Ischaemic postconditioning reduces apoptosis in experimental jejunal ischaemia in horses

Abstract

Background: Ischaemic postconditioning (IPoC) refers to brief periods of reocclusion of blood supply following an ischaemic event. This has been shown to ameliorate ischaemia reperfusion injury in different tissues, and it may represent a feasible therapeutic strategy for ischaemia reperfusion injury following strangulating small intestinal lesions in horses. The objective of this study was to assess the degree cell death, inflammation, oxidative stress, and heat shock response in an equine experimental jejunal ischaemia model with and without IPoC.

Methods: In this randomized, controlled, experimental in vivo study, 14 horses were evenly assigned to a control group and a group subjected to IPoC. Under general anaesthesia, segmental ischaemia with arterial and venous occlusion was induced in 1.5 m jejunum. Following ischaemia, the mesenteric vessels were repeatedly re-occluded in group IPoC only. Full thickness intestinal samples and blood samples were taken at the end of the pre-ischaemia period, after ischaemia, and after 120 min of reperfusion. Immunohistochemical staining or enzymatic assays were performed to determine the selected variables.

Results: The mucosal cleaved-caspase-3 and TUNEL cell counts were significantly increased after reperfusion in the control group only. The cleaved-caspase-3 cell count was significantly lower in group IPoC after reperfusion compared to the control group. After reperfusion, the tissue myeloperoxidase activity and the calprotectin positive cell counts in the mucosa were increased in both groups, and only group IPoC showed a significant increase in the serosa. Tissue malondialdehyde and superoxide dismutase as well as blood lactate levels showed significant progression during ischaemia or reperfusion. The nuclear immunoreactivity of Heat shock protein-70 increased significantly during reperfusion. None of these variables differed between the groups. The neuronal cell counts in the myenteric plexus ganglia were not affected by the ischaemia model.

Conclusions: A reduced apoptotic cell count was found in the group subjected to IPoC. None of the other tested variables were significantly affected by IPoC. Therefore, the clinical relevance and possible protective mechanism of IPoC in equine intestinal ischaemia remains unclear. Further research on the mechanism of action and its effect in clinical cases of strangulating colic is needed.

Fig. 1

Cleaved-caspase- and TUNEL positive cell counts in the intestinal mucosa. Individual value plots of positive cell counts in cells/mm² after immunohistochemical staining for cleaved-caspase-3 (a) and TUNEL (b) in the small intestinal mucosa from horses subjected to postconditioning (IPoC) and an untreated control group. The horizontal bar displays the mean. Significant differences are marked with an asterisk (* p < 0.05; ** p < 0.01). P = Pre-ischaemia, I = Ischaemia, R = Reperfusion, PR = proximal intestinal segment after reperfusion

Fig. 2

Calprotectin positive cell counts in the intestine. Individual value plots of the positive cell counts in cells/mm² after immunohistochemical staining for cytosolic calprotectin in the mucosa (a), submucosa (b), muscularis (c) and serosa (d) of an untreated control group and a group subjected to IPoC. There were no significant differences between the groups. The horizontal bar displays the mean. Significant differences between the time points are marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001). P = Pre-ischaemia, I = Ischaemia, R = Reperfusion, PR = proximal intestinal segment sampled after reperfusion

Fig. 3

Calprotectin immunoreactivity in the small intestine. Microscopic images of immunohistochemical staining for cytosolic calprotectin in the mucosa (a) and serosa (b) from the same intestinal sample taken after reperfusion, illustrating the intense positive staining of inflammatory cells in these locations (arrows). The scale bar represents 50 μm

Fig. 4

Heat Shock Protein-70 immunoreactivity score. Individual value plots of a semi-quantitative score assessing the immunoreactivity after immunohistochemical staining for Heat Shock Protein-70 of the small intestine from horses subjected to postconditioning (IPoC) and an untreated control group. The mucosal cytoplasm (a), the nuclei (b) and the muscularis propria (c) were scored separately. The horizontal bar displays the mean. Significant differences are marked with an asterisk (* p < 0.05; ** p < 0.01). P = Pre-ischaemia, I = Ischaemia, R = Reperfusion, PR = proximal intestinal segment after reperfusion

Fig. 5

Heat Shock Protein-70 immunoreactivity in the small intestinal mucosa. Microscopic images of the crypts and villi of the small intestinal mucosa after immunohistochemical staining for Heat Shock Protein-70. Panels a and b are from an ischaemic sample, c and d are from the reperfusion sample of the same horse, illustrating an increase in immunoreactivity. Representative examples of the stained enterocyte nuclei are marked (narrow arrow for weak stained nuclei, broad arrows for the intensely stained nuclei), the enterocyte cytoplasm shows a mild to moderate staining

Fig. 6

Hu and NOS stained neuronal counts in the myenteric plexus. Boxplot diagram of the Hu and NOS immunohistochemistry results. The left panels display the absolute and relative counts of the Hu stained neurons, and the right panels the NOS. The horizontal bar displays the median, the interquartile range is represented by the box, and the minimum and maximum by the whisker plots. IPoC = group undergoing postconditioning; P = pre-ischaemia; I = ischaemia; R = reperfusion

Fig. 7

Hu and NOS immunoreactivity in the myenteric plexus. Fluorescence microscopy image of a ganglion in the myenteric plexus, displaying the cytoplasmic staining for Hu-positive neurons (red), and NOS-positive neurons (green). Scale bar 100 μm

Fig. 8

SOD and MPO activity and MDA content in the small intestine. Individual value plots of superoxide dismutase activity (a), malondialdehyde content (b), and myeloperoxidase activity (c) measured in full thickness intestinal tissue from horses subjected to postconditioning (IPoC) and an untreated control group. The horizontal bar displays the median. Significant differences are marked with an asterisk (* p < 0.05; ** p < 0.01;). P = Pre-ischaemia, I = Ischaemia, R = Reperfusion, PR = proximal intestinal segment after reperfusion