ATF2/BAP1 Axis Mediates Neuronal Apoptosis After Subarachnoid Hemorrhage via P53 Pathway.

BACKGROUND: Neuronal apoptosis plays an essential role in the pathogenesis of brain injury after subarachnoid hemorrhage (SAH). BAP1 (BRCA1-associated protein 1) is considered to exert pro-apoptotic effects in multiple diseases. However, evidence supporting the effect of BAP1 on the apoptotic response to SAH is lacking. Therefore, we aimed to confirm the role of BAP1 in SAH-induced apoptosis. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect BAP1 expression in the cerebrospinal fluid. Endovascular perforation was performed in mice to induce SAH. Lentiviral short hairpin RNA targeting BAP1 mRNA was transduced into the ipsilateral cortex of mice with SAH to investigate the role of BAP1 in neuronal damage. Luciferase and coimmunoprecipitation assays were performed to investigate the mechanism through which BAP1 participates in hemin-induced SAH. RESULTS: First, BAP1 expression was upregulated in the cerebrospinal fluid of patients with SAH and positively associated with unfavorable outcomes. ATF2 (activating transcription factor-2) then regulated BAP1 expression by binding to the BAP1 promoter. In addition, BAP1 overexpression enhanced P53 activity and stability by reducing P53 proteasome-mediated degradation. Subsequently, elevated P53 promoted neuronal apoptosis via the P53 pathway. Inhibition of the neuronal BAP1/P53 axis significantly reduced neurological deficits and neuronal apoptosis and improved neurological dysfunction in mice after SAH. CONCLUSIONS: Our results suggest that the neuronal ATF2/BAP1 axis exerts a brain-damaging effect by modulating P53 activity and stability and may be a novel therapeutic target for SAH.

Factual and counterfactual learning in major adolescent depressive disorder, evidence from an instrumental learning study.

BACKGROUND: The incidence of adolescent depressive disorder is globally skyrocketing in recent decades, albeit the causes and the decision deficits depression incurs has yet to be well-examined. With an instrumental learning task, the aim of the current study is to investigate the extent to which learning behavior deviates from that observed in healthy adolescent controls and track the underlying mechanistic channel for such a deviation. METHODS: We recruited a group of adolescents with major depression and age-matched healthy control subjects to carry out the learning task with either gain or loss outcome and applied a reinforcement learning model that dissociates valence (positive v. negative) of reward prediction error and selection (chosen v. unchosen). RESULTS: The results demonstrated that adolescent depressive patients performed significantly less well than the control group. Learning rates suggested that the optimistic bias that overall characterizes healthy adolescent subjects was absent for the depressive adolescent patients. Moreover, depressed adolescents exhibited an increased pessimistic bias for the counterfactual outcome. Lastly, individual difference analysis suggested that these observed biases, which significantly deviated from that observed in normal controls, were linked with the severity of depressive symoptoms as measured by HAMD scores. CONCLUSIONS: By leveraging an incentivized instrumental learning task with computational modeling within a reinforcement learning framework, the current study reveals a mechanistic decision-making deficit in adolescent depressive disorder. These findings, which have implications for the identification of behavioral markers in depression, could support the clinical evaluation, including both diagnosis and prognosis of this disorder.

Engineered Metallacycle-Based Supramolecular Photosensitizers for Effective Photodynamic Therapy.

Although metallacycle-based supramolecular photosensitizers (PSs) have attracted increasing attention in biomedicine, their clinical translation is still hindered by their inherent dark toxicity. Herein, we report what to our knowledge is the first example of a molecular engineering approach to building blocks of metallacycles for constructing a series of supramolecular PSs (RuA-RuD), with the aim of simultaneously reducing dark toxicity and enhancing phototoxicity, and consequently obtaining high phototoxicity indexes (PI). Detailed in vitro investigations demonstrate that RuA-RuD display high cancer cellular uptake and remarkable antitumor activity even under hypoxic conditions. Notably, RuD exhibited no dark toxicity and displayed the highest PI value (≈406). Theoretical calculations verified that RuD has the largest steric hindrance and the lowest singlet-triplet energy gap (ΔEST , 0.61 eV). Further in vivo studies confirmed that RuD allows safe and effective phototherapy against A549 tumors.

Sodium-glucose cotransporter 2 inhibitor ameliorates high fat diet-induced hypothalamic-pituitary-ovarian axis disorders.

High-fat diet (HFD) consumption is known to be associated with ovulatory disorders among women of reproductive age. Previous studies in animal models suggest that HFD-induced microglia activation contributes to hypothalamic inflammation. This causes the dysfunction of the hypothalamic-pituitary-ovarian (HPO) axis, leading to subfertility. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a novel class of lipid-soluble antidiabetic drugs that target primarily the early proximal tubules in kidney. Recent evidence revealed an additional expression site of SGLT2 in the central nervous system (CNS), indicating a promising role of SGLT2 inhibitors in the CNS. In type 2 diabetes patients and rodent models, SGLT2 inhibitors exhibit neuroprotective properties through reduction of oxidative stress, alleviation of cerebral atherosclerosis and suppression of microglia-induced neuroinflammation. Furthermore, clinical observations in patients with polycystic ovary syndrome (PCOS) demonstrated that SGLT2 inhibitors ameliorated patient anthropometric parameters, body composition and insulin resistance. Therefore, it is of importance to explore the central mechanism of SGLT2 inhibitors in the recovery of reproductive function in patients with PCOS and obesity. Here, we review the hypothalamic inflammatory mechanisms of HFD-induced microglial activation, with a focus on the clinical utility and possible mechanism of SGLT2 inhibitors in promoting reproductive fitness.

Different gender-derived gut microbiota influence stroke outcomes by mitigating inflammation.

BACKGROUND AND PURPOSE: Stroke is associated with high disability and mortality rates and increases the incidence of organ-related complications. Research has revealed that the outcomes and prognosis of stroke are regulated by the state of the intestinal microbiota. However, the possibility that the manipulation of the intestinal microbiota can alter sex-related stroke outcomes remain unknown. METHODS: To verify the different effects of microbiota from different sexes on stroke outcomes, we performed mouse fecal microbiota transplantation (FMT) and established a model of ischemic stroke. Male and female mice received either male or female microbiota through FMT. Ischemic stroke was triggered by MCAO (middle cerebral artery occlusion), and sham surgery served as a control. Over the next few weeks, the mice underwent neurological evaluation and metabolite and inflammatory level detection, and we collected fecal samples for 16S ribosomal RNA analysis. RESULTS: We found that when the female mice were not treated with FMT, the microbiota (especially the Firmicutes-to-Bacteroidetes ratio) and the levels of three main metabolites tended to resemble those of male mice after experimental stroke, indicating that stroke can induce an ecological imbalance in the biological community. Through intragastric administration, the gut microbiota of male and female mice was altered to resemble that of the other sex. In general, in female mice after MCAO, the survival rate was increased, the infarct area was reduced, behavioral test performance was improved, the release of beneficial metabolites was promoted and the level of inflammation was mitigated. In contrast, mice that received male microbiota were much more hampered in terms of protection against brain damage and the recovery of neurological function. CONCLUSION: A female-like biological community reduces the level of systemic proinflammatory cytokines after ischemic stroke. Poor stroke outcomes can be positively modulated following supplementation with female gut microbiota.

Relevant mediators involved in and therapies targeting the inflammatory response induced by activation of the NLRP3 inflammasome in ischemic stroke.

The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome is a member of the NLR family of inherent immune cell sensors. The NLRP3 inflammasome can detect tissue damage and pathogen invasion through innate immune cell sensor components commonly known as pattern recognition receptors (PRRs). PRRs promote activation of nuclear factor kappa B (NF-κB) pathways and the mitogen-activated protein kinase (MAPK) pathway, thus increasing the transcription of genes encoding proteins related to the NLRP3 inflammasome. The NLRP3 inflammasome is a complex with multiple components, including an NAIP, CIITA, HET-E, and TP1 (NACHT) domain; apoptosis-associated speck-like protein containing a CARD (ASC); and a leucine-rich repeat (LRR) domain. After ischemic stroke, the NLRP3 inflammasome can produce numerous proinflammatory cytokines, mediating nerve cell dysfunction and brain edema and ultimately leading to nerve cell death once activated. Ischemic stroke is a disease with high rates of mortality and disability worldwide and is being observed in increasingly younger populations. To date, there are no clearly effective therapeutic strategies for the clinical treatment of ischemic stroke. Understanding the NLRP3 inflammasome may provide novel ideas and approaches because targeting of upstream and downstream molecules in the NLRP3 pathway shows promise for ischemic stroke therapy. In this manuscript, we summarize the existing evidence regarding the composition and activation of the NLRP3 inflammasome, the molecules involved in inflammatory pathways, and corresponding drugs or molecules that exert effects after cerebral ischemia. This evidence may provide possible targets or new strategies for ischemic stroke therapy.

The immune response of T cells and therapeutic targets related to regulating the levels of T helper cells after ischaemic stroke.

Through considerable effort in research and clinical studies, the immune system has been identified as a participant in the onset and progression of brain injury after ischaemic stroke. Due to the involvement of all types of immune cells, the roles of the immune system in stroke pathology and associated effects are complicated. Past research concentrated on the functions of monocytes and neutrophils in the pathogenesis of ischaemic stroke and tried to demonstrate the mechanisms of tissue injury and protection involving these immune cells. Within the past several years, an increasing number of studies have elucidated the vital functions of T cells in the innate and adaptive immune responses in both the acute and chronic phases of ischaemic stroke. Recently, the phenotypes of T cells with proinflammatory or anti-inflammatory function have been demonstrated in detail. T cells with distinctive phenotypes can also influence cerebral inflammation through various pathways, such as regulating the immune response, interacting with brain-resident immune cells and modulating neurogenesis and angiogenesis during different phases following stroke. In view of the limited treatment options available following stroke other than tissue plasminogen activator therapy, understanding the function of immune responses, especially T cell responses, in the post-stroke recovery period can provide a new therapeutic direction. Here, we discuss the different functions and temporal evolution of T cells with different phenotypes during the acute and chronic phases of ischaemic stroke. We suggest that modulating the balance between the proinflammatory and anti-inflammatory functions of T cells with distinct phenotypes may become a potential therapeutic approach that reduces the mortality and improves the functional outcomes and prognosis of patients suffering from ischaemic stroke.

Extracellular vesicle-derived miRNA as a novel regulatory system for bi-directional communication in gut-brain-microbiota axis.

The gut-brain-microbiota axis (GBMAx) coordinates bidirectional communication between the gut and brain, and is increasingly recognized as playing a central role in physiology and disease. MicroRNAs are important intracellular components secreted by extracellular vesicles (EVs), which act as vital mediators of intercellular and interspecies communication. This review will present current advances in EV-derived microRNAs and their potential functional link with GBMAx. We propose that EV-derived microRNAs comprise a novel regulatory system for GBMAx, and a potential novel therapeutic target for modifying GBMAx in clinical therapy.

Left Stellate Ganglion Ablation Inhibits Ventricular Arrhythmias through Macrophage Regulation in Canines with Acute Ischemic Stroke.

AIMS: To investigate the potential mechanism of ventricular arrhythmias (VAs) after acute ischemic stroke and explore the effects of left stellate gangling (LSG) ablation on VAs induced by stroke in canines. Materials and Methods: Twenty canines were randomly divided into the sham-operated group (n=6), AS group (n=7) and SGA group (n=7). Cerebral ischemic model was established in the AS group and the SGA group by right acute middle cerebral artery occlusion (MCAO). LSG ablation was performed in the SGA group as soon as MCAO. After 3 days, atrial electrophysiology and neural activity were measured in vivo. The levels of norepinephrine (NE) in plasma and ventricle were detected by ELISA. The levels of monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and NF-κB p65 in ventricle were detected by western blotting. The pro-inflammatory polarization of macrophages in ventricle was detected by immunofluorescence. Results: Higher ventricular tachycardia (VT) inducibility and lower ventricular fibrillation threshold (VFT) were observed in the AS group compared with those in the sham-operated group, associated with higher LSG activity and NE levels, increased number of M1 macrophages and secretion of inflammatory cytokines in ventricle (all P<0.001). Compared with the AS group, the SGA group had lower VT inducibility and higher VFT, combined with lower NE levels, and reduced number of M1 macrophages and secretion of inflammatory cytokines in ventricle (all P<0.001). Conclusion: LSG ablation could reduce VAs vulnerability after acute stroke by preventing the macrophages polarization and activation induced by sympathetic hyperactivity.

The Role of the Gut Microbiota in Coronary Heart Disease.

PURPOSE OF REVIEW: This review focuses on recent evidence examining the role gut microbiota play in coronary heart disease. It also provides a succinct overview of current and future therapies targeting the gut microbiota for coronary heart disease risk reduction. RECENT FINDINGS: A consensus has been reached that differences exist in the gut microbiotas of patients with coronary heart disease. Studies have shown that the gut microbiota is associated with obesity, diabetes, dyslipidemia, and hypertension, which are risk factors for coronary heart disease. The gut microbiota is involved in mediating basic metabolic processes, such as cholesterol metabolism, uric acid metabolism, oxidative stress, and inflammatory reactions, through its metabolites, which can induce the development of atherosclerosis and coronary heart disease. Interfering with the composition of gut microbiota, supplementing probiotics, and fecal donation are active areas of research to potentially prevent and treat coronary heart disease. Gut microbiota are causally associated with coronary heart disease. We analyzed the gut microbiota's effects on risk factors for coronary heart disease and studied the effects of gut microbiota metabolites on coronary heart disease. Gut microbiota is a potential target for preventing and treating coronary heart disease.

A highly specific probe for the imaging of inflammation-induced endogenous nitric oxide produced during the stroke process.

In this study, a turn-on two-photon fluorescent probe (Lyso-TP-NO) for nitric oxide (NO) was developed. It was synthesized using 4-ethylamino-1,8-naphthalimide as the two-photon fluorophore and N-methylaniline moiety as the reaction site. The probe and fluorophore were tested under one- and two-photon modes. The fluorescence intensity of the system was enhanced 23.1-fold after reacting with NO in the one-photon mode. However, the maximal two-photon action cross-section value of 200 GM was obtained under excitation at 840 nm. The probe exhibits high selectivity and sensitivity over other reactive oxygen species (ROS) and reactive nitrogen species (RNS), with a detection limit as low as 3.3 nM. The two-photon fluorescence imaging of living cells and mouse brain tissues can capture inflammation-induced endogenous NO production in lysosomes during stroke occurrence.

A pro-gastrin-releasing peptide imprinted photoelectrochemical sensor based on the in situ growth of gold nanoparticles on a MoS2 nanosheet surface.

Lamellar MoS2 nanosheets were successfully prepared by hydrothermal synthesis using 1-(3-mercaptopropyl)-3-methyl-imidazolium bromine (MIMBr) ionic liquid as a sulfur source and a morphology control agent, and sodium molybdate as a molybdenum source. Gold nanoparticles were assembled on the surface of MoS2 nanosheets by the in situ reduction of chloroauric acid at low temperatures to successfully fabricate AuNP/2D-MoS2 nanocomposites, thus improving photoelectrochemical response. AuNP/2D-MoS2 nanocomposites were used as photoelectrically active materials modified onto a glassy carbon electrode surface to construct a photoelectrochemical (PEC) sensor. Then, using 1-(N-pyrrolpropyl)-3-methyl-imidazolium bromine (PMIMBr) ionic liquid as a functional monomer and pro-gastrin-releasing peptide (Pro-GRP) as a template, a molecularly imprinted polymerized ionic liquid film was electrochemically deposited on an AuNP/2D-MoS2/GCE surface. Upon removing the templates, a molecularly imprinted photoelectrochemical sensor was constructed for the sensing of a tumor marker, pro-gastrin-releasing peptide. Experimental conditions including ascorbic acid concentration, polymerization conditions, incubation time, and pH value of the incubation solution have been optimized. Under the optimized conditions, the molecularly imprinted photoelectrochemical sensor can specifically detect the target protein Pro-GRP in the range of 0.02 ng mL-1-5 ng mL-1 with a detection limit of 0.0032 ng mL-1 (S/N = 3). The practicability of this photoelectrochemical sensor was demonstrated by accurately determining Pro-GRP in human serum samples.

NLRP3 inflammasome as a potential treatment in ischemic stroke concomitant with diabetes.

The NLRP3 (nucleotide-binding oligomerization domain-like receptor [NLR] family pyrin domain-containing 3) inflammasome is a member of the NLR family of innate immune cell sensors. These are crucial regulators of cytokine secretions, which promote ischemic cell death and insulin resistance. This review summarizes recent progress regarding the NLRP3 inflammasome as a potential treatment for ischemic stroke in patients with diabetes, two complicated diseases that often occur together. Stroke worsens glucose metabolism abnormalities, and the outcomes after stroke are more serious for diabetic patients compared with those without diabetes. Inflammation contributes to organ injury after ischemic stroke and diabetes. Recent research has focused on inhibiting the activation of inflammasomes and thus reducing the maturation of proinflammatory cytokines such as interleukin (IL)-1ß and IL-18. Studies suggest that inhibition of NLRP3 prevents or alleviates both ischemic stroke and diabetes. Targeting against the assembly and activity of the NLRP3 inflammasome is a potential and novel therapy for inflammasome-associated diseases, including ischemic stroke concomitant with diabetes.

MicroRNA-26b/PTEN Signaling Pathway Mediates Glycine-Induced Neuroprotection in SAH Injury.

Subarachnoid hemorrhage (SAH) is a form of stroke associated with high mortality and morbidity. Despite advances in treatment for SAH, the prognosis remains poor. We have previously demonstrated that glycine, a non-essential amino acid is involved in neuroprotection following intracerebral hemorrhage via the Phosphatase and tensin homolog (PTEN)/protein kinase B (AKT) signaling pathway. However, whether it has a role in inducing neuroprotection in SAH is not known. The present study was designed to investigate the role of glycine in SAH. In this study, we show that glycine can reduce brain edema and protect neurons in SAH via a novel pathway. Following a hemorrhagic episode, there is evidence of downregulation of S473 phosphorylation of AKT (p-AKT), and this can be reversed with glycine treatment. We also found that administration of glycine can reduce neuronal cell death in SAH by activating the AKT pathway. Glycine was shown to upregulate miRNA-26b, which led to PTEN downregulation followed by AKT activation, resulting in inhibition of neuronal death. Inhibition of miRNA-26b, PTEN or AKT activation suppressed the neuroprotective effects of glycine. Glycine treatment also suppressed SAH-induced M1 microglial polarization and thereby inflammation. Taken together, we conclude that glycine has neuroprotective effects in SAH and is mediated by the miRNA-26b/PTEN/AKT signaling pathway, which may be a therapeutic target for treatment of SAH injury.

Newly Detected Atrial Fibrillation after Acute Stroke: A Narrative Review of Causes and Implications.

Cardiac arrhythmias occur frequently in patients with acute stroke, with atrial fibrillation (AF) being the most common. Newly detected AF may lead to increased risk of ischemic stroke, which in turn generates stroke recurrence and adverse outcomes. Currently, most studies are focusing on the role of AF in ischemic stroke and attributing cryptogenic ischemic stroke to previously undetected AF. However, in these studies, subjects used to have neither symptoms of palpitation nor evidence of AF. A better understanding of this association will contribute to the management and therapy for patients after clinical decisions regarding stroke patients. Currently, the definition of newly detected AF has not come to an agreement, and the pathophysiology remains incompletely understood, possibly involving complex alterations in both the autonomic network and humoral regulation. Therefore, this review aims to introduce the definition and epidemiology of newly detected AF after stroke with updated information and elucidate the potential pathophysi-ology, such as autonomic imbalance, catecholamine surge, poststroke systematic inflammation, and microvesicles and microRNAs.

Role of Myeloid Lineage Cell Autophagy in Ischemic Brain Injury.

BACKGROUND AND PURPOSE: Inflammatory cells play a significant role in secondary injury after ischemic stroke. Recent studies have suggested that a lack of autophagy in myeloid cells causes augmented proinflammatory cytokine release and prolonged inflammation after tissue injury. In this study, we investigated the roles of myeloid cell autophagy in ischemic brain injury. METHODS: Focal cerebral ischemia was induced via transient middle cerebral artery occlusion in mice with autophagy-deficient myeloid lineage cells (Atg5flox/flox LysMCre+) and in their littermate controls (Atg5flox/flox). Infarct volume, neurological function, inflammatory cell infiltration, and proinflammatory cytokine expression levels were evaluated. RESULTS: Mice lacking autophagy in myeloid lineage cells had a lower survival rate for 14 days than control mice (20% versus 70%; P<0.05). Although there was no difference in infarct volume at 12 hours between the 2 groups, mice lacking autophagy in myeloid lineage cells had larger infarct volumes at later time points (3 and 7 days after reperfusion) with worse neurological deficit scores and lower grip test scores. There were a higher number of ionized calcium binding adaptor molecule 1-positive cells and cells expressing M1 marker CD16/32 in mice lacking autophagy in myeloid cells at the later time points. Moreover, these mice had higher expression levels of proinflammatory cytokines at later time points; however, there was no difference in ionized calcium binding adaptor molecule 1-positive cells or mRNA levels of proinflammatory cytokines at the earlier time point (12 hours after reperfusion). CONCLUSIONS: These data suggest that the lack of myeloid cell autophagy aggravates secondary injury by augmenting and prolonging inflammation after ischemic stroke without affecting the initial injury.

Engineering chimeric antigen receptor-T cells for cancer treatment.

Intratumor heterogeneity of tumor clones and an immunosuppressive microenvironment in cancer ecosystems contribute to inherent difficulties for tumor treatment. Recently, chimeric antigen receptor (CAR) T-cell therapy has been successfully applied in the treatment of B-cell malignancies, underscoring its great potential in antitumor therapy. However, functional challenges of CAR-T cell therapy, especially in solid tumors, remain. Here, we describe cancer-immunity phenotypes from a clonal-stromal-immune perspective and elucidate mechanisms of T-cell exhaustion that contribute to tumor immune evasion. Then we assess the functional challenges of CAR-T cell therapy, including cell trafficking and infiltration, targeted-recognition and killing of tumor cells, T-cell proliferation and persistence, immunosuppressive microenvironment and self-control regulation. Finally, we delineate tumor precision informatics and advancements in engineered CAR-T cells to counteract inherent challenges of the CAR-T cell therapy, either alone or in combination with traditional therapeutics, and highlight the therapeutic potential of this approach in future tumor precision treatment.

Bidirectional gut-brain-microbiota axis as a potential link between inflammatory bowel disease and ischemic stroke.

Emerging evidence suggests that gut-brain-microbiota axis (GBMAx) may play a pivotal role linking gastrointestinal and neuronal disease. In this review, we summarize the latest advances in studies of GBMAx in inflammatory bowel disease (IBD) and ischemic stroke. A more thorough understanding of the GBMAx could advance our knowledge about the pathophysiology of IBD and ischemic stroke and help to identify novel therapeutic targets via modulation of the GBMAx.

The Neuroprotective Roles of Sonic Hedgehog Signaling Pathway in Ischemic Stroke.

Ischemic stroke is characterized by high morbidity, mortality and disability rate worldwide. Because of its complexity in pathogenesis and lack of effective therapeutic strategies and drugs, great breakthrough has not yet been made in the treatment of cerebral ischemic stroke. Therefore, to explore a more effective and safer therapeutic strategy for cerebral ischemic stroke has been the focus of numerous researchers. Neuroprotective effects of sonic hedgehog (Shh) signaling pathway in ischemic stroke have been reported in recent studies, but have not been fully elucidated. In our review, we elaborate the roles of Shh signaling in ischemic stroke from different aspects, including oxidative stress, excitotoxicity, neuroinflammation, apoptosis, angiogenesis, neuroplasticity, neurogenesis, astrogliosis and oligodendrogenesis. Meanwhile, Shh signaling based therapeutic approaches for cerebral ischemic stroke are also included in our review. We hope it will benefit the readers to better understand the roles of Shh signaling pathway in cerebral ischemic stroke and provide more comprehensive insights for basic research and novel strategies for the clinical treatment of cerebral ischemic stroke.

Inhibition of miR-181a protects female mice from transient focal cerebral ischemia by targeting astrocyte estrogen receptor-α.

Whether the effect of miR-181a is sexually dimorphic in stroke is unknown. Prior work showed protection of male mice with miR-181a inhibition. Estrogen receptor-α (ERα) is an identified target of miR181 in endometrium. Therefore we investigated the separate and joint effects of miR-181a inhibition and 17ß-estradiol (E2) replacement after ovariectomy. Adult female mice were ovariectomized and implanted with an E2- or vehicle-containing capsule for 14d prior to 1h middle cerebral artery occlusion (MCAO). Each group received either miR-181a antagomir or mismatch control by intracerebroventricular injection 24h before MCAO. After MCAO neurologic deficit and infarct volume were assessed. Primary male and female astrocyte cultures were subjected to glucose deprivation with miR-181a inhibitor or transfection control, and E2 or vehicle control, with/without ESRα knockdown with small interfering RNA. Cell death was assessed by propidium iodide staining, and lactate dehydrogenase assay. A miR-181a/ERα target site blocker (TSB), with/without miR-181a mimic, was used to confirm targeting of ERα by miR-181a in astrocytes. Individually, miR-181a inhibition or E2 decreased infarct volume and improved neurologic score in female mice, and protected male and female astrocyte cultures. Combined miR-181a inhibition plus E2 afforded greater protection of female mice and female astrocyte cultures, but not in male astrocyte cultures. MiR-181a inhibition only increased ERα levels in vivo and in female cultures, while ERα knockdown with siRNA increased cell death in both sexes. Treatment with ERα TSB was strongly protective in both sexes. In conclusion, the results of the present study suggest miR-181a inhibition enhances E2-mediated stroke protection in females in part by augmenting ERα production, a mechanism detected in female mice and female astrocytes. Sex differences were observed with combined miR-181a inhibition/E2 treatment, and miR-181a targeting of ERα.