Second look and assessment of peritubal adhesions

In this study, rather than necropsy, the presence of peritoneal adhesions in specific sites was assessed laparoscopically 30 days post-injury using a standardized classification and quantification methodology that is based on the macroscopic appearance of adhesions and their distribution in different regions of the abdomen (Fig 1B). The sites were classified using an ordinal variable (on a 0 to 3 scale) derived from the Peritoneal Adhesion Index [21], and also received a score (on a 0 to 4 scale) based on the ratio of the area of adhesion to the area of injury [13]. The excised area of the peritoneum (about 8 x 10 cm) was considered the reference area (Fig 3) to determine the adhesion area score (Table 1).

The yellow dotted line represents the peritoneum area that was removed from the lateral wall (approximately 80 cm²). Both (A) and (B) exhibit adhesions that were classified as Peritoneal Adhesion Index raw score 2 (strong adhesions, sharp dissection) according to Coccolini et al., 2013 [21].

PAI (Peritoneal Adhesion Index) is the sum of the raw scores in all regions (Adapted from Coccolini et al., 2013) [21]: 0 No adhesions; 1 Filmy adhesions, blunt dissection; 2 Strong adhesions, sharp dissection; 3 Very strong vascularized adhesions, sharp dissection, damage hardly preventable. Area scores (adhesion area / injured area ratio): 0 (no adhesion); 1 (≤25% of initial injured area); 2 (>25% and <50% of initial injured area); 3 (50% - 75% of initial injured area) or 4 (>75% of initial injured area) [13]. The excised area of the sidewall peritoneum adjacent to each uterine horn was considered as a reference injured area (about 8 x 10 cm). Sites with no adhesions in all ten animals (all scores = 0) were not included in this table. The animals were numbered in order according the time of the first surgery (injury).

After evaluating adhesion scores under laparoscopic view, the distal uterine horn with oviduct (infundibulum, ampulla and isthmus) and ovary on each side were laparoscopically removed using an ultrasonic scalpel in order to minimize bleeding [25]. The harvested specimens were fixed in 10% neutral buffered formalin solution, and then embedded in paraffin, sectioned, and stained with hematoxylin-eosin for evaluation by a single experienced pathologist (L.C.). The histopathological assessment of the distal uterine horns, oviducts and ovaries from the ten 6½ month-old previously injured pigs were compared with those of a control group composed of six healthy 5½ months old pigs with no history of surgery, with a focus on the injury repair response and naturally occurring changes from 5½ to 6½ months.

In order to optimize the use of the animals according to the principles of the 3Rs (replacement, reduction, and refinement), upon completion of the experiment the pigs were used for training in surgical techniques and then euthanized by deep anesthesia followed by intravenous administration of 10 mL of 19.1% potassium chloride. The euthanasia of the animals was in accordance with the “Euthanasia Practice Directive of the National Council for Animal Experimentation Control (CONCEA)” and was also a requirement of the institutional committee on the ethical use of animals.