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leads to suppression of the hypothalamic-pituitary-adrenal (HPA) axis that can persist for months after steroids are withdrawn, leaving individuals vulnerable to life-threatening adrenal crises.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，超生理学的糖皮质激素暴露导致下丘脑 - 垂体 - 肾上腺（HPA）抑制撤回类固醇后可能会持续数月的轴，使人容易受到威胁生命肾上腺犯罪。人类的临床研究表明HPA轴功能障碍在长期立体定向后首先由下丘脑 - 垂体功能障碍介导适当的补偿性ACTH刺激。虽然哺乳动物HPA轴是高度保守的，但可靠的动物缺乏类固醇引起的抑制和恢复模型。这限制了我们对驱动的机制探测但可逆的HPA轴功能障碍在长期糖皮质激素退出后，这些可能既可以集中介导，又与简单的负反馈不同。这项研究寻求表征外源性，生理学糖皮质激素的持续时间与是时候进行功能性HPA轴恢复以及将这些过程驱动这些过程水平的分子变化的时间下丘脑，垂体和肾上腺。我们将以1、8或24治疗成人，男性C57BL/6J小鼠（n = 5/cohort）用车辆（DN）或地塞米松（Dex; 10 mcg/day = 〜35 mg氢化可的松等效/m2/day）进行周数周饮用水。然后，我们将每周对巴萨（昼夜节日峰和纳迪尔）进行每周评估，并进行压力诱导从类固醇抽出时期到功能恢复的记录，ACTH和CORT分泌物定义为这些ACTH和CORT水平与DN的时间点之间没有显着差异的时间点 ANOVA的动物进行了Dunnett的多重比较测试。为了测量压力引起的分泌，动物将接受胰岛素诱导的低血糖和cosyntropin刺激测试，以评估轴对潜在的，生理压力源以及对常见刺激的肾上腺皮质敏感性。每一个这些时间点将在最终功能评估后进行坏死，我们将产生。下丘脑，垂体和肾上腺切片，用于定量CRH，POMC和CYP11B1 mRNA表达，分别。接下来，我们将评估类固醇引起的抑制和恢复是否由顺序介导每种细胞类型的凋亡和有丝分裂。为此，我们将首先进行Tunel染色，然后进行 1）激活的caspase-3或2）Ki67与A）CRH，B）POMC或C）类固醇ki67 肾上腺，垂体或下丘脑切片中的11β-羟化酶。最后，我们将记录Zona Fasicurata 在DEX处理的，醛固酮合酶（AS）-CRE/MTMG（AS+/CRE ：：： R26R+/mtmg）小鼠，在类固醇戒断，HPA轴恢复和先前实验定义的几个中间时间点。这些研究将集体用于生成在这笔赠款范围之外，假设未来的机械性，表观基因组学研究。阐明小说彻底但可逆地抑制HPA轴的慢性糖皮质激素的机制可能最终导致降低肠胃植物肾上腺功能不全的频率或持续时间的干预措施。