Reperfusion Promotes Mitochondrial Dysfunction following Focal Cerebral Ischemia in Rats

Background and Purpose

Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia.

Methods

Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis.

Results

Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca²⁺ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period.

Conclusions

Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca²⁺-induced mitochondrial swelling. The mechanism of this swelling may be mediated by the upregulation of the Cyclophilin D protein, the destruction of the mitochondrial membrane potential and the generation of excessive reactive oxidative species.