Expression of growth factors on isolated brain microvessels and cerebral cortex sections.

a-c Representative confocal images of cerebral cortex isolated microvessels from EAE-affected mice, immunolabelled for CD31 (a, b) and for CD31/GFAP (c). a, b CD31 identifies the endothelial cells and appears localized at the junctional endothelial contacts (b, arrowheads). c GFAP+ astrocyte endfeet are recognizable in the close vicinity of the CD31-stained vessel wall. d-f Representative confocal images of cerebral cortex of EAE-affected WT mice processed for the detection of GFAP antibody/Vegfa mRNA and PDGFRβ antibody/Vegfr2 mRNA by dual RNAscope IHC/ISH. d GFAP+ perivascular astrocyte close to the vessel wall (V), and (e) perivascular astrocyte processes on a microvessel (V) show red fluorescent puncta corresponding to Vegfa ISH signal (arrows); note fluorescent puncta also in nuclei of neuron-like cells (arrowheads); f PDFGRα+ perivascular (PV), juxtavascular (JV) and parenchymal (Pa) OPCs show Vegfr2 ISH signal in the cell body (arrows). g, h Phase contrast microscopy images of the pelleted microvessels. i Real-time PCR analysis of mRNAs expression of Fgf2, Pdgfa and Tgfb in cerebral cortex of naïve and EAE-affected WT mice. The analysis by real-time PCR shows a significant increase in Fgf2 and Pdgfa levels in the isolated vessels of cerebral cortex from EAE-affected compared with WT mice. Instead, Tgfb expression shows no statistically significant difference. Nuclear counterstaining with TO-PRO-3 (a-c) and Sytox Green (d-f). Scale bars, a-f 25 μm and g, h 20 μm.