Aarhus University View Institution's Website 30 articles published in JoVE Medicine Semi-Automated Planimetric Quantification of Dental Plaque Using an Intraoral Fluorescence Camera Pernille Dukanovic Rikvold1, Yumi Chokyu Del Rey1, Karina Kambourakis Johnsen1, Sebastian Schlafer1 1Department of Dentistry and Oral Health, Section for Oral Ecology and Caries Control, Aarhus University This study presents a semi-automated digital image analysis procedure for the planimetric quantification of disclosed dental plaque based on images acquired with an intraoral fluorescence camera. The method allows for the rapid and reliable quantification of dental plaque in the research environment. Biology Isolation of Quiescent Stem Cell Populations from Individual Skeletal Muscles Zofija Frimand*1, Shubhangi Das Barman*1, Troels Rønn Kjær1, Ermelinda Porpiglia1, Antoine de Morrée1 1Department of Biomedicine, Aarhus University This protocol describes the isolation of muscle stem cells and fibro-adipogenic progenitors from individual skeletal muscles in mice. The protocol involves single muscle dissection, stem cell isolation by fluorescence activated cell sorting, purity assessment by immunofluorescence staining, and quantitative measurement of S-phase entry by 5-ethynyl-2´-deoxyuridine incorporation assay. Cancer Research Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples Josephine Geertsen Keller1,2, Karol Mizielinski3, Kamilla Vandsø Petersen1,2, Magnus Stougaard2,3,4, Birgitta R. Knudsen1,3, Cinzia Tesauro3 1Department of Molecular Biology and Genetics, Aarhus University, 2Department of Clinical Medicine, Aarhus University, 3VPCIR Biosciences ApS, 4Department of Pathology, Aarhus University Hospital A protocol for the sensitive and quantitative detection of topoisomerase 1 activity using the rolling circle enhanced enzyme activity detection assay is described. The method allows detection of topoisomerase 1 activity from purified components or cell/tissue extracts. This protocol has wide-ranging applications in any field involving detection of enzymatic activity. Biology Deep Vascular Imaging in the Eye with Flow-Enhanced Ultrasound Christian Damsgaard1,2, Henrik Lauridsen3 1Aarhus Institute of Advanced Studies, Aarhus University, 2Zoophysiology, Department of Biology, Aarhus University, 3Department of Clinical Medicine, Aarhus University We present a non-invasive ultrasound technique for generating three-dimensional angiographies in the eye without the use of contrast agents. Behavior Transition of Farm Pigs to Research Pigs using a Designated Checklist followed by Initiation of Clicker Training - a Refinement Initiative Anders F. Thomsen*1, Birgitte S. Kousholt*1 1Department of Clinical Medicine, AUGUST Centre, Aarhus University Refinement of porcine studies is achieved by introducing a standardized checklist and positive reinforcement training using a clicker. This work supports the collection of samples and the conduct of daily chores related to the animals. Medicine Closed Chest Biventricular Pressure-Volume Loop Recordings with Admittance Catheters in a Porcine Model Mads Dam Lyhne1,2, Jacob Gammelgaard Schultz1,2, Simone Juel Dragsbaek1,2, Jacob Valentin Hansen1,2, Christian Schmidt Mortensen1,2, Anders Kramer1,2, Jens Erik Nielsen-Kudsk1,2, Asger Andersen1,2 1Department of Cardiology Research, Aarhus University Hospital, 2Department of Clinical Medicine, Aarhus University Here we present a closed chest approach to admittance-based bi-ventricular pressure-volume loop recordings in pigs with acute right ventricular dysfunction. Behavior A Within-Subject Experimental Design using an Object Location Task in Rats Gülberk Bayraktar*1,2, Kristoffer Højgaard*1,3, Luc Nijssen1,4, Tomonori Takeuchi1,5 1Danish Research Institute of Translational Neuroscience (DANDRITE), Department of Biomedicine, Aarhus University, 2Universitätsklinikum Würzburg, Institut für Klinische Neurobiologie, 3Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, 4Faculty of Science, Radboud University, 5Center for Proteins in Memory - PROMEMO, Danish National Research Foundation, Department of Biomedicine, Aarhus University This protocol provides detailed steps for an object location task with four repetitions using the same cohort of rats. Weak and strong encoding can produce short- and long-term memories. The flexibility of the protocol with repetition can be beneficial for studies involving surgical operations by saving time and labor. Biochemistry Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using χCRAC Stuart W. McKellar1, Ivayla Ivanova1, Robert W. van Nues2, Ross A. Cordiner3, Mehak Chauhan1, Niki Christopoulou1, Will Worboys4, Andrew Langford4, Torben Heick Jensen3, Sander Granneman1 1Centre for Engineering Biology, University of Edinburgh, 2Institute of Cell Biology, University of Edinburgh, 3Department of Molecular Biology and Genetics, Aarhus University, 4UVO3 Ltd. Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps. Immunology and Infection Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection Anna H. F. Andersen1,2, Stine S. F. Nielsen1,2, Rikke Olesen1,2, Katharina Mack1, Frederik Dagnæs-Hansen3, Niels Uldbjerg1,4, Lars Østergaard1,2, Ole S. Søgaard1,2, Paul W. Denton1,2,5, Martin Tolstrup1,2 1Department of Clinical Medicine, Aarhus University, 2Department of Infectious Diseases, Aarhus University Hospital, 3Department of Biomedicine, Aarhus University, 4Department of Gynecology and Obstetrics, Aarhus University Hospital, 5Department of Biology, University of Nebraska at Omaha We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification. Immunology and Infection Monitoring Extracellular pH in Cross-Kingdom Biofilms using Confocal Microscopy Sebastian Schlafer1, Mathilde Frost Kristensen1 1Department of Dentistry and Oral Health, Aarhus University The protocol describes the cultivation of cross-kingdom biofilms consisting of Candida albicans and Streptococcus mutans and presents a confocal microscopy-based method for the monitoring of extracellular pH inside these biofilms. Chemistry Luminescence Lifetime Imaging of O2 with a Frequency-Domain-Based Camera System Maria Moßhammer*1, Vincent V. Scholz*2, Gerhard Holst3, Michael Kühl1,4, Klaus Koren5 1Marine Biological Section, Department of Biology, University of Copenhagen, 2Center for Electromicrobiology, Aarhus University, 3PCO AG, 4Climate Change Cluster, University of Technology Sydney, 5Aarhus University Centre for Water Technology, Section for Microbiology, Department of Bioscience, Aarhus University We describe the use of a novel, frequency-domain luminescence lifetime camera for mapping 2D O2 distributions with optical sensor foils. The camera system and image analysis procedures are described along with the preparation, calibration and application of sensor foils for visualizing the O2 microenvironment in the rhizosphere of aquatic plants. Medicine Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl Mathias Møler Thygesen1,2, Fredrik Guldbæk-Svensson1, Mikkel Mylius Rasmussen2, Henrik Lauridsen1 1Department of Clinical Medicine, Aarhus University, 2Department of Neurosurgery, Aarhus University Hospital This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum). Behavior Transcranial Direct Current Stimulation (tDCS) of Wernicke's and Broca's Areas in Studies of Language Learning and Word Acquisition Evgeny Blagovechtchenski1, Daria Gnedykh1, Diana Kurmakaeva1, Nadezhda Mkrtychian1, Svetlana Kostromina1, Yury Shtyrov1,2 1Laboratory of Behavioural Neurodynamics, St. Petersburg State University, 2Center of Functionally Integrative Neuroscience (CFIN), Department of Clinical Medicine, Aarhus University Here, we describe a protocol for using transcranial direct current stimulation for psycho- and neurolinguistic experiments aimed at studying, in a naturalistic yet fully controlled way, the role of cortical areas of the human brain in word learning, and a comprehensive set of behavioral procedures for assessing the outcomes. Behavior Spotlighting Customers' Visual Attention at the Stock, Shelf and Store Levels with the 3S Model Tobias Otterbring1,2, Erik Wästlund2, Poja Shams3 1Department of Management/MAPP, Aarhus University, 2Department of Psychology & CTF, Service Research Center, Karlstad University, 3Department of Business Administration & CTF, Karlstad University This article presents a new conceptualization of the in-store search process, the 3S Model, which captures customers’ visual attention at three distinct levels of analysis: Stock, Shelf, and Store. We illustrate the usefulness of our conceptualization through three eye-tracking studies, one from each level of analysis in the 3S Model. Immunology and Infection Interrogating Individual Autoreactive Germinal Centers by Photoactivation in a Mixed Chimeric Model of Autoimmunity Thomas R. Wittenborn1, Cecilia Hagert1, Søren E Degn1 1Department of Biomedicine, Aarhus University This protocol describes the generation of mixed murine bone marrow chimeras with spontaneous autoimmune germinal centers, in which autoreactive lymphocytes carry a photoactivatable green fluorescent protein (PA-GFP) reporter. This provides the ability to link cellular location in tissues with downstream molecular and functional analyses. Genetics Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells Yan Yan*1, Chi-Chih Chang*1, Morten T. Venø1,2, Colin R. Mothershead1, Junyi Su1, Jørgen Kjems1,2 1The Interdisciplinary Nanoscience Centre, Aarhus University, 2Department of Molecular Biology and Genetics, Aarhus University This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs. Medicine 2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum) Anita Dittrich1, Mathias Møller Thygesen1, Henrik Lauridsen1 1Department of Clinical Medicine, Aarhus University Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution. Genetics Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation Rikke H. Kofoed1, Cristine Betzer1, Søren Lykke-Andersen2, Ewa Molska2, Poul H. Jensen1 1DANDRITE - Danish Research Institute of Translational Neuroscience, Dept. of Biomedicine, Aarhus University, 2Dept. of Molecular Biology and Genetics, Aarhus University This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction. Cancer Research Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration Maria Riedel1, Martin F. Berthelsen1, Latifa Bakiri2, Erwin F. Wagner2, Martin K. Thomsen1 1Department of Clinical Medicine, Aarhus University, 2GDD, Cancer Cell Biology Program, National Cancer Research Center (CNIO) This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer. Neuroscience An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice Nastasia K-H Lim1,2, Visse Moestrup3,4, Xiao Zhang1, Wen-An Wang5, Arne Møller3,4,6, Fu-De Huang1,4 1Shanghai Advanced Research Institute, University of Chinese Academy of Sciences, Chinese Academy of Science, 2Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences, Chinese Academy of Science, 3Center of Functionally Integrative Neuroscience, Department of Clinical Medicine, Aarhus University, 4Sino-Danish Center for Education and Research (SDC), 5Department of Neurology, Xinhua Hospital Chongming Branch Affiliated to Shanghai Jiao Tong University School of Medicine, 6Department of Nuclear Medicine and PET-centre, Aarhus University Hospital This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord. Bioengineering Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition Jeremy J. Flint1,2, Kannan Menon2,3, Brian Hansen4, John Forder2,3,5, Stephen J. Blackband1,2,6 1Department of Neuroscience, University of Florida, 2McKnight Brain Institute, University of Florida, 3Department of Biomedical Engineering, University of Florida, 4Center for Functionally Integrative Neuroscience, Aarhus University, 5Department of Radiology, University of Florida, 6National High Magnetic Field Laboratory, Florida State University The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data. Medicine Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells Simone S. E. Nielsen1, Piotr Siupka1, Ana Georgian2, Jane E. Preston2, Andrea E. Tóth1, Siti R. Yusof2,3, N. Joan Abbott2, Morten S. Nielsen1 1Lundbeck Foundation Research Initiative on Brain Barriers and Drug Delivery, Department of Biomedicine, Aarhus University, 2 The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery. Medicine Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model Travis K. Mann-Gow*1, Troy R. Larson*1, Chrissie T. Wøien2, Thomas M. Andersen2, Karl-Erik Andersson3,4, Peter Zvara1,2 1Department of Surgery, University of Vermont, 2Department of Urology and Biomedical Laboratory, University of Southern Denmark, 3Wake Forest Institute for Regenerative Medicine, Wake Forest University, 4Institute for Clinical Medicine, Aarhus University This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization. Neuroscience Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents Mette Richner1, Sara B. Jager1, Piotr Siupka1, Christian B. Vaegter1 1Department of Biomedicine, Aarhus University Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage. Biochemistry SorLA and CLC:CLF-1-dependent Downregulation of CNTFRα as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry Jakob V. Larsen1, Claus M. Petersen1 1Department of Biomedicine, Aarhus University The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-α (CNTFRα), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA. Chemistry X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects Robert E. Dinnebier1, Andrea Fischer2, Gerhard Eggert2, Tomče Runčevski1, Nanna Wahlberg3 1Max-Planck-Institute for Solid State Research, 2State Academy of Art and Design Stuttgart, 3Center for Materials Crystallography, Department of Chemistry and iNANO, Aarhus University Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects. Immunology and Infection Ratiometric Imaging of Extracellular pH in Dental Biofilms Sebastian Schlafer1, Irene Dige1 1Department of Dentistry, Aarhus University A pH-sensitive ratiometric dye is used in combination with confocal laser scanning microscopy and digital image analysis to monitor extracellular pH in dental biofilms in real-time. Biology Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy Eva C. Arnspang1, Jennifer S. Koffman1, Saw Marlar1, Paul W. Wiseman2, Lene N. Nejsum1 1Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, 2Departments of Chemistry and Physics, McGill University This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells. Medicine The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice Mette Richner1, Ole J. Bjerrum2, Anders Nykjaer1, Christian B. Vaegter1 1The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, 2Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments. Medicine Basic Surgical Techniques in the Göttingen Minipig: Intubation, Bladder Catheterization, Femoral Vessel Catheterization, and Transcardial Perfusion Kaare S. Ettrup1,2, Andreas N. Glud2, Dariusz Orlowski2, Lise M. Fitting1, Kaare Meier1, Jens Christian Soerensen1, Carsten R. Bjarkam1,2, Aage K. Olsen Alstrup3 1Department of Neurosurgery, Aarhus University Hospital, 2Department of Neurobiology, Institute of Anatomy, Faculty of Health Sciences, Aarhus University, 3Positron Emission Tomography (PET) Centre, Aarhus University Hospital The use of domestic and miniature pigs in science has increased significantly in recent years. By demonstrating how to perform intubation, transurethral bladder catheterization, femoral artery and vein catheterization, as well as transcardial perfusion, we aim to further increase the value of Göttingen minipigs in biomedical research.