A lack of ovarian function increases neuroinflammation in aged mice

Abstract

Although several lines of evidence have indicated that menopause is associated with increased susceptibility to neurological disorders, the mechanisms involved in this phenomenon remain to be elucidated. Because neuroinflammation is a common feature of a number of brain diseases, we hypothesized that the cessation of ovarian functions and the consequent decrease in estrogen receptor (ER)-mediated antiinflammatory activity may represent a trigger for postmenopausal brain dysfunctions. The aim of the present study was to investigate the effects of aging and surgical menopause on the activity of ER in neuroinflammation. The present study shows that ER genes are expressed in the hippocampus, but ER transcriptional activity decreases significantly beginning at 12 months of age in intact and ovariectomized mice. With ovariectomy, we observe an age-dependent accumulation of mRNA encoding inflammatory mediators (e.g. TNFα, IL1β, and macrophage inflammatory protein-2) and changes in the morphology of astroglia and microglia. In addition, we show that aging itself is coupled with an exaggerated response to acute inflammatory stimuli with a major accumulation of TNFα, IL1β, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 mRNA in response to lipopolysaccharide administration. The response to acute inflammatory stimuli appears to be differentially modulated by the duration of hormone deprivation in 12-month-old mice. Taken together, the present results show that aging is associated with decreased ER activity, despite continuous ER synthesis, and that age-dependent neuroinflammation is strongly influenced by hormone deprivation.

Fig. 1.

The effects of aging on ERα and ERβ mRNA content in mouse hippocampus. Cycling mice were euthanized at metestrus, and the ERα (panel A) and ERβ (panel B) mRNA contents were measured using real-time PCR of tissue extracts from the hippocampi of sham and OVX female ERE-Luc mice. Data were analyzed with a two-way ANOVA followed by Bonferroni's post hoc test. *, P < 0.05; ** and °°, P < 0.01; and *** and °°°, P < 0.001; data are expressed as mean ± sem (n = 4–9).

Fig. 2.

ER activity in the brain decreases with aging. A (left panel), A gray-scale image of the section where the area of the hippocampus used for the measurement of photon emission is identified (white grid); central panel, pseudocolor images associated with photon emission, which were generated by a Night Owl LB981 image processor after 15 min of exposure to a charge-coupled device camera; right panel, semiquantitative analysis of the photon emission in the hippocampus. B, ER activity was measured in selected brain areas of ERE-Luc female mice by optical imaging. Data were analyzed with a two-way ANOVA followed by Bonferroni's post hoc test. + and *, P < 0.05; ** and ++, P < 0.01; and +++, P < 0.001; data are expressed as mean ± sem (n = 4–6).

Fig. 3.

The effects of aging on the expression of endogenous ER target genes. PgR (A), PTMA (B), and the 36b4 negative control (C) mRNA were measured in the hippocampi of female ERE-Luc mice using real-time PCR. Data were analyzed with a two-way ANOVA followed by Bonferroni's post hoc test. *, P < 0.05; data are expressed as mean ± sem (n = 6–8).

Fig. 4.

The expression of proinflammatory compounds in the hippocampi of female mice before and after OVX. TNFα, IL1β, MIP2, and MCP1 mRNA levels were measured using real-time PCR in the hippocampal tissue extracts of female ERE-Luc mice. Data were analyzed with a two-way ANOVA followed by Bonferroni's post hoc test *, °, and +, P < 0.05; **, P < 0.01; *** and °°°, P < 0.001; data are expressed as mean ± sem (n = 4–6). The experiment was repeated twice with two separate colonies of animals, and the results that are shown are the average of the two experiments.

Fig. 5.

Aging and OVX modify microglia and astrocyte immunoreactivity and morphology in the mouse hippocampus. Representative photomicrographs were taken from the hippocampi of sham (Aa, Cc, E, and G) or OVX (Bb, Dd, F, and H) mice at 6 months (Aa, Bb, E, and F) or 22 months of age (Cc, Dd, G, and H) and analyzed by immunohistochemistry for Mac-1 (A–D, a–d) or GFAP (E–H) expression. The scale bar equals 10 μm.

Fig. 6.

The effects of age and OVX on the response to an acute inflammatory stimulus. TNFα, IL1β, MIP2, MCP1, and IGF1 mRNA levels were measured using real-time PCR in the hippocampi of female ERE-Luc mice treated with LPS. Data are represented as the fold induction with respect to 3-month-old sham mice treated with vehicle (data not shown). Data were analyzed with a two-way ANOVA followed by Bonferroni's post hoc test unless otherwise stated. + and °, P < 0.05; ++ and °°, P < 0.01; +++, P < 0.001; #, P < 0.05; and ##, P < 0.01 (unpaired t test); data are expressed as mean ± sem (n = 4–8).