53 Translocator protein expression in multiple sclerosis combining longitudinal MRI monitoring of the evolution of individual lesions with TSPO PET. In the present study, TSPO expression in cells of the adaptive immune system was also investigated across the different lesion sub-types. A previous study has highlighted TSPO expression in several T-cell populations in blood54, but our study did not find a strong expression of TSPO in T-cells in the brain. On the other hand, we did see strong expression of TSPO in B-cells in active, chronic active, and inactive lesions. However, lymphocyte numbers are low, so these contribute only a negligible amount to the total TSPO+ cells and thus will not influence the TSPO PET signal significantly. TSPO expression in vascular endothelial cells in all blood vessels contributes to the diffuse TSPO PET signal in the healthy human brain85. Moreover, the endothelial component of TSPO PET, which is dependent on the vascular density in the brain, differs between regions, and may change with ageing and pathology85-87. We therefore investigated TSPO expression in endothelial cells by pixel count. In the white matter, endothelial TSPO expression did not contribute significantly to the total amount of TSPO expression (on average 5% of total TSPO pixels). However, as expected, we found an increase in endothelial contribution to TSPO expression relative to white matter as well as relative to grey matter lesions, which is consistent with the reported greater vascularity of grey verses white matter74 and the hypocellularity of grey matter lesions63. The abundance of TSPO expression in astrocytes in the centres of chronic active lesions may suggest a more important role for TSPO in the pathophysiology of multiple sclerosis than previous data suggested46. The human GFAP-driven conditional knockout of TSPO in mice resulted in significantly reduced severity of experimental autoimmune encephalomyelitis 88. In humans, activated microglia are capable of producing neurotoxic, reactive astrocytes in multiple sclerosis lesions89. The apparent effects of TSPO ligands to reduce pathology in animal disease models could be mediated through effects on astrocytes90,91. Similar to TSPO expression after cessation of lesion activity in multiple sclerosis, TSPO was found to be expressed long after recovery from experimental autoimmune encephalomyelitis and in cuprizone-mediated demyelination92,93, implicating a role of TSPO in regenerative processes such as remyelinating lesions, possibly through production of neurotrophic factors such as pregnenolone and progesterone94. These activities may be mediated by activated astrocytes. There are limitations to our study suggesting future work. HLA-DR, Iba1 and CD68 are often used as markers for microglia interchangeably. Although these markers may be co-expressed, some Iba1+ and CD68+ cells do not express HLA-DR95. This likely accounts for the TSPO+ cells with microglial morphology that did not express HLA-DR. These appear to constitute a fraction of up to 35% of the active lesions and the chronic active lesion rims, but confirmation of this is warranted. A more general problem is that characterisation of the complexity of microglial/macrophage activation phenotypes well ideally demands use of more than one or two markers. Future work employing cytokine expression assays and cell receptor markers simultaneously (e.g., using imaging mass cytometry) would add to their characterisation. In summary, our studies confirm that TSPO PET imaging provides a general marker of glial activation in multiple sclerosis, but emphasise that precise interpretations depend on the specific pathological context. With a single MRI scan, active, chronic active and inactive lesions cannot be distinguished well, leaving uncertainties regarding the interpretation of a paired TSPO PET scan. With other pathologies e.g., in Alzheimer’s disease96, the
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