Adhesion formation required the contact, abrasion, and clotted blood
A rat peritoneal strip ex vivo adhesion model detected visible adhesive bands when abraded peritoneal strips were sutured and subsequently cultured in the presence of clotted blood. [8] Here, we described a mouse peritoneal strip ex vivo adhesions model, with modifications made to the previously published experimental conditions and we show that adhesions can be reproducibly created. The key features of this mouse peritoneal strip ex vivo adhesions model were the use of gauze padding in tissue cassettes to prevent the unfolding of tissue strips (rather than sutures as used in the rat model) and the use of a standardised volume of clotted blood. Macroscopic adhesion bands were reproducibly observed after 72 hours. Adhesion formation was confirmed by histological assessment (H&E and PSR) and immunohistochemical staining (αSMA and Cx43).
Contact between the peritoneal surfaces is required for reproducible adhesion development. In pilot studies, we found suturing of the mouse peritoneal strips to be cumbersome, have the propensity to introduce additional grasping damage to the tissue, and the potential to introduce a foreign body reaction. As these factors could introduce variables to the model, we refined the approach to use a tissue histology cassette to prevent the abraded portions from unfolding while in culture. The cassette also helped to keep the strips in place during culture. The folded peritoneal strips were padded with sterile cotton gauze to increase contact between the opposing parietal surfaces. This simulated an approximation of two injured surfaces commonly performed in post-surgical in vivo adhesion experimental models.[11-14] By improving the positional stability, we standardised the location and area of adhesion creation, which resulted in an increase in the baseline incidence and extent of adhesions created [10]. Adhesions were found in all the mouse peritoneal strips cultured in the presence of the gauze, but only 4 out of 7 strips cultured in the without the gauze. The adhesions observed were consistent with “Type 1B adhesions” in humans.[9] This aligns with clinical practices, as minimising contact between two opposing traumatised regions after surgery is an important step, and also justifies the requirement to use clinical barrier agents targeting adhesion formation.[15]
The model may simulate bleeding that occurs during surgery and create tissue trauma from manipulations during surgical procedures through the presence of clotted blood and abraded surfaces, respectively. No adhesion bands were observed when an abrasion injury was absent in either the rat or mouse models, suggesting that this trauma is a key contributor to adhesion formation. Additionally, no adhesions were formed when abraded strips were cultured without clotted blood, suggesting that this is also an essential factor in the development of adhesions. The essential role of blood is in line with current understanding, as the blood facilitates the initial adhesion of two surfaces during early adhesion formation by providing a source of fibrin and immune cells [10, 16]. This observation is also consistent with surgical practices, where it is important to achieve haemostasis to prevent/minimise adhesion development in the patient.[17] Previously, there was no standardisation of the amount of clotted blood placed between the strips in the rat model [8], which introduced potential variations. A standardised volume of 200 μL was used in this study, and larger volumes of clotted blood did not increase the quality of adhesion created (data not shown).
Differences between the rat and mouse ex vivo models included: propensity to form adhesions in sub-optimal conditions, timings of adhesion development and appearance of the adhesions. For example, although no adhesions were detected in our model when mouse peritoneal strips cultured in the absence of clotted blood after 72 hours, the rat model reported adhesions detected as early as 48 hours in strips cultured under the same conditions.[8] This difference suggests that rat tissues may have a higher propensity to form adhesions. The differences observed may be due to interspecies variation in fibrinolytic activity [18, 19, 10]. In the murine strips with abrasion and clotted blood, the adhesive bands were observed after being in culture for 72 hours and the majority of adhesions were thin and filmy, requiring gentle traction to disrupt. In contrast, the adhesive bands described in the rat model, were observed as early as 24 hours and noted to be obvious, “dense and opaque” with appreciable vascularity [8].
Validation of adhesion formation with microscopic assessment
As the majority of the adhesive bands were thin and filmy and lysed upon unfolding of the strip, we preserved the adhesions in their physiological state by processing intact strips and paraffin embedding them en bloc for histological assessment. As it can be challenging to pinpoint the exact location of adhesions microscopically without staining, in each case 5 tissue sections were sampled from 5 positions at 500 μm intervals along the peritoneal strip. The presence of adhesions was confirmed by the observation of eosinophilic matrices between juxtaposing mesothelium and collagen deposition (inferred from the positive staining of PSR in these matrices) [20] Additionally, surrounding mesothelium peripheral to the matrices expressed high levels of αSMA. This may suggest mesothelial-to-mesenchymal transition[21] and sub-mesothelial fibroblast activation to the adhesion phenotype.[5] Notably, regions with no adhesions were negative for these markers. This demonstrates that these expression patterns were unique to abrasion and blood clot treatment.
Interesting, the mesothelial layer (assessed by H&E) was found to thicken over time in the tissues where adhesions formed, similar to that described in a model of post-surgical adhesions [22]. This thickening of mesothelium resembles what is observed in human patients, suggesting our model closely mimics the human condition [23-25]. Conversely, in conditions that did not result in adhesions (conditions 2 and 4; Table 1), the mesothelium remained thin even at the later time points. Considering the discontinuous nature of the mesothelium due to abrasion reduced over time, this suggests that some degree of healing had occurred. However, unlike normal inflammatory events, little or no infiltration of immune cells were observed. This could have limited the magnitude of the adhesion created .[10]
Considerations for future use of this model
Having a mouse model available provides the additional advantage of supporting researchers with investigations using genetically modified transgenic murine strains and widely available commercial immunological reagents and/or assay kits designed for murine systems.
However, due to the larger size of the rat peritoneal cavity it may present a more “cost effective” approach for research, with approximately 6 strips obtained from each rat compared to 3 strips from each mouse. Nevertheless, 3 strips are sufficient for assessment of multiple treatments on tissue from one animal. Furthermore, paired dose-response comparisons and intra-animal paired comparisons for different test articles can be accomplished with multiple strips [8].
A major limitation of any ex vivo model is the lack of blood circulation. One of the early inflammatory responses to the inflicted injury of the peritoneum is characterised with the infiltration of the immune cells [22, 15]. Both resident and infiltrating cells release a cocktail of inflammatory mediators [15]. Many of these mediators have been shown to play a major role in adhesion formation [26]. One of earliest cytokines present in the injured peritoneal cavity is IL-1, which causes human peritoneal mesothelial cells to proliferate [27]. However, due to the nature of the model, only tissue resident immune cells and those that are found in the clotted blood were present. These contribute very little to inflammation in the model.
The incidence of adhesions may be reported when assessing the efficacy of test articles in the mouse model, as an “adhesion-free outcome” is a meaningful result [10]. However, as most adhesions were thin and filmy, this may make the model too “permissive” such that all test articles become effective. On the contrary, the adhesive bands generated in the rat model were described to be dense, opaque, and vascularised. Although the number of abrasions was standardised, it is difficult to optimally apply consistent pressure during scalpel abrasion due to the flaccidity of the strip. This may account for the observed heterogeneity of adhesive bands created across the strip. Making microscopic assessment of adhesions and screening the efficacy of anti-adhesion agents challenging.
Interestingly, we observed that Cx43 protein levels were observed to be higher in regions where adhesions were present using this model (Figure 4e). This finding demonstrates that it is possible to study the development of adhesions, identify molecules of interest and test therapeutics. As far as we are aware this is the first report that Cx43 has a role in adhesion formation.