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Walker. Neuroimmunol Neuroinflammation 2020;7:194-214 I http://dx.doi.org/10.20517/2347-8659.2020.09 Page 195

markers of microglia with expression profiles that do not definitively fall into the pro-inflammatory or anti-
inflammatory classification. These additional markers include triggering receptor expressed on myeloid cells-2,
CD33 and progranulin, identified from genetic findings, colony stimulating factor-1 receptor, purinergic receptor
P2RY12, CD68 and Toll-like receptors. Further directions will be considered for addressing crucial issues.

Keywords: Neuropathology, RNA-sequencing, TREM2, microglia, activation states, immunohistochemistry

INTRODUCTION
Alzheimer’s disease (AD) and Parkinson’s disease (PD) have become the most significant and feared brain
diseases of elderly populations who are now enjoying longer lifespans due to more effective treatments
for cancer, cardiovascular and metabolic diseases. AD is the most common cause of cognitive decline and
[1]
dementia in elderly populations , while PD can lead to severe loss of mobility and independence, amongst
[2]
other features . Both diseases are significant causes of morbidity in elderly populations and share many
common pathological features involving the accumulation of aggregated proteins. In AD, it is extracellular
[3]
amyloid and neurofibrillary-associated phosphorylated tau neurites and tangles , while in PD, it is
[4]
aggregated/phosphorylated alpha-synuclein accumulated into pathological inclusions . These diseases
are distinctive on account of the degenerative changes occurring in different brain regions; however, one
common feature is the appearance of “activated” microglia within brain regions showing degenerative
changes. Inflammation has become one of the targets being investigated as treatment strategies for these
diseases, and the importance of studying microglia in relation to many different brain diseases is widely
[5-7]
appreciated .

Initial antibody-based observations on “activated” microglia in postmortem brain tissues were made
30 years ago and gave rise to the inflammatory hypotheses for neurodegeneration. This is illustrated in
Figure 1 and suggests that initial cell death or accumulation of aberrant/aggregated proteins [amyloid
beta (Aβ), tau or alpha-synuclein (α-syn)] results in proinflammatory activation of microglia, causing the
production of toxic and/or inflammatory cytokines. The resulting neurotoxicity would then accelerate
further inflammation, thus exacerbating the neurodegenerative process. These concepts developed in the
80’s and 90’s might now be considered imprecise based on more recent findings. However, it was from this
hypothesis that treatments for AD and PD with anti-inflammatory agents were developed and tested. This
approach was supported by data from epidemiological studies that patients who had long-term usage of
anti-inflammatory drugs for inflammatory conditions such as arthritis, had less dementia, which appeared
[8]
to support the inflammatory hypothesis of AD . However, although many anti-inflammatory compounds
and strategies have proven effective in AD animal models, clinical trials of AD patients have generally
[9]
shown no significant effect . The purpose of this review is to consider the approaches used to define
changes in microglial phenotypes and neuroinflammation in brain tissue, and discuss how the role of
microglia in neurodegeneration should be considered in light of a wider range of markers identified from
recent transcriptional profiling of microglia. The focus of this review will be on studies relating to AD, but
many of the concepts might be applicable to PD, multiple sclerosis (MS) or stroke. The aim for this review
is to bridge the gap between the studies that have analyzed transcription in large numbers of samples or
isolated cells with in situ studies in human brain samples with antibodies to define the microenvironments
of microglia in the specialized neuroanatomy of the human brain [10-12] . Ultimately, the best way to define
the microglia responsible for damaging inflammation in brain samples will be with a single or small panel
of markers that can be studied reliably in widely available types of pathological brain samples.

METHODOLOGY FOR INVESTIGATING MICROGLIAL PHENOTYPES
Antibody-based methodology for in situ localization of microglial antigens
[13]
Pioneering observations on microglia by Rio-Hortega used traditional metal-based histological stains to