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Benusa et al. Neuroimmunol Neuroinflammation 2020;7:248-63 I http://dx.doi.org/10.20517/2347-8659.2020.03 Page 251

express varying levels of keratin sulfate proteoglycan (KSPG) and these microglia are not uniformly
distributed throughout the brain, with high concentrations in the hippocampus, brainstem and olfactory
+
bulb while few were found in the cerebellum and cortex. The presence of these KSPG microglia was
independent of development though, since they were found in the same regions of both the neonatal and
adult CNS. Moreover, microglia have also been shown to respond differently to the same stimuli [63,64] .
Although consistent with the involvement of environmental cues in defining subclasses, these findings are
also consistent with microglia being intrinsically distinct and independent of environmental influences.

Heterogenic microglial morphology
Amoeboid vs. ramified microglia
Perhaps the most recognized heterogenic aspect of microglia is their morphology. Two main classes have
been identified - amoeboid-like, with few processes; and ramified, with numerous thin, highly-branched
processes. Following initial colonization of the embryonic CNS, the majority of microglia present with
an amoeboid-like morphology [65,66] . With CNS maturation, microglia transform their shape with brain
region specificity. In the steady state CNS, amoeboid-like microglia are more abundant in perivascular
white matter regions. In contrast, the extent of ramified microglia varies among regions with cerebellar
microglia presenting with a less ramified morphology compared to microglia in the cortex [38,67,68] .
[69]
Interestingly, Hanamsagar et al. reported heterogeneity with regard to sex as microglia from male
rodents presented with a greater and more complex process of arborization, and exhibited a greater change
in process morphology following LPS perturbation as compared to their female counterparts. With age,
and as the local environment changes, amoeboid-like microglia become more ramified while ramified
microglia transition into amoeboid-like microglia, exhibiting greater phagocytic activity and releasing
pro-inflammatory cytokines following pathologic insult [70,71] . Although the use of the amoeboid/ramified
classification provides a simple approach for discussion, microglial morphologies present a spectrum of
shapes and a two-class scheme is insufficient to accurately describe microglial morphologic differences.

Dark microglia
Recently, a new class of microglia was identified based on morphology. These microglia are “dark” based
[72]
on their electron dense cytoplasm and are observed in non-homeostatic conditions . Dark microglia
exhibit signs of oxidative stress including condensed cytoplasm and nucleoplasm (consistent with their
name), disrupted mitochondria and dilated endoplasmic reticulum, and are frequently observed extending
processes toward synaptic clefts consistent with a role in pathologic synaptic pruning. Although their
[72]
precise role remains to be fully determined, Bisht and colleagues have proposed that these cells constitute
a subclass of hyperactive microglia with dysregulated interactions with synapses. If correct, these cells may
play a critical role in the progression of a plethora of neurodegenerative diseases with known synaptic
[73]
loss including Alzheimer’s Disease [74,75] and multiple sclerosis [76,77] .
Axon initial segment-associated microglia
[78]
Baalman et al. have also presented evidence of a subset of microglia known as axon initial segment-
associated (AXIS) microglia . AXIS microglia, which comprise ~8% of cortical microglia, establish an
[78]
intimate association with the neuronal cell body and the proximal axon, in contrast to “satellite” microglia
that associate with the neuronal cell body and proximal dendrites instead . AXIS microglia, which are
[68]
initially observed at postnatal day 9 and persist through adulthood, contact both inhibitory and excitatory
neurons but present with a significant preference for axon initial segments (AISs) of excitatory pyramidal
[78]
neurons of layer V of the cortex . The function of AXIS microglia is not known but they may provide
trophic support for the neuron and the AIS.

Upon activation following a controlled cortical impact (CCI)-induced traumatic brain injury, the
association between CNS microglia and the AIS is lost, consistent with the regulation of microglial