68 Chapter 3 The number of microglia and macrophages expressing all three markers was approximately 18-fold greater in active lesions compared to control (P<0.0001) or to NAWM (P<0.0001) (Fig. 2F). This large increase is a combination of 1) an increase in the percentage of microglia and macrophages which are triple positive, as described above, and 2) an increase in microglia and macrophage density in active lesions of approximately 4-fold. In inactive lesions, the microglia and macrophage population was substantially smaller than in control and NAWM regions, and the percentage of triple positive cells was lower, too, contributing only approximately 15% of the microglia and macrophage population. In summary, most microglia and macrophages express IBA-1 and CD68 in control and NAWM, while in active MS lesions, many microglia and macrophages also express HLA-DR. Identity of TSPO+ cells in MS lesions As we reported previously11 TSPO+HLA- cells were observed in control, NAWM and lesional white matter (Fig. 3A). TSPO+HLA- cells are most abundant in the centres of chronic active lesions and in inactive lesions. We have previously shown that the TSPO+HLA- cells are astrocytes in these regions11. In other regions, the TSPO+HLA- population is approximately 30% lower, but, and the identity of these cells has not been made clear. Morphologically these TSPO+HLA- cells resemble microglia or macrophages. To test the hypothesis that these are indeed macrophages and/ or microglia, we performed triple-immunostaining for TSPO and different pairs of three microglial and macrophage markers; TSPO/HLA-DR/IBA (Fig. 3B), TSPO/HLA-DR/CD68 (Fig. 3C), and TSPO/IBA-1/CD68 (Fig. 3D). All three triple stains (Fig. 3E-G) showed an increase of triple positive cells in active lesion areas compared to control and NAWM. The triple stain for TSPO, IBA-1 and CD68 (Fig. 3J) showed that in control and NAWM and in the rims of chronic active lesions, almost all TSPO+ cells were microglia and macrophages. In control tissue, every TSPO+ cell was also double positive for IBA-1 and CD68 (n=36 cells / mm2) (Fig. 1S). In NAWM, every TSPO+ cell (n=35 cells / mm2) expressed at least one of IBA-1 or CD68, and most (88%) were double positive (Fig. 3J). In the rim of chronic active lesions, 98% of TSPO+ cells were double positive for IBA-1 and CD68. In active lesions, only 88% of TSPO+ cells expressed at least one of IBA-1 or CD68. However, the majority of the remaining 12% of cells which were IBA-1 and CD68 negative are likely to be microglia/macrophages, because the triple stains which included HLA-DR, instead of CD68, showed that only 6% of TSPO+ cells were HLA-DR and IBA-1 negative (Fig. 3H,I). As expected, the percentage of TSPO+ cells which were negative for microglia/macrophage markers was high in inactive lesions (75%) and in the centre of chronic active lesions (25%). These percentages are consistent with our previous work which identified these cells as astrocytes11. In summary, almost all TSPO+ cells in control, NAWM, active lesions, and the rims of chronic active lesions are microglia and/or macrophages, although, in the centres of chronic active lesions and inactive lesions, there is a substantial proportion of TSPO+ cells which are astrocytes. Figure 3. Co-localisation of TSPO with microglia/macrophage markers in white matter lesions in MS. Images of TSPO+HLA-DR- cells in active lesions in MS (a), and images showing colocalisation between TSPO and HLA-DR/IBA-1 (b), HLA-DR/CD68 (c), IBA-1/CD68 (d). Microglia markers are increased in active lesions (e-g). Percentages showing that nearly all TSPO+ cells in white matter MS lesions colocalise microglia/macrophage markers (h-j). **P<0.01, ***P<0.001, ****P<0.0001. Scale bar = 50 µm. NAWM = normal appearing white matter, CA = chronic active.
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