Introduction

It has been proven that intraperitoneal administration of chemotherapeutic agents destroys possible micrometastases, as well as cancer cells, that disseminate intraoperatively, and which can cause local recurrence, peritoneal carcinomatosis or liver metastases. Nevertheless, the intraperitoneal administration of agents can impair the healing process of the anastomoses [1–5].

Aim of this study was to assess the effects of the intraperitoneal administration of combined 5-fluorouracil with irinotecan on the healing process of large bowel anastomoses in rats.

Materials and methods

Sixty male Wistar rats were used for this study. All principles of laboratory animal care were followed. The animals were weighed on the day of the operation and before sacrifice. During surgery, a 1-cm colonic segment was resected 10 cm distally of the ileocecal valve. After the resection, an end-to-end anastomosis was performed using 8 interrupted sutures (6/0 polypropylene).

Before the operation, the rats were randomized in four groups. The drug dosages and frequency of administration were guided by internationally accepted treatment regimens of these drugs. Group 1 (control group): The rats received intraperitoneal normal saline (3 ml) intraoperatively, as well as on the first post-operative day. Group 2 (5-FU group): The rats received intraperitoneal 5-FU (20 mg/kg BW) intraoperatively, as well as on the first post-operative day. Group 3 (irinotecan group): The rats received intraperitoneal irinotecan (3 mg/kg BW) intraoperatively. Group 4 (combination group): The rats received intraperitoneal 5-FU (20 mg/kg BW) and irinotecan (3 mg/kg BW) intraoperatively. On the first post-operative day, they received intraperitoneal 5-FU (20 mg/kg BW).

Sacrifice and macroscopic assessment

All animals were killed on the eighth post-operative day, using intraperitoneal pentothal (300 mg/kg BW). Immediately after the killing, the anastomosis was assessed macroscopically for the presence or not of an anastomotic leak. The presence of abscesses at the anastomotic site and the extent of adhesions were also recorded. All observations were performed in a blind fashion by two examiners. The extent of adhesions was assessed using the scale of van der Hamm et al. [1] as follows: 0 = no adhesions, 1 = minimal adhesions, e.g. between anastomosis and the omentum, 3 = moderate adhesions, e.g. between anastomosis and a small bowel loop, 4 = extensive adhesions with the formation of perianastomotic abscess.

Bursting pressure and histological assessment

After sacrifice, a 5-cm segment of the transverse colon with the anastomosis at the centre was carefully resected along with the adhesions that had been formed. After this bowel segment was washed, the bursting pressure (meaning the pressure at which any leakage of saline or gross rupture of the bowel was noted) was measured, using a three-way catheter. Following the bursting pressure measurement, a 1-cm segment of the bowel with the anastomosis in the middle was resected. That segment was incised longitudinally and divided into two equal parts. The first part was placed in 4% formalin solution and was sent for histological evaluation, using the scale of Ehrlich and Hunt from 0 to 4, as modified by Philips et al. [2]. The grading was as follows: 0 = no evidence, 1 = occasional evidence, 2 = light scattering, 3 = abundant evidence and 4 = confluent cells or fibres. The histological assessment included inflammatory cell infiltration (white blood cell count), neoangiogenesis (new blood vessel development), fibroblast activity and collagen deposition. All histological assessments were carried out in a blind fashion by an experienced histopathologist. The second segment was used for the measurement of hydroxyproline. The process of the hydroxyproline measurement is described in relevant literature.

Statistical analysis

Continuous variables were summarized using means with 95% confidence intervals and medians with ranges from minimum to maximum. Plots were used for the graphical presentation of the results. For the analysis of the anastomotic leaks, the Fisher’s exact test and the χ2 test were employed. Differences amongst groups, with respect to non-normally distributed parameters, were tested using the Kruskal–Wallis test, whereas pairwise differences were compared using the Mann–Whitney test, at a Bonferroni-adjusted significance level. All reported P values were two-tailed with P < 0.05 considered as significant. The Bonferroni correction used with the Mann–Whitney test increased the statistical threshold at a P value of 0.008. All analyses were conducted using SPSS 12.0.

Results

None of the animals died before the 8th post-operative day. The anastomotic leak frequency is presented in Fig. 1 (P = 0.111).

Fig. 1
figure 1

Anastomotic leak frequency in the four groups. Fisher’s exact test: P = 0.111. χ2 test: P = 0.128

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Adhesion formation

Group 1 was the only group with anastomoses free of adhesions (33%). Neither did we observe any perianastomotic abscesses in the control group. The adhesion scores of all groups are presented in Table 1.

Table 1 Degree of adhesion formation in all groups according to the van der Hamm scale
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Bursting pressure

We observed statistically significant differences in the bursting pressures of the four groups (Kruskal–Wallis, P < 0.001). The lowest mean bursting pressure was observed in group 4 (110.2 mmHg) and the highest in the control group (270.87 mmHg). The bursting pressures of the anastomoses that leaked or dehisced before killing were valued as zero. These anastomoses were not included in the comparisons and the statistical calculations. The descriptive values of the bursting pressures in the four groups are presented in Fig. 2.

Fig. 2
figure 2

Bursting pressures (mmHg) in all four groups. Kruskal–Wallis test: P < 0.001. Pairwise group comparison using the Mann–Whitney test with the Bonferroni correction: 1 versus 2 (P < 0.001), 1 versus 3 (P < 0.001), 1 versus 4 (P < 0.001), 2 versus 3 (P > 0.200), 2 versus 4 (P = 0.003), 3 versus 4 (P = 0.003)

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Histological assessment/hydroxyproline

Signs of intestinal damage such as mucositis were not reported by the histopathologist during the anastomotic assessment. The control group showed the lowest mean inflammatory score (1.2) and the highest mean fibroblast activity and mean collagen deposition scores (3.47 and 2.87, respectively), as well as significantly increased hydroxyproline concentrations (P < 0.001). Table 2 presents descriptive values of all four histological parameters and the hydroxyproline concentration levels with their statistical comparison results.

Table 2 Descriptive values of histological parameters and hydroxyproline (scale of Erlich and Hunt, modified by Philips)
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Discussion

Peritoneal carcinomatosis is not uncommon in patients with colorectal cancer and has been reported to have a poor prognosis in the past. Since 1990, studies on cytoreductive surgery followed by early post-operative intraperitoneal chemotherapy (EPIC) or hyperthermic intraperitoneal chemotherapy (HIPEC) have prompted new treatment options for selected patients with advanced colorectal cancer [4–6]. The immediate post-operative, intraperitoneal administration of cytostatic drugs achieves high concentrations of these drugs in the peritoneum as well as in the liver, avoiding the side effects of systemic administration. Intraperitoneal chemotherapy has been associated with marked toxicity in the form of lethargy, weight loss and bacterial translocation [7]. Extensive surgical resection of macroscopic tumour is often followed by the construction of multiple bowel anastomoses. Immediate intraperitoneal chemotherapy puts the intestinal healing under increased risk. Various surgical complications have been described following intraperitoneal chemotherapy, the commonest being anastomotic leakage. Leakage rates up to 20% have been reported depending on anastomotic site, resection size and individual risks [7–10].

The combination of 5-FU with newer chemotherapeutic agents seems to improve the patient response rate to chemotherapy and thus results to an even better survival rate. The anastomotic dehiscence rate is possibly the most important indicator of anastomotic healing failure. Zacharakis et al. [9] observed anastomotic dehiscence in 30% of rats under 5-FU treatment, compared to 0% in the control group. The negative effect of 5-FU on the anastomotic bursting pressure has been shown by Kanellos et al. [10]. They have found significant bursting pressure decreases on anastomoses under the intraperitoneal influence of 5-FU and Leucovorin. In our experiment, the presence of intraperitoneal irinotecan or 5-FU significantly decreased the mechanical strength of the anastomoses, whereas the bursting pressures were further decreased when the two agents were combined (group 4 vs. control, P < 0.001).

The microscopic assessment of the anastomotic healing usually finds place by assessing histological parameters, such as inflammatory reaction, neoangiogenesis and collagen formation. It is known that chemotherapy agents affect the healing process by increasing the inflammatory reaction at the wound site. 5-FU has been found to increase inflammatory reaction at the anastomotic site. Furthermore, 5-FU decreases the fibroblast reproduction rate and their concentration [10–12]. Our study has shown 5-FU to negatively affect the anastomotic healing by increasing the inflammatory reaction, reducing neocollagen and fibroblasts and minimally reducing neoangiogenesis at the anastomotic site. Irinotecan had similar effects. It significantly increased the inflammatory reaction (P < 0.001), and it significantly reduced the neocollagen formation (P < 0.001), the fibroblast concentration (P < 0.001) and the hydroxyproline concentration (P < 0.001) when compared to the control group. The combined effects of the two agents were once again more detrimental on the anastomotic healing compared to when the agents were used as monotherapy. Inflammatory reaction in group 4 (5-FU and irinotecan) was significantly increased compared to all other groups. Neoangiogenesis was increased, but no significant results were found. The collagen formation, fibroblast concentration and hydroxyproline concentration were reduced compared to all groups, whereas the comparison with the control group was statistically significant.

In conclusion, the immediate, post-operative intraperitoneal administration of 5-fluorouracil or irinotecan had a negative effect on the healing process of the large bowel anastomoses in rats, with statistically significant results. Nevertheless, the negative effects of the combination of these two agents were statistically more significant compared to the single use of 5-FU or irinotecan.