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

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

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

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

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

Additional file 1: of Novel TNF receptor-1 inhibitors identified as potential therapeutic candidates for traumatic brain injury

Figure S1. Uninjured sham mice showed no significant drug-induced change in sleep compared to baseline or the vehicle treated group. (A) There was no significant treatment effect on sleep in uninjured sham mice when compared to their pre-treatment baseline sleep (F2,6 = 0.6759, p = 0.5436). (B) The change in cumulative sleep from vehicle-treated shams was calculated and there was no significant treatment effect on sleep in the C7-treated or SGT-treated shams (t4 = 1.689, p = 0.1665). (TIF 152 kb

Significant increase in post-traumatic sleep is independent of the time of day of the injury.

<p>Mice subjected to mild or moderate injury at 9:00 (<b>A</b>), following the dark/light transition showed significant increases in acute post-traumatic sleep compared to uninjured sham. A multivariate ANOVA and Bonferroni post-hoc analysis was used (mean ±SEM; sham n = 12; injured n = 17; F(1,25) = 15.95); *, p<0.05). Mice subjected to mild or moderate injury at 21:00 (<b>B</b>), following the light/dark transition also showed significant increases in acute post-traumatic sleep compared to sham. A multivariate ANOVA and Bonferroni post-hoc analysis was used (mean ±SEM; sham n = 5; injured n = 14; F(1,17) = 4.42; *, p<0.05). An increase in sleep is observed acutely following TBI and is observed over the course of the first 3 hours in injured mice compared to sham. After 3 hours, sleep began to normalize in the injured animals and became indistinguishable from sleep in the sham. Mean percent sleep of uninjured sham mice in the 9:00 group was significantly higher than the mean percent sleep of sham mice in the 21:00 group (F(1,15) = 6.303, p = 0.0240), as expected.</p

Diffuse TBI in the mouse disrupts acute post-traumatic sleep parameters compared to uninjured sham.

<p>(<b>A</b>) A multivariate ANOVA showed a significant increase in mean percent sleep over the first 6 hours post-injury compared to the uninjured sham (mean ±SEM; sham n = 16; injured n = 31; F(1, 45) = 6.545, p = 0.00007). After 6 hours post-injury, the mean percent sleep of injured mice normalized to sham mean percent sleep levels and remained comparable for 7 days post-injury (data not shown). (<b>B</b>) A detailed analysis of the acute post-traumatic sleep (in the first hour) following diffuse TBI indicated a significant time dependent effect on the increase in sleep. A multivariate ANOVA of the rolling average of the mean percent sleep over 5 min intervals showed post-traumatic sleep significantly increased over the first hour post-injury with a significant effect of time (mean ±SEM; sham n = 16; injured n = 31; F(11,495) = 8.22, p<0.0001) and group (mean ±SEM; sham n = 16; injured n = 31; F(1,45) = 37.00, p<0.0001). Bonferroni post hoc analysis was used (*, p<0.05). (<b>C</b>) Acutely post-injury, the brain-injured mice showed an increase in median bout length compared to shams. A multivariate ANOVA revealed an increase in bout length significant over the first 4 hours post-injury (mean ±SEM; sham n = 16; injured n = 31; F(1,45) = 2.9138, p = 0.032). This increase in bout length suggested that the increase in mean percent sleep observed acutely post-injury could result from mice sleeping for longer durations, as opposed to sleeping more bouts after diffuse TBI.</p