Development and validation of a mouse model to investigate post surgical pain after laparotomy

Abstract

Background: Postoperative pain following abdominal surgery is a significant obstacle to patient recovery, often necessitating high analgesic doses associated with adverse effects like cognitive impairment and cardiorespiratory depression. Reliable animal models are crucial for understanding the pathophysiology of post surgical pain and developing more effective pain-relieving strategies.

Methods: We developed a mouse model to replicate peritoneal trauma induced by abdominal surgery. 30 C57BL/6 mice underwent laparotomy, with half undergoing standardised peritoneal abrasion and the rest serving as controls. Mouse recovery was assessed using two validated scoring systems of surgical recovery: Post surgery Severity Assessment (PSSA) and Mouse Grimace Score (MGS). Blood samples were taken for cytokine analysis. Adhesions were evaluated on day 6, and peritoneal tissue was examined for healing markers.

Results: After laparotomy, all mice exhibited expected pain profiles. Mice with peritoneal abrasion had significantly higher PSSA (7.2 ± 1.2 vs 4.68 ± 0.82, p ≤ 0.001) and MGS scores (3.62 ± 0.74 vs 0.82 ± 0.40, p ≤ 0.05) with slower recovery. Serum inflammatory cytokine levels were significantly elevated in the abraded group, and adhesion formation was higher in this group. Immunohistochemical analysis showed significantly increased expression of α-SMA, CD31, CD68, and F4/80 in peritoneal tissue in the abraded group.

Discussion: A mouse model involving laparotomy and standardised peritoneal abrasion replicates the expected pathophysiological changes following abdominal surgery. It will be a useful model for better understanding the mechanisms of post surgical pain and developing improved pain-relief strategies. It also has utility for the study of intra-abdominal adhesion formation.

Key message: To understand the intricate relationship between peritoneal trauma-induced pain, cytokine response, and post-operative adhesion formation in mouse models for advancing therapeutic interventions and enhancing post-operative recovery outcomes.

Keywords: Adhesion; Cytokines; Mouse model; Pain; Peritoneal trauma; Post operative recovery.

Fig. 1

Abrasion examples for the mouse model. Animals were subjected to a 1 cm midline laparotomy. Left, peritoneum before abrasion. Right, erythematous reaction in the peritoneum following abrasion with a 100-grit Emery Cloth.

Fig. 2

Post-surgery severity and mouse grimace scores after laparotomy. Comparison of Post surgery Severity Assessment scores (A) and Mouse Grimace Scale scores (B) observed in abraded (black) with unabraded mice (red) following recovery from the intervention. Two-way repeated measures ANOVA was used for statistical analysis, each data point represents the mean score with error bars (± sd). * represents a statistically significant difference, p ≤ 0.05. *** represents a statistically significant difference, p ≤ 0.001.

Fig. 3

Adhesion scores results after schedule 1 of the animals. A) comparison of adhesion scores between the abraded and control mice groups. Each data point represents the mean adhesion score ± SD. ** represents a statistically significant difference using Student's t-test, p ≤ 0.01. B) Representative examples of adhesions in abraded group (left) and the lack of adhesion in the control group (right).

Fig. 4

Cytokine quantification results from serum samples. Quantification (pg/mL) of A) IL-1β, B) IL-2, C) IL-6, D) IL-10, E) IL-12 and F) TNF-α from serum samples taken at days 1, 3, and 5 after surgery. The black line shows cytokine levels in the abraded group, and the red line in the control group. Two-way repeated measures ANOVA was used for statistical analysis, * represents a statistically significant difference, p ≤ 0.05. *** represents a statistically significant difference, p ≤ 0.001.

Fig. 5

Immunohistochemistry results from peritoneal tissue samples. A) Percentage of positive cells expressing α-SMA, CD-31, CD68, and F4/80 from the abraded and control groups. Antibody expression was significantly higher in the abraded group. B) Examples of immunohistochemical staining for α-SMA, CD-31, CD68, and F4/80. Abraded samples showed greater immunoreactivity than control samples. ****, ***, * represents a statistically significant difference using Student's t-test p ≤ 0.0001, p ≤ 0.001, and p ≤ 0.05 respectively.

Appendix Fig. A1

Mouse Grimace Scale provided by the National Centre for the Replacement, Refinement and Reduction of Animals in Research [14].