Five rats were selected as donors and used for PRP preparation. Blood samples were obtained by cardiac puncture and 8.5 mL of blood from each donor was drawn into the acid-citrate-dextrose tubes (BD Vacutainer, BD, Franklin Lakes, NJ, USA). The tubes were then centrifuged at 2,500 rpm and 22℃ for 10 minutes. After the first spin, 3 layers were seen as follows; (1) light yellow plasma layer, (2) buffy coat layer, and (3) red blood cells layer (Fig. 1A). Plasma and buffy coat layers were taken carefully and drawn into another tube, then centrifuged at 3,500 rpm and 22℃ for 15 minutes. Finally, 1-mL PRP was harvested and mixed with 1-mL thrombine (Human Thrombin Lyophilized, Baxter, Cherry Hill, NJ, USA) and 0.5-mL calcium chloride in order to obtain the gel form of PRP (Fig. 1B).

Rats were divided equally into 3 groups (Fig. 2): Group 1: Control group; left colon was transected and end-to-end colonic anastomosis was performed, then hyperthermic (42℃) saline were perfused for 60 minutes. Group 2: Oxaliplatin alone group; left colon was transected and end-to-end colonic anastomosis was performed followed by HIPEC with oxaliplatin (77.5 mg/kg) at 42℃ and 5 mg/kg for 60 minutes. Group 3: Oxaliplatin and PRP group; left colon was transected, end-to-end colonic anastomosis was performed and PRP gel was applied to the anastomosis site, then HIPEC was performed with oxaliplatin (77.5 mg/kg) at 42℃ and 5 mg/kg for 60 minutes.

Ketamine hydrochloride (Ketalar, Park Devis, Istanbul, Turkey) at a dose of 50 mg/kg, and xylazine (Rompun, Bayer, Istanbul, Turkey) at a dose of 5 mg/kg were used for anesthesia. After skin shaving and skin preparation with 10% povidone iodine, a laparotomy was performed and the left colon was identified and transected. End-to-end anastomosis was performed with 5/0 polypropylene sutures (Prolene, Ethicon, Cincinnati, OH, USA). In group 3, PRP gel was applied all around the anastomosis line (Fig. 3). Inflow and outflow catheters were placed to the right supradiaphragmatic space and pelvis, respectively, then the abdomen was closed with separate 3/0 silk sutures. After closure of the abdomen oxaliplatin in 5% dextrose solution was perfused for 60 minutes by a roller pump in groups 2 and 3. The perfusate was heated by a heater plate and the temperature of the abdomen was monitored continuously with a digital thermometer. The animals were taken back to their cages and fed chow and water after the operation.

On the seventh day following surgery, the animals were reoperated on and both abdomen and colonic anastomosis were evaluated. The colon segment involving the anastomosis was removed and 2 rubber catheters were placed into the proximal and distal lumen of the excised segment. The catheters were fixed with 3/0 silk sutures and catheters were connected to a syringe pump and a manual sphygmomanometer to measure the anastomotic bursting pressure (ABP). The colon segment was insufflated with air by syringe pump and when the first air bubble was detected, the pressure value was recorded as ABP. After recording ABP, the colon segment was opened and divided into 2 pieces, one piece was to be used for histopathological analysis and therefore put into 10% formalin solution, and the other was stored at −20℃ for hydroxyproline assay. The rats were euthanized with a high dose of anesthetic agent.

All materials were fixed in formalin and embedded in paraffin. Haematoxylin-eosin stained sections were evaluated by one pathologist without knowledge of the group. An Olympus (BX51) light microscope was used. Verhofstad scale was used to evaluate the anastomotic healing by scoring the degrees of necrosis, neutrophils, macrophages, lymphocytes, edema, and mucosal epithelium [15].

Tissues were homogenized with liquid nitrogen and were soaked in a phosphate buffer solution (10 mM; pH, 7.2). Tissue samples were sonicated for 10 minutes at +4℃ with 220 V (METU electromechanical, Serial No. 30607, Berlin, Germany) before they were stored at −80℃ until assay. The homogenates, which were defrosted at room temperature, were later centrifuged at +4℃ for 5 minutes with 15,000 g. The supernatants were taken for hydroxyproline analysis. The concentrations of hydroxyproline in the sample were measured using a commercially available enzyme-linked immunosorbent Hydroxyproline Assay Kit (CEA621Ge, Cat. No.l180307228; Cloud-Clone Corp., TX, USA). This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to hydroxyproline was precoated onto a microplate. A competitive inhibition reaction was launched between biotin labeled hydroxyproline and unlabeled hydroxyproline (standards or samples) with the precoated antibody specific to hydroxyproline. After incubation the unbound conjugate was washed off. Next, avidin conjugated to horseradish peroxidase (HRP) was added to each microplate and incubated. The amount of bound HRP conjugate was in reverse proportion to the concentration of hydroxyproline in the sample. After addition of the substrate solution, the intensity of color developed was in reverse proportion to the concentration of hydroxyproline in the sample. The protein amount of each sample of the tissue homogenates was calculated with the Lowry method [16]. Tissue hydroxyproline values were expressed as ng per mg protein. All assays were conducted according to the manufacturer's instructions. The samples, which showed higher concentrations, were diluted and measured in duplicate.

Sample size was calculated by the statistical package (G^*Power software) from previous experimental rat studies data on intestinal anastomosis healing with a power of 80% α-error and 5% β-error, and it is estimated that at least 10 samples were needed for each group. Numerical data were presented as mean ± standard deviation. One-way analysis of variance test was used for analyzing the differences between the mean values of the parameters (ABP, tissue hydroxyproline level, and histological scores) and either Tukey honestly significant difference or Tamhane post hoc tests were used to analyze the differences between the groups. A P-value below 0.05 was considered statistically significant.

The ABP values of the groups are given in Table 1. In variant analysis, as expected, group 1 had higher ABP value than group 2 and 3 (P < 0.001 and P < 0.001, respectively). Group 2 had lower ABP than group 3 and the difference was significant (P < 0.001). These findings indicate that PRP gel application has a positive effect on ABP.

Table 1 shows the hydroxyproline levels of the groups. Group 2 had significantly lower hydroxyproline levels than groups 1 and 3 (P < 0.001). The difference between groups 1 and 3 was significant (P < 0.001). These data show that PRP application improved wound healing.

Verhofstad scale is a good guideline for evaluating anastomotic wound healing and is widely used in experimental studies [13151718]. In our study, histopathological analysis demonstrated that PRP application improved the anastomotic healing on a cellular basis by decreasing neutrophils and lymphocytes amounts, as well as degree of edema. Also, submucosal bridging was significantly better in group 1 and group 3 than in group 2 (Fig. 4) Detailed data analysis is shown in Tables 2 and 3.