**5. Conclusions**

*Antioxidants*

(apoptosis).

safely administered a thrombolytic agent [18], the possibility of cerebral hemor-

Cerebral capillary endothelial cells represent the fundamental structure of the blood-brain barrier [52]. These cells are polarized, containing both luminal (blood-facing) and abluminal (brain-facing) plasma membranes with distinct properties [21–23, 25, 26, 53–56]. The cells are held together by tight junctions that effectively inhibit paracellular transport [21], implying that the properties of the respective luminal and abluminal plasma membranes regulate transport across the blood-brain barrier. Based upon observations in other tissues [40, 57], we hypothesized that ischemia-reperfusion injury to cerebral capillary endothelial cells is due to oxidative injury associated with the formation of reactive oxygen species in the presence of elevated intracellular calcium during reperfusion [58–62]. This would require loss of intracellular antioxidant (e.g., glutathione) during prolonged ischemia, formation of free reactive oxygen species and uptake of calcium during re-oxygenation, and damage to mitochondria causing programmed cell death

To test this hypothesis, we began by determining if the cellular processes required for these mechanisms are functional in blood-brain barrier endothelial cells. We reasoned that intracellular glutathione, an endogenous antioxidant, would diffuse out of the cells during ischemia, utilizing passive carriers we had previously described on both plasma membranes [58]. We next provided evidence that both Na/H (NHE1) and Na/Ca (NCX 1) exchangers are present on the plasma membrane domains of these cells, and that conditions simulating ischemia and reperfusion result in elevated intracellular calcium due to activation of sodium-hydrogen ion antiport and reverse movement of sodium-calcium exchange (**Figure 3**). Cellular lysis following these events was significantly inhibited by adding the antioxidant γGlu-Cys [19, 20, 58], which is consistent with the formation of reactive oxygen species expected upon re-oxygenation in glutathione depleted cells. These findings reinforced our interpretation that a combination of elevated intracellular calcium and reactive oxygen species are involved in injury to cerebral capillary endothelial

Our studies further indicated that injury to brain capillaries involves two phases. An early increase in permeability to sucrose (**Figure 5**), a marker of paracellular transport, was associated with the formation of actin stress fibers inside the endothelial cells (**Figure 4**). This was, in turn, accompanied by the appearance of large intercellular holes (**Figure 4**), apparently due to the opening of tight junctions [63]. We reasoned that such a change could be due to activation of myosin light chain kinase in the presence of the elevated intracellular calcium that we previously observed (**Figure 3**). Since it has been shown that ischemia causes actin filaments to conjugate with ZO-1 [64], a tight junctional protein, force generated by contraction of the cytoskeleton could weaken the tight junctions and result in the formation of stress fibers. Indeed, treatment with a myosin light chain kinase inhibitor effectively reversed the effects of simulated ischemia and reperfusion on the formation of actin stress fibers, and the appearance of large intercellular holes (**Figure 4**). These morphological and functional changes in cerebral capillary endothelial cells occurred within a few hours of exposure to conditions simulating ischemia and reperfusion, and generally correlated with an early and reversible phase of altered permeability to the cerebral vasculature when exposed to conditions of ischemia and reperfusion

A second phase of injury to cerebral capillaries following transient ischemia and reperfusion involved apoptosis and endothelial cell death. In an initial set of experiments, sucrose permeability was measured across monolayers of cultured

rhage severely limits the treatment of stroke with tPA.

cells, under conditions of ischemia and reperfusion.

**210**

*in vivo* [64].

Together these studies support the interpretation that prolonged cerebral ischemia followed by reperfusion of oxygenated blood may cause oxidative damage to cerebral capillary endothelial cells consistent with loss of vascular integrity and hemorrhage. The mechanisms involve mitochondrial injury and apoptosis due to the formation of reactive oxygen species in the presence of elevated cellular calcium. This condition may be treated by using the antioxidant γGlu-Cys to buffer reactive oxygen species, and a Na/Ca inhibitor (reverse mode) to prevent calcium loading in cerebral capillary endothelial cells. The *in vivo* studies indicate that co-administering both drugs intravascularly 1 minute prior to reperfusion provides cytoprotection that reduces reperfusion injury to the cerebral vasculature. Thus, this may represent a means of prolonging the window of opportunity to use a thrombolytic agent for treatment of ischemic stroke, and reduce the occurrence of hemorrhagic transformation in a clinical setting.
