Effects of estradiol on ischemic factor-induced astrocyte swelling and AQP4 protein abundance.

In the early hours of ischemic stroke, cerebral edema forms as Na, Cl, and water are secreted across the blood-brain barrier (BBB) and astrocytes swell. We have shown previously that ischemic factors, including hypoxia, aglycemia, and arginine vasopressin (AVP), stimulate BBB Na-K-Cl cotransporter (NKCC) and Na/H exchanger (NHE) activities and that inhibiting NKCC and/or NHE by intravenous bumetanide and/or HOE-642 reduces edema and infarct in a rat model of ischemic stroke. Estradiol also reduces edema and infarct in this model and abolishes ischemic factor stimulation of BBB NKCC and NHE. There is evidence that NKCC and NHE also participate in ischemia-induced swelling of astrocytes. However, little is known about estradiol effects on astrocyte cell volume. In this study, we evaluated the effects of AVP (100 nM), hypoxia (7.5% O(2)), aglycemia, hypoxia (2%)/aglycemia [oxygen glucose deprivation (OGD)], and estradiol (1-100 nM) on astrocyte cell volume using 3-O-methyl-d-[(3)H]glucose equilibration methods. We found that AVP, hypoxia, aglycemia, and OGD (30 min to 5 h) each significantly increased astrocyte cell volume, and that estradiol (30-180 min) abolished swelling induced by AVP or hypoxia, but not by aglycemia or OGD. Bumetanide and/or HOE-642 also abolished swelling induced by AVP but not aglycemia. Abundance of aquaporin-4, known to participate in ischemia-induced astrocyte swelling, was significantly reduced following 7-day but not 2- or 3-h estradiol exposures. Our findings suggest that hypoxia, aglycemia, and AVP each contribute to ischemia-induced astrocyte swelling, and that the edema-attenuating effects of estradiol include reduction of hypoxia- and AVP-induced astrocyte swelling and also reduction of aquaporin-4 abundance.

Simultaneous determination of 6 L-arginine metabolites in human and mouse plasma by using hydrophilic-interaction chromatography and electrospray tandem mass spectrometry.

BACKGROUND: Macrophages and related cells are important cellular mediators of the innate immune system and play important roles in wound healing and fibrosis. Flux through different l-arginine metabolic pathways partially defines the functional behavior of macrophages. Methods to measure metabolites within the nitric oxide synthase/arginase pathways could provide insights into local and systemic inflammatory processes. METHODS: A targeted metabolomics approach was developed by using hydrophilic-interaction liquid chromatography and electrospray ionization-tandem mass spectrometry to simultaneously measure l-arginine, asymmetric dimethylarginine, symmetric dimethylarginine, l-citrulline, l-ornithine, and l-proline in plasma from humans and mice. RESULTS: All analytes were quantifiable in human and mouse plasma with a small volume (25 µL), minimal sample preparation, and no derivatization. Patients with high plasma concentrations of C-reactive protein and mice with acute inflammation induced by lipopolysaccharide had significant reductions of arginine metabolites in plasma compared with controls. CONCLUSIONS: This new assay uses plasma metabolomic measurements to help provide new insights into metabolic changes coupled to the innate immune response. We identified significant changes in arginine metabolism in both humans and mice following an inflammatory stimulus. These changes were associated with decreased plasma arginine metabolite concentrations and increased methylated arginine concentrations.

Production of proinflammatory cytokines and chemokines during neuroinflammation: novel roles for estrogen receptors alpha and beta.

Neuroinflammation is a common feature of many neurological disorders, and it is often accompanied by the release of proinflammatory cytokines and chemokines. Estradiol-17ß (E2) exhibits antiinflammatory properties, including the suppression of proinflammatory cytokines, in the central nervous system. However, the mechanisms employed by E2 and the role(s) of estrogen receptors (ERs) ERα and ERß are unclear. To investigate these mechanisms, we employed an in vivo lipopolysaccharide (LPS) model of systemic inflammation in ovariectomized (OVX) and OVX and E2-treated (OVX+E2) mice. Brain levels of proinflammatory cytokines (IL-1ß, IL-6, and IL-12p40) and chemokines (CCL2/MCP-1, CCL3/MIP-1α, CCL5/RANTES, and CXCL1/KC) were quantified in mice at 0 (sham), 3, 6, 12, and 24 h after infection using multiplex protein analysis. E2 treatment inhibited LPS-induced increases in all cytokines. In contrast, E2 treatment only suppressed CCL/RANTES chemokine concentrations. To determine whether ERα and ERß regulate brain cytokine and chemokine levels, parallel experiments were conducted using ERα knockout and ERß knockout mice. Our results revealed that both ERα and ERß regulated proinflammatory cytokine and chemokine production through E2-dependent and E2-independent mechanisms. To assess whether breakdown of the blood-brain barrier is an additional target of E2 against LPS-induced neuroinflammation, we measured Evan's blue extravasation and identified distinct roles for ERα and ERß. Taken together, these studies identify a dramatic cytokine- and chemokine-mediated neuroinflammatory response that is regulated through ERα- and ERß-mediated ligand-dependent and ligand-independent mechanisms.

Estradiol: a hormone with diverse and contradictory neuroprotective actions.

The concept that estrogens exert important neuroprotective actions has gained considerable attention during the past decade. Numerous studies have provided a deep understanding of the seemingly contradictory actions of estrogens. We realize more than ever that the effects of estrogens (with and without simultaneous or sequential progestins) are diverse and sometimes opposite, depending on the use of different estrogenic and progestinic compounds, on different delivery routes, on different concentrations, on treatment sequence, and on the age and health status of the women who receive hormone therapy. During the past few years, we have gained an increasing appreciation of the impact of estrogens on the immune system and on inflammation. In addition, we have learned that estrogens cannot only protect against cell death, but can also stimulate the birth of new neurons. Here we posit the concept that estrogen's modulation of the immune status may be the basic mechanism that underlies its ability to protect against neurodegeneration and its powerful neuroregenerative actions. We hope that this update will encourage even richer dialogues between basic and clinical scientists to ensure that future clinical studies fully consider the information that can be derived from basic science studies. Only then will we have a better understanding of the impact of hormones on the menopausal and postmenopausal period in a woman's life.

Cerebral microvascular endothelial cell Na/H exchange: evidence for the presence of NHE1 and NHE2 isoforms and regulation by arginine vasopressin.

Blood-brain barrier (BBB) Na transporters are essential for brain water and electrolyte homeostasis. However, they also contribute to edema formation during the early hours of ischemic stroke by increased transport of Na from blood into brain across an intact BBB. We previously showed that a luminal BBB Na-K-Cl cotransporter is stimulated by hypoxia, aglycemia, and AVP and that inhibition of the cotransporter by intravenous bumetanide significantly reduces edema and infarct in the rat middle cerebral artery occlusion (MCAO) model of stroke. More recently, we found evidence that intravenous cariporide (HOE-642), a highly potent Na/H exchange inhibitor, also reduces brain edema after MCAO. The present study was conducted to investigate which Na/H exchange protein isoforms are present in BBB endothelial cells and to evaluate the effects of ischemic factors on BBB Na/H exchange activity. Western blot analysis of bovine cerebral microvascular endothelial cells (CMEC) and immunoelectron microscopy of perfusion-fixed rat brain revealed that Na/H exchanger isoforms 1 and 2 (NHE1 and NHE2) are present in BBB endothelial cells. Using microspectrofluorometry and the pH-sensitive dye BCECF, we found that hypoxia (2% O(2), 30 min), aglycemia (30 min), and AVP (1-200 nM, 5 min) significantly increased CMEC Na/H exchange activity, assessed as Na-dependent, HOE-642-sensitive H(+) flux. We found that AVP stimulation of CMEC Na/H exchange activity is dependent on intracellular Ca concentration and is blocked by V(1), but not V(2), vasopressin receptor antagonists. Our findings support the hypothesis that a BBB Na/H exchanger, possibly NHE1 and/or NHE2, is stimulated during ischemia to participate in cerebral edema formation.

Neuroprotective effects of estrogens following ischemic stroke.

Our laboratory has investigated whether and how 17beta-estradiol (E(2)) protects the brain against neurodegeneration associated with cerebrovascular stroke. We have discovered that low, physiological concentrations of E(2), which are strikingly similar to low-basal circulating levels found in cycling mice, dramatically protect the brain against stroke injury, and consequently revealed multiple signaling pathways and key genes that mediate protective action of E(2). Here we will review the discoveries comprising our current understanding of neuroprotective actions of estrogens against ischemic stroke. These findings may carry far reaching implications for improving the quality of life in aging populations.

Estradiol is a potent protective, restorative, and trophic factor after brain injury.

Estrogens are a group of pleiotropic steroid hormones that exhibit diverse mechanisms of action in multiple physiologic systems. Over the past 30 years, biomedical science has begun to appreciate that endogenous estrogens and their receptors display important roles beyond the reproductive system. Our growing appreciation of novel, nonreproductive functions for estrogens has fundamentally contributed to our knowledge of their role in human health and disease. Recent findings from the Women's Health Initiative have caused clinicians and scientists to question whether estrogens are protective factors or risk factors. In light of the dichotomy between basic science and clinical studies, this review will attempt to reconcile differences between them. We will focus on studies from our laboratory and others highlighting the beneficial properties of the most abundant endogenous estrogen, 17beta-estradiol, using in vivo and in vitro models of cerebral ischemia and neuronal injury. These studies demonstrate that 17beta-estradiol powerfully protects the brain using multiple molecular mechanisms that promote: (1) decreased cell death, (2) increased neurogenesis, (3) an enhancement of neurotrophic support, and (4) the suppression of proinflammatory pathways.

The role of the brain in female reproductive aging.

In middle-aged women, follicular depletion is a critical factor mediating the menopausal transition; however, all levels of the hypothalamic-pituitary-gonadal (HPG) axis contribute to the age-related decline in reproductive function. To help elucidate the complex interactions between the ovary and brain during middle-age that lead to the onset of the menopause, we utilize animal models which share striking similarities in reproductive physiology. Our results show that during middle-age, prior to any overt irregularities in estrous cyclicity, the ability of 17beta-estradiol (E(2)) to modulate the cascade of neurochemical events required for preovulatory gonadotropin-releasing hormone (GnRH) release and a luteinizing hormone (LH) surge is diminished. Middle-aged female rats experience a delay in and an attenuation of LH release in response to E(2). Additionally, although we do not observe a decrease in GnRH neuron number until a very advanced age, E(2)-mediated GnRH neuronal activation declines during the earliest stages of age-related reproductive decline. Numerous hypothalamic neuropeptides and neurochemical stimulatory inputs (i.e., glutamate, norepinephrine (NE), and vasoactive intestinal peptide (VIP)) that drive the E(2)-mediated GnRH/LH surge appear to dampen with age or lack the precise temporal coordination required for a specific pattern of GnRH secretion, while inhibitory signals such as gamma-aminobutyric acid (GABA) and opioid peptides remain unchanged or elevated during the afternoon of proestrus. These changes, occurring at the level of the hypothalamus, lead to irregular estrous cycles and, ultimately, the cessation of reproductive function. Taken together, our studies indicate that the hypothalamus is an important contributor to age-related female reproductive decline.

Inducible nitric oxide synthase and estradiol exhibit complementary neuroprotective roles after ischemic brain injury.

Estradiol-17beta exerts profound neuroprotective actions following cerebral ischemia through multiple molecular mechanisms. To examine the putative anti-inflammatory mechanisms employed by estradiol during stroke, we explored the interactions between estradiol and inducible nitric oxide synthase (iNOS) in both wildtype and iNOS knockout (iNOSKO) female mice following permanent middle cerebral artery occlusion (MCAO). Female mice were ovariectomized and treated with estradiol. One week later, they were subjected to MCAO, and then killed 24 h later. Analysis of total, cortical and striatal infarct volumes confirmed that estradiol is neuroprotective in wildtype mice. Infarct volumes were also significantly smaller in female iNOSKO female mice, but estradiol did not further decrease injury. We found that one mechanism by which estradiol may act is by decreasing nitric oxide synthase 2 gene expression in the cortex and in the striatum of wildtype mice. These results show that the pro-inflammatory actions of iNOS exacerbate stroke-induced injury within the cortex and striatum, and that iNOS deletion is neuroprotective in ovariectomized and estrogen-replaced female mice.

Timing of estrogen therapy after ovariectomy dictates the efficacy of its neuroprotective and antiinflammatory actions.

Recent studies describing the seemingly contradictory actions of estrogens in ischemic stroke injury have led us to reevaluate the circumstances under which estrogen therapy (ET) provides benefits against cerebral stroke and decipher its mechanisms of action. One prominent feature that follows stroke injury is massive central and peripheral inflammatory responses. Evidence now suggests that postischemic inflammatory responses strongly contribute to the extent of brain injury, and 17beta-estradiol (E(2)) may protect the ischemic brain by exerting antiinflammatory actions. In an attempt to explain recently reported dichotomous effects of E(2) in stroke injury, we tested the hypothesis that an extended period of hypoestrogenicity both prevents E(2) from protecting the brain against ischemia and simultaneously suppresses its antiinflammatory actions. We report that E(2) exerts profound neuroprotective action when administered immediately upon ovariectomy, but not when administered after 10 weeks of hypoestrogenicity. Consistently, E(2) treatment given immediately at the time of ovariectomy attenuated central and peripheral production of proinflammatory cytokines after ischemic stroke. In contrast, E(2) did not suppress production of proinflammatory molecules when it was administered after 10 weeks postovariectomy. These results demonstrate that a prolonged period of hypoestrogenicity disrupts both neuroprotective and antiinflammatory actions of E(2). Our findings may help to explain the results of the Women's Health initiative that reported no beneficial effect of ET against stroke because the majority of the subjects initiated ET after an extended period of hypoestrogenicity.

Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors alpha and beta.

Neurogenesis persists throughout life under normal and degenerative conditions. The adult subventricular zone (SVZ) generates neural stem cells capable of differentiating to neuroblasts and migrating to the site of injury in response to brain insults. In the present study, we investigated whether estradiol increases neurogenesis in the SVZ in an animal model of stroke to potentially promote the ability of the brain to undergo repair. Ovariectomized C57BL/6J mice were implanted with capsules containing either vehicle or 17beta-estradiol, and 1 week later they underwent experimental ischemia. We utilized double-label immunocytochemistry to identify the phenotype of newborn cells (5-bromo-2'-deoxyuridine-labeled) with various cellular markers; doublecortin and PSA-NCAM as the early neuronal marker, NeuN to identify mature neurons, and glial fibrillary acidic protein to identify astrocytes. We report that low physiological levels of estradiol treatment, which exert no effect in the uninjured state, significantly increase the number of newborn neurons in the SVZ following stroke injury. This effect of estradiol is limited to the dorsal region of the SVZ and is absent from the ventral SVZ. The proliferative actions of estradiol are confined to neuronal precursors and do not influence gliosis. Furthermore, we show that both estrogen receptors alpha and beta play pivotal functional roles, insofar as knocking out either of these receptors blocks the ability of estradiol to increase neurogenesis. These findings clearly demonstrate that estradiol stimulates neurogenesis in the adult SVZ, thus potentially facilitating the brain to remodel and repair after injury.

Complex actions of sex steroids in adipose tissue, the cardiovascular system, and brain: Insights from basic science and clinical studies.

Recent publications describing the results of the Women's Health Initiative (WHI) and other studies reporting the impact of hormone therapy on aging women have spurred reexamination of the broad use of estrogens and progestins during the postmenopausal years. Here, we review the complex pharmacology of these hormones, the diverse and sometimes opposite effects that result from the use of different estrogenic and progestinic compounds, given via different delivery routes in different concentrations and treatment sequence, and to women of different ages and health status. We examine our new and growing appreciation of the role of estrogens in the immune system and the inflammatory response, and we pose the concept that estrogen's interface with this system may be at the core of some of the effects on multiple physiological systems, such as the adipose/metabolic system, the cardiovascular system, and the central nervous system. We compare and contrast clinical and basic science studies as we focus on the actions of estrogens in these systems because the untoward effects of hormone therapy reported in the WHI were not expected. The broad interpretation and publicity of the results of the WHI have resulted in a general condemnation of all hormone replacement in postmenopausal women. In fact, careful review of the extensive literature suggests that data resulting from the WHI and other recent studies should be interpreted within the narrow context of the study design. We argue that these results should encourage us to perform new studies that take advantage of a dialogue between basic scientists and clinician scientists to ensure appropriate design, incorporation of current knowledge, and proper interpretation of results. Only then will we have a better understanding of what hormonal compounds should be used in which populations of women and at what stages of menopausal/postmenopausal life.

Mechanisms of neuroprotection by estrogen.

Over the past decade our recognition that estrogens function as important neurotrophic and neuroprotective factors has grown rapidly. Accumulating evidence from basic science studies demonstrates that estrogens exert profound protective actions against various forms of neurodegenerative diseases and injury. Although a thorough understanding of the mechanisms underlying the protective effect of estrogens is far from complete, significant progress has been achieved through the use of in vivo as well as in vitro models. Here we review the results from our laboratory demonstrating that low physiological levels of estradiol therapy exert powerful protection against ischemic stroke-like injury. Using an animal model of cerebrovascular stroke and in vitro explant cultures, we have begun to decipher under what circumstances 17beta-estradiol protects against neuronal death and to uncover its mechanisms of action. In addition, we will review recent work demonstrating that estradiol may additionally enhance the ability of the adult brain to undergo repair by influencing the production of new neurons under neuropathological conditions, as well as by promoting an anti-inflammatory response. As we uncover the important protective roles of ovarian steroid hormones in brain disease and injury, we increasingly appreciate that the mechanisms by which estrogens achieve these effects are diverse and complex.

Differential modulation of estrogen receptors (ERs) in ischemic brain injury: a role for ERalpha in estradiol-mediated protection against delayed cell death.

Estradiol enhances plasticity and survival of the injured brain. Our previous work demonstrates that physiological levels of estradiol protect against cerebral ischemia in the young and aging brain through actions involving estrogen receptors (ERs) and alterations in gene expression. The major goal of this study was to establish mechanisms of neuroprotective actions induced by low levels of estradiol. We first examined effects of estradiol on the time-dependent evolution of ischemic brain injury. Because estradiol is known to influence apoptosis, we hypothesized that it acts to decrease the delayed phase of cell death observed after middle cerebral artery occlusion (MCAO). Furthermore, because ERs are pivotal to neuroprotection, we examined the temporal expression profiles of both ER subtypes, ERalpha and ERbeta, after MCAO and delineated potential roles for each receptor in estradiol-mediated neuroprotection. We quantified cell death in brains at various times after MCAO and analyzed ER expression by RT-PCR, in situ hybridization, and immunohistochemistry. We found that during the first 24 h, the mechanisms of estradiol-induced neuroprotection after MCAO are limited to attenuation of delayed cell death and do not influence immediate cell death. Furthermore, we discovered that ERs exhibit distinctly divergent profiles of expression over the evolution of injury, with ERalpha induction occurring early and ERbeta modulation occurring later. Finally, we provide evidence for a new and functional role for ERalpha in estradiol-mediated protection of the injured brain. These findings indicate that physiological levels of estradiol protect against delayed cell death after stroke-like injury through mechanisms requiring ERalpha.

Vasoactive intestinal polypeptide regulates dynamic changes in astrocyte morphometry: impact on gonadotropin-releasing hormone neurons.

Recent studies suggest that astrocytes modulate the GnRH-induced LH surge. In particular, we have shown that the surface area of astrocytes that ensheath GnRH neurons exhibits diurnal rhythms. Vasoactive intestinal polypeptide (VIP) influences numerous aspects of astrocyte function in multiple brain regions and is a neurotransmitter in the suprachiasmatic nucleus (SCN) that affects GnRH neurons. The goals of this study were to: 1) assess whether astrocytes that surround GnRH neurons express VIP receptors, 2) determine the effects VIP suppression in the SCN on the morphometry of astrocytes surrounding GnRH neurons, and 3) assess whether this effect mimics aging-like changes in surface area of astrocytes. Young rats were ovariectomized (d 0), implanted with cannulae into the SCN (d 5), injected with VIP antisense (antioligo) or random sequence oligonucleotides, implanted with capsules containing 17beta-estradiol dissolved in oil (d 7), and perfused at 0300, 1400, and 1800 h (d 9). Brains were processed for immunocytochemistry. Our results demonstrate that astrocytes in close apposition to GnRH neurons express VIP receptors. Antioligo treatment blocked diurnal rhythms in surface area of astrocytes ensheathing GnRH neurons. The absence of diurnal rhythms resembles observations in middle-aged rats. Together these findings suggest that the ability of the VIP-containing neurons in the SCN to relay diurnal information to GnRH neurons may be by influencing dynamic changes in the morphometry of astrocytes that surround GnRH neurons. Furthermore, the absence of a VIP rhythm in aging animals may lead to altered GnRH activity via astrocyte-dependent mechanisms.

Estrogens and cerebrovascular stroke: what do animal models teach us?

Recent findings from the Women's Health Initiative (WHI) suggest that hormone therapy (HT) and estrogen therapy (ET) increase the risk of stroke in postmenopausal women. These results were unexpected based upon many previous clinical, observational, and epidemiological studies and a large body of evidence that come from studies performed in animal models. Before we assume that these results are widely applicable to other hormone preparations and to all older postmenopausal women, we should consider whether the particular hormone preparations, the doses that were used, the age of the women, the length of time that they were postmenopausal prior to the initiation of treatment, and/or their health status may have been important factors in the results of this clinical trial. We believe that results of studies using animal models provide insights into why the result of the WHI should have been expected. Furthermore, results of basic science studies provide a strong rationale for the design of future clinical studies that will more accurately test the effects of ET/HT on the risk and outcomes of cerebrovascular stroke in middle-aged perimenopausal and early postmenopausal women. We will review data, predominantly from our laboratory, gathered over the past six years that lead us to this conclusion.

Are estrogens protective or risk factors in brain injury and neurodegeneration? Reevaluation after the Women's health initiative.

Estrogens are essential for normal reproductive function. In addition, they exert important, complex, and diverse nonreproductive actions on multiple tissues. Although accumulating evidence from basic science studies using animal models suggests that estradiol plays a critical neuroprotective role against multiple types of neurodegenerative diseases and injuries, recent clinical studies have reported either inconclusive or untoward effects of hormone therapy on the brain. We focus herein on the work that we have done during the past 6 yr that strongly suggests that low levels of estradiol therapy exert dramatic protective actions in the adult injured brain. Our results reveal that 17beta-estradiol slows the progression of this injury and diminishes the extent of cell death by suppressing apoptotic cell death pathways and enhancing expression of genes that optimize cell survival. Furthermore, we have found that estrogen receptors play a pivotal functional role in neuroprotection. Together, these results carry broad implications for the selective targeting of estrogen receptors in the treatment of neurodegenerative conditions resulting from disease or injury, particularly for aging, postmenopausal women.

Suppression of vasoactive intestinal polypeptide in the suprachiasmatic nucleus leads to aging-like alterations in cAMP rhythms and activation of gonadotropin-releasing hormone neurons.

Input from the suprachiasmatic nucleus (SCN) to gonadotropin-releasing hormone (GnRH) neurons is critical to the occurrence of regular cyclic GnRH secretion. It is thought that an essential neuropeptide in the SCN that communicates this cyclic information to GnRH neurons is vasoactive intestinal polypeptide (VIP) and that it may act through cAMP. We tested the hypothesis that (1) aging involves a blunting of cAMP diurnal rhythmicity in the SCN; (2) administration of antisense oligonucleotides (anti-oligos) against VIP, which produces an aging-like pattern in VIP, would lead to an aging-like suppression of cAMP; and (3) this in turn would lead to inhibition of the steroid-induced activation of GnRH neurons. We measured cAMP concentrations in the SCN and rostral preoptic nucleus throughout the day in young and middle-aged rats that were ovariectomized (OVX) or OVX and treated with estradiol. Our results show that cAMP concentrations exhibit a diurnal rhythm in young rats, and that this rhythm is totally abolished by the time rats are middle age. Administration of antisense oligonucleotides against VIP or random oligos suppresses VIP concentrations and abolishes the cAMP rhythm, leading to significantly reduced activation of GnRH neurons. Together, these findings strongly suggest that the SCN conveys diurnal information to GnRH neurons by driving VIP-dependent cAMP rhythms. In addition, aging involves deterioration in this VIP-driven rhythmicity, which impacts the ability of steroids to induce GnRH neuronal activation.

Hormone therapy: physiological complexity belies therapeutic simplicity.

The results of the Women's Health Initiative, a study anticipated to provide definitive answers about health benefits and risks of postmenopausal hormone therapy, have generated debate and confusion among clinicians, researchers, and the lay public. The ovarian hormones estrogen and progesterone, which decline at menopause, normally elicit complex tissue-specific responses throughout the body. Major advances are providing a detailed molecular definition of how that differential action is achieved. Here we review estrogen and progestin actions, discuss how effectively knowledge of steroid hormone endocrinology has been incorporated into clinical studies, and consider the impact on modern hormone therapy protocols and pharmaceutical development.

Estradiol attenuates programmed cell death after stroke-like injury.

Estradiol is a known neurotrophic and neuroprotective factor. Our previous work demonstrated that replacement with physiological concentrations of estradiol protects the cortex against middle cerebral artery occlusion (MCAO)-induced cell death. The cerebral cortex exhibits caspase-dependent programmed cell death (PCD) in many models of focal cerebral ischemia. We hypothesized that estradiol attenuates PCD during stroke injury. The current study explored the temporospatial pattern of markers of PCD, their relationship to the evolution of injury, and their modulation by estradiol. Rats were ovariectomized and treated with either estradiol or vehicle. One week later, rats underwent MCAO, and brains were collected at 1, 4, 8, 16, and 24 hr. We assessed the temporospatial evolution of infarction volume, DNA fragmentation, and levels of spectrin cleavage products in ischemic cortex. Estradiol led to a delay and attenuation of injury-mediated DNA fragmentation as early as 8 hr after MCAO. Estradiol also dramatically reduced the level of the 120 kDa caspase-mediated spectrin breakdown product (SBDP120) at 4 hr but not at 8 or 16 hr. The SBDP150, produced by caspase and calpain, showed peak levels at 16 hr but was not altered by estradiol. These results strongly suggest that estradiol protects the ischemic cortex by attenuating PCD, thereby reducing caspase activity, DNA fragmentation, and subsequently, overall cell death. These studies deepen our understanding of the mechanisms underlying estrogen-mediated neuroprotection.