Focal Ischemia

After identification of CFA and RFA, rats assigned to the Ischemia group had six 0.7-mm diameter holes drilled over the CFA contralateral to the reaching forelimb. Relative to bregma and using a stereotaxic coordinate system in which anterior and lateral directions are positive, injection sites were located distributed in two rows mediolaterally (+2.5-mm and +3.5-mm), along three anteroposterior locations (+1.5-mm, +0.5-mm, and -0.5-mm). A focal, cortical ischemic infarct was induced with the vasoconstrictor, endothelin-1 (ET-1; 0.3 µg ET-1 dissolved in 1 µl sterile saline (~120 pmol)). Each hole was injected with 0.33 µL of ET-1 through a glass micropipette (160 µm o.d.) attached to a Hamilton syringe with the tip inserted to a depth of 1.5-mm below the cortical surface. Injections were made at a rate of 4-nl/sec by a programmable motorized micro-injector. The injection pipette remained in place for an additional 5 minutes after injection to minimize backflow of ET-1 during pipette removal from the cortex.