70 Chapter 3 TSPO CONTROL ACTIVE P2RY12 HLA-DR TSPO / P2RY12 / HLA-DR TSPO TMEM119 HLA-DR TSPO / TMEM119 / HLA-DR A E D CONTROL ACTIVE G J M N I H L K B C F E F C B H I K L Figure 4. Microglia show a phenotypic shift in active lesions. Representative images of TSPO, P2ry12 and HLA-DR (a-f) in control white matter (a-c) and an active MS white matter lesion (d-f) showing both TSPO+P2ry12+HLA-DR- cells (b,e) as well as TSPO+P2ry12+HLA-DR+ cells (c,f). Representative images of TSPO, TMEM119 and HLA-DR (g-l) in control white matter (g-i) and an active white matter lesion (j-l) showing both TSPO+TMEM119+HLA-DR- cells (h,k) as well as TSPO+TMEM119+HLA-DR+ cells (i,l). Quantitative analysis showed a phenotypic shift and subsequent loss of P2ry12 expression in active lesion areas (m), as well as an increase in the cell density of microglia but a reduction in the percentage of TMEM119+ microglia/macrophages (n). Scale bar = 50 µm. NAWM = normal appearing white matter, CA = chronic active. Expression of homeostatic markers TMEM119 and P2ry12 by TSPO+HLA+ cells Expression of the homeostatic markers TMEM119 and P2ry12 is reported to be mostly limited to NAWM and inactive lesions19,20. To further characterise the relationship between TSPO expression and phenotypic markers, we performed triple staining of P2ry12 or TMEM119 in combination with HLA-DR and TSPO to identify whether TSPO+ cells expressed markers of both activated (HLA-DR) and homeostatic (TMEM119 and P2ry12) microglial cells. The triple stains showed that in control (Fig. 4A,G), NAWM, active (Fig. 4D,J), chronic active and inactive
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