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

All animal experiments were approved by the animal care and use committee of the School of Applied Science, Temasek Polytechnic.
1. Preparation of DMN
<ol>
	<li>Pipette 200 &#181;l 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.</li>
</ol>
2. Intraperitoneal Injection of DMN
<ol>
	<li>Use male Wistar rats with an average weight...

<ol>
	<li>Magee, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+liver+injury;+the+metabolism+of+dimethylnitrosamine.">Toxic liver injury; the metabolism of dimethylnitrosamine.</a> Biochem J. 64 (4), 676-682 (1956).</li><li>Pritchard, D. J., Butler, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-the+mechanism+of+cell+death+in+dimethylnitrosamine-induced+hepatotoxicity.">Apoptosis-the mechanism of cell death in dimethylnitrosamine-induced hepatotoxicity.</a> J Pathol. 158 (3), 253-260 (1989).</li><li>Madden, J. W., Gertman, P. M., Peacock, E. 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II. Electron immunohistochemical studies in rats with CCl4-induced cirrhosis.</a> Lab Invest. 53 (2), 166-186 (1985).</li><li>Pritchard, M. T., Apte, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+to+Study+Liver+Regeneration.">Models to Study Liver Regeneration.</a> Liver Regeneration. , 15-40 (2015).</li><li>Kountouras, J., Billing, B. H., Scheuer, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+bile+duct+obstruction:+a+new+experimental+model+for+cirrhosis+in+the+rat.">Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat.</a> Br J Exp Pathol. 65 (3), 305-311 (1984).</li><li>Marques, T. G., Chaib, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+experimental+models+for+inducing+hepatic+cirrhosis+by+bile+duct+ligation+and+carbon+tetrachloride+injection.">Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection.</a> Acta Cir Bras. 27 (8), 589-594 (2012).</li><li>Wasser, S., Tan, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+hepatic+fibrosis+in+the+rat.">Experimental models of hepatic fibrosis in the rat.</a> Ann Acad Med Singapore. 28 (1), 109-111 (1999).</li><li>George, J., Chandrakasan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+abnormalities+during+the+progression+of+hepatic+fibrosis+induced+by+dimethylnitrosamine.">Biochemical abnormalities during the progression of hepatic fibrosis induced by dimethylnitrosamine.</a> Clin Biochem. 33 (7), 563-570 (2000).</li><li>Shimizu, I., Ma, Y. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Sho-saiko-to,+a+Japanese+herbal+medicine,+on+hepatic+fibrosis+in+rats.">Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats.</a> Hepatology. 29 (1), 149-160 (1999).</li><li>Wang, Y., Gao, J., Zhang, D., Zhang, J., Ma, J., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+antifibrotic+effects+of+sorafenib+on+hepatic+stellate+cells+and+liver+fibrosis.">New insights into the antifibrotic effects of sorafenib on hepatic stellate cells and liver fibrosis.</a> J Hepatol. 53 (1), 132-144 (2010).</li><li>Machholz, E., Mulder, G., Ruiz, C., Corning, B. F., Pritchett-Corning, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+Restraint+and+Common+Compound+Administration+Routes+in+Mice+and+Rats.">Manual Restraint and Common Compound Administration Routes in Mice and Rats.</a> J Vis Exp. (67), (2012).</li><li>Lee, G., Goosens, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+Blood+from+the+Lateral+Tail+Vein+of+the+Rat.">Sampling Blood from the Lateral Tail Vein of the Rat.</a> J Vis Exp. (99), (2015).</li><li>Parkinson, C. M., O'Brien, A., Albers, T. M., Simon, M. A., Clifford, C. B., Pritchett-Corning, K. 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(54), (2011).</li><li>Ruehl-Fehlert, C., Kittel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55 (2-3), 91-106 (2003).</li><li>Ishak, K., Baptista, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+grading+and+staging+of+chronic+hepatitis.">Histological grading and staging of chronic hepatitis.</a> J Hepatol. 22 (6), 696-699 (1995).</li><li>Sant'Anna, L. B., Sant'Anna, N., Parolini, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+computer+assisted+image+analysis+for+identifying+and+quantifying+liver+fibrosis+in+a+experimental+model.">Application of computer assisted image analysis for identifying and quantifying liver fibrosis in a experimental model.</a> J Comput Interdiscip Sci. 2 (2), 139-148 (2011).</li></ol>

The authors declare that they have no competing financial interests.

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...

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 rats3,4.
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 steatosis5. It was shown that in early fibrosis, the collagen formed was more cross linked with type III being more prominent than type I6. In common with other causes, DMN-induced fibrotic changes were associated with an increase in Kupffer cells; hepatic macrophages that reside in the sinusoids. These cells morph into myofibroblasts and produce excessive amounts of extracellular matrix which is the primary problem in fibrosis7.
In terms of cellular signaling, Nakamura et al. demonstrated that TGF-&#946; plays a critical role in the progression of liver fibrosis8. They used an adenovirus expressing a truncated type II TGF-&#946; receptor, to specifically inhibit TGF-&#946; signaling. Liver fibrosis in these rats was spectacularly halted during DMN treatment when compared to control groups. Other studies have confirmed that suppression of TGF-&#946; leads to alleviation of liver fibrosis development9,10. The model was also used in a global gene profiling study to identify other fibrosis markers and proteins which could be used as drug targets for anti-fibrotic therapy11.
Other chemical agents used to induce liver fibrosis include thioacetamide (TAA) and carbon tetrachloride (CCl4). TAA was used first in rats and later in mice12. The advantages of this model include: ease of chemical administration in drinking water, the reproducibility of the model with characteristic micronodular cirrhosis and biochemical changes. The disadvantages include: the long time period of 3 months for liver fibrosis to develop and the lack of understanding of the molecular mechanism for induction of liver fibrosis. As for CCl4, its use has declined for the following reasons: it does not mimic human liver disease, has harmful effects on the ozone layer, causes pain and distress to animals, is very toxic to humans and requires extra precautions in its handling and disposal13,14.
Prolonged bile duct obstruction (by surgical intervention) as an experimental model for liver cirrhosis was first reported by Kountouras et al.15. This method is nontoxic to humans and animals. However, the time required for liver fibrosis to develop varies. A review of 30 reports by Marques et al.16, found that it took from seven days to four weeks after surgery for liver fibrosis to develop. The pathological changes described mimic those of human chronic biliary fibrosis and the model would be more suited for researchers interested in this area.
In summary, the intermittent administration of a constant dose of DMN in the rat over 4 weeks produces liver fibrosis that mimics the human disease. Dosed rats show progressive development of liver damage and parenchymal fibrosis4,17. Disease progression and severity can be monitored via blood samples or sacrifice of animals at specific time points and the effect is highly reproducible18. Thus the model has been widely used to study the mechanisms of liver fibrosis and cirrhosis as well as to screen for potential anti-fibrotic agents10,19,20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dimethylnitrosamine</td>
			<td>Wako</td>
			<td>147-03781</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sinopharm chemicals</td>
			<td>F63257009</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Masson trichome stains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline Blue</td>
			<td>Electron Microscopy Sciences</td>
			<td>#42755</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Fuschin</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT42685</td>
			<td></td>
		</tr>
		<tr>
			<td>Scarlet Red</td>
			<td>Electron Microscopy Sciences</td>
			<td>#26905</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphotungstic Acid Hydrate</td>
			<td>ALFA AESAR</td>
			<td>ALFA40116.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphomolybdic Acid Hydrate</td>
			<td>Sigma SG</td>
			<td>221856-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigert&#39;s Iron Hematoxilyn</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX Mounting Medium</td>
			<td>Merck</td>
			<td>HX066873</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processor</td>
			<td>Leica</td>
			<td>Leica TP 1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding machine</td>
			<td>Sakura</td>
			<td>&#160;Sakura Tissue Tek TEC5 Embedding System</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>Leica RM 2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Vet Test Analyzer</td>
			<td>Idexx</td>
			<td>Vet Test 8008</td>
			<td></td>
		</tr>
	</tbody>
</table>

the dimethylnitrosamine induced liver fibrosis model in the 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 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&#39;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.

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 ...

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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.

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 ...

The overall goal of this procedure is to produce an animal model of liver fibrosis that can be used to assess the efficacy of potential anti-fibrotic agents. This animal model can help answer key questions about the nature and development of fibrosis in the liver and how anti-fibrotic agents reduce the fibrotic process. Furthermore, this model mimics the complex physiological processes of human liver disease from early to late-stage fibrosis and can be studied at any point of interest of the disease.Individuals new to this method need to plan the workflow because the den-glis-nov animal model takes at least five weeks. After the liver has been harvested, the processing requires one or two weeks before especially stained sections are ready to be examined by the pathologist. DMN must be diluted from a stock to 10 milligrams per milliliter in PBS.When performing this dilution, be sure to work in a fume hood as the substance is a carcinogen. For the experiment, select male Wistar rats between 150 and 200 grams. Acclimate the rats to a housing facility with a normal climate for at least four days.Provide food and water ad libitum and measure their daily intake. On a weekly basis, measure the rat's body weight and take a blood sample. For the DMN treatment, at the fume hood, prepare and inject 10 milligrams per kilogram doses.Use a 25 gauge needle and intraperitoneally inject the lower-right quadrant of the abdomen. Inject the rats for three consecutive days and repeat this process weekly for four weeks. Always on the same three days, and importantly, adjust the DMN dose weekly over the four-week period.Euthanize the DMN-treated animals four to five days after their last injection. On a dissecting board, disinfect and moisten the abdominal skin with 70%ethanol. Then, using scissors, incise the skin from the anus to the chin and reflect it apart.Next, incise the abdominal wall from the anus to the zyphoid cartilage to expose the abdominal viscera. Check the organs for abnormalities such as inappropriate color, size, location, or fluids within the peritaneal cavity. Also probe the organs for their consistency and note any abnormal odors.Next, isolate the liver. Begin at the hilus, the diaphragm attachment point, and work around the liver from there, freeing it from surrounding ligaments and vessels. Then, transfer the liver to a dish and record its weight.Now, using a scalpel, from one lobe, make a five millimeter block that extends one to two centimeters into the lobe for sectioning. Then, make one or two more blocks, preferably from the same lobe, to serve as back-ups. The remaining liver can be stored frozen.Fix the tissue blocks in 10%forumlulin with at least ten times more fixative than tissue. After 24 or more hours of fixation, trim the liver samples and place them into cassettes. Then load the basket for the automated tissue processor.Fully submerge the basket into the first processing station filled with 10%buffered formulin. Then, continue with one-hour baths in an ethanol series followed by xylene, followed by paraffin. Next, warm up the embedding station for at least an hour.Once warm, transfer liver samples into the wax bath. After two to three minutes, use warmed forceps to transfer the cassette to a 65 degree Celsius hotplate. Then half-fill the mold with warm, liquid paraffin.Next, transfer the tissue out of the cassette and into the paraffin with the cut surface flat on the mold. Then, finish filling the mold with warmed paraffin. Mount the empty cassette on top of the paraffin to serve as a label and support.Then, place the mold on a four-deegree Celsius block to cool for 30 minutes. Once cooled, pop out the paraffin block. If the block cracks, melt it and repeat the embedding.Now, section the block on a microtome. Make ten micron thick sections until the tissue is visible. And then make five micron thick sections.Next, carefully transfer the sections to a 40-degree Celsius water bath, holding them at their edges. Let the sections float and lift them off the water onto clean glass slides. Now, place all the slides on a slide warmer at 37 degree Celsius and let them dry overnight.To begin, remove the paraffin and re-hydrate the tissues with xylene and decreasing concentrations of ethanol. Then, immerse the slides in iron hematoxylin for 10 minutes to stain the nuclei. After 10 minutes, briefly rinse the slides with water.Then, immerse the slides in Biebrich scarlet acid fuchsin for two minutes to stain the cytoplasm. Follow with a water rinse as before. Next, immerse the slides in freshly-made PTA for 10 minutes to promote the uptake of aniline blue.Logically, next, immerse the slides in aniline blue for 10 minutes to stain the collagen fibers. Now, rinse off the excess stain with water and transfer the slides to a 1%acetic acid bath for one minute. To render the color of the slide more delicate and transparent.Over four weeks of DMN treatment, male Wister rats lost weight and became less vigorous. Weight loss was first detectable after two weeks. Gradually, damage to the liver weakened the rats.The liver to body weight ratio was significantly lower in the DMN-treated animals. Grossly, the treated livers were smaller and harder when compared to controls. As expected, serum indicators of a hepatocyte damage, ALT and AST, were at elevated levels in the treated group.Histological examination was made using a standardized set of criteria. The DMN treatment led to progressive increase and expansion of fibrous septa with loss of hepatocytes. Collagen deposits are stained blue.There was fibrous expansion of portal areas with portal-to-portal bridging and portal-to-central bridging. By four weeks of treatment, there was cirrhosis with nodule formation. With the need for repeated injections, the correct dressing and technique of injections is important to a wide interaction of contamination and damage to internal organs.Don't forget that DMN is toxic, and all the injections should be done in the fume hood. Cutting good, five micromedial, thin sections on the microtome requires practice and patience, during the staining process. Ensure that the sections are kept moist at all times and transfer it from one staining solution to the next according to the specified timing.In summary, rats produced in this model, show progressive liver disease and pericardial fibrosis which can be monitored by biochemical states. We have studied various aspects of liver disease progression and tested potential anti-fibrotic agents using this model.

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JoVE Journal

Medicine

11.9K Görüntüleme.  Temasek Polytechnic. The overall goal of this procedure is to produce an animal model of liver fibrosis that can be used to assess the efficacy of potential anti-fibrotic agents. This animal model can help answer key questions about the nature and development of fibrosis in the liver and how anti-fibrotic agents reduce the fibrotic process. Furthermore, this model mimics the complex physiological processes of human liver disease from early to late-stage fibrosis and can be studied at any point of interest of the ...