Reproductive Senescent Females Research Articles

Reproductive Senescence and Ischemic Stroke Remodel the Gut Microbiome and Modulate the Effects of Estrogen Treatment in Female Rats.

Our previous work has shown that reproductively senescent (or middle-aged; 10-12-month-old) Sprague-Dawley female rats, that are naturally estrogen-deficient, have worse stroke outcomes as compared to normally estrous-cycling adult (5-6-month-old) females. Paradoxically, estrogen replacement to this middle-aged group exacerbates stroke outcomes, while it is neuroprotective in adult females. Recent studies reveal an important role for the gut microbiome and gut metabolites in cardiovascular health, including stroke outcomes. To determine whether gut dysbiosis underlies stroke severity in reproductive senescent females, and underlies the anomalous effects of estrogen on stroke, we compared the gut microbiota and gut metabolites pre and post stroke in (a) gonadally intact adult and middle-aged females, (b) in ovariectomized and estrogen-treated (OVX+E) adult and OVX+E middle-aged females, and (c) in middle-aged OVX+E females after fecal microbiome transfer. Our data show significant gut dysbiosis in reproductive senescent females at baseline and after stroke as indicated by an elevated ratio of the major phyla, Firmicutes/Bacteroidetes (F:B), reduced alpha diversity, and significant shifts in beta diversity as compared with adult females. Specific bacterial families were also altered as a result of reproductive aging, as well as gut metabolites, including elevated serum endotoxin levels and decreased short-chain fatty acids (SCFAs), with a concomitant increase in IL-17A, indicating that reproductive senescence significantly affects gut communities under pathologic conditions. Despite the differences in gonadally intact adult and middle-aged females, estrogen-treated ovariectomized (OVX+E) females of either age group displayed no differences in the major phyla, but there was increased abundance in specific bacterial taxa, including Prevotella and Lactobacillus. The SCFA butyrate was significantly reduced at baseline in the middle-aged OVX+E females, while circulating endotoxin LPS were elevated in this group after stroke, suggesting that gut metabolites were differently affected by estrogen treatment in the two age groups. A fecal transfer from adult OVX+E females to middle-aged OVX+E females significantly reduced infarct volume, improved behavioral recovery and transiently reduced IL-17A expression. These data provide the first evidence that microbial gut communities and metabolites are altered by reproductive senescence in female rats at baseline and after stroke, and suggest that estrogen may impact stroke recovery differently in adult and reproductive senescent females due to an age-specific effect on gut microbiota and metabolites.

Sexual dimorphism in inflammasome-containing extracellular vesicles and the regulation of innate immunity in the brain of reproductive senescent females

A woman's risk for stroke increases exponentially following the onset of menopause; however, the underlying mechanisms responsible for the increased risk remain unknown. The depletion of endogenous estrogen at menopause is known to activate the inflammatory response. Therefore, in this study we have used reproductively senescent (RS) rats to test the hypotheses that (1) inflammasome activation is significantly higher in the brain of RS females (RSF) as compared to their younger counterparts and age-matched senescent male rats, and that (2) RS triggers an innate immune response mediated in part by inflammasome-containing extracellular vesicles (EV) that originate in the female reproductive organs and then spreads to the brain. We tested these hypotheses using male and female Sprague–Dawley rats (Young: 6–7 months and RS: 9–13 months). Hippocampus, gonads and serum were collected. Additionally, cerebrospinal fluid (CSF) of pre- and post-menopausal women (ages 23 to 37 and 52 to 68) was purchased and extracellular vesicles (EV) were isolated from serum and CSF. The Inflammasome proteins caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and IL-1β were then resolved by immunoblotting. We found that inflammasome protein expression increased significantly in the analyzed tissues in RSF as compared to young females (YF), such difference was not present in age-matched male rat brains. Interestingly, we found that Nik-related kinase (NRK), which is present in female reproductive organs was present in the CSF and serum-derived EV, suggesting that the source of the EV seen in the brain during RS/menopause originate, in part, in the female reproductive organs. Thus, this study shows for the first time an involvement of the inflammasome originating in the female reproductive system as a contributor to inflammation in the brain that makes the peri-menopausal women's brain more susceptible to neurodegenerative diseases such as stroke.

The Neurotoxic Effects of Estrogen on Ischemic Stroke in Older Female Rats Is Associated with Age-Dependent Loss of Insulin-Like Growth Factor-1

Hormone therapy to postmenopausal females increases the risk and severity of ischemic stroke. Our previous work using an animal model of menopause (reproductive senescence) shows that middle cerebral artery occlusion (MCAo) causes a larger cortical-striatal infarct in this older acyclic group compared with younger females. Moreover, although estrogen treatment is neuroprotective in younger females, estrogen paradoxically increases infarct volume in acyclic females. We hypothesized that the neurotoxic effects of estrogen in older females occurs because of decreased availability of IGF-1, a neuroprotectant that decreases with advancing age and is downregulated by estrogen treatment. Our data show that plasma IGF-1 levels are significantly reduced in reproductive senescent females and further reduced by estrogen at all ages. The neuroprotective effect of estrogen on MCAo-induced cortical infarct volume in mature adult female is reversed by intracerebroventricular injections of IGF-1 receptor antagonist JB-1. Similarly, estrogens neurotoxic effects on cortical infarct volume in senescent females is attenuated by concurrent IGF-1 treatment, and reversed when IGF-1 is infused 4 h after the onset of ischemia (delayed IGF-1 treatment). Delayed IGF-1/estrogen treatment also suppressed ischemia-induced ERK1 phosphorylation, reduced protein oxidation, and stimulated an early increase in prostaglandin E(2) at the infarct site. IGF-1 treatment was only protective in senescent females that received estrogen, indicating that the neuroprotective actions of this peptide require interaction with the steroid hormone receptor. These data support the hypothesis that stroke severity in older females is associated with decreased IGF-1 and further indicate that short-term postischemic IGF-1 therapy may be beneficial for stroke.

A high cholesterol diet elevates hippocampal cytokine expression in an age and estrogen-dependent manner in female rats

Background While the effects of a proatherogenic diet have been widely studied in the context of systemic inflammation, much less is known about its effects on central or brain inflammation and its modulation with age. In this study, we examined the effect of a high cholesterol/choline diet in adult and older acyclic females to assess its impact on systemic and central inflammatory markers. Moreover, since the loss of ovarian hormones at menopause may predispose women to increased production of pro-inflammatory cytokines, we also tested the impact of estrogen replacement to adult and older females in diet-induced inflammation. Methods Ovariectomized adult female rats and older (reproductive senescent) female rats were replaced with estrogen or a control pellet and maintained thereafter on a diet containing either 4% cholesterol/1% choline or control chow for 10 weeks. Interleukin 1β (IL-1β) expression in the liver was used as a marker of systemic inflammation, while a panel of cytokine/chemokines were used to examine the effects of diet on the hippocampus. Results IL-1β expression was elevated in the liver of adult and reproductive senescent females fed with the high cholesterol diet, although this was restricted to groups that were ovariectomized and not replaced with estrogen. Estrogen-treated animals of both ages did not have elevated IL-1β levels when fed the high cholesterol diet. Diet-induced changes in cytokine/chemokine expression in the hippocampus however were critically age dependent and restricted to the reproductive senescent females. In this group, the high cholesterol diet led to an increase in interleukin (IL)-4, IL-6, IL-12p70, IL-13, RANTES (Regulated on Activation, Normal T Expressed and Secreted) and VEGF (vascular endothelial growth factor). Moreover, estrogen treatment to reproductive senescent females suppressed diet-induced expression of specific cytokines (RANTES, VEGF, IL-6) and attenuated the expression of others (IL-4, IL-12p70, and IL-13). Conclusions These data indicate that a proatherogenic diet presents a significant risk for central inflammation in older females that are deprived of estrogen treatment.

Reproductive age-related changes in the blood brain barrier: Expression of IgG and tight junction proteins

We previously demonstrated that there is a significantly greater transfer of intravenously-injected Evan's blue dye into the forebrain of acyclic (reproductive senescent) females compared to young adult females, indicating that blood brain barrier permeability is compromised in the reproductive senescent forebrain. The present study examined brain IgG expression and microvessel tight junction proteins to assess ovarian age-related changes in microvascular permeability, and further compared young and senescent females with age-matched males to distinguish changes attributable to age and reproductive senescence. Blood brain barrier breakdown are often associated with increased extravasation of plasma proteins and high levels of immunoglobulin G (IgG) in brain. In the present study, IgG expression was dramatically increased in the hippocampus and thalamus, but not the hypothalamus of reproductive senescent females compared to young adult females. In males, IgG expression was increased in all these regions in middle-aged animals (aged-matched to senescent females) as compared to young males (age-matched to the young adult females). Furthermore, the proportion of hippocampal microvessels with perivascular IgG immunoreactivity was significantly greater in reproductive senescent females as compared to young adult females, while middle-aged males and young adult males did not differ. The tight junctions between adjacent microvascular endothelial cells regulated by transmembrane proteins such as claudin-5 and occludin play a critical role in maintaining the blood brain barrier integrity. Increased hippocampal IgG expression in senescent females was paralleled by poor junctional localization of the tight junction protein claudin-5 in hippocampal microvessels. However, there was no difference in hippocampal claudin-5 localization between young adult and middle-aged males, indicating that dysregulation of this junctional protein was associated with ovarian aging. Parallel studies in human brain microvessels also revealed age-dependent disruption in claudin-5 distribution in post-menopausal women compared to pre-menopausal women. Collectively, these data support the hypothesis that constitutive loss of barrier integrity in the forebrain during reproductive senescence may be due, in part, to the selective loss of tight junction proteins in endothelial junctions.

Astrocytes from acyclic female rats exhibit lowered capacity for neuronal differentiation

Astrocytes comprise a large proportion of the central nervous system support cells and play a critical role in neural injury and repair. The present study examined the impact of ovarian aging using an ex vivo model system, where astrocytes were derived from the olfactory bulb of young, reproductively competent females and reproductive senescent females. Cellular morphology and the spatial pattern of laminin deposition was altered in astrocyte cultures derived from reproductive senescent females. Young adult astrocytes had a flattened polygonal shape with actin bundles at the cell edges, while reproductive senescent astrocytes had a contractile appearance with thick stress fibers visible throughout the cell. Moreover, in reproductive senescent astrocytes, BDNF was elevated with a concomitant reduction in expression of the BDNF receptor, TrkB. To examine the ability of astrocytes derived from young adult and reproductive senescent females to promote neuronal differentiation, neural progenitor cells (NPCs) were co-cultured with astrocytes derived from these groups. At day 4 in vitro, MAP-2(+) NPCs were located in smaller clusters when co-cultured with young adult astrocytes and in large clusters when co-cultured with older astrocytes. At days 6 and 10, neuronal differentiation was significantly reduced in reproductive senescent astrocyte-NPC co-cultures, as determined by NeuN(+) cell numbers and MAP-2(+) process lengths. Furthermore, estrogen only enhanced neuronal differentiation in young adult-NPC co-cultures. The ovarian age-related astrocyte phenotype thus limits the ability of this cell to promote neuronal differentiation in NPC populations and suggests that the astrocyte-mediated microenvironment in older acyclic females is less conducive to repair following neurovascular injury.

Reproductive age modulates the impact of focal ischemia on the forebrain as well as the effects of estrogen treatment in female rats.

While human observational studies and animal studies report a neuroprotective role for estrogen therapy in stroke, the multicenter placebo-controlled Women's Health Initiative (WHI) study concluded that hormone therapy increased the risk for stroke in postmenopausal women. The present study therefore tested the hypothesis that estrogen replacement would increase the severity of a stroke-like injury in females when this replacement occurs after a prolonged hypoestrogenic period, such as the menopause or reproductive senescence, but not when given to females that were normally cycling immediately prior to the hormone replacement. Two groups of female rats were used: multiparous females with normal but lengthened estrus cycles (mature adults), and older multiparous females currently in a persistent acyclic state (reproductive senescent). Animals were either used intact, or were bilaterally ovariectomized and immediately replaced with a 17beta-estradiol pellet or control pellet. Animals were subject to a forelimb placing test (a test for sensorimotor deficit) and thereafter to middle cerebral artery occlusion (MCAo) by stereotaxic injection of the vasoconstrictive peptide endothelin-1, adjacent to the MCA. One week after stroke, behavioral tests were performed again. Cortical and striatal infarct volume, measured from brain slices, was significantly greater in intact reproductive senescent females as compared to intact mature adults. Furthermore, estrogen treatment to ovariectomized mature adult females significantly reduced the cortical infarct volume. Paradoxically, estrogen treatment to ovariectomized reproductive senescent females significantly increased cortical and striatal infarct volumes as compared to control pellet replaced senescent females. Significant post-stroke behavioral deficit was observed in all groups on the side contralateral to the lesion, while senescent females also exhibited deficits on the ipsilateral side, in the cross-midline forelimb placement test. Using an animal model that approximates the natural ovarian aging process, these findings strongly support the hypothesis that the effectiveness of estrogen therapy in protecting brain health may depend critically on the time of initiation with respect to a female's reproductive status.

Effects of estrogen receptor agonists on regulation of the inflammatory response in astrocytes from young adult and middle-aged female rats

Estrogen has been shown to attenuate the inflammatory response following injury or lipopolysaccharide treatment in several organ systems. Estrogen's actions are transduced through two estrogen receptor sub-types, estrogen receptor (ER) -alpha and estrogen receptor-beta, whose actions may be overlapping or independent of each other. The present study examined the effects of ERα- and ERβ-specific ligands in regulating the inflammatory response in primary astrocyte cultures. Pre-treatment with 17β-estradiol (ERα/ERβ agonist), HPTE (ERα agonist/ERβ antagonist) and DPN (ERβ agonist) led to attenuation of IL-1β, TNFα, and MMP-9 in astrocyte media derived from young adult (3-4 mos.) and reproductive senescent female (9-11 mos., acyclic) astrocyte cultures, while pretreatment with PPT (ERα agonist) attenuated IL-1β (but not MMP-9) in both young and senescent-derived astrocyte cultures. Our previous work determined that 17β-estradiol was unable to attenuate the LPS-induced increase in IL-1β in olfactory bulb primary microglial cultures derived from either young adult or reproductive senescent females. In young adult-derived microglial cultures, the LPS-induced increase in IL-1β was not attenuated by pre-treatment with 17β-estradiol, PPT or HPTE. Interestingly, the ERβ agonist, DPN significantly decreased IL-1β following LPS treatment in young adult-derived microglia. Thus while both microglia and astrocytes synthesize and release inflammatory mediators, the present data shows that compounds which bind ERβ are more effective in attenuating proinflammatory cytokines in both cell types and may therefore be a more effective agent for future therapeutic use.

Estrogen's effects on central and circulating immune cells vary with reproductive age

Previous work from this lab has shown that estrogen attenuates inflammatory cytokine production following brain lesions in young adult female rats, but not in older, reproductive senescent females. The present study was designed to elucidate whether these effects result from estrogen's actions on brain-resident immune cells (microglia) or on circulating immune cells recruited to the brain from blood. Microglia, harvested from the olfactory bulbs of ovariectomized young adult and reproductive senescent animals, were pretreated with 17β-estradiol and subsequently with the bacterial endotoxin LPS. LPS treatment significantly increased the pro-inflammatory cytokine IL-1β in microglial cultures harvested from young and senescent females, but estrogen treatment had no effect on cytokine expression in either group. In young adult-derived microglia, LPS treatment also increased nitric oxide (NO), which was attenuated by estrogen, and MMP-9, which was not affected by estrogen. Reproductive senescent-derived microglia cultures had higher basal expression of NO and MMP-9 activity as compared to those from young adult microglial cultures, although LPS did not further stimulate these inflammatory markers. In blood cultures, LPS stimulated a dose-dependent increase in the inflammatory cytokine TNF-α expression in both young adult and reproductive senescent animals. Estrogen replacement significantly attenuated TNF-α induction by LPS in blood cultures derived from young adult females. Paradoxically, estrogen replacement increased LPS-induced TNF-α expression in blood cultures derived from reproductive senescent animals as compared to age-matched controls. The age and estrogen dependent effects on circulating immune cells found in whole blood cultures closely mimic the effects of estrogen on cytokine expression in the young and senescent animals that we reported in vivo, supporting the hypothesis that the immunosuppressive actions of estrogen replacement on neural injury may result from hormone-action on circulating immune cells.

Differential effects of estrogen in the injured forebrain of young adult and reproductive senescent animals

Estrogen attenuates neural damage resulting from a variety of experimental injury models in adult female rats. To determine whether estrogens neuroprotective actions are age-specific, the present study compared the effects of estrogen on young adult and reproductive senescent animals subject to excitotoxic injury to the forebrain. NMDA was injected bilaterally into the olfactory bulbs of estrogen and placebo-replaced young adult and reproductive senescent animals. Lysates of the olfactory bulb and its basal forebrain afferent, the horizontal limb of the diagonal band of Broca (hlDBB), harvested 24 h later were analyzed for expression of IL-1β, IL-10, and nerve growth factor (NGF). NMDA injections resulted in local activation of microglia and an increase in IL-1β. Estrogen replacement decreased IL-1β expression in young adult females, but paradoxically enhanced its expression in reproductive senescent females. Furthermore, bulb injury increased IL-1β production in the hlDBB of reproductive senescent animals although estrogen replacement was able to suppress lesion-induced expression of this cytokine. In both, the olfactory bulb and hlDBB, constitutive expression of the anti-inflammatory cytokine IL-10 was significantly higher while that of NGF was almost 50% lower in senescent animals as compared to young adults, indicating that aging preferentially altered Th2-type secretions. The present findings are consistent with our earlier observations that estrogen does not exert trophic effects in the aging forebrain and supports the hypothesis that estrogen treatment to reproductive senescent females may exacerbate neural injury.