Verification of p38β KO in microglia and brain.

<p>Primary microglia from mouse cortex were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056852#s2" target="_blank">Methods</a> and plated at 1×10⁴ cells/well in 48 well plates. Total RNA from microglia cultures (<b>A</b>) or from mouse cortical tissue (<b>B</b>) derived from WT (white bars) or p38β KO (black bars) mice was isolated, and the mRNA levels of different p38 MAPK isoforms were determined by qPCR. In both the microglia cultures and the brain tissues, p38β mRNA was readily measureable in WT mice but was not detected in the p38β KO mice. The p38α MAPK isoform in both microglia and cortex was expressed at much higher levels than any of the other isoforms, and there was no significant difference between the levels of p38α in either WT or p38β KO mice. The levels of p38δ mRNA were very low to undetectable in both WT and p38β KO mice. The expression of p38γ mRNA was slightly higher in microglia cultures from p38β KO mice compared to microglia from WT mice, but this difference was not seen in the cortical tissue samples. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056852#s3" target="_blank">Results</a> are expressed as fold change compared to p38β levels, and represent the mean ± SEM of two determinations.</p

Microglial p38β MAPK deficiency failed to protect cortical neurons against LPS-induced neurotoxicity in microglia/neuron co-culture.

<p>WT mouse primary cortical neurons were plated on coverslips at 5×10⁴/well, and were co-cultured with microglia (2×10⁴/well) from WT or p38β KO mice. Cells were treated with either vehicle or LPS (3 ng/ml) for 72 hr, followed by trypan blue exclusion assay to evaluate neuronal survival. LPS treatment induced significant neuronal death in WT microglia/WT neuron co-culture. Similar levels of neuronal death were seen in LPS-treated co-cultures of p38β KO microglia/WT neurons. Data represent the mean ± SEM from 2–4 independent experiments. ****p<0.0001 WT-veh vs. WT-LPS; ^####p<0.0001 KO-veh vs. KO-LPS.</p

CaMKIIβ KO show reduced anxiety-related behaviors.

<p>(<b>A</b>) In the elevated plus maze task, the CaMKIIβ^+/− and CaMKIIβ^−/− mice spent more time in the open arms and less time in the closed arms compared to the CaMKIIβ^+/+ mice. (<b>B</b>) The KO mice also spent more time in the center of the open field. (<b>C</b>) Using the visual cliff test, the CaMKIIβ KO mice showed no deficit in vision (depth perception). Data are presented as means ±SEM. ^‡p<0.0001 Number of mice per group is indicated on each graph.</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

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

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

CaMKIIβ KO mice have impaired cognitive function and nesting behavior.

<p>(<b>A</b>) None of the mice show a preference for two identical objects presented in the NOR training session. In the subsequent test session done after a 4 hour delay, the WT mice showed a preference for the novel object as indicated by increased D2 index, but the KO mice showed no preference for either object. (<b>B</b>) CaMKIIβ KO mice made a significantly lower quality nest compared to their littermates. (<b>C</b>) Representative photographs of the nests. Photographs on the bottom row are higher-power views of the nests at 24 h. *p<0.05, **p<0.01. Data are presented as means ±SEM. Number of mice per group is indicated on each graph.</p

Development and characterization of CaMKIIβ KO mice.

<p>(<b>A</b>) Diagram of the targeting vector plasmid used to construct the CaMKIIβ KO. (<b>B</b>) Representations of the CaMKIIβ wildtype genomic locus, the targeted selection allele, the conditional allele, and the KO allele are shown. Location of PCR primer sets used to detect the different alleles is indicated by the horizontal arrows. (<b>C</b>) Representative western blots show the lack of CaMKIIβ protein in the KO mice with no compensatory changes in CaMKIIα levels. Data are presented as means ±SEM. n = 3–4 mice per genotype, for each brain region.</p

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