Suppression and Recovery of the Murine Hypothalamic-Pituitary-Adrenal Axis after Exogenous Glucocorticoid Treatment

外源性糖皮质激素治疗后小鼠下丘脑-垂体-肾上腺轴的抑制和恢复

基本信息

批准号：
10609799
负责人：
Lindsey Sara Gaston
金额：
$ 8.11万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10609799
关键词：
Adrenal Cortex Hormones Adrenal Glands Adrenal gland hypofunction Adult American Analysis of Variance Animal Model Animals Apoptosis Apoptotic Automobile Driving Biological Assay Blood Glucose CASP3 gene CRH gene CYP11B2 gene Cells Chronic Clinical Research Corticosterone Corticotropin Cosyntropin DNA Nucleotidylexotransferase Dangerousness Dexamethasone Dose Exposure to Feedback Frequencies Functional disorder Future Glucocorticoids Grant Herpes zoster disease Histologic Human Hydrocortisone Hypoglycemia Hypothalamic structure Iatrogenesis Immunohistochemistry Individual Insulin Intervention Label Life Maintenance Measures Mediating Microscopy Mitosis Mitotic Mixed Function Oxygenases Modeling Molecular Morbidity - disease rate Mus Natural regeneration Neurons Patients Pharmacologic Substance Pituitary Gland Process Proliferating Recovery Recovery of Function Role Staining and Labeling Standardization Steroids Stimulus Stress Testing Time Tissue Harvesting Tissues Transferase Trauma Treatment Efficacy Vulnerable Populations Withdrawal Zona Glomerulosa biological adaptation to stress cell type circadian cohort drinking water epigenomics experimental study fluorescence imaging hypothalamic-pituitary-adrenal axis insight mRNA Expression male mouse model novel physiologic stressor prevent progenitor response transdifferentiation

项目摘要

PROJECT SUMMARY/ABSTRACT: Chronic, supraphysiologic glucocorticoid exposure leads to suppression of the hypothalamic-pituitary-adrenal (HPA) axis that can persist for months after steroids are withdrawn, leaving individuals vulnerable to life-threatening adrenal crises. Clinical studies in humans suggest that HPA axis dysfunction after withdrawal of long-term steroids is mediated first by hypothalamic-pituitary dysfunction followed by delayed adrenocortical recovery despite appropriate, compensatory ACTH stimulation. While the mammalian HPA axis is highly conserved, robust animal models of steroid-induced suppression and recovery are lacking. This has limited our understanding of the mechanisms driving protracted but reversible HPA axis dysfunction after withdrawal of long-term glucocorticoids, which are likely both centrally and adrenally mediated and distinct from simple negative feedback. This study seeks to characterize the relationship between the duration of exogenous, supraphysiologic glucocorticoid exposure and time to functional HPA axis recovery as well as the molecular changes driving these processes at the level of the hypothalamus, pituitary, and adrenal glands. We will treat adult, male C57BL/6J mice (n=5/cohort) for 1, 8, or 24 weeks with either vehicle (DN) or dexamethasone (DEX; 10 mcg/day=~35 mg hydrocortisone equivalent/m2/day) via drinking water. We will then perform weekly assessments of basal (circadian peak and nadir) and stress-induced ACTH and CORT secretion from the time of steroid withdrawal until functional recovery is documented, defined as the timepoint at which there are no significant differences between these ACTH and CORT levels vs. those of DN animals by ANOVA with Dunnett’s multiple comparisons test. To measure stress-induced secretion, animals will undergo both insulin-induced hypoglycemia and Cosyntropin stimulation testing to assess how the axis responds to a potent, physiologic stressor as well as adrenocortical sensitivity to a common stimulus. Animals from each of these timepoints will undergo necroscopy after the final functional assessment, from which we will generate hypothalamic, pituitary, and adrenal sections for quantification of Crh, Pomc, and Cyp11b1 mRNA expression, respectively. We will next assess whether steroid-induced suppression and recovery are mediated by sequential apoptosis and mitosis of each of these cell types. To do this, we will first perform TUNEL staining followed by immunohistochemistry for either 1) activated caspase-3 or 2) Ki67 co-localized with a) CRH, b) POMC, or c) steroid 11β-hydroxylase in adrenal, pituitary, or hypothalamic sections. Finally, we will document zona Fasiculata regeneration from zona Glomerulosa precursors in DEX-treated, aldosterone synthase (AS)-Cre/mTmG (AS+/Cre : : R26R+/mTmG) mice, with fluorescent microscopy of adrenals at the time of steroid withdrawal, HPA axis recovery, and several intermediate timepoints defined by the prior experiments. These studies will be used collectively to generate hypotheses for future mechanistic, epigenomic studies outside the scope of this grant. Elucidation of the novel mechanisms by which chronic glucocorticoids durably but reversibly suppress the HPA axis may ultimately lead to interventions that decrease the frequency or duration of iatrogenic secondary adrenal insufficiency.

项目摘要/摘要：慢性、超生理性糖皮质激素暴露会导致下丘脑-垂体-肾上腺 (HPA) 抑制类固醇停用后，轴可能会持续数月，使个体容易受到生命威胁人类临床研究表明，长期停用类固醇后，HPA 轴功能障碍。首先是由下丘脑-垂体功能障碍介导，然后是肾上腺皮质恢复延迟，尽管适当的、补偿性的 ACTH 刺激，而哺乳动物的 HPA 轴是高度保守、强健的动物。缺乏类固醇诱导的抑制和恢复模型，这限制了我们对这一现象的理解。长期停用糖皮质激素后导致长期但可逆的 HPA 轴功能障碍的机制，这可能是中枢和肾上腺介导的，与简单的负反馈不同。表征外源性、超生理性糖皮质激素暴露的持续时间与 HPA 轴功能恢复的时间以及驱动这些过程的分子变化我们将对成年雄性 C57BL/6J 小鼠（n=5/队列）进行 1、8 或 24 次治疗。使用媒介物 (DN) 或地塞米松 (DEX；10 微克/天=~35 毫克氢化可的松当量/平方米/天) 几周然后，我们将每周进行基础（昼夜节律峰值和最低点）和压力引起的评估。从类固醇停药到功能恢复记录的 ACTH 和 CORT 分泌，定义为 ACTH 和 CORT 水平与 DN 水平之间没有显着差异的时间点通过方差分析和 Dunnett 的多重比较测试来测量动物为了测量压力诱导的分泌，动物会接受胰岛素引起的低血糖和促肌钙蛋白刺激测试，以评估轴如何响应强烈的生理应激源以及肾上腺皮质对每种动物的共同刺激的敏感性。这些时间点将在最终功能评估后进行尸检，我们将根据评估结果生成下丘脑、垂体和肾上腺切片，用于量化 Crh、Pomc 和 Cyp11b1 mRNA 表达，接下来我们将分别评估类固醇诱导的抑制和恢复是否是由顺序介导的。为了做到这一点，我们首先进行 TUNEL 染色，然后进行这些细胞类型的凋亡和有丝分裂。免疫组织化学检测 1) 激活的 caspase-3 或 2) Ki67 与 a) CRH、b) POMC 或 c) 类固醇共定位最后，我们将记录肾上腺、垂体或下丘脑切片中的 11β-羟化酶。肾小球带前体在 DEX 处理的醛固酮合酶 (AS)-Cre/mTmG (AS+/Cre : : R26R+/mTmG) 小鼠，在类固醇停药时进行肾上腺荧光显微镜检查，HPA 轴恢复，以及先前实验定义的几个中间时间点将被共同用于生成。未来机制、表观基因组研究的假设超出了本次资助的范围。慢性糖皮质激素持久但可逆地抑制 HPA 轴的机制最终可能导致减少医源性继发性肾上腺皮质功能不全的频率或持续时间的干预措施。