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Medicine

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

Published: April 1, 2022 doi: 10.3791/63129
Automatic Translation
1, 1, 1, 2,4,5, 1, 2,3,4,6
1Department of Gastroenterology, the First Affiliated Hospital of Soochow University, 2MOE Engineering Center of Hematological Disease, Soochow University, 3Cyrus Tang Hematology Center, Soochow University, 4National Clinical Research Center for Hematologic Diseases, the First Affiliated Hospital of Soochow University, 5MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, 6Collaborative Innovation Center of Hematology, Soochow University

Summary

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.

Abstract

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.

Introduction

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.

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Protocol

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

  1. Fast a C57BL/6 wild-type mouse 8-12 h before the surgery.
  2. Prepare 5% (w/v) TC solution (93 mM) diluted into 0.9% sodium chloride. Filter the solution through a 0.22 µm filter.
  3. Autoclave the surgical instruments and materials, including forceps, scissors, blade holder, needle holder, hemostatic clip, vascular clamps, and sterile drapes, using a pressure sterilizer at a temperature of 121 °C for 30 min.
  4. Prepare a homemade microsyringe. To do this, manually install a disposable insulin tip and a prolonged plastic pipette tip onto a 25 µL flat tip Microliter syringe. Sterilize through irradiation.

2. Anesthesia and preoperative preparation

  1. Weigh 6- to 8-week-old male C57BL/6 wild-type mice (approximately 21-23 g). Induce general anesthesia with an intraperitoneal injection of 1% pentobarbital sodium solution at a dose of 50 mg/kg.
  2. Fix the mouse in the supine position on anirradiatedfoam flat plate with the tape.
  3. Prepare the operation region of each animal by removing its fur 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.
  4. Prepare a sterile surgical field with particular focus on the surgical area, consisting of the area prepared on the animal and the front of the surgeon.
    1. Disinfect the benchtop.
    2. Clean the incision site with multiple applications of surgical scrub solution. Wipe the skin two to three times with 0.5% iodophor and allow it dry for 1-2 min each time.
    3. Cover the mouse with sterile drapes.

3. Surgical procedure

NOTE: Figure 1 depicts the anatomy and morphology of the mouse pancreas before and after retrograde injection of TC into the biliopancreatic duct by microinjection.

  1. Make an approximate 2 to 3 cm long median abdominal incision.
  2. 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. Take care not to injure the intestinal wall or related vessels.
  3. Use 8-0 sutures to pass through the connection between the duodenum and pancreas around the papilla, and pull them to the left side of the mouse body to fix the duodenum.
  4. Gently pull the skin and the lower edge of the liver aside. Clamp the common hepatic duct, making sure not to clamp the portal vein or pancreas. Clamp the proximal biliopancreatic duct.
  5. Pierce the microinjector into the distal biliopancreatic duct retrogradely. Fix the needle tip near the duodenal papilla with a clamp. Infuse 10 µL of 5% TC into the pancreatic duct at a rate of 5 µL/min.
  6. Keep the biliopancreatic duct closed for 5 min to achieve a stable pressure. Then, pull out the microinjector and remove the clamps.
  7. Restore the duodenum to the original anatomical position and check the bleeding.
  8. Close the wound with 6-0 sutures and disinfect with 0.5% iodophor.

4. Recovery and postoperative management

  1. Recover the animals from anesthesia on a 37 °C thermal pad. Do not send the operated animals to the temporal postoperative room until all the animals have woken up.
  2. Administer 0.8 mL of 0.9% saline subcutaneously and slowly for fluid supplementation.
  3. Monitor the animals postoperatively for their general condition and signs of infections or illness.

5. Serological testing and histology evaluation

  1. At 24 h post-operation, anesthetize the mice with pentobarbital sodium using the same dose and injection route.
  2. Collect all the blood from the abdominal aorta (about 0.8 mL per mouse) and spin at 1,500 x g for 15 min to isolate the serum for further testing.
  3. Collect the pancreas tissues.
  4. Open the thoracic cavity to collect the lung tissues.
  5. Measure the biochemistry function indexes from the serum, including amylase, lipase, liver function (alanine transaminase (ALT) and aspartate transaminase (AST)), and kidney function (urea and creatine), using commercial kits according to the manufacturers' instructions.
  6. Homogenize the lung and pancreas tissues and measure the myeloperoxidase (MPO) level using a commercial kit according to the manufacturer's instructions.
  7. Open the thoracic cavity and perfuse with 20 mL of phosphate-buffered saline (PBS) twice, then collect the pancreas. Immerse into 4% paraformaldehyde solution for 24 h. Dehydrate in a series of alcohol concentrations and embed in paraffin.
  8. Use a microtome to prepare 5 µm thick pancreas tissue sections.
  9. Perform H&E staining for histological evaluation of the pancreas.
  10. Evaluate the pathological scores according to the criteria proposed by Schmidt et al.30.

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Representative Results

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 survival rates of the control and SAP mice were 100% (8/8) and 72.7%(8/11), respectively, at 72 h post-operation (Figure 3).

According to the H&E staining (Figure 4) and pathological score (Figure 5) results from pancreas sections, obvious edema (score: 3.14 ± 0.51 vs. 0.10 ± 0.08), inflammation (score: 1.41 ± 0.81 vs. 0), and necrosis (score: 3.03 ± 0.77 vs. 0) of the pancreas tissue could be observed in SAP mice relative to the control mice. Meanwhile no significant changes could be found on hemorrhage (score: 0.125 ± 0.31 vs. 0) in both groups of mice, resulting in a substantially elevated total pathology score in SAP mice (score: 7.72 ± 1.61 vs. 0.10 ± 0.08).

Moreover, compared to those of the control mice, significantly increased levels of amylase ([46,740.32 ± 12,801.30] U/L vs. [3,324.40 ± 753.66] U/L, p < 0.001), lipase ([545.02 ± 356.28] U/L vs. [18.32 ± 25.22] U/L, p < 0.05), and pancreas and lung MPO (pancreas: [5.18 ± 3.26] U/g vs. [0.71 ± 0.41] U/g, p < 0.05; lung: [11.70 ± 1.31] U/g vs. [1.56 ± 0.45] U/g, p < 0.001) were found in SAP mice (Figure 6).

In addition, compared with control mice, SAP mice demonstrated impaired liver and kidney functions, as illustrated by the following indexes: ALT ([164.36 ± 47.66] U/L vs. [77.15 ± 31.06] U/L, p < 0.001), AST ([222.35 ± 82.13] U/L vs. [116.61 ± 64.79] U/L, p < 0.01), blood urea nitrogen (BUN) ([37.59 ± 16.36] mM vs. [14.80 ± 3.74] mM, p < 0.001) and creatinine ([40.33 ± 11.53] µM vs. [28.66 ± 4.53] µM, p < 0.01) (Figure 7).

Figure 1
Figure 1. The anatomy and morphology of the mouse pancreas before and after retrograde injection of sodium taurocholate (TC) into the biliopancreatic duct. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Time course of the weight changes of the control (n = 8) and severe acute pancreatitis (n = 11) mice. Error bars represent the standard deviation (SD). Please click here to view a larger version of this figure.

Figure 3
Figure 3. Survival curve monitoring of the control (n = 8) and severe acute pancreatitis (n = 11) mice. The numbers in blue and red represent the numbers of surviving mice at a specific time point in the control and SAP groups, respectively. Please click here to view a larger version of this figure.

Figure 4
Figure 4. Histopathology of pancreatic sections from control and severe acute pancreatitis (SAP) mice. Black arrow indicates inflammatory cells infiltration, red arrow indicates bleeding, black inverted triangle indicates acinar necrosis. Please click here to view a larger version of this figure.

Figure 5
Figure 5. Histology scores. Histology scores (including edema, necrosis, inflammation, hemorrhage, and total scores) of the pancreas section from control and severe acute pancreatitis (SAP) mice. Each black dot represents the corresponding score from one mouse. ns: not significant; **p < 0.01; ***p < 0.001. Please click here to view a larger version of this figure.

Figure 6
Figure 6. Serological evaluation of amylase and lipase and determination of the pancreas and lung myeloperoxidase (MPO) activity. Significantly increased levels of amylase, lipase, and pancreas and lung MPO were found in SAP mice relative to control mice. One black dot represents one mouse. Error bars represent the standard deviation (SD). *p < 0.05; ***p < 0.001. Please click here to view a larger version of this figure.

Figure 7
Figure 7. Liver function indexes (ALT: alanine aminotransferase, and AST: aspartate aminotransferase) and kidney function indexes (creatinine and BUN: blood urea nitrogen), in control and severe acute pancreatitis (SAP) mice. Significantly increased levels of ALT, AST, BUN, and creatinine were found in SAP mice relative to control mice. One black dot represents one mouse. Error bars represent the standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001. Please click here to view a larger version of this figure.

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Discussion

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, and the major difference was that they used a pump for TC infusion, which may provide a more accurate infusion dose.

According to our experience, during the surgical process of the model establishment, the most important factors included full exposure of the biliopancreatic duct, smooth puncture of the microinjector, and injection of TC into the biliopancreatic duct and the pancreatic duct at a relatively constant speed. Attention should be given to the following points during surgery. First, the duodenum should be pulled out gently to avoid application of excessive force, which may result in duodenal necrosis. The opening of the biliopancreatic duct is located along the inner edge of the duodenum (also named the duodenal papilla), and the biliopancreatic duct can be subsequently located. If successful, the white duodenal papilla and tubules that shape the biliopancreatic duct and pass through the leaf-shaped pancreas should be identifiable. Adequate exposure of the biliopancreatic duct should facilitate the subsequent procedures. Second, moderate pulling force should be applied when fixing the duodenum to the lower left side of the mouse placed in the supine position using sutures, thereby straightening the biliopancreatic duct without tension loss. Excessive and insufficient pulling forces could result in tearing of the wall of the duodenum and inadequate exposure of the biliopancreatic duct, respectively, which could adversely affect the subsequent puncture. A vertical angle is recommended to ensure a smooth puncture when the duodenum is fixed to the lower left side using sutures. Then, during puncture, ensure that the puncture direction is parallel to the direction of the biliopancreatic duct. When puncturing, the inclined plane of the needle tip should be oriented downward to pass through the duodenum wall and subsequently enter the biliopancreatic duct, making sure to use appropriate force to avoid puncturing the biliopancreatic duct. After a successful puncture, no resistance should be felt when entering the biliopancreatic duct, and tension of the biliopancreatic duct wall should be felt when gently shaking the needle tip. Finally, during the procedure, the duodenum should be kept moist with warm normal saline to maintain its activity.

However, this method also has some limitations. First, reproducibility may be affected by different reactions to the TC from each mouse. Therefore, a paired comparison of corresponding results from each mouse could be performed to minimize these impacts. Second, injury of the duodenal papilla is a common phenomenon during the puncture process, which can also cause acute pancreatitis. Therefore, a longer training time may be required to develop adequate surgical skills to fulfill this modeling method. In addition, keeping the mice a suitable size could be beneficial for ensuring the surgical results.

Despite these limitations, this model is one of the most widely used and best characterized models to date for inducing AP and investigating novel therapies and possible molecular events during the process of AP. According to previous studies, SAP induced through TC has also been described to be suitable for target therapy evaluation because the severity achieved with this model is similar to human disease and can be easily reproduced. Quercetin intervention has been reported to attenuate pancreatic and ileal pathological damage in this SAP model via the inhibition of TLR4/MyD88/p38 MAPK and endoplasmic reticulum stress (ERS) activation31. The administration of CM4620, a selective Orai1 channel blocker, could significantly decrease the severity of this SAP model; prevention of trypsinogen activation, acinar cell death, NF-κB, and nuclear factor of activated T cells (NFAT) activation and inflammatory responses were shown to serve as the underlying mechanisms32. In addition to TC, taurolithocholic acid 3-sulfate (TLCS) is another bile acid frequently used in AP induction. As reviewed by Petersen et al.33, elevated calcium ion concentrations induced by TLCS in acinar cells could affect not only intracellular mitochondrial ATP production, but also other cells (acinar, stellate, and macrophages) located in the pancreas via calcium ion exocytosis; whereas, TC might exert indirect damage to acinar cells via its effect on periacinar stellate cells.

Therefore, the TC-induced SAP model provides excellent insights into the pathogenesis of AP and is a highly relevant tool for preclinically validating new drugs. Moreover, this model can be adapted to large animals to further evaluate the unpleasant and unexpected effects resulting from therapies for treating AP.

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Disclosures

None.

Acknowledgments

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 & Social Development Plan of Jiangsu Province (BE2018659).

Materials

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

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References

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Mouse Severe Acute Pancreatitis Model Retrograde Injection Sodium Taurocholate Biliopancreatic Duct Animal Ethics Committee Surgical Procedures Anesthesia Fast The Mouse Sodium Taurocholate Solution Surgical Instruments Forceps Scissors Blade Holder Needle Holder Hemostatic Clip Vascular Clamps Sterile Drapes Micro-syringe Sterilization General Anesthesia Pentobarbital Sodium Solution Supine Position Depilation Cream
Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct
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Zhou, X., Chen, H., Wei, X., He, Y., … More

Zhou, X., Chen, H., Wei, X., He, Y., Xu, C., Weng, Z. Establishment of a Mouse Severe Acute Pancreatitis Model using Retrograde Injection of Sodium Taurocholate into the Biliopancreatic Duct. J. Vis. Exp. (182), e63129, doi:10.3791/63129 (2022).

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