An Obstructive Chronic Pancreatitis Model Established Through Electrocoagulation

Summary

The present protocol describes a novel approach to modeling chronic obstructive pancreatitis, which involves visualization of the pancreatic duct and electrocoagulation of the pancreatic duct to cause pancreatic duct obstruction.

Abstract

Chronic pancreatitis (CP) is a severe inflammatory condition that can destroy the pancreas, lead to diabetes, and increase pancreatic cancer risk, severely impacting patients' quality of life. Addressing CP is a critical research focus in gastroenterology. We developed a new animal model to overcome the limitations of existing ones, using 8-week-old C57BL/6 male mice.

After exposing the pancreas through abdominal surgery on anesthetized mice, we clamped the common bile duct near the liver to prevent methylene blue from entering it. A capillary was inserted into the bile duct to inject the dye, and after the pancreatic duct was stained. After the pancreatic duct had been visualized, the pancreas was selected for electrocoagulation in the middle and lower 2/3 of the position between the common bile duct and the superior pancreaticoduodenal artery. Electrocoagulation was terminated when the blue staining solution could not be recognized. The abdomen was then closed, and the mice were normally raised. At 7, 14, 21, and 28 days post surgery, the pancreas were examined using HE staining, Masson staining, and immunohistochemistry, and blood serum was analyzed for biochemical parameters. The pathology after 14 days closely matched the characteristics of chronic pancreatitis, as did the serum biochemical parameters.

Introduction

Chronic pancreatitis (CP) is a severe progressive inflammatory disorder, leading to the destruction of the pancreas and manifesting as abdominal pain and digestive disorders. Over time, it can cause diabetes mellitus and increase the risk of pancreatic cancer. Establishing the animal model of CP and developing effective therapeutic strategies is thus crucial.

Pancreatic exocrine acinar cells produce and secrete a large number of digestive enzymes. When organelles and protein homeostasis regulation are disrupted, it can lead to improper activation of intracellular trypsinogen, ultimately leading to acinar cell damage and the development of pancreatitis¹.

Previous studies have shown that, murine models, especially those induced by repetitive cerulein injections² and surgical ligation of pancreatic ducts^3,4, have been instrumental in studying CP. Cerulein can induce acute pancreatitis that may transition to a chronic state, but its lack of specific pancreatic duct obstruction limits its clinical significance in simulating obstructive pancreatitis. Although surgical ligation better mimics the obstructive nature of human CP, it is highly invasive, difficult to perform, and often leads to severe systemic effects, hampering the study of localized ductal pathologies.

Addressing these limitations, a new obstructive CP model through electrocoagulation has been proposed. Similar to the principle of pancreatic duct ligation, it blocks the pancreatic duct but is easier to perform and has a higher success rate. Researchers have proposed that radiofrequency ablation in the main pancreatic duct of pigs is a safe and effective method for inducing pancreatic atrophy⁵, which also suggests that cutting off the main pancreatic duct with high-frequency electrocoagulation may produce similar effects. This technique involves retrograde pancreatic duct puncture and methylene blue solution injection for ductal staining, followed by electrocoagulation to induce localized ductal obstruction (Figure 1). This approach precipitates inflammation and fibrosis mimicking human obstructive CP while avoiding widespread inflammatory response and systemic complications. Compared to conventional models, the electrocoagulation model offers several advantages: it specifically targets the pancreatic duct, reduces variability, and mitigates systemic complications. It provides a more disease-specific, reproducible, and versatile approach to understanding the mechanisms underpinning ductal obstruction in CP and its role in disease progression.

Protocol

All animal experiments described were approved by the Naval Medical University's Committee on the Use and Care of Animals. Ensure that all surgical materials are sterile.

1. Laparotomy

1. Administer a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection for anesthesia induction. Monitor the anesthesia depth by assessing the loss of pedal reflex and corneal reflex. Provide additional doses (20% of the initial dose) as needed to maintain adequate anesthesia during the procedure. Confirm adequate depth of anesthesia by pinching the toes. Place the animal on a heating pad to maintain the body temperature.
2. Use a trimmer to shave the hair (~2 cm²) between the chest and lower abdomen. Disinfect the surgical area 3x with alternating rounds of chlorhexidine/iodine and alcohol.
3. Fix the mice to the surgical plate using surgical tape and apply sterile drapes.
4. Use scissors to make a 2 cm-long incision in the central abdomen, followed by a 1 cm incision between the upper abdomen and the xiphoid process.

2. Locating and exposing the pancreas

1. With the help of an abdominal expander, locate the area of the pancreas where electrocoagulation will be performed.
2. With the help of a sterile cotton swab, identify the duodenum from the rear of the left upper abdomen, and turn the duodenum to visualize the clear bile duct connecting the liver.

3. Tracing the surgical site

1. Use a microvascular clip to temporarily occlude the proximal common bile duct and prevent leakage of retrograde infusion into the liver.
2. Connect the polyethylene tube with an inner diameter of 0.25 mm and an outer diameter of 0.35 mm to the needle with an outer diameter of 0.25 mm. Use this device to insert it around the ampulla. Aim at the major papilla of the duodenum and insert the tube into it. Insert the catheter into the bile duct halfway and stop the insertion.
3. Start the infusion pump, and micropump 0.2% methylene blue solution (diluted in 0.9% saline) at a dose of 50 µL/10 g for 2 min at a rate of 50 µL/min. The pancreatic duct will gradually become blue.
4. At the end of the session, pull the polyethylene tube out and remove the microvascular clip.

4. Induction of the electrocoagulation procedure

1. With the assistance of sterile cotton swabs, fix the pancreas and direct the electrocoagulation knife at the blue part for electrocoagulation. The electrocoagulation site is in the middle and lower 2/3 between the common bile duct and the superior pancreaticoduodenal artery.
   1. To follow this study, use the following operating parameters of electrocoagulation: voltage : 220 V; electrode material: pure copper; treatment time: treatment in the position of the pancreas avoiding blood vessels for 2-3 s, until the blue of methylene blue marked position is replaced by yellow or brown after electrocoagulation; operation area size: depending on the mice, the electrocoagulation area is located on the back of the pancreas. Choose the position between the common bile duct and the anterior superior pancreaticoduodenal artery; temperature: 300 °C.
2. Terminate electrocoagulation when the blue staining solution is not seen.
3. Close the abdomen with 4-0 absorbable sutures and the skin with 4-0 nonabsorbent monofilament polypropylene sutures. Do not take more than 20 min between laparotomy and suturing.
4. Treat the control group in the same manner as the experimental mice, but ensure that the infusion components consist of methylene blue only. In the control group (Sham), administer the methylene blue injection, but do not perform electrocoagulation surgery.

5. Keeping animals alive

1. For postoperative wound care of the mouse after abdominal surgery, examine the incision daily for bleeding or redness: if bleeding occurs, apply gentle pressure with sterile gauze to stop it and then change the dressing, and if there is redness, apply erythromycin ointment once a day, while also taking measures to prevent the mouse from gnawing at the wound. Regarding analgesia, administer carprofen (5 mg/kg) via intraperitoneal injection immediately after surgery, then once every 24 h for 3 consecutive days.

6. Methods for analysis

1. Measure serum biochemical indexes and observe pathological changes in the pancreatic tissue on the 7th, 14th, 21^st, and 28th days of electrocoagulation.
2. Anesthetize the animal with a mixture of ketamine (80 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection and collect approximately 500 µL of blood through the orbital plexus.
3. Gently hold the skin on the back to facilitate a slight protrusion of the eyeball.
4. Place the end of the capillary in the corner of the eye and gently insert it under the eyeball at an angle of approximately 30°-45°. Rotate the capillary until blood starts to flow.
5. At the end of the collection, keep the eyelids closed by applying gentle pressure with gauze.
6. Centrifuge the serum (1,200 × g, 15 min) and remove the supernatant for the determination of amylase, bilirubin, and hyaluronic acid (step 6.10).
7. Anesthetize mice with ketamine-xylazine, confirm they are in a deep anesthetic state (no pedal reflex or corneal reflex), then perform cervical dislocation for euthanasia.
8. Place the euthanized mice on the operating table at 4 °C, and use scissors and forceps to make a "V" incision in the abdominal wall to expose the abdominal cavity. Remove the pancreas of the mice and divide the organ into three parts for staining.
9. Process the pancreas by fixing it in 4% polyformaldehyde solution and embed it into paraffin. Cut paraffin blocks of the pancreas into 0.5 mm-thick sections and stain them with hematoxylin and eosin (HE) as well as Masson's stain for visualization under a light microscope. Use the paraffin-embedded pancreatic tissue for immunohistochemical analysis.
10. Measure amylase (U/dL), bilirubin (mmol/L), and hyaluronic acid (µg/L) using commercially available kits according to the manufacturers' recommendations.

Results

The animals were divided into a control group (only injection of methylene blue for electric coagulation) and electric coagulation group, a total of 20 mice in each group. The mortality rate was 0% in the control group and 15% in the electric coagulation group; the success rate of building the model (number of live model mice) was 71%. In each group, a dozen animals were chosen for pathological assessment. Three mice in each group were randomly selected on the 7th, 14th, 21^st, and 28th day for HE, Masson, and immunohistochemical staining. The process of modeling is shown in Figure 1. After tracing with methylene blue, we electrocoagulated the location between the common bile duct and the anterior superior pancreaticoduodenal artery on the pancreas of mice. If the electrocoagulation area is selected in the middle and lower part of the position with an area less than one-third, the effect is likely to not meet expectations. However, if it exceeds 2/3, the mice are likely to die within 72 h of electrocoagulation, which is likely due to severe acute pancreatitis that occurs after the pancreatic duct is completely blocked.

From this, we conclude that on the 14th day after electrocoagulation, a histological image of chronic pancreatitis can be observed in the pancreas of mice (as shown in Figure 1), which is not completely blocked but formed after partial blockage of the pancreatic duct. The tissue on day 7 showed slight tissue edema, and the tissue did not show evidence of chronic pancreatitis. On day 14, HE staining of pancreatic tissue showed that pancreatic acinar cells were scattered. On the 21st day, the degree of inflammation of the acinar cells in pancreatic tissue gradually recovered. There were signs of reversal of the pathological changes in the tissue on day 28 (Figure 2). This could be due to insufficient pancreatic duct electrocoagulation during the electrocoagulation process, as well as the compensatory effect of the remaining pancreatic duct after partial pancreatic duct obstruction.

Masson staining showed that the degree of fibrosis in the operation group was significantly more severe than that in the sham operation group. Immunohistochemical scores for the staining of col-1 and (alpha-smooth muscle actin) SMA were higher (Figure 3). The presence of chronic pancreatitis in the pancreatic tissue was observed by pathological analysis on day 14, and the changes before and after were compared by HE staining only in pathological analysis thereafter. Through the analysis of the serum biochemical indicators, serum amylase, bilirubin, and hyaluronic acid concentrations were found to be higher in the electric coagulation group than in the control group, indicating the initiation of chronic pancreatitis (Figure 4).

Figure 1: Schematic illustration of obstructive chronic pancreatitis induced through electrocoagulation in mice. Please click here to view a larger version of this figure.

Figure 2: Hematoxylin-eosin staining of pancreatic tissues over time. Please click here to view a larger version of this figure.

Figure 3: Hematoxylin-eosin and Masson staining and Immunohistochemistry of the pancreas. Abbreviations: Col-1 = alpha-1 type 1 collagen; SMA = alpha-smooth muscle actin. Please click here to view a larger version of this figure.

Figure 4: Representative results of serum biochemical indices of mice in different periods. (A) Serum hyaluronic acid concentration (µg/L). (B) Serum bilirubin concentration (mmol/L). (C) Serum amylase concentration (U/dL). Please click here to view a larger version of this figure.

Discussion

The risk and severity of pancreatitis are determined by both genetic and environmental factors⁶. The complete cure of clinical pancreatitis is a goal in gastroenterology, with the creation of animal models being the first step. Various methods exist to establish these models, each with its advantages and disadvantages^7,8,9,10,11.

Surgeons have used high-frequency electrocoagulation to perform endoluminal thermal ablation (ETHA) in the main pancreatic duct to reduce pancreatic exocrine secretion, thereby reducing the incidence of postoperative pancreatic fistula (POPF) after pancreaticoduodenectomy (PD)¹². This suggests that the method of thermal ablation causing pancreatic atrophy has been implemented in clinical practice. We developed a new model of obstructive pancreatitis by using methylene blue to visualize the pancreatic duct and precisely electrocoagulate it.

C57BL/6 8-week-old male mice were selected for this experiment. Female mice have a reproductive cycle, and the levels of hormones in the body may have an impact on the experimental data. At 8 weeks of age, the mice were in the prime of life with mature body organs and good life health index, and the male mice of the same age were stronger in size. In the postoperative 1 month of observation, we sampled mouse pancreas on 7, 14, 21, and 28 days; the results obtained at 14 days most conform to the pathological characteristics of pancreatitis.

There are several caveats to this approach. First, the common bile duct should be clamped as close as possible to the hepatic hilum to facilitate the injection of the dye into the pancreatic duct. Second, attention should be paid to the speed and time of injection of methylene blue dye into the pancreatic duct. The pancreatic duct cannot be seen with too little dye solution, and too much dye solution will hinder the identification of the location of the main pancreatic duct in the surgical field. Third, electrocoagulation was terminated when the blue staining solution could not be seen. In these experiments, surgery is performed in the area indicated by the methylene blue stain, because the area of the mouse pancreas that is electrocoagulated will be different, which is why the methylene blue stain is used to track the pancreatic duct. Fourth, any bleeding on the pancreas during surgery should be stopped immediately. At the same time, the electrocoagulation knife should be used to cut the pancreas. If the electrocoagulation knife is stuck to the pancreas for any reason, the electrocoagulation time should be extended. This is the most common way to prevent the occurrence of serious postoperative complications caused by pancreatic leakage.

However, there are drawbacks to this method. The proficiency of the operator directly affects the experiment's outcome, and the uniformity of the surgery across different mice influences the model's success rate. This model simulates the effects of gallstones or pancreatic duct obstruction, leading to repeated bouts of acute pancreatitis, which eventually cause chronic pancreatitis. This could explain the elevated bilirubin levels. Therefore, this model can also be developed as an acute pancreatitis model based on the degree of electrocoagulation. Early experiments on the pancreas of mice 3 days post electrocoagulation showed characteristics of acute pancreatitis, but postoperative complications lowered the model's success rate for acute pancreatitis. However, it cannot be ruled out that the heat generated by the electrocautery during the electrocoagulation process may have spread to the main bile duct. The question of how to stably induce acute pancreatitis through electrocoagulation is worth exploring.

Compared to the traditional cerulein injection method, which requires repeated injections over several weeks, the electrocoagulation method saves time and is more convenient. While cerulein injection demonstrates excellent stability and ease of operation, electrocoagulation takes ~20 min and saves subsequent experimental time.

The development of new models aims to study the natural history of the disease and develop targeted therapies. Our new animal model helps simulate chronic pancreatitis caused by pancreatic duct obstruction in mice, including post endoscopic retrograde cholangiopancreatography pancreatitis (ERCP)¹³.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
16 mm microvascular clip	Stronger, Ningbo, China
Absorbable Suture	Yangzhou Jinhuan Medical Device Factory	4-0	-
Carprofen	Shanghai Macklin Biochemical Co., Ltd.	C830557-5g	-
Capillary tube	Tygon,US	AAQ(AAD)04091	0.25 x 0.3 mm
Electrocoagulation knife	Shun Ye MEDICAL Equipment Co., LTD, Ningbo, China	Shun Ye BDD-YE-DF-1
Eye scissors
Ketamine Hydrochloride Injection (Ketaset CIII)	Zoetis Inc.	S5564	-
Methylene blue staining solution	Solarbio, Shanghai, China	G1303
Non absorbent 4-0 monofilament polypropylene suture	Jinhuan, Shanghai, China
Operating microscope	Murzider, Guangdong, China	MSD203
Ophthalmic forceps
Round head swab	Olga Sherer, Huaximedical, China
Syringe needle	Minank, Zhejiang, China		0.25 mm
Tissue forceps
Tissue scissors	Shanghai Puxin Instrument Technology Corporation
Xylazine Hydrochloride Injection	Bayer Animal Health	XYL-50-20	-