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 of 150 - 200 g. Acclimate rats for 4 - 7 days before the start of the experiment.</li>
	<li>Maintain rats at room temperature of 22 &#177; 1 &#176;C, with 12 hr light and 12 hr dark cycles. Provide rat chow and water ad libitum.</li>
	<li>Measure food and water intakes daily. Measure body weight weekly.</li>
	<li>Calculate the amount of DMN for each rat based on a dose of 10 mg/kg body weight. Use a 1 ml syringe with a 25 gauge needle to draw the calculated volume of DMN solution into the syringe.</li>
	<li>Restrain the rat for intraperitoneal injection21.</li>
	<li>With the rat properly restrained, introduce the needle into the lower right quadrant of the abdomen, draw back on the plunger of the syringe (nothing should be aspirated) to check for the correct placement of needle within the abdominal space and slowly inject DMN solution.</li>
	<li>When the dose of DMN has been administered, slowly withdraw the needle and dispose into the sharps bin.</li>
	<li>Perform the intraperitoneal injections of DMN for 3 consecutive days a week for 4 weeks. Administer the injections at the same time each day. Re-calculate the dosage based on the body weight at the start of each week of injections.</li>
	<li>Collect blood from the tail vein weekly to measure biochemical parameters: alanine aminotransferase (ALT) and aspartate aminotransferase (AST)22.</li>
	<li>Measure serum ALT and AST using a commercial Vet Test Chemistry Analyzer.</li>
</ol>
3. Gross Examination and Harvest of Liver&#160;
<ol>
	<li>Euthanize the rats by intraperitoneal injection of pentobarbital at a dose of 100 mg/kg body weight. Ensure death by checking for the lack of heartbeat.</li>
	<li>Prepare for the post mortem and assess the condition of the animal as described by Parkinson et al.23 Place dead rats onto dissecting board in dorsal recumbency with the abdomen facing upwards.</li>
	<li>Disinfect and moisten the skin with 70% ethanol.</li>
	<li>Using scissors, incise the skin the full length of the ventrum from the anus to the chin, and reflect the skin. With the scissors, incise the abdominal wall from the anus to the xiphoid cartilage, to expose the abdominal viscera.</li>
	<li>Examine the abdominal organs in situ to check for any abnormalities. Note any changes in color, size, location of organs and the presence of fluid within the peritoneal cavity. Examine the consistency of organs and note the presence of any odors.</li>
	<li>Using scissors and forceps, dissect the entire liver free of its ligaments and attachments.
	<ol>
		<li>Begin at the hilus where it is attached to the diaphragm and work to free all the liver lobes of attachments. Carefully cut all ligaments and blood vessels.</li>
	</ol>
	</li>
	<li>Transfer the liver onto a petri dish and weigh the liver.</li>
	<li>Using a scalpel, cut 2 - 5 mm thick sections of liver lobes. For consistency, always take samples from the same liver lobe. Take a wedge about 5 mm diameter in thickness, from the right lateral lobe24 about 1 cm from the edge of the lobe.Immediately place into 10% buffered formalin for fixation. Ensure the tissue to formalin volume is 1:10 or more.</li>
	<li>Harvest and immediately freeze portions (using liquid nitrogen) of liver lobes at this point if required for other studies. As for (3.8), sample from the same liver lobe for consistency.</li>
</ol>
4. Processing, Embedding and Sectioning of the Liver
<ol>
	<li>Fix the liver samples in 10% buffered formalin for a minimum of 24 hr.</li>
	<li>After fixation, trim the liver, place into cassettes and load them into the basket of the automated tissue processor.</li>
	<li>Place the basket with cassettes into the first station which contains 10% buffered formalin. Ensure the cassettes are entirely submerged. They can remain in this chamber for up to 12 hr until the cycle begins.</li>
	<li>Program the tissue processor to rotate the cassettes for 1 hr each, through successive stations containing the following solutions: ethanol at concentrations of 50%, 70%, 90%, 100% (2 stations), xylene (2 stations) and liquid paraffin (2 stations).</li>
	<li>Transfer the liver samples into the wax bath of the embedding station. Ensure that the embedding machine is switched on at least one hour earlier.</li>
	<li>Using pre-warmed forceps (65 &#176;C), remove each cassette from the wax bath and place onto the warm plate (65 &#176;C) of the embedding machine. Dispense sufficient amount of liquid paraffin into the stainless steel base mold (pre-warmed to 65 &#176;C) till half full. Transfer the section of liver from the cassette into the mold. Ensure the specimen is placed with the cut surface (to be examined microscopically) flat on the bottom of the mold.</li>
	<li>Fill the mold completely with liquid paraffin, mount the empty cassette on top and place the mold onto the 4 &#176;C plate of the embedding machine to cool for at least 30 min.</li>
	<li>Check that the wax has cooled and solidified, and remove the paraffin block from the mold. The block should pop out easily and not stick or crack. If this happens, melt the block and repeat this step. The paraffin block can be sectioned once it solidifies. Blocks can be stored at room temperature for many years.</li>
	<li>Place the paraffin block into the microtome and section at 10 &#181;m thickness until the wax layer is removed sufficiently for the embedded liver tissue to be visible.</li>
	<li>Change the setting on the microtome to cut at 5 &#181;m thickness. Using forceps, pick up a well cut section by holding it at the edge and carefully float the section in the water bath at a temperature of 40 &#176;C.</li>
	<li>Gently place a clean glass slide under the floating section and lift it onto the surface of the glass slide. Place the slide onto a slide warmer at 37 &#176;C overnight.</li>
</ol>
5. Masson Trichrome Staining
<ol>
	<li>Before applying the stains, treat the slides to remove the wax and rehydrate the tissues as follows:
	<ol>
		<li>Immerse the sections in xylene for 5 min. Repeat 2x.</li>
		<li>Immerse the sections in 100% ethanol for 3 min. Repeat 1x.</li>
		<li>Immerse the sections for 1 min each in the following solutions of ethanol: 90%, 80% and 70%.</li>
	</ol>
	</li>
	<li>Rinse the slide under tap water to remove any existing ethanol on the slide.</li>
	<li>Immerse slides in Iron Hematoxylin for 10 min to stain the nucleus.</li>
	<li>Rinse the slides with water to remove the excess stain from the slide.</li>
	<li>Immerse the slides in Biebrich Scarlet-Acid Fuchsin for 2 min to stain the cytoplasm.</li>
	<li>Rinse the slides with water.</li>
	<li>Place the slides in Phosphotungstic/Phosphomolybdic Acid Solution for 10 min to promote the uptake of Aniline blue. Change the Phosphotungstic/Phosphomolybdic Acid Solution after 2 rounds of use.</li>
	<li>Place the slides in aniline blue solution for 10 min to stain the collagen fibers.</li>
	<li>Rinse the excess stain with water and place the slides in 1% acetic acid solution for 1 min to allow differentiation to take place to render the color of the slide more delicate and transparent.</li>
	<li>Rinse the slides with water and proceed to dehydrate the tissue sections. The dehydration steps are as follows:</li>
	<li>Immerse the slides successively for 10 sec each time, in the following concentrations of ethanol: 70%, 80%, 95% and 100%.</li>
	<li>Immerse the slides for 30 sec in 100% ethanol. Repeat 1x.</li>
	<li>Rinse the slides through 3 stations of xylene for 5 min each to remove ethanol.</li>
	<li>Mount a glass cover slip onto the specimen with mounting media (e.g., DPX mountant) and allow to dry. 
	Note: DPX is a synthetic resin that dries quickly, preserves stain and protects the tissue section.</li>
</ol>
6. Fibrosis Scoring on Masson&#39;s Trichome Stained Sections of Liver
<ol>
	<li>Have a veterinary pathologist assess the severity of fibrosis according to the fibrosis score25. It would be optimum if the fibrosis scoring is done by two or three pathologists independently in a blinded manner. In such a scenario, obtain a final score after discussion and group review to sort out any differences in scoring.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Score</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>0</td>
			<td>No fibrosis</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Fibrous expansion of some portal areas, with or without short fibrous septa</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Fibrous expansion of most portal areas, with or without short fibrous septa</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Fibrous expansion of most portal areas, occasional portal to portal (P-P) bridging&#160;</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Fibrous expansion of portal areas with marked bridging (portal to portal (P-P) as well as portal to central (P-C)&#160;</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Marked bridging (P-P and/or P-C) with occasional nodules (incomplete cirrhosis)</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Cirrhosis, probable or definite</td>
		</tr>
	</tbody>
</table>
Table 1: Fibrosis Score Used for Reading the Masson&#39;s Trichome Stained Liver Sections25.

<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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimethylnitrosamine-induced+hepatic+cirrhosis:+a+new+canine+model+of+an+ancient+human+disease.">Dimethylnitrosamine-induced hepatic cirrhosis: a new canine model of an ancient human disease.</a> Surgery. 68 (1), 260-267 (1970).</li><li>Jenkins, S. A., Grandison, A., Baxter, J. N., Day, D. W., Taylor, I., Shields, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dimethylnitrosamine-induced+model+of+cirrhosis+and+portal+hypertension+in+the+rat.">A dimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat.</a> J Hepatol. 1 (5), 489-499 (1985).</li><li>J&#233;z&#233;quel, A. M., Mancini, R., Rinaldesi, M. L., Macarri, G., Venturini, C., Orlandi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morphological+study+of+the+early+stages+of+hepatic+fibrosis+induced+by+low+doses+of+dimethylnitrosamine+in+the+rat.">A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat.</a> J Hepatol. 5 (2), 174-181 (1987).</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=Molecular+characteristics+of+dimethylnitrosamine+induced+fibrotic+liver+collagen.">Molecular characteristics of dimethylnitrosamine induced fibrotic liver collagen.</a> Biochim Biophys Acta, Protein Struct Mol Enzymol. 1292 (2), 215-222 (1996).</li><li>Winwood, P. J., Arthur, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kupffer+cells:+their+activation+and+role+in+animal+models+of+liver+injury+and+human+liver+disease.">Kupffer cells: their activation and role in animal models of liver injury and human liver disease.</a> Semin Liver Dis. 13 (1), 50-59 (1993).</li><li>Nakamura, T., Sakata, R., Ueno, T., Sata, M., Ueno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+transforming+growth+factor&#946;+prevents+progression+of+liver+fibrosis+and+enhances+hepatocyte+regeneration+in+dimethylnitrosamine-treated+rats.">Inhibition of transforming growth factor&#946; prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.</a> Hepatology. 32 (2), 247-255 (2000).</li><li>Shek, F. W., Benyon, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+can+transforming+growth+factor+beta+be+targeted+usefully+to+combat+liver+fibrosis?.">How can transforming growth factor beta be targeted usefully to combat liver fibrosis?.</a> Eur J Gastroenterol Hepatol. 16 (2), 123-126 (2004).</li><li>Wang, J. H., Shin, J. W., Son, J. Y., Cho, J. H., Son, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifibrotic+effects+of+CGX,+a+traditional+herbal+formula,+and+its+mechanisms+in+rats.">Antifibrotic effects of CGX, a traditional herbal formula, and its mechanisms in rats.</a> J Ethnopharmacol. 127 (2), 534-542 (2010).</li><li>Su, L. J., Hsu, S. L., Yang, J. S., Tseng, H. H., Huang, S. F., Huang, C. Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+gene+expression+profiling+of+dimethylnitrosamine-induced+liver+fibrosis:+from+pathological+and+biochemical+data+to+microarray+analysis.">Global gene expression profiling of dimethylnitrosamine-induced liver fibrosis: from pathological and biochemical data to microarray analysis.</a> Gene Expr. 13 (2), 107-132 (2006).</li><li>M&#252;ller, A., Machnik, F., Zimmermann, T., Schubert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioacetamide-induced+cirrhosis-like+liver+lesions+in+rats+usefulness+and+reliability+of+this+animal+model.">Thioacetamide-induced cirrhosis-like liver lesions in rats usefulness and reliability of this animal model.</a> Exp Pathol. 34 (4), 229-236 (1988).</li><li>Martinez-Hernandez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hepatic+extracellular+matrix.+II.+Electron+immunohistochemical+studies+in+rats+with+CCl4-induced+cirrhosis.">The hepatic extracellular matrix. 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. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Necropsy+and+Selected+Tissue+and+Sample+Collection+in+Rats+and+Mice.">Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice.</a> J Vis Exp. (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 ...

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.

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-dimethylnitrosamine-induced-liver-fibrosis-model-in-the-rat

Research

JoVE Journal

Medicine

11.9K Views.  Temasek Polytechnic. 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.

Video: The Dimethylnitrosamine Induced Liver Fibrosis Model in the Rat

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS) Procedure

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver metastases, the amount of liver tissue left behind after liver resection has become the main limiting factor of resectability of large or multiple liver tumors. A minimal amount of functional tissue is required to avoid the severe complication of post-hepatectomy liver failure, which has high morbidity and mortality. Inducing liver growth of the prospective remnant prior to resection has become more established in liver surgery, either in the form of portal vein embolization by interventional radiologists or in the form of portal vein ligation several weeks prior to resection. Recently, it was shown that liver regeneration is more extensive and rapid, when the parenchymal transection is added to portal vein ligation in a first stage and then, after only one week of waiting, resection performed in a second stage (Associating Liver Partition and Portal vein ligation for Staged hepatectomy = ALPPS). ALPPS has rapidly become popular across the world, but has been criticized for its high perioperative mortality. The mechanism of accelerated and extensive growth induced by this procedure has not been well understood. Animal models have been developed to explore both the physiological and molecular mechanisms of accelerated liver regeneration in ALPPS. This protocol presents a rat model that allows mechanistic exploration of accelerated regeneration.

Rat Model of the Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy ALPPS Procedure

Inducing rapid liver hypertrophy using Associating Liver Partition and Portal vein ligation for a Staged hepatectomy (ALPPS) has been proposed for resection of borderline resectable liver tumors. This model may elucidate mechanisms involved in rapid hypertrophy and allows testing of drugs that promote or block the acceleration of regeneration.

Recent clinical data support an aggressive surgical approach to both primary and metastatic liver tumors. For some indications, like colorectal liver ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

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

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

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