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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leads to suppression of the hypothalamic-pituitary-adrenal (HPA) axis that can persist for months after steroids are withdrawn, leaving individuals vulnerable to life-threatening adrenal crises.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.

项目总结/摘要：慢性、超生理性糖皮质激素暴露导致下丘脑-垂体-肾上腺（HPA）抑制轴，可以持续数月后，类固醇撤出，使个人容易受到危及生命的肾上腺危象人类临床研究表明，长期停用类固醇后HPA轴功能障碍首先由下丘脑-垂体功能障碍介导，随后是延迟的肾上腺皮质恢复，适当的、补偿性的促肾上腺皮质激素刺激。虽然哺乳动物HPA轴是高度保守的，但强健的动物HPA轴是一种非常重要的基因。缺乏类固醇诱导的抑制和恢复的模型。这限制了我们对在长期糖皮质激素停药后驱动持久但可逆的HPA轴功能障碍的机制，这可能是中枢和肾上腺介导的，并不同于简单的负反馈。本研究旨在表征外源性、超生理性糖皮质激素暴露持续时间与功能性HPA轴恢复的时间以及在细胞水平上驱动这些过程的分子变化下丘脑垂体和肾上腺我们将治疗成年雄性C57 BL/6 J小鼠（n=5/队列）1、8或24 通过以下途径给予溶媒（DN）或地塞米松（DEX; 10 mcg/天=约35 mg氢化可的松当量/m2/天）饮用水然后，我们将每周对基础（昼夜节律峰值和最低点）和压力诱导的记录从停用类固醇直至功能恢复的ACTH和CORT分泌，定义为这些ACTH和CORT水平与DN水平之间无显著差异的时间点通过ANOVA和Dunnett多重比较检验对动物进行分析。为了测量压力诱导的分泌，动物将接受胰岛素诱导的低血糖和促肾上腺皮质激素刺激试验，以评估轴如何响应一种强有力的生理应激源以及肾上腺皮质对共同刺激的敏感性。每种动物这些时间点将在最终功能评估后进行尸检，下丘脑、垂体和肾上腺切片，用于定量Crh、Pomc和Cyp 11b 1 mRNA表达，分别接下来我们将评估类固醇诱导的抑制和恢复是否是由序贯性的这些细胞类型中的每一种的凋亡和有丝分裂。为此，我们将首先进行TUNEL染色， 1）活化的半胱天冬酶-3或2）Ki 67与a）CRH、B）POMC或c）类固醇共定位的免疫组织化学肾上腺、垂体或下丘脑切片中的11β-羟化酶。最后，我们将记录束状带在DEX处理的醛固酮合成酶（AS）-Cre/mTmG（AS+/Cre：： R26 R +/mTmG）小鼠，在类固醇戒断、HPA轴恢复时用肾上腺荧光显微镜检查，由先前实验定义的几个中间时间点。这些研究将共同用于生成未来机制的假设，表观基因组研究的范围之外，这一补助金。对小说的阐释慢性糖皮质激素持久但可逆地抑制HPA轴的机制可能最终导致减少医源性继发性肾上腺功能不全的频率或持续时间的干预措施。