Background

Abdominal surgeries are lifesaving procedures. However, they can lead to post-surgical peritoneal adhesions, a fibrotic complication that arises from any insults within the peritoneal cavity. The peritoneal cavity and its organs are lined by the peritoneum, comprising a protective monolayer of mesothelial cells and subjacent connective tissue. Normally, the peritoneum provides a frictionless surface to ensure that intra-abdominal organs, such as the intestines, can move freely. However, the movement of organs is compromised in patients suffering from peritoneal adhesions. These are intra-abdominal scar bands forming between the abdominal wall and abdominal organs (Ellis et al., 1999; Hellebrekers and Kooistra, 2011; Zwicky et al., 2021). Peritoneal adhesions lead to severe adhesion-related complications, such as small bowel obstruction, chronic pelvic pain, and secondary infertility in women (Hellebrekers and Kooistra, 2011; Zwicky et al., 2021). This poses a major health burden for patients and challenges our health care systems (van Goor, 2007; ten Broek et al., 2013). Adhesion-related complications in the US health care system alone cost several billion dollars per year to treat (Sikirica et al., 2011). Currently, the only approved treatment for adhesions is the use of so-called anti-adhesive barriers. These are biocompatible barrier materials that can be inserted after abdominal surgery resulting in a slight reduction of adhesions. However, the beneficial effect of anti-adhesive barriers for patients is limited and does not rely on a specific molecular mechanism; thus, their regular use in daily practice is not supported (ten Broek et al., 2014; Huang and Ding, 2019; Strik et al., 2019; Fatehi Hassanabad et al., 2021). As such, it is essential to investigate the underlying molecular mechanisms of adhesion formation to develop specific prevention and treatment options in the future.

The current paradigm of adhesion formation states that peritoneal injury induces inflammation and coagulation, resulting in fibrin deposition (Hellebrekers and Kooistra, 2011). More recent results suggest that fibrin deposition may be accompanied by an aggregation reaction of GATA6⁺ peritoneal macrophages (Zindel et al., 2021b). The resulting clot of fibrin and macrophages is proposed to transform into stable adhesions by the process of extracellular matrix deposition by myofibroblasts (Fischer et al., 2020; Sandoval et al., 2016; Zindel et al., 2021a; Zwicky et al., 2021). This paradigm is largely based on results gained from research in rodents using surgical injury models. The most commonly used models have been described and reviewed elsewhere (Oncel et al., 2005; Whang et al., 2011; Kraemer et al., 2014; Bianchi et al., 2016; Sandoval et al., 2016; Tsai et al., 2018; Fischer et al., 2020; Zindel et al., 2021b). In mice, the predominant adhesion model includes the creation of a sterile, ischemic, button-shaped injury of the peritoneum. This model has been referred to as the peritoneal button (PB) model. Other models include the induction of peritoneal injury by abrasion, diathermia, or by introducing sterile foreign material. In summary, all these models rely on sterile peritoneal injuries to induce and study adhesion formation. However, peritoneal insult in clinical abdominal surgeries differs from those standardized, sterile mouse models. Most importantly, surgical trauma to the peritoneum may be accompanied by an acute microbial contamination. This frequently happens when small amounts of intestinal microbes are spilled during the resection and reconstruction phases of abdominal surgery procedures. In fact, a clinical study has linked bacterial peritonitis to an increased risk of subsequent admission due to post-surgical adhesions (Parker et al., 2005).

In a recent paper, we tested the hypothesis that sterile peritoneal injury and microbial contamination independently trigger adhesion formation (Zindel et al., 2021a). First, we confirmed that the PB model of sterile injury (Zindel et al., 2021b) reproducibly led to peritoneal adhesions. Interestingly, a similar amount of adhesions was induced using a modified cecal ligation and puncture (CLP) model. Unlike the standard CLP, which is widespread in sepsis research (Hubbard et al., 2005; Rittirsch et al., 2009; Dejager et al., 2011), in the modified CLP only a small part of the cecum was ligated. This decreased the amount of microbial contamination resulting in a non-lethal septic insult to the peritoneal compartment, which reproducibly led to the formation of peritoneal adhesions. Importantly, the combination of PB and CLP resulted in significantly more adhesions than each procedure alone, indicating that sterile injury and microbes synergistically trigger events that lead to adhesion formation. Surgical induction of bacterial contamination was further found to be interchangeable with administration of native or heat-inactivated cecal slurry (CS) (Starr et al., 2014) during laparotomy, resulting in comparable adhesion scores. In summary, we found that sterile injury and microbial contamination independently and synergistically cause peritoneal adhesions.

Here, we describe in detail the surgical procedures used in our previous publication (Zindel et al., 2021a). We show how adhesions can be caused by a modular system of surgically induced insults. These modules comprise peritoneal injury (PB), microbial contamination (CLP or CS), or a combination thereof (Figure 1A–C). We further describe how adhesions can be quantified using a clinical scoring system (Figure 1D, Table 1). Compared to preexisting methodologies of adhesion mouse models, the presented protocol is extended by the factor of intraperitoneal microbial contamination, an important driver of post-surgical adhesions (Zindel et al., 2021a). Apart from the use in post-surgical adhesion studies, we suggest that adapted forms of the presented modular mouse system could be applied in other research areas, such as abdominal infection or chronic pelvic pain research.

Table 1. Peritoneal adhesion index (PAI)

Figure 1. Overview of the proposed modular surgery model system for peritoneal adhesion induction in mice. A. Localized sterile surgical trauma is induced by generating one peritoneal button (PB) per abdominal quadrant. B. Polymicrobial contamination is surgically induced by cecal ligation and puncture (CLP). C. Polymicrobial contamination is alternatively induced by peritoneal administration of cecal slurry (CS). Combinations of the modular components PB (A), CLP (B), and CS (C) are used to synergistically induce adhesion formation in the mouse peritoneal cavity. D. Within seven days post-surgery, peritoneal adhesion severity is scored at six different locations for tenacity and vascularization (Table 1). These locations are: the four PB (1–4), the midline incision (5), and adhesions that occur between intestines (6). At each scoring location, peritoneal adhesions are assigned a grade between 0 (minimum) and 4 (maximum) according to the criteria (Table 1). The sum of the six scores yields the total peritoneal adhesion index (PAI).