204 Chapter 9 combinations of microglial markers and TSPO. This study showed that the majority of cells expressing TSPO in control, NAWM, and active lesions are likely to be microglia/macrophages as the majority colocalised with either HLA-DR, IBA-1 or CD68. We also confirmed that in the centre of chronic active lesions and inactive lesions up to 70% of the cells expressing TSPO did not express microglia/macrophage markers and were thus considered to be astrocytes based on their morphology and this was investigated in Chapter 2. Additionally, our studies showed that in control and NAWM tissue TSPO+ microglia primarily express IBA1 or CD68, indicating that microglia HLA-DR expression arises during microglial activation under neuroinflammatory conditions. Some TSPO+ microglia expressed both markers of homeostasis (P2ry12 and TMEM119) and HLA-DR suggesting that some microglia might be in a transitional state towards activation or vice versa. To extend the findings from the neuropathology study in MS to other neurodegenerative and neuroinflammatory diseases we performed a similar in-depth study in ALS. In ALS, a higher TSPO PET signal in motor cortex, supplementary motor and temporal cortex in ALS patients with bulbar onset compared to healthy controls using [18F]‐DPA‐714 has been reported54. In addition a study using [11C]‐PK11195 showed increased binding in motor cortex, pons, frontal lobe regions and the thalamus in ALS patients compared to healthy controls, regardless of bulbar or limb onset38. More recent studies utilizing [11C]‐PBR28 report increased binding of TSPO PET in the motor cortex, but also in upper regions of the corticospinal tracts55-57. More specifically, increased binding was found in precentral gyri for limb‐onset ALS patients and in the brain stem for bulbar‐onset ALS patients, indicating that glial activation is present in clinically relevant neuroanatomical areas55,56 and providing an atlas of the disease neuropathology with a close relationship to the clinical phenotype. Correlating altered PET binding with MRI has shown that increased binding in the precentral gyri was associated with cortical thinning, reduced fractional anisotropy (FA) and higher diffusivity58,59. Overall, PET studies have found similar affected regions in ALS patients (motor cortex and the upper corticospinal tracts), and there is some evidence that astrocytes are also affected in ALS patients60. Furthermore, while there is no lack of PET studies in ALS4 most do not focus on glial cells and have limitations that restrict the ability to draw strong conclusions about the immunopathology in ALS. In addition, few studies involve large sample sizes or follow their subjects longitudinally. In Chapter 4 we found that while microglia express TSPO in ALS, most of the TSPO expression was originating in astrocytes in the spinal cord. This finding was consistent when looking at short and medium disease duration and was present regardless of looking at the ventral horn or lateral columns – indicating a widespread increase of TSPO+ astrocytes in the ALS spinal cord. Additionally, also in ALS, we found both homeostatic and activated microglia expressing TSPO, indicating that TSPO expression in multiple microglia states is most likely not MS-specific. To further extend our neuropathology studies in MS and ALS we also examined the common neurodegenerative disease AD. In AD, the hippocampus is one of themost affected regions61,62. When looking at TSPO PET studies investigating the hippocampus in AD there are conflicting findings. Some studies have found increases in TSPO PET, while some studies have found no differences. These discrepancies might be due to differences in cohort size, methodology, disease duration, radioligands, or therapeutics of the patients. In Chapter 4 we show that there no differences in the numbers of TSPO+ cells either homeostatic or activated microglia or astrocytes in the hippocampus compared to control cases. In addition, classical amyloid plaques in AD were not associated with a high number of TSPO+ microglia.
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