CD13/NG2-based morphometry detects activated pericytes in EAE.

Representative confocal images of cerebral cortex sections double-immunolabelled for CD13 and NG2 (a-c, e-k) and for PDGFRβ and NG2 (d, h, l) to identify and quantify NG2+ activated pericytes in naïve (a-d) and EAE-affected (e-l) WT mice. A high number of hypertrophic, JV OPCs, which overexpress NG2 together with numerous CD13+/NG2+ activated pericytes are recognizable in cortical layers from 2 to 6 in EAE-affected (e-j), compared with naïve (a, b) WT mice. High magnification of single optical planes on ‘z’ axis (_z24 and _z04, _z21 in naïve and EAE-affected WT mice, respectively) localizes NG2 molecules on the plasma membrane around the cell nucleus, where the proteoglycan in part co-localizes with CD13 (c, g, k). PDGFRβ+/NG2+ pericytes are recognizible in cortical microvessels, NG2 molecules preferentially localize in cell bodies, whereas PDGFRβ in pericyte processes (d, h, l). m The bar chart shows a significant increase of activated pericytes in EAE-affected WT mice at 20 dpi, measured as the percentage of NG2+ pericytes over the total number of CD13+ pericytes ($p = 0.005; n = 4; unpaired Student’s t-test). n The percentageof NG2+ pericytes over the total number of PDGFRβ+ pericytes is similar in naïve and EAE-affected WT mice (p = 0.78; n = 4; unpaired Student’s t-test). EAE-affected WT mice (at 20 dpi: clinical score range = 2.0–3.5, mean number of counted PDGFRβ+ pericytes = 729 per mouse), EAE-affected NG2KO (at 20 dpi: clinical score range = 1.5–2.5, mean number of counted PDGFRβ+ pericytes = 459 per mouse). Nuclear counterstaining with TO-PRO-3. Scale bars, a-l 15 μm.