Histological Analysis and Infarct Volume Measurement

Animals were euthanised 24 h post-stroke onset. They were transcardially perfused with saline and their brains were removed and sectioned into 6 coronal slices using a rat brain matrix, each of 2 mm thickness.

Triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Missouri, USA) staining was performed to confirm the presence of ischaemic stroke by identification of infarcted tissue. The slices from each brain were incubated for 12 min at 37°C in 2% TTC. TTC was used for early confirmation of infarct, however, haematoxylin and eosin (H&E) staining was then used on the same tissue for infarct volume quantification. Infarct volume quantification was carried out using our routine procedure (27). Tissues were fixed, processed, paraffin embedded and cut at 10 μm coronal sections. Images were scanned using a high-resolution scanner (Aperio, Vista, CA, USA) and analyzed by an investigator blinded to treatment allocation. Infarct (corrected for oedema) was calculated (Aperio ImageScope) by subtracting the measured interhemispheric volume difference from the measured infarct volume for each side. Infarct volumes were corrected for oedema by applying the formula: corrected infarct volume (mm³) = infarct volume × (contralateral volume/ipsilateral volume). Oedema was calculated by infarct volume minus corrected infarct volume (6).