Brain edema model (cold lesion model). Mice were mounted in a stereotaxic frame (Narishige), after which the scalp was incised, subcutaneous tissue was retracted from the bone, and the skull was exposed. Using a drill, a circular craniotomy was then carried out over the right parietal cortex, extending from the lambda suture to bregma, and the resultant bone flap was lifted off to expose the underlying dura. The cold lesion was made using a copper cylinder (3 mm in diameter) that had been precooled with liquid nitrogen. The metal probe was lowered quickly onto the surface of the intact dura over the parietotemporal cortex under microscopic control and pressed down to a depth of 1 mm for 30 seconds (48).

To quantify the vascular permeability of brain vessels, 0.2 ml of sodium fluorescein at a concentration of 6 mg/ml in PBS was injected via the tail vein 24 hours after making the cold lesions. Thirty minutes later, the mice were anesthetized and perfused with PBS (20 ml) via the left cardiac ventricle to remove the fluorescent tracer from the vascular bed. To assess their fluorescence, brain hemispheres were homogenized in 0.5 M borate buffer (pH 10) and centrifuged (800 g) for 15 min at 4°C, after which the supernatant was added to 1.2 ml of ethanol to precipitate the proteins. The samples were again centrifuged, and the fluorescence in the supernatant was measured using a Multi-Detection Microplate Reader (49); the excitation and emission wavelengths were 330 nm and 485 nm, respectively.