**2. Dichotomy of microglial function: do we know which microglia are producing damaging inflammation and which are performing phagocytosis of damaging abnormal proteins**

Since the initial discoveries of activated microglia in AD and aging brains, the concepts and knowledge of what microglia are doing or could potentially be doing has progressed. The central role of microglia in brain, or macrophages in general, is to phagocytose and digest cellular waste products, which should include the extra-cellular Aβ that is deposited in AD and aging brains. An important question that is still unanswered today is "why are microglia not doing a better job of removing Aβ plaques?" Some concepts of microglial function in relation to AD came from transgenic mouse models using animals engineered to develop Aβ plaques in a manner similar to humans. These studies produced some conflicting results, but in general it was shown that microglia could be manipulated to achieve greater removal of plaque material, but in these mice, as in humans, microglial removal of plaque material is not efficient without some stimuli [26]. Again, we can ask does this apply to all microglia? Certain cytokine treatments affect microglia leading to reduced phagocytosis such that plaque material accumulates to a greater extent [27–29]. These studies illustrated how microglia can be activated to be more or less efficient at Aβ removal [30, 31]. This was particularly shown in Aβ-peptide immunized mice, which had produced a specific antibody response to plaque material. The coating of plaques with anti-Aβ immunoglobulin appeared to promote phagocytosis through engagement of the microglial IgG Fc receptors. Overall, these studies showed that microglia of a particular phenotype have the potential to remove Aβ; similar observations have come from human pathological studies in certain subjects who had received the Aβ vaccine [14].

**4. Practical issues involved in microglial phenotyping in human** 

the lack of panels of antibodies functional on available brain tissue samples.

Since the initial studies of increased HLA-DR expression by microglia in AD brains, in areas associated with pathology [9, 19, 36, 39–42], expression of a range of macrophage markers have been applied to AD brain tissues. These include beta II integrins (CD11a, b, and c and CD18 complement and phagocytic receptors), immunoglobulin Fc receptors (CD16, CD32, CD64) [11], lipopolysaccharide receptor CD14 [43], macrophage colony stimulating factor receptor-1 (CSF-1R; CD115) [44], type B scavenger receptor CD36 [45, 46], ferritin [47], signal regulatory

**4.1. Previous studies of microglial functional proteins in AD**

Success in classifying microglia in postmortem human autopsy tissue sections is primarily dependent on the antibodies being used for this purpose, but also the manner in which the brain tissue being studied was preserved. Many published studies of microglial markers for immunohistochemistry have been restricted to antibodies that produce strong immunoreactivity on extensively fixed tissue sections. This is particularly true for HLA-DR, which is the most widely used for human microglial studies, as available antibodies can produce vivid results on a wide range of preserved brain tissue. The following references are the first for HLA-DR and the most recent, spanning 30 years of studies [36, 37]. The function of HLA-DR in AD microglia is still unclear. This protein functions to present processed antigens to T lymphocytes that are not present in the AD microenvironment. The signaling that leads to upregulation of HLA-DR in AD microglia has not been defined. In recent years, the marker IBA-1, which recognizes an actin-binding protein involved in cytoskeletal reorganization and cell motility, has also been extensively used to identify microglia because of the availability of robust staining antibodies [38]. IBA-1 antibodies seem to recognize all microglia with limited upregulation in activated microglia, though this interpretation is also dependent on observations related to microglial morphology. The use of antibodies that produce strong results in tissue sections may have biased our understanding of microglial function in disease as many other antigenic markers are present, but suitable antibodies to reveal them in tissue are not available. Most useful markers of function are cell-surface glycosylated proteins whose antigenicity become significantly affected by fixation conditions and also by the degree of glycosylation. The most widely available tissues for many researchers are tissue blocks that have been formalin-fixed for extended periods and then embedded in paraffin (FFPE); this process includes treatments with xylene. These preservation methods strongly affect the ability of antibodies to recognize many antigens, but in particular cell-surface glycoproteins. The numbers of antibodies that are effective at antigen recognition on FFPE tissue are a small percentage of available antibodies. In addition, the use of FFPE tissue usually requires the application of antigen retrieval techniques for most antibodies to work; there are a range of these methods but their successful application is dependent on operator skill and can lack reproducibility. As mentioned, the applicability of M1 and M2-like schemes to classify microglia in human brain samples has been criticized as many of the classification antigens have not been proven in tissue microglia [35], however such schemes may have been prematurely discarded due to

Defining Microglial Phenotypes in Alzheimer's Disease http://dx.doi.org/10.5772/intechopen.75511 45

**autopsy brains**
