Functional Assessment of Stroke-Induced Regulation of miR-20a-3p and Its Role as a Neuroprotectant.

MicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17-92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.

Sex differences in miRNA as therapies for ischemic stroke.

MicroRNAs, a subset of non-coding RNAs, are present in virtually all tissues including body fluids and are global regulators of the transcriptome. In view of the expanding number of microRNAs and the large number of gene targets that each microRNA can potentially regulate, they have been compared to hormones in the scope of their effects. MicroRNA have been implicated as biomarkers for several diseases including stroke, as well as chronic conditions that are associated with stroke. Recent research has focused on manipulating miRNA to improve stroke outcomes. Although several miRNAs have been shown to have neuroprotective properties, the overwhelming majority of these studies have employed only male animals. This review will focus on two miRNAs, Let7f and mir363-3p, whose effectiveness as a stroke neuroprotectant is sex-specific.

Stroke triggers nigrostriatal plasticity and increases alcohol consumption in rats.

Excessive alcohol consumption is a known risk factor for stroke, but the effect of stroke on alcohol intake is unknown. The dorsomedial striatum (DMS) and midbrain areas of the nigrostriatal circuit are critically associated to stroke and alcohol addiction. Here we sought to explore the influence of stroke on alcohol consumption and to uncover the underlying nigrostriatal mechanism. Rats were trained to consume alcohol using a two-bottle choice or operant self-administration procedure. Retrograde beads were infused into the DMS or midbrain to label specific neuronal types, and ischemic stroke was induced in the dorsolateral striatum (DLS). Slice electrophysiology was employed to measure excitability and synaptic transmission in DMS and midbrain neurons. We found that ischemic stroke-induced DLS infarction produced significant increases in alcohol preference, operant self-administration, and relapse. These increases were accompanied by enhanced excitability of DMS and midbrain neurons. In addition, glutamatergic inputs onto DMS D1-neurons was potentiated, whereas GABAergic inputs onto DMS-projecting midbrain dopaminergic neurons was suppressed. Importantly, systemic inhibition of dopamine D1 receptors attenuated the stroke-induced increase in operant alcohol self-administration. Our results suggest that the stroke-induced DLS infarction evoked abnormal plasticity in nigrostriatal dopaminergic neurons and DMS D1-neurons, contributing to increased post-stroke alcohol-seeking and relapse.

Mir363-3p improves ischemic stroke outcomes in female but not male rats.

With age, stroke prevalence is higher, and stroke outcome, worse, in women. Thus there is an urgent need to identify stroke neuroprotectants for this population. Using a preclinical stroke model, our studies focused on microRNAs (miRNAs), a class of translational repressors, as neuroprotectants. Analysis of circulating miRNA in the acute phase of stroke indicated potential neuroprotective capacity for miR363. Specifically, mir363 is elevated in serum of adult female rats that typically have small infarct volumes, but is deficient in age-matched males or middle-aged males and females, groups that have greater stroke-associated impairment. To directly test the effect of mir363 on stroke outcomes, first, adult females were treated with antagomirs to mir363 post stroke and next, middle-aged females were treated with mimic to mir363-3p post stroke. Antagomir treatment to adult females significantly increased infarct volume and impaired sensory motor performance. Reciprocally, mir363 mimic to middle-aged females reduced infarct volume, preserved forebrain microvessels and improved sensory motor performance. In the early acute stroke phase, mir363-3p mimic reduced the expression and functional activity of caspase-3, a critical component of the apoptotic cell cascade. In contrast, mir363-3p mimic treatment had no effect on stroke outcomes or caspase regulation in young males. Collectively, these studies show that mir363 is neuroprotective for stroke in females and implicates caspase-3 as a sex-specific miRNA-sensitive node for recovery from ischemic stroke.

Sex Differences in the Impact of Shift Work Schedules on Pathological Outcomes in an Animal Model of Ischemic Stroke.

Circadian clock desynchronization has been implicated in the pathophysiology of cardiovascular disease and related risk factors (eg, obesity, diabetes). Thus, we examined the extent to which circadian desynchronization exacerbates ischemic stroke outcomes and whether its detrimental effects on stroke severity and functional impairments are further modified by biological sex. Circadian entrainment of activity rhythms in all male and female rats was observed during exposure to a fixed light-dark (LD) 12:12 cycle but was severely disrupted when this LD cycle was routinely shifted (12 h advance/5 d) for approximately 7 weeks. In contrast to the regular estrous cycles in fixed LD animals, cyclicity was abolished and persistent estrus was evident in all shifted LD females. The disruption of estrous cyclicity in shifted LD females was associated with a significant increase in serum estradiol levels relative to that observed in fixed LD controls. Circadian rhythm disruption exacerbated stroke outcomes in both shifted LD male and female rats and further amplified sex differences in stroke impairments. In males, but not females, circadian disruption after exposure to the shifted LD cycle was marked by high rates of mortality. In surviving females, circadian desynchronization after exposure to shifted LD cycles produced significant increases in stroke-induced infarct volume and sensorimotor deficits with corresponding decreases in serum IGF-1 levels. These results suggest that circadian rhythm disruption associated with shift work schedules or the irregular nature of our everyday work and/or social environments may interact with other nonmodifiable risk factors such as biological sex to modulate the pathological effects of stroke.

Histone methylation patterns in astrocytes are influenced by age following ischemia.

In animal models, middle-aged females sustain greater ischemia-induced infarction as compared to adult females. This age difference in infarct severity is associated with reduced functional capacity of astrocytes, a critical neural support cell. The impaired response of astrocytes following stroke in middle-aged females may be related to epigenetic alterations, including histone acetylation or methylation. The present study measured the activity of enzymes that regulate histone acetylation and methylation in cerebral cortical astrocytes of adult (6 month) and middle-aged (11+ month) female rats 48 h following middle cerebral artery occlusion. H3K4 histone methyltransferase activity was decreased in astrocytes from middle-aged females. The next experiment therefore examined H3K4me3 (transcriptional enhancer) and H3K9me3 (transcriptional repressor) in astrocytes from adult and middle-aged females using ChIP-seq analysis. Adult females had more enriched H3K4me3 peaks (304 vs. 26) at transcriptional start sites and fewer H3K9me3 enriched peaks than middle-aged females (4 vs. 22), indicating a pattern of less active chromatin in astrocytes in the older group following ischemia. DAVID clustering analysis of H3K4me3 enriched genes found several functional categories, including cell motility, regulation of apoptosis and the vascular endothelial growth factor (VEGF) pathway. H3K4me3 was enriched at the miR-17-20 cluster and VEGFa, and analysis of a separate set of astrocytes confirmed that VEGF protein expression and miR-20 mRNA expression were significantly greater following ischemia in adult females compared to middle-aged females. These data indicate that astrocytes display less active chromatin with aging and provide new insight into possible mechanisms for differences in stroke severity observed during aging.

Blood brain barrier and neuroinflammation are critical targets of IGF-1-mediated neuroprotection in stroke for middle-aged female rats.

Ischemia-induced cerebral infarction is more severe in older animals as compared to younger animals, and is associated with reduced availability of insulin-like growth factor (IGF)-1. This study determined the effect of post-stroke IGF-1 treatment, and used microRNA profiling to identify mechanisms underlying IGF-1's neuroprotective actions. Post-stroke ICV administration of IGF-1 to middle-aged female rats reduced infarct volume by 39% when measured 24h later. MicroRNA analyses of ischemic tissue collected at the early post-stroke phase (4h) indicated that 8 out of 168 disease-related miRNA were significantly downregulated by IGF-1. KEGG pathway analysis implicated these miRNA in PI3K-Akt signaling, cell adhesion/ECM receptor pathways and T-and B-cell signaling. Specific components of these pathways were subsequently analyzed in vehicle and IGF-1 treated middle-aged females. Phospho-Akt was reduced by ischemia at 4h, but elevated by IGF-1 treatment at 24h. IGF-1 induced Akt activation was preceded by a reduction of blood brain barrier permeability at 4h post-stroke and global suppression of cytokines including IL-6, IL-10 and TNF-α. A subset of these cytokines including IL-6 was also suppressed by IGF-1 at 24h post-stroke. These data are the first to show that the temporal and mechanistic components of post-stroke IGF-1 treatment in older animals, and that cellular components of the blood brain barrier may serve as critical targets of IGF-1 in the aging brain.

Circulating miRNA profiles provide a biomarker for severity of stroke outcomes associated with age and sex in a rat model.

Small non-coding RNA [miRNA (microRNA)] found in the circulation have been used successfully as biomarkers and mechanistic targets for chronic and acute disease. The present study investigated the impact of age and sex on miRNA expression following ischaemic stroke in an animal model. Adult (6 month) and middle-aged (11-12 months) female and male rats were subject to MCAo (middle cerebral artery occlusion) using ET-1 (endothelin-1). Circulating miRNAs were analysed in blood samples at 2 and 5 days post-stroke, and brain miRNAs were analysed at 5 days post-stroke. Although stroke-associated infarction was observed in all groups, infarct volume and sensory-motor deficits were significantly reduced in adult females compared with middle-aged females, adult males or middle-aged males. At 2 days post-stroke, 21 circulating miRNAs were differentially regulated and PCA (principal component analysis) confirmed that most of the variance was due to age. At 5 days post-stroke, 78 circulating miRNAs exhibited significantly different regulation, and most of the variance was associated with sex. A small cohort (five) of miRNAs, miR-15a, miR-19b, miR-32 miR-136 and miR-199a-3p, were found to be highly expressed exclusively in adult females compared with middle-aged females, adult males and middle-aged males. Predicted gene targets for these five miRNAs analysed for KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways revealed that the top ten KEGG pathways were related to growth factor signalling, cell structure and PI3K (phosphoinositide 3-kinase)/Akt and mTOR (mammalian target of rapamycin) signalling. Overall, the pattern of circulating miRNA expression suggests an early influence of age in stroke pathology, with a later emergence of sex as a factor for stroke severity.

Revisiting the timing hypothesis: biomarkers that define the therapeutic window of estrogen for stroke.

Significantly extended life expectancy coupled with contemporary sedentary lifestyles and poor nutrition has created a global epidemic of cardiovascular disease and stroke. For women, this issue is complicated by the discrepant outcomes of hormone therapy (HT) for stroke incidence and severity as well as the therapeutic complications for stroke associated with advancing age. Here we propose that the impact of estrogen therapy cannot be considered in isolation, but should include age-related changes in endocrine, immune, and nucleic acid mediators that collaborate with estrogen to produce neuroprotective effects commonly seen in younger, healthier demographics. Due to their role as modulators of ischemic cell death, the post-stroke inflammatory response, and neuronal survival and regeneration, this review proposes that Insulin-like Growth Factor (IGF)-1, Vitamin D, and discrete members of the family of non-coding RNA peptides called microRNAs (miRNAs) may be crucial biochemical markers that help determine the neuroprotective "window" of HT. Specifically, IGF-1 confers neuroprotection in concert with, and independently of, estrogen and failure of the insulin/IGF-1 axis is associated with metabolic disturbances that increase the risk for stroke. Vitamin D and miRNAs regulate and complement IGF-1 mediated function and neuroprotective efficacy via modulation of IGF-1 availability and neural stem cell and immune cell proliferation, differentiation and secretions. Together, age-related decline of these factors differentially affects stroke risk, severity, and outcome, and may provide a novel therapeutic adjunct to traditional HT practices.

An antagomir to microRNA Let7f promotes neuroprotection in an ischemic stroke model.

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke.

Vitamin D deficiency exacerbates experimental stroke injury and dysregulates ischemia-induced inflammation in adult rats.

Vitamin D deficiency (VDD) is widespread and considered a risk factor for cardiovascular disease and stroke. Low vitamin D levels are predictive for stroke and more fatal strokes in humans, whereas vitamin D supplements are associated with decreased risk of all-cause mortality. Because VDD occurs with other comorbid conditions that are also independent risk factors for stroke, this study examined the specific effect of VDD on stroke severity in rats. Adult female rats were fed control or VDD diet for 8 wk and were subject to middle cerebral artery occlusion thereafter. The VDD diet reduced circulating vitamin D levels to one fifth (22%) of that observed in rats fed control chow. Cortical and striatal infarct volumes in animals fed VDD diet were significantly larger, and sensorimotor behavioral testing indicated that VDD animals had more severe poststroke behavioral impairment than controls. VDD animals were also found to have significantly lower levels of the neuroprotective hormone IGF-I in plasma and the ischemic hemisphere. Cytokine analysis indicated that VDD significantly reduced IL-1α, IL-1ß, IL-2, IL-4, IFN-γ, and IL-10 expression in ischemic brain tissue. However, ischemia-induced IL-6 up-regulation was significantly higher in VDD animals. In a separate experiment, the therapeutic potential of acute vitamin D treatments was evaluated, where animals received vitamin D injections 4 h after stroke and every 24 h thereafter. Acute vitamin D treatment did not improve infarct volume or behavioral performance. Our data indicate that VDD exacerbates stroke severity, involving both a dysregulation of the inflammatory response as well as suppression of known neuroprotectants such as IGF-I.

Age-related severity of focal ischemia in female rats is associated with impaired astrocyte function.

In middle-aged female rats, focal ischemia leads to a larger cortical infarction as compared with younger females. To determine if stroke-induced cytotoxicity in middle-aged females was associated with impaired astrocyte function, astrocytes were harvested and cultured from the ischemic cortex of young and middle-aged female rats. Middle-aged astrocytes cleared significantly less glutamate from media as compared with young female astrocytes. Furthermore, astrocyte-conditioned media from middle-aged female astrocytes induced greater migration of peripheral blood monocyte cells (PBMCs) and expressed higher levels of the chemoattractant macrophage inflammatory protein-1 (MIP-1). Middle-aged astrocytes also induced greater migration of neural progenitor cells (NPCs), however, their ability to promote neuronal differentiation of neural progenitor cells was similar to young astrocytes. In males, where cortical infarct volume is similar in young and middle-aged animals, no age-related impairment was observed in astrocyte function. These studies show that the aging astrocyte may directly contribute to infarct severity by inefficient glutamate clearance and enhanced cytokine production and suggest a cellular target for improved stroke therapy among older females.

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.

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.

The PKC inhibitor, bisindolymaleimide, blocks DOI's attenuation of the effects of 8-OH-DPAT on female rat lordosis behavior.

Ovariectomized rats with bilateral cannulae near the ventromedial nucleus of the hypothalamus were hormonally primed with 10 microg estradiol benzoate and 500 microg progesterone. Sexually receptive females were infused bilaterally with 200 ng of the 5-HT(1A) receptor agonist, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT), or with a combination of 200 ng 8-OH-DPAT and 2000 ng of the 5-HT(2) receptor agonist, (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane HCl (DOI). 8-OH-DPAT inhibited lordosis behavior and DOI reduced this inhibition. However, if females were preinfused with the PKC inhibitor, bisindolymaleimide I hydrochloride (BIM), DOI's effect was eliminated. BIM's attenuation of the effects of DOI was time-dependent. When BIM was infused 90 min, but not 30 min, before the 5-HT receptor agonists, BIM eliminated DOI's protection against the lordosis-inhibiting effects of 8-OH-DPAT. A concentration of BIM as low as 10(-5) nmol in a 0.5 microl infusion volume was effective and there was little evidence of dose responsivity between 10(-5) and 10(-1) nmol of BIM. In contrast, prior infusion with vehicle or with 10(-7) nmol BIM had no impact on the female's response to the 5-HT receptor agonists. These findings allow the suggestion that DOI's ability to increase PKC may be responsible for attenuation of the effects of 8-OH-DPAT on lordosis behavior.

Mild restraint reduces the time hormonally primed rats spend with sexually active males.

The effect of 5 min of restraint on the time sexually-receptive females spend in the compartment of a sexually active male was examined. Ovariectomized females, hormonally primed with 10 microg estradiol benzoate and 500 microg progesterone (EP) or primed only with estradiol benzoate (EO) were used. After the restraint or home-cage experience, females were tested for 30 min in a chamber that allowed the female to escape to a small "burrow". Females, subjected to restraint, left the male's compartment faster and spent significantly less time in the male's compartment than did non-restrained females. This was true for both EP and EO females. When females were injected with the 5-HT(2B/2C)-receptor antagonist, SB 206553, 15 min before restraint, time spent in the male's compartment was even further reduced. However, additional studies indicated that it was the stress of the injection rather than the action of the drug that was responsible for the female's behavior. These findings are discussed in terms of their significance to the understanding of the female's reproductive response to stress and are compared to prior findings, where lordosis behavior was significantly reduced by restraint.