Name: the dimethylnitrosamine induced liver fibrosis model in the rat
Uploaded: 2016-06-17T14:15:00
Description: Watch this Scientific Journal Video about The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat at JoVE.com

The authors acknowledge the funding support from the Ministry of Education, Singapore, grant number MOE2010-IF-1-025.

Alle eksperimenter dyr blev godkendt af pasning af dyr og brug udvalg af School of Applied Science, Temasek Polytechnic. 1. Udarbejdelse af DMN <ol><li> Pipetter 200 pi DMN (1 g / ml stamopløsning) og tilsæt 19,8 ml PBS til fremstilling 10 mg / ml DMN opløsning til injektion. Bemærk: DMN er kræftfremkaldende. Brug en emhætte for at forberede DMN og udfører dyret injektioner. </li></ol> 2. intraperitoneal injektion af DMN <ol><li> Brug Wistar hanrotter med en gennemsnitli...

<ol>
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Vi har beskrevet en metode til at gøre en dyremodel af leverfibrose og vurdere sværhedsgraden af ​​leverfibrose. Det er vigtigt at levere den korrekte dosis af DMN og overholde tidsplanen for ugentlige intraperitoneale injektioner. Som eksperimentet skrider frem, er det afgørende at afveje rotterne og genberegne dosis ved starten af ​​hver uge af DMN injektioner. Husk, at DMN er giftigt og skal håndteres i stinkskab. Vi udfører de intraperitoneale injektioner i stinkskab så godt. Med behovet for gentagne i...

DMN er en potent leveren specifikke toksin. Dens stofskifte, væv fordeling, og evne til at forårsage skade på lever af rotter blev rapporteret af Magee 1, og mekanismen for hepatocyt skader og celledød ved apoptose blev beskrevet af Pritchard og Butler 2. Intermitterende indgivelse af denne forbindelse blev rapporteret at inducere leverfibrose hos hunde og rotter 3,4. De mekanismer og morfologiske forandringer af leverfibrose er blevet omfattende undersøgt ved anvendelse af denne model. I tidlige undersøgelser under anvendelse af rotte, 3-ugers behandling med DMN produceret centrilobulær hæmoragisk nekrose efterfulgt af mikronodulær cirrose uden steatose 5. Det blev vist, at i begyndelsen af fibrose, det dannede collagen var mere tværbundet med type III er mere fremtrædende end type I 6. Til fælles med andre årsager, blev DMN-induceret fibrotiske forandringer forbundet med en stigning i Kupffer-celler; hepatiske makrofager, der bor in sinusoider. Disse celler morph i myofibroblaster og producere store mængder af ekstracellulær matrix, som er det primære problem i fibrose 7. Med hensyn til cellulær signalering, Nakamura et al. Viste, at TGF-β spiller en kritisk rolle i progressionen af leverfibrose 8. De brugte et adenovirus, der udtrykker en trunkeret type II TGF-β-receptor, til specifikt at inhibere TGF-β-signalering. Leverfibrose i disse rotter spektarkulært standset under DMN behandling sammenlignet med kontrolgrupper. Andre undersøgelser har bekræftet, at undertrykkelse af TGF-β fører til lindring af leverfibrose udvikling 9,10. Modellen blev også anvendt i en global gen profilering undersøgelse for at identificere andre fibrose markører og proteiner, som kan anvendes som lægemiddelmål for anti-fibrotisk terapi 11. Andre kemiske midler, som anvendes til at inducere leverfibrose omfatter thioacetamid (TAA) og cArbon tetrachlorid (CCI4). TAA blev brugt først i rotter og senere i mus 12. Fordelene ved denne model omfatter: nem kemisk indgivelse i drikkevand, reproducerbarheden af ​​modellen med karakteristiske mikronodulær cirrose og biokemiske ændringer. Ulemperne omfatter: den lange periode på 3 måneder for leverfibrose at udvikle og manglende forståelse af den molekylære mekanisme for induktion af leverfibrose. Som for CCI4, har dens anvendelse faldet af følgende grunde: det ikke efterligne menneskelige leversygdom, har skadelige virkninger på ozonlaget, forårsager smerte og lidelse for dyrene, er meget giftige for mennesker og kræver ekstra forholdsregler i dens håndtering og bortskaffelse 13,14. Langvarig galdegang obstruktion (ved et kirurgisk indgreb) som en eksperimentel model for levercirrhose blev først rapporteret af Kountouras et al. 15. Denne metode er ikke-toksiske for mennesker og dyr. However, den nødvendige tid til leverfibrose at udvikle varierer. En gennemgang af 30 rapporter fra Marques et al. 16, fandt, at det tog fra syv dage til fire uger efter operation for leverfibrose at udvikle sig. De patologiske ændringer, der er beskrevet efterligner dem af human kronisk galde fibrose og modellen ville være mere velegnet til forskere interesseret i dette område. Sammenfattende den intermitterende indgivelse af en konstant dosis af DMN i rotten over 4 uger producerer leverfibrose, der efterligner den humane sygdom. Doserede rotter viser gradvise udvikling af leverskade og parenkymalt fibrose 4,17. Sygdomsprogression og sværhedsgraden kan overvåges via blodprøver eller ofring af dyr på bestemte tidspunkter, og effekten er meget reproducerbar 18. Således modellen har været meget anvendt til at undersøge mekanismerne ved leverfibrose og cirrose samt til at screene for potentielle anti-fibrotiske midler 10,19,20.

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			<td>Phosphotungstic Acid Hydrate</td>
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			<td>ALFA40116.4</td>
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			<td>Phosphomolybdic Acid Hydrate</td>
			<td>Sigma SG</td>
			<td>221856-25G</td>
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			<td>Weigert&#39;s Iron Hematoxilyn</td>
			<td>Merck</td>
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			<td>DPX Mounting Medium</td>
			<td>Merck</td>
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			<td>Tissue processor</td>
			<td>Leica</td>
			<td>Leica TP 1020</td>
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			<td>Embedding machine</td>
			<td>Sakura</td>
			<td>&#160;Sakura Tissue Tek TEC5 Embedding System</td>
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			<td>Leica</td>
			<td>Leica RM 2235</td>
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			<td>Vet Test Analyzer</td>
			<td>Idexx</td>
			<td>Vet Test 8008</td>
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the dimethylnitrosamine induced liver fibrosis model in the rat

Fire til seks uger gamle, blev Wistar-hanrotter anvendes til fremstilling af dyremodeller for leverfibrose. Processen kræver fire ugers indgivelse af 10 mg / kg dimethylnitrosamin (DMN), givet intraperitonealt tre på hinanden følgende dage om ugen. Intraperitoneale injektioner blev udført i stinkskab som DMN er et kendt hepatoxin og kræftfremkaldende. Modellen har flere fordele. For det første kan leverforandringer studeres sekventielt eller på bestemte stadier af interesse. For det andet kan den fase af leversygdom overvåges ved måling af serum-alanin-aminotransferase (ALT) og aspartataminotransferase (AST) enzymer. For det tredje kan sværhedsgraden af ​​leverskade på forskellige stadier bekræftes ved aflivning af dyr på udpegede tidspunkter, efterfulgt af histologisk undersøgelse af Massons trichome farvede levervæv. Efter fire ugers DMN dosering, den typiske fibrose score er 5 til 6 på Ishak skalaen. Modellen kan reproduceres konsekvent og har væretalmindeligt anvendt til at vurdere effektiviteten af ​​potentielle anti-fibrotiske midler.

DMN behandlede rotter tabe sig og bliver mindre kraftig med pjusket pels. Der er et betydeligt tab i den gennemsnitlige legemsvægte af DMN behandlede rotter; først påvises efter 2 uger af DMN behandling, og denne forskel er fortsat gennem uger 3 og 4 efter DMN behandling (figur 1a). Som modtager rotterne DMN løbet successive uger, leverskader får den til at blive mindre. Leveren indeks; som er den procent af levervægt på endelig kropsvægt var signifikant lavere f...

Watch this Scientific Journal Video about The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat at JoVE.com

The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

We describe a method to produce an animal model of liver fibrosis in the rat, and assess the degree of fibrosis by histological examination of the liver. The model can be used to study the development of liver disease as well as to test the efficacy of potential anti-fibrotic agents.

Den dimethylnitrosamin induceret leverfibrose Model i Rat

Four to six week old, male Wistar rats were used to produce animal models of liver fibrosis. The process requires four weeks of administration of 10 mg/kg ...

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Murine Precision-Cut Lever skiver som en Ex Vivo Model af leverbiologi

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Organ Iskæmi-Reperfusion Skade ved at simulere hæmodynamiske ændringer i rottelevertransplantation Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

En rottelungetransplantationsmodel af varm iskæmi / reperfusionsskade: optimeringer for at forbedre resultaterne

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

Establishing a Silicosis Rat Model via Exposure of Whole-Body to Respirable Silica

The major cause of silicosis is the inhalation of silica in the occupational environment. Despite some anatomical and physiological differences, rodent ...