TRANSCRIPTIONAL NETWORK ANALYSIS IDENTIFIES ACCELERATED NEUROPROTECTIVE EFFECTS OF CYCLOSPORINE A IN PIGLET MILD TRAUMATIC BRAIN INJURY MODEL

Introduction: Traumatic brain injury (TBI) is a leading cause of death and disability in young children. Mild TBI, which represents 80% of TBIs, can lead to prolonged symptoms lasting up to 25 years when sustained during critical periods of brain development (young child – adolescent). However, there is still no effective treatment for mTBI in children due to a limited understanding of the molecular mechanisms driving pathophysiological changes post-injury. A piglet is a well-developed animal model for pediatric mTBI due to similarities in brain anatomy. Previous work in our lab showed that a 20mg/kg/day dose of Cyclosporine A (CsA) infused over 24 hours (starting 6h post-mTBI) in a piglet model displays neuroprotective effects, including improved mitochondrial health and decreased injured brain volume. Understanding genes modulated by CsA to drive neuroprotection will assist with developing therapies for pediatric mTBI. Therefore, we hypothesized that CsA would modulate neuroprotective transcriptional profiles that positively regulate neuronal recovery post-injury.

Materials and

Methods: We explored genes modulated by CsA by profiling piglet frontal cortex (FC) and hippocampus + amygdala (HA) brain regions after a rapid non-impact head rotation (RNR) mTBI. 4-week-old female Yorkshire piglets were anesthetized and assigned to one of 4 groups: sham injury (N=18); RNR injury with brain perfused and collected at 24 hours (N=10) or 1 week (N=15); and RNR followed by 20mg/kg/day CsA infusion, starting at 6 hours post-injury (N=10). Brains were formalin-fixed paraffin-embedded and sectioned for RNA sequencing. Weighted gene co-expression network analysis (WGCNA) was conducted on 13,426 genes in both regions and all groups.

Results, Conclusions, and Discussions: WGCNA revealed 13 modules of co-expressed genes as depicted by the different colors in the module clustering dendrogram (Fig. 1A). Each module represented a network of genes displaying the effect of injury, time, and CsA treatment. Of all 13 modules, we identified a group of modules in both regions exhibiting accelerated effects of CsA treatment (Fig. 1B). Rather than restoring the expression scores of degenerative profiles at 24 hours post-injury, CsA treatment accelerated neuronal and immune recovery profiles in these modules to resemble the 1 week post-injury group (top 5 hub genes in purple and black boxes). Overall, our data highlights a mechanism of CsA in accelerating networks of co-expressed genes associated with neuronal and immune recovery post-injury. We found that a 20mg/kg/day dose of CsA, initiated 6 hours post-mTBI, accelerates improved neuronal and immune recovery processes post-injury. Identifying genes associated with the neuroprotective effects of CsA will help with the development of treatment for pediatric mTBI.

Acknowledgements (Optional): Funded by Georgia Research Alliance and NIH (R01NS097549, U01NS069545, R01NS039679).