44 Chapter 2 Results Heterogeneity of TSPO+ cells in multiple sclerosis lesions Expression and localisation of TSPO+ cells were investigated in NAWM and in active, chronic active and inactive white matter lesions from brains and spinal cord of people with multiple sclerosis and in control tissue from people who died of non-neurological diseases (Fig. 1AF). TSPO immunostaining had a punctate appearance across the cytoplasm of cells in both the controls and in cells from multiple sclerosis tissue, as is expected with a mitochondrial protein. The density of TSPO+ cells per mm2 was a five-fold greater in active white matter lesions (P<0.0001) and four-fold greater in rims of chronic active brain lesions (P=0.0001) compared to control white matter. Compared to NAWM, the density of TSPO+ cells was a mean of two-fold higher for active lesions (P=0.004) and chronic active lesions (P=0.0170, Fig. 1G). We next investigated the characteristics of the TSPO+ cells. HLA-DR+ cells expressing TSPO were 11- to 14-fold more abundant in active lesions and chronic active lesion rims compared to the control white matter (active: P=0.0220, CA rim: P=0.0022; Fig. 1H) and 16- to 21-fold greater than NAWM (active: P=0.0196, CA rim: P=0.0019, Fig. 1H). TSPO+HLA-DR- cells with an astrocytic morphology expressing GFAP were found in all lesion sub-types (Fig. 1I-N). Between 15- to 20-fold more TSPO+GFAP+ cells were observed in all lesion sub-types relative to control white matter (P<0.0001, Fig. 1O). The relative number of TSPO+GFAP+ cells in lesions was five to seven-fold greater than in the NAWM (P<0.0001, Fig. 1O). HLA-DR+ microglia/macrophages expressing TSPO accounted for a mean of 40% of total TSPO+ cells in active lesions and in the rims of chronic active lesion (Fig. 1P) and GFAP+ astrocytes constituted about 25% of TSPO+ cells in active lesions and in the rims of chronic active lesions. However, in the centre of chronic active and in inactive lesions TSPO+GFAP+ astrocytes represented as many as 65% of the TSPO+ cells although it must be emphasised that the center of these lesions are hypocellular relative to the chronic lesion rims or the NAWM. In active lesion areas, a few cells were found co-expressing TSPO and Olig2, an oligodendrocyte marker (Fig. 1P, insert). TSPO+ astrocytes did not only colocalise with GFAP but also with vimentin, glut-1 and S100β (Fig. 1Q-T). Most of the remaining fraction of TSPO+ cells that did not express HLADR or GFAP had microglial or macrophage morphology but were not further characterised. To examine if the same is true of lesions in early multiple sclerosis we used biopsy material from rapidly expanding active white matter lesions from 4 cases of multiple sclerosis. All samples also showed strong expression of TSPO in HLA-DR+ macrophages and microglia (Fig. 1U), as well as in scattered GFAP+ astrocytes (Fig. 1V). Together, microglia and to a lesser extent astrocytes in multiple sclerosis lesions appeared to account for most of the TSPO+ cells in lesions and NAWM in the multiple sclerosis brains. TSPO+ vascular endothelial cells also were identified commonly but made up less than 5% of the TSPO+ cells. Vascular TSPO expression patterns in multiple sclerosis lesions were not different from their expression in blood vessels of the NAWM and in control tissue.
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