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Medical university of innsbruck

16 ARTICLES PUBLISHED IN JoVE

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Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats
Robert Sucher *1, Rupert Oberhuber *1, Christian Margreiter 1, Guido Rumberg 1, Rishi Jindal 2, WP Andrew Lee 2, Raimund Margreiter 1, Johann Pratschke 1, Stefan Schneeberger 1, Gerald Brandacher 1
1Department of Visceral, Transplant, and Thoracic Surgery, Daniel Swarovski Research Laboratory, Innsbruck Medical University, 2Department of Surgery, Division of Plastic and Reconstructive Surgery, University of Pittsburgh Medical Center

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

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Medicine

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique
Rupert Oberhuber *1, Benno Cardini *1, Markus Kofler 1, Paul Ritschl 1, Robert Oellinger 1, Felix Aigner 1, Robert Sucher 1, Stefan Schneeberger 1, Johann Pratschke 1, Gerald Brandacher 2, Manuel Maglione 1
1Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, 2Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.

Herein, we provide additional details of this technique to supplement the video.

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Neuroscience

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala
Sabine Schönherr 1, Anna Seewald 1, Yu Kasugai 1, Daniel Bosch 2, Ingrid Ehrlich 2, Francesco Ferraguti 1
1Department of Pharmacology, Medical University of Innsbruck, 2Hertie Institute for Clinical Brain Research and Centre for Integrative Neuroscience, University of Tübingen

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

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Immunology and Infection

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood
Wilfried Posch 1, Cornelia Lass-Flörl 1, Doris Wilflingseder 1
1Division of Hygiene and Medical Microbiology, Medical University of Innsbruck

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

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Medicine

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique
Benno Cardini *1, Rupert Oberhuber *1, Sven R Hein 1, Rebecca Eiter 1, Martin Hermann 2, Markus Kofler 1,3, Stefan Schneeberger 1, Gerald Brandacher 1,4, Manuel Maglione 1
1Center of Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Medical University Innsbruck, 2Department of Anesthesiology and Critical Care Medicine, Medical University Innsbruck, 3Department of Cardiac Surgery, Medical University Innsbruck, 4Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

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Cancer Research

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples
Maximilian Boesch *1,2,3, Elisabeth Hoflehner 2,3, Dominik Wolf 2,3,4, Guenther Gastl 2, Sieghart Sopper *2,3
1Institute of Immunobiology, Kantonsspital St. Gallen, 2Internal Medicine V, Medical University of Innsbruck, 3Tyrolean Cancer Research Institute (TKFI), 4Medical Clinic III, Oncology, Hematology, Immunoncology and Rheumatology, University Clinic Bonn (UKB)

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

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Developmental Biology

Detection of Residual Donor Erythroid Progenitor Cells after Hematopoietic Stem Cell Transplantation for Patients with Hemoglobinopathies
Roman Crazzolara 1, Gabriele Kropshofer 1, Michael Steurer 2, Sieghart Sopper 2,3, Wolfgang Schwinger 4
1Department of Pediatrics, Medical University Innsbruck, 2Department of Internal Medicine V (Hematology & Oncology), Medical University Innsbruck, 3Tyrolean Cancer Research Institute, 4Department of Pediatrics, Medical University Graz

Quantification of donor-derived cells is required to monitor engraftment after stem cell transplantation in patients with hemoglobinopathies. A combination of flow cytometry-based cell sorting, colony formation assay, and subsequent analysis of short tandem repeats may be used to assess the proliferation and differentiation of progenitors in the erythroid compartment.

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Immunology and Infection

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector
Sarah Wilmschen 1, Zoltan Banki 1, Dorothee von Laer 1, Janine Kimpel 1
1Division of Virology, Department of Hygiene, Microbiology, Social Medicine, Medical University of Innsbruck

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

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Biochemistry

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay
Ingo Bauer 1, Angelo Pidroni 1, Özgür Bayram 2,3, Gerald Brosch 1, Stefan Graessle 1
1Division of Molecular Biology, Biocenter, Medical University of Innsbruck, 2Biology Department, Maynooth University, 3Maynooth University Human Health Research Institute

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

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Biochemistry

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions
Andreas O. Wagner *1, Rudolf Markt *1, Mira Mutschlechner *1, Nina Lackner *1, Eva M. Prem *1, Nadine Praeg 1, Paul Illmer 1
1Department of Microbiology, Universität Innsbruck

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

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Neuroscience

Brain Death Induction in Mice Using Intra-Arterial Blood Pressure Monitoring and Ventilation via Tracheostomy
Paul V. Ritschl *1,2,3, Lena Hofhansel *2,4, Bernhard Flörchinger 5, Rupert Oberhuber 2, Robert Öllinger 1, Johann Pratschke 1, Katja Kotsch 6
1Department of Surgery Campus Charité Mitte/Campus Virchow-Klinikum, Charité - Universitätsmedizin Berlin, 2Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, 3Charité Clinician Scientist Program, Berlin Institute of Health, 4Department of Psychiatry, Psychotherapy and Psychosomatics, RWTH Aachen University, 5Department of Cardiothoracic Surgery, University Medical Center Regensburg, 6Department of General, Visceral and Vascular Surgery, Charité - Universitätsmedizin Berlin

We present a murine model of brain death induction in order to evaluate the influence of its pathophysiological effects on organs as well as on consecutive grafts in the context of solid organ transplantation.

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Chemistry

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials
Andrew J. Chetwynd *1, Wei Zhang *2, Klaus Faserl *3, James A. Thorn 4, Iseult Lynch 1, Rawi Ramautar 2, Herbert H. Lindner 3
1School of Geography Earth and Environmental Sciences, University of Birmingham, 2Biomedical Microscale Analytics, Leiden University, 3Institute of Medical Biochemistry, Medical University of Innsbruck, 4AB Sciex UK Ltd

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis – mass spectrometry approach.

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Medicine

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion
Christoph R. Behem 1, Till Friedheim 1, Sabine H. Wipper 2, Hans O. Pinnschmidt 3, Michael F. Graessler 1, Catharina Gaeth 4, Hannes Holthusen 1, Adina Rapp 5, Timo Suntrop 1, Josephina Haunschild 6, Christian D. Etz 6, Constantin J. C. Trepte 1
1Department of Anesthesiology, Center of Anesthesiology and Intensive Care Medicine, University Medical Center Hamburg-Eppendorf, 2University Department for Vascular Surgery and Department of Operative Medicine, Medical University of Innsbruck, 3Department of Medical Biometry and Epidemiology, University Medical Center Hamburg-Eppendorf, 4Department of Vascular Medicine, University Heart and Vascular Center Hamburg (UHZ), 5Department of Cardiology, Rostock University Medical Center, 6University Department for Cardiac Surgery, Heart Center Leipzig

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

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Environment

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples
Rudolf Markt 1, Mathias Wunderer 1, Eva Maria Prem 1, Mira Mutschlechner 1, Nina Lackner 2, Andreas Otto Wagner 1
1Department of Microbiology, Universität Innsbruck, 2Department of Hygiene and Medical Microbiology, Medical University of Innsbruck

A method for the extraction of cofactor F420 from pure cultures was optimized for the liquid chromatographic separation and analysis of F420 tail length in pure culture and environmental samples.

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Medicine

Transmission Electron Microscopy: A Surgical Pathology Tool for Neuroblastoma
Consolato M. Sergi *1,2,3,4, Janice Patry *1, Harry Coenraad 1, Jeff McClintock 1, Rod Nicholls 1, Hans-Jörg Steiner 5, Gregor Mikuz 5
1Anatomic Pathology Division of EORLA, Children's Hospital of Eastern Ontario, University of Ottawa, 2Department of Laboratory Medicine and Pathology, University of Alberta, 3Department of Pediatrics, University of Alberta, Stollery Children's Hospital, 4Department of Orthopedics, Tianyou Hospital, Wuhan University of Science and Technology, 5Institute of Pathology, Medical University of Innsbruck

Pediatric small round blue cell tumors are an intriguing and challenging collection of neoplasms. Therefore, transmission electron microscopy (TEM) and professional knowledge of pediatric tumors can be extremely valuable in surgical pathology. Here, we present a protocol to perform TEM for diagnosing neuroblastoma, one of the most common solid tumors in childhood.

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Medicine

Electrically Evoked Stapedius Reflex Measurements in Cochlear Implantation and Its Application in the Postoperative Fitting Process
Maria Magdalena Mair 1, Ruth Schröcksnadel 1, Kurt Stephan 2, Josef Seebacher 2, Philipp Zelger 2, Franz Muigg 1, Timo Gottfried 3, Annett Franke-Trieger 4, Joachim Schmutzhard 3
1Department for Hearing, Speech and Voice Disorders, Tirol-Kliniken GmbH, 2Department for Hearing, Speech and Voice Disorders, Medical University of Innsbruck, 3Department of Otorhinolaryngology, Medical University Innsbruck, 4Saxonian Cochlear Implant Centre, Department of Otorhinolaryngology, Head and Neck Surgery, Faculty of Medicine (and University Hospital) Carl Gustav Carus, Technical University of Dresden

The present protocol describes the measurement of the electrically evoked stapedius reflex (eSR) via cochlear implant (CI). Two applications are discussed: intraoperative detection of eSR for verification of the coupling between the cochlear implant and the auditory nerve and postoperative measurement of eSR thresholds (eSRT) for CI fitting.

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