TTC staining and measurement of the cortical infarct volume

To examine tissue viability and evaluate the infarct size, TTC staining was conducted. One day after MCAO/R injury, the animals were deeply anesthetized and transcardially perfused with 100 ml of ice-cold saline with 0.5% sodium nitrite and 10 U/ml heparin. The rat brains were rapidly extracted and sliced into 2 mm thick coronal sections using a Rat Brain Blocker (David Kopf Instruments, CA, USA). The coronal sections at the level of caudoputamen where the anterior commissure was observed (approximately 3 mm caudal from the bregma) were incubated in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich Inc., MO, USA) for 10 min at 37 °C and fixed with 10% formalin solution after staining. After taking a photograph with a digital camera (NX200, Samsung, Seoul, Korea), ImageJ (Ver 1.45) software (http://rsb.info.nih.gov/nih-image) was used for infarct volume analysis, as described previously¹⁹. The size of the infarct volume (n = 5) was measured as follows,

where V_infarct is the infarct volume of the brain, A_contra is the area of the contralateral brain, A_ipsi is the area of the ipsilateral brain, and A_infarct is the area of the infarct brain. We also measured the infarct volumes with p-FMMD (n = 5) and T2 MRI (n = 5). Statistical significance was assessed by independent Student’s t-test and one-way ANOVA followed by Dunnett’s test. Data are represented as the mean ± standard error of the mean (SEM). Values of p < 0.05 were considered significant.