Technische Universitat Munchen View Institution's Website 16 articles published in JoVE Immunology and Infection Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model Vicky Garcia-Hernandez1, Philipp-Alexander Neumann2, Stefan Koch3,4, Renae Lyons1, Asma Nusrat1, Charles A. Parkos1 1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation. Genetics Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection Eric K. Hubner1,2, Christian Lechler1, Thomas N. Rösner1, Birgit Kohnke-Ertel1, Roland M. Schmid1, Ursula Ehmer1 1Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 2Department of Pneumology, Center for Medicine, Medical Center University of Freiburg Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver. Biochemistry Modification and Functionalization of the Guanidine Group by Tailor-made Precursors Tobias G. Kapp*1, Maximilian Fottner*1, Horst Kessler1 1Department of Chemistry, Technische Universität München A protocol for the synthesis of alkyl-modified guanidines based on the use of the corresponding precursors is presented. Biology Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis Broder Rühmann1, Jochen Schmid1, Volker Sieber1 1Chair of Chemistry of Biogenic Resources, Technische Universität München We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections. Developmental Biology Imaging Subcellular Structures in the Living Zebrafish Embryo Peter Engerer1, Gabriela Plucinska1,2, Rachel Thong1,3, Laura Trovò1, Dominik Paquet4,5,6, Leanne Godinho1 1Institute of Neuronal Cell Biology, Technische Universität München, 2Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3Faculty of Biology, Ludwig-Maximilians-Universität-München, 4Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München, 5German Center for Neurodegenerative Diseases, 6Laboratory of Brain Development and Repair, The Rockefeller University Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. Biology Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles Harald Lahm*1, Stefanie A. Doppler*1, Martina Dreßen1, Klaudia Adamczyk1, Marcus-André Deutsch1, Hanna Ulrich2, Matthias Schiemann2,3, Rüdiger Lange1,4, Markus Krane1,4 1Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München, 2Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, 3Clinical Cooperation Groups: "Antigen-specific Immunotherapy" and "Immune Monitoring", Helmholtz Center Munich (Neuhererg), Technische Universität München, 4DZHK (German Center for Cardiovascular Research) – Partner site Munich Heart Alliance This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species. Engineering Investigating Single Molecule Adhesion by Atomic Force Spectroscopy Frank W. S. Stetter1,2, Sandra Kienle1,2, Stefanie Krysiak1,2, Thorsten Hugel1,2 1Physik-Department E22a, Technische Universität München, 2IMETUM, Technische Universität München A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided. Medicine Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia Yves Harder1, Daniel Schmauss1, Reto Wettstein2, José T. Egaña1, Fabian Weiss1, Andrea Weinzierl1, Anna Schuldt1, Hans-Günther Machens1, Michael D. Menger3, Farid Rezaeian4 1Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universität München, 2Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital of Basel, 3Institute for Clinical and Experimental Surgery, University of Saarland, 4Division of Plastic and Hand Surgery, University Hospital Zurich The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses. Bioengineering Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time Xosé Deán-Ben1, Thomas Felix Fehm1,2, Daniel Razansky1,2 1Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, 2Faculty of Medicine, Technische Universität München We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice. Bioengineering A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo Thilo L. Schenck1, Myra N. Chávez1, Alexandru P. Condurache2, Ursula Hopfner1, Farid Rezaeian3, Hans-Günther Machens1, José T. Egaña1,4 1Department of Plastic Surgery and Hand Surgery, University Hospital rechts der Isar, Technische Universität München, 2Institute for Signal Processing, University of Lübeck, 3Department of Plastic Surgery and Hand Surgery, University Hospital Zürich, 4FONDAP Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo. Medicine Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay Ihsan Ekin Demir1, Elke Tieftrunk1, Karl-Herbert Schäfer2, Helmut Friess1, Güralp O. Ceyhan1 1Department of Surgery, Klinikum rechts der Isar, Technische Universität München, 2Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level. Medicine Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots Markus T. Berninger1,2, Gabriele Wexel3, Ernst J. Rummeny2, Andreas B. Imhoff1, Martina Anton3, Tobias D. Henning*2,4, Stephan Vogt*1 1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation. Medicine Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model Markus T. Berninger1,2, Gabriele Wexel3, Ernst J. Rummeny2, Andreas B. Imhoff1, Martina Anton3, Tobias D. Henning*2,4, Stephan Vogt*1 1Department of Orthopaedic Sports Medicine, Klinikum rechts der Isar der Technischen Universität München, 2Department of Radiology, Klinikum rechts der Isar der Technischen Universität München, 3Institute of Experimental Oncology and Therapy Research, Klinikum rechts der Isar der Technischen Universität München, 4Department of Radiology, Uniklinik Köln An experimental technique for the treatment of chondral defects in the rabbit's knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method. Biology Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures Vera Wenzel1, Daniela Roedl1, Johannes Ring2, Karima Djabali1 1Department of Dermatology and Institute for Medical Engineering, Technische Universität München, 2Department of Dermatology and Allergology, Technische Universität München We report a method to isolate naïve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog. Neuroscience Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction Monika S. Brill1, Petar Marinković1, Thomas Misgeld1,2,3,4 1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings. Medicine Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology Lucia M.A. Crane1, George Themelis2, K. Tim Buddingh1, Niels J. Harlaar1, Rick G. Pleijhuis1, Athanasios Sarantopoulos2, Ate G.J. van der Zee3, Vasilis Ntziachristos2, Gooitzen M. van Dam1 1Department of Surgery, Division of Surgical Oncology, University Medical Center Groningen, 2Helmholtz Zentrum, Technical University Munich, 3Department of Obstetrics and Gynaecology, University Medical Center Groningen Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.