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 ble utført i samsvar med politikken til Institutional Animal Care og bruk komité der det er aktuelt. 1. Forberedelse <ol><li> Klargjør stabilisering media med 75% Dulbeccos modifiserte Eagle-medium (DMEM), 25% Hanks &#39;Balanced Salt Solution (HBSS). Bland godt, delmengde og oppbevar ved -20 ° C inntil bruk. </li><li> Klargjør fosfatbufret saltvann (PBS) og sterilisere i autoklav. Oppbevar løsningen ved romtemperatur. Varm PBS til 37 ° C før forsøket (5 ml per br...

<ol>
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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 mengde bevis støtter eksistensen av blod retinal barriere (BRB) 1-5 og sin likhet til blod-hjerne barrieren (BBB) ​​6,7. Kompromiss av BBB er tett knyttet kausalt eller som en diagnostisk markør for å kroniske neurodegenerative sykdommer slik som Alzheimers sykdom (AD) 8,9 og akutte lidelser så som delirium 10. Mekanistiske innsikt i disse patologi og funn for potensielle narkotika mål er generelt hemmet av begrenset tilgjengelighet og nettverk intricacy av hjernen. Alternativer som in vivo avbildning 11, hjerne organotypiske kultur 12, primære cellekulturer 13,14 og co-kultur systemer 15 har blitt generert. Men de fleste av disse modellene krever spesielle instrumenter, lange eksperimentelle perioder eller flere markører for å identifisere celler. Funksjonelle og strukturelle likheter mellom BBB og BRB, samt en korrelasjon mellom dysfunctions av de to har blitt hevdet 16-19. I tillegg er det lettere tilgang, vel-definerte celletyper og en lagdelt struktur tillot godt karakterisert netthinnen som et vindu til hjernen. De strukturelle og funksjonelle identiteter BBB og BRB gjenstår å bli sammenlignet i detalj. Imidlertid, retinale sykdommer, spesielt BRB brudd, har også vært tett assosiert med progresjonen av forskjellige sykdommer, inklusive diabetes, 18-19 og 21,22 AD. Det er således av interesse å etablere et BRB dysfunksjon system ikke bare for å avgrense den mekanisme, men også for å screene potensielle medikamenter. I denne rapporten er en protokoll som har BRB dysfunksjon ved hjelp av en enkel akutt retinal kultur utvikles og presenteres. Økt BBB permeabilitet og AD-lignende patologiske endringer har blitt etablert i en hjerne organotypiske kultur inkuberes med histamin, en pro-inflammatorisk megler 12. Derfor, i presenterte system, histamin var ApplIED til ex vivo retinal kultur å indusere BRB dysfunksjon. Netthinne fra flere arter, for eksempel Mus musculus og Bos Taurus, har blitt testet. På grunn av sin kommersielle tilgjengelighet og likhet til menneskelig vev, ble friske svin øyeepler utnyttes til å tilby de dataene som presenteres her. Etter inkubering med histamin og / eller andre stoffer, ble netthinne bearbeidet for bedømmelse ved farging av flere proteiner 12, slik som immunoglobulin G (IgG), en av hovedkomponentene i blodet; glial fibrillary surt protein (GFAP), en kjent markør for glial aktivisering; og microtubule-assosiert protein 2 (MAP2), et nevron-spesifikk cytoskeletal protein viktig for microtubule montering. Videre er den lagdelte strukturen av netthinnen tillater en detaljert analyse av prosesser av Muller celler og ganglion-celler, slik som endringer i deres bredde og kontinuitet. Dermed flere andre parametre er tilgjengelige for å vurdere konsekvensene avBRB brudd på et tidlig stadium, og å evaluere reversering effekten av potensielle behandlinger også. I denne protokollen, er potensielle reverseringseffekter av medikamenter vist evaluert fra tre perspektiver: lekkasje av blodårer (BVS), aktivering av gliaceller og skaden-responsen av neuronale celler. Flere kvantifisering fremgangsmåter blir benyttet, for eksempel, ekspresjonsnivået er vist ved intensiteten av immunofarging, bredde måling av en prosess og kontinuitet av neuronale prosesser som vises ved en forsterkning filter. For bedre å illustrere metoden og å bidra til å tolke resultatene, lipoxin A4 (LXA4), en forbindelse endogent syntetisert i respons til inflammatoriske skader og dempe endotelial dysfunksjon 23, har blitt valgt for demonstrasjonsformål.

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			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</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 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>
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			<td height="21" style="height:21px;width:291px;">GFAP Antibody</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>
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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>
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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>
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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 presenterer en lav pris, tidsbesparende og lett-å-bruke system for å vurdere potensielle behandlinger som kan beskytte mot BRB brudd indusert av histamin. IgG er begrenset innenfor fartøyene i kontroll hinnen (figur 1A), men lekker ut av blodårene ved histamin eksponering (figur 1B), bekrefter at modellen er opprettet. LXA4 ble valgt for å demonstrere screening av medikamenter mot BRB ...

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 lav-kost, lett-å-bruke og kraftig system etablert for å vurdere potensielle behandlinger som kan bli forbedret blod retinal barriere brutt indusert av histamin. Blodkar lekkasje Muller celleaktivering og kontinuiteten av neuronale prosesser blir benyttet for å vurdere skaden respons og dens reversering med en potensiell medikament, lipoxin A4.

En akutt retinal modell for evaluering Blood Netthinne Barrier på brudd og mulige narkotika for 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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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

Oksygenindusert retinopati modell for iskemiske retinale sykdommer hos gnagere

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 induksjonssystem for avledning av 3D retinal vev 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 ...