Name: cecal ligation and puncture-induced sepsis as a model to study autophagy in mice
Uploaded: 2014-02-09T22:00:00
Description: Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

Dette arbejde blev støttet af NIH tilskud P01 HL108801, R01-HL60234, R01-HL55330, R01-HL079904 til AMK Choi. S. Ryter fået støtte løn fra Lovelace Respiratory Research Institute.

Bemærk: Den Institutional Animal Care og brug Udvalg på Brigham and Women &#39;s Hospital / Harvard Medical School-området godkendt følgende procedurer. 1.. Cecal ligatur og Puncture Brug mus af den samme baggrund (C57BI / 6), mand, 8-10 uger gamle. Hunmus er mere modstandsdygtige end mænd mod sepsis-induceret dødelighed. Mus ældre end 8 uger producerer mindre variable resultater end yngre mus i form af overlevelse efter CLP. Ca. N = 10 mus ...

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Den største fordel ved CLP er, at det giver forskerne at undersøge sepsis forskellige sværhedsgrader (dvs. fra lav-til midt-og high-grade). Sværhedsgraden af induceret sepsis påvirkes af længden af coecum ligeret (som den vigtigste faktor), størrelsen af nålen, brugt til punktur og antallet af huller udført 15. Desuden kan musen stamme og køn indflydelse på sværhedsgraden af sepsis, flere stammer er mere modtagelige end andre, og mænd er generelt mere modtagelige end hunnerne 19,20....

Sepsis, systemisk inflammatorisk respons på infektion, er en førende dødsårsag i kritisk-syge patienter 1. Intra-abdominale infektioner, der ofte fører til polymikrobiale sepsis, tegner sig for 20% af sepsistilfælde, som har en betydelig dødelighed på op til 60% 2. Sepsis-associeret dødelighed primært skyldes multi-organsvigt med fiasko efterfølgende orgel 3,4. Yderligere undersøgelse af patogene mekanisme af denne sygdom er et presserende behov for at fremme udviklingen af ​​nye og mere effektive behandlingsformer. Cecal ligatur og punktering (CLP)-metoden er en almindeligt anvendt procedure for modellering sepsis in vivo. Som coecum er fuld af bakterier, dets punktur resulterer i polymikrobielle peritonitis, translokation af bakterier i blodet (bakteriæmi), septisk shock, multi-organsvigt og i sidste ende død 5. Det er generelt accepteret, at CLP afspejler den kliniskevirkeligheden mere præcist end tidligere teknikker, såsom injektion af endotoxin eller endda oprensede bakterier i gnavere Således CLP betragtes som den gyldne standard (omend ikke uden begrænsninger) 6 for den eksperimentelle induktion og dermed undersøgelse af patogenesen af sepsis. I denne monografi beskriver vi protokoller, designet til at vurdere, om patogene mekanismer sepsis omfatter autofagi. Autophagy, en evolutionært bevaret cellulær proces, letter omsætningen af beskadigede proteiner og organeller, såsom mitokondrier og spiller en vigtig rolle ved udskillelsen af intracellulære patogener, herunder bakterier 7,8. Under autophagy, er cytosoliske proteiner eller organeller afsondret i dobbelt membran-bundne vesikler kaldet autophagosomes, som efterfølgende leveres til lysosomerne for nedbrydningen 9. En række proteiner er blevet identificeret som mammale homologer af autophagy-relaterede gener (ATG),oprindeligt identificeret i gær, der regulerer processen autofagi. Omdannelsen af mikrotubuli-associeret protein-1 let kæde 3B (LC3B) (homolog af Atg8) fra LC3B-I (fri form) til LC3B-II (phosphatidylethanolamin-konjugeret form) er et stort skridt i autophagosome formation 9. Autophagic dysfunktion er forbundet med aldring og menneskelige sygdomme, herunder kræft og neurodegenerative sygdomme 10. Desuden autofagi påvirker medfødte og adaptive immunitet, såsom antigen-præsentation, lymfocyt udvikling og cytokinsekretion af immunceller 8. Det synes således rimeligt, at autofagi også kan spille en rolle i systemisk inflammatorisk respons på infektion (dvs. i sepsis). Til dato er blevet beskrevet flere metoder til at vurdere den rolle autophagy i vævsskade in vivo. Disse omfatter anvendelse af grønne fluorescensprotein (GFP)-LC3 udtrykkende mus og kvantificering af autophagosomes i væv ved elektronmikroskopi (disse to metoder er beskrevet i denne monografi). Yderligere metoder omfatter kvantificering af autophagic protein udtryk i vævshomogenater, og analysen af autophagic flux (som beskrevet andetsteds) 11-13. Målet med denne gennemgang er at give nuværende protokoller til vurdering autofagi in vivo i forbindelse med eksperimentel sepsis.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >GFP-LC3 Transgenic Mice</td>
			<td >Riken (Japan)</td>
			<td >RBRC00806</td>
			<td >GFP-LC3#53</td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >Henry-Schein</td>
			<td >568-0606</td>
			<td>Xylazine HCl Injection Vet</td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >Henry-Schein</td>
			<td >995-2949</td>
			<td>Ketaset Inj 100 mg/ml</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A405-20</td>
			<td>Histology Grade</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A407-1</td>
			<td>For Sterilizatiion</td>
		</tr>
		<tr >
			<td >silk surgical sutures 6-0</td>
			<td >Owens &#38; Minor</td>
			<td >2300-0078OG, 017624</td>
			<td></td>
		</tr>
		<tr >
			<td >buprenorphine-HCl</td>
			<td >Henry-Schein</td>
			<td >614-5157</td>
			<td>Buprenex Ampules</td>
		</tr>
		<tr >
			<td >paraformaldehyde (37%) solution</td>
			<td >JT Baker</td>
			<td >S898-09</td>
			<td></td>
		</tr>
		<tr >
			<td >xylenes</td>
			<td >Fisher</td>
			<td>X3P-1GAL </td>
			<td></td>
		</tr>
		<tr >
			<td >anti-GFP monoclonal antibody</td>
			<td>Life Technologies</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoescht</td>
			<td >Sigma</td>
			<td>944403</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >OCT</td>
			<td >VWR scientific</td>
			<td >25608-930</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan Black</td>
			<td >Santa Cruz</td>
			<td >sc-203760</td>
			<td ></td>
		</tr>
		<tr >
			<td >EM grade Glutaraldehyde 2.5% in sodium cacodylate</td>
			<td >Electron microscopy Sciences</td>
			<td>15960</td>
			<td></td>
		</tr>
		<tr >
			<td >propylene oxide</td>
			<td >Sigma</td>
			<td>240397</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar 100 resin</td>
			<td >Agar scientific</td>
			<td > R1045</td>
			<td></td>
		</tr>
		<tr >
			<td >dodecenylsuccinic anhydride</td>
			<td >Sigma</td>
			<td>46346</td>
			<td></td>
		</tr>
		<tr >
			<td >methylnadic anhydride</td>
			<td >Sigma</td>
			<td>45359</td>
			<td></td>
		</tr>
		<tr >
			<td >N-benzyldimethylamine</td>
			<td >Sigma</td>
			<td>185582</td>
			<td></td>
		</tr>
	</tbody>
</table>

cecal ligation and puncture-induced sepsis as a model to study autophagy in mice

Eksperimentel sepsis kan induceres i mus ved anvendelse cecal ligatur og punktering (CLP)-metoden, som forårsager polymikrobielle sepsis. Her er en protokol, forudsat at fremkalde sepsis af varierende sværhedsgrad i mus ved hjælp af CLP teknik. Autophagy er en grundlæggende væv reaktion på stress og patogeninvasion. To nuværende protokoller for at vurdere autofagi in vivo i forbindelse med eksperimentelle sepsis også præsenteret her. (I) Transgene mus, som udtrykker grønt fluorescensprotein (GFP)-LC3 fusionsprotein underkastes CLP. Lokaliseret forbedring af GFP signal (puncta), som analyseret enten ved immunohistokemiske eller konfokale assays kan anvendes til at påvise øget autophagosome dannelse og dermed ændret aktivering af autofagi vej. (II) Udvidet autophagic vacuole (autophagosome) dannelse pr vævsareal (som en markør for autofagi stimulation) kan kvantificeres ved hjælp af elektronmikroskopi. Studiet af autophagic reaktioner sepsis er en kritisk component at forstå de mekanismer, som væv reagerer på infektion. Forskningsresultater på dette område kan i sidste ende bidrage til at forstå patogenesen af ​​sepsis, som repræsenterer et stort problem i intensivbehandling medicin.

Bakteriæmi er til stede i mus så tidligt som 6 timer, efter CLP-induceret sepsis 14. Kliniske tegn på sepsis (herunder kuldegysninger, takypnø og nedsat motorisk aktivitet) vises cirka 12 timer efter indgrebet. Mus udsat for CLP begynder at dø på omkring 18 timer efter induktion af bughindebetændelse. Jo mere alvorlig er den sepsis mere øget er dødeligheden 15. I detaljer, high-grade sepsis forårsager 100% mortalitet inden for 2-3 dage, mens mid-grade sepsis resulterer i omkring 60% døde...

Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Eksperimentel sepsis kan induceres i mus ved hjælp af den cecal ligatur og punktering (CLP) metode. Nuværende protokoller for at vurdere autofagi in vivo i forbindelse med CLP-induceret sepsis præsenteres her: En protokol til måling autofagi anvendelse af (GFP)-LC3 mus og en protokol til måling af autophagosome dannelse ved elektronmikroskopi.

Cecal ligatur og Punktering-induceret sepsis som en model til at studere Autophagy i mus

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