45 Translocator protein expression in multiple sclerosis R Glut-1/TSPO/DAPI Vimentin/TSPO/DAPI Q S100β/TSPO/DAPI S T Vimentin/TSPO/GFAP U V CON WM NAWM Active CA rim CA centre Inactive 0 250 500 750 1000 1250 TSPO+GFAP+ cells / mm2 **** **** **** **** **** **** **** **** CON WM NAWM Active CA rim CA centre Inactive 0 250 500 750 1000 1250 TSPO+HLA-DR+ cells / mm2 * ** * ** CON WM NAWM Active CA rim CA centre Inactive 0 250 500 750 1000 1250 TSPO+ cells / mm2 **** **** *** * NAWM B Control A TSPO/HLA-DR Active C CA rim D CA centre E Inactive F G NAWM J Control I Active K CA rim L O P CA centre M Inactive N TSPO/GFAP GFAP/TSPO/HLA-DR H OLIG2/TSPO TSPO/HLA-DR TSPO/GFAP Figure 1. Astrocyte and microglial expression of TSPO in white matter lesions. Representative images of TSPO expression in control (A, I) and multiple sclerosis lesions (B-F, J-N); NAWM (B, J), active (C, K), CA rim (D, L) and centre (E, M), and inactive (F, N) lesions. Expression of TSPO in HLA-DR- cells (black arrowheads; inserts C-F). Quantitative analysis of number of TSPO+ cells showed a significant increase up to five times in active and in the rim of chronic active lesions compared to control and NAWM (G). An 11- to 14-fold increase in TSPO+HLA-DR+ cells was found in active and the rim of chronic active lesions compared to control and NAWM (H). A five-fold increase in TSPO+GFAP+ cells was found throughout all lesion stages compared to control and NAWM contributing up to 25% of the TSPO+ cells (I-O, inserts). An overview of cellular TSPO signal of astrocytes and activated microglia macrophages (P) and oligodendrocytes (P, white arrowheads, insert). Representative images of astrocytic markers with TSPO expression in one multiple sclerosis lesion (Q-T). Biopsy material showing high TSPO expression in HLA-DR+ and GFAP+ cells (U,V, insert). *=P<0.05; **=P<0.01; ***=P<0.001; ****=P<0.0001. Scale bars (A-F, I-N, P, U, V) = 50 μm, (Q-T) = 12.5 μm. Inserts are digitally zoomed in to 800x., NAWM = normal appearing white matter, A =active, CA = chronic active, IA = inactive. TSPO expression in microglial phenotypes To investigate further whether TSPO expression in microglial cells defined a functionally specific phenotype, we tested for co-expression with more specific markers for microglia and their polarisation. TMEM119 is reported to differentiate activated microglia from myeloid-derived macrophages and has been used to identify CNS-resident microglia. P2RY12 is a marker that identifies homeostatic microglia in normal white matter. Markers for pro-
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