*2.1.5 Culturing cerebral capillary endothelial cells*

*Antioxidants*

**2. Materials and methods**

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

technique in rats to simulate ischemic stroke.

*2.1.2 Transport measurements in membrane vesicles*

*2.1.3 Western blot analysis of membrane vesicles*

a significant number of eligible patients bleed anyway [18]. Thus, preventing injury to the cerebral vasculature when using a thrombolytic agent to induce reperfusion is of great importance. In this study, we provide evidence that reperfusion injury is associated with *oxidative* damage to brain capillary endothelial cells in the presence of elevated calcium. Furthermore, we show that the antioxidant γ-glutamylcysteine [19, 20] (a precursor of glutathione) together with an agent to prevent calcium sequestration inhibit oxidative injury to brain capillaries when co-administered

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

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.

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

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.

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

immediately prior to inducing reperfusion for ischemic stroke.

*2.1.1 Preparation of brain capillary endothelial membrane vesicles*

using radiolabeled tracers and a rapid filtration technique [27, 28].

*2.1.4 Polymerase chain reaction analysis of cerebral capillaries*

**198**

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 cAMP, as previously described [33–37].
