A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Summary

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).

Abstract

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.

Introduction

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 hypertension^6,7. 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 patients^9,10,11. 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 tissue^10,12. 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.

Protocol

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

1. Prepare 10% ethanol in 0.9% saline. Dissolve stock TNBS (see Table of Materials) in 10% ethanol to a final concentration of 7.5 mM by adding 7.5 µL of TNBS into 1 mL of 10% ethanol.
   CAUTION: TNBS presents a chemical hazard. Prepare the solution inside a fume hood and use personal protective equipment such as gloves, goggles, and a lab coat to avoid direct contact with TNBS.
2. Load 50 µL of 0.75% TNBS solution into an insulin syringe with a 31-gauge needle. Load 50 µL of 10% ethanol in saline into a syringe of the same size as vehicle control. Place the syringes on ice and protect them from light until needed.

2. Mouse preparation and surgery

1. Shave the hair from the abdominal surgical area.
2. Inject one pre-emptive dose of the analgesic (e.g., buprenorphine 0.1 mg/kg i.p.) before surgery.
3. Induce and maintain the mouse under general anesthesia with 1.5-2% isoflurane and 1 L/min of oxygen. Confirm the anesthetization by pinching the toes and observing the animal for a lack of reflex.
4. Place the mouse on a heated surgical pad during surgery. Apply veterinary ointment on each eye when the mouse is under anesthesia.
5. Disinfect the surgical site by wiping the surgical area 3x with 2% iodine, followed by 70% alcohol (Table of Materials).
6. Perform a laparotomy with micro scissors to generate a 0.5-1 cm incision.
7. Gently expose the duodenum and locate the common bile duct using cotton swabs (Table of Materials).
8. Place a straight micro hemo clip (Table of Materials) over the proximal common duct to prevent the flow of TNBS or vehicle solutions into the liver and the gallbladder (Figure 1A, B).
9. Gently expose the duodenum and insert the needle into the pancreatic duct through the papilla of Vater.
10. Once the needle is inside the duct, place a curved micro hemo clip (Table of Materials) over the duodenum surrounding the needle (Figure 1A) to secure the needle in place and prevent the injected solution from entering the duodenum.
11. Gradually infuse the solution (TNBS or vehicle) into the pancreatic duct over the course of one min.
    NOTE: TNBS needs to be infused slowly over one min of time, and it is easy to control the infusion speed when the pancreas is perfused with an insulin syringe with a 5/16 inch and 31G needle. Keep the hand as stable as possible to avoid pricking the bile duct. If TNBS is successfully injected, yellow color may be visible inside the pancreas.
12. After infusion, carefully remove the micro clamp near the liver, and then remove the micro clamp holding the needle and the duodenum.
13. Carefully return the duodenum to its original position.
14. Leave 0.5 mL of warm sterile saline (36-37 °C) in the abdominal cavity before closure, to help the duodenum return to its original position and assist with the recovery of peristalsis.
15. Close the incision in the muscle layer using continuous suture with a 5-0 stitch. Close the skin using interrupted suture with a 4-0 stitch.
16. Place the cage containing mice on a heating pad to allow recovery from the anesthesia.
17. Confirm that the mice are warm and capable of spontaneous movement before returning them to the holding room.
18. Continue to provide an analgesic (e.g., buprenorphine 0.1 mg/kg i.p.) every 12 h and supplemental heat for 48 h post-surgery.

3. Monitoring mouse behavior

1. Remove sutures at day 7 post-surgery.
2. Monitor mouse health and behavior daily during the first-week post-surgery. Watch for signs of distress such as vocalizing, hunched back posture, or reduced locomotion. Measure the body weight every other day.
3. Use Von Frey monofilaments (VFFs) to measure abdominal mechanical hypersensitivity before, and 2, 3 weeks post-surgery as described^9,14.
   1. Apply VFFs of different applied forces in the ascending order to the upper abdominal area 10x every 1-2 s. Consider the raising, retraction, or licking of the abdomen (withdrawal response) as a positive response.
   2. Apply a stronger stimulus if a positive response is not observed, and a weaker stimulus if a positive response is observed. The withdrawal threshold is the force at which the mouse responds 50% of the time.

4. Collection and histological analysis of pancreatic tissue

1. Sacrifice the mice under anesthesia by cervical dislocation, and carefully dissect the pancreas from the intestine and other organs.
2. Fix the pancreas in 10% paraformaldehyde for 24 h, embed in paraffin, cut tissue sections of 5 µm thickness, and place them on glass slides for staining.
3. Perform hematoxylin-eosin, and Masson's trichrome staining using standard methods as previously reported⁴.

Representative Results

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 studies^9,10.

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.

Discussion

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 inflammation^10,15,16. 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.

Materials

Name	Company	Catalog Number	Comments
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