This experimental protocol was approved by the Animal Care and Use Committee of the Terumo Corporation. Thirty male Sprague-Dawley rats (7 weeks old, weighing between 240-270 g) were used. They were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). They were housed in plastic cages for 10 days before surgery under standard laboratory conditions (temperature, 20-25°C; humidity, 40-60%; and 12-hour lighting cycle). They had ad libitum access to a standard laboratory diet and water.
2.2 Study groups
Three study groups were included: control group (n = 10), where no treatment was applied to the hepatic resection surface and surrounding organs (hepatic resection only).; AdSpray group (n = 10), where AdSpray was applied at a volume of 1 mL to a 4 × 5 cm area of the hepatic resection surface and surrounding organs; and a Seprafilm group (n = 10), where a 4 × 5 cm piece of Seprafilm was applied to the hepatic resection surface and surrounding organs.
2.3 Surgical procedure
The same surgeon (M.K.) performed all operations, and T.A. assisted with the operations. Rats were anesthetized with 3% isoflurane. Enrofloxacin (5 mg/kg) was administered subcutaneously for the prevention of intraoperative infections. The abdomens of the rats were shaved, disinfected, and opened by a 5-cm midline incision. The left lateral liver lobe was taken out of the abdominal cavity and resected transversely using surgical scissors to remove the end of the lobe, with an approximate width of 3.5 cm. Hemostasis of the hepatic resection surface was achieved using gauze and electrocautery. After hemostasis was achieved, the rats were randomly assigned to three groups.
For abdominal closure, the abdominal wall was sutured with 4-0 nylon sutures, and the skin was sutured using clips. The sutured area of the skin was disinfected, and the rats were returned to the housing cages. When the animals woke up, buprenorphine (0.01 mg/kg) was administered subcutaneously for pain relief. After surgery, all animals were administered an antibiotic (5 mg/kg of enrofloxacin, subcutaneously every 12 h) and an analgesic (0.01 mg/kg of buprenorphine, subcutaneously every 12 h) for 2 days.
2.4 Evaluation of adhesion prevention
At 7 days post-surgery, the animals were euthanized by exsanguination, and necropsies were performed. After confirmation of death, a U-shaped abdominal incision was made from both costal margins, and macroscopic adhesion assessment was performed. Adhesion assessment was performed by M.T. and T.A. in a blinded manner. The ratio of the length of the adhesion sites to the length of the resected site was calculated (Fig. 1A).
2.5 Histopathological examination
The left lateral lobe was removed without adhesiolysis and fixed in 10% neutral-buffered formalin. The left lateral lobe was divided into five parts including the resected region. The tissues were embedded in paraffin and cut into thin sections (4 μm), which were hematoxylin-eosin stained and then stained with Sirius Red for the identification of collagen fibers (fibrous tissue). Mesothelial cells were immunohistochemically stained with monoclonal mouse anti-human mesothelial cell antibodies (1:50, clone HBME-1, DAKO). After reacting with a biotin-conjugated secondary antibody, the cells were treated with horse radish peroxidase-conjugated streptavidin, followed by the addition of diaminobenzidine for color development and counterstaining with hematoxylin.
The specimens were observed microscopically by M.T and H.H. in a blinded manner. The degree of necrosis, inflammation, adherence of foam cells to the resection surface, coverage of the resection surface by a fibrous layer, regeneration of mesothelial cells, and vascular proliferation were evaluated based on grade assessment (0, nil; 1, slight; 2, mild; 3, moderate; and 4, marked). Hematoxylin-eosin stained specimens were evaluated about the necrotic area of the resection surface (necrosis) and the number of inflammatory cells (inflammation), vascular (vascular proliferation) and foam cells (adherence of foam cells to the resection surface). Sirius Red stained specimens were evaluated about the thickness of fibrous layer (coverage of the resection surface by a fibrous layer). HBME-1 stained specimens were evaluated about the coverage area and maturity of mesothelial cells (regeneration of mesothelial cells). The fibrous layer on the resection surface was graded in specimens without adhesions. In addition, the regeneration of mesothelial cells was also graded in specimens without adhesions as no mesothelial regeneration occurred in areas of adhesion.
2.6 Statistical analyses
The incidence of adhesions was evaluated using the chi-squared test (with continuity correction). The extent of adhesion was evaluated using a Steel-Dwass multiple comparison test. Grades for necrosis, inflammation, adherence of foam cells to the resection surface, and vascular proliferation were evaluated using a Steel-Dwass multiple comparison test. Coverage of the resection surface by a fibrous layer and regeneration of mesothelial cells were compared between the AdSpray and Seprafilm groups by using the Wilcoxon paired comparison test. P-values <0.05 were considered statistically significant. Statistical analyses were performed using EXSUS Ver. 7.7.1 in an SAS system (release 9.1).