302 Chapter 13 Study Outcomes The primary study outcome was macroscopic scores including anastomotic adhesion and leakage scores. Secondary outcomes were bursting pressure, histological evaluation of the anastomosis (inflammation, fibroblast activity, neoangiogenesis and oedema) and changes in body weight. Macroscopic Evaluation After euthanasia of the animal, adhesions to the anastomotic site were assessed according to the method described by van der Ham et al. 21, in which the following classification was used: (0) no adhesions; (1) minimal adhesions, mainly between the anastomosis and omentum; (2) moderate adhesions, i.e., between omentum and the anastomotic site and between the anastomosis and a loop of the small bowel; and (3) severe and extensive adhesions, including abscess formation. The adhesion score was calculated per group by summing the scores per animal. Subsequently, AL was scored using a four-score system 22. The latter is categorized as (1) no AL, (2) small abscess < 1 cm3 at the anastomotic site, (3) large abscess of >1 cm3 at the anastomotic site and (4) complete dehiscence with (fecal) peritonitis. Bursting Pressure Anastomotic strength was assessed by measuring the bursting pressure (Supplementary Figure S2), based on previously described methods 22, 23. In short, a 4 cm segment of the colon including the anastomosis was resected en bloc, without removal of adherend adhesions to prevent iatrogenic damage. A plastic tube was inserted in the proximal end and ligated with polypropylene 6-0 suture (Prolene 6-0, Ethicon, Inc., Johnson & Johnson). The part distal of the anastomosis was clamped. The resected colon segment was immersed in water, while air was infused using a balloon connected to a manometer (Digitron, part of Rototherm Group). The pressure (mBar) was manually increased by pumping up the balloon and inflating the colon. Bursting pressure was defined as the intraluminal pressure at which air leakage was initially observed from the anastomosis. Tissue Preparation and Histological Evaluation After measuring the bursting pressure, the colon tissue including the anastomosis was placed in formalin. All samples were paraffinized within one week. From each formalin-fixed, paraffin-embedded (FFPE) tissue specimen, a 5 μm section was cut and stained with standard hematoxylin-eosin (H&E). Infiltration of inflammatory cells, fibroblast activity, oedema and neoangiogenesis at the anastomotic site were assessed by an experienced animal pathologist (MG). Inflammatory parameters were scored based on the modified 0-to-4 Ehrlich and Hunt numerical scale: 0 = no evidence, 1 = occasional evidence, 2 = light scattering, 3 = abundant evidence and 4 = confluent cells or fibers 24, 25. All other characteristics were score on a 0-to-3 scale, meaning 0 = no evidence, 0.5 = minimal, 1 = mild, 2 = moderate and 3 = severe. Additionally, all other abdominal organs were collected as well and placed in formalin in case of need for further histological examination.
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