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.

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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><tbody><tr><td>Dimethylnitrosamine</td><td>Wako</td><td>147-03781</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>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&#039;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>Tissue processor</td><td>Leica</td><td>Leica TP 1020</td><td></td></tr><tr><td>Embedding machine</td><td>Sakura</td><td>&amp;nbsp;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-dimethylnitrosamine-induced-liver-fibrosis-model-in-the-rat

Research

JoVE Journal

Medicine

12.0K 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

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Developmental Biology

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

A Multimodal Imaging Approach Based on Micro-CT and Fluorescence Molecular Tomography for Longitudinal Assessment of Bleomycin-Induced Lung Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure.
The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

We describe a non-invasive multimodal imaging approach based on Micro-CT and fluorescence molecular tomography for longitudinal assessment of the mouse lung fibrosis model induced by double intratracheal instillation of bleomycin.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which ...

Immunology and Infection

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Bioengineering

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

Generation of a Rat Model of Acute Liver Failure by Combining 70% Partial Hepatectomy and Acetaminophen

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and detoxifying functions of the liver. In most irreversible liver damage cases, orthotropic liver transplant (OLT) remains the only available treatment. To study the therapeutic potential of a treatment for ALF, its prior testing in an animal model of ALF is essential. In the current study, an ALF model in rats was developed by combining 70% partial hepatectomy (PHx) and injections of acetaminophen (APAP) that provides a therapeutic window of 48 h. The median and left lateral lobes of the liver were removed to excise 70% of the liver mass and APAP was given 24 h postsurgically for 2 days. Survival in ALF-induced animals was found to be severely decreased. The development of ALF was confirmed by altered serum levels of the enzymes alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP); changes in prothrombin time (PT); and assessment of the international normalized ratio (INR). Study of the gene expression profile by qPCR revealed an increase in expression levels of genes involved in apoptosis, inflammation, and in the progression of liver injury. Diffused degeneration of hepatocytes and infiltration of immune cells was observed by histological evaluation. The reversibility of ALF was confirmed by the restoration of survival and serum levels of ALT, AST, and ALP after intrasplenic transplantation of syngeneic healthy rat hepatocytes. This model presents a reliable alternative to the available ALF animal models to study the pathophysiology of ALF as well as to evaluate the potential of a novel therapy for ALF. The use of two different approaches also makes it possible to study the combined effect of physical and drug-induced liver injury. The reproducibility and feasibility of current procedure is an added benefit of the model.

The acute liver failure animal model developed in the current study presents a feasible alternative for the study of potential therapies. The current model employs the combined effect of physical and drug-induced hepatic injury and provides a suitable time window to study the potential of novel therapies.

Acute liver failure (ALF) is a clinical condition caused by various etiologies resulting in the loss of metabolic, biochemical, synthesizing, and ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

Biology

Using Nicotine in a Silica-Exposed Mouse Model to Promote Lung Epithelial-Mesenchymal Transition

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role of nicotine, the primary addictive ingredient in cigarettes, in silicosis development is unclear. The mouse model employed in this study was simple and easily controlled, and it effectively simulated the effects of chronic nicotine ingestion and repeated exposure to silica on lung fibrosis through epithelial-mesenchymal transition in human beings. In addition, this model can help in the direct study of the effects of nicotine on silicosis while avoiding the effects of other components in cigarette smoke.
After environmental adaptation, mice were injected subcutaneously with 0.25 mg/kg nicotine solution into the loose skin over the neck every morning and evening at 12 h intervals over 40 days. Additionally, crystalline silica powder (1-5 &#181;m) was suspended in normal saline, diluted to a suspension of 20 mg/mL, and dispersed evenly using an ultrasonic water bath. The isoflurane-anesthetized mice inhaled 50 &#181;L of this silica dust suspension through the nose and were awoken via chest massage. Silica exposure was administrated daily on days 5-19.
The double-exposed mouse model was exposed to nicotine and then silica, which matches the exposure history of workers who are exposed to both harmful factors. In addition, nicotine promoted pulmonary fibrosis through epithelial-mesenchymal transformation (EMT) in mice. This animal model can be used to study the effects of multiple factors on the development of silicosis.

This study describes a mouse model to study the synergistic effect of nicotine on the progression of pulmonary fibrosis in experimental silicosis mice. The dual-exposure mouse model simulates the pathological progression in the lung after simultaneous exposure to nicotine and silica. The methods described are simple and highly reproducible.

Smoking and exposure to silica are common among occupational workers, and silica is more likely to injure the lungs of smokers than non-smokers. The role ...