Procedures and animal care complied with principles formulated by the National Society for Medical Research (animal facility agreement: n° B75-18-03, experimentation authorization n° 75-101, APAFiS#8724). Eight-week-old male Wistar rats weighing 280 g were purchased from Janvier laboratory.
Model of acute mesenteric ischemia/reperfusion
A total of 28 rats were randomized into four groups: an ischemia/reperfusion (I/R) group treated with saline perfusion (I/R controls, 7 rats), an ischemia/reperfusion group treated with P8RI perfusion (P8RI group, 7 rats), and a sham-operated group (sham group, 7 rats). A group of sham-operated rats treated by P8RI perfusion (sham-P8RI group, 7 rats) was added in order to assess for potential histological changes within the normal bowel epithelium in response to P8RI.
Rat body temperature was maintained at 37.5 °C by a heated surgical table. Non-fasting rats were anesthetized by intraperitoneal injection of 2 mg/kg of urethane, and analgesia was performed by subcutaneous injection of buprenorphine (0.05 mg/kg). Cannulation of the right jugular vein and right carotid artery were performed for perfusion, blood sampling, and arterial pressure measurements. After laparotomy, the superior mesenteric artery (SMA) was exposed but not occluded in the sham group. In the control and P8RI groups, the SMA was clamped. The clamp was removed after 30 min of ischemia, followed by 4 h of reperfusion. A single 3.5-mg/kg intravenous bolus of P8RI was administered 5 min before clamping SMA and was followed by a continuous perfusion at 3.5 mg/kg/h until the end of reperfusion. The same procedure was performed for the control group, except for the perfusion of P8RI, which was substituted by an equivalent volume of saline perfusion. The experimenter was blinded with regard to the treatment administered. Arterial blood pressure and diuresis were monitored throughout the experiment. Blood and peritoneal fluid were sampled in EDTA tubes before the laparotomy and 30 min after clamping the SMA. Blood samples were also collected every hour during the reperfusion, and peritoneal fluid samples after 2 and 4 h of reperfusion. Samples were centrifuged for 30 min at 16,000g. Supernatants were used for assays. At the end of the procedure, a total enterectomy was performed after rats were euthanized. The intestinal luminal content was collected for the quantification of intestinal bleeding. Histological analysis was performed on a central section of 2-cm-long jejunum specimen as previously recommended [14]. The remaining small bowel was homogenized. Homogenates were centrifuged for 30 min at 16,000g, and supernatants were used for assays.
Assessment of mesenteric ischemia/reperfusion-induced intestinal injury
Histological analysis of the intestinal mucosa
Highly glycosylated mucins and nuclei were stained respectively with Alcian Blue and nuclear fast red on transverse paraffin-embedded sections of the small intestine. Histological evaluation was performed using the grading system described by Chiu et al. [15], as described in Additional file 1 (Table S1).
Morphometric analysis of the small bowel
A morphometric evaluation of histological gut sections was performed using QWin software to determine the luminal area, epithelial and muscular layer areas, and the overall surface area, as described in Additional file 1 (Figure S1).
Intestinal bleeding
The intestinal bleeding was assessed by the quantification of heme concentration in the small intestine luminal content using formic acid, as described in Additional file 1.
Neutrophil activation in the small bowel tissue
Neutrophil activation was assessed by the intestinal tissue concentrations of matrix metalloproteinase-9 (MMP-9) and myeloperoxydase (MPO), using enzyme-linked immunosorbent assays, as described in Additional file 1.
Assessment of mesenteric ischemia/reperfusion-induced neutrophil activation in plasma
Neutrophil activation in plasma was assessed by the plasma concentration of MMP-9 using an enzyme-linked immunosorbent assay, as described in Additional file 1.
Assessment of mesenteric ischemia/reperfusion-induced bacterial translocation
Bacterial translocation was assessed by the quantification of plasma DNA from Escherichia coli at 4 h of reperfusion after the onset of mesenteric ischemia, using a real-time PCR technique, as described in Additional file 1.
Assessment of soluble CD31 in plasma
The shedding of CD31 into plasma was assessed by measuring soluble plasma CD31 at repeated time points before, during ischemia, and during the reperfusion period. Within this timeframe, raised levels of CD31 can only derive from the cleavage and shedding of the membrane-anchored molecule. The assay was based on the use of cytometric polystyrene beads. A polyclonal antibody targeting rat CD31 (R&D, #AF3628) was immobilized onto COOH-magnetic beads (MC10035-01, BioRad). CD31 capture beads were then incubated with the EDTA-plasma (diluted 1:6) from individual rats and each time point for 90 min at room temperature. After repeated washing, the soluble circulating CD31 that was captured by the beads was revealed with using a phycoerythrin-labeled monoclonal antibody (clone TLD-3A12) directed to the most membrane-distal portion of rat CD31. Median fluorescent intensity was analyzed from a total of 100 beads per sample on a BioPlex 200® System (BioRad). A standard curve was obtained using serial dilutions of recombinant CD31.
Sample size calculation
The study was designed with 90% power to detect a relative 50% difference in epithelial layer area between I/R controls and P8RI group. Statistical testing was performed at the two-tailed level of 0.05 using a t test. Based on preliminary data indicating that mean epithelial layer area after mesenteric ischemia/reperfusion was 100 μm2 (SD ± 27), sample size calculation indicated seven rats per group.
Statistical analysis
Quantitative data are expressed as medians with interquartile range (IQR) or means ± SEM. Mann-Whitney U test, two-way ANOVA with post hoc multiple comparison Bonferroni tests and Spearman correlation were performed as appropriate. Principal component analysis was performed to study how I/R conditions (controls, P8RI-treated, and sham-operated) impact the relationships (correlations) between the epithelial area, neutrophil activation, CD31 cleavage, intestinal bleeding, intestinal tissue injury, and bacterial translocation.