Name: establishment of a mouse severe acute pancreatitis model using retrograde injection of sodium taurocholate into the biliopancreatic duct
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Description: Watch this Scientific Journal Video about Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct at JoVE.com

We are grateful for the support from the following grants: a Translational Research Grant of NCRCH [2020WSA01], a KJXW Scientific Grant from Suzhou Commission of Health for Young Scholars [KJXW2020002], a Science and Technology Plan of Suzhou City (SKY2021038 and SKJY2021050), a grant from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and a Primary Research &#38; Social Development Plan of Jiangsu Province (BE2018659).

All experiments involving animals were approved by the Animal Ethics Committee of Soochow University. All surgical procedures were performed under full anesthesia. Analgesics were not used to avoid interference with the natural course of the disease according to previous literatures28,29. Approval for the lack of analgesia was also granted by the Animal Ethics Committee of Soochow University.
1. Preparation
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<ol>
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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=Classification+of+acute+pancreatitis-2012:+revision+of+the+Atlanta+classification+and+definitions+by+international+consensus.">Classification of acute pancreatitis-2012: revision of the Atlanta classification and definitions by international consensus.</a> Gut. 62 (1), 102-111 (2013).</li><li>Munir, 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=Advances+in+immunomodulatory+therapy+for+severe+acute+pancreatitis.">Advances in immunomodulatory therapy for severe acute pancreatitis.</a> Immunology Letters. 217, 72-76 (2020).</li><li>Peery, A. 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=Burden+of+gastrointestinal+disease+in+the+United+States:+2012+update.">Burden of gastrointestinal disease in the United States: 2012 update.</a> Gastroenterology. 143 (5), 1179-1187 (2012).</li><li>Hines, O. J., Pandol, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+severe+acute+pancreatitis.">Management of severe acute pancreatitis.</a> BMJ. 367, 6227 (2019).</li><li>James, T. W., Crockett, 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=Management+of+acute+pancreatitis+in+the+first+72+hours.">Management of acute pancreatitis in the first 72 hours.</a> Current Opinion in Gastroenterology. 34 (5), 330-335 (2018).</li><li>Silva-Vaz, P., 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=Murine+models+of+acute+pancreatitis:+a+critical+appraisal+of+clinical+relevance.">Murine models of acute pancreatitis: a critical appraisal of clinical relevance.</a> International Journal of Molecular Sciences. 20 (11), 2794 (2019).</li><li>Hyun, J. J., Lee, H. S. <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+pancreatitis.">Experimental models of pancreatitis.</a> Clinical Endoscopy. 47 (3), 212-216 (2014).</li><li>Renner, I. G., Wisner, J. R., Rinderknecht, H. <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+exogenous+secretin+on+ceruletide-induced+acute+pancreatitis+in+the+rat.">Protective effects of exogenous secretin on ceruletide-induced acute pancreatitis in the rat.</a> Journal of Clinical Investigation. 72 (3), 1081-1092 (1983).</li><li>Niederau, C., Ferrell, L. D., Grendell, J. 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M., Werner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+alcohol-induced+pancreatic+injury+is+similar+with+intravenous+and+intragastric+routes+of+alcohol+administration.">Acute alcohol-induced pancreatic injury is similar with intravenous and intragastric routes of alcohol administration.</a> Pancreas. 43 (1), 69-74 (2014).</li><li>Huang, W., 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=Fatty+acid+ethyl+ester+synthase+inhibition+ameliorates+ethanol-induced+Ca2+-dependent+mitochondrial+dysfunction+and+acute+pancreatitis.">Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.</a> Gut. 63 (8), 1313-1324 (2014).</li><li>Sun, 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=NRF2+mitigates+acute+alcohol-induced+hepatic+and+pancreatic+injury+in+mice.">NRF2 mitigates acute alcohol-induced hepatic and pancreatic injury in mice.</a> Food and Chemical Toxicology. 121, 495-503 (2018).</li><li>Kui, 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=New+insights+into+the+methodology+of+L-arginine-induced+acute+pancreatitis.">New insights into the methodology of L-arginine-induced acute pancreatitis.</a> PLoS One. 10 (2), 0117588 (2015).</li><li>Creutzfeldt, W., Schmidt, H., Horbach, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+effects+of+a+trypsin+inhibitor+(Trasylol)+on+Enzyme+activities+and+morphology+in+taurocholate+and+calciphylaxis+pancreatitis+of+the+rat+(a+contribution+to+the+pathogenesis+of+pancreatitis).">Studies on the effects of a trypsin inhibitor (Trasylol) on Enzyme activities and morphology in taurocholate and calciphylaxis pancreatitis of the rat (a contribution to the pathogenesis of pancreatitis).</a> Klin Wochenschr. 43, 15-22 (1965).</li><li>Liu, Z. H., 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=A+simple+taurocholate-induced+model+of+severe+acute+pancreatitis+in+rats.">A simple taurocholate-induced model of severe acute pancreatitis in rats.</a> World Journal of Gastroenterology. 15 (45), 5732-5739 (2009).</li><li>Cavdar, 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=Controversial+issues+in+biliary+pancreatitis:+when+should+we+perform+MRCP+and+ERCP.">Controversial issues in biliary pancreatitis: when should we perform MRCP and ERCP.</a> Pancreatology. 14 (5), 411-414 (2014).</li><li>Perides, G., van Acker, G. J., Laukkarinen, J. M., Steer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+acute+biliary+pancreatitis+induced+by+retrograde+infusion+of+bile+acids+into+the+mouse+pancreatic+duct.">Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.</a> Nature Protocols. 5 (2), 335-341 (2010).</li><li>Orabi, A. I., 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=Cluster+of+differentiation+38+(CD38)+mediates+bile+acid-induced+acinar+cell+injury+and+pancreatitis+through+cyclic+ADP-ribose+and+intracellular+calcium+release.">Cluster of differentiation 38 (CD38) mediates bile acid-induced acinar cell injury and pancreatitis through cyclic ADP-ribose and intracellular calcium release.</a> Journal of Biological Chemistry. 288 (38), 27128-27137 (2013).</li><li>Fanczal, 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=TRPM2-mediated+extracellular+Ca(2+)+entry+promotes+acinar+cell+necrosis+in+biliary+acute+pancreatitis.">TRPM2-mediated extracellular Ca(2+) entry promotes acinar cell necrosis in biliary acute pancreatitis.</a> Journal of Physiology. 598 (6), 1253-1270 (2020).</li><li>Huang, W., 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=Caffeine+protects+against+experimental+acute+pancreatitis+by+inhibition+of+inositol+1,4,5-trisphosphate+receptor-mediated+Ca2++release.">Caffeine protects against experimental acute pancreatitis by inhibition of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release.</a> Gut. 66 (2), 301-313 (2017).</li><li>Zhang, 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=Dehydrocholic+acid+ameliorates+sodium+taurocholate-induced+acute+biliary+pancreatitis+in+mice.">Dehydrocholic acid ameliorates sodium taurocholate-induced acute biliary pancreatitis in mice.</a> Biology and Pharmaceutical Bulletin. 43 (6), 985-993 (2020).</li><li>Hagiwara, S., 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=Antithrombin+III+prevents+cerulein-induced+acute+pancreatitis+in+rats.">Antithrombin III prevents cerulein-induced acute pancreatitis in rats.</a> Pancreas. 38 (7), 746-751 (2009).</li><li>Hagiwara, S., 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=Danaparoid+sodium+prevents+cerulein-induced+acute+pancreatitis+in+rats.">Danaparoid sodium prevents cerulein-induced acute pancreatitis in rats.</a> Shock. 32 (1), 94-99 (2009).</li><li>Wittel, U. 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=Taurocholate-induced+pancreatitis:+a+model+of+severe+necrotizing+pancreatitis+in+mice.">Taurocholate-induced pancreatitis: a model of severe necrotizing pancreatitis in mice.</a> Pancreas. 36 (2), 9-21 (2008).</li><li>Barlass, U., 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=Morphine+worsens+the+severity+and+prevents+pancreatic+regeneration+in+mouse+models+of+acute+pancreatitis.">Morphine worsens the severity and prevents pancreatic regeneration in mouse models of acute pancreatitis.</a> Gut. 67 (4), 600-602 (2018).</li><li>Wu, D., 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=A+systematic+review+of+NSAIDs+treatment+for+acute+pancreatitis+in+animal+studies+and+clinical+trials.">A systematic review of NSAIDs treatment for acute pancreatitis in animal studies and clinical trials.</a> Clinical Research in Hepatology and Gastroenterology. 44, 100002 (2020).</li><li>Schmidt, 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=A+better+model+of+acute+pancreatitis+for+evaluating+therapy.">A better model of acute pancreatitis for evaluating therapy.</a> Annals in Surgery. 215 (1), 44-56 (1992).</li><li>Junyuan, Z., 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=Quercetin+protects+against+intestinal+barrier+disruption+and+inflammation+in+acute+necrotizing+pancreatitis+through+TLR4/MyD88/p38MAPK+and+ERS+inhibition.">Quercetin protects against intestinal barrier disruption and inflammation in acute necrotizing pancreatitis through TLR4/MyD88/p38MAPK and ERS inhibition.</a> Pancreatology. 18 (7), 742-752 (2018).</li><li>Waldron, R. 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=The+Orai+Ca(2+)+channel+inhibitor+CM4620+targets+both+parenchymal+and+immune+cells+to+reduce+inflammation+in+experimental+acute+pancreatitis.">The Orai Ca(2+) channel inhibitor CM4620 targets both parenchymal and immune cells to reduce inflammation in experimental acute pancreatitis.</a> Journal of Physiology. 597 (12), 3085-3105 (2019).</li><li>Petersen, O. H., Gerasimenko, J. V., Gerasimenko, O. V., Gryshchenko, O., Peng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+calcium+and+ATP+in+the+physiology+and+pathology+of+the+exocrine+pancreas.">The roles of calcium and ATP in the physiology and pathology of the exocrine pancreas.</a> Physiological Reviews. 101 (4), 1691-1744 (2021).</li></ol>

The TC-induced SAP model is an excellent research tool. As shown in this study, this model is very easily realized in general labs without employing specific devices. When used in combination with histology and biochemical analysis, it provides a cost- (inexpensive reagents) and time-saving (24 h time window) approach for inducing and evaluating AP. Adjusting the concentration of TC also offers the possibility of producing mild and moderate AP. Perides et al. also employed TC to induce SAP in mice20</su...

Acute pancreatitis (AP) is an acute inflammation of the pancreas characterized by obstruction of the main pancreatic duct with subsequent ductal distension and pancreas autodigestion by its abnormally activated enzymes. Its clinical manifestations include local or systemic inflammation, abdominal pain, and elevation of serum amylase1,2. According to the severity classification3, AP can present in mild, moderate, and severe forms, and among them, severe acute pancreatitis (SAP) is the most concerning condition due to its high mortality rate of more than 30%4. In the United States, AP is one of the most common reasons for hospitalization, affecting over 200,000 patients5. Moreover, AP, especially SAP, is increasing annually and affecting younger age groups6. However, there is a lack of effective treatment options in current clinical practice6,7. Therefore, it is necessary to explore the molecular mechanisms involved in AP, thereby facilitating treatment improvement.
Well-established experimental animal models are required for studying the mechanisms involved in AP and evaluating the effectiveness of different treatment modalities. With the easy accessibility of transgenic and knockout strains and their small size, which minimizes the doses of drugs required for in vivo evaluation, mice are preferred for AP research. Therefore, several models of AP have been developed in mice8,9.
Working from a mild pancreatitis rat model induced through the intravenous administration of caerulein10, Niederau et al. developed a SAP mouse model presented with acinar cell necrosis induced using the same drug and injection route11. Although this model possesses several advantages, including noninvasiveness, rapid induction, wide reproducibility, and applicability, the major disadvantage is that only a mild form of AP is developed in most cases, thereby limiting its clinical relevance. Alcohol is considered one of the major etiologic factors of AP; however, Foitzik et al. reported that it causes pancreatic injury only when combined with other factors, such as exocrine hyperstimulation12. Moreover, although alcohol-induced AP models developed via different administration routes, and drug doses have been reported13,14,15, their major disadvantage is the difficulty in reproducing them. Intraperitoneal administration of L-arginine can also induce AP in mice16; however, its low clinical relevance hinders its application. Taurocholate, a bile salt, was first proposed by Creutzfeld et al. in 1965 for inducing a condition resembling human AP via pancreatic duct infusion17. Although controversies exist regarding its clinical relevance in pathophysiology18,19, taurocholate-induced pancreatitis remains an indispensable model for SAP.
As this model is simple to realize and is also effective in mice, it is now one of the most used AP models for small animal in vivo studies. Perides et al. employed sodium taurocholate (TC) to induce SAP in mice20, providing insights to understand its pathology. Combined with genetic modification techniques, this model has allowed us to confirm several specific genes involved in AP. For example, Bicozo et al. showed that a knockout of the CD38 gene protected against a model of TC-infusion pancreatitis and attributed the mechanisms to alterations in intracellular Ca2+ signaling21. Fanczal et al. investigated the physiological implication of TRPM2 expression in the plasma membrane of mouse pancreatic acinar and ductal cells, and demonstrated reduced severity of TC-induced SAP in TRPM2 knockout mice22. Furthermore, this model also provides a simple and effective way to test many novel drugs in vivo. For instance, this method enabled validation of the therapeutic effects of caffeine23, dehydrocholic acid24, and various antioxidants and anticoagulants25,26. This evidence demonstrates the versatility of the TC-induced SAP model. Although Wittel et al. described a similar mouse model27, a lack of details on the implementation procedures could result in an inability to reproduce the findings. In this article, we focus on methods using a simple homemade microsyringe and study TC-induced SAP, thereby providing possible guidance not only for further study of the pathogenesis and treatment of AP, but also for a perfectly adaptable experimental method for many other substances.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% iodophor</td>
			<td>Shanghai Likang Disinfectant</td>
			<td>310102</td>
			<td>4 mL/mouse</td>
		</tr>
		<tr>
			<td>0.9% sodium chloride</td>
			<td>Sinopharm Group Co., Ltd.</td>
			<td>10019318</td>
			<td>0.8 mL/mouse</td>
		</tr>
		<tr>
			<td>1% Pentobarbital sodium</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td>0.2 -0.25 mL/mouse</td>
		</tr>
		<tr>
			<td>25 &#956;L flat tip Microliter syringe</td>
			<td>Gaoge, Shanghai</td>
			<td>A124019</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td>Beyotime, Nantong, China</td>
			<td>P0099-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>5% sodium taurocholate (TC)</td>
			<td>Aladdin</td>
			<td>S100834-5g</td>
			<td>10 &#956;L/SAP mouse</td>
		</tr>
		<tr>
			<td>6-0 Sterile nylon microsuture with threaded needle (1/2 circle)</td>
			<td>Cheng-He</td>
			<td>20093</td>
			<td></td>
		</tr>
		<tr>
			<td>75% alcohol</td>
			<td>Sinopharm Group Co., Ltd.</td>
			<td>10009218</td>
			<td>4 mL/mouse</td>
		</tr>
		<tr>
			<td>8-0 Sterile nylon microsuture with threaded needle (3/8 circle)</td>
			<td>Cheng-He</td>
			<td>19064</td>
			<td></td>
		</tr>
		<tr>
			<td>ALT Activity Assay Kit</td>
			<td>EPNK, Anhui, China</td>
			<td>ALT0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylase Assay Kit</td>
			<td>EPNK, Anhui, China</td>
			<td>AMY0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled small bulldog clamp with 12 mm jaw (3 cm)</td>
			<td>Cheng-He</td>
			<td>HC-X022</td>
			<td></td>
		</tr>
		<tr>
			<td>aspen shavings or shreds for mouse bedding</td>
			<td>Beijing Vital River Laboratory Animal Technology</td>
			<td>VR03015</td>
			<td></td>
		</tr>
		<tr>
			<td>AST Activity Assay Kit</td>
			<td>EPNK, Anhui, China</td>
			<td>AST0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Urea Nitrogen (BUN) Assay Kit</td>
			<td>EPNK, Anhui, China</td>
			<td>BUN0011</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mouse</td>
			<td>Beijing Vital River Laboratory Animal Technology</td>
			<td>213</td>
			<td></td>
		</tr>
		<tr>
			<td>Creatine Assay Kit</td>
			<td>EPNK, Anhui, China</td>
			<td>CRE0012</td>
			<td></td>
		</tr>
		<tr>
			<td>Feature microtome blade</td>
			<td>Beyotime, Nantong, China</td>
			<td>E0994</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic Forceps (9.5 cm, Curved)</td>
			<td>JZ, Shanghai Medical Instruments Co. Ltd.</td>
			<td>JC3901</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipase Assay Kit</td>
			<td>Jiancheng, Nanjing, China</td>
			<td>A054-2-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica biosystem, Germany</td>
			<td>RM2245</td>
			<td></td>
		</tr>
		<tr>
			<td>Mindray biochemistry analyzer</td>
			<td>Mindray, Shenzhen, China</td>
			<td>BS-420</td>
			<td></td>
		</tr>
		<tr>
			<td>MPO Assay Kit</td>
			<td>Jiancheng, Nanjing, China</td>
			<td>A044-1-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal mouse chow</td>
			<td>Trophic, Nantong, China</td>
			<td>LAD 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Beyotime, Nantong, China</td>
			<td>C0221A</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight micro-bulldog clamp with 5 mm jaw (1.5 cm)</td>
			<td>JZ, Shanghai Medical Instruments Co. Ltd.</td>
			<td>W40130</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight or curved forceps (11.0 cm)</td>
			<td>Cheng-He</td>
			<td>HC-X091A or HC-X090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Scissors (10.0 cm)</td>
			<td>Cheng-He, Ningbo, China</td>
			<td>HC-J039102</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Centrifuge</td>
			<td>Thermo Scientific, USA</td>
			<td>Multifuge X1R</td>
			<td></td>
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establishment of a mouse severe acute pancreatitis model using retrograde injection of sodium taurocholate into the biliopancreatic duct

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a lack of effective treatments in the current clinical practice. With the easy accessibility of transgenic and knockout strains and their small size, which allows minimal doses of drugs required for in vivo evaluation, a well-established experimental model in mice is preferred for AP research. Moreover, SAP induced through sodium taurocholate (TC) is currently one of the most widely used and best characterized models. This model has been investigated for novel therapies and possible molecular events during the process of AP. Here, we present the generation of an AP mouse model using sodium taurocholate and a simple homemade microsyringe. Moreover, we also provide the methodology for the subsequent histology and serological testing.

By carefully following the instructions above, we obtained a mean surgery duration of approximately 40 min. The mice were slightly inactive and had lost approximately 0.5-1.75 g, 0.85-1.85 g and 0.5-4.73 g of weight at 24 h, 48 h and 72 h post-operation, respectively (Figure 2).
From the time of surgery completion to 24 h post-operation, as the disease developed, the mice became inactive and showed slow responses and actions.
The surviv...

Watch this Scientific Journal Video about Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct at JoVE.com

Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct

A mouse model of severe acute pancreatitis is described herein. The procedure presented here is very rapid, simple, and accessible, thereby potentially allowing the study of the molecular mechanisms and different therapeutic interventions in acute pancreatitis in a convenient way.

The prevalence of acute pancreatitis (AP), especially severe acute pancreatitis (SAP), is increasing in younger age groups annually. However, there is a ...

The overall goal of this video is to establish a mouse model of acute pancreatitis through retrograde injection of sodium taurocholate into biliopancreatic duct. All experiments involving animals were approved by the Animal Ethics Committee of Soochow University. All surgical procedures were performed under full anesthesia.Analgesics were not used in order not to interfere with the natural course of the disease, which was evidenced by previous literatures and approved by the Animal Ethics Committee of Soochow University. Fast the mouse eight to 12 hours before the surgery. Prepare 5%sodium taurocholate solution filters through a 0.22 microfilter.Autoclave the surgical instruments and materials, including forceps, scissors, blade holder, needle holder, hemostatic clip, vascular clamps, and the sterile drapes. The micro-syringe was sterilized through irradiation. With the mouse, induce a general anesthesia with intraperitoneal injection of 1%pentobarbital sodium solution at a dosage of 50 milligram per kilogram.Fix the mouse in the supine position on an irradiated foam flat plate with the tape. Remove fur of a mouse with a depilation cream from the xiphoid process level down to the connecting line of the bilateral anterior superior iliac spine, with the left and right sides parallel to the front axillary line. Wipe the skin two to three times with 0.5%iodophor and let it dry for one to two minutes each time.Cover the mouse with sterile drapes. Cut open skin layer and then open subcutaneously. Make an approximate two to three centimeters long median abdominal incision.Locate the duodenum from left to right along the stomach. Gently pull the duodenum out and to the left side using surgical forceps and a disinfectant-soaked cotton swab to expose the pancreas, the biliopancreatic duct, and the duodenal papilla. Use 8-0 sutures to pass through the connection between the duodenum and pancreas around the papilla, pull the suture to the left side of the mouse body to fix the duodenum.Gently pull the skin and the lower edge of the liver aside. Clamp the common hepatic duct. Pierce the microinjector into the distal biliopancreatic duct retrogradely.Fix the needle tip near the duodenal papilla with a clamp. Infuse 10 microliters of 5%sodium taurocholate into the biliopancreatic duct at a rate of 5 microliters per minute. Keep the biliopancreatic duct closed for 5 min to achieve a stable pressure.Remove the clamps. Pull out the microinjector, remove the other clamp and fix the suture. Restore the duodenum to the original anatomical position and check the bleeding.Close the wound with 6-0 sutures. Remove the sterile drape. Disinfect the incision with 0.5%iodophor.Recover the animals from anesthesia on a 37 degree Celsius thermal pad. Administer 0.8 milliliter of 0.9%saline subcutaneously for fluid supplementation. At 24 hour post-operation, anesthetize the mouse with pentobarbital sodium using the same dose and injection route.Collect the whole blood from the abdominal aorta and spin at 1, 500 x g for 15 minutes to isolate the serum for serological testing. Collect the pancreas and the lung tissues for histology evaluation. Immerse the pancreas into 4%paraformaldehyde solution for 24 hours.Dehydrate in a series of alcohol concentrations and embed in paraffin. Use a microtome to prepare 5 micro thick pancreas tissue sections. Perform H&E staining for the histological evaluation of the pancreas.The mice were slightly inactive and had weight lost at 24 hours post-operation compared to pre-operation. The survival rates of the control and SAP mice were 100%and 72.7%respectively, at 72 hours post-operation. According to the H&E staining results from pancreas sections, pulpal necrosis of the pancreas tissue could be observed in SAP mice while no significant changes could be found in control mice.Moreover, significantly increased levels of amylase, lipase, pancreas, and lung MPO activity were found in SAP mice compared to control mice. In addition, significantly elevated levels of the liver and kidney indexes including ALT, AST, BUN, and creatinine were also found. Using retrograde injection of sodium taurocholate into biliopancreatic duct, severe acute pancreatitis could be induced in mice.Well exposure of the biliopancreatic duct to ensure the successful puncture. During the puncture, ensure the appropriate angle and force to avoid puncturing of the biliopancreatic duct. TC induced SAP model descried here is a suitable model to study the mechanisms involved in severe acute pancreatitis.It is an economic model using less expensive reagents and this video helps training the novice surgeon faster.

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Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

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 ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

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 ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...