A Translational Surgical Porcine Model for Postoperative Intra-Abdominal Adhesion Formation

Summary

This protocol describes a porcine model of postoperative intra-abdominal adhesion formation using Yucatan mini pigs undergoing open abdominal surgery with bowel resection. The approach reliably induces intra-abdominal adhesions and provides a platform for evaluating adhesion development and testing anti-adhesion strategies.

Abstract

A porcine model of postoperative intra-abdominal adhesion formation was established using Yucatan mini pigs. The protocol combines midline laparotomy, small bowel resection with two-layer primary anastomosis, and a unilateral, parietal peritoneal abrasion in the format of an open abdominal surgical procedure. Adhesion formation was assessed four weeks postoperatively using established gross and histologic scoring criteria, with evaluations performed by blinded observers. Adhesions developed in all animals using this model and were multifocal, involving bowel loops, between the bowel and abdominal wall, involving the peritoneum overlying other organs in the abdomen (e.g. the liver), and operative sites, with variable severity. Histological analysis at four weeks demonstrated adhesions composed predominantly of extracellular matrix, fibroblasts, and blood vessels, consistent with a remodeling-phase wound healing tissue phenotype. This model is relevant for the study of abdominal adhesion fibrosis biology and/or the translational evaluation of candidate anti-adhesion therapeutics. By integrating a clinically relevant intestinal surgical procedure with a defined peritoneal injury and a standardized assessment strategy, this protocol provides a reproducible approach for inducing and evaluating postoperative intra-abdominal adhesions in a large animal model.

Introduction

Postoperative adhesions are a major source of morbidity following abdominal and pelvic surgery, developing in up to 93% of patients^1,2, with approximately 20% requiring hospitalization within five years for adhesion-related complications^3,4. Peritoneal injury arises from abdominal surgical procedures as well as intra-abdominal irritation (infection or chemical exposure) or chronic inflammatory conditions such as inflammatory bowel disease. The resulting adhesions can lead to a wide range of clinical consequences, including recurrent bowel obstructions sometimes requiring additional surgeries, or causing infertility and chronic pain^5,6,7. Adhesion formation can be particularly severe following multi-visceral resections for malignancy, such as cytoreductive surgeries, chronic inflammation, or bowel perforation, where extensive peritoneal injury amplifies the fibrotic response. Additionally, adhesive disease can significantly complicate subsequent surgical interventions, often making it impossible to proceed with a minimally invasive approach or causing inadvertent iatrogenic injury to the bowel or other organs.

Despite their clinical significance, the development of effective anti-adhesion strategies is limited - likely hindered in part by limitations in preclinical models that recapitulate the anatomy, physiology, and wound-healing responses seen in humans⁸. Rodent models are widely used due to their availability, experimental tractability, and reliable adhesion generation^9,10,11. However, their small peritoneal cavity, thin mesentery, and distinct inflammatory milieu can limit translational relevance^8,12. Such models have contributed to the development of anti-adhesion approaches that demonstrated robust efficacy in animal studies but demonstrated limited or context-dependent clinical benefit in humans^{8,13,14,15,16,17}. In addition, human-comparable surgical procedures such as bowel resection and anastomosis are difficult to model in a translationally relevant manner in rodents, and species-specific differences in drug metabolism further limit clinical extrapolation¹⁸.

Large animal models, particularly swine, are widely used to study abdominal processes due to their anatomical and physiological similarity to humans and their comparable surgical handling, providing a platform to study both adhesion formation and test therapeutic interventions^19,20,21,22. Existing porcine adhesion models have provided important insights, yet many focus on procedure-specific adhesion formation and often lack standardized procedures limiting their applicability particularly to severe adhesions arising after open intestinal surgery^{23,24,25,26,27}.

To address these limitations, we developed a porcine model of postoperative intra-abdominal adhesions that integrates midline laparotomy, small bowel resection, and a unilateral, parietal peritoneal abrasion. This multifocal peritoneal strategy recapitulates adhesion-promoting insults commonly encountered during abdominal surgical procedures and produces adhesions of different strengths and locations at the four-week timepoint with guaranteed adhesions on the side of the abdomen where directed parietal abrasion is performed as well as stochastic adhesions throughout the abdomen. By incorporating a clinically relevant intestinal surgical procedure with a defined and reproducible peritoneal injury, this model overcomes key limitations of prior porcine systems and provides a consistent platform for studying postoperative adhesion formation and evaluating anti-adhesion interventions.

Protocol

Ethics Statement

All porcine studies were conducted under protocols approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) protocol #34052 as previously described²⁸.

1. Animal Husbandry

1. Obtain Yucatan mini pigs (recommended 9- to 12-kg, 7 to 9 weeks old) from a verified vendor. We recommend n = 4 pigs per cohort. House animals in pairs in light- and temperature-controlled rooms.
2. Provide standard porcine chow ad libitum. The animals should have access to a continuous supply of water to avoid dehydration.
   NOTE: Animals were fasted for 12 h prior to surgery and terminal harvest.
3. Monitor and record body weight and general health at regular intervals per institutional standards.

2. Anesthesia and Preoperative Preparation

1. Sedate pigs in their home enclosures using intramuscular Xylazine (2.2 mg/kg, intramuscular (IM)) and Ketamine (12 mg/kg IM). Obtain intravenous (IV) access. Carefully transport animals to the surgical suite once sedation is deemed adequate by a veterinarian. Please refer to the Supplementary Table for anesthesia and analgesia information, as protocols may vary by institution.
2. Once in the surgical suite, secure the animal's airway via endotracheal intubation. Maintain anesthesia throughout the procedure with isoflurane administered in oxygen via a precision vaporizer through an anesthesia circuit equipped with waste gas scavenging under the supervision of a licensed veterinarian.
3. Administer intravenous (IV) fluids, anti-nausea medications (Cerenia 1-2 mg/kg IV), vasoconstricting medications, and inotropes as needed to treat intraoperative hypotension to avoid bowel hypoperfusion and causing potential risk of anastomotic leak. Please see Supplementary Table for full medication details, regimens will vary per institution.
4. Apply ophthalmic ointment to lubricate eyes and prevent corneal abrasions. Administer cefazolin (25 mg/kg, IV) or equivalent antibiotic 1 hour prior to incision to prevent a wound infection. Administer an intraoperative analgesic regimen according to institutional veterinary standards. A reference regimen is provided in the Supplementary Table and includes a combination of Carpofen (25 mg/kg IV), Buprenorphine ER (0.18 mg/kg IV).

3. Surgical Procedure

1. Position the animal supine on a heating pad to maintain normothermia. Measure oxygen saturations (SpO₂) via pulse oximeter, pulse with a heart rate monitor, and temperature every 10 mins.
2. Remove hair from the abdomen using electric clippers, then prepare the skin with a single round of alcohol swabs followed by gauze soaked in povidone-iodine. Blot the excess antiseptic solution away from the abdomen using a sterile towel.
3. Drape the abdomen using sterile towels and surgical drapes to maintain sterility throughout the procedure. Infiltrate lidocaine (2 mg/kg subcutaneously (SQ)) into the abdominal wall at the planned incision site using a 25-gauge needle.
4. Perform a midline laparotomy by making a full-thickness incision through the skin, subcutaneous tissue, and abdominal wall musculature along the linea alba to enter the peritoneal cavity using a 10-blade scalpel. Place abdominal wall retractors and eviscerate the intestines.
5. Identify a region of the bowel ~10-15 cm proximal to the ileocecal valve and resect a 2.5-cm (1-inch) segment of the small bowel using scissors. Resect the associated mesentery with electrocautery for hemostasis of the mesenteric blood vessels. Close the two open bowel ends using interrupted 3-0 silk sutures to achieve a watertight closure. Perform a two-layer, side-to-side primary anastomosis (functional end-to-end). Reconstruct the inner layer of the lumen with a synthetic 3-0 absorbable suture in a running fashion and the outer layer with a running 3-0 non-absorbable suture. Attempt to minimize bowel spillage throughout the procedure.
6. Gently abrade a 3 x 5 cm section of the parietal peritoneum along the right abdominal sidewall using sterile gauze or an electrocautery scratch pad until the peritoneal surface appears frayed or there is sub serosal edema in the abdominal wall (10-15 strokes with medium pressure). The left abdominal sidewall can be left unabraded to serve as a technical control although using this model stochastic adhesions form throughout the abdomen in a manner similar to human disease.
7. Reposition the bowel into the abdominal cavity and remove the abdominal wall retractors. Irrigate the abdomen with 1 L of warmed sterile saline, evacuate all fluid and confirm hemostasis (no active bleeding) prior to closure.
8. Close the fascia with a running 2-0 absorbable suture. Close the skin with a running 2-0 or 3-0 non-absorbable baseball stitch and cover the incision with a sterile dressing. The average duration of the procedure was 1 hour.
9. Administer Atipamezole (0.2 mg/kg IV) at the end of the procedure to reverse sedation prior to extubating. Extubate animals upon return of spontaneous respiration. Monitor vital signs (pulse, oxygen saturation, temperature, blood pressure) continuously until fully recovered from anesthesia.

4. Postoperative Care

1. Administer Carprofen (25 - 50 mg) twice a day by mouth (PO) the first 3 days after the surgical procedure and then as needed (PRN) for pain. Maintain the animals on a liquid high protein diet (1.5 bottles twice a day) for the first 3 postoperative days.
   NOTE: Of note, although routine suture removal at 10–14 days postoperatively is common practice, sutures were left in place in animals without signs of wound infection or dehiscence to avoid an additional anesthetic exposure prior to the planned 4-week harvest.
2. Administer amoxicillin (10 mg/kg) twice a day by mouth for one week then PRN for signs of wound infection (redness, warmth, swelling with or without drainage).
3. Check on animals twice a day for appetite, activity, bowel function and wound integrity. If concerned about infection, discuss antibiotic dosage and duration with the veterinarian. The sterile dressing should be left on for the first 3 days after the surgical procedure to minimize risk of wound infection. Record weight in 2 - 3 day intervals to ensure weight maintenance postoperatively.
4. Transition animals to solid chow beginning on postoperative day 4 (POD4). Bridge solid chow intake with supplemental liquid high protein diet until tolerating sufficient calories from solid chow alone.

5. Euthanasia

1. Euthanize animals at 4 weeks under veterinary supervision prior to necropsy and tissue harvest using an intravenous euthanasia solution according to institutional and AVMA standards.
2. Assess adhesions grossly. Immediately collect tissues and blood samples for downstream analyses. Dispose of all biological and pharmaceutical waste according to your institution's Environment Health and Safety guidelines.

Results

The proposed porcine postoperative adhesion model (Figure 1) was combined with an adapted scoring system to grade adhesions in real time²⁸. Adhesion scoring was completed independently by 3 team members blinded to experimental conditions at the 4-week timepoint on a representative cohort of n=4 pigs; the scores were averaged for a final adhesion score per animal (Figure 2A). Intra-abdominal adhesions formed in 100% of animals evaluated on...

Discussion

The proposed porcine adhesion model provides a clinically relevant and reliable method for modeling intra-abdominal adhesions. In contrast to focal or single-site injury models, the combination of midline laparotomy, small bowel resection with anastomosis, and standardized parietal peritoneal abrasion was intentionally selected to recapitulate the multifactorial peritoneal insults encountered during open abdominal surgical procedures. The controlled abrasion of the parietal peritoneum along the abdominal sidewall mimics ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animals
Female Yucatan mini pigs, 9 to 12 kg in body weight (7 to 9 weeks old)	S&S Farms		8
Surgical Supplies
DMI4000 B microscope	Leica Microsystems		Histology
Materials
Euthasol	Virbac		Euthanasia
Eosin-Y Alcoholic Stain	Azer Scientific	Cat#ES709	Histology
Frosted slides	Fisher Scientific	Cat#12-550-400	Histology
Mayer′s Hematoxylin Solution	Sigma-Aldrich	Cat#MHS80	Histology
Medications
Amoxicillin	Covetrus	59629	Infection prevention 10mg/kg PO BID x1
Ancef/Cefazolin	Covetrus	84773	Pre-op infection control 25 mg/kg IV
Atipamezole	Covetrus	82124	Sedation Reversal Agent 0.2 mg/kg
Atropine	Covetrus	74760	Bradycardia 0.04mg/kg
Bupernorphine Extended Release (ER)	Wedgewood Pharamcy		Analgesia 0.18 mg/kg IV
Carprofen - intraoperative	Dechra		Analgesia 25 mg/kg IV
Carprofen - post-operative	Dechra		Analgesia 25-50mg PO PRN
Cerenia	Zoetis		Anti-nausea 1-2 mg/kg IV once intraoperatively
Isoflurane	Dechra		Sedation
Lidocaine 2%	VetOne	V1 510212	Analgesia 2 mg/kg SQ prior to incision
Ketamine	Dechra		Anesthsia & Analgesia 12 mg/kg IM
Dobutamine	Hospira		Dosing and titration per anesthesiologist
Povidone-iodine (Betadine)	ValleyVet	12265	Antiseptic
Xylazine (Rompun)	Dechra	GW l400000	Sedation/Analgesia 2.2 mg/kg IM
Surgical Supplies
3-0 PDS (polydioxanone) Suture	Ethicon	Cat#Z316H	Anastamosis Quantity - 2
3-0 Silk Suture (Pop-off)	Ethicon	Cat#1679H	Anastamosis Quantity - 1 pack of pop off
0-0 PDS (polydioxanone) Suture	Ethicon	Cat#Z990G	Linea Alba closure Quantity - 1
3-0 Prolene Suture	Ethicon	Cat#8684G	Skin Closure Quantity - 1
Bovie Electrocautery	Grayline Medical Supply	Cat#E2516	Homeostasis and cutting
Standard set of sterile abomdinal surgical instruments with abdominal wall retractors like Army/Navy's or Richardson reactors.
3M Tegaderm + Pad	3M	Cat#3593	Surgical Dressing
High protein liquid diet	Ensure		Diet 1.5 bottles BID
Software
BioRender	Agreement number SH28Z3OOKI		Figure generation
Adobe Illustrator	Adobe	v29.8.1	Figure generation
FIJI	NIH		Figure generation
*Note: AI tools were used only for minor language and grammar edits
*BID - two times a day,  IV - intravenous, IM - intramuscular, PO - per os/ by mouth, SQ - subcutaneous, PRN - as needed