Mechanisms and blood-based biomarkers of intergenerational neurobehavioral effects of general anesthetics

每年有数以亿计的患者暴露在全身麻醉剂(GAS)中，从而产生可遗传的影响 天然气是最重要的公共卫生问题。有必要研究气体和酒精的遗传效应 开发预防性疗法也显示出前所未有的增长，特别是在工业化国家， 神经发育障碍。这些疾病中的大多数原因尚不清楚，许多更常见。 在男性身上。为了形成关于这一主题的临床研究的基础，拟议的临床前项目将测试以下内容 关于GAS可遗传效应的机制、治疗工具和生物标志物的假说： 1)GA诱导的类固醇应激激素皮质酮(CORT)的分泌在 亲代生殖细胞(F0代)的表观遗传学变化以及后代(F1代)的异常 2)GABA能GA引起K+-2Cl-(KCC2)Cl-输出体的损伤，导致 GA诱导的F0需要抑制的GABAAR信号的损伤和皮质醇分泌的增加 神经行为缺陷；3)F1男性更容易受到伤害，因为F0气体通过修饰男性- 特定的F1脑部阳刚化。目的1：确定KCC2和CORT在启动 七氟醚(Sevo)的代际效应。临床使用的四种部分重叠的GAS 作为药理工具，赛沃、异丙酚、氯胺酮和依托咪酯将被用来确定这些作用 KCC2和Cort.F0代大鼠在出生后56天、58天和60天暴露于GAS，并在 P85以产生后代。F0和F1大鼠将在高架加迷宫中进行评估，预脉冲抑制 惊吓*，莫里斯水迷宫，恐惧强化惊吓，强迫游泳试验，通过评估休息和 应激诱导的下丘脑-垂体-肾上腺(HPA)轴活动*。F0和F1全基因组DNA甲基化 将使用生殖细胞和海马体，以及F0和F1海马体和外周血单核细胞中的RNA-seq 为了深入了解表观基因组学和转录学机制(*=将被评估为潜在的 未来的人体研究)。目标2：确定父母较短的Sevo暴露是否不足以诱导 F0神经行为效应可以诱导F1效应，并决定了Cl-转运体和糖皮质激素的作用 受体(GRs)。假设：较短的F0 Sevo暴露足以上调F0 HPA轴和 对生殖系进行重新编程，导致F1缺陷。KCC2促进或Na+-K+-Cl-(NKCC1)和GR抑制 治疗效果。目的3：确定双亲性别偏向代际效应的机制 暴露在Sevo中。假设：F1男性更脆弱，因为F0 Sevo会影响睾酮(T)-- 有规律的大脑男性化。因为T通过17β-雌二醇(E_2)作用于雌激素受体α(ER-α)和 通过T激活的雄激素受体(AR)，我们假设F1 ERα-/-(但不是ERβ-/-)雄性和/或F1野生型 出生时接受AR拮抗剂氟他胺治疗的型男性受影响较小。KCC2增强或NKCC1 抑制GR可减轻F0Sevo诱导的F1男性脑男性化机制的改变。

Hundreds of millions of patients are exposed to general anesthetics (GAs) each year, making the heritable effects of GAs a public health issue of paramount importance. The need to investigate the heritable effects of GAs and develop preventative therapies is also indicated by an unprecedented rise, particularly in industrialized countries, of neurodevelopmental disorders. The origin of most of these disorders is unknown and many are more common in males. To form the basis for clinical studies on this topic, the proposed preclinical project will test the following hypotheses regarding the mechanisms, therapeutic tools, and biomarkers pertaining to heritable effects of GAs: 1) GA-induced secretion of the steroid stress hormone corticosterone (CORT) is essential in the initiation of epigenetic changes in parental germ cells (F0 generation) and, by extension, abnormalities in offspring (F1 generation); 2) both GABAergic GA-induced impairment of the K+-2Cl- (KCC2) Cl- exporter, resulting in impairment of inhibitory GABAAR signaling, and an increase in CORT secretion are required for GA-induced F0 neurobehavioral defects; and 3) F1 males are more vulnerable because F0 GAs act via modification of male- specific F1 brain masculinization. Aim 1: Determine the roles of KCC2 and CORT in the initiation of intergenerational effects of sevoflurane (SEVO). Four clinically used GAs with partially overlapping mechanisms of action, SEVO, propofol, ketamine and etomidate, will be used as pharmacological tools to determine the roles of KCC2 and CORT. The F0 rats will be exposed to GAs on postnatal day (P) 56, P58, and P60 and mated on P85 to generate offspring. The F0 and F1 rats will be evaluated in the elevated plus maze, prepulse inhibition of startle*, Morris water maze, fear-potentiated startle, and forced swimming test and by assessing resting and stress-induced hypothalamic-pituitary-adrenal (HPA) axis activity*. Genome-wide DNA methylation in F0 and F1 germ cells and hippocampi and RNA-seq in F0 and F1 hippocampi and peripheral blood monocytes* will be used to gain insight into epigenomic and transcriptomic mechanisms (* = will be evaluated as potential biomarkers for future human studies). Aim 2: Determine whether shorter parental SEVO exposure that is not sufficient to induce F0 neurobehavioral effects can induce F1 effects, and determine the roles of Cl- transporters and glucocorticoid receptors (GRs). Hypotheses: Shorter F0 SEVO exposure is sufficient to upregulate the F0 HPA axis and reprogram the germline, inducing F1 defects. KCC2 enhancement or Na+-K+-Cl- (NKCC1) and GR inhibition have therapeutic effects. Aim 3: Determine the mechanisms of male-biased intergenerational effects of parental exposure to SEVO. Hypotheses: F1 males are more vulnerable because F0 SEVO affects testosterone (T)- regulated brain masculinization. Because T acts through 17β-estradiol (E2) at estrogen receptor α (ERα) and via T-activated androgen receptors (ARs), we hypothesize that F1 ERα-/- (but not ERβ-/-) males and/or F1 wild type males treated with the AR antagonist flutamide at birth will be less affected. KCC2 enhancement or NKCC1 and GR inhibition will alleviate the F0 SEVO-induced changes in F1 male mechanisms of brain masculinization.