Inhibition of microRNA-210 suppresses pro-inflammatory response and reduces acute brain injury of ischemic stroke in mice

doi:10.1016/j.expneurol.2017.10.024

Experimental Neurology

Volume 300, February 2018, Pages 41-50

https://doi.org/10.1016/j.expneurol.2017.10.024 Get rights and content

Highlights

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Inhibition of miR-210 reduced stroke-induced cerebral infarction and edema.
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Inhibition of miR-210 ameliorated the behavioral deficits after stroke.
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Inhibition of miR-210 suppressed post-stroke inflammatory reaction.
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MiR-210 inhibitor posttreatment reduced infarction and behavioral deficits.
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MiR-210 inhibitor posttreatment improved long-term behavioral recovery after stroke.

Abstract

Stroke is a leading cause of mortality and chronic neurologic disability. Yet, the successful treatment remains limited. In this study, we investigated the efficacy and the mechanism of a novel treatment, microRNA-210 (miR-210) inhibition, in protecting acute ischemic brain injury in adult mice. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in adult male C57BL/6 mice. MiR-210-LNA (miR-210 inhibitor) or the negative control was administered via intracerebroventricular injection 24 h prior or 4 h after MCAO. Cerebral infarction volume and behavioral deficits were determined 48 h after MCAO. The expression of inflammation-related genes and infiltration/activation of various immune cells in the brain were assessed by RT-qPCR, flow cytometry, and immunohistochemistry. Acute ischemic stroke significantly increased miR-210 levels in the brain, which was abolished by miR-210-LNA administered prior to MCAO. Pre- and post-MCAO treatments with miR-210-LNA significantly decreased cerebral infarction and ameliorated behavioral deficits induced by MCAO. Long-term behavioral recovery was also improved by miR-210-LNA post-treatment. At the same time, inhibition of miR-210 significantly reduced the expression of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (CCL2 and CCL3), but had no significant effect on anti-inflammatory factors (TGF-β and IL-10). In addition, MCAO-induced macrophage infiltration and microglial activation in the brain were inhibited by the miR-210-LNA treatment. In summary, inhibition of miR-210 suppresses pro-inflammatory response and reduces brain damage in the acute phase of ischemic stroke, providing new insight in molecular basis of a novel therapeutic strategy of miR-210 inhibition in the treatment of acute ischemic stroke.

Introduction

Stroke is the leading cause of adult chronic disability and the fifth leading cause of death in the United States (Benjamin et al., 2017). About $36.5 billion are spent due to stroke every year (Benjamin et al., 2017), and this cost will be steadily rising over the next 40 years (Howard and Goff, 2012). Unfortunately, our ability to treat stroke remains extremely limited. Thus, it has been urgent to explore novel and effective treatment strategies for stroke.

Growing evidence has been reported that recently discovered microRNAs (miRNAs) are essential mediators in neural development and diverse neurological diseases (Fiore et al., 2008), including ischemic stroke (Jeyaseelan et al., 2008, Rink and Khanna, 2011). MiRNAs are a class of non-coding RNAs, participating in post-transcriptional gene regulation. Mature miRNAs are single-stranded nucleotides of ~ 21–22 in length and can bind to 3′-untranslated region (3′UTR) of target mRNAs, leading to translation repression and mRNA degradation (Boyd, 2008, Filipowicz et al., 2008). A group of miRNAs are upregulated by hypoxia, termed 'hypoxamirs' (Chan and Loscalzo, 2010). It is possible that hypoxamirs are of importance in stroke, as hypoxia is a crucial pathogenetic element of ischemic stroke that caused by occlusion of cerebral blood supply. Among hypoxamirs, miR-210 is a 'master hypoaxmir' that is robustly induced by hypoxia in all species including humans and all cell types investigated up to date (Kulshreshtha et al., 2007, Pulkkinen et al., 2008).

Few studies reported the role of miR-210 in ischemic stroke and the results were somewhat controversial. Brain miR-210 levels were shown to be upregulated at 24 h and downregulated at 48 h in transient ischemic stroke rats (Jeyaseelan et al., 2008). Another study, using the same stroke model, reported that post-stroke brain cortex miR-210 levels continually increased to 7 days (Lou et al., 2012). In addition, it was demonstrated that upregulation of miR-210 induced pro-apoptotic gene caspase 3 expression and increased endothelial cell apoptosis (Chan et al., 2009). In contrast, miR-210 overexpression appeared anti-apoptotic in PC12 cells and neural progenitor cells under oxygen-glucose deprivation (Chio et al., 2013) or hypoxia alone (Wang et al., 2013). These studies imply that the role of miR-210 may vary in different cell types and treatments. Nonetheless, the cause and effect role of miR-210 in brain injury in vivo in the setting of acute ischemic stroke remains undetermined.

Our previous study indicated that inhibition of miR-210 provided a neuroprotective effect in a neonatal rat hypoxic-ischemic encephalopathy (HIE) model (Ma et al., 2016). However, the effect of miR-210 inhibition on the outcome of adult ischemic stroke, especially in the acute phase, remains unclear. Herein, we provide evidence that inhibition of miR-210 suppresses pro-inflammatory response and reduces ischemic brain injury in the acute phase of stroke in adult mice, suggesting a novel therapeutic strategy of the miR-210 blockade in the treatment of acute ischemic stroke.

Section snippets

Experimental animals and group allocation

Total 85 eight-week-old male C57BL/6 mice were purchased from Charles River Laboratories (Portage, MI). Animals were maintained at 20 ± 2 °C and housed in a 12-h light-dark cycle with access to food and water ad libitum. Mice were randomly divided into 2 major groups and 7 subgroups using a sequence of computer-generated random numbers. Two major groups include 1) negative control (n = 41); and 2) miR-210-LNA treatment (n = 44). Experimental subgroups were listed as followed: Group 1: infarction and

MiR-210-LNA pretreatment reduced brain infarct volume and brain edema in mice with MCAO

We first measured brain miR-210 levels after MCAO in both negative control and miR-210-LNA pretreated groups. As shown in Fig. 1A, compared to the contralateral hemisphere, MCAO significantly increased miR-210 levels in the ipsilateral hemisphere in the control group, which was blocked by the miR-210-LNA treatment. Brain infarction and correlated edema were evaluated by MRI T2 weighted image 48 h after stroke. Compared to the negative control group, the infarct volume was significantly reduced

Discussion

The present study demonstrated that inhibition of miR-210 by miR-210-LNA significantly reduces brain damage and ameliorates behavioral deficits in the acute phase of ischemic stroke. In addition, miR-210 inhibition decreased post-ischemic inflammation response. Of importance, the finding that miR-210-LNA posttreatment 4 h after stroke confers neuroprotection similar to that seen in the pretreatment suggests that silencing miR-210 may present as a novel and potential therapeutic intervention in

Conclusion

The present investigation identifies a novel cause-and-effect mechanism of miR-210 in regulating pro-inflammatory response and brain injury in the acute phase of ischemic stroke in a mouse model. Although it may be difficult to translate directly these findings into humans, several lines of evidence support the significance of the present study in clinical implications. Thus, mature miR-210 is identical between humans and mice, and hypoxia increases miR-210 in cells and tissues of all species

Acknowledgements

A portion of this research used the Loma Linda University School of Medicine Advanced Imaging and Microscopy Core, a facility is supported in part by the National Science Foundation through the Major Research Instrumentation program of the Division of Biological Infrastructure Grant No. 0923559 and the Loma Linda University School of Medicine.

Sources of funding

This work was supported in part by the National Institutes of Health grants HL118861 (LZ) and NS103017 (LZ).

Disclosures

None.

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Research PaperInhibition of microRNA-210 suppresses pro-inflammatory response and reduces acute brain injury of ischemic stroke in mice

Highlights

Abstract

Introduction

Section snippets

Experimental animals and group allocation

MiR-210-LNA pretreatment reduced brain infarct volume and brain edema in mice with MCAO

Discussion

Conclusion

Acknowledgements

Sources of funding

Disclosures

Lab. Investig.

Cell Metab.

Biochim. Biophys. Acta

Mol. Ther.–Nucleic Acids

Neurobiol. Dis.

FEBS Lett.

Antivir. Res.

Stem Cell Res.

Heart disease and stroke statistics-2017 update: a report from the American Heart Association

Circulation

MicroRNA-210: a unique and pleiotropic hypoxamir

Cell Cycle

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

Arch. Toxicol.

miR-210: more than a silent player in hypoxia

IUBMB Life

Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?

Nat. Rev. Genet.

Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4 + T cells compared

J. Immunol.

Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice

Science

Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation

Stroke

Temporal and spatial dynamics of cerebral immune cell accumulation in stroke

Stroke

Noninvasive quantification of brain edema and the space-occupying effect in rat stroke models using magnetic resonance imaging

Stroke

Research Paper
Inhibition of microRNA-210 suppresses pro-inflammatory response and reduces acute brain injury of ischemic stroke in mice