**3.4 Evidence for calcium-mediated alteration of the cytoskeleton**

Cultured blood-brain barrier endothelial cells were exposed to conditions simulating ischemia and reperfusion, as described above. Following treatment, the cytoskeletal component actin was stained with phalloidin (0.05 mg/ml) [41] and examined using confocal laser microscopy. Incubating cells for 120 minutes under control conditions revealed the usual configuration of actin, which forms an organized ring of fibrils just inside the plasma membrane. Incubating under ischemic conditions either did not alter this configuration at all, or had a relatively minor effect on cytoskeletal arrangement. However, exposing the cells to ischemic conditions for 90 minutes, followed by 30 minutes of control treatment (reperfusion) caused a remarkable reorganization of the actin (**Figure 4**), which assumed the usual stress fiber configuration associated with damage and increased permeability characteristics [44]. An inhibitor of calcium activated myosin light chain kinase [50] (Sigma, 0.1 μM) prevented the appearance of stress fibers observed during reperfusion (**Figure 4**).

#### **3.5 Evidence for alterations of permeability characteristics**

Cultured cerebral capillary endothelial cells were incubated in transwells under conditions simulating ischemia (90 minutes) and reperfusion (30 minutes), and permeability properties of the endothelial barrier were measured by quantifying unidirectional flux of 14C-sucrose across the endothelium [42]. **Figure 5** shows that selective inhibition of Na/H exchange, a brain capillary endothelial carrier shown earlier to contribute to calcium uptake (**Figure 3**) and structural damage (**Figure 4**) during reperfusion, caused a significant reduction in the observed permeability to sucrose.

#### **3.6 Evidence for mitochondrial dysfunction**

**Figure 6** shows that 1 μM Cyclosporin A (CsA), an inhibitor of mitochondrial damage and apoptosis [42, 43, 51], significantly reduced sucrose permeability across

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**Figure 5.**

**Figure 4.**

*from 4 sets of monolayers for each treatment.*

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

brain capillary endothelial cell monolayers associated with simulated ischemia (90 minutes) and reperfusion (30 minutes). In addition, **Figure 7** confirms that ischemia (30 minutes) followed by reperfusion (24 hours) greatly increased caspase 3 activity that is typically associated with apoptosis [51], and that this was significantly reduced with DMA (100 μM), an inhibitor of Na/H antiport and calcium loading (**Figure 3**). A specific inhibitor of caspase 3 activity (z-VAD-FMK, 10 μM)

*An inhibitor of Na/H antiport (100 μM DMA, R + DMA) significantly reduced the permeability (% of control) to 14C-sucrose across cultured brain capillary endothelial cells observed during 90 minutes ischemia/30 minutes reperfusion (reperfusion). \*P = 0.05. Values are mean ± SD, n = 4 0bservations.*

*Cultured blood-brain barrier cells were stained for actin with phalloidin following incubation under conditions of ischemia and reperfusion. Normally the actin forms a concentrated ribbon inside the plasma membrane, and ischemia alone causes only minor changes, if any. Simulated reperfusion (30 minutes) following ischemia (90 minutes), however, causes a random and diffuse rearrangement of actin, termed stress fibers (reperfusion without inhibitor). This pattern is typical of cell damage and enhanced permeability of passive solute markers through tight junctions. An inhibitor of calcium-activated myosin light chain kinase (sigma, 0.1 μM) completely prevented the appearance of stress fibers (reperfusion with inhibitor). These are representative fields* 

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

#### **Figure 4.**

*Antioxidants*

**Figure 3.**

**3.4 Evidence for calcium-mediated alteration of the cytoskeleton**

appearance of stress fibers observed during reperfusion (**Figure 4**).

**3.5 Evidence for alterations of permeability characteristics**

**3.6 Evidence for mitochondrial dysfunction**

Cultured blood-brain barrier endothelial cells were exposed to conditions simulating ischemia and reperfusion, as described above. Following treatment, the cytoskeletal component actin was stained with phalloidin (0.05 mg/ml) [41] and examined using confocal laser microscopy. Incubating cells for 120 minutes under control conditions revealed the usual configuration of actin, which forms an organized ring of fibrils just inside the plasma membrane. Incubating under ischemic conditions either did not alter this configuration at all, or had a relatively minor effect on cytoskeletal arrangement. However, exposing the cells to ischemic conditions for 90 minutes, followed by 30 minutes of control treatment (reperfusion) caused a remarkable reorganization of the actin (**Figure 4**), which assumed the usual stress fiber configuration associated with damage and increased permeability characteristics [44]. An inhibitor of calcium activated myosin light chain kinase [50] (Sigma, 0.1 μM) prevented the

*Simulating ischemia-reperfusion in cultured brain capillary endothelial cells resulted in a significant increase in intracellular sodium (panel a) during ischemia (120 minutes), that was maintained during ischemia followed by reperfusion (90/30 minutes). The rise observed during ischemia/reperfusion was prevented by inhibiting Na/H exchange (100 μM DMA). The same results were observed when measuring intracellular calcium (panel B). The rise in calcium during ischemia/reperfusion was significantly reduced with a specific inhibitor of the reverse movement of Na/Ca exchange (20 μM KB-R 7943). Intracellular calcium was also significantly reduced by DMA (100 μM (not shown). \*P < 0.05, different from control; + P < 0.05, different from ischemia/reperfusion. Values are mean ± SD. Measurements are made from 50 cells randomly chosen to represent each treatment.*

Cultured cerebral capillary endothelial cells were incubated in transwells under conditions simulating ischemia (90 minutes) and reperfusion (30 minutes), and permeability properties of the endothelial barrier were measured by quantifying unidirectional flux of 14C-sucrose across the endothelium [42]. **Figure 5** shows that selective inhibition of Na/H exchange, a brain capillary endothelial carrier shown earlier to contribute to calcium uptake (**Figure 3**) and structural damage (**Figure 4**) during reperfusion, caused a significant reduction in the observed permeability to sucrose.

**Figure 6** shows that 1 μM Cyclosporin A (CsA), an inhibitor of mitochondrial damage and apoptosis [42, 43, 51], significantly reduced sucrose permeability across

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*Cultured blood-brain barrier cells were stained for actin with phalloidin following incubation under conditions of ischemia and reperfusion. Normally the actin forms a concentrated ribbon inside the plasma membrane, and ischemia alone causes only minor changes, if any. Simulated reperfusion (30 minutes) following ischemia (90 minutes), however, causes a random and diffuse rearrangement of actin, termed stress fibers (reperfusion without inhibitor). This pattern is typical of cell damage and enhanced permeability of passive solute markers through tight junctions. An inhibitor of calcium-activated myosin light chain kinase (sigma, 0.1 μM) completely prevented the appearance of stress fibers (reperfusion with inhibitor). These are representative fields from 4 sets of monolayers for each treatment.*

**Figure 5.**

*An inhibitor of Na/H antiport (100 μM DMA, R + DMA) significantly reduced the permeability (% of control) to 14C-sucrose across cultured brain capillary endothelial cells observed during 90 minutes ischemia/30 minutes reperfusion (reperfusion). \*P = 0.05. Values are mean ± SD, n = 4 0bservations.*

brain capillary endothelial cell monolayers associated with simulated ischemia (90 minutes) and reperfusion (30 minutes). In addition, **Figure 7** confirms that ischemia (30 minutes) followed by reperfusion (24 hours) greatly increased caspase 3 activity that is typically associated with apoptosis [51], and that this was significantly reduced with DMA (100 μM), an inhibitor of Na/H antiport and calcium loading (**Figure 3**). A specific inhibitor of caspase 3 activity (z-VAD-FMK, 10 μM)

#### **Figure 6.**

*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.*

#### **Figure 7.**

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

served as an internal control. The non-mitochondrial caspase 8 pathway for apoptosis showed no significant response in these cells.

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

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

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per group).

**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

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

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

indicate that injury to cultured brain capillary endothelial cells exposed to ischemia/reperfusion is significantly reduced in the presence of known antioxidants [19, 20, 45] including native glutathione, and its precursors *N*-acetylcysteine, and

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

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

γ-glutamylcysteine.

indicate that injury to cultured brain capillary endothelial cells exposed to ischemia/reperfusion is significantly reduced in the presence of known antioxidants [19, 20, 45] including native glutathione, and its precursors *N*-acetylcysteine, and γ-glutamylcysteine.
