Epigenetic and transcriptomic profiling of neural stem cells after midazolam exposure

Here we show that early-life exposure to midazolam (MDZ), a widely used drug in pediatric anesthesia, persistently alters chromatin accessibility and expression of quiescence-associated genes in neural stem cells (NSCs) in mouse hippocampus. The alterations led to a continuous restriction of NSC proliferation toward adulthood, resulting in reduction of neurogenesis associated with the impairment of hippocampal-dependent memory functions. Moreover, we found that voluntary exercise restored hippocampal neurogenesis accompanied by the normalization of MDZ-perturbed transcriptome and ameliorated declined cognitive ability in MDZ-exposed mice. Nestin-EGFP mice were injected with 10mg/kg of MDZ or dH2O. RNA-seq and ATAC-seq was performed using EGFP-positive NSCs and their progenies isolated from Nestin-EGFP mice at 10-day-old (P10) and 8-week-old (8W) mice with or without voluntary exercise (RUN). For ChIP-seq analysis, Egr1 was transduced in cultured NSC and collected samples were then subjected to ChIP assay using anti-FLAG antibody..

本研究证实，生命早期暴露于咪达唑仑（midazolam, MDZ）——一种儿科麻醉领域广泛应用的药物——会持续改变小鼠海马体神经干细胞（neural stem cells, NSCs）的染色质可及性及静息相关基因的表达水平。该改变会持续限制神经干细胞增殖至成年阶段，最终导致神经发生水平下降，并伴随海马依赖型记忆功能受损。此外，本研究发现自愿跑轮运动可恢复咪达唑仑暴露小鼠的海马神经发生水平，同时使受MDZ扰动的转录组恢复正常，并改善其衰退的认知能力。实验中，我们以10mg/kg剂量的咪达唑仑或双蒸水（dH2O）对巢蛋白增强绿色荧光蛋白（Nestin-EGFP）小鼠进行注射。分别于小鼠出生后10日龄（P10）及8周龄（8W）时，分离携带EGFP阳性的神经干细胞及其子代细胞，针对是否进行自愿跑轮运动（RUN）的各组样本开展RNA测序（RNA-seq）与转座酶可及性测序（ATAC-seq）。针对染色质免疫共沉淀测序（ChIP-seq）分析，我们将早期生长反应因子1（Egr1）转染至体外培养的神经干细胞，随后使用抗FLAG抗体开展染色质免疫共沉淀实验并收集对应样本。