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

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

CaMKIIβ KO mice have a developmental delay in body weight.

<p>(<b>A</b>) CaMKIIβ^+/+ and CaMKIIβ^−/− mice were weighed at birth, or starting at weaning (3 weeks old) until 4 months of age. n = 5–13/time point. (<b>B</b>) Echo MRI was used to determine lean/fat mass in 12 week old mice. No genotype effect was seen in the amount of water (<b>C</b>) or in the amount of food (<b>D</b>) consumed, or in respiratory exchange ratio (<b>E</b>). *p<0.05, **p<0.01. Data are presented as means ±SEM. n = 4–6 per group in panels B–E.</p

CaMKIIβ KO have impaired motor function.

<p>(<b>A</b>) CaMKIIβ KO mice show impaired forelimb grip strength but not hindlimb grip strength. The CaMKIIβ were impaired on the rotorod (<b>B</b>) and on the balance beam (<b>C</b>). No difference in spontaneous movement was seen in the open field task (<b>D</b>), but spontanous activity in the running wheel was decreased (<b>E</b>). *p<0.05, **p<0.01. Data are presented as means ±SEM. Number of mice per group is indicated on each graph.</p

CaMKIIβ immunohistochemistry.

<p>Representative images of the CaMKIIβ IHC staining for one mouse from each genotype is shown at approximately 1 mm lateral intervals from midline. In the WT (+/+) mouse, CaMKIIβ staining is highest in the olfactory bulb, but is also apparent in the cerebellum, cortex, hippocampus, striatum, and substantia nigra. The heterozygous mice show an intermediate level of staining. The KO mice show a lack of CaMKIIβ staining throughout the brain.</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

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

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

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