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.

Alla protokoll utfördes i enlighet med den politik som Institutional Animal Care och användning kommittén i förekommande fall. 1. Beredning <ol><li> Förbereda stabiliseringen media med 75% Dulbeccos Modified Eagle Medium (DMEM), 25% Hanks balanserade saltlösning (HBSS). Blanda väl, delprov och förvara vid -20 ° C fram till användning. </li><li> Förbered Fosfatbuffrad saltlösning (PBS) och sterilisera i autoklav. Lagra lösningen vid rumstemperatur. Värma PBS till 37 ° C innan...

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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 växande mängd bevis stöder förekomsten av blod retinal barriären (BRB) 1-5 och dess likhet med blod-hjärnbarriären (BBB) ​​6,7. Kompromiss av BBB har tätt kopplade kausalt eller som en diagnostisk markör för kroniska neurodegenerativa sjukdomar såsom Alzheimers sjukdom (AD) 8,9 och akuta tillstånd såsom delirium 10. Mekanistiska insikter i dessa sjukdomar och upptäckter för potentiella läkemedelsmål i allmänhet hindras av den begränsade tillgängligheten och nätverk intrikat av hjärnan. Alternativ som in vivo imaging 11, hjärna organotypic kultur 12, primära cellkulturer 13,14 samt co-odlingssystem 15 har genererats. Men de flesta av dessa modeller kräver speciella instrument, långa experimentella perioder eller flera markörer för att identifiera celler. Funktionella och strukturella likheter mellan BBB och BRB samt ett samband mellan dysfunctions av två ha kunnat hävda 16-19. Dessutom har lättare tillgång till väldefinierade celltyper och en skiktad struktur tillät väldefinierad näthinnan som ett fönster till hjärnan. De strukturella och funktionella identiteter BBB och BRB återstår att jämföras i detalj. Emellertid retinala patologier, särskilt BRB brott, har också tätt associerad med framskridandet av olika sjukdomar, inklusive diabetes 18-19 och AD 21,22. Således är det av intresse att upprätta ett BRB dysfunktion systemet inte bara för att beskriva mekanismen utan även för att screena potentiella läkemedel. I denna rapport, är ett protokoll som gör det möjligt BRB dysfunktion med hjälp av en enkel akut retinal kultur utvecklas och presenteras. Ökad BBB permeabilitet och AD-liknande patologiska förändringar har fastställts i en hjärna organotypic kultur inkuberades med histamin, en pro-inflammatoriska medlare 12. Därför, i den presenterade systemet, histamin var applerat till ex vivo retinal kultur för att inducera BRB dysfunktion. Näthinnor från flera arter, såsom Mus musculus och Bos Taurus, har testats. På grund av deras kommersiella tillgänglighet och likhet med mänsklig vävnad, var färska svin ögonglober utnyttjas för att ge de uppgifter som rapporteras här. Efter inkubering med histamin och / eller andra läkemedel, var näthinnorna bearbetas för utvärdering genom immunfärgning för flera proteiner 12, såsom immunglobulin G (IgG), en av de viktigaste komponenterna i blodet; glia fibrillärt surt protein (GFAP), en välkänd markör för glial aktivering; och mikrotubuli-associerat protein 2 (MAP2), ett neuronspecifikt cytoskelettprotein viktigt för mikrotubuli montering. Dessutom tillåter den skiktade strukturen av näthinnan en detaljerad analys av de processer Müller celler och ganglieceller, såsom förändringar i bredd och kontinuitet. Således flera ytterligare parametrar finns tillgängliga för att bedöma konsekvenserna avBRB brott på ett tidigt stadium och att utvärdera återföring effekterna av potentiella behandlingar. I detta protokoll, potentiella återföring effekterna av avskärmade läkemedel utvärderas ur tre perspektiv: läckage av blodkärl (BvS), aktivering av gliaceller och skade respons av neuronala celler. Flera kvantifieringsmetoder utnyttjas, till exempel, expressionsnivån visas genom intensiteten av immunfärgning, breddmätning av en process och kontinuitet i neuronala processer som visas av en förstärkningsfilter. För att bättre illustrera förfarandet och hjälpa till att tolka resultaten, lipoxin A4 (LXA4), en förening endogent syntetiserat som svar på inflammatoriska skador och dämpa endoteldysfunktion 23, har valts för demonstrationssyfte.

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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>
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			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</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>
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			<td height="21" style="height:21px;width:291px;">MAP2 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>
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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>
			<td style="width:269px;">Nikon</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 presenterar en låg kostnad, tidseffektiv och enkel att använda system för att utvärdera potentiella behandlingar som kan skydda mot BRB brott induceras av histamin. IgG begränsas inom kärlen i kontroll näthinnan (Figur 1A), men läcker ut i blodkärlen vid histamin exponering (Figur 1B), bekräftar att modellen framgångsrikt etablerat. LXA4 valdes för att demonstrera screening för...

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 låg kostnad, är enkel att använda och kraftfullt system inrättas för att utvärdera potentiella behandlingar som kan lindra blod retinal barriärbrott inducerad av histamin. Blodkärlsläckage, Müller-cellsaktivering och kontinuiteten i neuronala processer används för att bedöma skadorna respons och dess återföring med ett potentiellt läkemedel, lipoxin A4.

En akut retinal modell för utvärdering Blood Retinal Barrier överträdelse och potentiella läkemedel för 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 -Metod för utvärdering av Gut-blod-barriären och Metabolism i levern av bakterieflora 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

Syreinducerad retinopatimodell för ischemiska näthinnesjukdomar hos gnagare

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 för härledning av 3D retinal vävnader från mänskliga pluripotenta stamceller

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