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Page 252 Benusa et al. Neuroimmunol Neuroinflammation 2020;7:248-63 I http://dx.doi.org/10.20517/2347-8659.2020.03

Figure 1. Frequency of microglial-AIS contact is not altered in LPS-induced neuroinflammation. Female c57black6 mice were given a
single intraperitoneal injection of LPS (5 mg/kg) or vehicle (0.9% saline, 10 mL/kg). Confocal z-stacks spanning an optical thickness of
25 μm, using a pinhole of 1 Airy disc unit and Nyquist sampling (optical slice thickness, 0.48 μm), were collected from neocortical layer
V for each of six sections (spanning 1.1 mm anterior to the bregma to 2.5 mm posterior to the bregma) per mouse, resulting in 12 images
per animal (n = 4-6 animals per treatment group). Microglial-AIS contact was quantitatively analyzed at 6 h-2 weeks post-injection in
a blind manner using Volocity™ 3D Image Analysis Software, allowing each confocal z-stack to be observed in three dimensions. The
number of microglia, AISs, and contact points in each double immunolabeled z-stack was counted manually. Contact points along the
six edges of the z-stacks were excluded from analysis. A-F: double immunolabeling of Iba-1 and AnkG revealed that microglia (Iba-1,
green) contact AISs (AnkG, red) (white arrows) in the cortex of saline- and LPS-injected mice; G: the mean ± SEM of microglia making
AIS contact per FOV in saline- and LPS-treated mice as a percent of saline controls. Quantitation of confocal z-stacks revealed that
~45% of microglia contact AISs in the cortex of saline-injected control mice. Contact was defined by co-localization of Iba-1 and AnkG
and included process touching (A, yellow single asterisk), process alignment (A, yellow double asterisk), and process wrapping (F,
yellow triple asterisk) as defined by 3D analysis. No change in the percent of microglia making contact was observed throughout the
course of LPS-induced neuroinflammation; H: the mean ± SEM of the number of Microglia/FOV. A significant increase in the number
of microglia was observed at 3 days post-LPS injection. Data were statistically compared by one-way ANOVA where mean differences
were significant as assessed using Tukey’s post hoc analysis. An asterisk indicates a significant difference (P < 0.05) from saline. Scale
bar = 20 μm. LPS: lipopolysaccharide; FOV: field of view; AISs: axon initial segments

function and response by the local environment. Interestingly, our laboratory has also reported contact
between microglia and the AIS [79,80] . Using three dimensional (3D) analysis encompassing multiple types
of contact, which was defined by colocalization of ionized calcium binding adaptor molecule 1 (Iba-1)
and AnkyrinG, termed (1) process touching, (2) process wrapping, or (3) process alignment [Figure 1], we
found that ~45% of microglia in cortical layer V contact AISs. In contrast to the loss of contact observed
[79]
following CCI injury, we observed a maintained [Figure 1], and even increased , association between the
microglia and the AIS following inflammatory insults of LPS injection and EAE induction, respectively. The
difference in AXIS microglial responses to insult is intriguing and requires further study to fully elucidate
microglial response to pathology.

Herein, we have reviewed several subclasses of microglia that have been defined based on morphology;
however, it is unclear if these subclasses are truly distinct, or if they are merely the consequence of
artificial classifications based on techniques used for identification, and loose criteria for defining
[29]
subtypes (reviewed ). If the latter is the case, then there is likely considerable overlap among these