Sorbonne Universites 24 articles published in JoVE Immunology and Infection Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts Kristina Irsch1,2,3, Kate Grieve1,2, Marie Borderie2, Maëlle Vilbert1,2,4, Karsten Plamann4,5, Djida Ghoubay1,2, Cristina Georgeon2, Vincent Borderie1,2 1Vision Institute, Sorbonne University, UM 80 / INSERM, UMR_S 968 / CNRS, UMR_7210, 2Quinze Vingts National Ophthalmology Hospital, 3Laboratory of Ophthalmic Instrument Development, The Wilmer Eye Institute, Johns Hopkins University School of Medicine, 4Laboratory for Optics and Biosciences (LOB), École polytechnique, 5LOA - ENSTA Paris, École polytechnique We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty. Developmental Biology Targeted Laser Ablation in the Embryo of Saccharina latissima Samuel Boscq1, Stéphanie Dutertre2, Ioannis Theodorou1, Bénédicte Charrier1 1UMR8227, CNRS / Sorbonne University, 2Univ Rennes, CNRS, Inserm, Biosit UAR 3480 US_S 018, MRic Core Facility The destruction of specific cells in the embryo is a powerful tool for studying cellular interactions involved in cell fate. The present protocol describes techniques for the laser ablation of targeted cells in the early embryo of the brown alga Saccharina latissima. Neuroscience Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy Hortense de Calbiac*1,2, Adriana Dabacan*3, Raul Muresan3, Edor Kabashi1,2, Sorana Ciura1,2 1University Paris Descartes Hospital Necker-Enfants Malades, Institut Imagine, 2Institut du Cerveau et de la Moelle épinière - ICM, Sorbonne Universités Paris, 3Transylvanian Institute of Neuroscience (TINS) Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene. Developmental Biology Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay Natasja G. J. Costermans1,2, Enrico M. Daldello1,2,3, Ria J. Marathe1,2, Marco Conti1,2 1Center for Reproductive Sciences, University of California at San Francisco, 2Department of Obstetrics Gynecology and Reproductive Sciences, University of California at San Francisco, 3Present affiliation: Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS, Sorbonne Université This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation. Medicine Halogenated Agent Delivery in Porcine Model of Acute Respiratory Distress Syndrome via an Intensive Care Unit Type Device Raiko Blondonnet1,2, Bertille Paquette1,2, Jules Audard1,2, Ridvan Guler1,2, François-Xavier Roman1,2, Ruoyang Zhai2, Corinne Belville2, Loïc Blanchon2, Thomas Godet1, Emmanuel Futier1,2, Jean-Etienne Bazin1, Jean-Michel Constantin3, Vincent Sapin2,4, Matthieu Jabaudon1,2,5 1Department of Perioperative Medicine, CHU Clermont-Ferrand, 2GReD, CNRS, INSERM, Université Clermont Auvergne, 3GRC 29, AP-HP, DMU DREAM, Department of Anesthesiology and Critical Care, Pitié-Salpêtrière Hospital, Sorbonne University, 4Department of Biochemistry and Molecular Genetics, CHU Clermont-Ferrand, 5Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University Medical Center We describe a model of hydrochloric acid-induced acute respiratory distress syndrome (ARDS) in piglets receiving sedation with halogenated agents, isoflurane and sevoflurane, through a device used for inhaled intensive care sedation. This model can be used to investigate the biological mechanisms of halogenated agents on lung injury and repair. Biology Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells Hugo Lachuer1,2,3, Pallavi Mathur1,2,3, Kevin Bleakley4, Kristine Schauer1,2,3 1Unité Mixte de Recherche 144 CNRS, Molecular Mechanisms of Intracellular Transport group, Institut Curie, 75005 Paris, France, 2PSL Research University, Paris, France, 3Sorbonne Université, Paris, France, 4INRIA, Université Paris-Sud, PSL Live imaging of lysosomal exocytosis on micropatterned cells allows a spatial quantification of this process. Morphology normalization using micropatterns is an outstanding tool to uncover general rules about the spatial distribution of cellular processes. Biology A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft Océane Blaise*1,2, Constance Duchesne*1,2, Sébastien Banzet2, Antoine Rousseau1, Nadira Frescaline1,2 1Laboratoire de Physique des Plasmas, École Polytechnique, Sorbonne Université, CNRS, 2Institut de Recherche Biomédicale des Armées, Clamart, INSERM UMRS-MD The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse. Neuroscience Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures Melina Thetiot1, Rémi Ronzano1, Marie-Stéphane Aigrot1, Catherine Lubetzki1, Anne Desmazières1 1Sorbonne Université, Inserm, CNRS, Institut du Cerveau et de la Moelle épinière, ICM-GH Pitié-Salpétrière Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system. Neuroscience Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices Fanny Etienne1,2,3, Vincenzo Mastrolia1,2,3, Luc Maroteaux1,2,3, Jean-Antoine Girault1,2,3, Nicolas Gervasi*1,2,3, Anne Roumier*1,2,3 1Institut National de la Santé et de la Recherche Médicale (INSERM), UMR-S 1270 Paris, France, 2Sorbonne Université, Paris, France, 3Institut du Fer à Moulin, Paris, France Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice. Genetics Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders Sébastien Dussaud1,2, Corinne Pardanaud-Glavieux1, Claire Sauty-Colace1, Philippe Ravassard1 1UPMC Univ Paris 06, INSERM U1127, CNRS UMR 7225, Institut du Cerveau et de la Moelle épinière, ICM, Sorbonne Universités, 2UPMC Univ Paris 06, INSERM UMRS1166, Institute of Cardiometabolism and Nutrition, Sorbonne Universités Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte. Developmental Biology Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells Karim Ben M'Barek1,2,3, Walter Habeler1,2,3, Alexandra Plancheron1,2,3, Mohamed Jarraya4, Olivier Goureau5, Christelle Monville1,2 1U861, I-Stem, Association Française contre les Myopathies (AFM), Institut National de la Santé et de la Recherche Médicale (INSERM), 2I-Stem, Association Française contre les Myopathies (AFM), Centre pour L’Etude des Cellules Souches (CECS), 4Banque de tissus humain, Hôpital Saint Louis, Assistance Publique - Hôpitaux de Paris (AP-HP), 5Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012 We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models. Developmental Biology Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models Amélie Slembrouck-Brec*1, Céline Nanteau*1, José-Alain Sahel1, Olivier Goureau1, Sacha Reichman1 1Institut de la Vision, Sorbonne Université, INSERM, CNRS, F-75012 Paris, France The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research. Neuroscience Single Cell Multiplex Reverse Transcription Polymerase Chain Reaction After Patch-clamp Gabrielle Devienne*1, Benjamin Le Gac*1, Juliette Piquet*1, Bruno Cauli1 1UPMC Univ Paris 06, INSERM, CNRS, Neuroscience Paris Seine - Institut de Biologie Paris Seine (NPS - IBPS), Sorbonne Université This protocol describes the critical steps and precautions required to perform single cell multiplex reverse transcription polymerase chain reaction after patch-clamp. This technique is a simple and effective method to analyze the expression profile of a predetermined set of genes from a single cell characterized by patch-clamp recordings. Immunology and Infection Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry Elodie Martin1,2,3, Mohamed El-Behi1,2,3, Bertrand Fontaine1,2,3,4, Cecile Delarasse1,2,3 1Inserm U 1227, CNRS UMR 7225, 2Sorbonne Universités, UPMC, University of Paris, 3Institut du Cerveau et de la Moelle épinière, ICM, 4AP-HP, Hôpital de la Pitié Salpêtrière; This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells. Developmental Biology In Vitro Differentiation of Mature Myofibers for Live Imaging Mafalda R. Pimentel1, Sestina Falcone2, Bruno Cadot2, Edgar R. Gomes1,2 1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, 2Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6 Muscle cells are among the most complex eukaryotic cells. We present a protocol for the in vitro differentiation of highly mature myofibers that allows for genetic manipulation and clear imaging during all developmental stages. Neuroscience Protocol for Isolating the Mouse Circle of Willis Justine Claire Hur1, Régis Blaise1, Isabelle Limon1 1UPMC Univ Paris 06, Sorbonne Universités We describe here a reproducible protocol for isolating the mouse circle of Willis. Biology The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo Anne-Sophie Perrin-Terrin1,2, Florine Jeton1,3, Aurelien Pichon1,3,4, Alain Frugière2, Jean-Paul Richalet1,3, Laurence Bodineau2, Nicolas Voituron1,3 1Sorbonne Paris Cité, Laboratory “Hypoxia & Lung” EA2363, University Paris 13, 2UPMC Univ Paris 06, INSERM, UMR_S1158 Neurophysiologie Respiratoire Expérimentale et Clinique, Sorbonne Universités, 3Laboratory of Excellence GR-Ex, 4Laboratory MOVE (EA 6314), University of Poitiers Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo. Neuroscience Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo Tristan Altwegg-Boussac1,2,3,4, Séverine Mahon1,2,3,4, Mario Chavez1,2,3,4, Stéphane Charpier1,2,3,4, Adrien E. Schramm1,2,3,4 1Inserm U1127, 2CNRS UMR 7225, 3UPMC Univ Paris 06, UMR S 1127, Sorbonne Universités, 4Institut du Cerveau et de la Moelle épinière (ICM) This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition. Medicine Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets Audrey Courboulin1, Chantal Kang2, Olivier Baillard3,4,5, Sebastien Bonnet1, Pierre Bonnet6 1Department of Medicine, Pulmonary Hypertension Research Group (CRIUCPQ), Laval University, 2Institut National de la Recherche Agronomique, 3Université Diderot Paris, Sorbonne Paris Cité, 4Hôpital Lariboisière, Physiologie clinique Explorations Fonctionnelles, 5INSERM U 965, 6Service de Cardiologie, Centre Hospitalier Universitaire Tours Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension. Medicine Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease Ophélie Vacca1,2,3, Brahim El Mathari1,2,3, Marie Darche1,2,3, José-Alain Sahel1,2,3, Alvaro Rendon1,2,3, Deniz Dalkara1,2,3 1Department of Therapeutics, Institut de la Vision, Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968, 2INSERM, U968, 3CNRS, UMR_7210 To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability. Immunology and Infection Tracking Mouse Bone Marrow Monocytes In Vivo Pauline Hamon1, Mathieu Paul Rodero1, Christophe Combadière1, Alexandre Boissonnas1 1Centre d'Immunologie et des Maladies Infectieuses (CIMI), INSERM, U1135, CNRS, ERL 8255, Sorbonne Universités, UPMC Univ Paris 06, CR7 Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism. Developmental Biology Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes Estelle Hirsinger1, João Emanuel Carvalho2, Christine Chevalier1,3, Georges Lutfalla4, Jean-François Nicolas1, Nadine Peyriéras5, Michael Schubert2 1Département de Biologie du Développement et Cellules Souches, Institut Pasteur, 2Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités, 3Equipe Epigenetic Control of Normal and Pathological Hematopoiesis, Centre de Recherche en Cancérologie de Marseille, 4Unité de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR5235/DAA/cc107/Université Montpellier II, 5Plateforme BioEmergences IBiSA FBI, CNRS-NED, Institut de Neurobiologie Alfred Fessard We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations. Immunology and Infection Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling Laura Megrelis1,2,3, Jérôme Delon1,2,3 1Inserm U1016, Institut Cochin, 2Cnrs, UMR8104, 3Université Paris Descartes, Sorbonne Paris Cité Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events. Bioengineering Remote Magnetic Actuation of Micrometric Probes for in situ 3D Mapping of Bacterial Biofilm Physical Properties Olivier Galy1, Kais Zrelli1, Patricia Latour-Lambert2, Lyndsey Kirwan3, Nelly Henry1,3 1Physicochime Curie, CNRS UMR 168, Institut Curie, Sorbonne Universités, UPMC, 2Unité de Génétique des Biofilms, Institut Pasteur, 3Laboratoire Jean Perrin, CNRS UMR 8237, Sorbonne Universités, UPMC This paper shows an original methodology based on the remote actuation of magnetic particles seeded in a bacterial biofilm and the development of dedicated magnetic tweezers to measure in situ the local mechanical properties of the complex living material built by micro-organisms at interfaces.