Biology subjects

Gaston, L. S.

Publications and source records attributed to Gaston, L. S..

3 recordsLinked to original sources

bioRxiv2025

Rapid hypothalamic-pituitary recovery after chronic glucocorticoid therapy enables strategies that prevent adrenal suppression

Glucocorticoid-induced adrenal insufficiency (GIAI) can persist for months after discontinuation of chronic corticosteroid therapy, placing patients at risk for life-threatening adrenal crises. This prolonged suppression has been attributed primarily to delayed restoration of hypothalamic-pituitary signaling based on indirect measures of central axis activity. To identify the rate-limiting site of hypothalamic-pituitary-adrenal (HPA) axis recovery, we evaluated the timing of functional and histologic recovery at each node of the axis following 8 weeks of dexamethasone (DEX) treatment in adult, male mice. DEX administration fully suppressed HPA axis activity. Unexpectedly, within one week of DEX withdrawal, hypothalamic Crh mRNA and plasma ACTH rebounded above control levels, whereas corticosterone (CORT) remained suppressed for an additional seven weeks. DEX-treated adrenals were markedly atrophic and contained large clusters of lipid-filled macrophages. Even after adjusting for macrophage content, CORT secretion was disproportionately low relative to the remaining adrenocortical cell mass despite supraphysiologic ACTH stimulation. The adrenal is thus the principal site of post-withdrawal GIAI, involving adrenocortical cell loss and a superimposed defect in steroidogenesis. We next tested whether preserving adrenal trophic signaling during glucocorticoid exposure could prevent GIAI. Adrenal function recovered more slowly in mice treated with DEX and daily cosyntropin (a synthetic ACTH analog) compared to those treated with DEX alone. In contrast, mice with non-suppressible endogenous ACTH due to targeted hypothalamic deletion of the glucocorticoid receptor maintained normal adrenal architecture and steroidogenic capacity despite prolonged DEX treatment. Pharmacologic treatments that mimic sustained trophic signaling to the adrenal during chronic glucocorticoid treatment may thus prevent GIAI.

physiology↗

bioRxiv2024

Absent, but not glucocorticoid-modulated, corticotropin-releasing hormone (Crh) regulates anxiety-like behaviors in mice

The hypothalamic-pituitary-adrenal (HPA) axis is a well characterized endocrine response system. Hypothalamic Crh in the paraventricular nucleus of the hypothalamus (PVH) initiates HPA axis signaling to cause the release of cortisol (or corticosterone in rodents) from the adrenal gland. PVH-specific deletion of Crh reduces anxiety-like behaviors in mice. Here we report that manipulation of PVH Crh expression in primary adrenal insufficiency or by dexamethasone (DEX) treatment do not alter mouse anxiety behaviors. In Experiment 1, we compared wildtype (WT) mice to those with primary adrenal insufficiency (MrapKO) or global deletion of Crh (CrhKO). We analyzed behaviors using open field (OF) and elevated plus maze (EPM), PVH Crh mRNA expression by spatial transcriptomics, and plasma ACTH and corticosterone after a 15-minute restraint test with ELISAs. EPM analysis showed CrhKO mice were less anxious than WT and MrapKO mice, and MrapKO mice had no distinguishing behavioral phenotype. In Experiment 2, we evaluated HPA axis habituation to chronically elevated Crh expression by comparing mice treated with 5-8 weeks of DEX with those similarly treated followed by DEX withdrawal for 1 week. All mice regardless of genotype and treatment showed no significant behavioral differences. Our findings suggest that reduced anxiety associated with low Crh expression requires extreme deficiency, perhaps outside of those PVH Crh neurons negatively regulated by glucocorticoids. If these findings extend to humans, they suggest that increases in Crh expression with primary adrenal insufficiency, or decreases with exogenous glucocorticoid therapy, may not alter anxiety behaviors via modulation of Crh expression.

neuroscience↗

bioRxiv2024

Harmonizing terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) with multiplexed iterative immunofluorescence enriches spatial contextualization of cell death

Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) is an essential tool for the detection of cell death in tissues. Although TUNEL is not known to be compatible with multiplexed spatial proteomic methods, harmonizing TUNEL with such methods offers the opportunity to delineate cell-type specific cell death labeling and precise spatial contextualization of cell death in complex tissues. Here we evaluated variations of the TUNEL assay for their compatibility with a multiplexed immunofluorescence method, multiple iterative labeling by antibody neodeposition (MILAN), in two different tissues and injury models for cell death, acetaminophen-induced hepatocyte necrosis and dexamethasone-induced adrenocortical apoptosis. Using a commercial Click-iT-based assay as a standard, TUNEL signal could be reliably produced independent of antigen-retrieval method, with tissue-specific minor differences in signal-to-noise. In contrast, proteinase K treatment consistently reduced or even abrogated protein antigenicity, while pressure cooker treatment consistently enhanced protein antigenicity for the targets tested. Antibody-based TUNEL protocols using pressure-cooker antigen retrieval were MILAN erasure-compatible thus enabling harmonization of TUNEL with MILAN. As many as four staining cycles could be performed without loss of subsequent TUNEL signal, while first-round TUNEL did not influence protein antigenicity in subsequent rounds. We conclude this harmonized assay performs comparably to an established commercial assay, but preserves protein antigenicity, thus enabling versatile integration with multiplexed immunofluorescence using MILAN. We anticipate this harmonized protocol will enable broad and flexible integration of TUNEL into multiplexed spatial proteomic assays, thus vastly enhancing the spatial contextualization of cell death in complex tissues.

pathology↗