Original Contributions

Genome-Wide Analysis of the Circulating miRNome After Cerebral Ischemia Reveals a Reperfusion-Induced MicroRNA Cluster

Stefan Uhlmann, Eva Mracsko, Ehsan Javidi, Sarah Lamble, Ana Teixeira, Agnes Hotz-Wagenblatt, Karl-Heinz Glatting, Roland Veltkamp
https://doi.org/10.1161/STROKEAHA.116.013942
Stroke. 2017;48:762-769
Originally published February 13, 2017

Abstract

Background and Purpose—Circulating microRNAs (miRNAs) are emerging biomarkers for stroke because of their high stability in the bloodstream and association with pathophysiologic conditions. However, the circulating whole-genome miRNAs (miRNome) has not been characterized comprehensively in the acute phase of stroke.

Methods—We profiled the circulating miRNome in mouse models of acute ischemic and hemorrhagic stroke by next-generation sequencing. Stroke models were compared with sham-operated and naive mice to identify deregulated circulating miRNAs. Top-ranked miRNAs were validated and further characterized by quantitative reverse transcription polymerase chain reaction.

Results—We discovered 24 circulating miRNAs with an altered abundance in the circulation 3 hours after ischemia, whereas the circulating miRNome was not altered after intracerebral hemorrhage compared with sham-operated mice. Among the upregulated miRNAs in ischemia, the top-listed miR-1264/1298/448 cluster was strongly dependent on reperfusion in different ischemia models. A time course experiment revealed that the miR-1264/1298/448 cluster peaked in the circulation around 3 hours after reperfusion and gradually decreased thereafter.

Conclusions—Alteration of the miRNome in the circulation is associated with cerebral ischemia/reperfusion, but not hemorrhage, suggesting a potential to serve as biomarkers for reperfusion in the acute phase. The pathophysiological role of reperfusion-inducible miR-1264/1298/448 cluster, which is located on chromosome X within the introns of the serotonin receptor HTR2C, requires further investigation.

Introduction

Micro-RNAs (miRNAs) are a family of small noncoding RNAs with an important role as post-transcriptional regulators of gene expression in many biological processes and diseases,1,2 including stroke.3–8 In addition to cerebral expression, miRNAs circulate in the blood where they are remarkably stable against degradation.9,10 These circulating miRNAs may serve as biomarkers because they can also be amplified and readily quantified in a clinical setting and are expressed earlier than proteins. Using circulating miRNAs as a tool for diagnostic and prognostic assessment and even as potential therapeutic targets for stroke is a rapidly evolving field.11 Previous clinical12–15 and experimental16,17 studies focused mainly on delayed phases after stroke and thus the pattern of circulating miRNAs in the first hours after different types of stroke has not been explored. Moreover, none of these experimental studies investigated the circulating whole-genome miRNAs (miRNome) in mouse models of stroke to distinguish between ischemic and hemorrhagic stroke in the early phase.

We analyzed the circulating miRNome in the acute phase of experimental mouse models of ischemic stroke (IS) and intracerebral hemorrhage (ICH).

Methods

Animals

The study was conducted in accordance with national guidelines for the use of experimental animals. All experimental procedures were approved by the governmental committees (animal care committee, Regierungspräsidium Karlsruhe, Germany) and within the German Animal Welfare Act and were performed according to the ARRIVE (Animal Research: Reporting in Vivo Experiments) guidelines. Age-matched (8- to12-week) male C57BL/6 mice obtained from Janvier Laboratories (France) were used for the experiments. In a single cage, 4 specific pathogen-free mice were housed in the animal facility of the University of Heidelberg. All mice were kept on a standard 12-hour light/dark cycle and had free access to food and water.

Animal Numbers

In this exploratory study, we randomly assigned a total of 25 mice into 5 groups (as shown in Figure I in the online-only Data Supplement) to deep sequence the circulating miRNome: (1) middle cerebral artery occlusion (MCAO; n=5); (2) MCAO-sham (n=5); (3) ICH (n=5); (4) ICH-sham (n=5); and (5) naive mice (n=5) to evaluate the sham surgery for artificially deregulated miRNAs caused by the surgical procedure. Afterward, the results after IS were validated with a total amount of 15 animals and assigned into 3 groups: (1) MCAO (n=5); (2) MCAO-sham (n=5); and (3) naive mice (n=5). To evaluate the effect of ischemia duration on induction of the miR-1264/1298/448 cluster, the mice underwent: (1) 30-minute MCAO (n=3); (2) 90-minute MCAO (n=3); (3) sham surgery (n=3); and (4) no surgery (n=3). Time course experiments to evaluate the kinetics of the circulating miRNA cluster after MCAO after reperfusion (0 minutes [n=3], 30 minutes [n=3], 150 minutes [n=3], 300 minutes [n=3], 720 minutes [n=3], and 1440 minutes [n=3]) were compared with sham surgery (n=3). To determine the dependence of the miRNA cluster on reperfusion, we measured the abundance of the miRNA cluster in a permanent intraluminal occlusion model (n=3) and compared this group with transient MCAO (n=3) and sham surgery (n=3). In addition, electrocoagulated mice (n=5) were compared with sham-operated mice (n=5).

Filament Model

In the filament-induced ischemia experiments, we conducted a transient focal cerebral ischemia for 30 or 90 minutes, as previously described.18 Briefly, mice were anesthetized with 2% isoflurane in O2, and left-sided MCAO was performed by advancing a nylon-coated monofilament with a diameter of 0.22 mm (Doccol, MA, USA) through the left common carotid artery. For laser Doppler measurements, we placed the probe (P403; Perimed, Sweden) 3 mm lateral and 1 mm posterior to the bregma and obtained relative perfusion units (Periflux4001; Perimed). Only animals in which relative cerebral blood flow dropped <25% of preischemic baseline after MCAO were included in the analysis. Afterward, mice were reanesthetized and the filament was removed after 30 minutes or 90 minutes of ischemia. During surgery, a body temperature of 37°C was feedback controlled by using a heating pad. The preparation for the sham surgery was identical, but the filament was not inserted into the vessel. All mice received carprofen subcutaneously at 5.0 mg/kg as an analgesic for postoperative pain.

Coagulation Model

In some experiments, we induced permanent focal cerebral ischemia by coagulation of the left middle cerebral artery distal from the origin of the lenticulostriate arteries as previously described.19 Briefly, after making a 1-cm skin incision between the left eye and the ear, the temporal muscle was removed and a hole was drilled through the temporal skull. The dura mater was removed, and the middle cerebral artery was occluded permanently using a bipolar electrocoagulation forceps (Erbotom, Erbe, Germany). Sham operation was identical to the operation described above except for the coagulation of the exposed artery.

ICH Model

To simulate ICH in mice, we used the blood infusion model as previously described.20 Briefly, after placing the anesthetized animals in a stereotactic frame with a mouse adaptor (Model 51625; Stoelting, USA), and a borehole was drilled at 2.2 mm left and 0.2 mm anterior to the bregma. Using an infusion system containing a 26-gauge needle connected to a 50-μL microsyringe (Hamilton, Switzerland), 20 μL of autologous blood was infused into the striatum at a depth of 3.7 mm. Sham operation was conducted by the same procedure and duration including placement of the microneedle but without injection of fluid.

RNA Extraction

The blood was collected from the retrobulbar plexus after complete anesthesia of mice by intraperitoneal injection of ketamine/xylazine (120 and 16 mg/kg) and euthanized thereafter. In addition, brains were removed as well, immediately frozen and 20-µm–thick coronal cryosections were cut every 400 µm and stained with 6cresyl violet in case of MCAO. Blood was incubated for 30 minutes at room temperature to allow clotting. Blood serum was isolated by spinning at 600g for 15 minutes at 15°C. The serum was additionally spun at 16 000g for 10 minutes at 10°C to remove debris and residual cells. Afterward the samples were stored at −80°C until assayed. To exclude hemolytic samples, we assessed the absorbance at λ=414 nm by NanoDrop. All samples were excluded from the analysis with an absorbance value >0.1 as a cutoff to distinguish hemolyzed and nonhemolyzed serum.21 miRNAs were extracted with the miRNeasy Micro Kit in case of samples used for miRNA-seq or miRNeasy Mini Kit (both from Qiagen, Germany) according to the manufacturer’s instructions after lysis and homogenization in TRIzol from 200 µL serum. Synthetic Caenorhabditis elegans miRNAs (cel-miR-39) containing 1.6×108 copies/μL were supplemented to the serum before starting the RNA isolation procedure. These spiked-in RNAs served as an exogenous control to monitor the efficiency of RNA extraction, cDNA synthesis, and quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Quantitative RT-PCR

Measurement of the miRNA expression was performed with TaqMan gene-specific MicroRNA Assays on the ABI Prism 7900HT from Applied Biosystems (Darmstadt, Germany). The TaqMan MicroRNA RT Kit from Applied Biosystems was used to reverse transcript mature miRNAs to cDNA. The TaqMan qRT-PCR was performed in triplicates for each miRNA in 96-well plates. Data were normalized to the exogenous control (cel-miR-39) and analyzed according to the ΔΔCT method.22

cDNA Library Preparation, Sequencing, and Data Analyses

Small RNA sequencing libraries were prepared using the NEBNext Multiplex Small RNA kit according to the manufacturer’s instructions with a fixed volume of 6 µL of small RNAs rather than an equal RNA concentration. Each of the 25 libraries was prepared with a unique barcode that allows to pool the samples into one flow cell (composed of 2 lanes) for sequencing. The amplified barcoded cDNA libraries underwent a size selection at ≈140 bp (corresponding to adaptor-ligated miRNAs) using a 6% polyacrylamide gel and then subjected to 50-bp paired-end sequencing on an Illumina HiSeq 2500 platform. We used an in-house computer pipeline in the HUSAR system23 with our curated noncoding RNA database to remove sequencing adapters, align to the mouse genome (mouse38) with bowtie, permitting one mismatch between the sequencing reads, and assign miRNA annotations to the reads. To annotate miRNAs, we used a combination of publicly available databanks, including miRBase24 (http://www.mirbase.org), Ensembl25 (http://www.ensembl.org), and Rfam26 (http://rfam.xfam.org). Reads shorter than 15 nucleotides and longer than 35 nucleotides were filtered, given that these were unlikely to map to mature miRNAs.

miRNA Target Prediction

Conserved mRNA targets among vertebrates of the identified miRNA were determined using target prediction databases TargetScanMouse release 6.227 (http://www.targetscan.org) with default parameters. The DAVID gene annotation tool28 (http://david.abcc.ncifcrf.gov/tools.jsp) was used with default parameters to provide further information about the corresponding functions of the genes.

Statistical Analyses

All analyses were performed by an investigator blinded to the group assignments. Statistical significance was determined by unpaired, 2-tailed Student t test for comparison between 2 groups, and ANOVA for multiple comparisons among 3 or more groups with post hoc Tukey test. The accepted significance level was ***P<0.001, **P<0.01, and *P<0.05 in all kinds of statistical comparisons in our study. The comparison of the miRNA read counts were done by DESeq229 and with the normalization offered by these tools.

Results

Profiling of the Circulating miRNome in the Acute Phase of Stroke

To investigate the circulating miRNome in the first hours after the onset of stroke, we used 2 well-characterized mouse models of IS and ICH (Figure 1A, upper) and measured the abundance of miRNAs in the blood serum by next-generation sequencing 180 minutes after MCAO (Figure 1A, lower). After the onset of IS in the 30-minute filament MCAO model, we identified a total of 24 circulating miRNAs that had a significantly different abundance level (Bonferroni-corrected DESeq P<0.01) in the sera of mice undergoing MCAO compared with sham-operated (MCAO-sham; Figure 1B and Table I in the online-only Data Supplement). In contrast, no significantly over- or underexpressed circulating miRNAs were identified in the ICH model compared with sham-operated (ICH-sham) animals (Table II in the online-only Data Supplement). We observed a bunch of deregulated miRNAs between ICH-sham and naive mice (Table III in the online-only Data Supplement), suggesting that the surgical procedure in the ICH model caused these differences. The expression profiles of the circulating miRNome for the different groups have been deposited in NCBI’s Gene Expression Omnibus with the accession number GSE84216, and brain sections are given in Figure II in the online-only Data Supplement.

Figure 1.
Figure 1.

Analysis of the circulating whole-genome miRNAs (miRNome) in the early phase of cerebral ischemia and intracerebral hemorrhage (ICH). A, Upper, Schematic drawing of used filament middle cerebral artery (MCA) occlusion (MCAO) model and autologous blood injection model to simulate ischemic stroke (IS) and ICH, respectively. Representative coronal sections are shown below for each model after 180 minutes after the model-specific surgery. In IS, the cresyl violet–stained section is showing a subcortical infarct and in ICH the unstained section is showing an intracerebral bleeding in the striatum. A, Lower, Experimental protocol to investigate the abundance of circulating miRNAs after experimental IS and ICH. We transiently occluded the middle cerebral artery for 30 minutes in a filament model of IS and collected the blood serum after 150-minute reperfusion. In the same manner, we collected the blood serum in the model of ICH on 180 minutes after blood infusion. B, Profiling of the miRNA content in the serum samples using next-generation sequencing revealed 24 significantly deregulated circulating miRNAs after IS. miRNA abundance is shown as log2-fold change and Bonferroni-corrected P values compared with sham-operated mice (n=5 per group; Student t test for every population; *P<0.05, **P<0.01, ***P<0.001). ACC indicates arteria carotis communis; ACE, arteria carotis externa; and ACI, arteria carotis interna.

Subsequently, we focused on the deregulated circulating miRNAs after IS. To confirm our next-generation sequencing results, the expression of the 3 highest ranking miRNAs (fold change cutoff of >2), namely miR-1298-5p, miR-448-3p, and miR-1264-3p, was examined by qRT-PCR in a second, independent set of samples. All 3 miRNAs were successfully validated with a strong increase in the miRNA fold change after MCAO compared with naive or sham-operated mice (Figure 2A).

Figure 2.
Figure 2.

Validation of the 3 top-ranked circulating miRNAs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A, miR-1264-3p, miR-1298-5p, and miR-448-3p and as the 3 top-ranked circulating miRNAs were quantified by qRT-PCR. Data were normalized with spiked-in cel-miR-39. miRNA fold change of the middle cerebral artery occlusion (MCAO) group with 30-minute ischemia and 150-minute reperfusion was compared with the sham-operated and naive (nonoperated) group (n=5 per group; 1-way ANOVA for every population; ***P<0.001), and the data are represented as mean±SD. B, Upper, Schematic representation shows the genomic organization (I–VI denotes exons) of the 3 top-ranked circulating miRNAs after ischemic stroke. miR-1264/1298/448 are clustered within the introns of the serotonin receptor HTR2C on the chromosome X. B, Lower, miRNA sequence with its seed regions (in bold) of the 3 members of the miR-1264/1298/448 cluster.

To identify the chromosomal location of these miRNAs, we used the ENSEMBL genome browser25 and found that their genes are organized as an miRNA cluster (miR-1264/1298/448) within the introns of the serotonin receptor HTR2C on chromosome X in the murine and human genome (Figure 2B).

MicroRNA Cluster miR-1264/1298/448 Upregulation Depends on Ischemia Duration and Reperfusion Time

To determine the effect of the duration of cerebral ischemia on the identified circulating miR-1264/1298/448 cluster, we measured their abundance after 30 and 90 minutes of ischemia followed by 150 minutes of reperfusion (Figure 3). We observed an almost doubling of the miRNA fold change of each member of the miR-1264/1298/448 cluster after 90 minutes compared with 30 minutes ischemia: miR-1264 (µ=122±17 versus µ=77±7; P=0.164; t test), miR-1298 (µ=230±26 versus µ=150±20; P=0.175; t test), and miR-448 (µ=838±103 versus µ=448±48; P=0.035; t test). This suggests that the upregulation of these miRNAs in the circulation could be related to the duration of ischemia.

Figure 3.
Figure 3.

Effect of ischemia duration on induction of the circulating miR-1264/1298/448 cluster. The miRNA level of the circulating miR-1264/1298/448 cluster was quantified by quantitative reverse transcription polymerase chain reaction after 30-minute and 90-minute middle cerebral artery occlusion (MCAO) followed by 150-minute reperfusion and compared with sham-operated and nonoperated (naive) mice (n=3 per group; 1-way ANOVA for every population; ***P<0.001; the data are represented as mean±SD).

Next, we performed a time course analysis after 30-minute MCAO and characterized the abundance of the miR-1264/1298/448 cluster after 0, 30, 150, 300, 720, and 1440 minutes of reperfusion (Figure 4). We observed a rapid increase after reperfusion with a peak level at around 150 minutes. Afterward, all 3 miRNAs gradually decreased over time, and after 1440 minutes (24 hours) the miRNA-level returned to the level observed immediately before reperfusion.

Figure 4.
Figure 4.

Kinetics of the circulating miR-1264/1298/448 cluster after focal ischemia and reperfusion. Time course analysis of the miR-1264/1298/448 cluster within the bloodstream by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 30 minutes of brain ischemia (middle cerebral artery occlusion [MCAO]) followed by 0, 30, 150, 300, 720, and 1440 minutes reperfusion (n=3 per group; Student t test for every population; *P<0.05, **P<0.01, ***P<0.001; the data are represented as mean±SD).

MicroRNA Cluster miR-1264/1298/448 Is Induced by Reperfusion

In a next step, we rigorously tested the abundance of this miRNA cluster in mice undergoing permanent filament-induced MCAO with no reperfusion. Therefore, we measured the miRNA level of the miR-1264/1298/448 cluster in the blood serum after 180 minutes of ischemia without reperfusion and compared the results to mice undergoing 30-minute ischemia and 150-minute reperfusion (transient MCAO; Figure 5A). We observed only a marginal increase of the miR-1264/1298/448 cluster in mice subjected to filament permanent MCAO without reperfusion compared with the strong increase in the filament transient MCAO group with reperfusion. To further explore whether the induction of miR-1264/1298/448 cluster in the circulation after cerebral ischemia depends on reperfusion, we used another model of IS where permanent focal ischemia is induced by coagulation of the distal middle cerebral artery. This leads to an ischemic lesion of similar size compared with 30-minute filament MCAO, but the lesion is located in the cortex instead of the striatum. Similarly, we found no increase of members of the miR-1264/1298/448 cluster in the bloodstream after coagulation permanent MCAO compared with the sham-operated control group (Figure 5B).

Figure 5.
Figure 5.

Abundance of the miR-1264/1298/448 cluster in the circulation after permanent ischemia without reperfusion. A, The miRNA level of the miR-1264/1298/448 cluster was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after extensive permanent filament middle cerebral artery occlusion (MCAO) by qRT-PCR. Mice subjected to filament-induced MCAO for 180 minutes without reperfusion (permanent MCAO [pMCAO]) were compared with 30-minute ischemia with 150-minute reperfusion (transient MCAO [tMCAO]) and sham-operated mice (n=3 per group; 1-way ANOVA for every population; ***P<0.001). B, The miRNA level of the miR-1264/1298/448 cluster was measured 180 minutes after coagulation-induced pMCAO by qRT-PCR and compared with sham-operated mice (n=5 per group; Student t test for every population; *P<0.05). The depicted data are represented as mean±SD.

Discussion

The present study aimed to characterize the circulating miRNome in different murine stroke subtypes. The major novel findings are that (1) only IS but not ICH leads to the deregulation of 24 circulating miRNAs, including the miR-1264/1298/448 cluster in the early phase of stroke; (2) upregulation of the miR-1264/1298/448 cluster is an indicator of reperfusion after ischemia; (3) the miR-1264/1298/448 cluster is transiently upregulated early after reperfusion; and (4) the extent of this reperfusion-dependent upregulation depends on the duration of ischemia.

Using next-generation sequencing, we detected 24 deregulated circulating miRNAs in a 30-minute filament model of IS followed by 150 minutes of reperfusion. In contrast, we did not identify any circulating miRNAs associated with ICH. Previous studies in patients and rats described the deregulation of circulating miRNAs in later phases (6 hours to 14 days) after ICH.12,17,30 For instance, Zheng et al30 reported an miRNA signature in the blood plasma within 6 hours after ICH, which they used to differentiate ICH patients with or without hematoma enlargement. Of note, P values were not corrected for the false discovery rate in the study of Zheng et al.30 We would have a few deregulated miRNAs after ICH as well (ie, miR-34c-5p, miR-3099-3p, miR-615-3p, miR-6538, miR-219a-2-3p, miR-9-3p, and miR-9-5p) without stringent Bonferroni-corrected P values. In another report, Liu et al17 quantified the circulating miRNome in murine blood infusion ICH models (fresh and lysed blood) and identified a subset of significantly deregulated miRNA (with false discovery rate–corrected P values) after 24 hours. Taken together, our data with no deregulated circulating miRNAs after acute ICH and the reported detection of miRNAs in later phases of ICH suggest a delayed deregulation of circulating miRNAs after brain hemorrhage.

We identified a bunch of circulating miRNA, including the miR-1264/1298/448 cluster as the most abundant circulating miRNAs, in the early phase after IS. The hampered upregulation of miR-1264/1298/448 cluster in 2 permanent ischemia models suggests that the increased abundance of this miRNA cluster in the bloodstream depends on established reperfusion of ischemic brain tissue. Accordingly, this miRNA cluster could serve as a marker of reperfusion after ischemia. Early reperfusion after cerebral ischemia is critical for improved patient outcome and can nowadays be achieved by pharmacological31 or mechanical endovascular32 means. Biomarkers to monitor reperfusion in IS are scarce and nervous system–specific metabolite N-acetylaspartate (NAA) is, to our knowledge, the only reported blood-based biomarker of early reperfusion after brain ischemia.33 Our findings may be translationally relevant in clinical scenarios where patients present with clinical syndromes, suggesting large-vessel occlusion but unknown patency of the occluded artery. Alternatively, the identified cluster may have the potential to assess microcirculatory perfusion after successful vessel recanalization. In contrast, the miRNA cluster is unlikely to be useful as a parameter to distinguish between ischemia before reperfusion and ICH in the hyperacute phase. Clearly, the potential usefulness of the miR-1264/1298/448 cluster or other circulating miRNAs should be evaluated in the much more heterogeneous scenario of clinical stroke.

The functional role of circulating extracellular miRNAs is currently debated and may include intracellular, as well as intercellular, communication. Moreover, the cellular origin of circulating miRNAs is frequently unknown. miRNAs may be actively secreted by cells or maybe released into the circulation as a consequence of apoptotic or necrotic cell death.34 We identified members of the miR-200 family (miR-141, miR-200a, miR-200b, and miR-200c) and miR-183 family (miR-182 and miR-183) to be upregulated after IS. Both these miRNA families may have a stroke-related functional role in neuronal cells. They are upregulated in the brain 3 hours after ischemic preconditioning, and transfection of these miRNAs had neuroprotective effects in vitro.6 On the contrary, these miRNA families were downregulated in the brain during reduced oxygen consumption in vivo, and inhibition in vitro led to an increased resistance of cells against ischemia.35,36 To shed light on a functional role of the novel identified reperfusion-inducible miR-1264/1298/448 cluster, we performed a KEGG pathway analysis using the DAVID functional annotation software (v6.7) with predicted and conserved target genes of these miRNAs. This analysis suggests that among the most significantly enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways for miR-1264-3p, miR-1298-5p and miR-448-3p are neurotrophin signaling, adherens junction, and axon guidance (Table IV in the online-only Data Supplement). Therefore, the functional role of this miRNA cluster maybe in brain remodeling and neural regeneration after IS.

Our study has strengths and limitations. We used deep sequencing of the circulating miRNome, which has the advantage of allowing identification of both known and unknown miRNAs.37 This methodology is high-throughput and offers a high dynamic range and specificity. Moreover, we used recently adapted RNA sequencing protocols,38,39 which allows to analyze from a low amount of starting material (ie, 2–5 ng). Another strength of our study is the usage of experimental mouse models of cerebral ischemia and hemorrhage, and the comparison with respective sham-operated and naive controls. A limitation of our study is that it used next-generation sequencing only at a single time point, whereas subsequent analyses were based on qRT-PCR of the previously identified deregulated miRNA cluster. Thus, we may have missed circulating miRNAs that were deregulated at later time points.

In conclusion, our study provides new insights into the circulating miRNome in the acute phase of different stroke subtypes. The miR-1264/1298/448 cluster may serve as a biomarker to monitor reperfusion after cerebral ischemia, but further investigation is necessary to locate its cellular origin and understand its functional role.

Sources of Funding

We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics (funded by Wellcome Trust grant reference 090532/Z/09/Z and Medical Research Council Hub grant G0900747 91070) for the generation of the Sequencing data.

Disclosures

None.

Footnotes

  • Received May 2, 2016.
  • Revision received November 30, 2016.
  • Accepted December 12, 2016.

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  • Genome-Wide Analysis of the Circulating miRNome After Cerebral Ischemia Reveals a Reperfusion-Induced MicroRNA Cluster
    Stefan Uhlmann, Eva Mracsko, Ehsan Javidi, Sarah Lamble, Ana Teixeira, Agnes Hotz-Wagenblatt, Karl-Heinz Glatting and Roland Veltkamp
    Stroke. 2017;48:762-769, originally published February 13, 2017
    https://doi.org/10.1161/STROKEAHA.116.013942
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