Injury and sex dependent observations on animal weights, righting times, and estrous cycle.
Measurements of animal weight, right time were measured in all animals immediately prior or after surgical procedures respectively, and two-way ANOVA was conducted to examine the effects of injury and sex (Fig. 1). For animal weight, there is a significant main effect of sex (p < 0.0001), with no significant effect of injury and no significant injury*sex interaction (Fig. 1a). For righting time, there were significant main effect of injury (p < 0.0001), sex (p = 0.0224) and injury*sex interaction (p = 0.0488) (Fig. 1b). Posthoc showed CCI-Male animals had significantly longer right time than CCI-Female animals (p = 0.014), while there were no differences in righting times between Sham-Male Sham-Female groups. Estrous cycle was determined in female animals only immediately prior to surgical procedures. Chi-square test showed no significant differences between estrous phases between female animals between the sham and CCI group (Fig. 1c).
Synaptic hippocampal neurogranin expression is reduced up to 4 weeks post-injury.
Changes in synaptic hippocampal expression of Ng was analyzed by two-way ANOVA to examine the effects of injury and sex at each time-point (Fig. 2a, b). At 24 hours post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction in Ng expression. At 1, 2 and 4 weeks post-injury, there is a significant main effect of injury (p < 0.0001, p = 0.0054, p = 0.0019, respectively), with no significant effect of sex and no significant injury*sex interaction.
Phosphorylated Neurogranin demonstrates sex and injury differences.
Changes in P-Ng synaptic hippocampal expression was analyzed by two-way ANOVA to examine the effects of injury and sex at each time-point (Fig. 3a, b). At 24 hours post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction in P-Ng expression. At 1 and 2 weeks post-injury, there is a significant main effect of injury (p = 0.0015, p = 0.0441, respectively), with no significant effect of sex and no significant injury*sex interaction. At 4 weeks post-injury there was no effect of injury; however, there was significant effect of sex (p = 0.0260) and a significant injury*sex interaction (p = 0.0266). Post-hoc comparisons shows significant differences between the male-CCI group from the male-sham group (p = 0.0413), female-sham group (p = 0.0407) and female-CCI group (p = 0.0141).
P-Ng synaptic expression was then normalized to total protein to determine a P-Ng/Ng ratio (Fig. 3c), which accounts for CCI-induced changes in synaptic Ng expression. At 24 hours and 2 weeks post-injury, there was no significant main effect of injury or sex and no significant injury*sex interaction in the P-Ng/Ng ratio. At 1 week post-injury there was a significant main effect of injury (p = 0.0306) with no significant effect of sex and no significant injury*sex interaction. At 4 weeks post-injury, there was a significant main effect of injury (p = 0.006), significant main effect of sex (p = 0.0308) and a significant injury*sex interaction (p = 0.0301). Post-hoc comparisons shows significant differences between the male-CCI group from the male-sham group (p = 0.001), female-sham group (p = 0.0011) and female-CCI group (p = 0.0175).
CaMKII expression shows sex and injury differences.
The effects of injury and sex on synaptic hippocampal P-CaMKII expression was determined by two-way ANOVA at each time-point (Fig. 4a, b). At 24 hours and 1 week post-injury, there is a significant main effect of injury (p = 0.0317, p = 0.0105, respectively), with no significant effect of sex and no significant injury*sex interaction in P-CaMKII expression. At 2 weeks post-injury there was a significant main effect of injury (p = 0.0003), significant main effect of sex (p = 0.0130) and a significant injury*sex interaction (p = 0.0130). Posthoc comparisons shows significant differences between the Female-CCI group from the male and female sham groups (p = 0.0004 and p = 0.0006, respectively), as well as significantly differences from the Male-CCI group (p = 0.0053). At 4 weeks post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction.
The effects of injury and sex on synaptic hippocampal α-CaMKII was determined by two-way ANOVA at each time-point (Fig. 4a, c). For synaptic expression, at 24 hours post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction. 1 week post-injury, there is a significant main effect of injury (p < 0.0001), with no significant effect of sex and no significant injury*sex interaction. At 2 weeks post-injury there was a significant main effect of injury (p < 0.0001), significant main effect of sex (p = 0.0376) and a significant injury*sex interaction (p = 0.0376). Post-hoc comparisons show significant differences between the Female-CCI group from both male and female sham groups (p = 0.0001 and p = 0.0002, respectively), as well as significant differences from the Male-CCI group (p = 0.0247). At 4 weeks post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction.
P-CaMKII synaptic expression was then normalized to α-CaMKII expression to determine changes in the P-CaMKII/α-CaMKII ratio (Fig. 4d). At 24 hours, 1 and 4 weeks post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction. At 2 weeks post-injury, there was no main effect of injury, however there was a significant main effect of sex (p = 0.0306) and a significant injury*sex interaction (p = 0.0325). Post-hoc comparisons show a significant difference between the Female-CCI and Male-CCI group (p = 0.0195).
CaM shows injury effect at 1 week post-injury.
The effects of injury and sex on synaptic hippocampal CaM expression was determined by two-way ANOVA at each time-point (Fig. 5a, b). At 24 hours, 2 and 4 weeks post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction. At 1 week post-injury, there is a significant main effect of injury (p = 0.0023), with no significant effect of sex and no significant interaction.
PSD-95 recovers from injury effect by 2 weeks post-injury.
The effects of injury and sex on synaptic hippocampal PSD-95 expression was determined by two-way ANOVA at each time-point (Fig. 6a, b). At 24 hours and 1 week post-injury, there is a significant main effect of injury (p = 0.0003, p = 0.0001, respectively), with no significant effect of sex and no significant injury*sex interaction. At 2 and 4 weeks post-injury, there was no significant main effect of injury or sex, and no significant injury*sex interaction.