Elsevier

Experimental Neurology

Volume 264, February 2015, Pages 1-7
Experimental Neurology

Regular Article
Post-stroke treatment with miR-181 antagomir reduces injury and improves long-term behavioral recovery in mice after focal cerebral ischemia

https://doi.org/10.1016/j.expneurol.2014.11.007Get rights and content

Highlights

  • Post-stroke treatment with miR-181a antagomir reduces infarction size.

  • Post-stroke treatment with miR-181a antagomir reduces long term neurobehavioral deficits.

  • Both ICV and IV administrations are effective.

  • Post-stroke treatment with miR-181a antagomir reduces inflammation.

  • miR-181a antagomir administered after stroke onset targets BCL2 and XIAP.

Abstract

miR-181 has deleterious effects on stroke outcome, and reducing miR-181a levels prior to middle cerebral artery occlusion (MCAO) was shown previously to be protective. Here we tested the effect of post-ischemic treatment with miR-181a antagomir by intracerebroventricular and intravenous routes of administration on infarct size, neurological outcome, inflammatory response and long term behavioral outcome. Post-treatment with miR-181a antagomir significantly reduced infarction size, improved neurological deficits and reduced NF-κB activation, numbers of infiltrating leukocytes and levels of Iba1. Targets affected by miR-181a antagomir administered after stroke onset include BCL2 and X-linked inhibitor of apoptosis protein (XIAP). Post-treatment with miR-181a antagomir significantly improved behavioral outcome assessed by rotarod at one month. These findings indicate that post-treatment with miR-181a antagomir has neuroprotective effects against ischemic neuronal damage and neurological impairment in mice, and the protection is long lasting including recovery of motor function and coordination over one month. The ability to protect the brain with post-treatment with miR-181a antagomir with long lasting effect makes this a promising therapeutic target and may be an innovative and effective new approach for stroke therapy.

Introduction

Stroke is one of the leading causes of death worldwide and the leading cause of long-term neurological disability. Although many clinical stroke trials have been completed, the only clinically efficacious treatment to date is thrombolysis (Blakeley and Llinas, 2007). Suggested reasons for the many failures include the complex interplay among multiple signaling pathways and intracellular organelles, interaction between different cell types, and the potentially short therapeutic window for neuroprotection after stroke.

MicroRNAs (miRs) are a class of small, non-coding RNAs. Mature miRs are generated from primary miR transcripts by sequential endonucleolytic processing and act as posttranscriptional regulators of gene expression including in the setting of cerebral ischemia (for review see Ouyang et al. (2013)). Many miRs exist in families. The miR-181 family contains four highly conserved members, miR-181a, miR-181b, miR-181c and miR-181d, which are derived independently from 6 precursors located on 3 chromosomes as identified using TargetScan (http://targetscan.org).

Our previous study reported increased injury with increased levels of miR-181 and a protective effect of reducing miR-181a levels using antagomir when administered the day prior to middle cerebral artery occlusion (MCAO) in male mice (Ouyang et al., 2012b). Reduced levels of miR-181 were associated with reduced oxidative stress in in vitro ischemia (Ouyang et al., 2012a). Previously validated targets of miR-181 include the ER stress protein GRP78 (Ouyang et al., 2012b) and three antiapoptotic proteins, BCL2, MCL1 (Ouyang et al., 2012a) and XIAP (Hutchison et al., 2013). While treatment before stroke provides evidence that anti-miR-181 can protect in acute stroke, treatment after stroke onset will be needed in most cases, as patients often present hours after stroke onset. Thus to assess potential translational relevance we tested the effect of post-ischemic treatment with miR-181a antagomir administered by intracerebroventricular (ICV) or intravenous (IV) injection in a transient focal cerebral ischemia model. We assessed both short term and long term outcomes with post-treatment, including rotarod neurobehavioral assessment.

Section snippets

miRNA-181a antagomir

miRNA-181a antagomir and a negative control (mismatched (MM)-miR-181a antagomir) were from Thermo Scientific (Hudson, New Hampshire, USA) and the sequences are:AntagomirmiR181aMAGWA000005mA.*.mC.*.mU.mC.mA.mC.mC.mG.mA.mC.mA.mG.mC.mG.mU.mU.mG.mA.mA.mU.*.mG.*.mU.*.mU.*.3ChlMMAntagomirmiR181aMAGWA00006mA.*.mG.*.mU.mC.mA.mG.mC.mG.mA.mG.mA.mG.mC.mC.mU.mU.mG.mA.mU.mU.*.mG.*.mU.*.mU.*.3ChlmN=2OMethylnucleotideN=AorCorGorU;*=phosphorothioatelinkage.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

RT-qPCR for miRNA quantitation in the

Post-stroke ICV treatment with miR-181a antagomir reduces infarct volume and reduces acute neurological deficit

Antagomir treatment reduced miR-181a in the brain to very low levels 24 h later by quantitative RT-PCR analysis (Fig. 2A). Using the same dose of miR-181a antagomir as used previously for pretreatment (Ouyang et al., 2012b), antagomir or MM was administered ICV 2 h after MCAO. The infarction size after miR-181a antagomir treatment (31.9 ± 3.6%) compared to MM control group (49.4 ± 2.3%) was reduced at about 35% (p < 0.05) at 48 h reperfusion (Fig. 2B) and the neurologic deficit at 24 h was significantly

Discussion

The main findings in this study are (1) post-stroke treatment with miR-181a antagomir has neuroprotective effects against ischemic neuronal damage, and improves long-term neurobehavioral recovery in mice; (2) both ICV and IV administrations were effective; (3) miR-181a antagomir administered shortly after stroke reduces evidence of inflammation and (4) miR-181a antagomir increases levels of BCL2 and XIAP but not GRP78 when given during reperfusion.

Previous studies have reported that miR-181a

Sources of funding

Grant sponsor: NIH; grant numbers: NS053898 and NS084396.

Disclosures

None.

Acknowledgments

The authors thank Siwei Liu for the help with Westerns, Jeong-Mi Moon for the help with RT-PCR, and Elysa Hall and William Magruder for the help in preparing the manuscript.

References (24)

  • G. Chen

    MicroRNA-181a sensitizes human malignant glioma U87MG cells to radiation by targeting Bcl-2

    Oncol. Rep.

    (2010)
  • W.D. Dietrich

    Intraischemic but not postischemic brain hypothermia protects chronically following global forebrain ischemia in rats

    J. Cereb. Blood Flow Metab.

    (1993)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    View full text