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20 ARTICLES PUBLISHED IN JoVE

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Biology

Engineering Cell-permeable Protein
Bernhard Münst 1, Christoph Patsch 1, Frank Edenhofer 1
1Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, University of Bonn - Life & Brain Center and Hertie Foundation

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

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Neuroscience

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro
Gabriela Oana Bodea 1, Sandra Blaess 1
1Institute of Reconstructive Neurobiology, University of Bonn

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

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Medicine

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion
Se-Chan Kim 1, Olaf Boehm 1, Rainer Meyer 2, Andreas Hoeft 1, Pascal Knüfermann 1, Georg Baumgarten 1
1Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Germany, 2Institute of Physiology, University of Bonn, Germany

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

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Medicine

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation
Tim O. Vilz 1, Marcus Overhaus 1, Burkhard Stoffels 1, Martin von Websky 1, Joerg C. Kalff 1, Sven Wehner 1
1Department of Surgery, University of Bonn

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

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Medicine

Orthotopic Small Bowel Transplantation in Rats
Koji Kitamura *1,2, Martin W. von Websky *1, Ichiro Ohsawa 1, Azin Jaffari 1, Thomas C. Pech 1, Tim Vilz 1, Sven Wehner 1, Shinji Uemoto 2, Joerg C. Kalff 1, Nico Schaefer 1
1Department of Surgery, University of Bonn, Germany, 2Department of Surgery, Kyoto University Hospital

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

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Neuroscience

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration
Christina Müller 1,2, Stefan Remy 1,2
1NRW Research Group 'Dendritic integration in the CNS', Department of Epileptology, University of Bonn, 2Neuronal Networks Group, Deutsches Zentrum für Neurodegenerative Erkrankungen e.V. (DZNE)

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

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Neuroscience

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells
Sandra Meyer 1,2, Philipp Wörsdörfer 1, Katharina Günther 1, Marc Thier 2,3, Frank Edenhofer 1,2
1Institute of Anatomy and Cell Biology, University of Würzburg, 2Institute of Reconstructive Neurobiology, University of Bonn, 3German Cancer Research Center, Heidelberg

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

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Medicine

A Step by Step Protocol for Subretinal Surgery in Rabbits
Sami Al-Nawaiseh 1, Fabian Thieltges 1, Zengping Liu 1,2, Claudine Strack 1, Ralf Brinken 1, Norbert Braun 3, Marc Wolschendorf 3, Arvydas Maminishkis 5, Nicole Eter 4, Boris V. Stanzel 1,6
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology, National University of Singapore, 3Geuder AG, 4Department of Ophthalmology, University of Münster, 5Section on Epithelial and Retinal Physiology and Disease, National Eye Institute/National Institutes of Health, 6Surgical Retina Department, Singapore National Eye Centre

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

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Medicine

A Model to Simulate Clinically Relevant Hypoxia in Humans
Lars Eichhorn 1, Florian Kessler 1, Volker Böhnert 2, Felix Erdfelder 1, Anja Reckendorf 3, Rainer Meyer 4, Richard K. Ellerkmann 1
1Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Bonn, 2Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, 3Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, 4Institute of Physiology 2, University of Bonn

Hypoxia simulation in humans has usually been performed by inhaling hypoxic gas mixtures. For this study, apneic divers were used to simulate dynamic hypoxia in humans. Additionally, physiological changes in desaturation and re-saturation kinetics were evaluated with non-invasive tools such as Near-Infrared-Spectroscopy (NIRS) and peripheral oxygenation saturation (SpO2).

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Developmental Biology

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes
Alexandra Raulf *1, Nadine Voeltz *1, Daniel Korzus 1, Bernd K. Fleischmann 1, Michael Hesse 1
1Institute of Physiology I, University of Bonn

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

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Developmental Biology

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells
Olivia Krefft *1, Ammar Jabali *1, Vira Iefremova 1, Philipp Koch 1,2,3, Julia Ladewig 1
1Institute of Reconstructive Neurobiology, University of Bonn, 2Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, 3Hector Institute for Translational Brain Research (HITBR gGmbH)

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

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Medicine

Assessment of Plasma Coagulation on Liver Tissue in a Large Animal Model In Vivo
Tim R. Glowka 1, Pascal Paschenda 2, Michael Czaplik 3, Jörg C. Kalff 1, René H. Tolba 2
1Department of Surgery, University of Bonn, 2Institute for Laboratory Animal Science & Experimental Surgery, RWTH Aachen University, 3Department of Anesthesiology, RWTH Aachen University

Here we present a protocol to experimentally assess plasma coagulation in liver tissue in vivo. In a porcine model, microcirculation is examined by laser Doppler, coagulation depth is measured histologically, temperature at coagulation site by infrared thermometer and thermographic camera, and duct sealing effect is documented by burst pressure experiments.

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Medicine

A Closed-chest Model to Induce Transverse Aortic Constriction in Mice
Lars Eichhorn *1, Christina Katharina Weisheit *1, Christopher Gestrich 2, Konrad Peukert 1, Georg Daniel Duerr 2, Muhammad Ajmal Ayub 3, Felix Erdfelder 1, Florian Stöckigt 4
1Department of Anaesthesiology, University Hospital Bonn, 2Department of Cardiac Surgery, University Hospital Bonn, 3Institute of Physiology 2, University of Bonn, 4Department of Medicine - Cardiology, University Hospital Bonn

Here, we present a protocol of Transverse Aortic constriction (TAC) via a lateral thoracotomy. This technique is a minimally invasive, closed chest surgical procedure aiming to simulate pressure overload and heart failure in mice utilizing standard TAC laboratory settings.

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JoVE Journal

Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities
Pascal Heimer *1, Thomas Schmitz *1, Charlotte A. Bäuml *1, Diana Imhof 1
1Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

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Chemistry

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs
Stefanie Lindner *1, Christian Steinebach *2, Hannes Kehm 1, Martin Mangold 2, Michael Gütschow 2, Jan Krönke 1
1Department of Internal Medicine III, University Hospital Ulm, 2Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

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Biochemistry

Extraction and Quantification of Soluble, Radiolabeled Inositol Polyphosphates from Different Plant Species using SAX-HPLC
Philipp Gaugler 1, Verena Gaugler 1, Marília Kamleitner 1, Gabriel Schaaf 1
1Department of Plant Nutrition, Institute of Crop Science and Resource Conservation, University of Bonn

Here we describe strong anion exchange high-performance liquid chromatography of [3H]-myo-inositol-labeled seedlings which is a highly sensitive method to detect and quantify inositol polyphosphates in plants.

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Medicine

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases
Ivan Seah *1,2, Zengping Liu *1,3,4, Daniel Soo Lin Wong 1, Wendy Wong 2, Graham E. Holder 1,2,5, Veluchamy Amutha Barathi 1,4,6, Gopal Lingam 1,2,4, Xinyi Su 1,2,3,4, Boris V. Stanzel 1,7,8
1Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 2Department of Ophthalmology, National University Hospital, Singapore, 3Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 4Singapore Eye Research Institute (SERI), 5UCL Institute of Ophthalmology, 6Academic Clinical Program in Ophthalmology, Duke-NUS Medical School, 7Macula Center Saar, Eye Clinic Sulzbach, Knappschaft Hospital Saar, 8Department of Ophthalmology, University of Bonn

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

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Medicine

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence
Leon von der Emde *1, Merten Mallwitz *1, Frank G. Holz 1, Kenneth R. Sloan 2, Thomas Ach 1
1Department of Ophthalmology, University of Bonn, 2Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

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Biology

Cost-Efficient Transcriptomic-Based Drug Screening
Jacqueline Leidner 1,2, Heidi Theis 3, Michael Kraut 3, Alice Ragogna 1, Marc Beyer 1,3,4, Joachim Schultze 1,2,3, Jonas Schulte-Schrepping 1,2, Caterina Carraro 1,2, Lorenzo Bonaguro 1,2
1Systems Medicine, German Center for Neurodegenerative Diseases (DZNE) eV., 2Genomics & Immunoregulation, LIMES Institute, University of Bonn, 3PRECISE Platform for Single Cell Genomics and Epigenomics, DZNE and University of Bonn, 4Immunogenomics & Neurodegeneration, German Center for Neurodegenerative Diseases (DZNE) eV.

This protocol describes a workflow from ex vivo or in vitro cell cultures to transcriptomic data pre-processing for cost-effective transcriptome-based drug screening.

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Biology

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato
Lamis Osama Anwar Abdelhakim 1, Barbora Pleskačová 1, Natalia Yaneth Rodriguez-Granados 2, Rashmi Sasidharan 2, Lucia Sandra Perez-Borroto 3, Sophia Sonnewald 4, Kristina Gruden 5, Ute C. Vothknecht 6, Markus Teige 7, Klára Panzarová 1
1PSI (Photon Systems Instruments), spol. s r.o. Drasov, 2Plant Stress Resilience, Institute of Environmental Biology, Utrecht University, 3Plant Breeding, Wageningen University and Research, 4Department of Biology, Biochemistry, Friedrich-Alexander Universität Erlangen-Nürnberg, 5Department of Biotechnology and Systems Biology, National Institute of Biology, 6Plant Cell Biology, Institute of Cellular and Molecular Botany, University of Bonn, 7Department of Functional & Evolutionary Ecology, University of Vienna

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

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