Measurement of Steroid Concentrations in Brain Tissue: Methodological Considerations

It is well recognized that steroids are synthesized de novo in the brain (neurosteroids). In addition, steroids circulating in the blood enter the brain. Steroids play numerous roles in the brain, such as influencing neural development, adult neuroplasticity, behavior, neuroinflammation, and neurodegenerative diseases such as Alzheimer’s disease. In order to understand the regulation and functions of steroids in the brain, it is important to directly measure steroid concentrations in brain tissue. In this brief review, we discuss methods for the detection and quantification of steroids in the brain. We concisely present the major advantages and disadvantages of different technical approaches at various experimental stages: euthanasia, tissue collection, steroid extraction, steroid separation, and steroid measurement. We discuss, among other topics, the potential effects of anesthesia and saline perfusion prior to tissue collection; microdissection via Palkovits punch; solid phase extraction; chromatographic separation of steroids; and immunoassays and mass spectrometry for steroid quantification, particularly the use of mass spectrometry for “steroid profiling.” Finally, we discuss the interpretation of local steroid concentrations, such as comparing steroid levels in brain tissue with those in the circulation (plasma vs. whole blood samples; total vs. free steroid levels). We also present reference values for a variety of steroids in different brain regions of adult rats. This brief review highlights some of the major methodological considerations at multiple experimental stages and provides a broad framework for designing studies that examine local steroid levels in the brain as well as other steroidogenic tissues, such as thymus, breast, and prostate

Steroid Concentrations in Plasma, Whole Blood and Brain: Effects of Saline Perfusion to Remove Blood Contamination from Brain

The brain and other organs locally synthesize steroids. Local synthesis is suggested when steroid levels are higher in tissue than in the circulation. However, measurement of both circulating and tissue steroid levels are subject to methodological considerations. For example, plasma samples are commonly used to estimate circulating steroid levels in whole blood, but steroid levels in plasma and whole blood could differ. In addition, tissue steroid measurements might be affected by blood contamination, which can be addressed experimentally by using saline perfusion to remove blood. In Study 1, we measured corticosterone and testosterone (T) levels in zebra finch (Taeniopygia guttata) plasma, whole blood, and red blood cells (RBC). We also compared corticosterone in plasma, whole blood, and RBC at baseline and after 60 min restraint stress. In Study 2, we quantified corticosterone, dehydroepiandrosterone (DHEA), T, and 17β-estradiol (E2) levels in the brains of sham-perfused or saline-perfused subjects. In Study 1, corticosterone and T concentrations were highest in plasma, significantly lower in whole blood, and lowest in RBC. In Study 2, saline perfusion unexpectedly increased corticosterone levels in the rostral telencephalon but not other regions. In contrast, saline perfusion decreased DHEA levels in caudal telencephalon and diencephalon. Saline perfusion also increased E2 levels in caudal telencephalon. In summary, when comparing local and systemic steroid levels, the inclusion of whole blood samples should prove useful. Moreover, blood contamination has little or no effect on measurement of brain steroid levels, suggesting that saline perfusion is not necessary prior to brain collection. Indeed, saline perfusion itself may elevate and lower steroid concentrations in a rapid, region-specific manner

Local glucocorticoid regulation in avian and murine lymphoid organs

Glucocorticoids are steroid hormones that circulate in the blood to coordinate organismal physiology. They have pleiotropic effects, regulating metabolic, cardiovascular, neural, and immune function. While glucocorticoids are classically thought to be secreted exclusively by the adrenal glands, evidence suggests that different organs may be able to autonomously regulate their local glucocorticoid levels via local production. Local production may be important when circulating glucocorticoids are low or absent, such as in early life of altricial young, which are unable to care for themselves. Immune (lymphoid) organs are particularly interesting candidates for tissue-specific regulation of glucocorticoid levels, as glucocorticoids are necessary for early-life immune development in altricial young. In this dissertation, I present a series of studies using birds and mice to examine whether tissue- specific regulation of glucocorticoids occurs in lymphoid organs. In brief, I report that a) glucocorticoids are locally elevated in lymphoid organs of newly-hatched altricial but not precocial birds, b) glucocorticoids are locally elevated in lymphoid organs of neonatal altricial mice, and c) lymphoid organs of both neonatal and adult mice synthesize glucocorticoids from other steroid precursors. Local glucocorticoid production in lymphoid organs may function to ensure production of functional lymphocytes, and factors that alter lymphoid glucocorticoid levels may play a role in programming the overall immune reactivity.Science, Faculty ofZoology, Department ofGraduat

Single-Cell Resolution and Quantitation of Targeted Glucocorticoid Delivery in the Thymus

Summary: Glucocorticoids are lipid-soluble hormones that signal via the glucocorticoid receptor (GR), a ligand-dependent transcription factor. Circulating glucocorticoids derive from the adrenals, but it is now apparent that paracrine glucocorticoid signaling occurs in multiple tissues. Effective local glucocorticoid concentrations and whether glucocorticoid delivery can be targeted to specific cell subsets are unknown. We use fluorescence detection of chromatin-associated GRs as biosensors of ligand binding and observe signals corresponding to steroid concentrations over physiological ranges in vitro and in vivo. In the thymus, where thymic epithelial cell (TEC)-synthesized glucocorticoids antagonize negative selection, we find that CD4+CD8+TCRhi cells, a small subset responding to self-antigens and undergoing selection, are specific targets of TEC-derived glucocorticoids and are exposed to 3-fold higher levels than other cells. These results demonstrate and quantitate targeted delivery of paracrine glucocorticoids. This approach may be used to assess in situ nuclear receptor signaling in a variety of physiological and pathological contexts. : Glucocorticoids signal via the GR, a ligand-dependent transcription factor, and paracrine glucocorticoid signaling occurs in the thymus. Taves et al. use chromatin-associated GRs as biosensors to estimate glucocorticoid concentrations in vitro and in vivo. In the thymus, antigen-signaled CD4+8+TCRhi cells are targeted by epithelial cell-synthesized glucocorticoids to promote positive selection. Keywords: Cyp11b1, glucocorticoids, glucocorticoid receptor, lymphocytes, nuclear receptors, paracrine, steroids, steroidogenesis, transcription facto

Radioimmunoassay specifications.

<p>Note. n/a  =  not assessed in these tissue types.</p

Steroid concentrations in plasma, whole blood, and brain.

<p>Corticosterone and DHEA levels in plasma, whole blood, and brain (caudal telencephalon, cTel) of sham-perfused subjects. *p≤0.05, ***p≤0.001.</p

Brain steroid concentrations after saline or sham perfusion.

<p>(<b>A</b>) Corticosterone, (<b>B</b>) DHEA, (<b>C</b>) testosterone, and (<b>D</b>) 17β-estradiol concentrations were measured in male zebra finch brain regions after saline perfusion or sham perfusion (control). Rostral telencephalon  =  rTel, caudal telencephalon  =  cTel, diencephalon  =  Dien, cerebellum  =  Cb. *p≤0.05, **p≤0.01.</p