El apoyo del Banco Leonístico de Delaware Valley fue muy valiosa para este trabajo. Anticuerpo monoclonal de ratón contra ICP8 primaria fue una especie de regalo del doctor David Knipe (Harvard Medical School). También agradecemos al Dr. Stephen Jennings (Drexel University College of Medicine) y el Dr. Peter Laibson (Wills Eye Institute) útil para los debates y la experiencia.

Todos los reactivos y equipos se compraron estéril o se esterilizaron antes del procedimiento. En este artículo se describen los pasos para la implantación del modelo ex vivo a partir de la vista pre-enucleado. En nuestros experimentos, utilizamos ojos frescos enteros de jóvenes (8-12 semanas) conejos albinos (Pel-Freez Biologicals, Rogers, AR). Además, utilizamos córneas humanas obtenidas a partir del banco de ojos local. Si se realiza enucleación usted mismo, tenga cuidado de no dañar la córnea o el limbo durante el procedimiento. Protocolos de conejo enucleación se puede encontrar en otra parte 2. Parte 1: La escisión del botón Corneoscleral <ol><li> Moje una estrecha franja de gasa con PBS y se envuelve alrededor del globo ocular para facilitar la celebración durante el procedimiento. </li><li> Utilice un bisturí afilado para hacer una incisión en la esclerótica tangencial, de aproximadamente 0,5 cm de distancia del limbo. </li><li> Use tijeras afiladas para extender la incisión y completamente cortar el corneoscleral botón. </li><li> Una vez que el botón corneoescleral se aísla, se burlan de lejos el iris mientras sostiene el borde escleral con fórceps. Evite dañar el endotelio o ejercer una tensión excesiva en la córnea. </li></ol> Parte 2: Introducción de la córnea en cultivo organotípico <ol><li> Córneas explantados se cultivaron en medio esencial mínimo (MEM) suplementado con no aminoácidos esenciales (1X), L-glutamina (2 mM), penicilina (200 U / ml) y estreptomicina (200 ug / ml). Un agente antifúngico, tal como anfotericina, también se puede añadir si es necesario. </li><li> Preparar una solución al 1% de agarosa en medio de cultivo y mantenerlo listo a 55 ° C. </li><li> Enjuague completamente la córnea en PBS estéril que contiene penicilina (200 U / ml) y estreptomicina (200 ug / ml). </li><li> Coloque el lado epitelial de la córnea hacia abajo en un pocillo de una placa estéril lugar. </li><li> Añadir el medio de agarosa que contiene a la concavidad endotelial de la córnea justo lo suficiente parallenar por completo. Permitir ~ 1 min para que la agarosa se solidifique. </li><li> Coloque la córnea con el medio de cultivo de soporte de gel de andamio lado epitelial hacia arriba en una placa de cultivo tisular de 35 mm y añadir a cubrir la superficie epitelial. </li><li> La córnea puede ser cultivado de este modo por más de una semana, con cambios de medio cada 48 horas. </li></ol> Parte 3: La infección por HSV-1 y el tratamiento con medicamentos experimentales <ol><li> Añadir 1 ml de medio que contiene la cantidad deseada de virus en una placa de cultivo tisular de 35 mm. Por lo general infectan con 1x10 4 PFU de la cepa KOS de HSV-1 por córnea. Los títulos valores se determinan mediante la realización de ensayos de placas en monocapas de CV-1. </li><li> Coloque la córnea, junto con el lado de agarosa andamio epitelial hacia abajo en el medio que contiene virus. </li><li> Coloque la córnea en una incubadora de cultivo de tejido para infectar durante 1 hora con agitación intermitente cada 10-15 minutos. </li><li> Después de 1 hora, aspirar el virus-contaiNing medio y enjuague 2-3 veces con PBS para eliminar cualquier partícula viral residual. </li><li> Coloque la córnea vuelve a su posición anterior y se añade medio de cultivo fresco para cubrir la superficie epitelial. </li><li> Los tratamientos experimentales se puede iniciar antes o después de la infección, dependiendo de la naturaleza del experimento. </li><li> El medio de cultivo se cambió cada 48 horas. </li></ol> Parte 4: Toma de Muestras <ol class="lalpha"><li> Medio para el análisis de la placa de ensayo. Mezclar bien el medio y utilizar de inmediato para un ensayo de placa o almacenar a -80 ° C para su uso posterior si la recolección de muestras en diferentes puntos temporales. </li><li> ADN, ARN, proteína o células de epitelio corneal <ol><li> Retire el andamio de agarosa y enjuague bien la córnea con PBS. </li><li> Escindir el anillo de tejido de la esclerótica de la córnea para asegurar que el material epitelial corneal sólo se recoge. </li><li> Ucantar un escalpelo, raspar las células epiteliales de la córnea en el tampón apropiado, en función del tipo de material a ser aislado. Para el aislamiento de ADN y ARN, se utiliza el kit DNeasy Blood &amp; Tissue y el kit RNeasy Mini, respectivamente (QIAGEN, Hilden, Alemania). Para la recolección de los lisados ​​de proteínas, se utiliza tampón Laemmli. Para el análisis de citometría de flujo de células epiteliales corneales, se pre-incuban las córneas en una pequeña cantidad de tripsina para desprender las células y, a continuación desalojarlos en tripsina con un escalpelo. Desde raspado el epitelio corneal no produce cantidades iguales de tejido, es importante incluir controles adecuados en el análisis de las muestras. Usamos ARNr 18S como un transcrito de referencia en QRT-PCR, GAPDH como un gen de referencia en qPCRs, y nucleolina como control de carga para las transferencias Western. </li></ol></li><li> Las muestras de tejidos para inmunohistoquímica o inmunofluorescencia <ol><li> Retire el andamio de agarosa y enjuagar el maízea bien con PBS. </li><li> Dependiendo de su objetivo experimental, el uso de la córnea para hacer un bloque de tejido en parafina o un bloque de tejido congelado, como se muestra en el vídeo. Seguimos procedimientos estándar en la preparación de secciones de tejido corneal. </li></ol></li></ol> Los resultados representativos La mayoría de los métodos disponibles para el estudio de células en cultivo de tejidos puede ser fácilmente adaptado para el uso en las córneas infectadas con HSV-1 ex vivo. Aquí se presentan resultados representativos de algunas de las técnicas más comunes aplicables al estudio de la infección por HSV-1 - qPCR (Figura 1 D), QRT-PCR (Figura 1B), Western blot (Figura 1F), la placa de ensayo (Figura 1 C), y tejido de inmunofluorescencia (Figura 1E). <img src="/files/ftp_upload/3631/3631fig1.jpg" alt="Figura 1" /> Figura 1. Análisis de las córneas infectadas con HSV-1 v exivo. (A) Representación esquemática del modelo ex vivo de aguda corneal epitelial infección HSV-1. (BF) Análisis de córneas infectadas utilizando el método descrito en este artículo de vídeo. Explantados córneas de conejo fueron infectadas con 1x10 4 PFU / córnea de la cepa KOS de HSV-1 y se cultivaron en presencia (PAA) o ausencia (Mock) de ácido fosfonoacético (400 ug / ml), un inhibidor conocido de la replicación del HSV. (B) El ARN total se recogieron en los puntos de tiempo indicados, y la transcripción viral se evaluó mediante qRT-PCR con cebadores para HSV-1 polimerasa. Primers contra 18S rRNA fueron utilizados como una referencia. n = 3. Las barras de error indican ± SEM. (C) El medio de cultivo se recogió a 48 hpi, y la producción de partículas infecciosas se evaluó mediante ensayo en placa sobre monocapas de CV-1. (D) El ADN total se recogió a 48 hpi, y la replicación del genoma viral se evaluó en un ensayo de qPCR con los cebadores para el HSV-1 del genoma. PrIMers contra GAPDH se utiliza como referencia. n = 3. Las barras de error indican ± SEM. (E) Mock tratados con córneas se flash congelado a 48 hpi, se seccionaron, y se analizaron por inmunofluorescencia indirecta para la presencia de una proteína viral temprana (ICP8) dentro del epitelio infectado. Los núcleos se tiñen con Höchst 33258. (F) Proteína lisados ​​se recogieron a las 48 hpi, y la expresión de una proteína viral (ICP0) se evaluó mediante Western blot. BCA ensayo se utilizó para garantizar la igualdad de carga de muestras. hpi = horas después de la infección.

<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>

No hay conflictos de interés declarado.

En este artículo de vídeo se describe un método para el estudio agudo corneal epitelial infección HSV-1 en un modelo ex vivo. Este modelo es un escalón útil entre la in vitro y la modelos in vivo, ya que permite la validación fisiológicamente precisa de los resultados de cultivo de células y, por lo tanto, limita la cantidad de experimentación animal que debe ser realizada. Además, el modelo ex vivo ofrece una oportunidad única y valiosa para estudiar la infecció...

<table border="1"><tbody><tr><td> Reactivos / Materiales </td><td> Empresa </td><td> Catálogo # </td></tr><tr><td> Medio Esencial Mínimo (MEM) </td><td> Mediatech </td><td> 10 a 010-CV </td></tr><tr><td> No aminoácidos esenciales (solución 100x) </td><td> Mediatech </td><td> 25 a 025-CI </td></tr><tr><td> L-Glutamina </td><td> Mediatech </td><td> 25 a 005-CI </td></tr><tr><td> La penicilina G sal de sodio </td><td> Sigma </td><td> P3032 </td></tr><tr><td> Sal de sulfato de estreptomicina </td><td> Sigma </td><td> S9137 </td></tr><tr><td> Agarosa </td><td> Invitrogen </td><td> 15510-027 </td></tr><tr><td> Tampón fosfato salino (PBS) </td><td> Preparada en laboratorio </td><td> N / A </td></tr><tr><td> Tripsina EDTA (1x) &lt;/ Td&gt; <td> Mediatech </td><td> 25 a 053-CI </td></tr><tr><td> Sangre y DNeasy Tissue Kit </td><td> QIAGEN </td><td> 69504 </td></tr><tr><td> RNeasy Mini Kit </td><td> QIAGEN </td><td> 74104 </td></tr><tr><td> Tampón de Laemmli </td><td> Preparada en laboratorio </td><td> N / A </td></tr><tr><td> Temperatura óptima de corte (OCT) Compuesto </td><td> Tissue-Tek </td><td> 4583 </td></tr><tr><td> Ojos enteros conejo (joven) </td><td> Pel-Freez </td><td> 41211-2 </td></tr><tr><td> Las córneas humanas y los ojos enteros </td><td> Banco de Ojos Local </td><td> N / A </td></tr><tr><td> 12 Pues Porcelana Plata al contado </td><td> Suministro de Avogadro Lab </td><td> 1877 </td></tr><tr><td> 35 mm placa de cultivo de tejidos </td><td> Halcón </td><td> 353001 </td></tr><tr><td> 6 BienPlaca de cultivo de la célula </td><td> Greiner Bio-One </td><td> 657160 </td></tr><tr><td> Criomolde 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

La queratitis herpética es una de las patologías más graves asociadas con el virus del herpes simple de tipo 1 (HSV-1). La queratitis herpética es actualmente la principal causa de ceguera tanto córnea-derivado y las infecciones asociadas-en el mundo desarrollado. Presentación típica de la queratitis por herpes incluye la infección del epitelio corneal y el estroma a veces córnea más profunda y el endotelio, lo que conduce a patologías corneales tales como cicatrices permanentes, adelgazamiento, y la opacidad 1. Corneal infección HSV-1 es tradicionalmente estudiado en dos tipos de modelos experimentales. El modelo in vitro, en el que las monocapas de cultivo de células epiteliales corneales infectadas en una placa de Petri, ofrece simplicidad, alto nivel de replicabilidad, experimentos rápidos y los costos relativamente bajos. Por otro lado, el modelo in vivo, en el que los animales tales como conejos o ratones se inoculan directamente en la córnea, ofrece una fisiológica altamente sofisticadosistema, pero tiene costos más altos, más largos experimentos, cuidado de animales necesario, y un mayor grado de variabilidad. En este artículo de vídeo, se proporciona una demostración detallada de un modelo ex vivo de nuevo corneal epitelial infección HSV-1, que combina las ventajas tanto de la vitro y en modelos in vivo en el. El modelo ex vivo utiliza córneas intactas organotypically mantenidas en cultivo e infectadas con HSV-1. El uso del modelo ex vivo permite conclusiones altamente fisiológicamente basadas, sin embargo, es bastante barato y requiere dedicación de tiempo comparable a la del 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

En este artículo de vídeo se describe el uso de un nuevo<em&gt; Ex vivo</em&gt; Modelo de herpes simplex virus tipo agudas que la infección del epitelio corneal.

Ex Vivo organotípicos Modelo corneal aguda de herpes simplex virus tipo epitelial que la infección

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 vistas.  Drexel University College of Medicine. En este artículo de vídeo se describe el uso de un nuevo Ex vivo Modelo de herpes simplex virus tipo agudas que la infección del epitelio corneal.

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 cultivo in vivo de la Plenaria, los cerebros en desarrollo 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 ...

Investigación

Neurociencias

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 la Producción de la Bombilla órgano vomeronasal y accesorios olfativa Intacto

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.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo 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.

Observación del Movimiento ciliar de las células epiteliales del plexo coroideo<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 Disección de circuitos del miedo en rodajas de cerebro

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.

Imágenes 3D de alta resolución de la infección por el virus de la rabia en el tejido cerebral con disolventes

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 Visualización del crecimiento de axones y la dinámica de los conos de crecimiento en cultivos agudos de cortes cerebrales embrionarios 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.

Cultivos organotípicos de corteza humana adulta como modelo ex vivo para el trasplante y validación de células madre humanas

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

Imágenes multimodales basadas en OCT de ojos de donantes humanos para la investigación de la DMAE

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 destacado: Imágenes multimodales ex vivo basadas en OCT de ojos de donantes humanos para la investigación de la degeneración macular relacionada con la edad  

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 Imágenes multimodales basadas en OCT de ojos de donantes humanos para la investigación de la degeneración macular relacionada con la edad

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