Il sostegno della Banca degli Occhi Lions del Delaware Valley era preziosa per questo lavoro. Anticorpo di topo primario monoclonale contro ICP8 era un gentile dono del Dr. David Knipe (Harvard Medical School). Ringraziamo anche il dottor Stephen Jennings (Drexel University College of Medicine) e il Dr. Peter Laibson (Wills Eye Institute) per le discussioni utili e competenze.

Tutti i reagenti sono stati acquistati ed attrezzature sterili o sono stati sterilizzati prima della procedura. In questo articolo viene descritta la procedura di impostazione del modello ex vivo inizia con un pre-enucleato occhi. Nei nostri esperimenti, usiamo freschi bulbi oculari interi da giovani (8-12 settimane) conigli albini (Pel-Freez Biologicals, Rogers, AR). Inoltre, utilizziamo cornee umane ottenute dalla banca degli occhi locale. Se si esegue l&#39;enucleazione te stesso, fare attenzione a non danneggiare la cornea o il limbo nel corso della procedura. Protocolli di enucleazione Coniglio può essere trovato altrove 2. Parte 1: L&#39;escissione del pulsante corneosclerale <ol><li> Bagnare una stretta striscia di garza con PBS e avvolgerlo intorno al bulbo oculare per facilitare la presa durante il procedimento. </li><li> Utilizzare un bisturi affilato per fare una tangenziale incisione nella sclera, circa 0,5 cm di distanza dal limbus. </li><li> Paio di forbici affilate per estendere l&#39;incisione e completamente delle accise, la cpulsante orneoscleral. </li><li> Una volta che il pulsante corneosclerale è isolato, prendere in giro via il diaframma mentre si tiene il cerchio sclerale con una pinza. Evitare di danneggiare l&#39;endotelio o esercitare sollecitazioni eccessive sulla cornea. </li></ol> Parte 2: Introduzione di Cornea in Cultura organotipiche <ol><li> Cornee espiantate vengono coltivate in Medium minimo (MEM) supplementato con amminoacidi non essenziali (1X), L-glutammina (2 mM), penicillina (200 U / ml) e streptomicina (200 mcg / ml). Un agente antifungino, come amfotericina, possono anche essere aggiunti, se necessario. </li><li> Preparare una soluzione di 1% agarosio in mezzo di coltura e tenerlo pronto a 55 ° C. </li><li> Sciacquare la cornea in PBS sterile contenente penicillina (200 U / ml) e streptomicina (200 mcg / ml). </li><li> Posizionare il lato epiteliale della cornea giù in un pozzetto di una piastra sterile posto. </li><li> Aggiungere l&#39;agarosio mezzo contenente la concavità endoteliale della cornea quanto basta perriempirlo completamente. Permettono ~ 1 min per l&#39;agarosio a solidificare. </li><li> Posizionare la cornea con il terreno di coltura di supporto gel lato epiteliale scaffold su in un piatto di coltura tissutale 35 millimetri e aggiungere per coprire la superficie epiteliale. </li><li> La cornea può essere coltivato in questo modo per più di una settimana, con cambiamenti medi ogni 48 ore. </li></ol> Parte 3: L&#39;infezione da HSV-1 e il trattamento con farmaci sperimentali <ol><li> Aggiungere 1 ml di terreno contenente la quantità desiderata di virus in un piatto di coltura tissutale 35 millimetri. Di solito infettare con 1x10 4 PFU del ceppo KOS HSV-1 per cornea. I nostri titoli azionari sono determinati con saggi di placca sulla CV-1 monostrati. </li><li> Posizionare la cornea con il lato agarosio scaffold epiteliale giù nel terreno contenente virus. </li><li> Posizionare la cornea in una coltura tissutale incubatore di infettare per 1 ora con dondolo intermittente ogni 10-15 min. </li><li> Dopo 1 ora, aspirare il virus-conteNing medie e sciacquare 2-3 volte con PBS per rimuovere eventuali particelle residue virali. </li><li> Posizionare la cornea nella sua posizione precedente e aggiungere mezzo fresco coltura per coprire la superficie epiteliale. </li><li> Trattamenti sperimentali può essere iniziata prima o dopo l&#39;infezione, a seconda della natura dell&#39;esperimento. </li><li> Terreno di coltura viene cambiato ogni ora 48. </li></ol> Parte 4: Raccolta dei campioni <ol class="lalpha"><li> Media per l&#39;analisi della placca dosaggio. Miscelare il mezzo di bene e utilizzarlo immediatamente per un saggio di placca o conservare a -80 ° C per un uso successivo, se il prelievo dei campioni in momenti diversi. </li><li> DNA, RNA, proteine, o cellule di epitelio corneale <ol><li> Rimuovere il patibolo agarosio e sciacquare bene la cornea con PBS. </li><li> Accise l&#39;anello di tessuto sclerale dalla cornea per garantire che solo i materiali corneale viene raccolto. </li><li> Ucantare un bisturi, raschiare le cellule epiteliali corneali nel buffer appropriata, a seconda del tipo di materiale da isolare. Per l&#39;isolamento di DNA e RNA, si usa il Kit DNeasy Blood &amp; Tissue e il kit RNeasy Mini, rispettivamente (Qiagen, Hilden, Germania). Per la raccolta dei lisati proteici, usiamo tampone Laemmli. Per l&#39;analisi di citometria a flusso di cellule epiteliali corneali, si pre-incubare le cornee in una piccola quantità di tripsina per allentare le cellule, e quindi rimuoverli in tripsina con un bisturi. Poiché raschiare l&#39;epitelio corneale non produce la stessa quantità di tessuto, è importante comprendere controlli appropriati quando si analizzano i campioni. Usiamo rRNA 18S come una trascrizione di riferimento in RT-PCR, GAPDH come gene di riferimento in qPCRs, e nucleolina come controllo di carico per Western blot. </li></ol></li><li> I campioni di tessuto per immunoistochimica o immunofluorescenza <ol><li> Rimuovere il patibolo agarosio e sciacquare il maissincerarsi bene con PBS. </li><li> Seconda obiettivo sperimentale, utilizzare la cornea per fare un blocco di tessuto paraffina o un blocco di tessuto congelato, come mostrato nel video. Seguiamo le procedure standard per la preparazione sezioni di tessuto corneale. </li></ol></li></ol> Risultati rappresentativi La maggior parte dei metodi disponibili per studiare le cellule in coltura tissutale può essere facilmente adattato per l&#39;uso in cornee infettati con HSV-1 ex vivo. Visualizzati sono risultati rappresentativi per alcune delle più comuni tecniche applicabili allo studio HSV-1 - qPCR (Figura 1D), qRT-PCR (Figura 1B), Western blot (figura 1F), saggio di placca (Figura 1C), e tessuto immunofluorescenza (Figura 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Figura 1" /> Figura 1. Analisi di cornee infettati con HSV-1 ex vIvo. (A) Rappresentazione schematica del modello ex vivo di acuta corneale HSV-1. (BF) Analisi delle cornee infette con il metodo descritto in questo articolo video. Cornee espiantate coniglio sono stati infettati con 1x10 4 PFU / cornea del ceppo KOS HSV-1 e coltivate in presenza (PAA) o assenza (Mock) di acido fosfonoacetico (400 mcg / ml), noto inibitore della replicazione di HSV. (B) RNA totale è stato raccolto nei punti temporali indicati, e trascrizione virale è stata valutata mediante RT-PCR con primer per HSV-1 polimerasi. Primers contro 18S rRNA sono stati utilizzati come riferimento. n = 3. Le barre di errore indicano ± SEM. (C) Il terreno di coltura è stato raccolto in 48 hpi, e la produzione di particelle infettive è stata valutata mediante saggio di placca su monostrati CV-1. (D) DNA totale è stato raccolto in 48 hpi, e la replicazione del genoma virale è stata valutata in un saggio qPCR con primer per il genoma HSV-1. Premporizzatori contro GAPDH sono stati utilizzati come riferimento. n = 3. Le barre di errore indicano ± SEM. (E) Mock-trattati cornee sono stati rapidamente congelati a 48 hpi, sezionato, e analizzati mediante immunofluorescenza indiretta per la presenza di una proteina virale precoce (ICP8) entro l&#39;epitelio infetto. Nuclei sono contrastate con Höchst 33.258. (F) lisati proteici sono stati raccolti a 48 hpi, e l&#39;espressione di una proteina virale (ICP0) è stata valutata mediante Western blot. BCA saggio è stato utilizzato per garantire caricamento parità di campioni. HPI = ore post infezione.

<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>

Nessun conflitto di interessi dichiarati.

In questo video articolo si descrive un metodo per lo studio acuta corneale HSV-1 in un modello ex vivo. Questo modello è un passo utile tra la in vitro e in vivo, perché permette di convalida fisiologicamente accurata dei risultati di coltura cellulare e, quindi, limita la quantità di sperimentazione animale che deve essere eseguita. Inoltre, il modello ex vivo offre un&#39;opportunità unica e preziosa per lo studio epiteliale infezione da herpes in intatti cornee umane....

<table border="1"><tbody><tr><td> Reagenti / Materiali di consumo </td><td> Azienda </td><td> Catalogo # </td></tr><tr><td> Medium minimo (MEM) </td><td> Mediatech </td><td> 10-010-CV </td></tr><tr><td> Aminoacidi non essenziali (soluzione 100x) </td><td> Mediatech </td><td> 25-025-CI </td></tr><tr><td> L-Glutammina </td><td> Mediatech </td><td> 25-005-CI </td></tr><tr><td> Penicillina G sale di sodio </td><td> Sigma </td><td> P3032 </td></tr><tr><td> Streptomicina solfato di sale </td><td> Sigma </td><td> S9137 </td></tr><tr><td> Agarosio </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> Soluzione tampone fosfato (PBS) </td><td> Preparato in laboratorio </td><td> N / A </td></tr><tr><td> Tripsina EDTA (1x) &lt;/ Td&gt; <td> Mediatech </td><td> 25-053-CI </td></tr><tr><td> Sangue DNeasy e kit di tessuti </td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Mini Kit </td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Laemmli buffer </td><td> Preparato in laboratorio </td><td> N / A </td></tr><tr><td> Temperatura ottimale di taglio (OCT) Composto </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Occhi coniglio intero (Young) </td><td> Pel-Freez </td><td> 41211-2 </td></tr><tr><td> Le cornee umane e Occhi Whole </td><td> Eye Banca Locale </td><td> N / A </td></tr><tr><td> 12 Beh porcellana Piastra Spot </td><td> Lab di Avogadro di alimentazione </td><td> 1877 </td></tr><tr><td> 35 millimetri tessuto piatto Cultura </td><td> Falco </td><td> 353001 </td></tr><tr><td> 6 BeneColture Cellulari Piastra </td><td> Greiner Bio-One </td><td> 657160 </td></tr><tr><td> Cryomold Intermedio </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

Cheratite da herpes è una delle patologie più gravi associati al virus herpes simplex di tipo 1 (HSV-1). Cheratite Herpes è attualmente la principale causa di cecità sia cornea-derivata e associata ad infezione nel mondo sviluppato. Presentazione tipica di cheratite da herpes include infezione dell&#39;epitelio corneale e talvolta più profonda stroma corneale e endotelio, portando a tali patologie corneali permanenti come cicatrici, assottigliamento e opacità 1. Corneale HSV-1 infezione è tradizionalmente studiato in due tipi di modelli sperimentali. Il modello in vitro, in cui vengono coltivate infettato monostrati di cellule epiteliali corneali in una capsula di Petri, offre semplicità, elevato livello di replicabilità, esperimenti veloci e costi relativamente bassi. D&#39;altra parte, il modello in vivo, in cui vengono inoculati animali come conigli o topi direttamente nella cornea, offre una fisiologica altamente sofisticatosistema, ma ha costi più elevati, più lunghi esperimenti, per la cura degli animali necessari, e un maggior grado di variabilità. In questo articolo video, forniamo una dimostrazione dettagliata di un nuovo modello ex vivo di corneale HSV-1, che unisce i punti di forza sia in vitro e in vivo. Il modello ex vivo utilizza cornee intatte organotypically mantenute in coltura e infettati con HSV-1. L&#39;uso del modello ex vivo permette conclusioni altamente fisiologicamente basati, tuttavia è piuttosto costoso e richiede impegno di tempo paragonabile a quella del modello 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

In questo video articolo si descrive l&#39;uso di una nuova<em&gt; Ex vivo</emModello&gt; di infezione acuta da herpes simplex virus di tipo I infezione corneale epiteliale.

Ex Vivo Modello organotipiche corneale di acuta epiteliali Herpes simplex virus di tipo I Infezione

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 ...

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Research

JoVE Journal

Neuroscience

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

12.6K Views.  Drexel University College of Medicine. In questo video articolo si descrive l'uso di una nuova Ex vivo di infezione acuta da herpes simplex virus di tipo I infezione corneale epiteliale.

Video: 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.

Ex coltura vivo di Whole, in via di sviluppo Brains Drosophila

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

Ricerca

Neuroscienze

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 preparati della lampadina vomeronasale Organo e accessori olfattivo intatto

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.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

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.

L&#39;osservazione del movimento ciliare delle cellule epiteliali del plesso coroide<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 optogenetic dissezione di circuiti paura nel cervello fette

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.

Imaging 3D ad alta risoluzione dell'infezione da virus della rabbia nel tessuto cerebrale con la rimozione del solvente

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.

Sul posto Visualizzazione delle dinamiche dei coni di crescita e crescita degli assoni in colture di fette di cervello embrionali ex vivo acute

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.

Colture organotipiche della corteccia umana adulta come modello ex vivo per il trapianto e la validazione di cellule staminali umane

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

Imaging multimodale basato su OCT di occhi di donatori umani per la ricerca sull'AMD

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.

Autore in primo piano: Imaging multimodale ex vivo basato su OCT di occhi di donatori umani per la ricerca sulla degenerazione maculare legata all'età  

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 Imaging multimodale basato su OCT di occhi di donatori umani per la ricerca sulla degenerazione maculare legata all'età

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