Age-at-Injury Determines the Extent of Long-Term Neuropathology and Microgliosis After a Diffuse Brain Injury in Male Rats

Traumatic brain injury (TBI) can occur at any age, from youth to the elderly, and its contribution to age-related neuropathology remains unknown. Few studies have investigated the relationship between age-at-injury and pathophysiology at a discrete biological age. In this study, we report the immunohistochemical analysis of na&iuml;ve rat brains compared to those subjected to diffuse TBI by midline fluid percussion injury (mFPI) at post-natal day (PND) 17, PND35, 2-, 4-, or 6-months of age. All brains were collected when rats were 10-months of age (n = 6&ndash;7/group). Generalized linear mixed models were fitted to analyze binomial proportion and count data with R Studio. Amyloid precursor protein (APP) and neurofilament (SMI34, SMI32) neuronal pathology were counted in the corpus callosum (CC) and primary sensory barrel field (S1BF). Phosphorylated TAR DNA-binding protein 43 (pTDP-43) neuropathology was counted in the S1BF and hippocampus. There was a significantly greater extent of APP and SMI34 axonal pathology and pTDP-43 neuropathology following a TBI compared with na&iuml;ves regardless of brain region or age-at-injury. However, age-at-injury did determine the extent of dendritic neurofilament (SMI32) pathology in the CC and S1BF where all brain-injured rats exhibited a greater extent of pathology compared with na&iuml;ve. No significant differences were detected in the extent of astrocyte activation between brain-injured and na&iuml;ve rats. Microglia counts were conducted in the S1BF, hippocampus, ventral posteromedial (VPM) nucleus, zona incerta, and posterior hypothalamic nucleus. There was a significantly greater proportion of deramified microglia, regardless of whether the TBI was recent or remote, but this only occurred in the S1BF and hippocampus. The proportion of microglia with colocalized CD68 and TREM2 in the S1BF was greater in all brain-injured rats compared with na&iuml;ve, regardless of whether the TBI was recent or remote. Only rats with recent TBI exhibited a greater proportion of CD68-positive microglia compared with naive in the hippocampus and posterior hypothalamic nucleus. Whilst, only rats with a remote brain-injury displayed a greater proportion of microglia colocalized with TREM2 in the hippocampus. Thus, chronic alterations in neuronal and microglial characteristics are evident in the injured brain despite the recency of a diffuse brain injury.</p

Rod microglia: a morphological definition.

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

Rod microglia: elongation, alignment, and coupling to form trains across the somatosensory cortex after experimental diffuse brain injury

Abstract Background Since their discovery, the morphology of microglia has been interpreted to mirror their function, with ramified microglia constantly surveying the micro-environment and rapidly activating when changes occur. In 1899, Franz Nissl discovered what we now recognize as a distinct microglial activation state, microglial rod cells (Stäbchenzellen), which he observed adjacent to neurons. These rod-shaped microglia are typically found in human autopsy cases of paralysis of the insane, a disease of the pre-penicillin era, and best known today from HIV-1-infected brains. Microglial rod cells have been implicated in cortical ‘synaptic stripping’ but their exact role has remained unclear. This is due at least in part to a scarcity of experimental models. Now we have noted these rod microglia after experimental diffuse brain injury in brain regions that have an associated sensory sensitivity. Here, we describe the time course, location, and surrounding architecture associated with rod microglia following experimental diffuse traumatic brain injury (TBI). Methods Rats were subjected to a moderate midline fluid percussion injury (mFPI), which resulted in transient suppression of their righting reflex (6 to 10 min). Multiple immunohistochemistry protocols targeting microglia with Iba1 and other known microglia markers were undertaken to identify the morphological activation of microglia. Additionally, labeling with Iba1 and cell markers for neurons and astrocytes identified the architecture that surrounds these rod cells. Results We identified an abundance of Iba1-positive microglia with rod morphology in the primary sensory barrel fields (S1BF). Although present for at least 4 weeks post mFPI, they developed over the first week, peaking at 7 days post-injury. In the absence of contusion, Iba1-positive microglia appear to elongate with their processes extending from the apical and basal ends. These cells then abut one another and lay adjacent to cytoarchitecture of dendrites and axons, with no alignment with astrocytes and oligodendrocytes. Iba1-positive rod microglial cells differentially express other known markers for reactive microglia including OX-6 and CD68. Conclusion Diffuse traumatic brain injury induces a distinct rod microglia morphology, unique phenotype, and novel association between cells; these observations entice further investigation for impact on neurological outcome.</p

Rod microglia morphology and alignment post-FPI.

<p>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.</p

Rod microglia compared to Nissl's Stäbchenzellen illustration.

<p>A) Franz Nissl's illustration of Stäbchenzellen (rod cells) observed in patients suffering from syphilitic paralysis, as depicted by Spielmeyer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097096#pone.0097096-Spielmeyer1" target="_blank">[5]</a>. 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.</p

Retraction of rod microglial branches post-injury.

<p>(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).</p

Retraction of planar processes gives the illusion of elongation.

<p>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).</p

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

<p>(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).</p

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

<p>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.</p

Ratios of morphological data for rod microglia.

<p>To further distill the changes observed in microglia morphology post-injury, ratios of the cell length∶width and soma length∶width analyzed. These data indicated that the most dynamic changes were occurring with the microglial processes, which were also analyzed as ratios of number of polar∶planar branches, average and total length of polar∶planar branches.</p><p>All significance is time post-injury compared to sham; *p<0.05, ***p<0.001 and ****p<0.0001.</p