L&#39;appui de la Banque d&#39;yeux des Lions du Delaware Valley a été inestimable pour ce travail. Anticorps de souris monoclonal primaire contre ICP8 était un gentil cadeau du Dr David Knipe (Harvard Medical School). Nous remercions également le Dr Stephen Jennings (Drexel University College of Medicine) et le Dr Peter Laibson (Wills Eye Institute) pour des discussions utiles et d&#39;expertise.

Tous les réactifs et du matériel ont été achetés stérile ou ont été stérilisés avant la procédure. Cet article décrit les étapes de mise en place du modèle ex vivo en commençant par un œil énucléé avant. Dans nos expériences, nous utilisons de nouveaux globes oculaires entières de jeunes (8-12 semaines) lapins albinos (Pel-Freez Biologicals, Rogers, AR). En outre, nous utilisons des cornées humaines obtenues à partir de la banque des yeux locale. Si vous effectuez vous-même l&#39;énucléation, prenez soin de ne pas endommager la cornée ou du limbe lors de la procédure. Protocoles énucléation de lapin peuvent être trouvés ailleurs 2. Partie 1: excision du bouton cornéosclérale <ol><li> Mouiller une étroite bande de gaze avec du PBS et l&#39;enrouler autour du globe oculaire pour faciliter sa préhension lors de la procédure. </li><li> Utiliser un scalpel pour faire une incision dans la sclère tangentielle, environ 0,5 cm du limbe. </li><li> Utiliser des ciseaux bien aiguisés pour prolonger l&#39;incision et complètement exciser la cbouton orneoscleral. </li><li> Une fois sur le bouton cornéosclérale est isolé, loin taquiner l&#39;iris tout en maintenant le bord scléral avec une pince. Éviter d&#39;endommager l&#39;endothélium ou exerçant un stress excessif sur la cornée. </li></ol> Partie 2: Introduction de la cornée en culture organotypique <ol><li> Cornées explantées sont cultivées dans un milieu essentiel minimum (MEM) additionné de non-acides aminés essentiels (1X), L-glutamine (2 mM), la pénicilline (200 U / ml) et de streptomycine (200 ug / ml). Un agent antifongique, tels que l&#39;amphotéricine, peuvent également être ajoutés si nécessaire. </li><li> Préparer une solution à 1% d&#39;agarose dans un milieu de culture et le tenir prêt à 55 ° C. </li><li> Rincer soigneusement la cornée stérile pénicilline PBS contenant (200 U / ml) et de streptomycine (200 ug / ml). </li><li> Placez le côté cornée épithéliale dans un puits d&#39;une plaque à godets stérile. </li><li> Ajouter le support d&#39;agarose contenant de la concavité endothéliale de la cornée juste assez pourle remplir complètement. Permettez ~ 1 min pour le agarose à se solidifier. </li><li> Placez la cornée avec le milieu de culture soutenir gel échafaudage côté épithélial vers le haut dans une boîte de culture tissulaire de 35 mm et d&#39;ajouter à couvrir la surface épithéliale. </li><li> La cornée peut être cultivé de cette manière depuis plus d&#39;une semaine, avec tous les changements de milieu h 48. </li></ol> Partie 3: L&#39;infection par le HSV-1 et le traitement avec des médicaments expérimentaux <ol><li> Ajouter 1 ml de milieu contenant la quantité souhaitée de virus dans une boîte de culture de tissus 35 mm. En général, nous infecter avec 1x10 4 PFU de la souche KOS de HSV-1 par la cornée. Nos titres boursiers sont déterminés par des essais de plaque sur des monocouches de CV-1. </li><li> Placer la cornée avec le côté échafaudage agarose épithéliale vers le bas dans le milieu contenant le virus. </li><li> Placez la cornée dans un incubateur de culture tissulaire pour infecter pendant 1 heure avec balancement intermittent toutes les 10-15 minutes. </li><li> Après 1 heure, aspirer le virus contaiNing moyennes et rincer 2-3 fois avec du PBS pour éliminer les particules résiduelles virales. </li><li> Placer la cornée dans sa position précédente et ajouter un milieu de culture frais pour couvrir la surface de l&#39;épithélium. </li><li> Traitements expérimentaux peuvent être commencée avant ou après l&#39;infection, en fonction de la nature de l&#39;expérience. </li><li> Le milieu de culture est changé tous les 48 h. </li></ol> Partie 4: Prélèvement des échantillons <ol class="lalpha"><li> Moyen pour l&#39;analyse dosage de la plaque. Mélangez bien le support et l&#39;utiliser immédiatement pour un essai de plaque ou de conserver à -80 ° C pour une utilisation ultérieure si la collecte d&#39;échantillons à différents moments. </li><li> ADN, ARN, protéines, ou des cellules de l&#39;épithélium cornéen <ol><li> Retirer l&#39;échafaud agarose et rincer la cornée et avec du PBS. </li><li> L&#39;excision de la bague de tissu scléral de la cornée afin de faire en sorte que seul matériau épithéliale cornéenne est recueillie. </li><li> Uchanter un scalpel, gratter les cellules épithéliales cornéennes dans le tampon approprié, en fonction du type de matériau devant être isolé. Pour l&#39;isolement de l&#39;ADN et de l&#39;ARN, on utilise le kit DNeasy Blood &amp; Tissue et le kit RNeasy Mini, respectivement (QIAGEN, Hilden, Allemagne). Pour la collecte des lysats protéiques, nous utilisons tampon de Laemmli. Pour l&#39;analyse par cytométrie en flux des cellules épithéliales cornéennes, nous pré-incuber les cornées dans une petite quantité de trypsine pour détacher les cellules, puis les déloger en trypsine avec un scalpel. Depuis racler l&#39;épithélium cornéen ne donne pas des quantités égales de tissu, il est important d&#39;inclure des contrôles appropriés lors de l&#39;analyse des échantillons. Nous utilisons ARNr 18S comme une transcription de référence dans qRT-PCR, GAPDH comme gène de référence dans RCQP et nucléoline comme un contrôle de chargement pour Western blot. </li></ol></li><li> Des échantillons de tissus pour l&#39;immunohistochimie ou immunofluorescence <ol><li> Retirer l&#39;échafaud agarose et rincer le maïseo bien avec du PBS. </li><li> En fonction de votre objectif expérimental, utilisez la cornée afin de faire un bloc de paraffine de tissu ou d&#39;un bloc de tissu congelé, comme le montre la vidéo. Nous suivre les procédures standard lors de la préparation des coupes de tissus cornéens. </li></ol></li></ol> Les résultats représentatifs La plupart des méthodes disponibles pour l&#39;étude des cellules en culture de tissus peut être facilement adapté pour une utilisation dans la cornée in vivo infectées par le HSV-1 ex. On voit ici des résultats représentatifs pour quelques-unes des techniques les plus communes applicables à l&#39;étude de l&#39;infection HSV-1 - qPCR (figure 1D), qRT-PCR (figure 1B), Western blot (figure 1F), dosage de la plaque (figure 1C), et tissus par immunofluorescence (figure 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Figure 1" /> Figure 1. Analyse des cornées infectées par le HSV-1 v exivo. Représentation schématique (A) du modèle ex vivo de épithéliale cornéenne aiguë infection HSV-1. (BF) Analyse des cornées infectées en utilisant la méthode décrite dans cet article la vidéo. Cornées de lapin explantés ont été infectées par le 1x10 4 UFP / cornée de souche KOS de HSV-1 et cultivées en présence (AAP) ou l&#39;absence (Mock) d&#39;acide phosphonoacétique (400 pg / ml), un inhibiteur connu de la réplication du HSV. (B) L&#39;ARN total ont été recueillies aux points de temps indiqués, et la transcription virale a été évaluée par qRT-PCR avec des amorces pour le HSV-1 polymérase. Primaires contre l&#39;ARNr 18S ont été utilisés comme référence. n = 3. Les barres d&#39;erreur indiquent ± SEM. (C) Le milieu de culture a été recueilli à 48 hpi, et la production de particules infectieuses a été évaluée par dosage de plaque sur des monocouches de CV-1. (D) L&#39;ADN total a été recueilli à 48 hpi, et la réplication du génome viral a été évaluée dans un test PCR quantitative avec des amorces pour le génome de HSV-1. PrIMERS contre GAPDH ont été utilisés comme référence. n = 3. Les barres d&#39;erreur indiquent ± SEM. (E) Maquette cornées ont été traités par flash-gelé à 48 hpi, sectionnées et analysés par immunofluorescence indirecte pour la présence d&#39;une protéine virale précoce (ICP8) dans l&#39;épithélium infecté. Les noyaux sont contre-Höchst 33258. (F) lysats protéiques ont été recueillies à 48 hpi, et l&#39;expression d&#39;une protéine virale (ICP0) a été évaluée par Western blot. BCA a été utilisé pour assurer un chargement égal d&#39;échantillons. hpi = heures après l&#39;infection.

<ol>
	<li>Kaye, S., Choudhary, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+keratitis.">Herpes simplex keratitis.</a> Prog. Retin. Eye Res. 25, 355-380 (2006).</li><li>Holmberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enucleation+of+exotic+pets.">Enucleation of exotic pets.</a> Journal of Exotic Pet Medicine. 16, 88-94 (2007).</li><li>Remeijer, L., Osterhaus, A., Verjans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+herpes+simplex+virus+keratitis:+the+pathogenesis+revisited.">Human herpes simplex virus keratitis: the pathogenesis revisited.</a> Ocul. Immunol. Inflamm. 12, 255-285 (2004).</li><li>Foreman, D. M., Pancholi, S., Jarvis-Evans, J., McLeod, D., Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+organ+culture+model+for+assessing+the+effects+of+growth+factors+on+corneal+re-epithelialization.">A simple organ culture model for assessing the effects of growth factors on corneal re-epithelialization.</a> Exp. Eye Res. 62, 555-564 (1996).</li><li>Xu, K. P., Li, X. F., Yu, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+organ+culture+model+for+assessing+epithelial+responses+to+surfactants.">Corneal organ culture model for assessing epithelial responses to surfactants.</a> Toxicol. Sci. 58, 306-314 (2000).</li><li>Klausner, E. A., Peer, D., Chapman, R. L., Multack, R. F., Andurkar, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+gene+therapy.">Corneal gene therapy.</a> J. Control Release. 124, 107-133 (2007).</li><li>Halford, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICP0+antagonizes+Stat+1-dependent+repression+of+herpes+simplex+virus:+implications+for+the+regulation+of+viral+latency.">ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency.</a> Virol. J. 3, 44 (2006).</li><li>Bhattacharjee, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+treatment+of+ocular+HSK+with+a+human+apolipoprotein+E+mimetic+peptide+in+a+mouse+eye+model.">Effective treatment of ocular HSK with a human apolipoprotein E mimetic peptide in a mouse eye model.</a> Invest Ophthalmol Vis. Sci. 49, 4263-4268 (2008).</li><li>Novitskaya, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difficulties+imaging+herpes+simplex+keratitis+with+fluorescein+isothiocynate-labeled+anti-HSV-1+antibodies+in+an+ex+vivo+model.">Difficulties imaging herpes simplex keratitis with fluorescein isothiocynate-labeled anti-HSV-1 antibodies in an ex vivo model.</a> Cornea. 28, 421-425 (2009).</li><li>Sharma, A., Shimeld, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+immunofluorescence+to+diagnose+herpes+simplex+virus+keratitis+in+mice.">In vivo immunofluorescence to diagnose herpes simplex virus keratitis in mice.</a> Br. J. Ophthalmol. 81, 785-788 (1997).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Dans cet article, vidéo, nous décrivons une méthode pour étudier aiguë épithélium cornéen infection HSV-1 dans un modèle ex vivo. Ce modèle est un tremplin utile entre le in vitro et dans des modèles in vivo de l&#39;, car elle permet de valider physiologiquement précise des résultats de culture cellulaire et, par conséquent, limite le montant de l&#39;expérimentation animale qui doit être effectuée. En outre, le modèle ex vivo fournit une occasion unique et...

<table border="1"><tbody><tr><td> Réactifs / fournitures </td><td> Entreprise </td><td> Catalogue # </td></tr><tr><td> Minimum Essential Medium (MEM) </td><td> Mediatech </td><td> 10-010-CV </td></tr><tr><td> Non-acides aminés essentiels (solution 100x) </td><td> Mediatech </td><td> 25-025-CI </td></tr><tr><td> L-Glutamine </td><td> Mediatech </td><td> 25 à 005-CI </td></tr><tr><td> Sel de sodium de pénicilline G </td><td> Sigma </td><td> P3032 </td></tr><tr><td> Sel de sulfate de streptomycine </td><td> Sigma </td><td> S9137 </td></tr><tr><td> Agarose </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> Phosphate Buffered Saline (PBS) </td><td> Préparé dans le laboratoire </td><td> N / A </td></tr><tr><td> Trypsine EDTA (1x) &lt;/ Td&gt; <td> Mediatech </td><td> 25 à 053-CI </td></tr><tr><td> Sang DNeasy Tissue Kit &amp; </td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Mini Kit </td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Tampon Laemmli </td><td> Préparé dans le laboratoire </td><td> N / A </td></tr><tr><td> Température optimale de coupe (PTOM) composé </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Yeux lapin entier (Young) </td><td> Pel-Freez </td><td> 41211-2 </td></tr><tr><td> Les cornées humaines et Yeux entiers </td><td> Banque des yeux locale </td><td> N / A </td></tr><tr><td> 12 Plaque Bien spot Porcelaine </td><td> Alimentation de laboratoire d&#39;Avogadro </td><td> 1877 </td></tr><tr><td> Boîte de 35 mm Tissue Culture </td><td> Faucon </td><td> 353001 </td></tr><tr><td> 6 BienPlaque de culture cellulaire </td><td> Greiner Bio-One </td><td> 657160 </td></tr><tr><td> Cryomold intermédiaire </td><td> Tissue-Tek </td><td> 4566 </td></tr></tbody></table>

ex vivo organotypic corneal model of acute epithelial herpes simplex virus type i infection

Kératite herpétique est une des pathologies les plus sévères associés au virus de l&#39;herpès simplex de type 1 (HSV-1). Kératite herpétique est actuellement la principale cause de cécité cornéenne deux dérivés et associée à l&#39;infection dans le monde développé. Présentation typique de kératite herpétique comprend également l&#39;infection de l&#39;épithélium cornéen et parfois le stroma cornéen profond et l&#39;endothélium, conduisant à de telles pathologies cornéennes permanents comme des cicatrices, l&#39;éclaircissage et l&#39;opacité 1. Cornée infection HSV-1 est traditionnellement étudiée dans deux types de modèles expérimentaux. Le modèle in vitro, dans lequel monocouches de culture de cellules épithéliales cornéennes sont infectées dans une boîte de Pétri, offre simplicité, niveau élevé de reproductibilité, des expériences rapides et des coûts relativement faibles. D&#39;autre part, le modèle in vivo, dans lesquelles les animaux comme les lapins ou les souris sont inoculés directement dans la cornée, propose un état ​​physiologique hautement sophistiquésystème, mais coûte plus cher, plus les expériences, les soins aux animaux nécessaires, et un plus grand degré de variabilité. Dans cet article, vidéo, nous fournissons une démonstration détaillée d&#39;un modèle ex vivo nouvelle épithélium cornéen infection HSV-1, qui combine les forces des deux in vitro et la le des modèles in vivo. Le modèle ex vivo utilise des cornées intactes organotypically maintenues en culture et infectées par le HSV-1. L&#39;utilisation du modèle ex vivo permet de tirer des conclusions très base physiologique, mais il est assez peu coûteux et exige un engagement de temps comparable à celle du modèle in vitro.

Watch this Scientific Journal Video about Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection at JoVE.com

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Dans cet article, nous décrivons vidéo l&#39;utilisation d&#39;une nouvelle<em&gt; Ex vivo</em&gt; Modèle de l&#39;herpès simplex virus de type aiguës je cornée infection épithéliale.

Ex Vivo modèle organotypique cornéenne aiguë de l&#39;herpès simplex virus type épithéliales je infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

ex-vivo-organotypic-corneal-model-acute-epithelial-herpes-simplex

Research

JoVE Journal

Neuroscience

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

12.6K vues.  Drexel University College of Medicine. Dans cet article, nous décrivons vidéo l'utilisation d'une nouvelle Ex vivo Modèle de l'herpès simplex virus de type aiguës je cornée infection épithéliale.

Vidéo: Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

La culture ex vivo de plénier, en développement chez la drosophile Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Recherche

Neurosciences

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex Vivo préparatifs de l&#39;ampoule Vomeronasal organes et accessoire olfactive Intact

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Une<em&gt; Ex Vivo</em&gt; Induite par laser Spinal Cord Injury modèle pour évaluer les mécanismes de la dégénérescence axonale dans en temps réel

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

Observation du Mouvement ciliaire des cellules épithéliales plexus choroïde<em&gt; Ex Vivo</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogénétiques Dissection de circuits Peur en tranches cérébrales

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ tissues provides pivotal data on disease development and progression. Using conventional microscopy methods, immunolabeling is mostly restricted to thin sections obtained from paraffin-embedded or frozen samples. However, the limited 2D image plane of these thin sections may lead to the loss of crucial information on the complex structure of an infected organ and the cellular context of the infection. Modern multicolor, immunostaining-compatible tissue clearing techniques now provide a relatively fast and inexpensive way to study high-volume 3D image stacks of virus-infected organ tissue. By exposing the tissue to organic solvents, it becomes optically transparent. This matches the sample&#8217;s refractive indices and eventually leads to a significant reduction of light scattering. Thus, in combination with long free working distance objectives, large tissue sections up to 1 mm in size can be imaged by conventional confocal laser scanning microscopy (CLSM) at high resolution. Here, we describe a protocol to apply deep-tissue imaging after tissue clearing to visualize rabies virus distribution in infected brains in order to study topics like virus pathogenesis, spread, tropism, and neuroinvasion.

Novel, immunostaining-compatible tissue clearing techniques like the ultimate 3D imaging of solvent-cleared organs allow the 3D visualization of rabies virus brain infection and its complex cellular environment. Thick, antibody-labeled brain tissue slices are made optically transparent to increase imaging depth and to enable 3D analysis by confocal laser scanning microscopy.

Imagerie 3D haute résolution de l'infection par le virus de la rage dans un tissu cérébral éclairci par solvant

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

In situ Visualisation de la croissance des axones et de la dynamique des cônes de croissance dans des cultures de tranches cérébrales embryonnaires ex vivo aiguës

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Cultures organotypiques du cortex humain adulte comme modèle ex vivo pour la transplantation et la validation de cellules souches humaines

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

Imagerie multimodale basée sur l’OCT de l’œil d’un donneur humain pour la recherche sur la DMLA

Ex Vivo OCT-Based Multimodal Imaging of Human Donor Eyes for Research into Age-Related Macular Degeneration

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging could add prognostic value to laboratory findings. In this work, ex vivo OCT and MMI were applied to human donor eyes prior to retinal tissue sectioning. The eyes were recovered from non-diabetic white donors aged &#8805;80 years old, with a death-to-preservation time (DtoP) of &#8804;6 h. The globes were recovered on-site, scored with an 18 mm trephine to facilitate cornea removal, and immersed in buffered 4% paraformaldehyde. Color fundus images were acquired after anterior segment removal with a dissecting scope and an SLR camera using trans-, epi-, and flash illumination at three magnifications. The globes were placed in a buffer within a custom-designed chamber with a 60 diopter lens. They were imaged with spectral domain OCT (30&#176; macula cube, 30 &#181;m spacing, averaging = 25), near-infrared reflectance, 488 nm autofluorescence, and 787 nm autofluorescence. The AMD eyes showed a change in the retinal pigment epithelium (RPE), with drusen or subretinal drusenoid deposits (SDDs), with or without neovascularization, and without evidence of other causes. Between June 2016 and September 2017, 94 right eyes and 90 left eyes were recovered (DtoP: 3.9 &plusmn; 1.0 h). Of the 184 eyes, 40.2% had AMD, including early intermediate (22.8%), atrophic (7.6%), and neovascular (9.8%) AMD, and 39.7% had unremarkable maculas. Drusen, SDDs, hyper-reflective foci, atrophy, and fibrovascular scars were identified using OCT. Artifacts included tissue opacification, detachments (bacillary, retinal, RPE, choroidal), foveal cystic change, an undulating RPE, and mechanical damage. To guide the cryo-sectioning, OCT volumes were used to find the fovea and optic nerve head landmarks and specific pathologies. The ex vivo volumes were registered with the in vivo volumes by selecting the reference function for eye tracking. The ex vivo visibility of the pathology seen in vivo depends on the preservation quality. Within 16 months, 75 rapid DtoP donor eyes at all stages of AMD were recovered and staged using clinical MMI methods.

Pleins feux sur l’auteur : Imagerie multimodale ex vivo basée sur l’OCT d’yeux de donneurs humains pour la recherche sur la dégénérescence maculaire liée à l’âge  

Laboratory assays can leverage prognostic value from the longitudinal optical coherence tomography (OCT)-based multimodal imaging of age-related macular degeneration (AMD). Human donor eyes with and without AMD are imaged using OCT, color, near-infrared reflectance scanning laser ophthalmoscopy, and autofluorescence at two excitation wavelengths prior to tissue sectioning.

Ex Vivo Imagerie multimodale basée sur l’OCT des yeux de donneurs humains pour la recherche sur la dégénérescence maculaire liée à l’âge

A progression sequence for age-related macular degeneration (AMD) learned from optical coherence tomography (OCT)-based multimodal (MMI) clinical imaging ...