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. 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 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 "generalized hypersensitivity" 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.
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
2. Mouse preparation and surgery
3. Monitoring mouse behavior
4. Collection and histological analysis of pancreatic tissue
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 μL or 50 μL. Injection of TNBS in a volume of 50 μL 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 prepared TNBS stored on ice and used within one hour of drug preparation also yielded better outcome compared to TNBS prepared longer than one hour. With these improvements, the mortality of recipients was controlled and remained under 10%.
Body weight loss is one of the characteristics of CP. Mice in the control group lost around 6% of their original body weight during the first 3 days after surgery, then gradually recovered (104.6% of original body weight at day 21) (Figure 1C). In contrast, mice receiving TNBS lost on average about 15% of their original body weight during the first 5 days and regained weight afterward (99.8% of original body weight at day 21) (Figure 1C). In addition, compared to controls, TNBS mice showed increased abdominal mechanical hypersensitivity at 2 and 3 weeks after TNBS injection (Figure 1D), which was likely associated with increased abdominal pain10.
To confirm that the bile duct TNBS infusion effectively induced pancreatic changes mimicking human CP, we collected pancreatic tissues from TNBS or vehicle-treated control mice at 3 weeks post-surgery. Both size, and weight per body weight of the pancreas were significantly reduced in TNBS mice compared with controls (Figure 2A,B), suggesting marked pancreatic atrophy consistent with the findings in humans with severe and long-term CP. In addition, the pancreas from the control mice appeared normal without obvious morphological changes, while TNBS mice showed vacuolization with massive loss of acinar cells replaced by fat cell infiltration and fibrosis (Figure 2C). These findings were consistent with reports from other studies9,10.
Figure 1: TNBS bile duct infusion for CP mice generation. (A) Illustration of bile duct injection. (B) Bile duct after injection of 50 μL of ink. (C) Averages of body weight change in mice receiving TNBS or vehicle. (D) Abdominal response threshold in TNBS and control mice at 3 weeks after infusion. Data were analyzed using available analysis software (e.g., GraphPad 8.2.1). Data are presented as mean ± SEM. Differences between groups were analyzed using the Student's t-test, ** p < 0.01 was considered statistically significant. Please click here to view a larger version of this figure.
Figure 2. Characterization of CP in TNBS-treated mice. (A) Micrographs of the pancreas from control (CTR) and TNBS mice. (B) The average pancreas weight divided by mouse bodyweight in CTR and TNBS mice. (C) Hematoxylin and eosin staining of pancreas sections of CTR and TNBS mice. Scale bar =100 μm. Data are presented as mean ± SEM. **, p < 0.01 by Student's t-test. Please click here to view a larger version of this figure.
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 <10%. For example, the increased drug volume (from 35 μL to 50μL) can ensure the drugs reach the whole pancreas. Using an insulin syringe and a smaller needle size (31G) reduces potential damage to the pancreatic duct and the leakage of bile into the gallbladder or the abdomen, which most likely would cause mouse death within the first several days after surgery. Clamping both ends of the bile duct can prevent TNBS from leaking to the gallbladder and the intestine, which may cause mortality. The hemo clips restrain TNBS within the injected pancreas, improving the efficacy while reducing damages to other tissues. In addition, TNBS is not stable at temperatures above 0 °C. Therefore, by using freshly prepared TNBS, CP induction achieved stable results.
This protocol effectively induces CP in male C57BL/6J mice at 8-12 weeks of age and generates a model that mimics major symptoms of chronic pancreatitis, including bodyweight loss, pancreatic atrophy, fibrosis, and likely abdominal pain. Compared to other CP mouse models, the TNBS model is widely used to evaluate analgesic effects in addition to inflammation10,15,16. The other advantage of the TNBS model is that the drug is injected directly into the pancreas, which reduces damage to other organs that may interfere with the study17. This TNBS CP mouse model can be used to study pathogenesis as well as treatment options together with other chronic pancreatitis models.
One limitation of this study is that only mice between 8-10 weeks of age were used. Since mice at different ages may have difference pancreas sizes, which consequently affect TNBS-CP development. Therefore, whether mice at different ages/sizes should be given a different dose of TNBS needs to be tested. Nevertheless, this study demonstrates the procedures to successfully perform pancreatic duct infusion that may help with studies focused on pancreatic diseases.
The authors have nothing to disclose.
This study was supported by the Department of Veterans Affairs (VA-ORD BLR&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.
10% Neutral buffered formalin v/v | Fisher Scientific | 23426796 | |
Alcohol prep pads, sterile | Fisher Scientific | 22-363-750 | |
Animal Anesthesia system | VetEquip, Inc. | 901806 | |
Buprenorphine hydrochloride, injection | Par Sterile Products, LLC | NDC 42023-179-05 | |
Centrifuge tubes, 15 mL | Fisher Scientific | 0553859A | |
Ethanol, absolute (200 proof), molecular biology grade | Fisher Scientific | BP2818500 | |
Extra fine Micro Dissecting scissors 4” straight sharp | Roboz Surgical Instrument Co. | RS-5882 | |
Graefe forceps 4” extra delicate tip | Roboz Surgical Instrument Co. | RS-5136 | |
Heated pad | Amazon | B07HMKMBKM | |
Hegar-Baumgartner Needle Holder 5.25” | Roboz Surgical Instrument Co. | RS-7850 | |
Insulin syringe with 31-gauge needle | BD | 324909 | |
Iodine prep pads | Fisher Scientific | 19-027048 | |
Isoflurane | Piramal Critical Care | NDC 66794-017-25 | |
Micro clip applying forceps 5.5” | Roboz Surgical Instrument Co. | RS-5410 | |
Micro clip, straight strong curved 1x6mm | Roboz Surgical Instrument Co. | RS-5433 | |
Micro clip, straight, 0.75mm clip width | Roboz Surgical Instrument Co. | RS-5420 | |
Picrylsulfonic acid solution, TNBS, 1M in H2O | Millipore Sigma | 92822-1ML | |
Polypropylene Suture 4-0 | Med-Vet International | MV-8683 | |
Polypropylene Suture 5-0 | Med-Vet International | MV-8661 | |
Sodium chloride, 0.9% intravenous solution | VWR | 2B1322Q | |
Surgical drape, sterile | Med-Vet International | DR1826 | |
Tissue Cassette | Fisher Scientific | 22-272416 | |
Von Frey filaments | Bioseb | EB2-VFF |