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

项目摘要/摘要： 慢性超生理糖皮质激素暴露导致下丘脑-垂体-肾上腺(HPA)抑制 在停用类固醇后可以持续几个月的轴心，使个人容易受到生命威胁 肾上腺危机。人类临床研究提示停用长期类固醇后HPA轴功能障碍 首先由下丘脑-垂体功能障碍介导，其次是肾上腺皮质恢复延迟，尽管 适当的代偿性促肾上腺皮质激素刺激。而哺乳动物的HPA轴是高度保守、健壮的动物 缺乏类固醇诱导的抑制和恢复的模型。这限制了我们对 长期停用糖皮质激素后导致持久但可逆的HPA轴功能障碍的机制， 这很可能是中枢和肾上腺调节的，与简单的负反馈不同。这项研究寻求 研究外源性超生理激素暴露时间与糖皮质激素之间的关系 HPA轴功能恢复的时间以及在水平上推动这些过程的分子变化 下丘脑、脑下垂体和肾上腺。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 使用赋形剂(DN)或地塞米松(地塞米松；10微克/天=~35毫克氢化可的松当量/平方米/天) 喝水。然后，我们将每周评估基础(昼夜节律峰值和最低点)和压力诱导 ACTH和皮质醇的分泌从类固醇停用到功能恢复被记录在案，定义为 这些ACTH和CORT水平与糖尿病肾病之间无显著差异的时间点 动物经方差分析和Dunnett‘s多重比较检验。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，以评估轴对 一种强大的生理应激源，以及肾上腺皮质对常见刺激的敏感性。来自每个国家的动物 这些时间点将在最终功能评估后接受尸检，我们将从这些评估中生成 下丘脑、垂体和肾上腺切片定量CRH、Pomc和Cyp11b1的mRNA表达， 分别进行了分析。接下来，我们将评估类固醇诱导的抑制和恢复是否是由 这些细胞类型中每一种的凋亡和有丝分裂。为此，我们将首先执行TUNEL染色，然后 1)激活的caspase-3或2)Ki67与a)CRH、b)POMC或c)类固醇共定位的免疫组织化学 11肾上腺、垂体或下丘脑切片中的β-羟基酶。最后，我们将记录舌状带 地塞米松处理的肾小球带前体在醛固酮合成酶(AS)-Cre/mTmG(AS+/Cre：： R26R+/mTmG)小鼠，在激素停用、HPA轴恢复时对肾上腺进行荧光显微镜观察，以及 由先前的实验定义的几个中间时间点。这些研究将被集体用于生成 未来机械论、表观基因组学研究的假设不在本基金的范围之内。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皮质功能不全的频率或持续时间的干预措施。

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leads to suppression of the hypothalamic-pituitary-adrenal (HPA) axis that can persist for months after steroids are withdrawn, leaving individuals vulnerable to life-threatening adrenal crises.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