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.

创伤性脑损伤（traumatic brain injury, TBI）是一种高发且可导致严重残疾的疾病，常继发创伤后癫痫（post-traumatic epilepsy, PTE），而目前针对该病仍缺乏有效的预防策略。比哌立登（biperiden）作为一种抗胆碱能药物，在PTE小鼠模型中已展现出确切疗效，是极具前景的候选干预手段。微小RNA（microRNAs, miRNAs）是一类小型调控RNA，可助力阐明PTE的生物学基础，同时可作为与TBI和PTE相关的外周检测生物标志物：这类RNA存在于可跨越血脑屏障的细胞外囊泡（extracellular vesicles, EVs）中，因此可在外周样本中被检测到。

本研究旨在探究TBI患者血清EV中的miRNA表达谱，及其与比哌立登治疗和PTE发生的关联。本研究依托一项双盲临床试验，共收集37名TBI患者在创伤后10天且启动治疗后的血液样本。其中18名患者接受比哌立登治疗，3人继发PTE；19名患者接受安慰剂治疗，2人继发PTE。

研究人员首先对血清EV进行粒径分布与蛋白质谱表征，随后对EV的miRNA组进行高通量测序。差异表达分析结果显示，继发PTE与未继发PTE的TBI患者之间，miRNA表达水平无显著差异。值得注意的是，与安慰剂组相比，接受比哌立登治疗的患者体内miR-9-5p的表达水平显著降低。该miRNA所调控的基因富集于应激反应通路，包括轴突生成与神经元死亡通路，这些通路均与PTE和TBI密切相关。上述研究结果提示，血清miR-9-5p可能作为比哌立登治疗的潜在标志物，其或许在TBI的转归过程中发挥作用。

总体实验设计：
在TBI发生后约12小时内，患者开始每6小时接受一次静脉输注的安慰剂或比哌立登。如临床试验NCT01048138所述，每名患者共接受40次给药（每次给药剂量为5 mg比哌立登）。在最后一次给药（比哌立登或安慰剂）后约10小时，采集每名患者的血液样本。血液经离心分离血清后，置于-80℃冷冻保存直至后续实验处理。随后分离血清细胞外囊泡并提取miRNA。采用Nanodrop测定miRNA浓度，之后构建miRNA文库并进行测序。分别对安慰剂组与比哌立登治疗组、继发与未继发PTE的患者，开展miRNA测序数据的差异表达分析。