71 Activated microglia do not upregulate translocator protein lesions P2ry12+TSPO+ cells as well as TMEM119+TSPO+ cells are found with (Fig. 4C,F,I,L) and without (Fig. 4B,E,H,K) HLA-DR expression. As expected, all TSPO+ cells in control and NAWM, which we demonstrated above are microglia or macrophages, express both P2ry12 and TMEM119. Also as expected, TSPO+ cells in active lesions and the rim of chronic active lesions, which again we have demonstrated above are microglia and macrophages, showed evidence of altered phenotype compared to control and NAWM. In these lesion areas, P2ry12 expression is lost in almost all cells (Fig. 4M), TMEM119 expression is lost in approximately 50% of cells, and HLA-DR expression increases so that it appears in almost all cells (Fig. 4N). TSPO pixels per cell do not change across regions with different microglial phenotypes and morphology We have shown that almost all TSPO+ cells in control, NAWM, active lesions and rim of chronic active lesions are microglia and macrophages. We have also shown that, as expected, microglia and macrophages in the active lesions and rim of chronic active lesions express activation markers. Therefore, the TSPO expression per cell was compared across the different regions of white matter to determine whether it is increased when the microglial cells are activated compared to when the cells are homeostatic. To do this, the number of TSPO+ pixels across 12,225 TSPO+ cells were analysed (Fig. 5A-C). The percentages of pixels that were TSPO+ were similar across all of the regions of the white matter (control, 0.036% (±0.015), NAWM, 0.033% (±0.008), Active lesions; 0.041% (±0.014), CA rim; 0.038% (±0.014)) and were not statistically significant (Fig. 5A). Furthermore, nearly all the cells in white matter lesions that express a microglia/macrophage marker are positive for TSPO (Fig. 5B). To further investigate the amount of TSPO expression in microglia/macrophages on a per cell basis, the morphology A B C D Percentage posi�ve for TSPO Marker CON NAWM Ac�ve CA Rim CA Centre Inac�ve HLA-DR 76,6 98,7 99,8 98,5 99,3 100 CD68 73,2 72,1 100 99,2 97,8 98,7 IBA-1 83,7 97,3 100 98,7 97,5 96,6 P2ry12 58,7 71,4 100 100 86,5 77,9 TMEM119 73,8 41,8 99,7 99,3 81,6 66,9 CD40 100 100 100 100 100 100 MR 99,3 97,4 99,8 99,9 99,8 99,2 Figure 5. TSPO pixel count does not change across regions with different microglial morphology. The overall TSPO+ pixels per TSPO+ cell did not change in active lesion areas compared to control white matter and NAWM p>0.05 (a). A table showing the percentages of microglia/macrophages that were positive for TSPO subdivided by lesion type (b). Microglia lose their complex morphology in active lesions, shown by a loss of intersections (c). No correlation was observed between the amount of TSPO+ area and the morphology of microglia in control, NAWM or active lesions (d). MR = mannose receptor, NAWM = normal appearing white matter, CA = chronic active.
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