Surgery

The right middle cerebral artery (MCAO) was slowly lifted with a tungsten hook attached to a micromanipulator (Maerzhaeuser Precision Micro-manipulator Systems, Fine Science Tools) and electrocoagulated. Both common carotid arteries were then occluded by tightening pre-positioned thread loops for 90 min. Throughout surgery, anesthesia was maintained by spontaneous inhalation of 1-1.5% halothane in a mixture of 75% nitrous oxide and 25% oxygen. Body temperature was controlled at 37°C by a Homeothermic Blanket System (Harvard Apparatus). The local changes in blood flow were monitored using a laser Doppler device (Perimed, Stockholm, Sweden), and blood gases were measured at several time points during ischemia. A decrease in laser Doppler signals to <20% of control values is considered to be successful MCA occlusion.

After 90 minutes, the common carotid arteries were re-opened. Subsequent to survival times of 14 or 28 days, the rats were deeply anesthetized with 2.5% halothane in 75% nitrous oxide and 25% oxygen, and perfused with neutral buffered saline followed by buffered 4% freshly depolymerized paraformaldehyde. The brain was removed, post-fixed in 4% buffered paraformaldehyde for 24 hr , cryoprotected in 15% glycerol prepared in 10 mmol/l phosphate buffered saline, flash-frozen in isopentane and stored at -70°C until sectioning