JoVE Logo

Sign In

University of Heidelberg

21 ARTICLES PUBLISHED IN JoVE

image

Biology

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators
Martin S Angst 1, Martha Tingle 1, Martin Schmelz 2,3, Brendan Carvalho 1, David C Yeomans 1
1Department of Anesthesia, Stanford University School of Medicine, 2Department of Anaesthesiology, University of Mannheim, 3Department of Anaesthesiology, University of Heidelberg

A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

image

Neuroscience

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth
Isabel Brachmann 1, Kerry L. Tucker 1
1Interdisciplinary Center for Neurosciences, Institute of Anatomy and Cell Biology, University of Heidelberg

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

image

Immunology and Infection

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling
Eike Kienle *1, Elena Senís *1, Kathleen Börner 2, Dominik Niopek 1, Ellen Wiedtke 1, Stefanie Grosse 1, Dirk Grimm 1
1Cluster of Excellence CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University, 2Department of Infectious Diseases, Virology, Heidelberg University

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

image

Neuroscience

Analyzing Murine Schwann Cell Development Along Growing Axons
Stephan Heermann 1,2, Kerstin Krieglstein 1,3
1Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg , 2Department of Neuroanatomy, University of Heidelberg, 3FRIAS, University of Freiburg

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

image

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization
Christian Erbel 1, Gregor Rupp 1, Christian M. Helmes 1, Mirjam Tyka 1, Fabian Linden 1, Andreas O. Doesch 1, Hugo A. Katus 1, Christian A. Gleissner 1
1Department of Cardiology, University of Heidelberg

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

image

Medicine

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology
Christian Erbel *1, Deniz Okuyucu *1, Mohammadreza Akhavanpoor 1, Li Zhao 1, Susanne Wangler 1, Maani Hakimi 2, Andreas Doesch 1, Thomas J. Dengler 3, Hugo A. Katus 1, Christian A. Gleissner 1
1Department of Cardiology, University of Heidelberg, 2Department of Vascular Surgery, University of Heidelberg, 3Department of Cardiology, SLK Hospital am Plattenwald

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

image

Chemistry

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry
Nikolai Hentze 1, Matthias P. Mayer 1
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

image

JoVE Journal

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach
Theresa L. M. Pohl 1, Elisabeth H. Schwab 1, Elisabetta A. Cavalcanti-Adam 1,2
1Department of Biophysical Chemistry, Institute for Physical Chemistry, University of Heidelberg, 2Max Planck Institute for Intelligent Systems at Stuttgart

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

image

Biology

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice
Christian Njoo 1, Celine Heinl 1, Rohini Kuner 1
1Institute for Pharmacology, University of Heidelberg

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

image

Behavior

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects
Rabih Moshourab 1,2, Henning Frenzel 2, Stefan Lechner 3, Julia Haseleu 2, Valérie Bégay 2, Damir Omerbašić 2, Gary R. Lewin 2
1Department of Anesthesiology and Intensive Care Medicine, Charite Universitätsmedzin, Campus Virchow Klinikum und Campus Charite Mitte, 2Department of Neuroscience, Molecular Physiology of Somatic Sensation, Max Delbrück Center for Molecular Medicine, 3Institute of Pharmacology, University of Heidelberg

Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man.

image

Developmental Biology

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System
Carlo A. Beretta 1,2,3, Nicolas Dross 2, Ulrike Engel 2, Matthias Carl 1
1Medical Faculty Mannheim, Department of Cell and Molecular Biology, Heidelberg University, 2COS and Nikon Imaging Center at the University of Heidelberg, Heidelberg University, 3Excellenzcluster CellNetworks, University of Heidelberg

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

image

Biology

Production of Human Norovirus Protruding Domains in E. coli for X-ray Crystallography
Mila M. Leuthold 1,2, Anna D. Koromyslova 1,2, Bishal K. Singh 1,2, Grant S. Hansman 1,2
1Schaller Research Group, University of Heidelberg and the German Cancer Research Center, 2Department of Infectious Diseases, Virology, University of Heidelberg

Here, we describe a method to express and purify high quality norovirus protruding (P) domains in E. coli for use in X-ray crystallography studies. This method can be applied to other calicivirus P domains, as well as non-structural proteins, i.e., viral protein genome-linked (VPg), protease, and RNA dependent RNA polymerase (RdRp).

image

Immunology and Infection

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry
Guido Wabnitz 1, Henning Kirchgessner 1, Yvonne Samstag 1
1Institute of Immunology, Section Molecular Immunology, University of Heidelberg

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

image

Biology

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry
Konstantin D. Bergmeister 1,3, Marion Gröger 2, Martin Aman 1, Anna Willensdorfer 1, Krisztina Manzano-Szalai 1, Stefan Salminger 1, Oskar C. Aszmann 1
1CD Laboratory for the Restoration of Extremity Function, Division of Plastic and Reconstructive Surgery, Department of Surgery, Medical University of Vienna, 2Core Facility Imaging, Core Facilities, Medical University Vienna, 3Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery, University of Heidelberg

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

image

Cancer Research

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer
Alexander M. Betzler 1, Susan Kochall 1, Linda Blickensdörfer 2, Sebastian A. Garcia 1, May-Linn Thepkaysone 1, Lahiri K. Nanduri 1, Michael H. Muders 3, Jürgen Weitz 1,4,5, Christoph Reissfelder 1, Sebastian Schölch 1,4,5
1Department of Gastrointestinal, Thoracic and Vascular Surgery, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 2Department of General, Gastrointestinal and Transplant Surgery, University of Heidelberg, 3Department of Pathology, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, 4German Cancer Consortium (DKTK), 5German Cancer Research Center (DKFZ)

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

image

Medicine

Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates
Nora Bruns 1, Susanne Blumenthal 1, Irmgard Meyer 1, Susanne Klose-Verschuur 1, Ursula Felderhoff-Müser 1, Hanna Müller 1,2
1Department of Pediatrics I, Neonatology, Pediatric Intensive Care, Pediatric Neurology, University Hospital Essen, University Duisburg-Essen, 2Division of Neonatology, Department of Pediatrics, University of Heidelberg

Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates.

image

Medicine

How to Administer Near-Infrared Spectroscopy in Critically ill Neonates, Infants, and Children
Nora Bruns 1, Julia Moosmann 2, Frank Münch 3, Christian Dohna-Schwake 1, Joachim Woelfle 4, Robert Cesnjevar 3, Sven Dittrich 2, Ursula Felderhoff-Müser 1, Hanna Müller 4
1Department of Pediatrics I - Neonatology, Pediatric Intensive Care, Pediatric Neurology, University Hospital Essen, University of Duisburg-Essen, 2Department of Pediatric Cardiology, University Hospital Erlangen, University of Erlangen-Nürnberg, 3Department of Pediatric Cardiac Surgery, University Hospital Erlangen, University of Erlangen-Nürnberg, 4Division of Neonatology and Pediatric Intensive Care, Department of Pediatrics, University Hospital Erlangen, University of Erlangen-Nürnberg

This protocol is designed to assist clinicians to measure regional tissue oxygenation at different body sites in infants and children. It can be used in situations where tissue oxygenation is potentially compromised, particularly during cardiopulmonary bypass, when using non-pulsatile cardiac-assist devices, and in critically ill neonates, infants and children.

image

JoVE Core

Cardiac Response to β-Adrenergic Stimulation Determined by Pressure-Volume Loop Analysis
Rebekka Medert *1,2, Lucas Bacmeister *1,2,3, Sebastian Segin *1,2,4, Marc Freichel 1,2, Juan E. Camacho Londoño 1,2
1Pharmakologisches Institut, Ruprecht-Karls-Universität Heidelberg, 2DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim, 3Department of Internal Medicine III, University of Heidelberg, 4Department of Anesthesiology, University Hospital RWTH Aachen

Here we describe a cardiac pressure-volume loop analysis under increasing doses of intravenously infused isoproterenol to determine the intrinsic cardiac function and the β-adrenergic reserve in mice. We use a modified open-chest approach for the pressure-volume loop measurements, in which we include ventilation with positive end-expiratory pressure.

image

Immunology and Infection

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples
Dario L. Frey *1,2, Matteo Guerra *1,2,3,4, Marcus A. Mall 1,2,5,6,7, Carsten Schultz 1,3,8
1Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), 2Dept. of Translational Pulmonology, University of Heidelberg, 3Molecular Medicine Partnership Unit (MMPU), European Molecular Biology Laboratory (EMBL), University of Heidelberg, 4Faculty of Biosciences, Collaboration for Joint Ph.D. Degree between EMBL and Heidelberg University, University of Heidelberg, 5Dept. of Pediatric Pulmonology, Immunology and Critical Care Medicine, Charité – Universitätsmedizin Berlin, 6Berlin Institute of Health (BIH), 7German Center for Lung Research (DZL), Associated Partner Site, Berlin, 8Dept. of Chemical Physiology and Biochemistry, Oregon Health and Science University

The protocols herein described provide a guide to visualize and quantify the activity of neutrophil proteases in human sputum. The applications of such analysis span from the evaluation of anti-inflammatory treatments, to biomarker validation, drug screening and large cohort clinical studies.

image

Bioengineering

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants
Kleopatra Rapti 1,2, Olena Maiakovska 1,2, Jonas Becker 1,2, Joanna Szumska 1,2, Margarita Zayas 1,2, Felix Bubeck 1,2, Jixin Liu 1,2, Emma Gerstmann 1,2, Chiara Krämer 1,2, Ellen Wiedtke 1,2, Dirk Grimm 1,2,3,4,5
1Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, 2BioQuant Center, BQ0030, University of Heidelberg, 3Cluster of Excellence CellNetworks, 4German Center for Infection Research (DZIF), 5German Center for Cardiovascular Research (DZHK)

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

image

Immunology and Infection

Immunofluorescence Imaging of Neutrophil Extracellular Traps in Human and Mouse Tissues
Lavinia Schoenfeld 1, Birgit Appl 1, Laia Pagerols-Raluy 1, Annika Heuer 3, Konrad Reinshagen 1, Michael Boettcher 1,2
1Department of Pediatric Surgery, University Medical Center Hamburg-Eppendorf, University of Hamburg, 2Department of Pediatric Surgery, University Medical Center Mannheim, University of Heidelberg, 3Division of Spine Surgery, Department of Trauma and Orthopedic Surgery, University Medical Center Hamburg-Eppendorf (UKE)

Neutrophil extracellular traps (NETs) are associated with various diseases, and immunofluorescence is often used for their visualization. However, there are various staining protocols, and, in many cases, only one type of tissue is examined. Here, we establish a generally applicable protocol for staining NETs in mouse and human tissue.

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved