Quantitative Analysis of Brain Tissue

Slides stained for VEGF, MMP-9, GLUT1, laminin/GFAP, and desmin/α-SMA were scanned at 40 × magnification to digitalised files using Aperio Scanscope AT Turbo (VEGF, MMP-9, GLUT1; Leica Biosystems, Germany) or Aperio Scanscope FL (laminin/GFAP, desmin/α-SMA; Leica Biosystems, Germany), and viewed with Aperio ImageScope (v12.3.3 for Windows, Leica Biosystems, Germany). Using these files, images were extracted from each of three separate, non-overlapping areas of 500 μm × 500 μm within each of four regions of interest in the brain: the cortical grey mater (Cx), the periventricular white matter (PVWM), the subcortical white matter (sCWM), and the subventricular zone (SVZ). Analysis of these images was performed using Image J software (v2.0.0 for Mac; National Institutes of Health, Bethesda, MD, USA) using previously published techniques [46 (link)]. Image analysis and quantification were undertaken by two researchers independently (AB and TY) on coded slides so that all analysis was blinded to the animal group.