TTC staining

Following 24 h of reperfusion, 8 rats/group were anesthetized and subsequently euthanized. The whole brain was rapidly removed from each rat, harvested and placed in a -20˚C freezer for 20 min. From the beginning of the optic chiasm, the coronal plane was sectioned at intervals of 2 mm, which yielded exactly five slices. The slices were incubated in 2% TTC phosphate buffer and immersed in a constant-temperature water bath in the dark at 37˚C for 15 min, with stirring performed every 5 min. After staining, brain slices were fixed with 4% paraformaldehyde at 4˚C for 24 h. Non-ischemic regions appeared red, whereas infarcted regions were pale. ImagePro Plus software version 6.0 (Media Cybernetics, Inc.) was used to calculate the non-ischemic red-stained TTC areas of the cerebral hemispheres on the normal and ischemic sides of each slice of brain. The corrected ischemic area value was obtained by subtracting the two areas in accordance with a previously described method (14) to eliminate the effect of cerebral edema on the infarction volume. The area of each ischemic cerebral hemisphere was multiplied by its thickness (2 mm) and this value was added to find the entire approximate cerebral infarction volume. Finally, this approximate value was divided by the normal hemisphere volume to calculate the infarction volume rate. As previously described (14), the following formula was used to calculate cerebral infarction volume rate: (Total contralateral hemisphere area-ipsilateral hemisphere area)/(total contralateral hemisphere area).