A specific inhibitor of apoptosis decreases tissue injury after intestinal ischemia-reperfusion in mice

Abstract

Purpose: Apoptosis is a stereotypical pathway of cell death that is orchestrated by a family of cysteine endoproteases called caspases. This study examined the effect of apoptosis inhibition with a specific caspase inhibitor on murine intestinal viability after ischemia-reperfusion (IR).

Methods: C57Bl6 X SV129 mice underwent segmental small bowel ischemia by vascular isolation of 10 cm of terminal ileum. In separate experiments, the ischemic time was varied from 30 to 130 minutes with a reperfusion interval of 6 hours. The degree of small bowel injury was quantified from 1 to 5 (increasing severity) by standardized, blinded histologic grading. The degree of apoptosis was assessed with a specific assay (terminal deoxyamcleotydil transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]) and quantified by calculating the apoptotic index (apoptotic cells/10 high-power fields). To evaluate for activation of interleukin-1beta converting enzyme we measured tissue mature interleukin-1beta levels using a specific enzyme-linked immunosorbent assay. To evaluate the effect of apoptosis inhibition on intestinal viability after IR, mice received 3.0 mg of the caspase inhibitor ZVAD (N-benzyloxycarbonyl Val-Ala-Asp-Ome-fluoromethylketone) subcutaneously before and after IR in five divided doses (n = 11), the same dose of ZFA (N-benzyloxycarbonyl Phe-Ala fluoromethylketone), a structurally similar molecule with no anticaspase activity (n = 9), or sham operation (n = 6).

Results: A linear relationship existed between ischemic interval and histologic grade (r = 0.69, P <.006). Increasing the ischemic interval from 0 to 50 minutes was associated with a fivefold increase in apoptotic index (P =.05). Ischemic bowel was measured to have an average of 57.3 +/- 7.8 pg/mL whereas normal bowel had an average of 1.8 +/- 0.5 pg/mL of mature interleukin-1beta present. Mice tolerated multiple injections of ZVAD and ZFA without signs of toxicity. Animals treated with ZVAD (apoptosis inhibitor) had little injury after 50 minutes of ischemia and 6 hours of reperfusion (injury grade 1.8) compared with sham controls (injury grade 1.2, P =.7) and had significantly less injury than mice treated with ZFA (placebo) (injury grade 3.0, P <.006).

Conclusions: Increasing ischemic interval in a segmental small bowel murine IR model is associated with increased histologic injury and augmented apoptosis as evidenced by increased TUNEL staining and interleukin-1beta converting enzyme activation. Inhibition of apoptosis with a specific caspase inhibitor significantly diminishes the degree of small bowel injury.