O apoio do Banco de Olhos do Lions de Delaware Valley foi inestimável para este trabalho. Anticorpo monoclonal contra o principal do mouse ICP8 foi um presente tipo de Dr. David Knipe (Harvard Medical School). Agradecemos também o Dr. Stephen Jennings (Drexel University College of Medicine) e Dr. Peter Laibson (Wills Eye Institute) para discussões úteis e conhecimentos.

Todos os reagentes e equipamentos foram comprados estéreis ou foram esterilizadas antes do procedimento. Este artigo descreve as etapas de implementação do modelo ex vivo começando com um olho pré-enucleado. Em nossos experimentos, utilizamos frescos olhos inteiros de jovens (8-12 semanas) coelhos albinos (Pel-Freez Biologicals, Rogers, AR). Além disso, usamos córneas humanas obtidos a partir do banco de olhos local. Se realizar a enucleação si mesmo, tome cuidado para não danificar a córnea ou o limbo durante o procedimento. Protocolos enucleação de coelho pode ser encontrada em outras duas. Parte 1: Excisão do botão córneo <ol><li> Molhe uma estreita faixa de gaze com PBS e envolvê-la em torno do globo ocular para facilitar a segurá-lo durante o procedimento. </li><li> Usar um bisturi afiado para fazer uma incisão na esclera tangencial, cerca de 0,5 cm de distância a partir do limbo. </li><li> Use uma tesoura afiada para estender a incisão e completamente especial sobre o consumo do corneoscleral botão. </li><li> Uma vez que o botão córneo é isolado, longe provocar a íris, segurando a borda escleral com uma pinça. Evitar danificar o endotélio ou exercer pressão excessiva sobre a córnea. </li></ol> Parte 2: Introdução de Córnea em Cultura organotípicas <ol><li> Córneas explantadas são cultivadas em Meio Essencial Mínimo (MEM) suplementado com não-aminoácidos essenciais (1X), L-glutamina (2 mM), penicilina (200 U / ml) e estreptomicina (200 ug / ml). Um agente antifúngico, tal como anfotericina, podem também ser adicionados, se necessário. </li><li> Prepara-se uma solução de 1% de agarose num meio de cultura e mantê-lo pronto a 55 ° C. </li><li> Enxaguar bem a córnea em PBS estéril contendo penicilina (200 U / ml) e estreptomicina (200 ug / ml). </li><li> Coloque o lado epitelial da córnea para baixo dentro de um poço de uma placa de mancha estéril. </li><li> Adicionar o meio de agarose contendo a concavidade endoteliais da córnea apenas o suficiente paraenchê-lo completamente. Permitir min ~ 1 para a agarose solidificar. </li><li> Coloque a córnea, com o meio de cultura de apoio lateral gel andaime epitelial para cima para um prato de cultura de tecido de 35 milímetros e adiciona-se a cobrir a superfície epitelial. </li><li> A córnea pode ser cultivado neste meio durante mais de uma semana, com mudanças de meio a cada 48 h. </li></ol> Parte 3: A infecção com HSV-1 e de tratamento com medicamentos experimentais <ol><li> Adicionar 1 ml de meio contendo a quantidade desejada de vírus em um prato de cultura de tecido 35 milímetros. Nós normalmente infectam com 1x10 PFU 4 de estirpe KOS HSV-1 por córnea. Os títulos de nossas ações são determinadas através da realização de ensaios de placa no CV-1 monocamadas. </li><li> Coloque a córnea em conjunto com o lado do andaime agarose epitelial para baixo para o meio contendo vírus. </li><li> Coloque a córnea num incubador de cultura de tecido para infectar durante 1 hora com agitação intermitente a cada 10-15 minutos. </li><li> Depois de 1 hora, aspirar o vírus contai-ning meio e lavar 2-3 vezes com PBS para remover quaisquer partículas residuais virais. </li><li> Coloque a córnea de volta para a sua posição anterior e adicionar o meio de cultura fresco para cobrir a superfície epitelial. </li><li> Os tratamentos experimentais pode ser iniciada antes ou após a infecção, dependendo da natureza do ensaio. </li><li> Meio de cultura é trocado a cada 48 horas. </li></ol> Parte 4: Coleta de Amostra <ol class="lalpha"><li> Meio de ensaio em placas de análise. Misturar bem o meio e utilizá-la imediatamente para um ensaio de placa ou armazenar a -80 ° C para uso posterior, se a recolha de amostras em diferentes pontos temporais. </li><li> DNA, RNA, proteínas ou células de epitélio da córnea <ol><li> Remover o andaime de agarose e enxaguar a córnea bem com PBS. </li><li> O anel de tecido escleral Excise da córnea para garantir que o material só epitelial da córnea é recolhida. </li><li> Ucantar um bisturi, raspar as células epiteliais da córnea no tampão apropriado, dependendo do tipo de material a ser isolado. Para o isolamento de DNA e RNA, usamos o Kit Blood &amp; Tissue Dneasy e o Kit Mini RNeasy, respectivamente (QIAGEN, Hilden, Alemanha). Para a coleta de lisados ​​de proteínas, usamos tampão Laemmli. Para a análise de citometria de fluxo de células epiteliais da córnea, é pré-incubar as córneas em uma pequena quantidade de tripsina para desprender as células, e, em seguida, desalojar-los para a tripsina com um bisturi. Uma vez que a raspagem do epitélio da córnea não produz a mesma quantidade de tecido, é importante incluir controlos adequados ao analisar as amostras. Usamos rRNA 18S como um transcrito de referência em qRT-PCR, GAPDH como gene de referência em qPCRs e nucleolin como um controlo de carga para transferência de Western. </li></ol></li><li> Amostras de tecido para imuno-histoquímica ou imunofluorescência <ol><li> Retire o andaime de agarose e lavar o milhoEa bem com PBS. </li><li> Dependendo do seu objectivo experimental, utilize a córnea para fazer um bloco de tecido ou de um bloco de parafina do tecido congelado, como mostrado no vídeo. Nós seguimos os procedimentos padrão quando se prepara cortes de tecido da córnea. </li></ol></li></ol> Resultados representativos A maior parte dos métodos disponíveis para o estudo de células em cultura de tecidos pode ser facilmente adaptado para o uso em córneas infectadas com HSV-1 in vivo ex. Mostrado aqui são resultados representativos para algumas das técnicas mais comuns aplicáveis ​​ao estudo da infecção por HSV-1 - qPCR (Figura 1D), qRT-PCR (Figura 1B), Western blot (Figura 1F), ensaio de placa (Figura 1C), e tecido de imunofluorescência (Figura 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Figura 1" /> Figura 1. Análise de córneas infectadas com HSV-1 ex vivo. (A) Representação esquemática do modelo ex vivo de aguda epitelial corneal HSV-1 infecção. (BF) Análise de córneas infectadas utilizando o método descrito neste artigo de vídeo. Córneas explantadas de coelho foram infectados com 1x10 4 PFU / córnea da estirpe KOS de HSV-1 e cultivadas na presença (PAA) ou ausência (Mock) de ácido fosfonoacético (400 ug / ml), um conhecido inibidor da replicação do HSV. (B) O ARN total foi colhido nos pontos de tempo indicados, e a transcrição viral foi avaliada por qRT-PCR com os iniciadores para o HSV-1 de polimerase. Primers contra rRNA 18S foram usados ​​como referência. n = 3. As barras de erro indicam ± SEM. (C) O meio de cultura foi recolhido a 48 hpi e a produção de partículas infecciosas foi avaliada por ensaio de placas em monocamadas de CV-1 (D). O ADN total foi recolhido a 48 hpi e a replicação do genoma viral foi avaliada em um ensaio qPCR com iniciadores para o HSV-1 do genoma. PrIMers contra GAPDH foram utilizados como referência. n = 3. As barras de erro indicam ± SEM. (E) Mock tratados com córneas foram rapidamente congeladas a-48 hpi, seccionados e analisados ​​por imunofluorescência indirecta para detectar a presença de uma proteína viral precoce (ICP8) dentro do epitélio infectado. Os núcleos são contrastadas com Höchst 33258. (F) Os lisados ​​de proteína foram recolhidas às 48 hpi e a expressão de uma proteína viral (ICP0) foi avaliada por Western blot. BCA ensaio foi utilizado para assegurar carga igual das amostras. IPH = horas após a infecção.

<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>

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Neste artigo de vídeo foi descrito um método para o estudo agudo epitelial da córnea infecção por HSV-1 em um modelo ex vivo. Este modelo é um degrau entre a utilidade in vitro e a modelos in vivo, uma vez que permite a validação fisiologicamente precisa dos resultados de cultura de células e, deste modo, limita a quantidade de experimentação animal, que deve ser realizada. Além disso, o modelo ex vivo proporciona uma oportunidade única e valiosa para estudar a i...

<table border="1"><tbody><tr><td> Reagentes / Suprimentos </td><td> Companhia </td><td> Catálogo # </td></tr><tr><td> Meio Essencial Mínimo (MEM) </td><td> Mediatech </td><td> 10-010 CV- </td></tr><tr><td> Não-aminoácidos essenciais (solução 100x) </td><td> Mediatech </td><td> 25-025 CI- </td></tr><tr><td> L-Glutamina </td><td> Mediatech </td><td> 25-005 CI- </td></tr><tr><td> Penicilina G Sal de sódio </td><td> Sigma </td><td> P3032 </td></tr><tr><td> O sal de sulfato de estreptomicina </td><td> Sigma </td><td> S9137 </td></tr><tr><td> Agarose </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> Tampão fosfato salino (PBS) </td><td> Preparado no laboratório </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 &amp; Kit Tecido </td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> Kit Mini RNeasy </td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Tampão de Laemmli </td><td> Preparado no laboratório </td><td> N / A </td></tr><tr><td> Temperatura ideal de corte composto (OCT) </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Inteiros olhos do coelho (jovem) </td><td> Pel-Freez </td><td> 41.211-2 </td></tr><tr><td> As córneas humanas e olhos inteiros </td><td> Banco de Olhos local </td><td> N / A </td></tr><tr><td> 12 Placa local bem Porcelana </td><td> Abastecimento de Avogadro Lab </td><td> 1877 </td></tr><tr><td> 35 placas de cultura de tecidos milímetros </td><td> Falcão </td><td> 353001 </td></tr><tr><td> 6 BemPlaca de cultura de células </td><td> Greiner Bio-One </td><td> 657160 </td></tr><tr><td> Cryomold Intermediário </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

Ceratite herpética é uma das mais graves patologias associadas com o vírus do herpes simplex tipo 1 (HSV-1). Ceratite herpética é actualmente a principal causa de cegueira córnea tanto derivados e infecção associada no mundo desenvolvido. Apresentação típica de ceratite herpética inclui infecção do epitélio da córnea e, por vezes, o estroma eo endotélio da córnea profunda, conduzindo a tais patologias corneanas permanentes como cicatrizes, desbaste, e opacidade 1. Corneal infecção HSV-1 é tradicionalmente estudada em dois tipos de modelos experimentais. O modelo in vitro, em que as monocamadas de culturas de células epiteliais da córnea são infectadas numa placa de Petri, oferece nível simplicidade, elevado de reprodutibilidade, experiências rápidas, e custos relativamente baixos. Por outro lado, o modelo in vivo, em que os animais, tais como coelhos ou ratos são inoculados directamente na córnea, oferece uma fisiológica altamente sofisticadosistema, mas tem custos mais elevados, experimentos mais longos, cuidado de animais necessário, e um maior grau de variabilidade. Neste artigo de vídeo, que proporcionam uma demonstração detalhada de um novo modelo ex vivo de corneal epitelial infecção HSV-1, que combina as vantagens de ambos os vitro e em modelos in vivo o. O modelo ex vivo utiliza córneas intactas organotypically mantidas em cultura e infectadas com HSV-1. A utilização do modelo ex vivo permite conclusões altamente fisiologicamente baseados, no entanto, é bastante barato e requer compromisso de tempo comparável à do modelo 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

Neste artigo de vídeo que descreve o uso de um novo<em&gt; Ex vivo</em&gt; Modelo de herpes simplex vírus tipo agudos eu infecção do epitélio da córnea.

Ex Vivo modelo organotípico córnea de Agudos Herpes simplex vírus tipo epiteliais eu Infecção

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

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Neuroscience

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

12.6K visualizações.  Drexel University College of Medicine. Neste artigo de vídeo que descreve o uso de um novo Ex vivo Modelo de herpes simplex vírus tipo agudos eu infecção do epitélio da córnea.

Vídeo: 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.

A cultura Ex vivo de todo, o desenvolvimento cerebral de 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 ...

Neurociência

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 preparações da Intacta Vomeronasal Órgão e acessórios bulbo olfatório

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.

Um<em&gt; Ex Vivo</em&gt; Induzida por laser Lesão Medular modelo para avaliar mecanismos de axonal Degeneration em tempo real

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.

Observação do movimento ciliar de células epiteliais do plexo coróide<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 optogenética Dissection de Circuitos medo no cérebro Slices

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.

Imagem latente 3D de alta resolução da infecção do vírus da raiva no tecido solvente-cancelado do cérebro

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 Visualização da Dinâmica do Cone de Crescimento e Crescimento do Axon em Culturas agudas de fatias de cérebro embrionário ex vivo

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.

Culturas organotípicas do córtex humano adulto como modelo ex vivo para transplante e validação de células-tronco humanas

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

Imagens multimodais baseadas em OCT de olhos de doadores humanos para pesquisa de DMRI

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.

Autor em destaque: Imagem multimodal baseada em OCT ex vivo de olhos de doadores humanos para pesquisa sobre degeneração macular relacionada à idade  

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 Imagem Multimodal Baseada em OCT de Olhos de Doadores Humanos para Pesquisa em Degeneração Macular Relacionada à Idade

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