Comparison of assay methods for quantifying sex hormone concentrations across the menstrual cycle in rhesus macaques.

Immunoassays have been the preferred method for steroid hormone analysis for more than 50 years. Automated immunoassays (AIAs) offer high-throughput, rapid data turnaround, and low cost for measuring steroid hormone concentrations. The application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for steroid quantification provides greater specificity and selectivity for individual steroids, the ability to simultaneously analyze multiple steroids, and high-throughput and automation. We compared AIA and LC-MS/MS for analysis of 17ß-estradiol (E2) and progesterone (P4) over the course of several menstrual cycles in 12 rhesus macaques (Macaca mulatta). Serum samples were collected every four days across four menstrual cycles from each monkey. AIAs were performed on a Roche cobas e411 analyzer. Analysis of E2 and P4 was performed by LC-MS/MS on a Shimadzu-Nexera-LCMS-8060 instrument. Scatter plots with Passing-Bablok regression showed excellent agreement between AIA and LC-MS/MS for both E2 and P4. Bland-Altman plots revealed no bias for either method; however, AIA overestimated E2 at concentrations >140 pg/ml and underestimated P4 at concentrations >4 ng/ml compared to LC-MS/MS. A comparison of testosterone (T) concentrations measured by AIA and LC-MS/MS in the same samples was also performed. In contrast to E2 and P4, AIA and LC-MS/MS yielded significantly different results for T concentrations, with AIA consistently underestimating concentrations relative to those obtained by LC-MS/MS. Well-characterized AIAs are an excellent tool for daily monitoring of monkey menstrual cycles or providing single data points requiring fast turnaround. In certain situations where AIA may provide inaccurate estimations of E2 and P4 concentrations, LC-MS/MS assays are preferable.

Age-related increase in the expression of 11ß-hydroxysteroid dehydrogenase type 1 in the hippocampus of male rhesus macaques.

Introduction: The hippocampus is especially susceptible to age-associated neuronal pathologies, and there is concern that the age-associated rise in cortisol secretion from the adrenal gland may contribute to their etiology. Furthermore, because 11ß-hydroxysteroid dehydrogenase type 1 (HSD11B1) catalyzes the reduction of cortisone to the active hormone cortisol, it is plausible that an increase in the expression of this enzyme enhances the deleterious impact of cortisol in the hippocampus and contributes to the neuronal pathologies that underlie cognitive decline in the elderly. Methods: Rhesus macaques were used as a translational animal model of human aging, to examine age-related changes in gene and protein expressions of (HSD11B1/HSD11B1) in the hippocampus, a region of the brain that plays a crucial role in learning and memory. Results: Older animals showed significantly (p < 0.01) higher base-line cortisol levels in the circulation. In addition, they showed significantly (p < 0.05) higher hippocampal expression of HSD11B1 but not NR3C1 and NR3C2 (i.e., two receptor-encoding genes through which cortisol exerts its physiological actions). A similar age-related significant (p < 0.05) increase in the expression of the HSD11B1 was revealed at the protein level by western blot analysis. Discussion: The data suggest that an age-related increase in the expression of hippocampal HSD11B1 is likely to raise cortisol concentrations in this cognitive brain area, and thereby contribute to the etiology of neuropathologies that ultimately lead to neuronal loss and dementia. Targeting this enzyme pharmacologically may help to reduce the negative impact of elevated cortisol concentrations within glucocorticoid-sensitive brain areas and thereby afford neuronal protection.

Pathological Markers of Alzheimer's Disease and Related Dementia in the Rhesus Macaque Amygdala.

Rhesus macaques develop amyloid-ß (Aß) plaques during old age, but it is unclear how extensively they express other pathological hallmarks of dementia. Here we used immunohistochemistry to examine expression of phosphorylated tau (pTau) protein and cytoplasmic inclusions of TAR DNA binding protein 43 kDa (TDP-43) within the amygdala of young and old males, and also in old surgically-menopausal females that were maintained on regular or obesogenic diets. Only one animal, a 23-year-old female, showed pTau expression and none showed TDP-43 inclusions. What genetic and/or environmental factors protect macaques from expressing more severe human neuro-pathologies remains an interesting unresolved question.

Effect of hormone replacement therapy on amyloid beta (Aß) plaque density in the rhesus macaque amygdala.

Background: Amyloid beta (Aß) plaque density was examined in the amygdala of rhesus macaques, to elucidate the influence of age, diet and hormonal environment. Methods: Luminex technology was used to measure cerebrospinal fluid (CSF) concentrations of Aß40 and Aß42 across three decades, while immunohistochemistry was used to examine Aß plaque density in the amygdala. Results: Aß40 was found to be the predominant isoform of Aß in the CSF, but neither Aß40 or Aß42 concentrations showed an age-related change, and the ratio of Aß42 to Aß40 showed only a marginal increase. Significantly fewer Aß plaques were detected in the amygdala of old ovariectomized animals if they received estradiol HRT (p < 0.001); similar results were obtained regardless of whether they had been maintained on a regular monkey chow for ∼48 months or on a high-fat, high-sugar, Western-style diet for ∼30 months. Conclusion: The results demonstrate that HRT involving estrogen can reduce Aß plaque load in a cognitive brain region of aged non-human primates. The results from this translational animal model may therefore have clinical relevance to the treatment of AD in post-menopausal women, whether used alone, or as a supplement to current pharmacological and monoclonal antibody-based interventions.