Schematic of the study design.

<p>Two cohorts of mice were used based on experimental outcome measures: (<b>A</b>) sleep recordings and (<b>B</b>) cortical samples and histology. (<b>A</b>) Mice were acclimated to piezoelectric sleep cages for 8 days while sample sleep recordings were monitored to test signal integrity. All mice received a midline craniotomy one day prior to brain or sham injury. Mice were divided into 2 groups based on the time of day they were subjected to injury (9:00, 21:00). Within each group, mice were selected at random and subjected to sham, mild (0.8 atm) or moderate (1.2–1.3 atm) diffuse brain injury by midline fluid percussion (mFPI) (n = 47). Following injury, mice were placed back into piezoelectric sleep cages and post-traumatic sleep was recorded for 7 days. (<b>B</b>) For biochemistry and histology, mice received a midline craniotomy one day prior to injury or sham injury. Mice were subjected to sham, or moderate (1.2–1.3 atm) diffuse brain injury (9:00) and cortical samples were retrieved at 1, 3, 9, 12, 24, 48, 168 hrs (n = 25). Tissue was also collected and prepared for histology 6 hrs post-injury (n = 3).</p

Figure 6

<p>(A) Temporal profile of IL-1β. The temporal profile indicated that levels in the cortex increase rapidly following moderate injury (9:00) as compared to uninjured sham. Levels of IL-1β peak at or near 9 hours post-injury and return to baseline levels by 12 hours post-injury (One-way ANOVA, mean ±SEM; sham n = 7; injured n = 22; F(7,21) = 6.474; p = 0.0004). Selected comparisons were made (Bonferroni post-hoc), asterisk denotes significance (*, p<0.05) compared to sham. (B, C, D) Microglia morphology, an indicator of microglia activation, was examined after mFPI in the mouse using Iba-1 immunohistochemistry. Iba-1 labels all microglia, however, tissue from a 6 hr sham (40×) (B) compared to a 6 hr mild injury (40×) (C) and a 6 hr moderate injury (40×) (D) show distinct differences in microglia morphology. Microglia in sham (B) demonstrated thin ramified processes (denoted by arrows) strongly contrasting the larger cell bodies and thicker processes (denoted by arrowheads) characteristic of activated microglia observed in the diffuse injured mouse (C, D).</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

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

The significant increase in post-traumatic sleep is observed acutely following both mild and moderate injury.

<p>A multivariate ANOVA showed a significant increase in mean percent sleep between injured mice and uninjured shams over the first 6-injury with no significant difference between mildly injured mice compared to moderately injured mice (mean ±SEM; sham n = 16; mild n = 16; moderate n = 15; F(2,44) = 3.4773, p = 0.00037).</p

Effects of MW151 on pSTAT3.

<p><b>(A)</b> Overview of <i>in vivo</i> TBI experimental design. (<b>B</b>) Representative example of pSTAT3 immunohistochemistry (IHC) in mFPI + veh -treated mice. Box indicates region shown at higher magnification in (<b>C</b>). Brown DAB staining is pSTAT3. Blue-green staining is a Methyl green counter stain. <b>(D)</b> Digital neuropathological quantification of pSTAT3⁺ nuclei in the cortex was done using the Aperio ScanScope and nuclear algorithm (n = 4 sham + veh, n = 9 mFPI + veh, n = 10 mFPI + MW151) (F_2,22 = 7.5286; p = 0.0037). *p<0.05, **p<0.001 compared to mFPI + veh. (mFPI = midline fluid percussion injury; veh = vehicle). <b>(E)</b> BV-2 cells were treated with veh or MW151 and stimulated with IFNγ (10μg/ml) for 60min, then cell lysates were harvested for western blot analysis. The data presented is a representative experiment (n = 2–3 samples per group), with the experiment replicated 3 times. (*p<0.05, **p<0.01 compared to IFNγ + veh). <b>(F)</b> BV-2 cells were treated with veh or MW151 and stimulated with IL-6 (1ng/ml) for 60min, then cell lysates were harvested for ELISA. The data presented is a representative experiment (n = 4–6 samples per group), with the experiment replicated 4 times. (***p<0.0001 compared to IL-6 + veh).</p

No effects of MW151 on BV-2 microglia cell engulfment of pH sensitive <i>E</i>. <i>coli</i> bioparticles.

<p><b>(A)</b> The pHrodo dye is non-fluorescent at neutral pH, but acidification, presumably in the cell phagosome, causes the dye to fluoresce in the red spectrum. Over the first 3h after adding the pHrodo-labeled bioparticles, the average mean intensity of the red calibrated unit (RCU) increased, but after 3h the RCU intensity plateaued. <b>(B)</b> At 3h, near the end of the linear phase of increasing RCU, the effect of treatment with vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (cytD, 1 μM) was quantified. The graph represents the average of three independent experiments (mean ± SEM, n = 3), each experiment carried out in 4 replicates for each treatment. **p<0.05 compared to saline vehicle. <b>(C)</b> Representative photographs of BV-2 cells treated with saline, 30μM MW151 or 1μM CytD treatment at 0, 3, and 6 hrs after the addition of the bioparticles. Images and data obtained using Incucyte Zoom at 20x objective.</p

No effects of MW151 on BV-2 microglia cell growth curves.

<p><b>(A)</b> Representative example of BV-2 cell growth curve over the first 60h after plating in a 96 well plate at 5,000 cells/well. <b>(B)</b> BV-2 cells were treated at 6h after plating, with vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (CytD, 1 μM). Each experiment was carried out in 8 replicates, with graph summarizing 3 independent experiments (mean ± SEM, n = 3). **p<0.01 compared to saline. <b>C)</b> Representative photographs of BV-2 cells treated with saline, 30μM MW151, or 1μM CytD at 0, 12, 24, 36 and 48 hrs after drug treatment. All imaging was done using Incucyte Zoom at 10x objective.</p

No effects of MW151 on BV-2 microglia cell migration into scratch wound.

<p><b>(A)</b> Representative graph of the rate of BV-2 cell migration into wound area, as determined by the percent confluency in the area left nearly devoid of cells after the scratch wound, and plotted as percent wound closure. <b>(B)</b> At 12h, during the linear phase of the wound closure, the effect of vehicle control (saline), MW151 (7.5, 15, or 30μM), or cytochalasin D (CytD, 1 μM) was quantified, as in (A), and plotted as percent of saline vehicle. The graph represents the average of three independent experiments (mean ± SEM, n = 3), each experiment carried out in 8 replicates for each treatment. **p<0.01 compared to saline vehicle. <b>(D)</b> Representative photographs of BV-2 cells migrating into scratch wound area with saline, 30μM MW151 or 1μM CytD treatment at 0, 6, 12, and 24 hrs after initial scratch. Blue lines indicate initial scratch wound area. Pink is wound area at each time point, calculated by Incucyte Zoom software. Images and data obtained using Incucyte Zoom at 10x objective.</p

Effects of MW151 on suppression of diffuse brain injury-induced IL-1β in the cortex.

<p><b>(A)</b> Overview of experimental design for dual administration, dose response experiment. <b>(B)</b> IL-1β was increased in the mFPI + veh group compared to sham + veh, and MW151 suppressed the injury-induced IL-1β increase at the three doses tested (F_4,39 = 5.4895; p = 0.0013) (n = 8 sham + veh; n = 12 mFPI + veh; n = 6 mFPI + MW151 0.5mg/kg; n = 6 mFPI + MW151 1.5mg/kg; n = 12 mFPI + MW151 5mg/kg). <b>(C)</b> Overview of experimental design for single administration, single dose experiment. (<b>D</b>) IL-1β was increased in the mFPI + veh group compared to sham + veh, and MW151 suppressed the injury-induced IL-1β increase compared to mFPI + veh (F_2,14 = 3.8882; p = 0.0499) (n = 3 sham + veh; n = 7 mFPI + veh; n = 5 mFPI + MW151 5mg/kg). ^#p<0.001 compared to sham + veh. *p<0.05, **p<0.001 compared to mFPI + veh. (mFPI = midline fluid percussion injury; veh = vehicle).</p