Name: a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)
Uploaded: 2014-06-06T14:15:00
Description: Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

Forfatterne anerkender det bidrag Hammond Bay Biological Station, Great Lakes Science Center, US Geological Survey. Vi takker også Dr. Melinda Frame ved Center for Advanced Microscopy ved Michigan State University for hendes tekniske support i laserscanning konfokal mikroskopi. Denne undersøgelse er støttet af tilskud fra De Store Søers Fiskerikommission til YWCD og WML.

1. Solution Preparation <ol><li> Lav 1 L 10x 0,1 M phosphatbuffer saltvand (PBS, pH 7,4): 26,2 g natriumphosphat (monobasisk), 115 g natriumphosphat (dibasisk), og 87,66 g NaCl nøjagtighed. Opløses i cirka 800 ml destilleret H 2 O, justere pH og bringe lydstyrken op til 1 l med destilleret H 2 O. <ol><li> Lav 1 L 0,1 M phosphatpuffer saltvand (pH 7,4): Tag 100 ml 10x PBS, og der tilsættes 900 ml destilleret H 2 O. </li><li> Lav 1 L 0,1 M fosfatbuffer saltvand (pH 7,4) med 0,1% Trito...

<ol>
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Denne protokol er modificeret ud fra en ny metode kaldet &quot;klarhed&quot; 12, som tværbinder intakt væv med polyacrylamid til dannelse af en nanoporøse hydrogel og derefter strimler væk plasmamembranen af vævet for at opnå optisk transparens og makromolekylære permeabilitet. &quot;Klarhed&quot; giver intakt væv billeddannelse af langtrækkende projektion og lokale kredsløb ledninger i nervesystemet. Denne nye metode kan anvendes til at visualisere hele galdevejen hos havlampret leveren under metam...

Havlampret udvikle sig gennem tre forskellige livsstadier 1,2. Larve havlampret (L) tilbringer mest tid i jordhuler som bundlevende filtratorer. Efter at have gået gennem syv metamorfe stadier af dramatiske ændringer i ydre morfologi og reorganisering i indre organer 3, de resulterende unge (JV) indtaste en parasitisk stadium, hvor de lever af blod og vævsvæsker fra host fisk, øge kropsmasse mere end 100 gange . Efter 1,0-1,5 years fodring på værten fisk i havet eller store søer, voksne ophøre fodring i det tidlige forår og migrere ud i floder for at gyde og derefter dø 1,2. Under metamorfose, det havlampret leveren mister galdeblære og hele galdetræet, en evolutionær mutantfænotypen der efterligner den menneskelige spædbarn sygdom galdeatresi. Infant galdeatresi er en sjælden pædiatrisk leversygdom med svære medicinske komplikationer 4,5,6,7, 8,9,10, men patogenese og ætiologien af galdeatresi er stort set ukendte 4. Patienter med galdeatresi dø inden for to år efter fødslen, medmindre kirurgisk indgreb (Kasai procedure) udføres 5. Efterfølgende disse patienter kræver omfattende kliniske ledelse og ofte levertransplantation 6. Mange teorier galdeatresi ætiopatogenese er blevet foreslået, såsom virusinfektion, medfødte misdannelser, autoimmune sygdomme og toksisk spot. Men bidraget fra hver til udviklingen af galdeatresi stadig usikkert 7,8,9,10. I modsætning til spædbørn, der lider patologisk galdeatresi undergår havlampret udviklingsmæssigt programmeret galdeatresi uden omfattende necroinflammation, fibrose eller cirrose 10. Dyrene kan lide forbigående cholestase under denne proces 10, men tilpasse sig denne udviklingsmæssige tilstandvia de novo-syntese og sekretion af galdesalte i tarmen efter udviklingsmæssige galdeatresi, udover kendte mekanismer, såsom reduktion af galdesalt syntese i leveren 11. Denne udviklingsproces i havlampret giver den eneste kendte mulighed for at undersøge udviklingen af ​​galdeatresi. En nyudviklet metode kaldet &quot;klarhed&quot; giver høj opløsning billeddannelse i komplekse pattedyr nervesystemer ved at omdanne intakt væv ind i en optisk transparent nanoporøse hydrogel 12. Brug havlampret lever og en modificeret CLARITY-protokollen, kan intakt væv billeddannelse af galde degeneration dokumenteres gennem leveren metamorfose.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40% acrylamide&#160;</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:119px;">161-0140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% bis-acrylamide&#160;</td>
			<td>Bio-Rad</td>
			<td>161-0142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ammonium persulfate&#160;</td>
			<td>Sigma</td>
			<td>A3678-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">boric acid</td>
			<td>Sigma</td>
			<td>B7901-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">saponin&#160;</td>
			<td>Sigma</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium dodecyl sulfate&#160;</td>
			<td>Sigma</td>
			<td style="width:119px;">L337-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (monobasic)</td>
			<td>Sigma</td>
			<td>04269-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium phosphate (dibasic)</td>
			<td>Sigma</td>
			<td>S5136-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol&#160;</td>
			<td>Sigma</td>
			<td>G9012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% paraformaldehyde&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH pellets&#160;</td>
			<td>EMD</td>
			<td>SX0590-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml centrifuge tubes</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting tools&#160;</td>
			<td style="width:200px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)

Galdeatresi er en sjælden sygdom i vorden, med en anslået 1 i 15.000 frekvens i det sydøstlige USA, men mere almindelig i østasiatiske lande, med en rapporteret frekvens på 1 i 5000 i Taiwan. Selvom meget er kendt om forvaltningen af ​​galdeatresi, dens patogenese er stadig undvigende. Den havlampret (Petromyzon marinus) giver en enestående mulighed for at undersøge mekanismen og progression af galde degeneration. Havlampret udvikle sig gennem tre forskellige livsstadier: larve, parasitter og voksne. Under overgangen fra larver til parasitisk juvenile, havlampret undergå metamorfose med dramatisk reorganisering og ombygning i ekstern morfologi og indre organer. I leveren er hele galdeveje tabt, herunder galdeblære og galdetræet. En nyudviklet metode kaldet &quot;klarhed&quot; blev ændret for at tydeliggøre hele leveren og krydset med tarmen i metamorfe havlampret. Denproces biliær degeneration blev visualiseret og skelnes under havlampret metamorfose ved hjælp af laserscanning konfokal mikroskopi. Denne metode giver et kraftfuldt værktøj til at studere galdeatresi i en unik dyremodel.

Flere vigtige udviklingsmæssige begivenheder forekommer i hepatobiliær systemet under havlampret metamorfose. Galdegangen og galdeblæren undergår apoptose og degenererede (figur 1). Ved at kombinere den modificerede afklaring metode og farvning med levercelle markør cytokeratin 19 (CK19, til stede i både cholangiocytes og hepatocytter før og efter metamorfose 13) og antiapoptotisk markør Bcl2 hjælp konfokal mikroskopi blev hele galdeveje fanget langs Z-aksen ( figur 2)</stron...

Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

En ny Afklaring metode til at synliggøre biliær Degeneration Under Lever Metamorphosis i havlampret ( Petromyzon marinus)

Havlampret miste galdeblære og galdeveje under metamorfose, en proces der ligner humant galdeatresi. En ny fiksering og afklaring metode (klarhed) blev modificeret til at visualisere hele galdetræet hjælp af laser konfokal mikroskopi. Denne metode giver et kraftfuldt værktøj til at studere galde degeneration.

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

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Biology

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

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