Protective effect of ischemic preconditioning on cold preservation and reperfusion injury associated with rat intestinal transplantation

Abstract

Objective: To define the protective effect of ischemic preconditioning on cold ischemia and reperfusion injury associated with intestinal transplantation, and the role of nitric oxide in this process.

Summary background data: Ischemia/reperfusion injury continues to be a significant obstacle in small bowel transplantation. Preconditioning is a mechanism that protects against this injury.

Methods: To study the capacity of preconditioning to prevent cold ischemia-associated injury and the inflammatory response associated with intestinal transplantation, the authors studied a control group of animals, cold ischemia groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS, and intestinal transplantation groups with or without previous preconditioning and with or without previous administration of L-NAME or NONOS.

Results: Histologic findings and the release of lactate dehydrogenase into the preservation solution showed that preconditioning protects against cold ischemic preservation-associated injury. Preconditioning also prevented the inflammatory response associated with intestinal transplantation, measured by the above parameters and by neutrophil recruitment in the intestine. Inhibition of nitric oxide eliminates the protective effect.

Conclusions: Preconditioning protects the intestinal grafts from cold preservation and reperfusion injury in the rat intestinal transplantation model. Nitric oxide is involved in this protection.

Figure 1. Histologic lesions of the rat small bowel corresponding to the different groups in this study. (A) Normal mucosa, 0 minutes of intestinal cold ischemia. (B) Extended subepithelial space along villi; representative of cold ischemia preconditioning + NAME + cold ischemia and cold ischemia + NAME (90 minutes of preservation) and preconditioning + transplant and transplant + NONOS (90 minutes of preservation followed by transplantation with 60 minutes of reperfusion). (C) Subepithelial space at the tip of some villi; preconditioning + cold ischemia and cold ischemia + NONOS (90 minutes of preservation). (D) Epithelial lifting along villus; cold ischemia and cold ischemia + NAME (4 hours of preservation) and transplant and transplant + NAME (90 minutes of preservation followed by a transplantation with 60 minutes of reperfusion). (E) Extended subepithelial space; preconditioning + cold ischemia and cold ischemia + NONOS (4 hours of preservation). (F) Denuded villi; preconditioning + NAME + cold ischemia (4 hours of preservation) and preconditioning + NAME + transplant (90 minutes of preservation followed by transplant with 60 minutes of reperfusion). Hematoxylin and eosin stain, ×127.

Figure 2. Lactate dehydrogenase (LDH) release (U/mL) as a function of the preservation time. CI: cold ischemia (small bowel extracted and preserved in cold Ringer’s lactate solution at 4°C for a maximum of 4 hours). Prec + CI: preconditioning before the cold ischemia (small bowel clamped for 10 minutes, followed by 5 minutes of reperfusion before preservation in cold Ringer’s lactate solution at 4°C for a maximum of 4 hours). Prec + NAME + CI: preconditioning + NAME (as in preconditioning group but with prior addition of L-NAME). CI + NAME: cold ischemia with previous L-NAME administration. CI + NONOS: cold ischemia with previous NONOS administration. * P < .05 vs. time 0 of the corresponding group. +P < .05 vs. Prec + CI.

Figure 3. Lactate dehydrogenase (LDH) release (U/mL) in blood in the following groups. Sham; Trp: 90 minutes of cold ischemia followed by a transplantation with 60 minutes of reperfusion; Prec + Trp: as in transplanted group but with preconditioning before cold ischemia; Prec + NAME + Trp: as in ischemic preconditioning group but with previous administration of L-NAME before preconditioning; Trp + NAME: as Trp but with NAME administration; Trp + NONOS: as Trp but with previous NONOS administration. * P < .05 vs. sham. +P < .05 vs. Prec + Trp.

Figure 4. Myeloperoxidase (MPO) activity (U/mg protein) in the following groups. Sham; Trp: intestinal transplantation; Prec + Trp: ischemic preconditioning; Prec + NAME + Trp: preconditioning + NAME; Trp + NAME: intestinal transplantation + NAME; Trp + NONOS: intestinal transplantation + NONOS. * P < .05 vs. sham. +P < .05 vs. Prec + Trp.

Figure 5. Reduced glutathione (GSH; nmol/μg protein) in the following groups. Sham; Trp: intestinal transplantation; Prec + Trp: ischemic preconditioning; Prec + NAME + Trp: preconditioning + NAME; Trp + NAME: intestinal transplantation + NAME; Trp + NONOS: intestinal transplantation + NONOS. * P < .05 vs. sham. +P < .05 vs. Prec + Trp.

Figure 6. Nitrate and nitrite tissue production (μmol/mg protein) in the intestine in the following groups. (A) Sham; preconditioning (10 minutes of ischemia followed by 5 minutes of reperfusion); Prec + NAME: preconditioning group plus L-NAME; CI: cold ischemia; Prec + CI; preconditioning before cold ischemia; Prec + NAME + CI: preconditioning followed by sustained ischemia with prior addition of L-NAME. (B) Sham; Trp: intestinal transplantation (90 minutes of cold ischemia followed by transplant with 60 minutes of reperfusion); Prec + Trp: ischemic preconditioning (as in transplanted group but with preconditioning before cold ischemia); Prec + NAME + Trp: as in ischemic preconditioning group but with previous administration of L-NAME before preconditioning. * P < .05 vs. sham.