Rod microglia: a morphological definition

Abstract

Brain microglial morphology relates to function, with ramified microglia surveying the micro-environment and amoeboid microglia engulfing debris. One subgroup of microglia, rod microglia, have been observed in a number of pathological conditions, however neither a function nor specific morphology has been defined. Historically, rod microglia have been described intermittently as cells with a sausage-shaped soma and long, thin processes, which align adjacent to neurons. More recently, our group has described rod microglia aligning end-to-end with one another to form trains adjacent to neuronal processes. Confusion in the literature regarding rod microglia arises from some reports referring to the sausage-shaped cell body, while ignoring the spatial distribution of processes. Here, we systematically define the morphological characteristics of rod microglia that form after diffuse brain injury in the rat, which differ morphologically from the spurious rod microglia found in uninjured sham. Rod microglia in the diffuse-injured rat brain show a ratio of 1.79 ± 0.03 cell length:cell width at day 1 post-injury, which increases to 3.35 ± 0.05 at day 7, compared to sham (1.17 ± 0.02). The soma length:width differs only at day 7 post-injury (2.92 ± 0.07 length:width), compared to sham (2.49 ± 0.05). Further analysis indicated that rod microglia may not elongate in cell length but rather narrow in cell width, and retract planar (side) processes. These morphological characteristics serve as a tool for distinguishing rod microglia from other morphologies. The function of rod microglia remains enigmatic; based on morphology we propose origins and functions for rod microglia after acute neurological insult, which may provide biomarkers or therapeutic targets.

Figure 1. Rod microglia compared to Nissl's Stäbchenzellen illustration.

A) Franz Nissl's illustration of Stäbchenzellen (rod cells) observed in patients suffering from syphilitic paralysis, as depicted by Spielmeyer . B) Representative Iba1-positive rod microglia in the sensory cortex 7 days post-FPI, cell length∶cell width ratio of 12∶1. Note the similarities in the morphological characteristics between the two images.

Figure 2. Rod microglia morphology and alignment post-FPI.

Representative images of Iba1-positive microglia in the cortex of sham-injured and FPI rats. Sham-injured microglia showed a sausage-like cell body but no polarization of processes. However, as early as 1 day post-injury the retraction of planar processes was evident giving rise to rod morphology. Rod microglia continued to retract their planar processes at day 2 post-injury, and by day 7 there were few planar processes visible. By day 28, the cell body had begun to become more rounded and planar processes were returning.

Figure 3. Retraction of planar processes gives the illusion of elongation.

Morphological features of rod microglia cells and soma after diffuse brain injury. Rod microglia do not elongate, but rather narrow, post-injury. (A, D) Representative 28 day post-FPI Iba1-positive rod microglia, 100x. The orange and blue arrows illustrate the measured length and width respectively (µm) of the cell (A) and soma (D). The population of rod microglial cell length (B) and width (C) is described by a box and whisker plot, similarly for the soma length (E) and width (F). Notches on the x-axis of each frequency histogram represent the bin center for each specific time point, the bin center is determined from the total frequency histogram. In all cases a Gaussian distribution was observed. Significance was calculated using the average mean values for each time point (n = 3/time point; *, P<0.05, one-way ANOVA).

Figure 4. Retraction of rod microglial branches post-injury.

(A, D) representative 28 day post-FPI Iba1-positive rod microglia, 100x. The orange and blue lines illustrate the measured primary polar and planar branches respectively. (B, E) Number of primary branches present on cell, polar and planar respectively. (C, F) Average length of the primary branches present on the cell (µm), polar and planar respectively. Populations described by a box and whisker plot. Notches of each frequency histogram represent the bin center for that specific time point. Unlike the average length of primary polar branches, the number of primary polar branches is heavily weighted to the left of the mean (negatively skewed). All other histograms show a Gaussian distribution. Significance was calculated using the mean values for each time point (n = 3/time point; *, P<0.05, one-way ANOVA).

Figure 5. Decrease in the number of rod microglial secondary branches post-injury.

(A, C) Representative 28 day post-FPI Iba1-positive rod microglia, 100x. The black lines illustrate the already measure primary branches. The orange and blue lines illustrate the secondary polar and planar branches respectively, secondary branches defined as those directly protruding off of a primary branch. (B) Number of secondary polar branches present on the cell. (D) Number of secondary planar branches present on the cell. Populations described by a box and whisker plot. Notches of each frequency histogram represent the bin center for that specific time point. In both cases a Gaussian distribution is observed. Significance was calculated using the average mean values for each time point (n = 3/time point; *, P<0.05, one-way ANOVA).

Figure 6. Schematic representation of rod microglia alignment and polarization post-injury.

In the sham animals, rod-like microglia are sporadically found throughout all regions of the brain, these cells had sausage-like soma with no defined alignment or polarization in any plane. Post-injury, rod microglia aligned within the S1BF of the cortex, perpendicular to the dural surface. Rod microglia in the diffuse-injured rat brain show a ratio of 1.79±0.03 cell length∶cell width at day 1 post-injury, which increases to 3.35±0.05 at day 7, compared to sham (1.17±0.02). The soma length∶width differs only at day 7 post-injury compared to uninjured sham. Once alignment has occurred, rod microglia form ‘trains’, hypothesized through migration, proliferation and /or differentiation of existing resting microglia. It is clear that cell to cell signals control the migration of microglia, and presumably control their morphology. Microglial function follows morphology, however, the role and signaling cascade for rod microglia has yet to be identified.