Intestinal injury can be reduced by intra-arterial postischemic perfusion with hypertonic saline

Abstract

Aim: To investigate the effect of local intestinal perfusion with hypertonic saline (HTS) on intestinal ischemia-reperfusion injury (IRI) in both ex vivo and in vivo rat models.

Methods: All experiments were performed on male Wistar rats anesthetized with pentobarbital sodium given intraperitoneally at a dose of 60 mg/kg. Ex vivo vascularly perfused rat intestine was subjected to 60-min ischemia and either 30-min reperfusion with isotonic buffer (controls), or 5 min with HTS of 365 or 415 mOsm/L osmolarity (HTS(365mOsm) or HTS(415mOsm), respectively) followed by 25-min reperfusion with isotonic buffer. The vascular intestinal perfusate flow (IPF) rate was determined by collection of the effluent from the portal vein in a calibrated tube. Spontaneous intestinal contraction rate was monitored throughout. Irreversible intestinal injury or area of necrosis (AN) was evaluated histochemically using 2.3.5-triphenyltetrazolium chloride staining. In vivo, 30-min ischemia was followed by either 30-min blood perfusion or 5-min reperfusion with HTS(365mOsm) through the superior mesenteric artery (SMA) followed by 25-min blood perfusion. Arterial blood pressure (BP) was measured in the common carotid artery using a miniature pressure transducer. Histological injury was evaluated in both preparations using the Chui score.

Results: Ex vivo, intestinal IRI resulted in a reduction in the IPF rate during reperfusion (P < 0.05 vs sham). The postischemic recovery of the IPF rate did not differ between the controls and the HTS(365mOsm) group. In the HTS(415mOsm) group, postischemic IPF rates were lower than in the controls and the HTS(365mOsm) group (P < 0.05). The intestinal contraction rate was similar at baseline in all groups. An increase in this parameter was observed during the first 10 min of reperfusion in the control group as compared to the sham-treated group, but no such increase was seen in the HTS(365mOsm) group. In controls, AN averaged 14.8% ± 5.07% of the total tissue volume. Administration of HTS(365mOsm) for 5 min after 60-min ischemia resulted in decrease in AN (5.1% ± 1.20% vs controls, P < 0.01). However, perfusion of the intestine with the HTS of greater osmolarity (HTS(415mOsm)) failed to protect the intestine from irreversible injury. The Chiu score was lower in the HTS(365mOsm) group in comparison with controls (2.4 ± 0.54 vs 3.2 ± 0.44, P = 0.042), while intestinal perfusion with HTS(415mOsm) failed to improve the Chiu score. Intestinal reperfusion with HTS(365mOsm) in the in vivo series secured rapid recovery of BP after its transient fall, whereas in the controls no recovery was seen. The Chiu score was lower in the HTS(365mOsm) group vs controls (3.1 ± 0.26 and 3.8 ± 0.22, P = 0.0079 respectively,), although the magnitude of the effect was lower than in the ex vivo series.

Conclusion: Brief intestinal postischemic perfusion with HTS(365mOsm) through the SMA followed by blood flow restoration is a protective procedure that could be used for the prevention of intestinal IRI.

Keywords: Acute mesenteric ischemia; Hypertonic saline; Intestinal ischemia-reperfusion injury; Intestinal perfusate flow rate; Perfusion; Superior mesenteric artery.

Figure 1

The sequential steps of the intestinal perfusion in vivo. A: The abdominal aorta (aa) is occluded by a clip, followed by cannulation of the vessel; the superior mesenteric artery (SMA) is clipped; B: The aortic clip is positioned above the SMA; the cannula is positioned at the level of SMA to allow its perfusion with hypertonic saline; the perfusate outflow is secured through the opened ileocecal branch (ib) of the superior mesenteric vein (SMV); the main trunk of SMV is clipped; C: The cannula is removed to allow blood flow through the SMA to resume simultaneously with the following procedures: the aortic clip is positioned as in Figure 1A; the clip occluding the SMV is removed, and bleeding from the ileocecal branch of the SMV is prevented by ligation.

Figure 2

Intestinal perfusate flow rate in the ex vivo rat experiments with 60-min ischemia followed by varying conditions of reperfusion. The groups tested were: sham-operated (no ischemia at all, n = 6); controls (30 min reperfusion with normotonic buffer, n = 5); 2 groups with either HTS^365mOsm (n = 5) or HTS^415mOsm (n = 5), both subjected to 5-min reperfusion with the respective hypertonic solution followed by 25 min reperfusion with normotonic buffer. The pre-ischemic values of Intestinal perfusate flow (IPF) rate were similar in all groups. No changes were apparent in the shams over the whole period, whereas IPF values were lower in all the ischemia-reperfusion groups, although the groups showed different patterns. HTS^415mOsm resulted in the most prominent decrease in the IPF rate, with a further progressive fall to the end of the experiment. In contrast, IPF rate was less affected in the control and HTS^365mOsm groups, to a similar degree in both, while an appreciable degree of stabilization took place towards the end of the postischemic period. Postischemic IPF rate recovery did not differ between the controls and the HTS^365mOsm group, whereas postischemic IPF rate values were significantly lower in the HTS^415mOsm group, compared to both the controls and the HTS^365mOsm group. Data are mean ± SD. ^aP < 0.05 vs sham; ^cP < 0.05 vs controls.

Figure 3

Area of necrosis within the isolated rat intestine after 60-min ischemia followed by the varying conditions of reperfusion. The area of necrosis (AN) is defined as the percentage of 2.3.5-triphenyltetrazolium chloride-negative in relation to the total intestinal tissue volume. Controls (reperfusion with normotonic saline) and the HTS^415mOsm group did not differ statistically regarding AN volume, whereas HTS^365mOsm administration resulted in a significantly lower value of AN. In both hypertonic saline (HTS) groups, the 5-min reperfusion with the appropriate hyperosmotic saline was followed by 25-min normotonic reperfusion. The data are presented as dot plots with median values.

Figure 4

The rate of low frequency intestinal contractions in the ex vivo experiments. In shams, the intestine was continuously perfused for 110 min. In controls (60-min ischemia followed by 30-min reperfusion), intestinal contraction rate was significantly higher over the initial 10 min of reperfusion in comparison with the shams. Intestinal reperfusion with hyperosmotic saline in the HTS^365mOsm group prevented the increase in postischemic intestinal contraction rate. Data are mean ± SD. ^aP < 0.05 vs sham. LF: Low frequency.

Figure 5

Histologically defined scores in the ex vivo (A) and in vivo (B) experiments. A, B: In the sham group (no ischemia at all, A) slight injury only is evident, probably due to the prolonged perfusion in the extracorporeal environment. Sixty-minute ischemia followed by 30-min normotonic reperfusion (controls) resulted in a significant injurious effect, which, however, was substantially less expressed in the HTS^365mOsm but not in the HTS^415mOsm group. The analogous difference between the controls and the HTS^365mOsm group is apparent in the in vivo experiments (B). The data are expressed as mean ± SD; ^aP < 0.05 vs controls.

Figure 6

Representative samples of arterial blood pressure recordings in anesthetized rats subjected to intestinal ischemia-reperfusion in vivo. A: Thirty-minute superior mesenteric artery (SMA) occlusion followed by 30-min reperfusion (controls) resulted in a persistent blood pressure (BP) decrease over the entire reperfusion period; B: Thirty-minute SMA occlusion finalized by aortic clamping followed immediately by 5-min SMA perfusion with hypertonic saline (365 mOsm/L), with a subsequent 25-min reperfusion with blood, resulted in a transient drop with a rapid restoration of BP up to the initial values. HTS: Hypertonic saline.