Total warm ischemia and reperfusion impairs flow in all rat gut layers but increases leukocyte-vessel wall interactions in the submucosa only

Abstract

Objective: To study the effect of warm ischemia and reperfusion (I/R) on local perfusion and leukocyte-vessel wall interactions in vivo in all small bowel layers, and to quantify small bowel tissue injury histologically and by measuring intestinal fatty acid binding protein (I-FABP) release from the enterocytes.

Summary background data: Gut injury as a result of I/R plays a pivotal role in a variety of clinical conditions, such as small bowel transplantation, heart or aortic surgery, and (septic) shock. The precise mechanism behind I/R injury and the role of microvascular changes remain unclear. The influence of warm I/R of the gut on microvascular parameters in the different gut layers has not been studied before.

Methods: Anesthetized Lewis rats were either subjected to 30 minutes of ischemia and 1 hour of reperfusion or sham-treated as controls. After ligating the inferior mesenteric artery, total warm ischemia was induced by clamping the superior mesenteric artery. Intravital video microscopic measurements were obtained at intervals. Tissue injury of the small bowel and other organs was histologically evaluated afterward. In addition, plasma levels of I-FABP were determined to measure enterocyte damage.

Results: After ischemia, mean red blood cell velocity decreased significantly in all layers of the small bowel, but no diameter changes were observed. Leukocyte-vessel wall interactions increased in the submucosa but not in the muscle layers. Plasma levels of I-FABP significantly increased from 30 minutes of reperfusion onward. The intestinal mucosa was severely injured; no histologic damage was detected in other tissues.

Conclusions: This is the first in vivo study showing that total warm ischemia of the rat gut impairs perfusion in the whole small bowel, whereas leukocyte-vessel wall interactions increase in the submucosal layer only. Therefore, the early inflammatory response to I/R seems to be limited to the submucosa. Both microvascular effects may have contributed to the severe morphologic and functional mucosal injury observed after I/R.

Figure 1. The small bowel microvascular bed, showing the six sites of intravital microscopic observation. Arterioles originating from the mesenteric arcade penetrate the muscular layers of the small bowel into the submucosa (C). Here they give rise to arterioles (1), which run mainly longitudinally. Right-angled branches originating from these longitudinal arterioles supply the submucosal capillary plexus (2) and the mucosa (D), whereas other branches ascend through the muscle layers, supplying the capillaries of the circular (3, B) and longitudinal (4, A) muscle layers. Blood from muscle capillaries is collected in venules, which in turn merge between the two muscle layers into larger venules (5). Venules are also formed at the base of the submucosal capillary bed; these in turn become the larger submucosal venules (6). Finally, the blood from the muscle and the submucosal layers is collected in venules, which return to the mesenteric arcade.

Figure 2. Mean red blood cell velocities (mRBCV) in arterioles (upper panel) and venules (middle panel) of the small bowel submucosa in the sham (solid line, ○) and ischemia group (broken line, •). In submucosal capillaries (lower panel), an arbitrary velocity index was determined. Medians with interquartile ranges (bars) are presented. The left side of the vertical line (start of reperfusion) represents the control period (baseline) before total warm ischemia (or sham). *P < .05, **P < .01, ***P < .001 for ischemia vs. sham.

Figure 3. Mean red blood cell velocities (mRBCV) in venules between the muscle layers (upper panel), capillaries of the longitudinal muscle layer (middle panel), and capillaries of the circular muscle layer (lower panel) of the small bowel in the sham (solid line, ○) and ischemia group (broken line, •). Medians with interquartile ranges (bars) are shown. The left side of the vertical line (start of reperfusion) represents the control period (baseline) before total warm ischemia (or sham). **P < .01, ***P < .001 for ischemia vs. sham.

Figure 4. Effect of warm ischemia and subsequent reperfusion (broken line, •) or sham (solid line, ○) on leukocyte rolling (upper panels), leukocyte adherence (middle panels), and total number of leukocytes interacting with the vessel wall (lower panels) in venules of the small bowel submucosa (left column) and between the muscle layers (right column). Medians with interquartile ranges (bars) are presented. The left side of the vertical line (start of reperfusion) represents the control period (baseline) before total warm ischemia (or sham). *P < .05, **P < .01, and ***P < .001 for ischemia vs. sham.

Figure 5. Median intestinal fatty acid binding protein (I-FABP) levels in serum at baseline (control) and at 30 and 60 minutes after the start of reperfusion in the ischemia and sham groups. Bars denote interquartile ranges. After ischemia, a large amount of I-FABP was released from the enterocytes, resulting in maximally detectable levels (i.e., >125 ng/mL). ***P < .001 for ischemia vs. sham.

Figure 6. Ileal histology after the sham procedure (A) and 30 minutes of ischemia and 1 hour of reperfusion (B). In the sham group no injury was found, whereas in the ischemia group grade 5 injury was observed (complete loss of villi). Bars = 100 μm.