The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

Summary

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.

Abstract

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 dimethylnitrosamine (DMN), given intraperitoneally for three consecutive days per week. Intraperitoneal injections were performed in the fume hood as DMN is a known hepatoxin and carcinogen. The model has several advantages. Firstly, liver changes can be studied sequentially or at particular stages of interest. Secondly, the stage of liver disease can be monitored by measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes. Thirdly, the severity of liver damage at different stages can be confirmed by sacrifice of animals at designated time points, followed by histological examination of Masson's Trichome stained liver tissues. After four weeks of DMN dosing, the typical fibrosis score is 5 to 6 on the Ishak scale. The model can be reproduced consistently and has been widely used to assess the efficacy of potential anti-fibrotic agents.

Introduction

DMN is a potent liver specific toxin. Its metabolism, tissue distribution, and ability to cause injury to livers of rats was reported by Magee¹, and the mechanism of hepatocyte damage and cell death by apoptosis was described by Pritchard and Butler². Intermittent administration of this compound was reported to induce liver fibrosis in dogs and rats^3,4.

The mechanisms and morphologic changes of liver fibrosis have been extensively investigated using this model. In early studies using the rat, 3-week treatment with DMN produced centrilobular hemorrhagic necrosis followed by micronodular cirrhosis without stea....

Protocol

All animal experiments were approved by the animal care and use committee of the School of Applied Science, Temasek Polytechnic.

1. Preparation of DMN

1. Pipette 200 µl of DMN (1 g/ml stock solution) and add 19.8 ml of PBS to prepare 10 mg/ml DMN solution for the injection.
   Note: DMN is carcinogenic. Use a fume hood to prepare the DMN and perform the animal injections.

2. Intraperitoneal Injection of DMN

1. Use male Wistar rats with an average weight of 150 - 200 g. Acclimate rats for 4 - 7 days before the start of the experiment.
2. Maintain rats at room temperature of 22 ± 1 °C,....

Results

DMN treated rats lose weight and become less vigorous with ruffled hair coat. There is significant loss in average body weights of DMN treated rats; first detectable after 2 weeks of DMN treatment, and this difference remains through weeks 3 and 4 after DMN treatment (Figure 1a). As the rats receive DMN over successive weeks, damage to the liver causes it to become smaller. The liver index; which is the percent of liver weight at final body weight was significantly lower .......

Discussion

We have described a method to make an animal model of liver fibrosis and to assess the severity of liver fibrosis. It is important to deliver the correct dose of DMN and adhere to the schedule of weekly intraperitoneal injections. As the experiment progresses, it is crucial to weigh the rats and re-calculate the dose at the start of each week of DMN injections. Keep in mind that DMN is toxic and needs to be handled in the fume cabinet. We perform the intraperitoneal injections in the fume cabinet as well. With the need f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dimethylnitrosamine	Wako	147-03781
Formalin	Sinopharm chemicals	F63257009
Ethanol	Sigma	64-17-5
Xylene	Fisher	1330-20-7
Masson trichome stains
Aniline Blue	Electron Microscopy Sciences	#42755
Acid Fuschin	Electron Microscopy Sciences	RT42685
Scarlet Red	Electron Microscopy Sciences	#26905
Phosphotungstic Acid Hydrate	ALFA AESAR	ALFA40116.4
Phosphomolybdic Acid Hydrate	Sigma SG	221856-25G
Weigert's Iron Hematoxilyn	Merck	1.15973.0002
DPX Mounting Medium	Merck	HX066873
Tissue processor	Leica	Leica TP 1020
Embedding machine	Sakura	Sakura Tissue Tek TEC5 Embedding System
Microtome	Leica	Leica RM 2235
Vet Test Analyzer	Idexx	Vet Test 8008