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Page 206 Walker. Neuroimmunol Neuroinflammation 2020;7:194-214 I http://dx.doi.org/10.20517/2347-8659.2020.09

and this was increased in pathology-associated microglia in AD brains [100] . This original finding of CSF1R
expression by microglia is now 25 years old and requires replication using modern microscopic techniques
to define the phenotypes of expressing microglia. We recently demonstrated increased expression of CSF1R
and CSF1 mRNA in AD brains that correlated with severity of pathology, but reduced expression of IL34
mRNA in these samples [101] . One study has shown that CSF1R can be expressed in neurons in lesioned rat
brains, but this finding has not been replicated [102] . Further characterization of CSF1R in AD tissue would
be helpful to map the sites of microglial proliferation in tissues. These studies could be combined with
markers and transcription factors associated with microglial proliferation (e.g., PU.1, Ki67).

CD14-Lipopolysaccharide receptor
A number of blood macrophage markers that have been defined are expressed at increased levels in
activated cells (reviewed [103] ). These defining studies have generally utilized strong activation agents such as
bacterial proinflammogens including lipopolysaccharide (LPS) and IFN-γ that induce strong inflammatory
responses for killing invading microorganisms. One of the activation receptors is CD14, along with Toll-like
receptor (TLR)-4, one of the components of the LPS receptor [104] . CD14 has been considered a constitutive
marker for macrophages and microglia, but it is noticeable that its expression in human AD brains has
only been described in a single study [105] . This study identified increased CD14 expression of plaque-
associated microglia. In Figure 4, we show examples of CD14 staining of microglia, but only in AD cases.
Studies characterizing the expression of CD14 in microglia freshly isolated from human brains showed
low levels of constitutive expression that increased significantly when isolated microglia were cultured for
2-4 days in vitro [106,107] . This culture-associated increase in CD14 mRNA expression was not observed for
TLR4, but culture-associated decreases of a number of genes (P2RY12, CX3CR1, TNFα, TGFβ HLA-DRA,
CD11b, FCγR3) were detected [107] . Increased expression of CD14 was detected by immunohistochemistry in
microglia and infiltrating monocytes in sections from cases with traumatic brain injury [108] .

The immunohistochemistry results presented here that follow up the earlier findings [105] suggest that using
CD14 as a marker to define pro-inflammatory activation phenotypes in human brain should be reassessed.
As had been encountered for other markers, successful CD14 staining was dependent on the antibody
o
used, and also the use of antigen-retrieval methods (pH 8.0, 80 C, 30 min). Other CD14 antibodies tested
did not produce this staining of microglia under the same conditions.

CD68
There has been some controversy in the literature about the status of CD68 (designated macrosialin for
rodents) as an activation or functional phagocytic marker. CD68 is a myeloid cell-specific lysosomal
associated membrane protein (LAMP) whose expression is increased in cells associated with elevated
phagocytic and degradative activity. Under appropriate conditions, by employing antigen retrieval methods
on free-floating sections, we can demonstrate CD68 positive structures in most microglia. We have recently
demonstrated that the majority of progranulin-positive, P2RY12-positive and TLR3-positive microglia
were CD68 positive to some extent, irrespective of whether the sections being examined were from
non-demented control or AD cases [83,91,109] . Without antigen retrieval, there was a noticeable decrease in
sensitivity of detection for CD68 with many microglia showing no reactivity (unpublished observations).
It is clear that being able to optimize the sensitivity of detection of CD68 (and other antigens) has a
significant effect on identifying the phenotypes of cells expressing this marker.

This is a good context to discuss semi-quantitative histological findings on the expression of a series of
microglial markers, including CD68, carried out in a large series of cases by Boche and colleagues [34,110,111] .
These investigators were able to examine the expression of microglial markers in samples from subjects
who had received the Aβ vaccine as treatment to produce circulating levels of Aβ antibodies [110] . There were
significantly decreased numbers of microglia positive for CD68, CD64, CD32 and MSR-1, but not IBA-1