Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
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Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

Todos los protocolos se llevaron a cabo de acuerdo con las políticas del Comité de Cuidado y Uso de Animales institucional en su caso. 1. Preparación <ol><li> Preparar los medios de estabilización con 75% de Eagle modificado por Dulbecco (DMEM), 25% solución salina equilibrada de Hanks (HBSS). Mezclar bien, alícuota y se almacena a -20 ° C hasta su uso. </li><li> Preparar el tampón fosfato salino (PBS) y esterilizar en autoclave. Almacenar la solución a temperatura ambiente. Calen...

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In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

Un creciente cuerpo de evidencia apoya la existencia de la barrera sangre de la retina (BRB) 1-5 y su similitud con la barrera hematoencefálica (BBB) ​​6,7. Compromiso de la BBB ha sido estrechamente vinculada causalmente o como un marcador de diagnóstico para enfermedades neurodegenerativas crónicas tales como la enfermedad de Alzheimer (AD) 8,9 y agudas condiciones tales como delirio 10. conocimientos mecánicos en estas patologías y descubrimientos de los posibles objetivos farmacológicos generalmente se ven obstaculizados por la accesibilidad y la red limitada complejidad del cerebro. Se han generado alternativas tales como imágenes in vivo 11, la cultura organotípicos cerebral 12, cultivos celulares primarios 13,14 y sistemas de co-cultivo 15. Sin embargo, la mayoría de estos modelos requieren instrumentos especiales, los períodos experimentales largos o múltiples marcadores para identificar las células. similitudes funcionales y estructurales entre BBB y BRB, así como una correlación entre la dysfunctions de los dos se han argumentado 16-19. Además, el acceso más fácil, tipos de células bien definidas y una estructura en capas han permitido a la retina bien caracterizado como una ventana al cerebro. Las identidades estructurales y funcionales de la BBB y BRB aún no se han comparado en detalle. Sin embargo, las patologías de la retina, especialmente la violación BRB, también han sido estrechamente asociado con la progresión de diversas enfermedades, incluyendo la diabetes 18-19 y 21,22 AD. Por lo tanto, es de interés para establecer un sistema de disfunción BRB no sólo para delinear el mecanismo, pero también para cribar fármacos potenciales. En este informe, un protocolo que permite la disfunción BRB mediante un sencillo cultura retiniana aguda es desarrollado y presentado. Aumento de la permeabilidad de la BHE y cambios patológicos AD-como se han establecido en una cultura organotípicos cerebro se incubaron con histamina, un mediador proinflamatorio 12. Por lo tanto, en el sistema presentado, la histamina era applIED hasta el cultivo ex vivo para inducir la disfunción retiniana BRB. Las retinas de varias especies, tales como Mus musculus y Bos Taurus, han sido probados. Debido a su disponibilidad comercial y semejanza con el tejido humano, se utilizaron ojos porcinos frescos para proporcionar los datos aquí presentados. Después de incubar con la histamina y / o otras drogas, las retinas se procesaron para su evaluación por la inmunotinción para varias proteínas de 12, tales como la inmunoglobulina G (IgG), uno de los principales componentes de la sangre; proteína ácida glial fibrilar (GFAP), un marcador conocido para la activación glial; y la proteína asociada a microtúbulos 2 (MAP2), una proteína del citoesqueleto neuronal específica esencial para el ensamblaje de los microtúbulos. Además, la estructura en capas de la retina permite un análisis detallado de los procesos de las células de Müller y células ganglionares, tales como cambios en su anchura y continuidad. Por lo tanto varios parámetros adicionales están disponibles para evaluar las consecuencias de laBRB incumplimiento en una etapa temprana y para evaluar los efectos de reversión de tratamientos potenciales. En este protocolo, los posibles efectos de reversión de los fármacos seleccionados se evalúan desde tres perspectivas: la fuga de los vasos sanguíneos (BVS), la activación de células gliales y el daño de respuesta de las células neuronales. Se utilizan varios métodos de cuantificación, por ejemplo, el nivel de expresión se muestra por la intensidad de la inmunotinción, la medición de la anchura de un proceso y la continuidad de los procesos neuronales se muestra por un filtro de mejora. Para ilustrar mejor el método y para ayudar a interpretar los resultados, la lipoxina A4 (LXA4), un compuesto sintetizado de forma endógena en respuesta a una lesión inflamatoria y atenuar la disfunción endotelial 23, ha sido elegido con fines de demostración.

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			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
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			<td></td>
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			<td height="21" style="height:21px;width:291px;">PFA</td>
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			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
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			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
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			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
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			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
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			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
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			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
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			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
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			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
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			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">1.45s</td>
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an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

Presentamos un sistema en tiempo eficiente y fácil de usar, de bajo costo para evaluar los tratamientos potenciales que podrían proteger contra la violación BRB inducida por la histamina. IgG se ve limitada dentro de los vasos de la retina de control (Figura 1A), pero se escapa de los vasos sanguíneos tras la exposición histamina (Figura 1B), lo que confirma que el modelo se ha establecido correctamente. <p class="jove_content" fo:keep-together....

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Un bajo costo, se establece un sistema fácil de usar y potente para evaluar posibles tratamientos que podrían aminorar incumplimiento barrera sangre retiniana inducida por la histamina. fuga de los vasos sanguíneos, la activación de células Müller y la continuidad de los procesos neuronales se utilizan para evaluar la respuesta al daño y su inversión con un fármaco potencial, la lipoxina A4.

Un modelo agudo de retina para evaluar sanguíneos de la retina barrera violación, así como potenciales fármacos para el tratamiento

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

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An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Un método In Vivo para evaluar la barrera intestinal-sangre y hígado metabolismo de la Microbiota de productos

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

Modelo de retinopatía inducida por oxígeno para enfermedades retinianas isquémicas en roedores

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Sistema de inducción organoide retiniana para la derivación de tejidos retinianas 3D a partir de células madre pluripotentes humanas

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...