rafael Rodriguez-Sosa*1, Ina Dobrinski1 1Department of Comparative Biology and Experimental Medicine, University of Calgary Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice. Biology Procedures for Rat in situ Skeletal Muscle Contractile Properties Brian R. MacIntosh1, Shane P. Esau1, R. John Holash1, Jared R. Fletcher1 1Faculty of Kinesiology, University of Calgary This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle’s contractile response at 37 degrees C with an intact circulation. Neuroscience Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation Rajiv Dixit1, Fuqu Lu2, Robert Cantrup1, Nicole Gruenig1,2, Lisa Marie Langevin1, Deborah M. Kurrasch2, Carol Schuurmans1 1Department of Biochemistry and Molecular Biology, Hotchkiss Brain Institute, Alberta Children’s Hospital Research Institute, University of Calgary, 2Department of Medical Genetics, Alberta Children’s Hospital Research Institute, Hotchkiss Brain Institute, University of Calgary In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina. Biology Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers Curtis C. Hughey1, Dustin S. Hittel1,2, Virginia L. Johnsen2, Jane Shearer1,2 1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry. Neuroscience Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats Aubrey A. Webb1,2, Brendan Kerr3, Tanya Neville3, Sybil Ngan4, Hisham Assem3 1CullenWebb Animal Neurology & Ophthalmology Center, Riverview, NB, 2Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, 3Department of Comparative Biology and Experimental Medicine, University of Calgary, 4Department of Neuroscience, University of Calgary Locomotion is often examined as a behavioural outcome in various models of disease in fields such as neuroscience and orthopedics. This video paper intends to describe a method for collecting ground reaction forces and kinematics from rats during unrestrained locomotion. Medicine Hyperinsulinemic-Euglycemic Clamp in the Conscious Rat Curtis C. Hughey1, Dustin S. Hittel1,2, Virginia L. Johnsen2, Jane Shearer1,2 1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 2Faculty of Kinesiology, University of Calgary The hyperinsulinemic-euglycemic clamp is the "gold standard" for the assessment of insulin action. Insulin is infused at a constant rate stimulating glucose uptake. The amount of exogenous glucose infused to counter this drop is indicative of insulin sensitivity. Here the procedure is performed on a conscious, unrestrained rat. Biology Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain John Jarrell1 1Department of Obstetrics and Gynecology, Faculty of Medicine, University of Calgary A demonstration of the bedside test for cutaneous allodynia and its clinical implications.