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KU Leuven

32 ARTICLES PUBLISHED IN JoVE

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Medicine

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation
Pieter Uvin 1,2,3, Wouter Everaerts 1,2,3, Silvia Pinto 2,3, Yeranddy A. Alpízar 2,3, Mathieu Boudes 1,2,3, Thomas Gevaert 1,2,3, Thomas Voets 2,3, Bernd Nilius 2,3, Karel Talavera 2,3, Dirk De Ridder 1,3
1Laboratory of Experimental Urology, Department of Development and Regeneration, KU Leuven, Belgium, 2Laboratory for Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Belgium, 3TRP Research Platform Leuven (TRPLe), KU Leuven, Belgium

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

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Medicine

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model
Marianne S. Carlon *1, Jaan Toelen *2, Marina Mori da Cunha 2, Dragana Vidović 1, Anke Van der Perren 3, Steffi Mayer 2, Lourenço Sbragia 2, Johan Nuyts 4, Uwe Himmelreich 5, Zeger Debyser 1, Jan Deprest 2
1Molecular Virology and Gene Therapy, KU Leuven, 2Department of Woman and Child, KU Leuven, 3Neurobiology and Gene Therapy, KU Leuven, 4Division of Nuclear Medicine, KU Leuven, 5Biomedical NMR Unit/ MoSAIC, KU Leuven

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

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Medicine

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction
Ryan Hodges 1,2, Masayuki Endo 1, Andre La Gerche 3, Elisenda Eixarch 4,5, Philip DeKoninck 1, Vessilina Ferferieva 3, Jan D'hooge 3, Euan M. Wallace 2, Jan Deprest 1
1Division Woman and Child, Department Women, University Hospitals Leuven, 2The Ritchie Centre, Monash Institute of Medical Research, Department of Obstetrics and Gynaecology, Monash University, Victoria, Australia, 3Department of Cardiovascular Sciences, Katholieke Universiteit Leuven, 4Fetal and Perinatal Medicine Research Group, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), 5Maternal-Fetal Medicine Department, ICGON, Hospital Clínic, Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

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Biology

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells
Catheleyne D'hondt 1, Bernard Himpens 1, Geert Bultynck 1
1Department of Cellular and Molecular Medicine, Laboratory of Molecular and Cellular Signaling, KU Leuven

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

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Education

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate
Jean-Marc Taymans 1, Fangye Gao 1, Veerle Baekelandt 1
1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven and Leuven Institute for Neuroscience and Disease (LIND)

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

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Education

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Jelle Caers 1, Katleen Peymen 1, Nick Suetens 1, Liesbet Temmerman 1, Tom Janssen 1, Liliane Schoofs 1, Isabel Beets 1
1Department of Biology, KU Leuven

The here described fluorescence-based calcium mobilization assay is a medium-throughput reverse pharmacology screening system for the identification of functionally activating ligand(s) of orphan G protein-coupled receptors (GPCRs).

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Medicine

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure
Ilayaraja Muthuramu 1, Marleen Lox 1, Frank Jacobs 1, Bart De Geest 1
1Department of Cardiovascular Sciences, Centre for Molecular and Vascular Biology, Catholic University of Leuven

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

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Medicine

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging
Soňa Kucharíková 1, Greetje Vande Velde 2, Uwe Himmelreich 2, Patrick Van Dijck 1
1Department of Molecular Microbiology, Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, VIB, KU Leuven, 2Biomedical MRI Unit/ MoSAIC, Department of Imaging & Pathology, KU Leuven

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

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Immunology and Infection

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions
Jorien Claes 1, Laurens Liesenborghs 1, Marleen Lox 1, Peter Verhamme 1, Thomas Vanassche 1, Marijke Peetermans 1
1Center for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

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Education

Simulation of Human-induced Vibrations Based on the Characterized In-field Pedestrian Behavior
Katrien Van Nimmen 1, Geert Lombaert 1, Guido De Roeck 1, Peter Van den Broeck 2
1Department of Civil Engineering, KU Leuven, 2Department of Civil Engineering, KU Leuven, Technology Campus Ghent

A protocol is presented for the characterization of the in-field pedestrian behavior and the simulation of the resulting structural response. Field-tests demonstrate that the in situ identified pacing rate and synchronization rate among the participants constitute an essential input for the simulation and verification of the human-induced loads.

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Medicine

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector
Anke Van der Perren 1, Cindy Casteels 2, Koen Van Laere 2, Rik Gijsbers 3,4, Chris Van den Haute 1,4, Veerle Baekelandt 1
1Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, 2Division of Nuclear Medicine, Leuven University Hospital - KU Leuven, 3Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, 4Leuven Viral Vector Core, KU Leuven

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

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Engineering

Nanostructured Ag-zeolite Composites as Luminescence-based Humidity Sensors
Eduardo Coutino-Gonzalez *1, Wouter Baekelant *1, Bjorn Dieu 1, Maarten B.J. Roeffaers 2, Johan Hofkens 1
1Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, 2Centre for Surface Chemistry and Catalysis, Department of Microbial and Molecular Systems, KU Leuven

A protocol for the synthesis of moisture-responsive luminescent Ag-zeolite composites is described in this report.

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Developmental Biology

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization
Katrien De Clercq *1, Aurélie Hennes *1, Joris Vriens 1
1Department of Development and Regeneration, Katholieke Universiteit Leuven (KU Leuven)

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

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Bioengineering

Medical-grade Sterilizable Target for Fluid-immersed Fetoscope Optical Distortion Calibration
Daniil I. Nikitichev *1, Dzhoshkun I. Shakir *1, François Chadebecq 1, Marcel Tella 1, Jan Deprest 1,2, Danail Stoyanov 3, Sébastien Ourselin 1, Tom Vercauteren 1
1Translational Imaging Group, CMIC, University College London, 2Department of Obstetrics and Gynecology, University Hospitals Leuven, 3Surgical Robot Vision Group, CMIC, University College London

This article describes the design and development of a sterilizable custom camera optical distortion calibration target for the peri-operative, fluid-immersed calibration of endoscopes during endoscopic interventions.

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Medicine

Use of a Central Venous Line for Fluids, Drugs and Nutrient Administration in a Mouse Model of Critical Illness
Sarah Derde 1, Steven Thiessen 1, Chloë Goossens 1, Thomas Dufour 1, Greet Van den Berghe 1, Lies Langouche 1
1Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven

This protocol describes a centrally catheterized mouse model of prolonged critical illness. We combine the cecal ligation and puncture method to induce sepsis with the use of a central venous line for fluids, drugs and nutrient administration to mimic the human clinical setting.

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Medicine

Transvaginal Mesh Insertion in the Ovine Model
Iva Urbankova 1,2, Geertje Callewaert 1,3, Nikhil Sindhwani 1, Alice Turri 1, Lucie Hympanova 1,2, Andrew Feola 1, Jan Deprest 1,3
1Centrum for Surgical Technologies, Department of Development and Regeneration, Clinical Specialties Research Groups, Faculty of Medicine, KU Leuven, 2Institute for the Care of Mother and Child and Third Faculty of Medicine, Charles University, Prague, 3Pelvic Floor Unit, University Hospitals KU Leuven

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

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Biochemistry

mRNA Interactome Capture from Plant Protoplasts
Zhicheng Zhang 1, Kurt Boonen 1, Meixia Li 1, Koen Geuten 1
1Department of Biology, KU Leuven

Here, we present an interactome capture protocol applied to Arabidopsis thaliana leaf mesophyll protoplasts. This method critically relies on in vivo UV crosslinking and allows for the isolation and identification of plant mRNA-binding proteins from a physiological environment.

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Distinguishing Allosteric Effects from Orthosteric Binding in Protein-Ligand Interactions
Arun Chandramohan 1, Srinath Krishnamurthy 1,2, Ganesh S Anand 1
1Department of Biological Sciences, National University of Singapore, 2Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven

Application of amide hydrogen-deuterium exchange mass spectrometry to map interactions of low affinity fragment and ligands is demonstrated. This protocol describes a method for distinguishing orthosteric binding from allosteric changes accompanying high affinity ligand and low-affinity fragment binding to target protein, Hsp90, and finds important applications in fragment-based drug design.

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Medicine

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
Sandra Claes 1, Thomas D'huys 1, Anneleen Van Hout 1, Dominique Schols 1, Tom Van Loy 1
1Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

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JoVE Journal

Assessment of Social Transmission of Food Preferences Behaviors
Ann Van der Jeugd 1, Rudi D'Hooge 1
1Laboratory of Biological Psychology, KU Leuven

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

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Biology

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4
Geert Schoofs 1, Anneleen Van Hout 1, Thomas D'huys 1, Dominique Schols 1, Tom Van Loy 1
1Laboratory of Virology and Chemotherapy, Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

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Developmental Biology

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues
Bartosz Ditkowski 1, Tiago R Veloso 1, Martyna Bezulska-Ditkowska 1,2, Andreas Lubig 3, Stefan Jockenhoevel 3, Petra Mela 3, Ramadan Jashari 4, Marc Gewillig 1, Bart Meyns 5, Marc F Hoylaerts 2, Ruth Heying 1
1Cardiovascular Developmental Biology, Department of Cardiovascular Sciences, KU Leuven, 2Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, KU Leuven, 3Department of Biohybrid & Medical Textiles, AME - Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, 4European Homograft Bank, Saint Jean Clinique, 5Division of Clinical Cardiac Surgery, Department of Cardiovascular Sciences, KU Leuven

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

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Neuroscience

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors
Shelly Fripont *1,2, Catherine Marneffe *1,2, Marika Marino *1,2, Melvin Y. Rincon 1,2, Matthew G. Holt 1,2,3
1VIB-KU Leuven Center for Brain and Disease Research, 2Department of Neuroscience, KU Leuven, 3Leuven Brain Institute

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer (≥1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

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Behavior

Investigating Pain-Related Avoidance Behavior using a Robotic Arm-Reaching Paradigm
Eveliina Glogan 1,2, Rena Gatzounis 1, Kristof Vandael 1,3, Mathijs Franssen 2, Johan W. S. Vlaeyen 1,2, Ann Meulders 1,2
1Experimental Health Psychology, Maastricht University, 2Research Group Health Psychology, KU Leuven, 3Laboratory of Biological Psychology, KU Leuven

Avoidance is central to chronic pain disability, yet adequate paradigms for examining pain-related avoidance are lacking. Therefore, we developed a paradigm that allows investigating how pain-related avoidance behavior is learned (acquisition), spreads to other stimuli (generalization), can be mitigated (extinction), and how it may subsequently re-emerge (spontaneous recovery).

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Neuroscience

Horizontal Hippocampal Slices of the Mouse Brain
Evelien Van Hoeymissen 1,2, Koenraad Philippaert 2, Rudi Vennekens 2, Joris Vriens *1, Katharina Held *1,2
1Laboratory of Endometrium, Endometriosis and Reproductive Medicine, Department of Development and Regeneration, KU Leuven, 2Laboratory of Ion Channel Research, VIB-KU Leuven Center for Brain and Disease Research, Leuven, Belgium and Department of Molecular Medicine, KU Leuven

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

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Medicine

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit
Andre Gie *1, Yannick Regin *1, Arianna Mersanne 1, Jaan Toelen 1
1Department of Development and Regeneration, KU Leuven

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

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Immunology and Infection

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging
Noémie Luyts 1, Greetje Vande Velde 2, Matthias Vanneste 1, Helene De Bruyn 1, Annelies Janssens 1, Natalie Verstraeten 3, Thomas Voets 1, Wouter Everaerts 4
1Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Center for Brain & Disease Research, Leuven, Belgium & Department of Cellular and Molecular Medicine, KU Leuven, 2Biomedical MRI, Department of Imaging & Pathology, KU Leuven, 3Belgium & KU Leuven Centre of Microbial and Plant Genetics, VIB-KU Leuven Center for Microbiology, Leuven, 4Laboratory of Experimental Urology, Department of Development and Regeneration, KU Leuven

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

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Medicine

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis
Senne Cuyx 1,2, Anabela S. Ramalho 1, Nikky Corthout 3,4, Steffen Fieuws 5, Eva Fürstová 6, Kaline Arnauts 7,8, Marc Ferrante 7,9, Catherine Verfaillie 8, Sebastian Munck 3,4, Mieke Boon 1,2, Marijke Proesmans 1,2, Lieven Dupont 10,11, Kris De Boeck 1,2, François Vermeulen 1,2
1Department of Development and Regeneration, Woman and Child Unit, CF research lab, KU Leuven, 2Department of Pediatrics, Pediatric Pulmonology, University Hospitals Leuven, 3VIB Bio Imaging Core, VIB-KU Leuven Center for Brain & Disease Research, 4Department for Neuroscience, KU Leuven, 5Interuniversity Center for Biostatistics and Statistical Bioinformatics, University of Leuven and University of Hasselt, 6Department of Pediatrics, 2nd Faculty of Medicine, Charles University and Motol University Hospital, 7Department of Chronic Diseases and Metabolism (CHROMETA), Translational Research Center for Gastrointestinal Disorders (TARGID), KU Leuven, 8Department of Development and Regeneration, Stem Cell Institute Leuven (SCIL), KU Leuven, 9Department of Gastroenterology and Hepatology, University Hospitals Leuven, KU Leuven, 10Department of Chronic Diseases, Metabolism and Ageing; Pneumology, KU Leuven, 11Department of Respiratory Diseases, University Hospitals Leuven

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.

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Immunology and Infection

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time
Merel Oeyen 1, Eef Meyen 1, Dominique Schols 1
1Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, KU Leuven

Here, we demonstrate the use of cell-based electrical impedance (CEI) as a very easy and straightforward method to study Zika virus infection and replication in human cells in real time. Furthermore, the CEI assay is useful for the evaluation of antiviral compounds.

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Medicine

Porcine Normothermic Isolated Liver Perfusion
Joris Blondeel 1,2, Nicholas Gilbo 1,2, Tine Wylin 1, Veerle Heedfeld 1, Diethard Monbaliu 1,2
1Department of Microbiology, Immunology and Transplantation, Laboratory of Abdominal Transplantation, KU Leuven, 2Department of Abdominal Transplant Surgery and Coordination, University Hospitals Leuven

The porcine model of liver normothermic machine perfusion (NMP), described here, can be successfully used to study NMP as a preservation strategy, a tool for viability assessment, and a platform for organ repair. It holds a high translational value, however it is technically challenging and labor-intensive.

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Medicine

Remotely Triggered LAD Occlusion Using a Balloon Catheter in Spontaneously Breathing Mice
Michiel Algoet *1, Matic Pusovnik *2, Hilde Gillijns 1, Sien Mestdagh 2, Janne Billiau 1, Ineke Artoos 1, Willy Gsell 2, Stefan P. Janssens 1, Uwe Himmelreich 2, Wouter Oosterlinck 1
1Department of Cardiovascular Sciences, KU Leuven, 2Biomedical MRI, Department of Imaging and Pathology, KU Leuven

This article presents a unique closed-chest technique for inducing myocardial ischemia-reperfusion injury (IRI) in mice. The presented method allows mice to breathe spontaneously while remotely inducing myocardial ischemia. This provides access to the animal for studying the dynamic processes of ischemia and reperfusion in situ and in real-time via noninvasive imaging.

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Bioengineering

Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction
Nathalie Van den Berghe 1, Elien Costermans 1, Samir Nuseibeh 1, Tine Brouns 1, Inge Van Hove 1, Benjamien Moeyaert 1, Els Henckaerts 1
1Trellis Research Group, Department of Cellular and Molecular Medicine, Department of Microbiology, Immunology and Transplantation, KU Leuven

Precise quantification of adeno-associated virus (AAV) vector genome copies is critical, but a standardized protocol has yet to be established. This protocol describes a validated method for preparing purified AAV samples and conducting digital droplet polymerase chain reaction (dd_PCR) to reliably quantify the viral genome titer.

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