Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

This protocol was reviewed and approved by the Ethics Committee at The First Affiliated Hospital of Anhui University of Science and Technology (Huainan, China) (Ethics Code: 2023-KY-905-001). The study followed the National Institutes of Health guidelines for the care and use of research rodents in all animal procedures. C57BL/6J adult mice weighing 20-30 g were used for the present study. The mice were housed in an animal laboratory for one week under controlled conditions (approximately 21 °C with a 12 h alternating day-night cycle). The mice had ad libitum access to food and water throughout. The details of the reagents and equipment used in the study are listed in the Table of Materials.

1. Animal preparation

1. Assign 84 healthy C57BL/6J mice to six groups, including the control group, Pancreatic injury (PI) I, PI II, PI III, PI IV, and PI V.
2. Prior to initiating the modeling procedure, label each group of mice with ear notches and allow them to fast for 12 h.

2. Preparation of induced drug diluent

1. Dissolve the Caerulein (1 mg) into 1 mL of PBS and refrigerate at -20 °C.
2. Measure and record the weight of the experimental mice 1 h before the intraperitoneal injection of the drug.
3. Extract the total mass of Caerulein required at a ratio of 50 µg/kg, based on the total weight of all mice.
4. Dilute the obtained Caerulein drug with PBS again.
   NOTE: The total volume of PBS dilution should be equivalent to 5 times the total weight value of all mice.The same dilution method was used to obtain LPS diluent.

3. Intraperitoneal injection

NOTE: Intraperitoneal injections were administered to each group of mice according to the protocol outlined in Supplementary Table 1 to induce the model. An additional 10 mice were grouped and treated observe the 7-day survival rates.

1. Grab and hold the mice in a way that the belly of the mice is facing up and the head is positioned lower than the tail to prevent damage to the organs when inserting the syringe.
2. Disinfect the mice's abdomen using 75% alcohol cotton balls.
3. Hold the syringe in the right hand (using a needle measuring ≤0.5/≥0.3) and insert the needle into the subcutaneous skin on either the left side of the abdominal white line.
   NOTE:Change to the right side for the next injection.
4. Once the needle has reached the subcutaneous layer, move it forward approximately 3-5 mm, and then insert the syringe needle into the abdominal cavity at a 45° angle. At this point, one should feel some resistance.
5. Keep the needle stationary and inject the substance slowly.
   NOTE: Do not exceed 5 times the weight value of the mice as the volume of a single intraperitoneal injection.
6. After the injection, pull out the needle and gently massage and press the injection site with a sterile cotton swab to fully diffuse the drug into the mice's abdominal cavity.
7. Use a new syringe to repeat with the next experimental mice.

4. Open-field behavioral ability testing

NOTE: 12 h after the last intraperitoneal injection, open-field behavioral ability testing was conducted to assess the total activity distance and immobility time of the mice.

1. Place four identical white boxes directly beneath the camera.
2. Connect the camera to the computer using a video cable.
3. Ensure the experimental box is clean and free from any odors before starting the experiment.
4. Position the animal in the middle of the grid, facing away from the experimenter, and allow it to adapt to the environment for 10 min.
5. Start the video tracking software and click on the File menu to create a new experiment.
6. Set up the monitoring for four simultaneous fields of view.
7. Mark the length and width of the experimental object that can move within the box in the real-time monitoring screen (30 cm × 30 cm).
8. Once the setup is complete, begin video surveillance and recording.
9. Record the activity of the mice for 15 min.
10. After the testing, remove the animal from the grid and return it to its cage. Thoroughly clean the grid using a sterilant containing chlorine dioxide.

5. Collecting and testing the peripheral blood of mice

1. Euthanize the mice (following institutionally approved protocol).
   1. Perform euthanasia on the experimental mice 36 h after the intraperitoneal injection. Measure and record the body weight of the mice prior to euthanasia.
      NOTE: Administer 0.18 mL of 10% chloral hydrate intraperitoneally, ensuring no reaction to stimulation of the toe or tail.
   2. With the left hand, secure the mice, and with the right hand, hold the scissors to trim the whiskers on one side of the mice.
   3. Gently press the skin around the eye to induce congestion and protrusion of the eyeball.
   4. Use curved forceps to grasp the eyeball and quickly remove it, collecting peripheral blood in a microcentrifuge tube.
      NOTE: Two different types of microcentrifuge tubes were used, one containing anticoagulant and the other without.
   5. Simultaneously, lightly press the mice's heart area with the middle finger of the left hand to increase the pumping speed of the heart.
2. Leave the peripheral blood without anticoagulant at room temperature for 30 min.
3. Measure the concentrations of serum amylase (Amy) and lipase (Lip) using an automated biochemical analyzer through the enzyme rate method and enzyme circulation method (following the manufacturer's instructions).
4. Determine the levels of inflammatory indicators such as Pro Calcitonin (PCT) in plasma using the chemiluminescence method using a commercial chemiluminescence imager (following the manufacturer's instructions).
5. Use ELISA kits to measure HMGB-1, IL-6, and TNF-α levels in mice serum (following the manufacturer's instructions).
6. Analyze the peripheral blood treated with anticoagulant using a fully automatic blood cell analyzer to assess the relevant parameters.

6. Collecting the pancreatic tissue and preparing a paraffin section

1. Place the mice in a supine position and secure them on a foam plate.
2. Shave and disinfect the area, then make an abdominal median incision and flip the small intestine tube to the right to fully expose the pancreas.
3. Disconnect the duodenum and pyloric duct and locate the small intestine beneath the pancreas. Fully free the pancreatic tissue along the intestinal duct.
4. Use toothless forceps to clamp the spleen and gently pull upwards.
   NOTE: Do not directly touch the pancreatic tissue, and avoid using excessive force during the entire dissociation process.
5. Sharply dissect the posterior pancreatic ligament tissue up to the head of the pancreas and disconnect the bile duct and blood vessels.
6. Remove the pancreatic tissue, pat dry the surface moisture with absorbent paper, weigh and record.
7. Fix half of the pancreatic tissue with 4% paraformaldehyde and store the other half in a refrigerator at -80 °C.
8. Prepare the pancreatic paraffin sections following the steps below.
   1. Clean the fixed pancreatic tissue with 75% alcohol and trim it to a size of approximately 0.5cm × 0.5cm.
   2. Immerse the tissue in 70% ethanol for 20 min, 80% ethanol for another 20 min, and 90% ethanol for 15 min.
   3. Treat the tissue twice with 95% ethanol for 15 min each time and then twice with 100% ethanol for 5 min each time.
   4. Subject the tissue to two rounds of 12 min and 5 min treatments with xylene solution for transparency.
   5. Soak the transparent tissue in a wax tank at 65 °C for 1 h.
   6. Embed the tissue in melted paraffin and allow it to cool. Obtain pancreatic paraffin sections with a slicer (thickness: 5 µm).
   7. Flatten the obtained paraffin slices in 45 °C water, mount them, and dry them (baking conditions: 40 °C for 14-16 h).

7. Hematoxylin and Eosin (H&E) staining

1. Place the pancreatic paraffin sections in xylene I and II for 30 min, then in anhydrous, 95%, 85%, and 75% alcohol for 5 min each, and ultrapure water for 5 min.
2. Stain the cell nucleus with hematoxylin for 160 s, then rinse with running water slowly.
3. Apply a hydrochloric acid alcohol differentiation solution for 5-10 s, then rinse with running water quickly.
4. Treat the sections with anhydrous alcohol for 5 min.
5. Incubate the cytoplasm with eosin for 30 s, then rinse with running water.
6. Immerse the sections in 75%, 85%, 95%, and 100% ethanol for 10 s each, then treat with xylene for 5 min to dehydrate.
7. Finally, seal the sections with neutral resin and observe under a microscope.
8. Perform the pathological scoring and standards¹³.
   NOTE: Determine the severity of pancreatitis based on indicators such as edema, acinar necrosis, bleeding, hemorrhage and fat necrosis, and inflammatory and perivascular inflammation.Use the previously published implementation guidelines for scoring¹³.

8. Immunohistochemical staining

1. Dewax the paraffin-embedded sections of pancreatic tissue in xylene and subsequently rehydrate them in a gradient of ethanol solutions.
2. Carry out endogenous enzyme-blocking using 3% H₂O₂ for 20 min after a 30 min membrane rupture.
3. Block the antibody with 5% bovine serum at room temperature for 30 min, then incubate overnight at 4 °C with a 1:1500 dilution of rabbit anti-HMGB1.
4. Rinse the sections with PBS, then incubate them in corresponding secondary antibodies for 30 min at 37 °C. After that, carry out DAB color development, hematoxylin counterstaining, and seal with neutral gum.

9. TUNEL method for detecting apoptosis in pancreatic sections

1. Dewax the pancreatic paraffin sections in xylene for 5 min, repeat twice and wash with gradient ethanol (100% for 5 min, 90% for 2 min, 70% for 2 min, and distilled water for 2 min).
2. Wash away excess liquid surrounding the paraffin sections using PBS. Treat each sample with 100 µL of Proteinase K, ensuring complete coverage of the tissue. Incubate the samples at 37 °C for 20 min. Subsequently, soak the samples in PBS 3 times, each time for 5 min.
   NOTE: The Proteinase K solution was prepared by diluting the original Proteinase K (200 µg/mL) solution with PBS at a ratio of 1:9 (volume), resulting in a final concentration of 20 µg/mL, excluding DNase. Thorough washing of Proteinase K is essential to avoid interference with subsequent labeling reactions.
3. Add a suitable amount of 3% H₂O₂ (diluted by PBS) onto the tissue to fully infiltrate it and incubate for 20 min. Rinse the tissue with PBS 3 times for 5 min each.
   NOTE: The paraffin sections should be kept moist. To inactivate the endogenous peroxidases in the tissue, the incubation time should not be too long to prevent false positives due to DNA breakage caused by 3% H₂O₂.
4. Cover the entire area of the sample to be tested with 50 µL of Equilibration buffer and incubate for 10 min.
5. Remove as much Equilibration buffer as possible. Then, add 56 µL of TdT Incubation buffer to each tissue sample and incubate for 1 h (at room temperature).
   NOTE: The slide should not be allowed to dry, and exposure to light should be avoided. TdT Incubation buffer was prepared according to the manufacturer's instructions (Recombinant TdT enzyme: Biotin-dUTP Labeling Mix: Equilibration Buffer = 1 µL: 5 µL: 50 µL).
6. Wash the tissue samples with PBS immediately. Rinse them 4 times for 5 min each. Gently remove any excess PBS solution around the samples with filter paper.
7. Add 100 µL of previously diluted Streptavidin-HRP reaction solution (Streptavidin-HRP: TBST = 1: 300) to each tissue sample for the Streptavidin-HRP reaction. Incubate for 30 min. Then, wash the samples with PBS and rinse them 3 times for 5 min each.
8. Perform DAB staining by adding 50 µL of DAB to each paraffin section. Observe the staining under a microscope in real-time. After positive staining appears, immediately place the slides in a humid box. Stop the reaction by washing it with pure water.
9. Immerse the slides in hematoxylin staining solution for 3-5 min, then rinse with pure water.
10. Differentiate in hematoxylin differentiation solution for about 2 s, followed by immediate rinsing with pure water.
11. Achieve blue coloration using hematoxylin rebluing solution for a few seconds, and rinse the slides clean with pure water.
    NOTE: Conduct microscopic examination after nuclear staining. If the staining is too dark, return the slides to the differentiation solution. If the staining is too light, restart the staining process from the nuclear staining step.
12. Dehydrate the samples with 4 rounds of fresh anhydrous ethanol for 5 min each. Then, soak them in butanol for 5 min and in xylene for 5 min. Finally, use fresh xylene for another 5 min.
13. Mount the slides using neutral gum. Allow them to air-dry naturally or dry them in a 60 °C oven.
14. Perform histological examination using a white light microscope. Apoptotic nuclei will appear brown.
15. Calculate the corresponding apoptotic index (AI).
    NOTE: Observe the slides in a double-blind manner. In TUNEL-positive slides, randomly select 5 positive areas under high magnification (400x), and count at least 100 acinar cells in each area to determine the percentage of positive cells. AI = (total number of apoptotic cells/total number of cells) × 100%.

10. Flow cytometry

1. Obtain fresh pancreatic tissues and thoroughly wash them after perfusing with PBS.
2. Prepare a 0.5% collagenase IV digestion solution and use sterile tissue scissors to adequately digest the pancreatic tissues.
3. Add a 5% BSA digestion termination solution depending on the condition of the pancreatic fragments and liquid turbidity, and centrifuge the mixture at low temperature for 5 min (~300 x g, 4 °C).
4. Discard the supernatant to terminate the digestion.
5. Prepare a cell culture medium containing phenyl methane sulfonyl fluoride (PMSF) and 2.5% fetal bovine serum cell suspension to resuspend the pancreatic cells.
6. Filter the pancreatic acinar cell suspensions through a 200-mesh nylon mesh to obtain cell suspensions through cell filtering.
7. Centrifuge the pancreatic acinar cell suspension at low temperature (follow the conditions as mentioned in step 10.3).
8. Discard the supernatant.
9. Wash the cells with pre-chilled PBS.
10. Gently resuspend the cells in pre-chilled 1x binding buffer through centrifugal resuspension.
11. Label the pancreatic acinar cells with Annexin V-FITC/PI according to the manufacturer's instructions after adjusting the cell concentration.
12. Use flow cytometry within 1 h to detect apoptosis in pancreatic acinar cells.

11. Western blot detection of Caspase-3 and HMGB-1

1. Extract the pancreatic protein.
   1. Extract 50 mg of pancreatic tissue and cut it into small fragments.
   2. Add 1 mL of RIPA lysis buffer (containing PMSF and phosphorylated protease inhibitor).
   3. Homogenize the pancreatic tissue on ice for 5 min using an electric grinder.
   4. Incubate the homogenized tissue on ice with gentle shaking for 2 h.
   5. Centrifuge the homogenate at 4 °C for 10 min at 4,500 x g.
   6. Collect the supernatant solution and store it at -80 °C.
2. Perform protein quantification.
   1. Dilute a certain volume of BSA standard sample (25 mg/mL) to a concentration of 0.5 mg/mL.
   2. Prepare a specific amount of BCA working solution by mixing 50 volumes of BCA solution A with 1 volume of BCA solution B, ensuring thorough and even mixing.
   3. Place the BSA standard sample into the standard sample holes on a 96-well plate in the order of 0, 1, 2, 4, 8, 12, 16, and 20 µL. Balance the volume of each hole with distilled water, resulting in a total volume of 20 µL.
   4. Add 2 µL of samples into the sample holes, then sequentially add 18 µL of distilled water for dilution (1:9).
   5. Fill each well with 200 µL of BCA working solution and allow it to stand at 37 °C for approximately 20-30 min.
   6. Measure the absorbance at a wavelength of 562 nm. Calculate the protein concentration of the samples based on the standard curve.
   7. Add a specific volume of RIPA cracking solution and diluting buffer to dilute the samples until the concentration reaches 5-10 µg/µL, which is considered appropriate.
   8. Store the samples at -20 °C after protein denaturation (100 °C; 10 min).
3. Perform the Western blotting.
   1. Make preparations for SDS-PAGE.
      1. Install the glue-making molds and inspect their sealing performance.
      2. Add TEMED to the 10% separation adhesive and mix evenly.
      3. Inject the mixture into the mold, accounting for approximately two-thirds of the volume, while avoiding bubbles.
      4. Add isopropanol to the mold and press the adhesive for about 40 min.
      5. Use the same program to configure the 5% concentrated adhesive.
      6. Pour out isopropanol and add the adhesive immediately.
      7. Place the comb vertically until the glue completely polymerizes.
   2. Perform the electrophoresis operation.
      1. Add the sample to be tested in the expected experimental control order, in reverse order, into the sampling holes of the SDS-PAGE adhesive.
      2. Simultaneously add the Protein Mark to determine the location of the target protein and internal reference protein.
      3. Place the sample in the electrophoresis tank after the sample addition is completed.
      4. Initially, run at 80 V for 30 min, followed by running the gel to separate the protein at 100 V for 60 min.
         NOTE: Attention should be paid to the position of bromophenol blue to avoid excessive electrophoresis.
   3. Perform the protein transfer.
      1. Cut the PVDF film at the top right corner to serve as a mark.
      2. Place the PVDF film in a methanol solution for 5-10 s to activate.
      3. Soak the PVDF film in an electrophoresis buffer for approximately 15 min.
      4. Carefully remove the gel after electrophoresis and trim excess gel, ensuring the gel remains wet throughout the process.
      5. Assemble the splint in the following order: negative plate → sponge → three layers of filter paper → gel → PVDF film → three layers of filter paper → sponge → positive plate.
         NOTE: Ensure there are no bubbles between the gel and PVDF film.
      6. Place the assembled clamp into the wet rotating groove (black to black) with ice cubes positioned around it.
      7. Turn on the power and initiate the film transfer at 400 mA for 100 min.
   4. Perform PVDF film sealing, and incubation of primary and secondary antibodies.
      1. Gently remove the PVDF film after the transfer and rinse with TBST for 10 min, repeating the process 3-5 times.
      2. Seal the PVDF film in 5% skim milk for 2 h.
      3. Remove the PVDF film and rinse with TBST for 10 min, repeating the process 4 times.
      4. Place the PVDF film and diluted primary antibody together in a refrigerator at 4 °C with gentle shaking and incubate overnight.
      5. Remove the PVDF film the next day and rinse with TBST for 10 min, repeating the process 4 times.
      6. Incubate the PVDF film in HRP-labeled secondary antibody diluent for approximately 2 h.
      7. Rinse the PVDF film with TBST for 10 min, repeating the process 4 times.
   5. Expose the film and analyze it.
      1. Apply the prepared ECL solution onto the film evenly.
      2. Expose the film in a dark room with an exposure meter for approximately 2-3 min and then store it.
      3. Conduct the analysis using image J software.