Simultaneous Stain with Neutral Red and Tetrazolium Salt for Experimental Cerebral Infarction Morphometry

Seo, Jung Ho; Park, Youn Kwan; Kim, Myung Hyun; Chong, Yong Gu; Chung, Heung Seob; Suh, Jung Kun; Lee, Hoon Kap; Chu, Jeong Wha; Lee, Ki Chan

Abstract

Serial changes in the size of infracted area induced by MCA occlusion(MCAO) were compared with Neutral Red(NR) and 2, 3, 5-Triphenyl tetrazolium chloride(TTC) stains. The differences in size of the infracted area as shown by the 2 stains and its significance were also evaluated. The experimental animals were divided into 7 troups, with each group consisting of rats;these groups were stained at 2, 4, 6, 8, 12, 24 and 48 hours after MCAO. After MCAO, NR was infused into the femoral vein, after which the brain was removed, the fraontal pole of the brain cut into 1.5mm sections, and each section photographed. Then, the NR-stained sections were immersed in TTC solution for 45 minutes and photographed. Results showed that the infracted area progressively increased according to time duration after MCAO(one-way ANOVA, p<0.01). Between 4 and 6 hour groups, the difference of the infracted area was greater than at any other timed groups, this being statistically significant(unpaired t-test, p<0.05). After 6 hours, the infracted area with NR stain became relatively stable. In contrast, however, the infracted area with TTC stain did not stabilize, but continued to increase up to 24 hours. Overall, the infracted area with NR stain was greater than with TTC stain in all the timed groups(paired t-test, p<0.05). As time progressed, the differences tended to decrease 48 hours post occlusion. In our study, serial changes of the ischemic penumbra area were evaluated by staining the ischemic area simultaneously with Neutral red and TTC stain. The results suggest that the ischemic penumbra area may still persist even after 48 hours post-MCAO.