Discovery and Validation of Circulating microRNAs as Biomarkers for Epileptogenesis after Experimental Traumatic Brain Injury–The EPITARGET Cohort

Abstract

Traumatic brain injury (TBI) causes 10–20% of structural epilepsies and 5% of all epilepsies. The lack of prognostic biomarkers for post-traumatic epilepsy (PTE) is a major obstacle to the development of anti-epileptogenic treatments. Previous studies revealed TBI-induced alterations in blood microRNA (miRNA) levels, and patients with epilepsy exhibit dysregulation of blood miRNAs. We hypothesized that acutely altered plasma miRNAs could serve as prognostic biomarkers for brain damage severity and the development of PTE. To investigate this, epileptogenesis was induced in adult male Sprague Dawley rats by lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least one unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Cortical pathology was analyzed by magnetic resonance imaging on day 2 (D2), D7, and D21, and by histology 6 months post-TBI. Small RNA sequencing was performed from tail-vein plasma samples on D2 and D9 after TBI (n = 16, 7 with and 9 without epilepsy) or sham operation (n = 4). The most promising miRNA biomarker candidates were validated by droplet digital polymerase chain reaction in a validation cohort of 115 rats (8 naïve, 17 sham, and 90 TBI rats [21 with epilepsy]). These included 7 brain-enriched plasma miRNAs (miR-434-3p, miR-9a-3p, miR-136-3p, miR-323-3p, miR-124-3p, miR-212-3p, and miR-132-3p) that were upregulated on D2 post-TBI (p < 0.001 for all compared with naïve rats). The acute post-TBI plasma miRNA profile did not predict the subsequent development of PTE or PTE severity. Plasma miRNA levels, however, predicted the cortical pathology severity on D2 (Spearman ρ = 0.345–0.582, p < 0.001), D9 (ρ = 0.287–0.522, p < 0.001–0.01), D21 (ρ = 0.269–0.581, p < 0.001–0.05) and at 6 months post-TBI (ρ = 0.230–0.433, p < 0.001–0.05). We found that the levels of 6 of 7 miRNAs also reflected mild brain injury caused by the craniotomy during sham operation (ROC AUC 0.76–0.96, p < 0.001–0.05). In conclusion, our findings revealed that increased levels of neuronally enriched miRNAs in the blood circulation after TBI reflect the extent of cortical injury in the brain but do not predict PTE development.