**3. Schemes for defining microglial function: limitations of M1 and M2 activation state definitions**

Phenotyping of macrophages and microglia progressed with the pioneering work of Gordon and colleagues who sought to develop schemes for classification of macrophages, first in mice and then in humans, by assigning activation states to the expression of different antigenic markers based on responses to defined activation stimuli [32, 33]. Much of this work employed gene expression profiling mRNA analysis since these techniques have fewer of the limitations associated with antibodies that will be discussed. These phenotyping schemes were also applied to microglia, both rodent and human. The scheme defined classical activation or M1 activation, as being the state of macrophages/microglia that have been stimulated with strong inflammatory agents such as lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Such activated cells will be expressing increased levels of cytokines and enzymes such as TNF-α, IL-1β, IL6, matrix metalloproteinase (MMP)-3 and MMP-9. The corollary to this is alternative activation or M2, which defines the markers and products of cells responding to anti-inflammatory cytokines such as IL-4 or IL-13. These cells have a reparative/neurotrophic phenotype and can produce growth factors. Such reparative M2 microglia also show increased phagocytosis. The M2 scheme was further subdivided into M2a (responses to IL4 or IL13), M2b (responses to immune complexes in combination with IL-1β or LPS) and M2c (responses to anti-inflammatory IL-10, TGFβ or glucocorticoids). It was shown that increased expression of the scavenger receptor CD163, a marker for M2c was upregulated in microglia in AD and Parkinson disease dementia cases. This is the first study showing a type of alternative activation in AD tissues by immunohistochemistry [34].

Many studies have tried to apply these schemes to tissue microglia but their validity has been contested [35]. The schemes are dependent on using defined stimuli, while in the degenerating AD brain, there will be many different stimuli (Aβ and tau in different conformations, reactive oxygen, cytokines, bioactive lipids, ATP/ADP, DNA, etc.) that will account for the heterogeneity of microglia responses in tissue. In recent years, there has been criticism that the M1 and M2 scheme is not applicable for tissue microglia as such defined microglia do not seem to exist in brain [35]. This may be correct as the microenvironment around every plaque and every neuron will be different, but to attempt to profile microglia does require some form of scheme, even an imperfect one, to relate to function. It will also be proposed that the limitations of the M1 and M2 classification schemes could be due to technical reasons as much as biological reasons.
