One hundred and fifty eight-months-old male SD rats (grade SPF), weighing between 250 and 280 g, were obtained from animal facility of Traditional Chinese Medicine University of Guangzhou (Certificate No. 44005800000296). The rats were raised in the clean grade feeding room of the laboratory animal center on the north campus of Sun Yet-sen University (License No. SYXK2012-0081). The room temperature of the feeding room ranged between 18˚C and 25˚C, and the relative humidity was between 45% and 55%. The rats took food and water freely, and the rat food was from the animal facility of Sun Yet-sen University. The pads for the feeding case were changed every two days, and the rats were used for experiment one week later. All the experiments were approved by the Experimental Animal Ethics Committee of Sun Yet-sen University.

Sham operation group: The rats fasted from food for 12 hours but drank water freely before the operation. They were given anesthesia by intraperitoneal injection of 10% chlorine aldehyde at a dose of 0.3 mL/100g body weight, and the abdominal cavity was opened along the middle line after disinfection and depilation; the cecum and ascending colon were freed from mesenterium after full exposure and injury to blood vessels was carefully avoided. Then cecum and ascending colon were placed back to the original position, and the incision was closed layer by layer.

CLP group: The operation was similar to that of the sham operation group, except the cecum was ligated in the middle of the root with 5/0 surgical suture. The perforated puncture at the cecum 1.0 cm distal to ligation was made with 18 G needle, and the blood vessel was avoided. The small amount of intestinal content overflow was forced out by cotton swab pressure to ensure the success of puncture. Then, the cecum was placed back to the original position, and the incision was closed layer by layer [18] .

CASP group: The beginning of the operation was similar to that of the sham operation group. After exposure of the ascending colon, a 18 GA venous indwelling needle (BD, US) was stitched through the antimesenteric wall into the lumen of the ascending colon and then fixed with 7/0 surgical suture placed approximately 10 mm from the ileocecal valve. Consecutively, the inner needle of the stent was removed, and the stent was cut at the prepared site. To ensure proper intraluminal positioning of the stent, the stool was milked from the cecum into the ascending colon and the stent until a small drop of stool appeared. The ascending colon and indwelling needle were placed back to the original position, and the incision was closed layer by layer [19] [20] .

Normal saline solution was injected subcutaneously into each rat at the concentration of 5 mL/100g body weight after the operation, and the rats were placed back in the cages; the body temperature was maintained by electrically heated blanket until they woke from anesthesia. The physical activity of the rats was observed after anesthetic recovery, and the food and water were provided for a free feed. The conditions of the rats were recorded every 12 hours.

One hundred and fifty SD rats were randomly classified into three groups: CLP (n = 50), CASP (n = 50), and sham operation groups (n = 50). Ten rats from each group were randomly selected after the operation as normal feeding and observed for one week to record the time of death. The rest of the rats were analyzed by re-opening the abdomen for direct observation, four rats from each group were randomly selected at the postoperative time of 12 hours, 24 hours, 48 hours, and 72 hours, respectively, under anesthesia by intraperitoneal injection of 10% chlorine aldehyde at a dose of 0.3 mL/100g body weight. Venous blood of 7 to 9 mL was collected under the sterile condition from thoroughly exposed inferior vena cava. Then, the rats were sacrificed by decapitating. The blood samples were used for bacterial culture assessments for inflammatory mediators and oxidative indicators. The ileum tissue of 2 to 3 cm was cut off and placed in Bouin solution (Spectrum Chemicals & Laboratory Products, Shanghai, China) and used for specimen section preparation.

Blood samples: 2 mL blood sample was spread onto Columbia blood agar (Becton Dickinson, Heidelberg, Germany) and incubated at 350˚C to 37˚C overnight; the strains and colonies of the bacterium were assessed by typical bacterial growth method. Another 2 mL blood sample was transferred to Heparin anticoagulant tube and analyzed by NAVO CCX blood gas analyzer (NAVO Biomedical, Waltham, US). The rest of the blood samples were stored at room temperature for two hours before centrifugation at a speed of 3400 rpm for 15 minutes, and then the serums were used for IL-6, TNF-α, MDA, NO, and T-SOD quantitation. The serum levels of IL-6 and TNF-α were measured by ELISA, MDA was measured by thiobarbituric acid (TBA) colorimetry, and NO and T-SOD were measured by the method of nitrate reductase activity determination and xanthine oxidase activity determination, respectively. Kits for IL-6 and TNF-α quantitation were from Ray Biotech (US); and kits for MDA, NO, and T-SOD quantitation were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All the measurements were carried out according to the manufacturer’s instruction.

Ileum tissue samples: The ileum tissue was separated and repeatedly washed with an isotonic saline solution, followed by fixation in Bouin solution for 48 hours. The ileum tissues were cut into pieces of 1 cm and embedded in paraffin for consecutive sections, the specimen was 5 µm thick and was used for HE staining; the pathological morphology was observed under a microscope (Nikon, Tokyo, Japan).

The data are presented as mean ± SD and processed by SPSS 13.0 software. The comparisons among multiple groups were analyzed by ANOVA, and the inter-group comparison was conducted by LSD analysis. Survival analysis was conducted by Kaplan-Meier, and inter-group comparison regarding survival was performed by log-rank analysis. P < 0.05 was considered as statistical significance.

The survival rate of sham operation group within one week was 100%, whereas, it was 40% and 60% for CLP and CASP groups, respectively. There was a significant difference in survival rate between the sham operation and CLP or CASP group (P < 0.05), there was no significant difference between the CLP and CASP groups (P > 0.05) (Figure 1).

・ Abdominal cavity infection

Chills, reduced activity, reduced food consumption, the closing of eyes, curl-

ing up of the body, piloerection, increased eye secretions, oliguria, and yellow urine were commonly observed in the rats from the CLP and CASP groups after operation; rats from the CLP group had even longer lasting symptoms. However, the rats from the sham operation group had none of the above symptoms.

It was observed from abdominal open operation that the swelling, congestion, adhesion, hemorrhage, reduced intestinal peristalsis were found in the rats from the CLP and CASP groups 12 hours after the operation; the aggravated signs were observed 24 and 48 hours after the operation. Moreover, these signs were more persistent in the rats from the CLP group. The rats that survived for 72 hours after operation had alleviated symptoms. Also, the congestion and gangrene at the distal parts of cecum in the CLP group could be found at different time points whereas the rats from the sham operation group had no such manifestation (Figure 2).

・ Venous blood bacterial culture

There was no bacterial colony formed in the venous blood sample from rats in the sham operation group compared to the rats in the CLP and CASP groups, which had bacterial colonies mostly at 24 and 48 hours after the operation. The positive rate of bacterial culture was 100% for CLP group at 24 and 48 hours and CASP at 24 hours. The major strains of the bacterium were Escherichia coli, Streptococcus spp., and bacteroidetes (Table 1).

・ Tissue oxygen metabolism

The blood lactic acid concentration was significantly increased in the CLP at

each time point compared with the sham operation group (P < 0.05). Also, the lactic acid increased considerably in CASP group from 12 hours to 48 hours but was restored at 72 hours post-operation. Compared with CLP group, the blood lactic acid level in CASP group was of no significant difference at 24 hours (P > 0.05) but significantly decreased after postoperative 48 hours (P < 0.05).

In comparison with sham operation group, the central venous oxygen saturation in the CLP and CASP groups was significantly decreased (P < 0.05) though there was no obvious difference between the two groups (P > 0.05) (Table 2).

・ Inflammation mediators

The serum concentration of IL-6 and TNF-α was significantly increased at each time point in the rats from CLP and CASP groups compared with those from the sham operation group (P < 0.05) (Table 2). However, there was no noticeable difference between the two groups (P > 0.05), except that the serum IL-6 was decreased in the CASP group at 72 hours (P < 0.05).

The peak concentrations of IL-6 and TNF-α were observed at 24 hours, and lasted till 48 hours after operation; a significant decline was only observed after 72 hours in the CLP group. The dynamic alteration of IL-6 and TNF-α in the CASP group was similar to that in CLP group (P > 0.05), except that the decline

*Comparison of CLP or CASP with the sham operation group at the same time point, i.e., *P < 0.05 vs. sham; # Comparison between CASP and CLP at the same time point. i.e., #P < 0.05 vs. CLP; Δ intra-group comparison between the time point of 24 h/48 h/72 h and 12 h, i.e., ΔP < 0.05 vs. 12 h; ▲ intra-group comparison between the time point of 48 h/72 h and 24 h, i.e., ▲P < 0.05 vs. 24 h; & intra-group comparison between 72 h and 48 h. i.e., &P < 0.05 vs. 48 h.

at postoperative 72 hours was even more significant (P < 0.05).

The morphology of ileum presented no significant difference between CLP and CASP groups at postoperative 12 hours, manifested as the integrated layers of ileum, with small amount of neutrophils or monocytes infiltration; the neutrophils infiltration was also found in mesangial adipose tissue. The increased neutrophils and monocytes infiltration was observed in each layer of the intestine in the rats from CLP and CASP groups at postoperative 24 hours; the mucosal edema, erosion, submucosal telangiectasia, and congestion were more significant in both groups, but more severe in the CLP group. The mucosal edema and erosion became severe in CLP group at postoperative 48 hours, while the inflammatory cells infiltration and mucosal edema were severe in CASP group; the erosion was not that significant. The mucosal atrophy in CLP and CASP was found 72 hours after the operation, and the inflammatory cells infiltration was still severe in CASP group (Figure 3).

As shown in Figure 4, the serum levels of MDA and NO in CLP and CASP groups were increased at each time point compared with the sham operation group while the T-SOD level was significantly decreased (P < 0.05). Compared with CLP, the serum level of MDA was significantly decreased at 72 hours after operation (P < 0.05), while the serum level of T-SOD was increased at 12 and 72 hours after operation in the CASP group (P < 0.05). Also, the serum level of NO was significantly decreased at all time points (P < 0.05) except at postoperative 12 hours (P > 0.05).