This study was supported by the Department of Veterans Affairs (VA-ORD BLR&#38;D Merit I01BX004536), and the National Institute of Health grants # 1R01DK105183, DK120394, and DK118529 to HW. We thank Dr. Hongju Wu for sharing technical experience.

All procedures were conducted with the approval of the Institutional Animal Care and Use Committees at the Medical University of South Carolina and the Ralph H. Johnson Medical Center. C57BL/6J male mice between 8-10 weeks of age were used in this study. Mice were housed under a standard 12 light/ 12 dark cycle with ad libitumaccess to food and water.
1. Preparation of TNBS solution for injection
<ol>
	<li>Prepare 10% ethanol in 0.9% saline. Dissolve stock TNBS (see Table of Material...

<ol>
	<li>Klauss, 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=Genetically+induced+vs.+classical+animal+models+of+chronic+pancreatitis:+a+critical+comparison.">Genetically induced vs. classical animal models of chronic pancreatitis: a critical comparison.</a> The Federation of American Societies for Experimental Biology Journal. 32, 5778-5792 (2018).</li><li>Liao, Y. 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=Histone+deacetylase+2+is+involved+in+&#181;-opioid+receptor+suppression+in+the+spinal+dorsal+horn+in+a+rat+model+of+chronic+pancreatitis+pain.">Histone deacetylase 2 is involved in &#181;-opioid receptor suppression in the spinal dorsal horn in a rat model of chronic pancreatitis pain.</a> Molecular Medicine Reports. 17 (2), 2803-2810 (2018).</li><li>Gui, 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=Trypsin+activity+governs+increased+susceptibility+to+pancreatitis+in+mice+expressing+human+PRSS1R122H.">Trypsin activity governs increased susceptibility to pancreatitis in mice expressing human PRSS1R122H.</a> The Journal of Clinical Investigation. 130 (1), 189-202 (2020).</li><li>Sun, 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=Adipose+Stem+Cell+Therapy+Mitigates+Chronic+Pancreatitis+via+Differentiation+into+Acinar-like+Cells+in+Mice.">Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.</a> Molecular Therapy. 25 (11), 2490-2501 (2017).</li><li>Aghdassi, A. 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=Animal+models+for+investigating+chronic+pancreatitis.">Animal models for investigating chronic pancreatitis.</a> Fibrogenesis and Tissue Repair. 4 (1), 26 (2011).</li><li>Scoggins, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53-dependent+acinar+cell+apoptosis+triggers+epithelial+proliferation+in+duct-ligated+murine+pancreas.">p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 279 (4), 827-836 (2000).</li><li>Bradley, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+duct+pressure+in+chronic+pancreatitis.">Pancreatic duct pressure in chronic pancreatitis.</a> The American Journal of Surgery. 144 (3), 313-316 (1982).</li><li>Zhao, J. B., Liao, D. H., Nissen, T. D. <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+pancreatitis:+can+it+be+translated+to+human+pain+study.">Animal models of pancreatitis: can it be translated to human pain study.</a> World Journal of Gastroenterology. 19 (42), 7222-7230 (2013).</li><li>Winston, J. H., He, Z. J., Shenoy, M., Xiao, S. Y., Pasricha, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+behavioral+changes+in+nociception+in+a+novel+rat+model+of+chronic+pancreatitis+for+the+study+of+pain.">Molecular and behavioral changes in nociception in a novel rat model of chronic pancreatitis for the study of pain.</a> Pain. 117 (1-2), 214-222 (2005).</li><li>Cattaruzza, 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=Transient+receptor+potential+ankyrin+1+mediates+chronic+pancreatitis+pain+in+mice.">Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 304 (11), 1002-1012 (2013).</li><li>Bai, Y., 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=Anterior+insular+cortex+mediates+hyperalgesia+induced+by+chronic+pancreatitis+in+rats.">Anterior insular cortex mediates hyperalgesia induced by chronic pancreatitis in rats.</a> Molecular Brain. 12 (1), 76 (2019).</li><li>Puig-Div&#237;, V., 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=Induction+of+chronic+pancreatic+disease+by+trinitrobenzene+sulfonic+acid+infusion+into+rat+pancreatic+ducts.">Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts.</a> Pancreas. 13 (4), 417-424 (1996).</li><li>Zhang, Y., 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=PAX4+Gene+Transfer+Induces+alpha-to-beta+Cell+Phenotypic+Conversion+and+Confers+Therapeutic+Benefits+for+Diabetes+Treatment.">PAX4 Gene Transfer Induces alpha-to-beta Cell Phenotypic Conversion and Confers Therapeutic Benefits for Diabetes Treatment.</a> Molecular Therapy. 24 (2), 251-260 (2016).</li><li>Ceppa, E. 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=Serine+proteases+mediate+inflammatory+pain+in+acute+pancreatitis.">Serine proteases mediate inflammatory pain in acute pancreatitis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 300 (6), 1033-1042 (2011).</li><li>Puig-Divi, V., 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=Induction+of+chronic+pancreatic+disease+by+trinitrobenzene+sulfonic+acid+infusion+into+rat+pancreatic+ducts.">Induction of chronic pancreatic disease by trinitrobenzene sulfonic acid infusion into rat pancreatic ducts.</a> Pancreas. 13 (4), 417-424 (1996).</li><li>Xu, G. Y., Winston, J. H., Shenoy, M., Yin, H., Pasricha, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+excitability+and+suppression+of+A-type+K++current+of+pancreas-specific+afferent+neurons+in+a+rat+model+of+chronic+pancreatitis.">Enhanced excitability and suppression of A-type K+ current of pancreas-specific afferent neurons in a rat model of chronic pancreatitis.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 291 (3), 424-431 (2006).</li><li>Drewes, A. M., 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=Pain+in+chronic+pancreatitis:+the+role+of+neuropathic+pain+mechanisms.">Pain in chronic pancreatitis: the role of neuropathic pain mechanisms.</a> Gut. 57 (11), 1616-1627 (2008).</li></ol>

All authors declare that they do not have conflict of interest.

Bile duct infusion of TNBS to induce chronic pancreatitis is technically challenging in mice, as up to 22.5% of mice can die within 3-4 days of drug infusion10. Here, this report refined the procedure based on previous studies and reduced early mouse mortality to &#60;10%. For example, the increased drug volume (from 35 &#956;L to 50&#956;L) can ensure the drugs reach&#160;the whole pancreas. Using an insulin syringe and a smaller needle size (31G) reduces potential damage to the pancreatic duct a...

Chronic pancreatitis (CP) is a chronic inflammatory disease characterized by the atrophy of the pancreas, fibrosis, abdominal pain, and eventual loss of both exocrine and endocrine functions1. Current medical and surgical treatments are not curative but are undertaken to relieve symptoms that are the consequence of the disease: refractory abdominal pain, endocrine and exocrine dysfunction. Therefore, more effective treatments are urgently needed2. Animal models provide an essential tool for developing a better understanding of the disease and investigating potential therapeutics3. Multiple mouse models for CP have been developed, of which cerulein and/or alcohol models are commonly used. Cerulein, an oligopeptide stimulating pancreatic secretion, has been shown to reproducibly induce a CP model featuring pancreatic atrophy, fibrosis, among others4. Another common model uses serial injections of L-arginine, which produces exocrine insufficiency similar to that observed in human patients5. CP can also be induced by complete or partial pancreatic duct ligation, as well as pancreatic duct hypertension6,7. Despite the variety of animal models available for CP, none of these models effectively reproduces the abdominal pain experienced by CP patients8.
Previous studies showed that local pancreatic injection of 2,4,6 -trinitrobenzene sulfonic acid (TNBS) replicates the persistent pain experienced by CP patients9,10,11. TNBS-treated mice demonstrated abdominal hypersensitivity and increased pain-related behaviors as well as a &#34;generalized hypersensitivity&#34; to painful stimuli, a phenomenon that has been observed in CP patients10. In addition to accurately mimicking CP pain, the TNBS model also replicates other pathological features of the human condition such as fibrosis, mononuclear cell infiltration, and replacement of acinar cells with fatty tissue10,12. However, TNBS infusion via bile duct is a technically challenging procedure in mice that may cause death. To our knowledge, there is no visual protocol to show how bile duct infusion is performed. In this article, we demonstrate the procedure of the bile duction infusion of TNBS to generate a CP mouse model. This procedure will help generate valuable animal models for the study of CP and other pancreatic diseases and can be used to infuse other materials (e.g., virus, cells) into the pancreas13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Neutral buffered formalin v/v</td>
			<td>Fisher Scientific</td>
			<td>23426796</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol prep pads, sterile</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Anesthesia system</td>
			<td>VetEquip, Inc.</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine hydrochloride, injection</td>
			<td>Par Sterile Products, LLC</td>
			<td>NDC 42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes, 15 mL</td>
			<td>Fisher Scientific</td>
			<td>0553859A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, absolute (200 proof), molecular biology grade</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra fine Micro Dissecting scissors 4&#8221; straight sharp</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps 4&#8221; extra delicate tip</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated pad</td>
			<td>Amazon</td>
			<td>B07HMKMBKM</td>
			<td></td>
		</tr>
		<tr>
			<td>Hegar-Baumgartner Needle Holder 5.25&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-7850</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe with 31-gauge needle</td>
			<td>BD</td>
			<td>324909</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine prep pads</td>
			<td>Fisher Scientific</td>
			<td>19-027048</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical Care</td>
			<td>NDC 66794-017-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip applying forceps 5.5&#8221;</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5410</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight strong curved 1x6mm</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5433</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro clip, straight, 0.75mm clip width</td>
			<td>Roboz Surgical Instrument Co.</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrylsulfonic acid solution, TNBS, 1M in H2O</td>
			<td>Millipore Sigma</td>
			<td>92822-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 4-0</td>
			<td>Med-Vet International</td>
			<td>MV-8683</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Suture 5-0</td>
			<td>Med-Vet International</td>
			<td>MV-8661</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride, 0.9% intravenous solution</td>
			<td>VWR</td>
			<td>2B1322Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape, sterile</td>
			<td>Med-Vet International</td>
			<td>DR1826</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Cassette</td>
			<td>Fisher Scientific</td>
			<td>22-272416</td>
			<td></td>
		</tr>
		<tr>
			<td>Von Frey filaments</td>
			<td>Bioseb</td>
			<td>EB2-VFF</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The bile duct infusion procedures were optimized to reduce mouse mortality associated with this procedure10. TNBS was first given in a total volume of 35 &#956;L or 50 &#956;L. Injection of TNBS in a volume of 50 &#956;L could reach the whole pancreas and induce a more homogeneous disease phenotype (Figure 1B). In addition, injection of TNBS using insulin syringe with 31G needle could better control infusion speed relative to regular syringes and needle sizes. Freshly...

Watch this Scientific Journal Video about A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion at JoVE.com

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

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. ...

Chronic pancreatitis is a complex disease involving pancreatic inflammation, fibrosis, abdominal pain, and other symptoms. The bile duct TNBS infusion model replicates the neuropathic pain observed in chronic pancreatitis patients. This bile duct TNBS infusion protocol has been optimized to induce the generation of a chronical pancreatitis model in mice.As bile duct drug fusion in mice is technically challenging, its visualization will be helpful to researchers new to the method. Before and two and three weeks after the surgery, use Von Frey monofilaments to apply different levels of force in ascending order to the upper abdominal area of an eight to 10 week old, C57 black six mouse, 10 times for one to two seconds per application. Consider raising retraction or licking of the abdomen as a positive response.Next, load an insulin syringe equipped with a 31 gauge needle with 50 microliters of a freshly prepared 0.75 percent TNBS solution. Load a second syringe with 50 microliters of 10 percent ethanol in saline as a vehicle control and place both syringes on ice protected from light. After confirming a lack of response to pedal reflex, remove the hair from the abdominal region of the anesthetized mouse by shaving or applying hair removal cream and place the mouse on a heated surgical pad under a dissecting microscope.Apply veterinary ointment to each eye, and disinfect the surgical site with three sequential two percent iodine and three 70 percent ethanol wipes use micro scissors to make a 0.5 to one centimeter incision in one continuous motion and use cotton swabs to gently expose the duodenum and to locate the common bile duct. To prevent the flow of the treatment into the liver and gallbladder, use the microscope to place a straight micro hemoclip over the proximal common duct, and insert the needle of the TNBS loaded syringe through the papilla of vater into the pancreatic duct. Use a curved micro hemoclip to secure the needle, and infuse the solution into the pancreatic duct over the course of one minute.When all of the solution has been delivered, carefully remove the micro clamps and confirm that the duodenum has returned to its original position. Before closure of the abdominal cavity, add 500 microliters of 36 to 37 degrees Celsius sterile saline to help the duodenum return to its original position and to assist with the recovery of peristalsis. Use a continuous suture with a five-O stitch to close the incision in the muscle layer, and close the skin using an interrupted suture with a four-O stitch.Then place the mouse in a recovery cage on a heating pad with monitoring until full recumbency. Monitor mouse health and behavior daily during the first week post-surgery for signs of distress, such as vocalizing, a hunched back posture or reduce locomotion and measure the body weight every other day. As observed, the injection of 50 microliters of ink as demonstrated results in the infiltration of the entire pancreas, allowing the induction of a more homogenous disease phenotype.In this representative analysis, in the first three days after surgery, the mice in the control group lost around six percent of their original body weight before gradually recovering and gaining over 100%of their original body weight by day 21. In contrast, mice that received TNBS lost about 15 percent of their original body weight during the first five days after surgery and regained just under 100 percent of their original body weight by day 21. Compared to control mice, the TNBS-injected animals showed an increased abdominal mechanical hypersensitivity during the second and third weeks after surgery.The size and weight per body weight of the pancreases, were significantly reduced in TNBS mice compared to pancreases harvested from control animals, suggesting a marked pancreatic atrophy in TNBS-treated animals consistent with the findings in humans with severe and longterm chronic pancreatitis. Histological analysis of pancreatic tissue sections from control ethanol-treated mice showed a normal morphology while tissue sections from TNBS-treated mice exhibited vacuous realization with a massive loss of acinar cells that were replaced by fat cell infiltration and fibrosis. Preventing the injected TNBS from entering the gal bladder and the intestine is important as a TNBS has tissue damaging effects.During the infusion, keep the hand as stable as possible to avoid puncture-acting that bile duct. In addition to inducing chronical pancreatitis development, this bile duct infusion mice art can be used for the development of the other pancreatic disease models. This TNBS chronic pancreatitis mouse model can be used to study both the pathogenesis and treatment of chronic pancreatitis as well as other pancreatic diseases.

a-mouse-model-for-chronic-pancreatitis-via-bile-duct-tnbs-infusion

Research

JoVE Journal

Medicine

3.7K 조회수.  Medical University of South Carolina. 만성 췌장염은 췌장 염증, 섬유증, 복통 및 기타 증상을 포함하는 복잡한 질병입니다. 담관 TNBS 주입 모델은 만성 췌장염 환자에서 관찰 된 신경 병증 통증을 복제합니다. 이러한 담관 TNBS 주입 프로토콜은 마우스에서 만성 췌장염 모델의 생성을 유도하도록 최적화되었다.마우스에 있는 담관 약 융합이 기술적으로 도전적이기 때문에, 그것의 시각화는 방법에 새로운 연?...