Quantification of Cerebral Infarction in Mice

At 48 h after HI, the pups were deeply anesthetized with isoflurane. The brain was immediately isolated after perfusion with 10 ml ice-cold phosphate buffered saline (PBS) and stored at −20°C for 20 min and subsequently was sliced into 1.5 mm coronal sections. The sections were immersed in 2% TTC solution (Sigma-Aldrich, USA) at 37°C for 20 min in the dark. After washing with distilled H₂O, the sections were fixed in 4% paraformaldehyde for 20 min and photographed. The infarcted portions were identified as the white areas without TTC staining. The infarcted areas of each section were traced and measured using Image-Pro Plus 6.0 (U.S. National Institutes of Health, Washington, DC) (24 (link)). The total infarct volume was calculated by summation of the representative infarct volume of each section. The infarction percentage was calculated as follows: ([total contralateral hemispheric volume] – [total ipsilateral hemispheric stained volume]) / (total contralateral hemispheric volume × 2) × 100% (19 (link), 25 (link)).