**2. Materials and methods**

To understand the mechanisms responsible for reperfusion injury to cerebral blood vessels, pertinent transport properties were first defined in isolated plasma membranes derived from brain capillary endothelial cells (i.e., the blood-brain barrier). This was followed by examining the effects of simulated ischemia and reperfusion on cultured blood-brain barrier endothelial cells, and testing the utility of experimental drugs in preventing cellular damage. Finally, the experimental therapeutic approach was tested *in vivo*, using the middle cerebral artery occlusion technique in rats to simulate ischemic stroke.

### **2.1** *In vitro* **studies**

#### *2.1.1 Preparation of brain capillary endothelial membrane vesicles*

Brain capillary endothelial cells are polarized and possess tight junctions (i.e., zonula occludens) [21]. Luminal (blood-facing) and abluminal (brainfacing) membrane vesicles were isolated from bovine brain capillary endothelial cells by methods that we have described in detail [10–12, 22, 23]. Briefly, cerebral capillaries were isolated from cow brains by homogenization, differential centrifugation, and separation on a column of glass beads. Following mild treatment with collagenase, the capillaries were further homogenized, and the endothelial membranes were separated in a discontinuous Ficoll gradient.

#### *2.1.2 Transport measurements in membrane vesicles*

The methods for quantifying transport measurements using membrane vesicles from brain capillary endothelial cells have been published by us [8, 9, 24–26]. Rates of substrate uptake by luminal and abluminal membrane vesicles were determined using radiolabeled tracers and a rapid filtration technique [27, 28].

#### *2.1.3 Western blot analysis of membrane vesicles*

NHE1 (Na/H antiporter, isoform 1) and NCX1 (Na/Ca exchanger, isoform 1) were identified in isolated plasma membranes from bovine brain capillary endothelial cells using immunoblotting with mouse monoclonal antibodies (Chemicon) and horseradish peroxidase-conjugated goat anti-mouse antibody, as previously described [29, 30]. The bands were analyzed by laser scanning densitometry.

#### *2.1.4 Polymerase chain reaction analysis of cerebral capillaries*

After mRNA was isolated from bovine cerebral capillaries [31], first-strand cDNA was synthesized using oligo-dT and AMV reverse transcriptase (Promega or

**199**

treatment.

treatment.

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

were scanned and quantified using NIH Image software.

*2.1.6 Simulating ischemia and reperfusion using cultured cells*

pre-equilibrated to the desired atmospheric conditions [39].

*2.1.7 Measuring intracellular calcium and sodium concentrations*

*2.1.8 Measuring actin stress fibers in cultured cells*

*2.1.5 Culturing cerebral capillary endothelial cells*

cAMP, as previously described [33–37].

Invitrogen), and sense and antisense primers (Sigma) were used to generate PCR products for NHE1, Na/K ATPase α2, and Na/K ATPase α3, as previously described [31, 32]. Sequencing was performed on both strands, using a commercial service. Quantitative Western blotting was done as previously described [30]. The bands

Cultured cerebral capillary endothelial cells were used as an *in vitro* model to measure the effects of simulated ischemia and reperfusion on blood-brain barrier function [33–37]. The capillary endothelial cells were isolated from bovine brain using the method of Meresse *et al*. [33], or purchased from Cell Systems Corporation (Kirkland, Washington). Cells were grown, maintained, seeded, and incubated in the presence of an astrocyte conditioned medium supplemented with

Cultured brain capillary endothelial cells were exposed to conditions simulating ischemia and reperfusion by incubating first at 37°C in an ischemic medium (without glucose, pH 6.8) equilibrated with an atmosphere of 95% N2 and 5% CO2, followed by simulated reperfusion in a control medium (5.6 mM glucose, pH 7.4) equilibrated with room air and 5% CO2 [38, 39]. To provide a constant environment, the cells were maintained in sealed chambers (Billups-Rothenberg, CA)

Intracellular calcium was quantified in cultured cerebral capillary endothelial cells by using a fluorescent probe and confocal laser microscopy, as previously described by us [39]. Cells were preloaded with 5 μM Fluo-4 [39] and treated under conditions of ischemia and reperfusion as described above. Calcium concentration was quantified by measuring emitted fluorescence [39, 40] at a wavelength of 494 nm in 50 randomly chosen (computer-assisted) cells, representing each

Intracellular sodium in cultured cerebral capillary endothelial cells was measured as previously described by us [39]. Cells were pre-treated with Sodium Green (5 μM), and the fluorescent signal was quantified by fluorescence microscopy. Measurements were made from 50 randomly chosen cells representing each

Cerebral capillary endothelial cells were grown on coverslips and exposed to conditions simulating normoxia, ischemia, and reperfusion as described above. Following treatment, the monolayers were washed in phosphate buffered saline (pH 7.4), fixed for 5 minutes in 3.7% buffered formaldehyde at room temperature, and rinsed again with the buffer [41]. A mixture of phalloidin (0.05 mg/ml buffer) and 1% dimethyl sulfoxide was added to the cells for 40 minutes at room temperature, in a humidified chamber. Following staining, the coverslips were washed with buffer and mounted in a mixture of 30% glycerol in 70% buffer (vol/vol). To determine the effects of calcium-mediated cytoskeletal activation, the cells were incubated in the presence of a myosin light chain kinase inhibitor (0.1 μM, Sigma).

*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*

Invitrogen), and sense and antisense primers (Sigma) were used to generate PCR products for NHE1, Na/K ATPase α2, and Na/K ATPase α3, as previously described [31, 32]. Sequencing was performed on both strands, using a commercial service. Quantitative Western blotting was done as previously described [30]. The bands were scanned and quantified using NIH Image software.
