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.

Alle protokoller blev udført i overensstemmelse med den politik, Institutional Animal Care og brug Udvalg givet fald. 1. Fremstilling <ol><li> Forbered stabilisering medier med 75% Dulbeccos modificerede Eagle medium (DMEM), 25% Hanks balancerede saltopløsning (HBSS). Bland godt, alikvot og opbevares ved -20 ° C indtil brug. </li><li> Forbered phosphatbufrede saltvand (PBS) og der steriliseres ved autoklavering. opløsningen ved stuetemperatur lagre. Opvarm PBS til 37 ° C før forsøge...

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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 -...

En voksende mængde beviser understøtter eksistensen af blod retina-barrieren (BRB) 1-5 og dens lighed med blodhjernebarrieren (BBB) ​​6,7. Kompromittering af BBB er blevet tæt forbundet kausalt eller som en diagnostisk markør til kroniske neurodegenerative sygdomme, såsom Alzheimers sygdom (AD) 8,9 og akutte tilstande, såsom delirium 10. Mekanistiske indsigt i disse patologier og opdagelser for potentielle lægemiddelmål er generelt hæmmet af den begrænsede tilgængelighed og netværk forviklinger af hjernen. Alternativer som in vivo imaging 11, hjerne organotypisk kultur 12, primære cellekulturer 13,14 og co-dyrkningssystemer 15 er blevet genereret. Men de fleste af disse modeller kræver særlige instrumenter, lange eksperimentelle perioder eller flere markører til at identificere celler. Funktionelle og strukturelle ligheder mellem BBB og BRB samt en sammenhæng mellem dysfunctions af de to har været fremført 16-19. Desuden har lettere adgang, veldefinerede celletyper og en lagdelt struktur tillod velkarakteriserede nethinden som et vindue til hjernen. De strukturelle og funktionelle identitet BBB og BRB mangler at blive sammenlignet i detaljer. Imidlertid retinale patologier, især BRB brud, har også været tæt forbundet med progressionen af forskellige sygdomme, herunder diabetes 18-19 og AD 21,22. Det er således af interesse for at etablere en BRB dysfunktion system, ikke kun at afgrænse den mekanisme, men også til at screene potentielle lægemidler. I denne rapport er en protokol, der gør det muligt BRB dysfunktion hjælp af en simpel akut retinal kultur udviklet og præsenteret. Øget BBB permeabilitet og AD-lignende patologiske ændringer er blevet etableret i en hjerne organotypisk kultur inkuberes med histamin, et pro-inflammatorisk mediator 12. Derfor, i den præsenterede system histamin var applIED til ex vivo retinal kultur at fremkalde BRB dysfunktion. Nethinder fra flere arter, såsom Mus musculus og Bos Taurus, er blevet testet. På grund af deres kommercielle tilgængelighed og lighed med humant væv, blev friske svin øjne udnyttes til at levere de rapporterede data her. Efter inkubering med histamin og / eller andre lægemidler, blev nethinder forarbejdet til evaluering ved immunofarvning for adskillige proteiner 12, såsom immunoglobulin G (IgG), en af de vigtigste bestanddele af blodet; glial fibrillært surt protein (GFAP), en velkendt markør for glial aktivering; og mikrotubulus-associeret protein 2 (MAP2), et neuron-specifikt cytoskeletprotein afgørende for mikrotubulus-samling. Endvidere den lagdelte struktur af nethinden tillader en detaljeret analyse af de processer af Müller celler og ganglieceller, såsom ændringer i deres bredde og kontinuitet. Således flere andre parametre er til rådighed til at vurdere konsekvenserne af denBRB brud på et tidligt tidspunkt, og at evaluere tilbageførsel virkninger af potentielle behandlinger samt. I denne protokol, er potentielle vending effekter af screenede lægemidler vurderes ud fra tre perspektiver: lækage af blodkar (BvS), aktivering af gliaceller og skaden-respons af neuronale celler. Adskillige kvantificeringsmetoder anvendes for eksempel ekspressionsniveauet vist ved intensiteten af ​​immunfarvning, bredde måling af en proces og kontinuitet i neuronale processer vist ved en forøgelse filter. For bedre at illustrere metoden og til at hjælpe med at fortolke resultaterne, lipoxin A4 (LXA4), en forbindelse endogent syntetiseret som reaktion på inflammatorisk skade og formildende endotel dysfunktion 23, er blevet valgt til demonstration.

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			<td height="21" style="height:21px;width:291px;">DMEM</td>
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			<td height="21" style="height:21px;width:291px;">HBSS</td>
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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>
			<td style="width:119px;"></td>
			<td></td>
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		<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>
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			<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>
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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>
			<td></td>
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		<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>
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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>
			<td></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.

Vi præsenterer en billig, tidsbesparende og nem at bruge systemet til at vurdere potentielle behandlinger, der kunne beskytte mod BRB brud fremkaldt af histamin. IgG er begrænset inden for de fartøjer i kontrol nethinden (figur 1A), men lækker ud af blodkarrene upon histamin eksponering (figur 1B), bekræfter, at modellen med succes er etableret. LXA4 blev valgt til at demonstrere screenin...

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

En billig, er nem at bruge og kraftfulde system, der etableres for at vurdere de potentielle behandlinger, som kunne lindre blod nethinde barriere brud fremkaldt af histamin. Blodkar lækage, Müller-celleaktivering og kontinuitet i neuronale processer anvendes for at vurdere skaden respons og dens tilbageførsel med et potentielt lægemiddel, lipoxin A4.

En akut Retinal Model for Evaluering Blood Retinal Barrier tilsidesættelse og potentielle lægemidler til behandling

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

En In Vivo metode til evaluering af Gut-blod barriere og lever metabolisme af mikrobiota produkter

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

Oxygen-induceret retinopati Model for iskæmiske retinale sygdomme hos gnavere

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

Retinal organoid induktionssystem til afledning af 3D Retinal-væv fra humane pluripotente stamceller

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