**3.9** *In vivo* **evidence for inhibition of apoptosis**

Cerebral cortical tissue was randomly sampled from the animals above (1 hour ischemia/24 hours reperfusion), and the TUNEL assay was performed to determine if apoptosis was occurring. Representative tissue from 2 of 4 animals in each group is shown in **Figure 9**. The left panels show cerebral tissue from stroked animals without the drugs. Comparable regions are shown in the right panels, representing tissue from stroked animals that also received the drugs just prior to reperfusion. The cerebral tissue from stroked animals without the drugs showed prominent

*Antioxidants*

**206**

**Figure 7.**

**Figure 6.**

served as an internal control. The non-mitochondrial caspase 8 pathway for apopto-

*Caspase 3 activity was expressed in cultured blood-brain barrier cells exposed to conditions simulating ischemia-reperfusion. Twenty-four hours of reperfusion following 30 minutes of ischemia resulted in a large increase in caspase 3 activity that was inhibited with DMA (100 μM). \*P < 0.05 from control; +P < 0.05 from I/ rep. I/rep is ischemia plus reperfusion; I/R Na inh is ischemia-reperfusion with DMA inhibition; I/R Cp inh is* 

*Inhibiting the mitochondrial permeability transition with Cyclosporin A (1 μM, R + CsA) significantly inhibited the sucrose permeability (% of control) observed during 30 minutes of reperfusion (R control),* 

*following 90 minutes of ischemia. \*P < 0.05. Values are mean ± SD, n = 3 observations.*

*ischemia-reperfusion with a specific caspase inhibitor. Values are mean ± SD, n = 3 observations.*

We have previously shown that the antioxidant γ-glutamylcysteine reduces injury to cultured brain capillary endothelial cells [58] under conditions of simulated ischemia/reperfusion. Cells were incubated for 1.5 hours under ischemic conditions, followed by 3 hours of simulated reperfusion. The presence of 1 mM γ-glutamylcysteine significantly inhibited release of lactate dehydrogenase (LDH) into the incubation medium, thus reducing cell lysis. Additional new studies in our laboratory confirm that the antioxidants glutathione and *N*-acetylcysteine significantly (P<0.05) inhibit LDH release from cultured brain capillary endothelial cells under the same circumstances. Compared to cultured cells incubated under conditions simulating ischemia (1.5 hours) and reperfusion (3 hours) in the *absence* of these antioxidants, 1 mM glutathione and 1 mM *N*-acetylcysteine inhibited mean LDH release by factors of 0.51 and 0.45, respectively. Collectively, the data

sis showed no significant response in these cells.

**3.7 Evidence for the protective role of antioxidants**

#### **Figure 8.**

*Rats were subjected to conditions of transient ischemic stroke using middle cerebral artery occlusion for1 hour, followed by 24 hours of reperfusion. One group of stroked animals received cytoprotective drugs (i.e., KB-R 7943 [10 mg/kg] and γ-glutamylcysteine [400 mg/kg]) 1 minute prior to reperfusion, and a second group of stroked animals were administered a placebo. Lateral cerebral cortical tissue was prepared for electron microscopy, and the cross-sectional area of mitochondria in cerebral capillary endothelial cells was measured morphometrically. Two of four stroked animals without the drugs areshown in the left panels of the figure, and two of four stroked animals receiving the drugs are shown in the right panels. It is apparent that mitochondria are swollen in cerebral capillary endothelial cells of stroked animals without the drugs (left panels, solid arrows), suggesting the mitochondrial permeability transition [43] associated with apoptosis [51]. In some cases, damaged mitochondria have been extruded into the capillary lumen (left panels, dashed arrows). By contrast, the mitochondria of stroked animals given the drugs appear normal (right panels). When compared to mitochondria of the unstroked contralateral hemisphere (internal control), the percent increase in crosssectional area was significantly (P = 0.0015) greater for the stroked animals not given the drugs (67 ± 15 vs. 13 ± 12, mean ± SD, n = 4 animals per group).*

nuclear staining indicative of apoptosis, both in cerebral capillaries (arrows) and neural tissue. A morphometric analysis of the number of stained nuclei per unit area for 4 animals in each group (without drugs vs. with drugs) showed a significant difference (3.16 ± 2.00 grains/mm2 vs. 0.39 ± 0.47 grains/mm2 , P = 0.036). No staining was observed in tissue from the contralateral, unstroked cerebral hemispheres.

#### **3.10** *In vivo* **evidence for inhibition of neurological dysfunction**

To examine the effects of the drugs on neurological behavior following ischemic stroke, the animals for each group above were observed following 1 hour of ischemia and 24 hours of reperfusion. **Table 1** indicates that all 4 of the stroked animals without the drugs showed neurological deficits. Two additional stroked animals not receiving the drugs died. By contrast, 3 of 4 stroked animals that were given the drugs at the time of reperfusion showed no signs of neurological deficit. Furthermore, none of the animals receiving the drugs died.

**209**

**4. Discussion**

**Table 1.**

*the drugs (3.1 6 ± 2.00 grains/mm2*

*Neurological behavior following transient ischemic stroke.*

**Figure 9.**

*Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke*

The purpose of this study was to define cellular mechanisms that contribute to reperfusion injury of blood vessels within the brain following thrombolysis for ischemic stroke, and to identify pharmacological agents that may be used to prevent cerebral bleeding associated with thrombolytic treatment for stroke. It is known that administering the thrombolytic agent tPA after approximately 3–4.5 hours of ischemia may cause reperfusion injury to brain capillaries [9–11] that can result in cerebral hemorrhage and death [12, 13]. Since approximately 95% of patients with ischemic strokes do not reach the hospital in time to be properly evaluated and

*Brain tissue from the above rats subjected to transient ischemic stroke (1 hour ischemia/24 hours reperfusion) was probed for evidence of apoptosis, using the TUNEL assay. Two of four stroke animals without and with the cytoprotective drugs (left and right panels, respectively) are depicted. In this figure, immunocytochemical nuclear reactions indicative of apoptosis are clearly visible in the animals without the drugs (left panels), but are not observed in the animals that were given the drugs (right panels). All four stroked animals without the drugs demonstrated apoptosis in blood-brain barrier endothelial cells (arrows) and neural tissue. Quantifying the number of stained nuclei per unit area showed a significant difference between animals without and with* 

*, mean ± SD, n = 4 animals per group, P = 0.036).*

 *vs. 0.39 ± 0.47 grains/mm2*

*DOI: http://dx.doi.org/10.5772/intechopen.84774*

*Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke DOI: http://dx.doi.org/10.5772/intechopen.84774*

#### **Figure 9.**

*Antioxidants*

**208**

**Figure 8.**

ference (3.16 ± 2.00 grains/mm2

*12, mean ± SD, n = 4 animals per group).*

nuclear staining indicative of apoptosis, both in cerebral capillaries (arrows) and neural tissue. A morphometric analysis of the number of stained nuclei per unit area for 4 animals in each group (without drugs vs. with drugs) showed a significant dif-

*Rats were subjected to conditions of transient ischemic stroke using middle cerebral artery occlusion for1 hour, followed by 24 hours of reperfusion. One group of stroked animals received cytoprotective drugs (i.e., KB-R 7943 [10 mg/kg] and γ-glutamylcysteine [400 mg/kg]) 1 minute prior to reperfusion, and a second group of stroked animals were administered a placebo. Lateral cerebral cortical tissue was prepared for electron microscopy, and the cross-sectional area of mitochondria in cerebral capillary endothelial cells was measured morphometrically. Two of four stroked animals without the drugs areshown in the left panels of the figure, and two of four stroked animals receiving the drugs are shown in the right panels. It is apparent that mitochondria are swollen in cerebral capillary endothelial cells of stroked animals without the drugs (left panels, solid arrows), suggesting the mitochondrial permeability transition [43] associated with apoptosis [51]. In some cases, damaged mitochondria have been extruded into the capillary lumen (left panels, dashed arrows). By contrast, the mitochondria of stroked animals given the drugs appear normal (right panels). When compared to mitochondria of the unstroked contralateral hemisphere (internal control), the percent increase in crosssectional area was significantly (P = 0.0015) greater for the stroked animals not given the drugs (67 ± 15 vs. 13 ±* 

was observed in tissue from the contralateral, unstroked cerebral hemispheres.

To examine the effects of the drugs on neurological behavior following ischemic stroke, the animals for each group above were observed following 1 hour of ischemia and 24 hours of reperfusion. **Table 1** indicates that all 4 of the stroked animals without the drugs showed neurological deficits. Two additional stroked animals not receiving the drugs died. By contrast, 3 of 4 stroked animals that were given the drugs at the time of reperfusion showed no signs of neurological deficit.

**3.10** *In vivo* **evidence for inhibition of neurological dysfunction**

Furthermore, none of the animals receiving the drugs died.

vs. 0.39 ± 0.47 grains/mm2

, P = 0.036). No staining

*Brain tissue from the above rats subjected to transient ischemic stroke (1 hour ischemia/24 hours reperfusion) was probed for evidence of apoptosis, using the TUNEL assay. Two of four stroke animals without and with the cytoprotective drugs (left and right panels, respectively) are depicted. In this figure, immunocytochemical nuclear reactions indicative of apoptosis are clearly visible in the animals without the drugs (left panels), but are not observed in the animals that were given the drugs (right panels). All four stroked animals without the drugs demonstrated apoptosis in blood-brain barrier endothelial cells (arrows) and neural tissue. Quantifying the number of stained nuclei per unit area showed a significant difference between animals without and with the drugs (3.1 6 ± 2.00 grains/mm2 vs. 0.39 ± 0.47 grains/mm2 , mean ± SD, n = 4 animals per group, P = 0.036).*


	-
	-
	-
	-

#### **Table 1.**

*Neurological behavior following transient ischemic stroke.*

#### **4. Discussion**

The purpose of this study was to define cellular mechanisms that contribute to reperfusion injury of blood vessels within the brain following thrombolysis for ischemic stroke, and to identify pharmacological agents that may be used to prevent cerebral bleeding associated with thrombolytic treatment for stroke. It is known that administering the thrombolytic agent tPA after approximately 3–4.5 hours of ischemia may cause reperfusion injury to brain capillaries [9–11] that can result in cerebral hemorrhage and death [12, 13]. Since approximately 95% of patients with ischemic strokes do not reach the hospital in time to be properly evaluated and

safely administered a thrombolytic agent [18], the possibility of cerebral hemorrhage severely limits the treatment of stroke with tPA.

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 (apoptosis).

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 cells, under conditions of ischemia and reperfusion.

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 *in vivo* [64].

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

**211**

clinical setting.

reperfusion (**Table 1**).

**5. Conclusions**

*Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke*

blood-brain barrier endothelial cells exposed to conditions simulating ischemia and reperfusion. Permeability was significantly reduced by co-incubating with cyclosporin A (**Figure 6**), an inhibitor of the mitochondrial permeability transition associated with the early stages of apoptosis [42, 43, 51]. Under similar conditions, cultured blood-brain barrier endothelial cells expressed a large increase in caspase 3 activity after 24 hours of simulated reperfusion following ischemia (**Figure 7**),

These *in vitro* findings were consistent with our working hypothesis which predicted that elevated intracellular calcium and reactive oxygen species would activate apoptosis in cerebral capillary endothelial cells exposed to conditions of ischemia and reperfusion. Furthermore, the results suggested that: (1) inhibiting reverse movement of Na/Ca exchange with KB-R7943 [65], and (2) replenishing lost antioxidant with γGlu-Cys [19, 20] would prevent reperfusion (oxidative) injury to brain capillaries. Since γGlu-Cys is a precursor of the antioxidant glutathione [59] lost during ischemia, and γGlu-Cys itself possesses antioxidant properties

[19, 20], it represents a reasonable antioxidant therapeutic in this setting.

The next logical step was to determine if drugs that prevent increased levels of intracellular calcium and reactive oxygen species in brain capillary endothelial cells would inhibit damage to cerebral capillaries *in vivo*. Thus, rats were exposed to middle cerebral artery occlusion to simulate ischemic stroke, after which the animals were either treated with a placebo (isotonic saline), or administered a combination of γGlu-Cys (400 mg/kg) and KB-R7943 (10 mg/kg) in isotonic saline that was infused intravenously approximately 1 minute prior to initiating reperfusion of cerebral blood flow. The rationale was that administering the combination of drugs immediately before re-establishing cerebral blood flow would protect the endothelial cells of cerebral capillaries from oxidative injury upon reperfusion. **Figure 8** indeed illustrates that such a therapeutic approach significantly inhibited morphological damage to cerebral capillary endothelial cells, including swelling of the mitochondria that is indicative of oxidative injury and the permeability transition that precedes apoptosis [51, 61, 62]. Furthermore, the Tunnel assay revealed that co-infusion of the drugs immediately before reperfusion inhibited the appearance of apoptosis in representative cerebral cortical tissue 24 hours after re-establishing blood flow to the brain (**Figure 9**). Finally, an assessment of neurological behavior confirmed that use of the drugs inhibited functional damage due to ischemia and

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

*DOI: http://dx.doi.org/10.5772/intechopen.84774*

indicating activation of the apoptotic pathway [51].

#### *Prevention of Oxidative Injury Associated with Thrombolysis for Ischemic Stroke DOI: http://dx.doi.org/10.5772/intechopen.84774*

blood-brain barrier endothelial cells exposed to conditions simulating ischemia and reperfusion. Permeability was significantly reduced by co-incubating with cyclosporin A (**Figure 6**), an inhibitor of the mitochondrial permeability transition associated with the early stages of apoptosis [42, 43, 51]. Under similar conditions, cultured blood-brain barrier endothelial cells expressed a large increase in caspase 3 activity after 24 hours of simulated reperfusion following ischemia (**Figure 7**), indicating activation of the apoptotic pathway [51].

These *in vitro* findings were consistent with our working hypothesis which predicted that elevated intracellular calcium and reactive oxygen species would activate apoptosis in cerebral capillary endothelial cells exposed to conditions of ischemia and reperfusion. Furthermore, the results suggested that: (1) inhibiting reverse movement of Na/Ca exchange with KB-R7943 [65], and (2) replenishing lost antioxidant with γGlu-Cys [19, 20] would prevent reperfusion (oxidative) injury to brain capillaries. Since γGlu-Cys is a precursor of the antioxidant glutathione [59] lost during ischemia, and γGlu-Cys itself possesses antioxidant properties [19, 20], it represents a reasonable antioxidant therapeutic in this setting.

The next logical step was to determine if drugs that prevent increased levels of intracellular calcium and reactive oxygen species in brain capillary endothelial cells would inhibit damage to cerebral capillaries *in vivo*. Thus, rats were exposed to middle cerebral artery occlusion to simulate ischemic stroke, after which the animals were either treated with a placebo (isotonic saline), or administered a combination of γGlu-Cys (400 mg/kg) and KB-R7943 (10 mg/kg) in isotonic saline that was infused intravenously approximately 1 minute prior to initiating reperfusion of cerebral blood flow. The rationale was that administering the combination of drugs immediately before re-establishing cerebral blood flow would protect the endothelial cells of cerebral capillaries from oxidative injury upon reperfusion. **Figure 8** indeed illustrates that such a therapeutic approach significantly inhibited morphological damage to cerebral capillary endothelial cells, including swelling of the mitochondria that is indicative of oxidative injury and the permeability transition that precedes apoptosis [51, 61, 62]. Furthermore, the Tunnel assay revealed that co-infusion of the drugs immediately before reperfusion inhibited the appearance of apoptosis in representative cerebral cortical tissue 24 hours after re-establishing blood flow to the brain (**Figure 9**). Finally, an assessment of neurological behavior confirmed that use of the drugs inhibited functional damage due to ischemia and reperfusion (**Table 1**).
