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

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leads to suppression of the hypothalamic-pituitary-adrenal (HPA) axis that can persist for months after steroids are withdrawn, leaving individuals vulnerable to life-threatening adrenal crises.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