72 Chapter 3 was investigated using the Sholl analysis. Using this approach we found no correlation between the complex morphological changes that microglia/macrophages undertake, as shown by a loss in intersections of microglia/macrophages in active lesions (Fig. 5C), and the amount of TSPO expression per cell (R2 = 0.006278, P = 0.3521) (Fig. 5D) when transitioning from an homeostatic to activated state. In summary, although the microglia and macrophages in active and rim of chronic active lesions express activation markers, the expression of TSPO at protein level in these cells is no different to the microglia in control and NAWM regions, which express homeostatic microglia/macrophage markers. Discussion TSPO is used widely as a target for PET imaging in studies of CNS inflammation. Many of these studies are interpreted assuming that TSPO is upregulated in activated glial cells such as microglia, macrophages and astrocytes with neuroinflammation21. This interpretation is based on studies of cellular expression of TSPO performed in rodents5-9. However, recent investigations have shown that TSPO expression does not increase in human microglia and macrophages in vitro after classical pro-inflammatory stimulation8, and that in the TSPO expression in microglia in the MS brain post mortem co-localizes with both classical proinflammatory and anti-inflammatory phenotypic markers11. Furthermore, an earlier study found that transformation of microglia to ameboid phagocytes is not necessary for maximal PK11195 binding22. In the current study, the expression of macrophage/microglia markers was examined in post mortem MS tissue to characterize the phenotype of cells expressing TSPO. We initially examined a large population of TSPO+ but HLA-DR- cells, presumed to be microglia based on morphology and the lack of GFAP expression11. Examination of IBA-1 and CD68 staining established that almost all cells were IBA-1+ or CD68+, and hence this population must be microglia/macrophages. To further characterize TSPO expression in microglia and macrophages, MS lesions were stained for combinations of microglia/macrophage markers with TSPO. Cells expressing TSPO and microglia/macrophage markers were most abundant in active lesion areas, as expected from PET data showing increased TSPO signal in lesions3,23-26. In control tissue, NAWM, active lesions and the rimof chronic active lesions very few TSPO+ cells were negative for themarkers IBA-1, CD68 or HLA-DR indicating that the majority of the TSPO+ cells in these areas are likely to be microglia. In the centre of chronic active lesions and in inactive lesion areas there were substantial numbers of TSPO+ cells that lacked expression of any microglial or macrophage marker. Indeed, up to 70% of TSPO+ cells do not colocalise with IBA-1, CD68 or HLA-DR in inactive lesions. This is consistent with our previous findings, showing that approximately 65% of the total population of TSPO+ cells in inactive lesions are astrocytes11,27,28. Although TSPO is also expressed in endothelial cells an can be found at low levels in oligodendrocytes and neurons, our data suggests that in the MS brain the largest proportion of TSPO PET signal in the white matter originates frommicroglia, except in the proportionally smaller volumes of inactive lesions, in which most of the signal must originate from astrocytes11,29-32. Microglia adopt intricate and complicated phenotypes that cannot be well defined by single markers19,33,34.Asexpected,usingarangeofmarkers,ourdatarevealedevidenceofaphenotypic
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