In this study, 6-week-old male Sprague-Dawley (SD) rats purchased from Koatech Inc., Republic of Korea, weighing 260–280 g were used. The SD rats were kept at the Animal Experiment Center of Hanyang University in a specific pathogen-free environment. Temperatures were kept at 23–25℃ and humidity was maintained at 50–60% with an artificial light/dark cycle of 12:12 h. The animal research protocol was approved by the Institutional Animal Care and Use Committee of Hanyang University (HY-IACUC-20-0003), and animal studies were conducted in accordance with the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guidelines .
Establishment of the abdominal adhesion model
The abdominal adhesion model was induced in rats using procedures based on previously described methods . The rats anesthetized with a combination of rompun (10 mg/kg, Bayer, Seoul, Korea) and zoletil 50 (30 mg/kg, Virbac SA, Carros, France). All rats underwent ventrotomy through a 3 cm skin incision in the midline of the abdomen. Three ischemic buttons were prepared within the parietal peritoneum by gripping a 5 mm parietal peritoneum button with a mosquito hemostat and connecting the bottom of the segment with a 4-O black silk suture (AILEE Co., Pusan, Korea) as described previously . The rats were divided randomly into three groups. Rats in the control group received no medication (n = 8). Rats in the pluronic gel group (n = 8) received only Pluronic F127 gel on the ischemic buttons, and rats in the G-CSF group (n = 8) received a mixture of Pluronic F127 gel and G-CSF (60 µg, Dong-A, Seoul, Korea) on the ischemic buttons (Fig. 1A). The incision was closed with a 3-O polyglycolic acid suture (AILEE Co.) for the fascia and a continuous 5-O polyglycolic acid suture (AILEE Co.) for the skin. After two weeks, the euthanasia of rats induced by carbon dioxide (CO2) inhalation to obtain samples for molecular analysis and to evaluate and score the adhesion.
Scoring for adhesion
At 2 weeks after surgery, all rats were assessed according to the standard adhesion scoring system [12–14]. The percentage of adhesions was calculated as the number of adhesions in a rat. The adhesion severity scale was characterized as follows: grade 0 = no adhesions, grade 1 = avascular or thin adhesion, grade 2 = dense or vascularized adhesion, and grade 3 = firm or cohesive attachment. The adhesion density was characterized as follows: grade 0 = no adhesions, grade 1 = adhesion was isolated from tissue without gentle traction, grade 2 = adhesion was isolated with mild traction, and grade 3 = adhesion required dissection as described previously . All rats were evaluated by two observers who were blinded to the animal groups.
RNA isolation and real-time PCR
RNA of the adhesion tissues was extracted by QiAzol Lysis reagent (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to test the concentration of each RNA sample. RNA was then reverse-transcribed with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. For quantitative real-time polymerase chain reaction (qRT-PCR) analysis, the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics, Indianapolis, IN, USA) and LightCycler 480 program (Roche) were used. The genes selected were substance P (SP), neurokinin 1 receptor (NK-1R), transforming growth factor-β1 (TGF-β1), and intracellular adhesion molecule-1 (ICAM-1). Then, qRT-PCR was performed using the LightCycler 480 program (Roche), under the following condition: amplification for 10 min at 95℃; 45 cycles of 95 ℃, 60 ℃, 72 ℃ for 10 s each; and dissociation for 15 s at 65 ℃. The crossing point (CP) value was calculated by the LightCycler 480 program (Roche), and the relative rate of change was calculated using the mRNA ratio of the target gene to that of glyceraldehyde-3-phosphate dehydrogenase.
Statistical analyses were conducted using the Statistical Package for the Social Sciences 24.0 software (IBM Co., Armonk, NY, USA). All data are expressed as the mean ± standard error (SE). The statistical difference between groups was analyzed using one-way analysis of variance for multiple comparisons, and post hoc multiple comparisons were performed with Tukey’s test (equal variances assumed). P-values less than 0.05 were considered statistically significant.