Poster U13 - Inhibition of p38 MAPK Phospho-signaling after TBI Modulates Microglial Markers and Cytokines in a Sex Dependent Manner

Introduction: Mild traumatic brain injury (mTBI) can lead to significant functional deficits, including depression, and anxiety, that can persist for months. Repetitive injuries sustained within a “window of vulnerability” after the initial insult can exacerbate these consequences compared to a single mTBI. Increasing evidence suggests that brain inflammatory processes consisting of phospho-signaling pathways and microglia activation mediate functional consequences. Previously, we reported that phosphorylation of the pro-inflammatory signaling molecule p38 MAPK was correlated with worse functional outcomes following a weight-drop mouse model of repetitive mTBI. In light of this finding, coupled with the established immunomodulatory role of p38 MAPK, we investigated the effect of acute p-p38 inhibition on cytokine expressions and microglia activation after repetitive mTBI. We hypothesized that acute inhibition of p38 MAPK phosphorylation would ameliorate microglial activation and cytokine expression after injury compared to controls.

Materials and

Methods: Adult male and female C57/BL6 mice were intraperitoneally injected with the p38 MAPK inhibitor SB239063 (SB) or vehicle 30 minutes following each of 5 once-daily weight-drop closed head injuries (CHIs) or sham injuries. Brain tissues were collected at 4-hr and 4-weeks after the final injury, followed by protein extraction in urea lysis buffer. A panel of 32 cytokines was quantified via Luminex multiplexed immunoassays. Microglial markers Tmem119 and CD68 were quantified by ELISA. All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee.

Results, Conclusions, and Discussions: In females, acute p38 inhibition resulted in reduced injury-induced elevated expression of the macrophage inflammatory protein 1a (MIP-1a), a pro-inflammatory molecule that has been implicated in the development of neuroinflammation after brain injury, at 4-hr post injury (Wilcox, p< 0.05; Figure 1a)). Indeed, recent studies have demonstrated that MIP-1a 1) was essential for the recruitment of neutrophils and glial cells, and 2) was able to regulate synaptic plasticity mechanisms critical for learning and memory functions. Similar trends were observed in MIP-1a, another class of MIP protein found elevated in damaged brain tissues, but did not reach significance (Wilcox, p>0.05; Figure 1b). In contrast, in males, the impact of acute p-p38 MAPK inhibition on cytokine changes was less pronounced, suggesting a distinct effect on cytokine expressions.
Coupled with the known role of microglia in mediating molecular and functional consequences following brain injury, we next investigated the effect of SB treatment on microglia. In females, the microglial homeostatic marker Tmem119 significantly increased at 4-hr post-injury, suggesting increased acute microglial recruitment, followed by a decrease at 4-week (Wilcox, p< 0.05; Figure 2b, d), implying a prolonged activation of the recruited microglia. Additionally, mononuclear phagocyte activation marker CD68 significantly increased at 4 weeks post-injury (Wilcox, p< 0.05l; Figure 2c, e), highlighting the sustained increase in microglial activation. Importantly, both Tmem119 and CD68 changes in females were attenuated by acute p38 MAPK inhibition (Wilcox, p< 0.05; Figure 2b-d), potentially indicating the role of p38 phosphorylation in driving both acute and longer-term microglial changes following repetitive mTBI. While males showed fewer significant microglial changes with injury, acute p-p38 MAPK inhibition decreased injury-associated upregulation of CD68 at 4-hr post-injury (Wilcox, p< 0.05; Figure 2c), suggesting an immediate effect on microglial activation. Together, these data suggest shared effects of transient inhibition of p38 MAPK phosphorylation on microglia activation across sex, but on pro-inflammatory chemokines uniquely in females.

In summary, our findings suggest that the p38 MAPK signaling pathway may modulate the brain's inflammatory response post-injury in a sex-specific manner. Furthermore, acute SB treatment may serve as a potential therapeutic strategy targeting undelaying brain inflammation following repetitive mTBI.

Acknowledgements (Optional): This project was supported by the National Institutes of Health R01 NS115994.