**3.8** *In vivo* **evidence for a strategy to prevent reperfusion injury**

The purpose of the following experiments was to confirm that the mechanisms revealed by our cellular and molecular studies could be used to design a therapeutic approach in whole animals for the treatment of reperfusion injury to the brain capillaries, following transient ischemic stroke. The *in vitro* data suggested that two key factors are responsible for cellular injury and disruption of brain capillaries: (1) a rise in intracellular calcium due to reverse movement of the Na/Ca exchanger during reperfusion, and (2) depletion of endogenous antioxidant activity during the prolonged ischemia, resulting in an elevation of reactive oxygen species upon reperfusion. This led to our conclusion that a reasonable treatment for damage to brain capillary endothelial cells following transient ischemia would be to administer an inhibitor of reverse movement of Na/Ca exchange to prevent the rise in intracellular calcium, and to buffer reactive oxygen species by restoring antioxidant activity within the cells.

To test this hypothesis, middle cerebral artery occlusion (MCAO) was performed on Long-Evans female rats, involving 1 hour of ischemia to the left cerebral hemisphere, and 24 hours of reperfusion. One group of animals received KB-R7943 (10 mg/kg) and γ-glutamylcysteine (400 mg/kg) in 1 ml of isotonic saline solution 1 minute prior to reperfusion (IV, femoral vein), while the other group was administered a placebo (isotonic saline). Following treatment, tissue from the lateral cortex was prepared for electron microscopy, and the cross-sectional area of mitochondria in blood-brain barrier endothelial cells was quantified using morphometric techniques. The contralateral (right) hemisphere served as an internal control for non-ischemic tissue. The data is represented by electron micrographs in **Figure 8** from 2 of 4 animals in each group. As observed in the left panels of **Figure 8** depicting tissue exposed to ischemia/reperfusion without the drugs, mitochondria of blood-brain barrier endothelial cells (solid arrows) are noticeably swollen and abnormal in appearance, indicative of the mitochondrial permeability transition associated with apoptosis [43, 51]. In some instances, mitochondria have actually been extruded into the lumen (dashed arrows) of the capillary, indicating extensive cell damage. In the right panels showing tissue from 2 of 4 stroked animals administered the drugs, the mitochondria are normal, and the blood-brain barrier endothelial cells look healthy. When comparing the groups of animals, morphometric measurements of endothelial mitochondrial size revealed a highly significant (P = 0.0015) increase in swelling in stroked animals without the drugs vs. those with the drugs, expressed as a percent change in cross-sectional area from the control contralateral hemisphere (67 ± 15 vs. 13 ± 12, mean ± SD, n = 4 animals per group).
