miR-9-5p is downregulated in serum extracellular vesicles of patients treated with biperiden after traumatic brain injury
Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE, and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10 days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with 3 subjects developing PTE, and 19 received placebo, with 2 developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings suggest serum miR-9-5p as a possible marker for biperiden treatment, which may play a role in TBI resolution. Overall design: Within approximately 12 hours following TBI, patients began receving, every 6 hours, either placebo or biperiden, intravenously. A total of 40 doses (5 mg of biperiden per dose) were administered per patient, as described in the clinical trial NCT01048138. Approximately 10 hours after the last biperiden or placebo dose, blood samples were collected from each patient. Serum was obtained after centrifugation, then frozen and maintained in -80C until processing. Serum extracellular vesicles (EVs) were isolated, and microRNAs (miRNAs) were extracted. miRNA concentration was determined using nanodrop, and miRNA libraries were then prepared and sequenced. Differential expression analysis of miRNA-Seq was performed between placebo- and biperiden-treated patients, as well as between patients that did or did not develop PTE.