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

每年有数亿患者暴露于全身麻醉药（GAs）， GAs是一个至关重要的公共卫生问题。需要调查遗传效应的遗传的气体和 发展预防性疗法也表明，特别是在工业化国家， 神经发育障碍大多数这些疾病的起源尚不清楚，而且许多更常见 在男性中。为了形成关于该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GA遗传效应的机制、治疗工具和生物标志物的假设： 1)GA诱导的类固醇应激激素皮质酮（CORT）的分泌在启动 亲代生殖细胞（F0代）的表观遗传变化，以及后代（F1代）的异常 2）GABA能GA诱导的K+-2Cl-（KCC 2）Cl-输出蛋白受损，导致 抑制性GABAAR信号传导的损伤和CORT分泌的增加是GA诱导的F0 神经行为缺陷;和3）F1雄性更脆弱，因为F0 GAs通过修饰雄性- 特异性F1脑雄性化。目的1：确定KCC 2和CORT在启动细胞凋亡中的作用。 七氟烷（SEVO）的代际效应。4种临床使用的GA，具有部分重叠的机制 SEVO、丙泊酚、氯胺酮和依托咪酯将用作药理学工具，以确定其作用 KCC 2和CORT。F0代大鼠将在出生后第56天（P）、第58天和第60天暴露于GA，并在 P85产生后代。将在高架十字迷宫中评价F0和F1大鼠， 惊吓 *、Morris水迷宫、恐惧增强惊吓和强迫游泳试验，并通过评估休息和 应激诱导下丘脑-垂体-肾上腺（HPA）轴活动 *。F0和F1中的全基因组DNA甲基化 将使用F0和F1代生殖细胞和外周血单核细胞中的RNA-seq * 以深入了解表观基因组和转录组机制（* =将被评估为潜在的生物标志物， 未来的人类研究）。目的2：确定不足以诱导的较短亲代SEVO暴露是否 F0神经行为效应可诱导F1效应，并决定Cl-转运体和糖皮质激素的作用 受体（GR）。假设：较短的F0 SEVO暴露足以上调F0 HPA轴， 重新编程生殖细胞，诱导F1缺陷。KCC 2增强或Na+-K+-Cl-（NKCC 1）和GR抑制， 治疗效果目的3：确定父母性别偏好代际效应的机制 暴露于SEVO。假设：F1雄性更脆弱，因为F0 SEVO影响睾酮（T）- 调节大脑男性化。因为T通过17β-雌二醇（E2）作用于雌激素受体α（ERα）， 通过T激活的雄激素受体（AR），我们假设F1 ERα-/-（而不是ERβ-/-）雄性和/或F1野生型 在出生时用AR拮抗剂氟替卡松治疗的2型男性受影响较小。KCC 2增强或NKCC 1 而GR抑制可减轻F0代SEVO诱导的F1代雄性脑雄性化机制的改变。

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.