Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00
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.

Tutti i protocolli sono stati effettuati nel rispetto delle norme del Comitato di cura e l&#39;uso di animali istituzionale, se del caso. 1. Preparazione <ol><li> Preparare il supporto di stabilizzazione con il 75% di Dulbecco Modified Eagle Medium (DMEM), il 25% Hanks &#39;soluzione salina bilanciata (HBSS). Mescolare bene, aliquotare e conservare a -20 ° C fino al momento dell&#39;uso. </li><li> Preparare il tampone fosfato (PBS) e sterilizzare in autoclave. Conservare la soluzione a te...

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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 crescente corpo di prove sostiene l&#39;esistenza di barriera retinica sangue (BRB) 1-5 e la sua somiglianza con la barriera emato-encefalica (BBB) ​​6,7. Compromesso della BBB è stata strettamente legata causalmente o come marcatore diagnostico per le malattie neurodegenerative croniche come il morbo di Alzheimer (AD) 8,9 e acuti condizioni come il delirio 10. intuizioni meccanicistici in queste patologie e scoperte per i potenziali bersagli farmacologici sono generalmente ostacolati dalla limitata accessibilità e network complessità del cervello. Alternative come l&#39;imaging in vivo 11, cervello organotipica cultura 12, colture cellulari primarie 13,14 e sistemi di co-coltura di 15 sono stati generati. Tuttavia, la maggior parte di questi modelli richiede strumenti speciali, lunghi periodi sperimentali o più marcatori per identificare le cellule. somiglianze funzionali e strutturali tra BBB e BRB nonché una correlazione tra la dysfunctions dei due sono stati sostenuto 16-19. Inoltre, un più facile accesso, tipi cellulari ben definiti e una struttura stratificata hanno consentito la retina ben caratterizzato come una finestra al cervello. Le identità strutturali e funzionali della BBB e BRB ancora essere confrontato nel dettaglio. Tuttavia, patologie retiniche, soprattutto la breccia BRB, sono stati strettamente associati con la progressione di varie malattie, compreso il diabete 18-19 e AD 21,22. Pertanto, è di interesse per stabilire un sistema disfunzione BRB non solo per delineare il meccanismo ma anche per schermo potenziali farmaci. In questo rapporto, un protocollo che consente BRB disfunzione utilizzando una semplice cultura della retina acuta è sviluppato e presentato. Aumento della permeabilità BBB e cambiamenti patologici AD-simili sono stati stabiliti in una cultura organotipica cervello incubate con l&#39;istamina, un mediatore pro-infiammatorio 12. Pertanto, nel sistema presentato, istamina era applied alla cultura della retina ex vivo per indurre disfunzioni BRB. Retine da diverse specie, come il Mus musculus e Bos taurus, sono stati testati. Grazie alla loro disponibilità commerciale e somiglianza con tessuti umani, bulbi oculari suina fresca sono stati utilizzati per fornire i dati qui riportati. Dopo incubazione con istamina e / o altri farmaci, le retine sono state preparate per la valutazione mediante immunocolorazione per diverse proteine ​​12, come immunoglobuline G (IgG), uno dei principali componenti del sangue; proteina silicea fibrillare gliale (GFAP), un indicatore ben noto per l&#39;attivazione gliale; e proteine ​​associate ai microtubuli 2 (MAP2), una specifica dei neuroni del citoscheletro proteina essenziale per l&#39;assemblaggio dei microtubuli. Inoltre, la struttura a strati della retina consente un&#39;analisi dettagliata dei processi di cellule Müller e cellule gangliari, quali i cambiamenti nella loro larghezza e continuità. Così diversi parametri aggiuntivi sono disponibili per valutare le conseguenze dellaBRB violazione in una fase iniziale e per valutare gli effetti di inversione di potenziali trattamenti pure. In questo protocollo, i potenziali effetti di inversione dei farmaci schermati vengono valutati da tre prospettive: la fuoriuscita di vasi sanguigni (BVS), l&#39;attivazione delle cellule gliali e il danno-risposta delle cellule neuronali. Diversi metodi di quantificazione sono utilizzati, per esempio, il livello di espressione dimostra intensità della immunocolorazione, misura della larghezza di un processo e la continuità dei processi neuronali mostrati da un filtro accessorio. Per meglio illustrare il metodo e per interpretare i risultati, Lipoxin A4 (LXA4), un composto endogeno sintetizzato in risposta al danno infiammatorio e attenuando la disfunzione endoteliale 23, è stata scelta a scopo dimostrativo.

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			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
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			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></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.

Vi presentiamo un basso costo, sistema di time-efficiente e facile da usare per valutare i potenziali trattamenti che potrebbero proteggere contro la violazione BRB indotta da istamina. IgG è vincolato all&#39;interno dei vasi in retina di controllo (Figura 1A), ma le perdite fuori dei vasi sanguigni su esposizione istamina (Figura 1B), a conferma che il modello è stata stabilita. LXA4 è st...

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

A basso costo, il sistema facile da usare e potente è stabilito per valutare i potenziali trattamenti che potrebbero migliorare barriera emato-retinica violazione indotta da istamina. perdita dei vasi sanguigni, l&#39;attivazione delle cellule Müller e la continuità dei processi neuronali sono utilizzati per valutare la risposta al danno e la sua inversione con un potenziale farmaco, Lipoxin A4.

Un modello acuta della retina per la valutazione sanguigni della retina barriera breccia e potenziali farmaci per il trattamento

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

Un metodo In Vivo per valutare la Gut-barriera e metabolismo epatico del Microbiota prodotti

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

Modello di retinopatia indotta dall'ossigeno per le malattie retiniche ischemiche nei roditori

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 di induzione organoide retinica per la derivazione di tessuti retinici 3D da cellule staminali pluripotenti umane

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