52 Chapter 2 likely reflects the fundamentally different measurements of antibody binding (pixel counts) and autoradiography (ligand binding); the antibody detects the C terminus of the TSPO and autoradiography the binding of the ligand in the active site of the TSPO. Autoradiography has greater sensitivity because each molecule of radioligand contributes to the signal, whereas for immunohistochemistry a pixel is only TSPO+ if a threshold is reached. Hence when the levels of TSPO are low, and thus the signal of DAB positivity is very low, differences between autoradiography and the pixels counts may arise. Increased TSPO expression was also observed in leukocortical lesions in multiple sclerosis but not in other grey matter lesions75. This may well reflect the greater microglia and astrocyte activity in the white matter; pure cortical lesions in post mortem tissues show little or no inflammation57,76,77, although early lesions have been associated with prominent microglial activation78. Leukocortical lesions, which can have a white matter inflammatory component, may show differences in behaviour with inflammatory processes diffusing into the cortical grey matter. Similar considerations may hold with active leptomeningeal inflammation79,80. The spatial resolution of PET is low relative to cortical thickness, making confident assignment of TSPO PET signal to cortex (rather than adjacent white matter or meninges) problematic. While populations of patients undoubtedly show heterogeneity of grey matter pathology, our observations here raise questions about the interpretation of widespread TSPO PET signal in the cortical region as arising from cortical lesions81, as opposed to meningeal79 or leukocortical inflammation. Highly localised relative increases in TSPO signal in the cortex82, may represent a more rare, relatively acute cortical lesion, rather than what is more typically defined post mortem. Increased TSPO expression also has commonly been attributed to activated microglia/ macrophages and there is often an implication that the activation is pro-inflammatory64. Recently, we have shown in vitro that, although TSPO gene and protein expression and TSPO radioligand binding increases in rodent microglia and macrophages with inflammatory stimulation, relative TSPO gene expression in human microglia and TSPO ligand binding in humanmonocyte derivedmacrophages are reducedwith pro-inflammatory activation53. Here, we show that the increased TSPO expression in multiple sclerosis lesions primarily represents an intermediate activation state in which cells expressed both CD40 and CD20655,62; microglia solely expressing the pro-inflammatory marker CD40 or the anti-inflammatory marker mannose receptor CD206 were sparsely represented. This new observation thus confirms in vitro data suggesting that TSPO expression signal does not distinguish between microglial phenotypes in human tissue53. To the extent that increased TSPO PET signal can be used as a marker of microglia, it thus should be interpreted as reflecting increased microglial density rather than a change in activation state. In principle, chronic active lesions showing a high expression of TSPO in the rims and a lower expression in the core could be distinguished from either active lesions that show high expression of TSPO throughout their volume or from inactive lesions with low cell density and low TSPO expression. Those chronic active lesions with increased rim TSPO expression might identify those that will expand slowly over time. The neuropathological active lesions with homogeneously high TSPO expression may define those most likely to decrease in volume over time83,84. However, in practice, this discrimination could be confounded by the limited signal resolution of PET (typically 3-5 mm linearly) and consequent local signal averaging across commonly encountered lesions. This question deserves further investigations, e.g., by
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