Low Flow versus No Flow: Ischaemia Reperfusion Injury Following Different Experimental Models in the Equine Small Intestine

Abstract

In experimental studies investigating strangulating intestinal lesions in horses, different ischaemia models have been used with diverging results. Therefore, the aim was to comparatively describe ischaemia reperfusion injury (IRI) in a low flow (LF) and no flow (NF) model. Under general anaesthesia, 120 min of jejunal ischaemia followed by 120 min of reperfusion was induced in 14 warmbloods. During ischaemia, blood flow was reduced by 80% (LF, n = 7) or by 100% (NF, n = 7). Intestinal blood flow and oxygen saturation were measured by Laser Doppler fluxmetry and spectrophotometry. Clinical, histological, immunohistochemical and Ussing chamber analyses were performed on intestinal samples collected hourly. Tissue oxygen saturation was significantly lower in NF ischaemia. The LF group exhibited high variability in oxygen saturation and mucosal damage. Histologically, more haemorrhage was found in the LF group at all time points. Cleaved-caspase-3 and calprotectin-stained cells increased during reperfusion in both groups. After NF ischaemia, the tissue conductance was significantly higher during reperfusion. These results aid in the selection of suitable experimental models for future studies. Although the LF model has been suggested to be more representative for clinical strangulating small intestinal disease, the NF model produced more consistent IRI.

Keywords: apoptosis; colic; horse; inflammation; intestine; ischaemia; reperfusion.

Figure 1

Timeline of the experiment and associated sampling. P = pre-ischaemia; I0 = immediately following induction of ischaemia; I1 = after 60 min ischaemia; I2 = after 120 min of ischaemia; R0 = immediately following reperfusion; R1 = after 60 min of reperfusion; R2 = after 120 min of reperfusion; RC = remote control segment after reperfusion.

Figure 2

Exemplary images of different grades of intestinal discolouration following ischaemia and reperfusion.

Figure 3

Box-plot diagram of the intestinal microperfusion measured by LDS in arbitrary units (AU) during the different time points. P, pre-ischaemia; I0, immediately after induction of ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R0, immediately following reperfusion; R1, 60 min reperfusion; R2, 120 min reperfusion. The horizontal bar displays the median, the interquartile range is represented by the box, and the minimum and maximum by the whisker plots. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01; p *** < 0.001). There were no significant differences between the groups.

Figure 4

Box-plot diagram of the intestinal StO₂ by MS in% during the different time points. P, pre-ischaemia; I0, immediately after induction of ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R0, immediately following reperfusion; R1, 60 min reperfusion; R2, 120 min reperfusion. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01; p *** < 0.001), significant differences between the groups are indicated with a horizontal bar and accompanying hash (# p < 0.05).

Figure 5

Box-plot diagram of the intestinal haemoglobin measured by MS in arbitrary units (AU) during the different time points. P, pre-ischaemia; I0, immediately after induction of ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R0, immediately following reperfusion; R1, 60 min reperfusion; R2, 120 min reperfusion. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001), significant differences between the groups are indicated with a horizontal bar and accompanying hash (### p < 0.001).

Figure 6

Clinical appearance of the jejunum after being subjected to two hours of different segmental ischaemia: low flow ischaemia (a) displaying intense purple discolouration, and no flow ischaemia (b) with mild purple discolouration and vessel-associated pale areas.

Figure 7

Box-plot diagrams of EPS (a) and mucosal HS (b) evaluated according to modified Chiu-score of the LF and NF group for the different intestinal samples. P, pre-ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R1, 60 min reperfusion; R2, 120 min reperfusion. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01), significant differences between the groups are indicated with a horizontal bar and accompanying hash (# p < 0.05; ## p < 0.01; ### p < 0.001).

Figure 8

Box-plot diagram of the cleaved caspase-3-positive cells/mm² in the mucosa of jejunum subjected to either LF or NF ischaemia at different time points. P, pre-ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R1, 60 min reperfusion; R2, 120 min reperfusion; RC, remote control sample at reperfusion. Significant differences are marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 9

Box-plot diagram of positive cell counts in cells/mm² after immunohistochemical staining for cytosolic calprotectin in the mucosa (a), submucosa (b), muscularis (c) and serosa (d) of jejunum subjected to either LF or NF ischaemia at different time points. P, pre-ischaemia; I1, 60 min ischaemia; I2, 120 min ischaemia; R1, 60 min reperfusion; R2, 120 min reperfusion; RC, remote control sample at reperfusion. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001), significant differences between the groups are indicated with a horizontal bar and accompanying hash (# p < 0.05).

Figure 10

Box-plot diagrams of the short circuit currents (I_sc) following the addition of (a) Alanine; or (b) Glucose during pre-ischaemia (P), following 120 min of ischaemia (I2) and after 120 min of reperfusion (R2) in horses subjected to low and NF ischaemia. The horizontal bar displays the median, the interquartile range is represented by the box, and the minimum and maximum by the whisker plots. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01).

Figure 11

Box-plot diagram of the tissue conductance (G_t) during pre-ischaemia (P), following 120 min of ischaemia (I2) and after 120 min of reperfusion (R2) in jejunum subjected to either LF or NF ischaemia. Significant differences of the time points compared to pre-ischaemia are marked with an asterisk (* p < 0.05; ** p < 0.01; *** p < 0.001), significant differences between the groups are indicated with a horizontal bar and accompanying hash (## p < 0.01).