The authors gratefully acknowledge Bertha Delgado, Department of Pathology, Soroka Medical Center, Faculty of Health Sciences, Ben-Gurion University of the Negev, for her help in the laboratory as well as in the histology analysis.

The experiments were conducted according to the recommendations of the Declarations of Helsinki and Tokyo and to the Guidelines for the Use of Experimental Animals of the European Community. The experiments were approved by the Animal Care Committee of Ben-Gurion University of the Negev.
NOTE: The CCl4 model has been generated and used in a previous study17. The protocol timeline is demonstrated in Table 1.
1. Preparing rats for the experimental procedure
NOTE: Select adult male Sprague Dawley rats weighing 300&#8722;350 g.
<ol>
	<li>Obtain approval for experiments from Institutional Animal Care and Use Committee (IACUC).</li>
	<li>Maintain rats at room temperature (22 &#176;C &#177; 1 &#176;C), with 12 h light and 12 h dark cycles. Provide rat chow and water ad libitum.</li>
	<li>Perform all experiments between 6:00 a.m. and 12:00 p.m.</li>
	<li>Shave the rat and disinfect the skin with alcohol.</li>
</ol>
2. Determination of serum GOT, GPT, and TB baseline levels
<ol>
	<li>Anesthesia
	<ol>
		<li>Prepare a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.</li>
		<li>Anesthetize the rat with 2% isoflurane. Confirm that the rat is fully anesthetized by observing the movement and pedal reflex in response to external stimuli. 
		NOTE: Use 1&#8722;5% isoflurane for anesthesia induction and maintenance.</li>
	</ol>
	</li>
	<li>Cannulate the tail vein with a 22 G catheter.</li>
	<li>Collect a 0.5 mL blood sample at baseline. Ensure that the blood volume retrieved does not exceed IACUC guidelines.</li>
	<li>Perform blood biochemical analysis including the measurements of serum GOT, GPT and TB, as previously described18. 
	NOTE: Examinations of liver enzymes and TB level were carried out in the biochemical laboratory of Soroka Medical Center. Blood samples were analyzed using a fluorescence method on a chemistry analyzer (Table of Materials).</li>
</ol>
3. Induction of acute liver injury in rats
CAUTION: Exposure to high concentrations of CCl4, including absorption through vapor or skin, can have negative effects on the central nervous system and cause degeneration of the liver and kidneys. Prolonged exposure can cause coma or death.
<ol>
	<li>Prepare a 50% solution of CCl4 (Table of Materials) by mixing CCl4 with olive oil as a vehicle in a 1:1 ratio. 
	NOTE: The solution should be prepared according to IACUC guidelines for non-pharmaceutical grade compounds.</li>
	<li>Induce hepatotoxicity in vivo by CCl4 administration via an orogastric tube.
	<ol>
		<li>Insert a 16 G orogastric tube (3 inches deep) through the oral cavity of the rat.</li>
		<li>Expose the rat to different doses of CCl4 by injecting a syringe with one of the following diluted solutions into the rat&#8217;s stomach: 1 mL/kg (mild ALI), 2.5 mL/kg (moderate ALI), or 5 mL/kg (severe ALI) of the 50% solution. For the sham-operated control group, expose the rat to 5 mL/kg olive oil only.</li>
	</ol>
	</li>
</ol>
4. Determination of serum GOT, GPT, and TB levels after 24 h
<ol>
	<li>Anesthesia
	<ol>
		<li>Prepare a continuous isoflurane administration system to induce anesthesia. Make sure the vaporizer system is filled with isoflurane.</li>
		<li>Anesthetize the rats with 2% isoflurane. Confirm that the rat is fully anesthetized by observing the movement and pedal reflex in response to external stimuli.</li>
	</ol>
	</li>
	<li>Collect blood samples at 24 h from CCl4 exposure.</li>
	<li>Perform blood biochemical analysis including measurements of serum GOT, GPT and TB.</li>
</ol>
5. Liver collection for histological examination
<ol>
	<li>Euthanize the rat by replacing the inspired gas mixture with 20% O2/80% CO2. Ensure that CO2 is delivered at a predetermined rate in accordance with IACUC guidelines.</li>
	<li>Ensure death by checking for lack of heartbeat and confirm by a secondary method in accordance with IACUC guidelines.</li>
	<li>Place the rat on a dissecting board with its dorsal surface facing down and abdomen facing up. Shave the abdomen of the rat.</li>
	<li>With a scalpel, incise the full length of the ventrum skin from the anus to the chin. Separate the skin. Incise the abdominal wall with a scalpel from the anus to the xyphoid cartilage, exposing the abdominal viscera.</li>
	<li>Using scissors and forceps, isolate the liver by dissecting it from its ligaments and attachments. Starting at the liver hilum, carefully perform a hepatectomy by releasing all the liver lobes from attachments. Dissect and cut away all ligaments and blood vessels.</li>
	<li>Transfer the liver into a Petri dish. Fix the liver in a 4% buffered formaldehyde solution (Table of Materials) for at least 24 h.</li>
</ol>
6. Histological examination
<ol>
	<li>Sample preparation
	<ol>
		<li>After fixation, cut the sample into 5 &#956;m thick slice series by microtome sectioning.</li>
		<li>Gently place the slices on glass slides with a soft brush, 1 slice per slide.</li>
		<li>Perform H&#38;E staining as previously described19.</li>
	</ol>
	</li>
	<li>Examine the slices under a microscope at 200x magnification using a 20 mm objective lens (Table of Materials). 
	NOTE: The liver sections should be graded by a specialized pathologist blinded to the treatment protocol. A score of 0 indicates no liver abnormalities, 1&#8211;2 indicates mild liver injury, 3&#8211;4 indicates moderate liver injury, and 5&#8211;6 indicates severe liver injury20,21,22.</li>
</ol>

<ol>
	<li>Hoofnagle, J. H., Carithers, R. L., Shapiro, C., Ascher, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fulminant+hepatic+failure:+Summary+of+a+workshop.">Fulminant hepatic failure: Summary of a workshop.</a> Hepatology. 21 (1), 240-252 (1995).</li><li>Rakela, J., Lange, S. M., Ludwig, J., Baldus, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fulminant+hepatitis:+Mayo+clinic+experience+with+34+cases.">Fulminant hepatitis: Mayo clinic experience with 34 cases.</a> Mayo Clinic Proceedings. 60 (5), 289-292 (1985).</li><li>Riordan, S. M., Williams, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+hepatic+encephalopathy.">Treatment of hepatic encephalopathy.</a> New England Journal of Medicine. 337, 473-479 (1997).</li><li>Bernuau, J., Rueff, B., Benhamou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fulminant+and+subfulminant+liver+failure:+Definitions+and+causes.">Fulminant and subfulminant liver failure: Definitions and causes.</a> Seminars in Liver Disease. 6 (2), 97-106 (1986).</li><li>Lidofsky, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+transplantation+for+fulminant+hepatic+failure.">Liver transplantation for fulminant hepatic failure.</a> Gastroenterology Clinics of North America. 22 (2), 257-269 (1993).</li><li>Auzinger, G., Wendon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intensive+care+management+of+acute+liver+failure.">Intensive care management of acute liver failure.</a> Current opinion in critical care. 14 (2), 179-188 (2008).</li><li>Wu, Q., 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=Protection+of+regenerating+liver+after+partial+hepatectomy+from+carbon+tetrachloride+hepatotoxicity+in+rats:+Roles+of+mitochondrial+uncoupling+protein+2+and+ATP+stores.">Protection of regenerating liver after partial hepatectomy from carbon tetrachloride hepatotoxicity in rats: Roles of mitochondrial uncoupling protein 2 and ATP stores.</a> Digestive Diseases and Sciences. 54 (9), 1918-1925 (2009).</li><li>Tu&#241;&#243;n, M. J., Alvarez, M., Culebras, J. M., Gonz&#225;lez-Gallego, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+animal+models+for+investigating+the+pathogenesis+and+therapeutic+strategies+in+acute+hepatic+failure.">An overview of animal models for investigating the pathogenesis and therapeutic strategies in acute hepatic failure.</a> World Journal of Gastroenterologyl. 15 (25), 3086-3098 (2009).</li><li>van de Kerkhove, M. P., Hoekstra, R., van Gulik, T. M., Chamuleau, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+animal+models+of+fulminant+hepatic+failure+in+artificial+and+bioartificial+liver+support+research.">Large animal models of fulminant hepatic failure in artificial and bioartificial liver support research.</a> Biomaterials. 25 (9), 1613-1625 (2004).</li><li>Butterworth, R. F., 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=Experimental+models+of+hepatic+encephalopathy:+ISHEN+guidelines.">Experimental models of hepatic encephalopathy: ISHEN guidelines.</a> Liver International. 29 (6), 783-788 (2009).</li><li>Zhang, B., Gong, D., Mei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+regenerating+liver+after+partial+hepatectomy+from+carbon+tetrachloride+hepatotoxicity+in+rats:+Role+of+hepatic+stimulator+substance.">Protection of regenerating liver after partial hepatectomy from carbon tetrachloride hepatotoxicity in rats: Role of hepatic stimulator substance.</a> Journal of Gastroenterology and Hepatology. 14 (10), 1010-1017 (1999).</li><li>Ugazio, G., Danni, O., Milillo, P., Burdino, E., Congiu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+protection+against+carbon+tetrachloride+toxicity.+I.+prevention+of+lethal+effects+by+partial+surgical+hepatectomy.">Mechanism of protection against carbon tetrachloride toxicity. I. prevention of lethal effects by partial surgical hepatectomy.</a> Drug and Chemical Toxicology. 5 (2), 115-124 (1982).</li><li>Taniguchi, M., Takeuchi, T., Nakatsuka, R., Watanabe, T., Sato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+process+in+acute+liver+injury+and+regeneration+induced+by+carbon+tetrachloride.">Molecular process in acute liver injury and regeneration induced by carbon tetrachloride.</a> Life Science. 75 (13), 1539-1549 (2004).</li><li>Gordis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+metabolites+of+carbon+tetrachloride.">Lipid metabolites of carbon tetrachloride.</a> Journal of Clinical Investigation. 48 (1), 203-209 (1969).</li><li>Albrecht, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+RNA+synthesis+in+experimental+hepatogenic+encephalopathy.">Cerebral RNA synthesis in experimental hepatogenic encephalopathy.</a> Journal of Neuroscience Research. 6 (4), 553-558 (1981).</li><li>Terblanche, J., Hickman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+fulminant+hepatic+failure.">Animal models of fulminant hepatic failure.</a> Digestive Diseases and Sciences. 36 (6), 770-774 (1991).</li><li>Gruenbaum, B. F., 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=Cell-free+DNA+as+a+potential+marker+to+predict+carbon+tetrachloride-induced+acute+liver+injury+in+rats.">Cell-free DNA as a potential marker to predict carbon tetrachloride-induced acute liver injury in rats.</a> Hepatology International. 7 (2), 721-727 (2013).</li><li>Juricek, J., 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=Analytical+evaluation+of+the+clinical+chemistry+analyzer+Olympus+AU2700+plus.">Analytical evaluation of the clinical chemistry analyzer Olympus AU2700 plus.</a> Biochemia Medica. 20 (3), 334-340 (2010).</li><li>Feldman, A. T., Wolfe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+processing+and+hematoxylin+and+eosin+staining.">Tissue processing and hematoxylin and eosin staining.</a> Methods in Molecular Biology. 1180, 31-43 (2014).</li><li>Wang, T., 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=Protective+effects+of+dehydrocavidine+on+carbon+tetrachloride-induced+acute+hepatotoxicity+in+rats.">Protective effects of dehydrocavidine on carbon tetrachloride-induced acute hepatotoxicity in rats.</a> Journal of Ethnopharmacology. 117 (2), 300-308 (2008).</li><li>Ye, X., 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=Hepatoprotective+effects+of+coptidis+rhizoma+aqueous+extract+on+carbon+tetrachloride-induced+acute+liver+hepatotoxicity+in+rats.">Hepatoprotective effects of coptidis rhizoma aqueous extract on carbon tetrachloride-induced acute liver hepatotoxicity in rats.</a> Journal of Ethnopharmacology. 124 (1), 130-136 (2009).</li><li>Wills, P. J., Asha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+lygodium+flexuosum+(L.)+sw.+extract+against+carbon+tetrachloride-induced+acute+liver+injury+in+rats.">Protective effect of lygodium flexuosum (L.) sw. extract against carbon tetrachloride-induced acute liver injury in rats.</a> Journal of Ethnopharmacology. 108 (3), 320-326 (2006).</li><li>Senior, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+for+hepatotoxicity:+What+is+the+predictive+value+of+liver+"function"+tests.">Monitoring for hepatotoxicity: What is the predictive value of liver "function" tests.</a> Clinical Pharmacology &amp; Therapeutics. 85 (3), 331-334 (2009).</li><li>Karmen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+note+on+the+spectrometric+assay+of+glutamic-oxalacetic+transaminase+in+human+blood+serum.">A note on the spectrometric assay of glutamic-oxalacetic transaminase in human blood serum.</a> Journal of Clinical Investigation. 34 (1), 131-133 (1955).</li><li>Hubner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+liver+damage+caused+by+direct+action+of+carbon+tetrachloride+in+vivo+and+in+vitro.">Ultrastructural liver damage caused by direct action of carbon tetrachloride in vivo and in vitro.</a> Virchows Archiv fur Pathologische Anatomie und Physiologie und fur Klinische Medizin. 339 (3), 187-197 (1965).</li><li>Newsome, P. N., Plevris, J. N., Nelson, L. J., Hayes, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+fulminant+hepatic+failure:+A+critical+evaluation.">Animal models of fulminant hepatic failure: A critical evaluation.</a> Liver Transplantation. 6 (1), 21-31 (2000).</li></ol>

The authors have nothing to disclose.

In this protocol, CCl4 is used as a liver toxin to induce ALI in rats. ALI is characterized by loss of hepatic parenchyma and subsequent dysregulation of the liver&#8217;s metabolic and synthetic functions. Drugs, viruses, toxins, autoimmune diseases, metabolic diseases, and vascular disorders all induce hepatocyte death, and the subsequent inflammatory response contributes to the pathogenesis of ALI.
The initial insult to the liver leads to cytokine production, chemokine release, a...

The frequency of toxic insults to the liver, especially due to alcohol and drug abuse, is increasing. Acute liver injury (ALI) is associated with high mortality rates and has caused clinical concerns1,2. Toxic injury leads to death signaling pathways in the liver, resulting in hepatocyte apoptosis, necrosis, or pyroptosis. ALI plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis3,4. Although recent research has increased our knowledge about the physiological and pathological changes accompanying hepatic failure, it has not completely explained the pathomolecular features that affect the mechanisms of cell death. Furthermore, no medications are currently available to reverse the progressive deterioration in ALI patients. Currently, the only significantly effective treatment is liver transplantation5,6.
In order to investigate the mechanism and pathophysiology of ALI and to test different hepatoprotective strategies, different animal models are used to induce ALI. A preferable animal model of ALI should mimic the pathological process of the disease via a reliable, validated, inexpensive and easy to apply method. Examples of experimental models include hepatotoxic agents, surgical procedures such as total or partial hepatectomy, complete or transient devascularization, and infective procedures7,8,9. Known hepatotoxic substances include galactosamine, acetaminophen, thioacetamide, azoxymethane and CCl4. Of these, CCl4 is widely used although it has not yet been well characterized10,11,12,13.
CCl4 is an organic colorless liquid compound with a sweet smell and almost no flammability at lower temperatures. Exposure to high concentrations of CCl4 can cause damage to the central nervous system, including deterioration of the liver and kidneys. CCl4 induces ALI through its biotransformation in the liver, which forms reactive oxygen species. This occurs via the P450 cytochrome enzyme 2E1, forming an active metabolite and resulting in cell damage by macromolecule binding, enhancement of lipid peroxidation and disturbance of intracellular calcium homeostasis14. In addition, the CCl4 model can be used to stimulate the astrocytes at the level of RNA synthesis15. This hepatotoxin has been administered by the intraperitoneal, intraportal, oral, and intragastric routes16.
In this protocol, we describe in detail CCl4-induced ALI in rats via an orogastric tube. This method induces robust and reproducible ALI that can be used to investigate the pathogenesis of ALI. Determination of liver disease severity is monitored by measurement of serum glutamate-pyruvate transaminase (GPT), glutamic oxaloacetic transaminase&#160;(GOT) enzymes and total bilirubin (TB) as well as definitive histological diagnosis by hematoxylin and eosin (H&#38;E) stained liver tissues. Exposure to CCl4 through an intragastric access allows for a practical, inexpensive, minimally invasive method with minimal hazard risk.

<table><tbody><tr><td>22 G catheter BD Neoflon TM</td><td>Becton Dickinson Infusion Therapy AB</td><td></td><td></td></tr><tr><td>4% buffered formaldehyde solution</td><td>Sigma - Aldrich lab materials technologies</td><td></td><td></td></tr><tr><td>BD Microtainer SST TM Tubes</td><td>Becton Dickinson and Company</td><td></td><td></td></tr><tr><td>Carbone tetrachloride</td><td>Sigma - Aldrich lab materials technologies</td><td>CAS 56-23-5</td><td></td></tr><tr><td>Isofluran, USP 100%</td><td>Piramamal Critical Care, Inc</td><td></td><td></td></tr><tr><td>Olympus AU2700 Chemistry-Immuno Analyzer</td><td>Olympus (MN, USA)</td><td></td><td>Analysis of blood samples was done by the fluorescence method</td></tr><tr><td>Olympus BX 40 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>RAT Feeding Needles</td><td>ORCHID SCIENTIFICS</td><td></td><td></td></tr><tr><td>SYRINGE SET 1 and 2 ml MEDI -PLUS</td><td>Shandong Zibo Shanchuan Medical Instruments Co., Ltd</td><td></td><td></td></tr></tbody></table>

inducing acute liver injury in rats via carbon tetrachloride (ccl4) exposure through an orogastric tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

The TB, GOT, and GPT levels significantly increased 24 h after inducing ALI (more at higher CCl4 doses) compared to sham-operated controls (p &#60; 0.001) (Figure 1). The levels of TB, GOT, and GPT at baseline were normal and were not significantly different than sham-operated controls. At 24 h, all three interventional groups, 1 mL/kg CCl4 (1, 1&#8722;2), 2.5 mL/kg CCl4 (3, 3&#8722;4), and 5 mL/kg CCl4 (4, 4&#8722;5.75), had a significantly higher...

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Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

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11.2K Views.  Ben-Gurion University of the Negev. Ik wil ons werk presenteren over geïnduceerde acute leverletsel bij ratten via blootstelling aan koolstoftetrachloride via orogastrische buis. Selecteer volwassen mannelijke Sprague-Dawley ratten met een gewicht van 300 tot 350 gram. Zorg voor rat chow en water ad libitum.Voer alle experimenten uit tussen zes uur 's zes en 12 uur. De dierenverzorging komedie van de Ben-Gurion Universiteit van de Negev, Israël goedgekeurd alle behandeling en testen procedure gebruikt in dit protocol. ...

Video: Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

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 ...

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.

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 ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

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 ...

Mouse Model of Oleic Acid-Induced Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette smoke, infectious agents, and fatty acids can induce ARDS. Animal models can mimic the complex pathomechanism of the ARDS. However, each of them has limitations. Notably, oleic acid (OA) is increased in critically ill patients with harmful effects on the lung. OA can induce lung injury by emboli, disrupting tissue, altering pH, and impairing edema clearance. OA-induced lung injury model resembles various features of ARDS with endothelial injury, increased alveolar permeability, inflammation, membrane hyaline formation, and cell death. Herein, induction of lung injury is described by injecting OA (in salt form) directly into the lung and intravenously in a mouse since it is the physiological form of OA at pH 7. Thus, the injection of OA in the salt form is a helpful animal model to study lung injury/ARDS without causing emboli or altering the pH, thereby getting close to what is happening in critically ill patients.

The present protocol describes a lung injury model in mice using oleic acid to mimic acute respiratory distress syndrome (ARDS). This model increases the inflammatory mediators on edema and decreases lung compliance. Oleic acid is used in the salt form (oleate) since this physiological form avoids the risk of embolism.

Acute respiratory distress syndrome (ARDS) is a significant threat to critically ill patients with a high fatality rate. Pollutant exposure, cigarette ...

Immunology and Infection

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

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 ...

Vinyl Chloride and High-Fat Diet as a Model of Environment and Obesity Interaction

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although several studies have investigated the role of VC as a direct hepatotoxicant, the concept that VC modifies sensitivity of the liver to other factors, such as nonalcoholic fatty liver disease (NAFLD) caused by high-fat diet (HFD) is novel. This protocol describes an exposure paradigm to evaluate the effects of chronic, low-level exposure to VC. Mice are acclimated to low-fat or high-fat diet one week prior to the beginning of the inhalation exposure and remain on these diets throughout the experiment. Mice are exposed to VC (sub-OSHA level: &#60;1 ppm) or room air in inhalation chambers for 6 hours/day, 5 days/week, for up to 12 weeks. Animals are monitored weekly for body weight gain and food consumption. This model of VC exposure causes no overt liver injury with VC inhalation alone. However, the combination of VC and HFD significantly enhances liver disease. A technical advantage of this co-exposure model is the whole-body exposure, without restraint. Moreover, the conditions more closely resemble a very common human situation of a combined exposure to VC with underlying nonalcoholic fatty liver disease and therefore support the novel hypothesis that VC is an environmental risk factor for the development of liver damage as a complication of obesity (i.e., NAFLD). This work challenges the paradigm that the current exposure limits of VC (occupational and environmental) are safe. The use of this model can shed new light and concern on the risks of VC exposure. This model of toxicant-induced liver injury can be used for other volatile organic compounds and to study other interactions that may impact the liver and other organ systems.

The goal of this protocol was to develop a murine model of low-level toxicant exposure that does not cause overt liver injury but rather exacerbates pre-existing liver damage. This paradigm better recapitulates human exposure and the subtle changes that occur upon exposure to toxicant concentrations that are considered safe.

Vinyl chloride (VC), an abundant environmental contaminant, causes steatohepatitis at high levels, but is considered safe at lower levels. Although ...

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 &#181;L/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice.

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

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.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl₄) Exposure Through an Orogastric Tube

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