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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 17β-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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Neurons in the cornu ammonis 1 (CA1) region of the hippocampus are vulnerable to cerebral ischemia, while dentate gyrus (DG) neurons are more resistant. This effect is mediated by local astrocytes, and may reflect differences in subregional hippocampal expression of miR-29a. We investigated the role of miR-29a on survival of hippocampal astrocytes cultured selectively from CA1 and DG in response to glucose deprivation (GD). CA1 astrocytes exhibited more cell death and a greater decrease in miR-29a than DG astrocytes. A reciprocal change was observed in the mitochondrial voltage dependent cation channel-1 (VDAC1), a regulator of mitochondria and target of miR-29a. In CA1 astrocytes, increasing miR-29a decreased VDAC1 and improved cell survival, while knockdown of VDAC1 improved survival. Finally, the protective effect of miR-29a was eliminated by inhibition of miR-29a/VDAC1 binding. These findings suggest that the selective vulnerability of the CA1 to injury may be due in part to a limited miR-29a response in CA1 astrocytes, allowing a greater increase in VDAC1-mediated cellular dysfunction in CA1 astrocytes., (Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Background: Isoflurane induces cell death in neurons undergoing synaptogenesis via increased production of pro-brain-derived neurotrophic factor (proBDNF) and activation of postsynaptic p75 neurotrophin receptor (p75). Astrocytes express p75, but their role in neuronal p75-mediated cell death remains unclear. The authors investigated whether astrocytes have the capacity to buffer increases in proBDNF and protect against isoflurane/p75 neurotoxicity., Methods: Cell death was assessed in day in vitro (DIV) 7 mouse primary neuronal cultures alone or in co-culture with age-matched or DIV 21 astrocytes with propidium iodide 24 h after 1 h exposure to 2% isoflurane or recombinant proBDNF. Astrocyte-targeted knockdown of p75 in co-culture was achieved with small-interfering RNA and astrocyte-specific transfection reagent and verified with immunofluorescence microscopy. proBDNF levels were assessed by enzyme-linked immunosorbent assay. Each experiment used six to eight replicate cultures/condition and was repeated at least three times., Results: Exposure to isoflurane significantly (P < 0.05) increased neuronal cell death in primary neuronal cultures (1.5 ± 0.7 fold, mean ± SD) but not in co-culture with DIV 7 (1.0 ± 0.5 fold) or DIV 21 astrocytes (1.2 ± 1.2 fold). Exogenous proBDNF dose dependently induced neuronal cell death in both primary neuronal and co-cultures, an effect enhanced by astrocyte p75 inhibition. Astrocyte-targeted p75 knockdown in co-cultures increased media proBDNF (1.2 ± 0.1 fold) and augmented isoflurane-induced neuronal cell death (3.8 ± 3.1 fold)., Conclusions: The presence of astrocytes provides protection to growing neurons by buffering increased levels of proBDNF induced by isoflurane. These findings may hold clinical significance for the neonatal and injured brain where increased levels of proBDNF impair neurogenesis.

Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only proapoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury., (Copyright © 2013 Wiley Periodicals, Inc.)

Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+) astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.

Ischemic stroke remains a devastating cerebrovascular disease that accounts for a high proportion of mortality and disability worldwide. MicroRNAs (miRNAs) are a class of small non-coding RNAs that are responsible for regulation of post-transcriptional gene expression, and growing evidence supports a role for miRNAs in stroke injury and recovery. The current study examined the role of miR-182 in experimental stroke using both in vitro and in vivo models of ischemic injury. Brain levels of miR-182 significantly increased after transient middle cerebral artery occlusion (MCAO) in mice and in primary astrocyte cultures subjected to combined oxygen–glucose deprivation/reperfusion (OGD/R) injury. In vivo, stroke volume and neurological score were significantly improved by pre-treatment with miR-182 antagomir. Astrocyte cultures stressed with OGD/R resulted in mitochondrial fragmentation and downregulation of cortactin, an actin-binding protein. Inhibition of miR-182 significantly preserved cortactin expression, reduced mitochondrial fragmentation and improved astrocyte survival after OGD/R. In parallel, lipopolysaccharide (LPS)-induced nitric-oxide release in astrocyte cultures was significantly reduced by miR-182 inhibition, translating to reduced injury in primary neuronal cultures subjected to conditioned medium from LPS-treated astrocytes. These findings identify miR-182 and/or cortactin as potential clinical targets to preserve mitochondrial structure and mitigate neuroinflammation and cell death after ischemic stroke. [ABSTRACT FROM AUTHOR]