*2.2.2 Measuring brain capillary mitochondrial morphology*

Mitochondrial damage indicative of reperfusion injury was measured in cerebral capillary endothelial cells of rats exposed for 1 hour to cerebral ischemia, followed by 24 hours of reperfusion. Cerebral ischemia and reperfusion was accomplished

**201**

(Neurolucida).

*2.2.3 Measuring brain apoptosis*

*2.2.4 Measuring neurological behavior*

**2.3 Analysis of the data**

**2.4 Vertebrate animals**

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

using the MCAO model of ischemic stroke. Stroked animals not treated with drugs were infused intravascularly (IV, femoral vein) with 1 ml of physiological saline approximately 1 minute prior to initiating reperfusion. Treated animals were co-administered γGlu-Cys (400 mg/kg) and KB-R7943 (10 mg/kg) IV in 1 ml of physiological saline 1 minute before reperfusion. The mitochondrial permeability transition was assessed by measuring a typical change in ultrastructural morphology characterized by swelling [47]. Following 60 minutes of transient ischemia and 24 hours of reperfusion, the animals were killed, and brain cortical tissue adjacent to the putamen was sampled and treated with a fixative and prepared for electron microscopy. Fixation involved 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer, and tissue blocks were osmicated, dehydrated in an ethanol series, cleared in propylene oxide, and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined on a Jeol JEM-1230 (HC) electron microscope. Measurements of mitochondrial cross sectional area in cerebral capillary endothelial cells were determined by examining electron micrographs and using computer-assisted morphometry. Briefly, random samples including blood-vessels from the outer cerebral cortical zones of the ipsilateral and contralateral sides were photographed for each animal. Five random samples included 3 blood vessels, 9 endothelial cells, and 45 mitochondria from each of 4 animals per treatment. These samples were measured by outlining the mitochondria with an electronic pen, and compiling the data with commercial stereological software

Evidence of apoptosis in the cerebral cortex of the above animals exposed to ischemia/reperfusion (1 hour/24 hours) was determined by using the Apop-Tag Kit (Oncor). Tissue sections were randomly sampled from the cortex, and prepared for immunocytochemistry using the Tunnel assay [48]. The number of stained nuclei were quantified per unit area, as determined with the Neurolucida program.

After 1 hour of ischemia and 24 hours of reperfusion, the above animals were observed for obvious neurological deficits indicated by behavioral changes.

For the measurements collectively described in this study, values were expressed as an average ± SD or SE. Means were compared by Student's t test, a paired t test, or an ANOVA, depending upon the conditions. Mean values were considered signifi-

Attention was focused on motor deficits, torticollis, and obvious paresis.

cantly different if the probability of their being the same was 0.05 or less.

Plasma membrane vesicles derived from cerebral capillary endothelial cells were isolated from cow brains obtained at a local slaughterhouse immediately after death. The cows were killed for food according to Federal and State laws, and not for the purposes of these experiments. Cultured cerebral capillary endothelial cells originated either from the fresh cow brains described above, or were purchased commercially (Cell Systems Corporation). The middle cerebral artery occlusion procedure *in vivo* was done using rats in the Blood–Brain Barrier Lab at Oregon

*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*

using the MCAO model of ischemic stroke. Stroked animals not treated with drugs were infused intravascularly (IV, femoral vein) with 1 ml of physiological saline approximately 1 minute prior to initiating reperfusion. Treated animals were co-administered γGlu-Cys (400 mg/kg) and KB-R7943 (10 mg/kg) IV in 1 ml of physiological saline 1 minute before reperfusion. The mitochondrial permeability transition was assessed by measuring a typical change in ultrastructural morphology characterized by swelling [47]. Following 60 minutes of transient ischemia and 24 hours of reperfusion, the animals were killed, and brain cortical tissue adjacent to the putamen was sampled and treated with a fixative and prepared for electron microscopy. Fixation involved 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer, and tissue blocks were osmicated, dehydrated in an ethanol series, cleared in propylene oxide, and embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate, and examined on a Jeol JEM-1230 (HC) electron microscope. Measurements of mitochondrial cross sectional area in cerebral capillary endothelial cells were determined by examining electron micrographs and using computer-assisted morphometry. Briefly, random samples including blood-vessels from the outer cerebral cortical zones of the ipsilateral and contralateral sides were photographed for each animal. Five random samples included 3 blood vessels, 9 endothelial cells, and 45 mitochondria from each of 4 animals per treatment. These samples were measured by outlining the mitochondria with an electronic pen, and compiling the data with commercial stereological software (Neurolucida).

### *2.2.3 Measuring brain apoptosis*

*Antioxidants*

ness of 4 microns.

endothelium.

of 510 nm.

(γGlu-Cys).

**2.2** *In vivo* **studies**

external carotid artery.

*2.2.2 Measuring brain capillary mitochondrial morphology*

*2.1.9 Measuring permeability of cultured cells*

*2.1.10 Measuring caspase activity of cultured cells*

*2.1.11 Measuring lysis/death of cultured cells*

*2.2.1 Middle cerebral artery occlusion*

The tissue was observed and evaluated using laser confocal microscopy, at a thick-

To quantify functional injury to brain capillaries, 14C-sucrose permeability was measured across a monolayer of cerebral capillary endothelial cells [42], under conditions simulating ischemia and reperfusion as described above. Cyclosporin A (1 μM) was used as an inhibitor of the mitochondrial permeability transition [43, 44], to determine if this process is associated with reperfusion injury to the

Caspase 3 activity was measured fluorometrically using an oncogene caspase-3 activity kit, as previously described [39]. Cultured blood-brain barrier cells were lysed, and the lysate was added to the caspase substrate: L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide (DEVD) tagged with the fluorescent molecule 7-amino-4-trifluoromethyl coumarin (AFC). Cleaved AFC was fluorescent, and was quantified at an excitation wavelength of 390 nm, and an emission wavelength

Lactate dehydrogenase (LDH) release was used to determine cell lysis, and was measured colorimetrically at a wave-length of 490 nm with a commercial kit (Promega). The effectiveness of various antioxidants [19, 20, 45] in preventing cell death was tested by incubating the cells in the presence (1 mM) and absence of glutathione (GSH), N-acetylcysteine (NAC), and gamma-glutamylcysteine

Ischemia and reperfusion were simulated in rats using the middle cerebral artery occlusion (MCAO) technique [46], by placing a thread in the left cerebral artery to reduce blood flow in the left hemisphere, and withdrawing it to re-establish circulation. Briefly, adult female Long-Evans rats (250–300 g) were anesthetized with isoflurane (5% induction and 2% for maintenance). The pterygopalatine artery and branches of the carotid artery were cauterized on the left side, after which a 3 centimeter length of 3.0 Dermalon suture (blunt tip) was introduced in a retrograde direction into the external carotid artery. It was advanced cranially in the internal carotid artery for 23 mm, as measured from the bifurcation of the common carotid artery. This model reduced cerebral blood flow to about 10% of control in the core of the ischemic area. Reperfusion was achieved by withdrawing the thread into the

Mitochondrial damage indicative of reperfusion injury was measured in cerebral capillary endothelial cells of rats exposed for 1 hour to cerebral ischemia, followed by 24 hours of reperfusion. Cerebral ischemia and reperfusion was accomplished

**200**

Evidence of apoptosis in the cerebral cortex of the above animals exposed to ischemia/reperfusion (1 hour/24 hours) was determined by using the Apop-Tag Kit (Oncor). Tissue sections were randomly sampled from the cortex, and prepared for immunocytochemistry using the Tunnel assay [48]. The number of stained nuclei were quantified per unit area, as determined with the Neurolucida program.

### *2.2.4 Measuring neurological behavior*

After 1 hour of ischemia and 24 hours of reperfusion, the above animals were observed for obvious neurological deficits indicated by behavioral changes. Attention was focused on motor deficits, torticollis, and obvious paresis.
