Establish the small intestinal ischemia-reperfusion injury model in rats and the experimental design

This study proceeded after being reviewed and approved by the Institutional Animal Care and Use Committee in accordance with the ethical principles provided by the Experimental Animal Laboratory of School of Medicine, SUN Yat-sen University. Forty-eight healthy Sprague-Dawley rats (weighing 200–250 g) were randomly divided into four groups. Each of which contained 12 rats, the group I (the ischemia time was 60 min), group II (the ischemia time was 75 min), group III (the ischemia time was 90 min), and group IV (the ischemia time was 120 min). Laboratory temperature was kept at 25–27°C. Surgery was conducted under general anesthesia with intra-peritoneal sodium pentobarbital (45 mg/kg) after they had been fasted for 18 h. The rats' abdomens were opened and their superior mesenteric artery (SMA) were found and clamped for 60, 75, 90 and 120 min respectively. Then the clamp was released and abdominal membrane, muscle and skin were sutured gradually. In addition, 5% Cefoperazone was injected intra-peritoneal to avoid wound infection. Animals were housed individually in wire-bottomed cages, free to eat water and food. The survival rates in each group were observed during the 1^st day to the 7^th day after intestine ischemia/reperfusion.

Based on the above result, One hundred and twenty healthy Sprague-Dawley rats (200–250 g) were randomly divided into five groups. Each of which contained 24 rats, Sham-operated group(group S), model group(group M), Ketotifen treated group(group K), cromolyn sodium treated group(group C) and compound 48/80 treated group(group CP). Intestinal damages were induced by clamping the superior mesenteric artery for 75 minutes based on the above study. Group K, C, and CP were treated with ketotifen (Sigma; USA) 1 mg·kg^-1, CS (ICN; USA)50 mg·kg^-1, and CP (Sigma; USA) 0.75 mg·kg^-1 via caudal vein at 5 min before reperfusion, respectively, while group S and M were treated with the same volume of saline. Then the clamp was released and abdominal membrane, muscle and skin were sutured gradually. In addition, 5% Cefoperazone was injected intra-peritoneal to avoid wound infection. Animals were housed individually in wire-bottomed cages, free to eat water and food. The surviving rats in group K, C, and CP were treated with ketotifen 1 mg·kg^-1, CS 50 mg·kg^-1, and CP 0.75 mg·kg^-1 via caudal vein once daily for 3 days after reperfusion respectively, while group S and M were treated with the same volume saline.

Survival rates

The survival rates in each group were observed during the 1^st day to the 3^rd day after intestine ischemia/reperfusion. The state, action, drinking and eating of each surviving rat was also recorded.

Preparation of specimens and measurements

The surviving rats were sacrificed by anesthetic overdose. 8 rats in each group except only 3 rats in group CP were paunched rapidly on the 3^rd day after reperfusion. 2 mL blood was obtained from the inferior vena cava, frozen at -20°C for 5 minutes and centrifuged for 15 minutes at 4,000 r/min. Supernatants were transferred into fresh tubes for evaluation of concentration of glutamic-oxaloacetic transaminase (AST), glutamic pyruvic transaminase (ALT), the ratio of AST compare ALT (S/L), total protein (TP), albumin (ALB), globulin (GLB), the ratio of ALB compare GLB (A/G), phosphocreatine kinase (CK), lactate dehydrogenase (LDH), urea nitrogen (BUN) and creatinine (CRE) through automatic biochemistry analyzer(abbott, USA).

Intestine histology

A 0.5–1.0 cm intestinal segment was cut 5 cm from the terminal ileum and fixed in 4% formaldehydum polymerisatum, then embedded in paraffin for sectioning. The segment was then stained with hematoxylin-eosin. Damages of intestinal mucosa were evaluated by two different histopathologist according to the criteria of Chiu's method [9]. Criteria of Chiu grading system consists of 5 subdivisions according to the changes of villus and gland of intestinal mucosa: grade 0, normal mucosa; grade 1, development of subepithelial Gruenhagen's space at the tip of villus; grade 2, extension of the space with moderate epithelial lifting; grade 3, massive epithelial lifting with a few denuded villi; grade 4, denuded villi with exposed capillaries; and grade 5, disintegration of the lamina propria, ulceration and hemorrhage.

Transmission Electron Microscopy

Another 0.5 cm intestinal segment cut 5 cm from the terminal ileum were immersed and fixed in 2.5% glutaraldehyde overnight at 4°C and washed three times in PBS. They were then postfixed in aqueous 1% OsO₄ and 1% K₃Fe (CN)₆ for 1 hour. Following three times of PBS washing, the tissue was dehydrated through a graded series of 30 to 100% ethanol and 100% propylene oxide and immersed in 1:1 mixture of propylene oxide and Polybed 812 epoxy resin for 1 hour. The infiltration solution was then changed to 100% resin. After 24 hours of infiltration, the tissue was embedded in molds and cured at 37°C overnight, followed by additional hardening at 65°C for 2 days. Ultrathin (70 nm) sections were collected on 200-mesh copper grids and stained with 2% uranyl acetate in 50% methanol for 10 minutes, followed by 1% lead citrate for 7 minutes. Sections were photographed using a Hitachi H-600 transmission electron microscope (TOSHIBA, Japan) at 80 kV onto electron microscope film.

Lung histology

A median sternotomy was performed. The harvested right middle lobe of the lung was fixed in 4% formaldehydum polymerisatum. Paraffin-embedded sections (5 μm) were stained with hematoxylin-eosin and evaluated blindly by two different histopathologist.

Detection of concentration of protein in intestine

Another segment of 10 cm intestine was cut 5 cm from terminal ileum. The small intestine was washed with frozen saline and dried with suction paper at 4°C. Intestinal tissues were homogenized with normal saline. Intestinal protein (content) was quantified by the Bradford method with a BSA standard kit, provided by Shenerg Biocolor BioScience & Technolgy Company, Shanghai, China.

Detection of the levels of TNF-α, IL-1β, IL-6 and IL-10 in the intestine

Intestinal tissues were homogenized with normal saline, frozen at -20°C for 5 minutes and centrifuged for 15 minutes at 4,000 r/min. Supernatants were transferred into fresh tubes. The levels of TNF-α, IL-1β, IL-6 and IL-10 were measured using a bead-based immunofluorescence assay (Luminex Inc. Austin, TX, USA)using multiplex cytokine reagents supplied by Linco International, USA. Briefly, antibody-coupled beads were incubated with the samples (antigen), followed by incubation with biotinylated detection antibody and streptavidin-phycoerythrin, respectively. A broad sensitivity range of standards (Linco International), ranging between 1.95 and 32 000 pg/ml were used to help enable the quantization of a dynamic wide range of cytokine concentrations and provide the greatest sensitivity. This captured bead-complexes were then read by the Luminex fluorescent bead-based technology Luminex™ 200 Liquid Array (Luminex Corporation Austin, TX, USA) with a flow-based dual laser detector with real-time digital signal processing to facilitate the analysis of up to 100 different families of colour-coded polystyrene beads and allow multiple measurements of the sample ensuring in the effective quantification of cytokines. The levels of TNF-α, IL-1β, IL-6 and IL-10 in the intestine were indicated as picogram per milligram of protein.

Immunohistochemical detection of tryptase in intestine

Five-micron-thick sections were prepared from paraffin-embedded intestinal tissues. After deparaffinization, endogenous peroxidase was quenched with 3% H₂O₂ in deionised water for 10 minutes. Nonspecific binding sites were blocked by incubating the sections in 10% normal rabbit serum for 1 hour. The sections were then incubated with polyclonal rat anti-mast cell tryptase (dilution 1: 50) for 30 minutes at 37°C, followed by incubating with biotinylated goat-anti-rat IgG at room temperature for 10–15 minutes. After 3 times rinsing of the sample with PBS for 5 minutes, the horseradish-peroxidase-conjugated streptavidin solution was added and incubated at room temperature for 10–15 minutes. The antibody binding sites were visualized by incubating with diaminobenzidine-H₂O₂ solution. Sections incubated with PBS instead of the primary antibody were used as negative controls. Brownish granules in the cytoplasm were recognized as positive staining for tryptase. We calculated the tryptase positive mast cells in 5 representative areas at 400× magnification by Image-Pro Plus 5.0 (USA)

Statistics

Data were expressed as mean ± SD. Analysis of variance was performed using SPSS 11.0 software. One-way analysis of variance was used for multiple comparison. Bonferroni test was used for intra-group comparison or Tamhane's T2 test was used if equal variances was not assumed. Chi-Square test was used to determine the significance of differences of the survival rates. Differences were considered significant when P value was less than 0.05.

Changes of survival rate after different ischemia time

The 1^st to 7^th day's survival rates after 60 minutes' intestine ischemia were from 83.3% to 75%. The 1^st to the 7^th day's survival rates after 75 minutes' intestine ischemia were from 50% to 41.6%. The 1^st to 7^th day's survival rates after 90 minutes' intestine ischemia were from 33.3% to 16.7%, while the 1^st to 7^th day's survival rates after 120 minutes' intestine ischemia were from 16.7% to zero. The survival rates on the 3^rd day groups I, II, III and group IV were 83.3%, 41.6%, 25%, and 0, respectively. There were almost no animals died in the 4^th to the 7^th day after reperfusion. (Fig. 1)

Changes in survival rates and states after operation

The surviving rats in group S recovered more vigorously and vitally on the 3^rd day after 75 min intestine ischemia. The survival states of the rats in group M, K and C also recovered vigorously and vitally while those in group CP recovered less vigorously and vitally.

The survival rates in group S were higher than those in the ischemia-reperfusion groups, and the 3^rd day's survival rates in group M were lower than those in group K and C while they were higher than those in group CP (P < 0.05), there was no significant difference between group K and group C(P > 0.05). [3^rd day's survival rates: group S 92%(22/24), group M 42%(10/24), group K 75%(18/24), group C 75%(18/24), group CP 12.5%(3/24)] (Fig. 2)

Changes of serum biochemical indicator in the survival rats

The concentration of AST in group CP was higher than that in group M, K, and C(P < 0.05) (Table 1); and the concentration of CRE in group CP was higher than that in group M (P < 0.05) (Table 1).

There were no significant differences in group M, C and group K (P > 0.05). (Table 1)

Changes of levels of TNF-α, IL-1β, IL-6 and IL-10 in intestine in the survival rats

Compared with group M, the levels of IL-6 and TNF-α in group K decreased significantly (P < 0.05). (Table 2)

There were no significant differences between group K and group C(P > 0.05). (Table 2)

Changes of intestinal mucosa under light microscope and Chiu's score in the survival rats

The villus and glands were normal. No inflammatory cell infiltration was observed in mucosal epithelial layer in sham group. Slight edema of mucosa villus and infiltration of few necrotic epithelial inflammatory cells neutrophil leukomonocyte were found in mucosa epithelial layer in M, K, C and CP groups. (Fig 3)

There were no significant differences in Chiu's score among the five groups on the 3^rd day (P > 0.05). (Table 2)

Changes of counts of IMMC and ultrastructure in the survival rats

There were no significant difference in the number of IMMC among the five groups in the survival rats(P > 0.05) (Fig. 4, Table 2). The ultrastructure of IMMC was normal in the sham group. There were abundant vacuoles with reduced number of granules in their endochylema in groups M and CP. There were fewer swollen granules in IMMC homogeneity in groups C and K (Fig. 5).

Changes of lung histology in the survival rats

The lung histological structure was normal in group S, while the lung tissues were obviously damaged with edema, hemorrhage, and inflammatory cell infiltration in groups M and CP, and the injury was ameliorated in groups C and K. (Fig 6)