**7. Conclusions**

replicated. Immunohistochemistry is not a quantitative technique due to the amplification technologies used along with the non-availability of standards for comparison, but the studies by Boche and colleagues using semi-quantitative measures deserve mention [10, 14, 66, 67]. Using a large series of brain samples and the expression of different markers, including CD64, MSR-A, CD68, HLA-DR and IBA-1, it was shown that microglia could be subtyped depending on their profile. These studies set the standards for microglial profiling in human brains. These studies employed two measures for analysis; the area of immunoreactivity and the numbers of positive cells. These studies attempted to assign phagocytic function or activation function to the microglia in relation to the type of pathology. One interesting observation was the lack of significant correlation between expression levels of these different antigens by microglia. These markers are related to different functions, with CD64, MSR-1, and C68 being related to phagocytosis, HLA-DR with antigen presentation and IBA-1 with microglial motility. Studies of TREM-2 and CD33 in AD brains demonstrated upregulated expression in AD brains, but both receptors induce inhibitory signaling when activated [55, 56]. There is evidence that upregulation of such activated microglial proteins encountered in disease tissue could be to have an inhibitory effect on inflammation, not amplifying inflammatory

In recent years, gene discovery methodology (RNA sequencing, microarrays, single cell sorting) has been applied to studies of microglia. One particularly interesting marker identified is the purinergic ADP/ATP receptor P2RY12. This was shown to be highly expressed by microglia (human and rodent) compared to macrophages. In addition, it appears to be a marker of non-activated microglia as expression becomes rapidly downregulated upon inflammatory activation with LPS [68]. P2RY12 has been defined as a M2 marker as it is unregulated in vitro in human microglia by treatment with IL-4 [69]. A common concept over the years about neuroinflammation and AD is that widespread proinflammatory activation is a significant and extensive feature. The expression that the brain is on fire has been used more than once in review articles of AD inflammation. If inflammation is widespread in pathological affected tissues, one would expect that P2RY12 expression would be very low or absent in AD brains. Our preliminary findings show that this is not the situation; there were many P2RY12 microglia in regions with severe AD pathology. Although western blot and mRNA studies showed no difference in expression of this gene between AD and control samples, however by immunohistochemistry, there was a very specific distribution of P2RY12 positive and negative microglia in brains in relation to pathological

The potential benefits for complete definition of microglial phenotypes by immunohistochemistry in human brains could be significant. The development of effective inflammationfocused therapies for AD requires the identification of therapeutic targets that are relevant

**5.1. Where are the non-activated microglia in AD or aging brains?**

responses.

50 Alzheimer's Disease - The 21st Century Challenge

structures.

**6. Future directions**

Microglia represent approximately 10% of the total cell population in human brain, but it is now appreciated how complex their responses are to pathological stimuli and for maintaining healthy neurons. Treating pathological inflammation in AD with broad spectrum agents (e.g., cyclooxygenase inhibitors) may do more harm than good. If the microglial responses to pathology are highly dependent on the microenvironment; for example one microglia may be producing excess TNF-α while an adjacent one is attempting to remove the pathological stimuli, then treatments need to be targeted appropriately. This will only be possible if the microglia actively involved in AD can be adequately profiled.
