Discovery and validation of circulating microRNAs as biomarkers for epileptogenesis after experimental traumatic brain injury – the EPITARGET cohort

Purpose: 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. In this study, we conducted high throughput small RNA-Seq analysis from tail-vein plasma samples collected from a rat model of PTE to discover miRNA biomarker candidates that could serve as prognostic biomarkers for brain damage severity and the development of PTE. Methods: Epileptogenesis was induced in adult male Sprague-Dawley rats by the lateral fluid-percussion-induced TBI. Epilepsy was defined as the occurrence of at least 1 unprovoked seizure during continuous 1-month video-electroencephalography monitoring in the sixth post-TBI month. Small RNA-seq was performed from tail-vein plasma samples collected from a subset of 20 animals (4 sham-operated controls, 7 TBI rats with epilepsy, 9 TBI rats without epilepsy) at 2 days and 9 days post-TBI. RNA was extracted from 200 μl plasma using an miRNeasy Mini Kit. Small RNA-Seq was conducted by GenomeScan (Leiden, the Netherlands). Small RNA library preparation was performed using the Illumina TruSeq Small RNA Sample Prep Kit. Single-end sequencing was performed on the Illumina HiSeq 4000.研究目的：创伤性脑损伤（Traumatic brain injury, TBI）可导致10%~20%的结构性癫痫，占所有癫痫病例的5%。目前创伤后癫痫（Post-traumatic epilepsy, PTE）缺乏预后生物标志物，这是阻碍抗致痫治疗开发的主要障碍。本研究针对创伤后癫痫大鼠模型的尾静脉血浆样本开展高通量小RNA测序分析，旨在发掘可作为脑损伤严重程度及创伤后癫痫发生预后生物标志物的微小RNA（miRNA）候选靶点。 研究方法：通过侧方流体冲击诱导法，在成年雄性斯普拉格-道利（Sprague-Dawley）大鼠体内构建创伤性脑损伤致痫模型。于创伤性脑损伤造模后第6个月，通过连续1个月的视频脑电图监测，将出现至少1次无诱因癫痫发作者定义为癫痫模型大鼠。本研究从20只大鼠的亚群中采集造模后第2天及第9天的尾静脉血浆样本，该亚群包含4只假手术对照大鼠、7只创伤后癫痫模型大鼠及9只创伤后无癫痫模型大鼠，并对样本进行小RNA测序。使用miRNeasy迷你试剂盒从200 μL血浆中提取RNA。小RNA测序工作由荷兰莱顿的GenomeScan公司完成。小RNA文库构建采用Illumina TruSeq小RNA样本制备试剂盒，在Illumina HiSeq 4000测序平台上完成单端测序。