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Benusa et al. Neuroimmunol Neuroinflammation 2020;7:23-39 I http://dx.doi.org/10.20517/2347-8659.2019.28 Page 27

consistent with other studies demonstrating that numbers of microglia/macrophages correlate to EAE
severity [27,72-74] . However, the cellular mechanisms by which microglia/infiltrating macrophages promote
disease progression and whether these cells play differential roles in initiating demyelination or promoting
[6]
repair remain unknown [61,92,93] . Yamasaki et al. began to elucidate the roles these cell types play in the
disease course of EAE and their differential roles in myelin disruption. Serial block-face scanning electron
microscopy of mice, in which the resident microglia fluoresced green and the infiltrating monocyte-derived
macrophages fluoresced red, was utilized to distinguish the two inflammatory cell populations and to
[6]
investigate their role in demyelination . It was demonstrated that both microglia and infiltrating peripheral
monocyte-derived macrophages contact the axo-glial unit at the NOR in the spinal cord of EAE-induced
mice at disease onset [Figure 1B] . They found that most (73%) of the NOR investigated (both intact and
[6]
[6]
disrupted) were physically contacted by some sort of macrophage . Interestingly, microglial association
with the axo-glial unit was limited, while monocyte-derived infiltrating macrophage contact at the NOR
[6]
was more extensive . Monocyte-derived macrophage processes were found extended between the myelin
and axolemma, potentially uprooting paranodal contacts and initiating demyelination [Figure 1B] . In
[6]
contrast, microglial processes contacted the axo-glial unit at the NOR, but the microglial processes did
not extend beneath the axolemma and, instead, appeared to primarily interact with adjacent macrophages
[6]
and appeared to be involved in debris clearance [Figure 1B] . Gene expression profiles supported that
infiltrating monocyte-derived macrophages were highly phagocytic and pro-inflammatory, whereas
[6]
microglia demonstrated a suppressed cellular metabolism and activation phenotype . These findings
suggest that, at disease onset, infiltrating macrophages initiate active demyelination while microglia
perform myelin debris clearance, a function that supports tissue regeneration and affects the maturation of
oligodendrocyte progenitor cells .
[3]
The differential mechanisms underlying microglial contact at the NOR is still to be fully determined. It
was shown that C-C chemokine receptor type 2 (CCR2), a chemokine receptor essential for monocyte
recruitment to CNS tissues during immune-mediated inflammation [94,95] , was important for recognition
[6]
of disrupted NOR by infiltrating monocyte-derived macrophages . Mice lacking CCR2 demonstrated
reduced NOR contact by monocyte-derived macrophages and significantly less demyelination at EAE
onset. Interestingly, CCR2-deficient mice displayed similar nodal pathology during the pre-onset stage of
EAE (post-EAE induction but prior to onset of motor clinical symptoms), suggesting that inflammatory
nodal disruption could be reversible if monocyte-derived macrophages were prevented from initiating
[6]
demyelination at those sites .

MICROGLIAL CONTACT WITH THE AIS
Microglia contact the AIS during normal development and throughout life, indicating that these cells likely
play a role in the regulation of AIS structure and/or function in both the developing and mature CNS
[45]
[Figure 1A] . A recent study utilizing both EAE and cuprizone models of MS to assess MS-related axonal
injury and their underlying mechanisms found that inflammatory microglia and/or Macrophages physically
contact the AIS . It was found that the AIS is a primary target in disease pathogenesis of EAE . In this
[27]
[27]
study, mice were induced with either myelin-oligodendrocyte glycoprotein + EAE or cuprizone and AIS
integrity of cortical neurons was assessed using immunohistochemical techniques. The integrity of the
AIS was assessed by immunolabeling for ankyrinG (AnkG), a protein critical for AIS establishment and
maintenance [96-98] . Upon EAE induction, it was found that the number and length of AISs were significantly
[27]
reduced and that the number of disrupted AISs was associated with disease severity and progression .
This loss of AIS integrity, however, was not associated with demyelination, neuronal death, or axonal
damage, rather appeared to be mediated by inflammatory factors [27-29] . Specifically, AIS disruption was
preceded by microglial morphological changes suggestive of enhanced reactivity and increased contact by
[27]
Iba-1 positive inflammatory cells but occurred independently of demyelination . The nature of microglial
interaction with the AIS changed substantially following EAE, transitioning from microglial process