Impact of microRNAs on ischemic stroke: From pre- to post-disease

Abstract

Stroke is the number one cause of neurological dysfunction in adults and has a heavy socioeconomic burden worldwide. The etiological origins of ischemic stroke and resulting pathological processes are mediated by a multifaceted cascade of molecular mechanisms that are in part modulated by posttranscriptional activity. Accumulating evidence has revealed a role for microRNAs (miRNAs) as essential mediators of posttranscriptional gene silencing in both the physiology of brain development and pathology of ischemic stroke. In this review, we compile miRNAs that have been reported to regulate various stroke risk factors and pre-disease mechanisms, including hypertension, atherosclerosis, and diabetes, followed by an in-depth analysis of miRNAs in ischemic stroke pathogenesis, such as excitotoxicity, oxidative stress, inflammation, apoptosis, angiogenesis and neurogenesis. Since promoting or suppressing expression of miRNAs by specific pharmaceutical and non-pharmaceutical therapies may be beneficial to post-stroke recovery, we also highlight the potential therapeutic value of miRNAs in clinical settings.

1. Introduction

Ischemic stroke is a leading cause of death and disability, resulting in over six million deaths per year worldwide (Mendis et al., 2015; Zhang et al., 2016b). Ischemic stroke occurs when cerebral blood flow is interrupted, usually due to arterial thrombosis or embolism. Despite decades of research, treatment for ischemic stroke is limited to thrombolytic therapy and the management of symptoms (dela Pena et al., 2015). However, research has revealed much of the pathophysiology of this disease, including the phases of injury, patterns of gene expression and physiological factors that can exacerbate or ameliorate the eventual outcome.

The immediate effects of blood deprivation include energy failure and excitotoxicity, followed shortly thereafter by edema and blood brain barrier (BBB) disruption (Yemisci et al., 2015). However, the damage is not limited to the ischemic core region, as dying neurons will release pro-apoptotic and pro-inflammatory factors into the adjacent brain parenchyma, killing neurons in the penumbral areas (Dirnagl et al., 1999). Penumbra can be protected by timely and efficient therapeutic interventions that can limit the extent of the infarction and improve the function and structural integrity of the cells in the penumbra (Hillis and JC, 2015; Boers et al., 2016).

In addition to de facto ischemia, reperfusion also damages the post-ischemic brain by multiple mechanisms, including inflammation and oxidative stress (Lakhan et al., 2009; Manzanero et al., 2013; Arumugam et al., 2016; Chen et al., 2016). Inflammatory processes begin within hours after the insult when the resident microglia and dying neurons release pro-inflammatory molecules and other damage-associated molecular patterns and, concomitantly, endothelial cells express cell adhesion molecules, including intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 (Huck et al., 2015). These changes promote trans-endothelial migration of peripheral immune cells, including macrophages and neutrophils, to the ischemic region that potentiate inflammation by further release of pro-inflammatory molecules and reactive oxygen species (ROS). At the same time, a lack of energy leads to failure of ionic pumps, promoting vascular/cellular edema and increasing intracranial pressure. Damaged or dysfunctional mitochondria also release ROS that mediate lipid peroxidation, nucleic acid damage and enzyme inhibition. All of these factors exacerbate post-stroke secondary brain damage. Inflammation and oxidative stress are not the only events that promote cellular and molecular post-ischemic damage, but they represent two promising leads for therapeutic targets for the treatment and prevention of secondary brain damage after stroke (Dirnagl et al., 1999; Nakka et al., 2016). Stroke leads to an acute phase of cellular damage, followed by a chronic phase of limited plasticity and regeneration. Both of these phases provide opportunities for therapeutic intervention by potentiating neuronal survival and recovery. Despite promising efficacy in experimental animals, most if not all of the neuroprotective therapies tested failed to translate to the clinic (Kikuchi et al., 2014). There might be many reasons for this, including inappropriate animal models, differences in species, timing of drug delivery, and age/sex differences. Hence, identifying and testing newer therapeutic targets is paramount for future clinically translatable stroke therapies.

Non-coding RNAs, particularly microRNAs (miRNAs), are among the many classes of molecules that cause functional alterations before, during and after ischemic stroke (see Section 2). The miRNAs are capable of targeting a variety of pathways due to redundancy of their targets; a single miRNA can target multiple mRNAs, and most mRNAs can be targeted simultaneously by multiple miRNAs (Lim et al., 2005). It is well established that the expression levels of many miRNAs are altered after stroke in the blood and brain of both rodents and humans (Jeyaseelan et al., 2008; Dharap et al., 2009; Sorensen et al., 2014; Li et al., 2015c). Furthermore, miRNAs can be modulated by external agents to improve functional outcome after stroke (Saugstad, 2015; Xu et al., 2015; Liu da et al., 2016). In addition, induction of ischemic tolerance by preconditioning (PC) also alters the levels of many miRNAs (Dharap and Vemuganti, 2010; Lusardi et al., 2010; Wakai et al., 2016). The goal of this review is to present a detailed overview of the current knowledge of miRNAs in cerebral ischemia with special attention paid to miRNAs that modulate stroke-associated risk factors and pathologic mechanisms of post-stroke brain damage (Fig. 1).

Fig. 1.

miRNAs play important roles in pre- and post-stroke pathologic processes. The aberrant expression of miRNAs has diagnostic potential pre- and post-stroke. Numerous miRNAs are associated with risk factors of stroke, including hypertension, diabetes and atherosclerosis. They also trigger or inhibit a series of pathophysiological responses post-stroke, including excitotoxicity, oxidative stress, inflammation, BBB damage, neuronal apoptosis, neurogenesis and angiogenesis. Moreover, some specific miRNAs regulate the pathophysiological progress of ischemic stroke by regulating various mechanisms simultaneously.

2. miRNA biogenesis and function

The miRNAs are a group of small noncoding RNAs that have been shown to modulate protein expression at the post-transcriptional level. Since their discovery in C. elegans in 1993 (Lee et al., 1993), over 30,000 unique miRNA sequences have been discovered in over 200 fully sequenced species (Kozomara and Griffiths-Jones, 2014). The miRNA structure, function, biogenesis, and mechanisms of action have been extensively studied over the past decade.

The miRNAs are transcribed as single-stranded primary miRNAs (pri-miRNA) that are several kilobases long and contain one or more stem-loop structures, which are cleaved by the nuclear RNase III Drosha (Lee et al., 2002, 2003). These cleaved stem-loops are called precursor miRNAs (pre-miRNAs), which are exported into cytosol by Exportin-5, further processed by an RNase III called Dicer to release the miRNA duplexes, and bound by an argonaute (AGO) protein. The less stable strand of the duplex will be digested, and the other strand will be released as an 18 to 22 nucleotide long mature miRNA which will be incorporated into an RNA-induced silencing complex (RISC) in which AGO proteins are a key component. The RISC complex guides the mature miRNAs to bind at the consensus seed sequences in the 3′UTRs of the target cytosolic mRNAs (Diederichs and Haber, 2007). Binding of a miRNA either prevents the translation of or promotes the degradation of the mRNAs (Winter et al., 2009).

In addition to this canonical biogenesis pathway, instances of Drosha- and Dicer-independent miRNA biogenesis have also been reported. Transcriptional intermediates represent the most common form of Drosha-independent biogenesis in which pre-miR stem-loops are generated during mRNA, tRNA or small nucleolar RNA processing (Berezikov et al., 2007; Ender et al., 2008; Chong et al., 2010). Rarely, pre-miRs can be transcribed directly (Xie et al., 2013), and in some cases, short hairpin RNAs can be processed similarly to that of pre-miRs. Dicer activity is required to release almost all of the known miRNAs, but miR-451 is a notable exception. Drosha processes pri-miR-451 into a stem-loop that is too short to be recognized by Dicer; thus, pre-miR-451 is bound directly to AGO2, which uses its slicer activity to produce mature miR-451 (Miyoshi et al., 2005; Cifuentes et al., 2010). Interestingly, miRNAs are implicated in a wide variety of diseases and have been shown to be required for proper physiological function. Many pathological states are known to alter miRNA profiles and functions, and understanding those changes and developing modalities to rectify them might lead to novel therapeutic strategies (van Empel et al., 2012). The following sections discuss the experimental evidence for miRNAs implicated in development, disease progression, and potential biomarkers for ischemic stroke and related pathologies.

3. microRNAs in brain development: implications for stroke

In mammals, the brain shows a very high level and activity of many miRNAs that display region-specific expression (Sempere et al., 2004; Bak et al., 2008). Studies employing conditional knockouts of Dicer (RNAse essential for miRNA biogenesis) demonstrated the indispensable functional significance of miRNAs in controlling processes that include cellular differentiation, proliferation, synaptic morphogenesis, and vascular formation and thus orchestrate brain development (Schratt et al., 2006; Davis et al., 2008; Wang et al., 2008; Huang et al., 2010b; McLoughlin et al., 2012; Pollock et al., 2014). miRNAs were also shown to be expressed temporally with developmental milestones in the mammalian brain (Miska et al., 2004; Sempere et al., 2004). Notably, some of the developmentally regulated miRNAs are also known to be activated following cerebral ischemia, indicating an overlap between brain development and brain injury.

The miR-17-92 cluster is involved in maintaining appropriate populations of progenitors and radial glial cells by regulating the proliferation-related genes phosphatase and tensin homolog and T-box brain protein 2 (Bian et al., 2013). Following experimental stroke in mice, the miRNA-17-92 cluster was shown to be robustly upregulated and promoted neural progenitor proliferation (Liu et al., 2013b). The miR-124 cluster is known to determine neuronal fate as its overexpression results in forced neural differentiation of progenitors (Smirnova et al., 2005; Krichevsky et al., 2006; Silber et al., 2008; Akerblom et al., 2012). Knockout of miR-124 prevents proper axonal development and leads to reduced brain size and abnormalities in neuroanatomical structures during development (Sanuki et al., 2011). Following cerebral ischemia, altered miR-124 levels are implicated in a wide array of aberrant cellular processes, including inflammation, edema, apoptosis, and neurogenesis (Liu et al., 2011, 2015b; Sun et al., 2013; Doeppner et al., 2016). In zebrafish, miR-142-3p mediates vascular integrity and developmental angiogenesis by repressing vascular endothelial cadherin (Lalwani et al., 2012). Overexpression of miR-142-3p was shown to reduce vascular integrity and cause cerebral hemorrhage, whereas inhibition of miR-142-3p led to abnormal vascular remodeling (Lalwani et al., 2012). Focal ischemia was shown to increase miR-142-3p levels in rodents (Liu et al., 2013a), indicating that it may play a role in post-ischemic neurovascular remodeling. Similarly, miR-126 is also a primary mediator of vascular growth and development in the brain by targeting vascular endothelial growth factor (VEGF) (Fish et al., 2008; Wang et al., 2008; Zhou et al., 2016). Rodent studies have demonstrated that miR-126 inhibits VEGF and retinal neovascularization following ischemia (Bai et al., 2011; Ye et al., 2014). Furthermore, levels of circulating miR-126 were identified as a biomarker for ischemic stroke in humans (Long et al., 2013).

4. microRNAs involved in stroke comorbidities

Stroke is associated with a variety of comorbidities, notably, hypertension, atherosclerosis and diabetes (Ku Mohd Noor et al., 2016; Ostergaard et al., 2016). Hypertension is the number one risk factor for stroke as it decreases vessel elasticity and hence can potentially lead to rupture, causing hemorrhagic stroke (Hasan et al., 2011). Atherosclerotic progression results in stenosis of arteries, clot formation and potential plaque rupture, all of which are common etiologies in stroke in humans (Kim et al., 2015). Diabetes has multiple phenotypes, but diabetic hyperglycemia in particular exacerbates neuronal death and worsens post-ischemic outcome (Kaarisalo et al., 2005; Dave et al., 2011). miRNAs are known to modulate all of these stroke risk factors (Park et al., 2013; Shi et al., 2015; Feinberg and Moore, 2016).

4.1. microRNAs and hypertension in relation to stroke

In the aorta of spontaneously hypertensive rats (SHRs), miR-155 levels were shown to be negatively correlated with blood pressure (Xu et al., 2008). The miR-155 levels were also reported to be lower in peripheral blood mononuclear cells of stage 1 hypertensive patients (Ceolotto et al., 2011). Furthermore, endothelial nitric oxide (NO) synthase and angiotensin II type 1 receptor (AT1R) were observed to be targets of miR-155, indicating its role in vasorelaxation and the renin-angiotensin system (Zheng et al., 2010; Sun et al., 2012). Thus, miR-155 seems to be an important miRNA that might modulate stroke incidence by controlling blood pressure. A few other miRNAs, including miR-125a/b-5p, miR-22, and miR-487b, were also shown to control blood pressure. miR-125a/b-5p targets endothelin-1 (a potent vasoconstrictor) in vascular endothelial cells (Li et al., 2010), and miR-22 targets chromogranin A, which increases catestatin that in turn regulates blood pressure (Mahapatra, 2008; Friese et al., 2013). In support of the role of miR-22, a study showed that when SHR rats were treated with antagomiR-22, there was a decrease in blood pressure (Zhu et al., 2013b). In addition, miR-487b was observed to be upregulated in the aorta of Sprague-Dawley rats with angiotensin II-induced hypertension and was linked to down-regulation of the anti-apoptotic insulin receptor substrate 1, resulting in damage of aortic adventitial fibroblasts (Nossent et al., 2013). Furthermore, nanostring miRNA sequencing identified 24 miRNAs expressed differentially in the brain stem between hypertensive SHR and normotensive WKY rats (DeCicco et al., 2015), and deep sequencing identified 30 miRNAs upregulated in human microvascular endothelial cells that were thought to have a putative role in hypertension (Kriegel et al., 2015).

4.2. microRNAs and diabetes in relation to stroke

Diabetes is a major comorbid condition for stroke in humans. Increased blood glucose can lead to plaque buildup and thrombus formation, potentially precipitating stroke (Zhang et al., 2013; Mozaffarian et al., 2016). A recent study showed that, in patients with type 2 diabetes who undergo stroke, there was a significant downregulation of miR-223 and upregulation of miR-144 (Yang et al., 2016a). In the platelets from diabetes patients, miR-223 and miR-146a were observed to decrease following stroke (Duan et al., 2014). In rats with streptozotocin-induced diabetes subjected to transient focal ischemia, administration of bone-marrow stromal cells (BMSCs) from diabetic rats was reported to be more neuroprotective than that of BMSCs from non-diabetic rats. This was shown to be due to decreased levels of miR-145 leading to derepression of its targets ATP-binding cassette transporter 1 (ABCA1) and insulin-like growth factor 1 in the diabetic BMSCs (Cui et al., 2016). Hyperglycemia can also affect brain miRNAs in the absence of ischemic stroke. For example, let-7a was shown to be involved in glucose metabolism (Zhu et al., 2011a; Perez et al., 2013) by repressing apoptosis signal regulating kinase 1 in microglia (Song and Lee, 2015). Another study identified downregulation of miR-200a/b and miR-466a/d-3p in neural stem cells of streptozotocin-induced diabetic mice (Shyamasundar et al., 2013).

4.3. microRNAs involved in atherosclerosis

Atherosclerosis is associated with miRNA alterations in the vessel wall, serum, and immune cells, suggesting their role in atherosclerotic progression (Santovito et al., 2015). Downregulation of some miRNAs, such as miR-320a (targeting a serum response factor required for VEGF signaling), miR-143/145 (targeting ABCA1) and miR-92a (targeting Kruppel-like factor 2 that modulates shear stress genes), protects blood vessels in atherosclerosis (Wu et al., 2011; Sala et al., 2014; Chen et al., 2015a). miR-181a is downregulated in monocytes of obese patients (Hulsmans et al., 2012), and treatment with a miR-181a precursor attenuates oxidized low-density lipoprotein-mediated inflammation in bone marrow-derived dendritic cells by targeting c-Fos (Wu et al., 2012). Furthermore, miR-155 was implicated in the inflammatory progression associated with atherogenesis, and it was shown to be atheroprotective by targeting the pro-inflammatory transcription factor Ets1 and AT1R in human endothelial cells (Zhu et al., 2011b). Knockdown of miR-155 in hyperlipidemic mice showed an increase in atherosclerotic progression, possibly by targeting colony stimulating factor 1 receptor or MAP kinases (Donners et al., 2012; Zhu et al., 2012; Wei et al., 2015). Macrophage-specific miR-155 was shown to downregulate Fas-associated death-domain-containing protein (Zhu et al., 2013a) as well as inflammatory factors, such as tumor necrosis factor alpha (TNFα), by targeting calcium-regulated heat stable protein 1 (Li et al., 2016). Furthermore, nuclear factor kappa B (NF-κB), which is a target of miR-155, controls miR-155 transcription in a feedback loop (Wu et al., 2014). In human macrophages, oxidized low-density lipoprotein stimulation increased miR-155 levels, and inhibition of miR-155 resulted in exacerbated lipid uptake and overall inflammatory burden (Huang et al., 2010a). In contrast, a few studies also implicated miR-155 in promotion of atherosclerotic progression by downregulation of the anti-inflammatory factors HMG box-transcription protein 1, B-cell lymphoma-6 (Bcl-6), and suppressor of cytokine signaling 1 (Nazari-Jahantigh et al., 2012; Tian et al., 2014; Yang et al., 2015a).

In human endothelial cell cultures, the miR-221/222 cluster was shown to target many inflammation-related controlling molecules, including transcription factor Ets1, peroxisome proliferator-activated receptor gamma coactivator 1 alpha, adiponectin receptor 1, and signal transducer and activator of transcription 5A, and hence might help to control atherogenesis (Dentelli et al., 2010; Zhu et al., 2011b; Chen et al., 2015b; Qin et al., 2015; Xue et al., 2015), and the miR-221/222 cluster targets Kip1, Kip2 and c-kit that to promote proliferation of vascular smooth muscle cells (Liu et al., 2009, 2012b). Clinically, miR-221/222 levels are seen to be decreased in the more advanced lesions, such that complete loss is associated with plaque rupture (Bazan et al., 2015), and a preclinical investigation of vascular calcification in rodents demonstrated that miR-221/222 upregulation can exacerbate aortic calcification (Mackenzie et al., 2014). The sum of these investigations indicates a potentially complex and cell-specific role for miRNAs in atherosclerosis progression.

5. Role of microRNAs in post-stroke brain damage

Cerebral ischemia/reperfusion injury induces a complex pathophysiological cascade that includes a wide range of aberrant cellular processes. In the ischemic phase, reduced blood supply rapidly leads to failure of ionic gradients, excitotoxicity and neuronal death. During the reperfusion phase, the return of oxygen contributes to oxidative stress, and the restoration of blood introduces factors that promote inflammation and edema, thereby further increasing the vulnerability of the affected tissue to neurodegeneration (White et al., 2000). The expression levels of hundreds of miRNAs were shown to be altered after transient focal ischemia after as early as 30 min and as late as 7 days of reperfusion (Jeyaseelan et al., 2008; Dharap et al., 2009; Liu et al., 2010, 2013a; Yuan et al., 2010; Hunsberger et al., 2012). This section will discuss the putative mechanisms downstream of those miRNAs in post-ischemic pathophysiology.

5.1. microRNAs and post-ischemic excitotoxicity

During ischemia, excessive release of glutamate and the concomitant failure of glutamate transporters lead to glutamate receptor overactivation and excitotoxic neuronal death (Paschen, 1996; Castillo et al., 2016). In the hippocampus, miR-223 overexpression was shown to lower the levels of glutamate receptor 2 (GluR2) and N-methyl-d-aspartate (NMDA) receptor subunit 2B (NR2B) and prevent neuronal death following transient global ischemia (Harraz et al., 2012). Furthermore, knockout of miR-223 enhanced NR2B and GluR2 expression, increased miniature excitatory postsynaptic currents, exacerbated memory deficits and increased hippocampal neuronal death (Harraz et al., 2012). Circulating miR-223 levels were shown to be increased after stroke in rodents (Dharap et al., 2009) and were positively associated with reduced severity and infarct volume in humans following ischemic stroke (Wang et al., 2014e). Similarly, exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group, and was positively correlated with NIHSS scores. Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (Chen et al., 2017). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes (Chen et al., 2017). Whereas miR-223 appears to prevent ischemia-induced excitotoxicity, miR-125b is implicated in exacerbating excitotoxicity by increasing NMDA receptor subunit unit NR2A (Edbauer et al., 2010). Both in vitro and in vivo studies show that upregulation of NR2A promotes NMDA-mediated neuronal death in cortical neurons (Morikawa et al., 1998; von Engelhardt et al., 2007).

Rapid removal of glutamate from the synaptic cleft is required to prevent excitotoxicity, and this is mediated by glutamate transporters, such as astrocytic glutamate transporter 1 (GLT-1) (Raghavendra et al., 2000; Yeh et al., 2005). A correlation between increased miR-107 and decreased GLT-1 levels was demonstrated following cerebral ischemia (Yang et al., 2014, 2015d). In neuronal cells subjected to hypoxia, blocking miR-107 prevented GLT-1 downregulation, glutamate accumulation and apoptosis (Yang et al., 2014). In contrast, in vivo studies showed that miR-107 inhibition exacerbates infarction after permanent focal ischemia in rodents, indicating that miR-107 might be neuroprotective (Li et al., 2015d). However, the role of miR-107 after ischemia might be complex as it targets the RNAse Dicer, which is essential for miRNA processing (Li et al., 2015d).

Exosomes that transport miRNAs might be critical for maintaining neuronal–glial cross-talk, which is essential for maintaining glutamate homeostasis after ischemia (Lachenal et al., 2011). Morel et al. (2013) showed that miR-124a transferred from neurons to astrocytes by exosomes increases GLT-1 protein levels, potentially by an indirect mechanism. Injection of antagomiR-124a into adult mouse brain was further shown to reduce GLT-1 expression and glutamate uptake (Morel et al., 2013). Upregulation of miR-124 was also shown to be associated with reduced expression of various glutamate receptors and subunits including α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)2, AMPA3 and GluR2, indicating additional miR-124 targets involved in glutamate signaling (Dutta et al., 2013; Ho et al., 2014). However, the role of miR-124a is controversial as some studies have shown that it can also protect the brain following ischemia (Doeppner et al., 2013; Sun et al., 2013; Zhu et al., 2014). The levels of miR-124 after ischemia were shown to be increased by Sun et al. (2013), whereas another study exhibited substantial reduction of miR-124 following focal ischemia in rats (Liu et al., 2011). miR-181a was also shown to regulate excitotoxicity by targeting both glutamate receptors and glutamate transporters. Overexpression of miR-181a was shown to reduce GluR2 expression and the frequency of miniature excitatory postsynaptic currents (Saba et al., 2012). Interestingly, miR-181a levels were shown to be increased in the core but decreased in the areas of brain that are resistant to ischemic injury (penumbra after focal ischemia and hippocampal dentate gyrus after global ischemia) (Yuan et al., 2010; Ouyang et al., 2012b). Furthermore, blocking miR-181a was shown to protect the brain and improve functional outcome following ischemia (Ouyang et al., 2012b; Moon et al., 2013; Xu et al., 2015). miR-181a was also shown to decrease GLT-1 levels (Moon et al., 2013). Roles of miRNAs in post-ischemic excitotoxicity are shown in Fig. 2.

Fig. 2.

Mechanisms of miRNAs control post-ischemic excitotoxicity, oxidative stress and apoptosis. miR-223, -107, -125b, -124, and -181a control the release of glutamate by regulating glutamate transporters, including GluR2, NR2A, NR2 B and GLT-1. Furthermore, miR-124, -21, -25, -181a, -15, -497, and -29b decrease or increase the expression of apoptosis-related proteins (Fas, Fas ligand, Bc1-2, etc.) to control neural apoptosis. Moreover, miR-424 and miR-23a-3p execute the antioxidant function following ischemic stroke by regulating the expression of antioxidant protein, and protection of mitochondrial function was observed by miR-210 inhibitor treatment. The activation and inhibition are indicated by an arrow line and a T-shape line, respectively.

5.2. microRNAs and post-stroke oxidative stress

An imbalance between free radical production and antioxidant activity leads to oxidative stress, which is a major pathologic mechanism of secondary brain damage after cerebral ischemia (Walder et al., 1997; Guldiken et al., 2009). Both ischemia and reperfusion are known to promote the formation of ROS, which include superoxide anion, hydrogen peroxide (H₂O₂), hydroxyl radical, singlet oxygen and peroxynitrite (Abramov et al., 2007; Radak et al., 2014; Jung et al., 2015). Increased ROS levels damage neurons by promoting mitochondrial dysfunction, calpain activation, inflammatory signaling and apoptosis, which determine the infarct volume and functional recovery following cerebral ischemia (Warner et al., 2004; Chen et al., 2011b). Although emerging evidence shows that miRNAs play an essential role in regulating the balance between oxidants and antioxidants after cerebral ischemia, the direct mRNA targets involved have yet to be identified. Overexpression of miR-424, miR-23a-3p and miR-99a was shown to attenuate oxidative stress and hence can protect the brain after an ischemic insult (Zhao et al., 2014; Liu et al., 2015a; Tao et al., 2015b). Treatment with agomiR-424 reduced the infarct volume and increased the expression of the transcription factor nuclear factor erythroid 2-related factor (Nrf2), which is known to be anti-inflammatory/anti-oxidant and neuroprotective (Shih et al., 2005; Liu et al., 2015a). AgomiR-424 treatment also enhanced manganese superoxide dismutase (Mn-SOD) expression and reduced ROS levels. In vitro, the protective effect of miR-424 against neuronal oxidative stress was shown to be attenuated by Nrf2 knockdown or SOD inhibition, confirming the antioxidant mechanism of action of miR-424 (Liu et al., 2015a). AgomiR-23a-3p treatment also decreased infarct volume by increasing Mn-SOD and reducing the levels of NO and 3-nitrotyrosine (3-NT) (Zhao et al., 2014). In vitro, miR-23a-3p was further shown to decrease H₂O₂-induced cell death by reducing the production of NO and 3-NT and promote the activity of SOD in neuro-2a cells (Zhao et al., 2014). Overexpression of miR-99a also protected neuro-2a cells after H₂O₂ treatment and reduced the infarct volume following cerebral ischemia (Tao et al., 2015b).

Hypoxic and ischemic insults were shown to induce mir-210 expression via hypoxia-inducible factor 1 alpha (HIF1-α) in both in vivo and in vitro conditions (Pulkkinen et al., 2008; Lou et al., 2012; Qiu et al., 2013). Recently, miR-210 expression was shown to be induced following vagus nerve stimulation (VNS) (Jiang et al., 2015), a procedure that protects the brain against cerebral ischemia/reperfusion injury by regulating cellular redox status (Ekici et al., 2013; Jiang et al., 2014). Knockdown of miR-210 attenuated neuronal death and the antioxidant stress response effects of VNS in the cortex following transient middle cerebral artery occlusion (MCAO) (Jiang et al., 2015). Although the specific mechanism by which miR-210 mediates protection against post-ischemic oxidative stress has not yet been elucidated, miR-210 was previously shown to target many mRNAs that code proteins involved in mitochondrial function, metabolism, and cell survival (Chan and Loscalzo, 2010) and hence might have a pleiotropic neuroprotective effect after ischemia. The role of miRNAs in post-ischemic oxidative stress is shown in Fig. 2.

5.3. microRNAs and post-ischemic inflammation

Inflammation after cerebral ischemia involves a complex pathological progression starting with the activation of microglial cells, infiltration of circulating leukocytes (such as neutrophils, lymphocytes and macrophages) and release of pro-inflammatory mediators by ischemic and immune cells (Hallenbeck, 1996; Zheng and Yenari, 2004; Wu et al., 2015). Although inflammation starts within minutes after focal ischemia, neutrophil infiltration, which is the hallmark of inflammation, occurs within 3 h and lasts until 24 h of reperfusion (Liu et al., 2001). Increased levels of pro-inflammatory molecules, such as chemokines and cytokines, also follow this timeline after stroke (Liu et al., 1994; Wang et al., 1997; Clark et al., 1999). Experimental models have shown that inhibition of neutrophilic inflammatory mechanisms reduces neurodegeneration and improves functional outcome after cerebral ischemia (Prestigiacomo et al., 1999; Zhang et al., 2003). However, none of the anti-inflammatory therapies showed efficacy in humans after stroke (Enlimomab Acute Stroke Trial Investigators, 2001; Becker, 2002; Krams et al., 2003).

Many miRNAs were shown to modulate inflammatory signaling after cerebral ischemia. A major mechanism by which miRNAs regulate inflammation is through targeting the expression of cytokines in immune cells. Overexpression of miR-22 in cortical neuronal cultures was shown to decrease the pro-inflammatory cytokines TNF-α and interleukin (IL)-6 and increase the anti-inflammatory cytokine IL-10 following oxygen and glucose deprivation (OGD) (Yu et al., 2015). Furthermore, miR-22 was also shown to decrease transient focal ischemia-induced inflammation, infarct volume, and neurological deficits (Yu et al., 2015). Modulation of inflammation by miR-22 involves suppression of pro-inflammatory signaling by targeting the nuclear receptor coactivator 1, an NF-κB coactivator (Yu et al., 2015). Preventing NF-κB expression has previously been shown to induce neuroprotection following cerebral ischemia (Buchan et al., 2000; Ueno et al., 2001; Xu et al., 2012). Other miRNAs are also linked to the NF-κB pathway in stroke paradigms, although the specific targeted mRNAs remain unclear. For example, miR-181a overexpression increases the NF-κB activator glucose-regulated protein 78 and exacerbates stroke injury (Nakajima et al., 2011; Ouyang et al., 2012b). Post-ischemic treatment with antagomiR-181a was shown to decrease NF-κB activation, leukocyte infiltration, infarct size and long-term behavioral deficits following MCAO (Xu et al., 2015). Alternately, overexpression of miR-203 inhibited NF-κB signaling by downregulating myeloid differentiation factor 88 (MyD88), a key downstream adapter of toll-like receptors, and the interleukin-1 receptor, which mediates NF-κB nuclear translocation. Furthermore, miR-203 was shown to suppress the production of pro-inflammatory cytokines IL-8 and TNF-α and thereby attenuate post-ischemia inflammation and decrease neuronal death following OGD (Yang et al., 2015c).

Stroke patients with high systemic inflammatory markers display poorer functional outcomes (Elkind et al., 2004; McColl et al., 2007). Analysis of miRNAs in serum samples from patients with acute ischemic stroke showed significant reduction in miR-124 and miR-9 levels, which were associated with a larger infarct volume and increased levels of pro-inflammatory molecules, including matrix metalloproteinase-9 (MMP-9) and high-sensitivity C-reactive protein (hs-CRP), in plasma (Liu et al., 2015b). MMP-9 degrades the basal lamina of the BBB, and hs-CRP is a pro-inflammatory molecule that correlates with stroke severity (Kamada et al., 2007; Youn et al., 2012). In vitro studies further showed that overexpression of miR-124 decreased cytokines, such as TNF-α, and inhibited microglial activation in experimental allergic encephalomyelitis models (Ponomarev et al., 2011). Being the primary resident innate immune cells in the brain, microglia is highly sensitive to inflammatory signals following an ischemic insult (Benakis et al., 2014). Once activated, microglia releases ROS, pro-inflammatory chemokines and cytokines and proteolytic enzymes to mediate brain damage (Kim et al., 2013; Benakis et al., 2014). Although a moderate activation of microglia is necessary for plasticity and clearing debris, their overactivation leads to neurotoxicity and brain damage after cerebral ischemia (Del et al., 2007). As many miRNAs regulate microglial activation, the post-ischemic brain can be protected by modulating them. In a rodent model of permanent MCAO, miR-424 overexpression inhibited microglial activation and reduced infarct size (Zhao et al., 2013). Additionally, miR-424 was shown to suppress the activation of microglial cultures by translational repression of cellcycle activators, including cyclin D1, cell division cycle 25A and cell division protein kinase 6 (Zhao et al., 2013). OGD-induced microglial activation was shown to require the MyD88 adapter, and targeting and repression of MyD88 by miR-203 prevented microglial activation and neuronal injury after OGD (Yang et al., 2015c). Furthermore, intracerebral administration of miR-203 mimics MCAO-downregulated MyD88 and decreases inflammation and secondary brain damage (Yang et al., 2015c). miR-181a also plays a role in microglial activation in addition to its ability to regulate NF-κB, as described earlier. Post-ischemic treatment with antagomiR-181a decreased the levels of the microglia-specific protein Iba1 and reduced neurodegeneration following MCAO (Xu et al., 2015). There is evidence that miR-181-mediated effects on inflammation following cerebral ischemia could potentially extend to astrocytes as well. In an in vitro model of inflammation using cultured astrocytes, inhibition of miR-181 led to enhanced production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1b, IL-8, and high mobility group box-1 protein), whereas overexpression of miR-181 increased the expression of anti-inflammatory cytokine IL-10 (Hutchison et al., 2013). Alterations in miR-132, miR-146a, and miR-155 have also been observed in cultured astrocytes following the induction of inflammatory signaling, where miR-132 was shown to mediate negative feedback regulation of IL-1β and IL-6 by targeting interleukin-1 receptor-associated kinase 4 (Kong et al., 2015). Overall, these studies indicate that miRNAs regulate post-ischemic neuroinflammation (Table 1).

Table 1.

miRNAs mediate inflammation response post-ischemic injury. In vivo, miR-9, -124 and -181a regulate post-stroke inflammation by reducing hc-CRP level, promoting the infiltration of leukocytes, and activating the NF-κB pathway, respectively. Moreover, miR-22 in neurons attenuates inflammation by increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines. In addition, miR-181a increases inflammation by activating the NF-κB pathway, whereas miR-181 and miR-132 decrease inflammation by increasing anti-inflammatory cytokines and decreasing pro-inflammatory cytokines in astrocytes. miR-124, -424, -181, and -203 take part in the inflammatory response by regulating proliferation or activation of microglia after ischemic stroke. The activation and inhibition are indicated by an arrow.

Type of research	MiR of interest	Species/ celltype	Upstream regulators	Inflammatory factors	Target nuclear factors	Reference
Clinical studies	miR-9, miR-124	Human	N/A	↓CRP	N/A	Liu et al. (2015a, b)
In vivo studies	miR-181a	Mouse	N/A	↑GRP78	N/A	Ouyang et al. (2012b)
	miR-181a	Mouse	N/A	↑MPO, Iba1	↑NF-κB	Xu et al. (2015)
	miR-203	Mouse	N/A	↓MyD88, IL-8, TNF-α	↓NF-κB	Yang et al. (2015c)
	miR-424	Mouse	N/A	↓TNF-α, Iba1, Cyclin D1, CDK6, CDC25A	N/A	Zhao et al. (2013)
In vitro studies	miR-22	Neuron	N/A	↓IL-6, TNF-α, COX-2, iNOS; ↑IL-10	↓NCOA1, ↑ NF-κB	Yu et al. (2015)
	miR-203	Microglia	↓MyD88	↓IL-8, TNF-α	N/A	Yang et al. (2015c)
	miR-424	Microglia	N/A	↓TNF-α, IL-1 β, Cyclin D1, CDK6, CDC25A	N/A	Zhao et al. (2013)
	miR-124	Microphage	N/A	↓TNF-α, iNOS	N/A	Ponomarev et al. (2011)
	miR-181a	Astrocyte	N/A	↑GRP78	N/A	Ouyang et al. (2012b)
	miR-181	Astrocyte	N/A	↓TNF-α, IL-1 β, IL-6, IL-8, HMGB1; ↑IL-10	N/A	Hutchinson et al., 2013
	miR-132	Astrocyte	↑IRAK4	↓IL-1 β, IL-6	N/A	Kong et al. (2015)

5.4. microRNAs, BBB damage and edema after stroke

Post-stroke brain edema leads to physiological deterioration by increasing intracranial pressure and by reducing access to the blood supply within cerebral tissues (Nag et al., 2009). Stroke-induced edema can be vasogenic, where BBB damage leads to extracellular accumulation of fluid, or cytotoxic due to intracellular fluid accumulation (Michinaga and Koyama, 2015). At the physiological level, brain edema has been associated with various factors, such as endothelial dysfunction, MMPs, and aquaporins (AQPs).

Several miRNAs have been shown to be involved in stroke-induced brain edema via direct or indirect mechanisms (Rom et al., 2015; Bukeirat et al., 2016). Endothelial cells are enriched with various miRNAs that are thought to control the BBB function under normal and pathologic states (Suarez and Sessa, 2009). In a rat permanent MCAO model, miR-150 upregulation was shown to lead to BBB permeability, and overexpression of miR-150 in microvascular endothelial cells decreased the expression of claudin-5, a major tight junction protein leading to increased endothelial permeability and cell death following OGD (Fang et al., 2016). These effects were reversed knockdown of angiopoietin receptor Tie-2 (a miR-150 target), and treatment with miR-150 antagomiR prevented BBB disruption and reduced post-stroke degeneration, potentially by regulating endothelial survival (Fang et al., 2016). When cerebral endothelial cells were subjected to OGD, expression of peroxisome proliferator-activated receptors-δ (PPAR-δ) was decreased with a concomitant increase in miR-15a, which represses anti-apoptotic Bcl-2 (Yin et al., 2010a). PPAR-d induced downregulation of miR-15a resulted in decreased apoptosis and DNA fragmentation in isolated cerebral microvessels, which led to amelioration of BBB disruption and a decrease in infarct size after MCAO (Yin et al., 2010a).

Stroke is known to induce MMPs, which damage the integrity of endothelial cells, resulting in increased BBB permeability (Seo et al., 2012). In particular, MMP-9, which is the major isoform that promotes BBB damage, was observed to be induced in astrocytes, microglia, neurons and endothelial cells after cerebral ischemia (Planas et al., 2001; Lee et al., 2004). In a rat global ischemia model, levels of miR-21 and MMP-9 were shown to increase significantly over a 24-h period in the hippocampus, and suppression of miR-21 resulted in downregulation of MMP-9 (Deng et al., 2013). The mechanistic relation between miR-21 and MMP-9 is not clearly known but might be an indirect effect of targeting an upstream controller of MMP-9 by miR-21. Interestingly, pharmacological inhibition of mitogen-activated protein kinase was shown to abrogate the ischemia-induced increase in both miR-21 and MMP-9, showing the involvement of the extracellular-regulated protein kinase pathway (Deng et al., 2013).

AQPs are a family of water channel proteins that modulate fluid transportation across plasma membranes and play a crucial role in maintaining both intracellular and extracellular water homeostasis (Lehmann et al., 2004; Sepramaniam et al., 2012). To date, at least 13 subtypes of AQPs have been identified, and AQP1, AQP4 and AQP9 are most abundant in central nervous system (CNS) with AQP4 having the highest levels (Papadopoulos and Verkman, 2013). AQP4 is found in astrocytic end feet that line the BBB and is considered to be a key regulator and a major contributor of vasogenic edema after focal ischemia (Manley et al., 2000; Yao et al., 2015; Chu et al., 2016). MiR-130a was shown to be a transcriptional repressor of the AQP4 M1 transcript, which codes for the AQP4 isoform that displays the greatest water permeability (Sepramaniam et al., 2012). Hence, suppression of miR-130 upregulated AQP4 M1 transcript and its protein and further led to reduced infarct volume (Sepramaniam et al., 2012). For miR-320 targets AQP1 and AQP4, its overexpression reduced and inhibition increased AQP1 and AQP4 protein levels after focal ischemia (Sepramaniam et al., 2010). Similarly, inhibition of miR-320 reduced infarct volume and the accumulation of water in the brain 1 day after MCAO by upregulating AQP1 and AQP4 (Sepramaniam et al., 2010). Interestingly, AQP4 was also shown to be a target of miR-29b, and overexpression of miR-29b in a mouse model of focal ischemia resulted in decreased AQP4 expression, reduced BBB disruption, curtailed edema and reduced infarct size (Wang et al., 2015). Furthermore, miR-29b was significantly downregulated in white blood cells after stroke in humans and was negatively correlated with stroke severity (Wang et al., 2015). All these studies show that miRNAs control the spatial or temporal expression of AQPs and thus edema after stroke.

Several studies have also shown that miRNAs modulate brain edema by indirect mechanisms. For example, miR-210 inhibition was shown to significantly increase brain water content following neonatal hypoxia, and overexpression of miR-210 resulted in marked reduction of edema (Zhao et al., 2016). Increases in miR-124 and miR-375 were shown to decrease edema following MCAO upon exposure to lithium or the phytoestrogen calycosin, respectively (Wang et al., 2014d; Doeppner et al., 2016). Overexpression of miR-424 was also shown to decrease brain edema and infarct size after MCAO (Zhao et al., 2013), but miR-424 has also been shown to regulate targets involved in angiogenesis, oxidative stress and microglial activation (Chamorro-Jorganes et al., 2011; Zhao et al., 2013; Liu et al., 2015a). Therefore, several mechanisms could contribute to the ability of miRNAs to control edema and swelling following stroke (Fig. 3).

Fig. 3.

Involvement of miRNAs in regulating brain blood barrier integrity and brain edema post-stroke. miR-15a and miR-150damage the integrity of the BBB by inducing endothelial cell apoptosis and tight junction injury. miR-124 and miR-9 decrease the MMP-9 level to protect the integrity of the basal lamina, but miR-21 induces basal lamina injury by increasing the expression of MMP-9. miR-130, -29b and -320 reduce the accumulation of water by targeting AQP1 and AQP4. Moreover, miR-210, -424 and -325 can decrease ischemia-induced brain edema with unclear mechanisms. The activation and inhibition are indicated by an arrow line and a T-shape line, respectively.

5.5. microRNAs and post-ischemic apoptotic pathways

The penumbra can potentially recover by therapeutic intervention, but based on the timing of therapy, excitotoxicity, oxidative stress and inflammation may promote neuronal apoptosis in the penumbra within hours to days following an ischemic insult (Broughton et al., 2009). Mechanistic studies have established that decreasing pro-apoptotic proteins or enhancing pro-survival proteins protect the brain after cerebral ischemia (Nakka et al., 2008). Recently, several miRNAs were shown to target the translation of proteins of both intrinsic and extrinsic apoptotic pathways and thus alter the outcome after stroke. Importantly, many miRNAs are known to target the anti-apoptotic protein Bcl-2. For example, the miR-15 cluster upregulated following focal ischemia targets Bcl-2, and thus, miR-15 inhibition increases Bcl-2 protein levels and thus protects both endothelial and neuronal cells leading to decreased infarct size and reduced vascular impairment after focal ischemia (Yin et al., 2010a; Shi et al., 2013). miR-497, induced after focal ischemia, also targets Bcl-2 and treatment with antagomiR-497 increased Bcl-2 levels accompanied by reduced infarct volume (Yin et al., 2010b). Following global cerebral ischemia, suppressing miR-181a has been shown to increase Bcl-2 levels and reduce CA1 neuronal loss in the hippocampus (Moon et al., 2013). Furthermore, reducing miR-181a in primary astrocyte cultures led to upregulation of both Bcl-2 and myeloid cell leukemia-1 (Mcl-1) protein levels and diminished mitochondrial dysfunction and apoptosis in response to glucose deprivation (Ouyang et al., 2012a). In cortical neurons, miR-124 agomiRs increased anti-apoptotic Bcl-2 and Bcl-xl expression without altering the expression of pro-apoptotic Bax or Bad and thus protected neurons against OGD (Sun et al., 2013). mir-29b has been shown to repress multiple pro-survival members of the Bcl-2 family, including Bcl-2, Mcl-1, and Bcl-w (Bcl2L2) (Mott et al., 2007; Shi et al., 2012). miR-29b expression was significantly upregulated in rat brain following transient focal ischemia and also promoted neuronal death in cortical neuronal cultures (Shi et al., 2012). Overexpression of Bcl-w rescued neuronal cells against miR-29b-induced death, indicating that miR-29b may promote neuronal death by suppressing Bcl-w and thus the ensuing apoptosis.

Although the majority of miRNAs identified thus far target the intrinsic apoptotic pathway, in vitro studies revealed that miR-21, miR-25, miR-181c regulate TNF signaling in the extrinsic apoptotic pathway. In cultured cortical neurons, overexpression of miR-21 decreased Fas ligand expression and prevented apoptosis following OGD (Buller et al., 2010). Similarly, miR-25 overexpression in neural cell lines prevented OGD-induced apoptosis by decreasing Fas levels (Zhang et al., 2016a). In microglial cultures, OGD increased TNF-α expression while concomitantly suppressing miR-181c expression (Zhang et al., 2012a). Interestingly, miR-181c was shown to suppress TNF-α and partially prevented neuronal apoptosis following OGD (Zhang et al., 2012a). All these studies indicate the potential of miRNAs in promoting or preventing apoptosis after cerebral ischemia (Fig. 2).

In addition to apoptosis, autophagy also plays a critical role in the viability of neurons under hypoxic conditions. An increasing number of evidence has revealed that miRNAs is extensively involved in the ischemic stroke via affecting the pathophysiological process of autophagy. miR-30a and miR-30c are members of miR-30 family, they are critical regulators of neural survival and death in hypoxic condition from previous studies (Ouzounova et al., 2013). In vivo study revealed that miRNA-30a in the brain was upregulated in model of permanent focal cerebral ischemia, while was significantly downregulated in ischemia/reperfusion injury models. Suppression of miR-30a attenuated the neural death and improved behavioral outcome of mice with ischemic stroke through enhancing beclin 1-mediated autophagy (Wang et al., 2014b). The similar result was observed in ischemia/reperfusion injury of spinal cord in rat model, increasing the expression of miR-30c can aggravated the ischemia/reperfusion injury of spinal cord through suppressing the Beclin 1-mediated autophagy (Li et al., 2015a). A recent study found that long noncoding RNAs Malat1 play an important role in ischemia/reperfusion or OGD BMECs. The following study found Malat1 was an endogenous sponge to downregulate the expression of miR-26b, whose potential target was an autophagy-related genes ULK2. So, suppressing the expression of miRNA-26b can promote the BMEC autophagy and survival under OGD condition (Li et al., 2017). Besides, miR-207, a downregulated miRNA after ischemic stroke, can reduce the number of autophagosome and increase the number of autophagic vacuoles in ischemic cortical area, which decreased the infarct volume and improved neurological deficits (Tao et al., 2015a).

5.6. microRNAs and post-stroke neurogenesis

Stroke has been shown to induce neurogenesis in stem cell niches in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG), and the newly born cells migrate to areas of ischemic damage and differentiate into mature neurons (Sailor et al., 2003; Wiltrout et al., 2007; Devaraju et al., 2013; Lin et al., 2015; Zhang et al., 2016b). The miRNAs miR-17-92 and miR-124 are known to regulate neurogenesis during development. The miR-17-92 cluster was observed to be upregulated in neural progenitor cells following focal ischemia in adult mice, and overexpression of miR-17-92 increased proliferation in both cultured progenitor cells and the SVZ following ischemic stress (Liu et al., 2013b). Alternatively, suppression of miR-18a or miR-19a of the miR17-92 cluster abrogated cell proliferation and increased cell death (Liu et al., 2013b). The c-Myc transcription factor has previously been shown to activate expression of miRNAs of the miR-17-92 cluster, including miR-17-5p and miR-20a (O'Donnell et al., 2005). In SVZ neural progenitor cells, ischemia was shown to promote the binding of c-Myc to the promoter region of miR-17-92 and upregulate miR-17-92 expression (Liu et al., 2013b). Furthermore, the sonic hedgehog protein was shown to enhance miR-17-92 following MCAO, potentially via c-Myc signaling (Liu et al., 2013b). A recent study showed miR-17-92 cluster–enriched exosome significantly improved functional recovery and enhanced of oligodendrogenesis, neurogenesis, and neurite remodeling/neuronal dendrite plasticity in the ischemic boundary zone of rats. The underlying mechanism possibly involve targeting phosphatase and tensin homolog to activate the PI3 K/protein kinase B/mechanistic target of rapamycin/glycogen synthase kinase 3β signaling pathway (Xin et al., 2017a). MiR-124 is a crucial regulator of the developing brain and is also constitutively expressed in mature neurons of the adult brain (Delaloy et al., 2010). Focal ischemia was shown to reduce miR-124 expression in neural progenitor cells of the SVZ, and transfection of miR-124 decreased the ischemia-induced proliferation by repressing Jagged-1, which modulates Notch (Liu et al., 2011). Notch signaling maintains the neural stem cell niche of the SVZ (Hitoshi et al., 2002; Androutsellis-Theotokis et al., 2006) and is also required for the induction of neurogenesis after stroke (Wang et al., 2009a, 2009b). Both miR-210 and Notch are upregulated in the ischemic brain, and miR-210 overexpression was shown to significantly increase Notch expression (Lou et al., 2012). It was also shown that miR-210 overexpression robustly promotes neurogenesis in the adult brain (Zeng et al., 2014). Recent studies have shown that miR-210 overexpression increases neural progenitor proliferation and enhances neurobehavioral outcomes following MCAO (Zeng et al., 2016). miR-210 was also shown to be downregulated in the blood of stroke patients and is positively correlated with prognosis (Zeng et al., 2011, 2016), demonstrating the biomarker potential of miR-210 in clinical settings. Mesenchymal stromal cells (MSCs) with overexpressed miR-133b significantly improve functional recovery in MCAO rats and that exosomes generated from MSCs mediate the therapeutic benefits for stroke (Xin et al., 2013). The following study found exosomes isolated from MSCs with overexpressed miR-133b exert increased functional improvement, and neurite remodeling/brain plasticity in the ischemic boundary area (Xin et al., 2017b). The protective mechanism involved exosomes from MSCs mediate the miR-133b transfer to astrocytes and neurons as a vehicle, which regulate target gene expression (connective tissue growth factor and ras homolog gene family member A), subsequently promote neurite remodeling and functional recovery after stroke in rat (Xin et al., 2013, 2017b). (Fig. 4).

Fig. 4.

miRNAs regulate neurogenesis and angiogenesis of post-stroke brain reorganization. miR-17-92 promotes neurogenesis through increased proliferation of neural progenitor cells following ischemic stroke. mR-124 decreases the ischemia-induced proliferation of neural progenitor cells by repressing Jagged-1 and the Notch signaling pathway. miR-210 promotes post-stroke neurogenesis by activating the Notch pathway. Several miRNAs, such as miR-210, -107, -140-5p and -376b-5p, modulate post-stroke angiogenesis by regulating the VEGF pathway. miR-155 and miR-124 promote the post-stroke angiogenesis targeting Rheb and REST, respectively. The activation and inhibition are indicated by an arrow line and a T-shape line, respectively.

5.7. microRNAs and post-stroke angiogenesis

Recent studies have shown the involvement of several miRNAs in post-ischemic angiogenesis, which is critical to restore blood supply to ischemic regions to promote recovery and plasticity after stroke (Liu et al., 2014b). Molecular mechanisms that underlie angiogenesis in post-stroke conditions involve a complicated process regulated by angiogenic factors, such as VEGF, netrins, fibroblast growth factor-2 and platelet-derived growth factor, which promote endothelial sprouting, vascular development and pericyte proliferation and migration (Saharinen et al., 2008; Augustin et al., 2009; Beenken and Mohammadi, 2009; Ferrara, 2009; Gaengel et al., 2009; Lu et al., 2012; Morancho et al., 2015; Yin et al., 2015). Multiple hypoxia-induced miRNAs have been shown to modulate post-stroke angiogenesis via regulating VEGF. It was shown that miR-107 induced by hypoxic conditions via HIF-1 promoted angiogenesis in various cell lines (Chen et al., 2011a, 2013). Further studies showed that miR-107 increased angiogenesis by inducing the expression of endogenous VEGF by downregulating Dicer-1 (Li et al., 2015d). Increased expression of miR-107 was also shown to promote angiogenesis in the penumbra, and treatment with antagomiR-107 reduced capillary density in the penumbra and increased the infarct volume after focal ischemia (Li et al., 2015d). miR-210 upregulated in the ischemic brain was also shown to contribute to angiogenesis by increasing VEGF levels in adult mice (Lou et al., 2012; Zeng et al., 2014). Overexpression of miR-210 activated the Notch signaling pathway, which was shown to cause endothelial cell migration and formation of capillary-like structures in cultured endothelial cells (Lou et al., 2012). In contrast, miR-376b-5p repressed MCAO-induced angiogenesis and was shown to inhibit angiogenesis in vitro by targeting the HIF-1α-mediated VEGF-A/Notch1 signaling pathway (Li et al., 2014b). VEGF-A expression can also be decreased by miR-140-5p, which directly targets the 3′ UTR of VEGF-A (Sun et al., 2016). Following MCAO, miR-140-5p expression is decreased and VEGF-A levels are increased, and in endothelial cultures, miR-140-5p inhibited proliferation, migration, and tube formation after OGD (Sun et al., 2016), indicating an inhibitory effect of miR-140-5p on angiogenesis.

Other miRNAs have been shown to regulate post-ischemic angiogenesis through pathways not associated with VEGF. For instance, miR-124 overexpression initiated neurovascular changes that led to increased angiogenesis 8 weeks following induction of MCAO, potentially through Usp14-dependent degradation of RE1-silencing transcription factor (REST) (Doeppner et al., 2013). Inhibition of miR-155 reduced infarct size, maintained microvascular integrity, preserved capillary tight junctions and improved blood flow in the penumbra following distal MCAO by targeting Rheb, which stabilizes zonula occludens-1 and the tight junctions (Caballero-Garrido et al., 2015). Taken together, changes in miRNA expression following stroke can contribute to neuronal protection by promoting angiogenesis. Similar to the miRNAs discussed in other ischemic mechanisms, increasing protective miRNAs and inhibiting harmful miRNAs may decrease ischemic brain damage and contribute to clinical recovery. It has been mentioned above that miR-145 was involved in the regulation of blood glucose metabolism, which also plays an important role in MCAO rats with diabetes. In vitro study revealed that BMSCs derived from type 1 diabetes rats (DM-BMSCs) increased capillary tube formation and axonal outgrowth in cultured primary cortical neurons, while was attenuated by the overexpression of miR-145. Similarly, DM-BMSC treatment significantly improved functional outcome, increased vascular and white matter remodeling by decreasing serum miR-145 expression in MCAO rat with type 1 diabetes. So, down-regulation of miR-145 may play beneficial effects in the neuro-restoration and DM-BMSCs’ functional in MCAO rats with type 1 diabetes (Cui et al., 2016) (Fig. 4).

6. microRNAs and ischemic tolerance

PC-induced ischemic tolerance is a phenomenon where a sublethal ischemic insult prepares various organs, including brain, heart, liver and kidney, for a subsequent severe ischemic insult (Liu et al., 1992, 2014a; Tsutsui et al., 2013; Rachmat et al., 2014; Zhao and Nowak, 2015; Ma et al., 2016). PC has been repeatedly shown to globally alter miRNA profiles in experimental models (Sun et al., 2015a). For example, cerebral ischemic PC in SHR rats was shown to modulate the expression of several miRNAs after as early as 3 h, and the changes were sustained for at least 3 days (Dharap and Vemuganti, 2010). Importantly, miRNAs that target neurotoxic pathways are induced, and miRNAs that target recovery pathways are reduced after PC, indicating that they promote an environment to protect the brain against a damaging stroke (Dharap and Vemuganti, 2010). Ischemic PC in adult mice also altered the levels of many miRNAs, and importantly, repression of miR-132 and other miRNAs that target methyl-CpG binding protein 2, a transcription regulator implicated in synaptic plasticity, is thought to be neuroprotective (Lusardi et al., 2010). Another study also confirmed that ischemic PC in adult mice alters miRNA profiles and, in particular, showed that increased expression of miR-200 family members target prolyl hydroxylase 2, a protein that prevents accumulation of HIF-1α (Lee et al., 2010).

Ischemic PC has also been shown to alter miRNA levels and protect against ischemia/reperfusion injury in the rodent heart (Yin et al., 2009; Varga et al., 2014). Ventricular injection of purified miRNAs extracted from the preconditioned mouse heart reduced myocardial infarction in naïve mice, indicating the important role of miRNAs to PC-induced cardioprotection (Yin et al., 2009). Notably, the pro-survival miRNA miR-21 was consistently reported to be elevated in the preconditioned rodent heart, and its levels were inversely correlated with the expression of its targets, programmed cell death factor 4 (Dong et al., 2009; Cheng et al., 2010; Duan et al., 2012) and Fas ligand, that promote apoptosis (Sayed et al., 2010). Similarly, ischemic PC in the mouse heart showed induction of miR-24 levels resulting in repression of Bim and CCAAT enhancer-binding protein homologous protein that promote apoptosis, leading to decreased myocardial infarction (Qian et al., 2011; Wang and Qian, 2014). Ischemic post-conditioning, which is also cardioprotective, was shown to alter cardiac miRNA profiles (Varga et al., 2014). Additionally, stem cells subjected to PC have been shown to be more protective against cardiac ischemia than that of untreated stem cells due to exosomal delivery of miRNAs, such as miR-210 and miR-22 (Kim et al., 2012; Feng et al., 2014).

Exposure to chemical agents, such as polyphenols and inhalable anesthetics, has been shown to induce protection against ischemia in a manner comparable to that of ischemic PC (Kitano et al., 2007; Lopez et al., 2015). Some of these agents, such as sevoflurane, isoflurane and resveratrol, have also been shown to alter miRNA expression (Goto et al., 2014; Lu et al., 2015; Ye et al., 2016). Sevoflurane PC has been shown to protect PC12 cells against hypoxia by decreasing the pro-apoptotic miRNA miR-101a and increasing the anti-apoptotic miRNA miR-34b (Hermeking, 2010; Hu et al., 2012; Wang et al., 2014a; Sun et al., 2015b). Sevoflurane PC was shown to induce protection against focal ischemia in rodents by repressing miR-15b, which targets the transcript for the anti-apoptotic protein Bcl2 (Shi et al., 2013). Isoflurane PC has been shown to upregulate the neuroprotective miRNA miR-203 in the brains of adult rats (Cao et al., 2012). Isoflurane treatment was also shown to increase miR-21 levels in rodents to protect cardiomyocytes from lactate-mediated oxidative stress as well as ischemia-reperfusion injury (Olson et al., 2015; Qiao et al., 2015). Resveratrol has been shown to precondition the brain against ischemia (Raval et al., 2006; Dong et al., 2008). Resveratrol-induced ischemic tolerance requires sirtuin 1(which deacetylates histones as well as other proteins, including protein kinase c epsilon), brain-derived neurotrophic factor, mitochondrial uncoupling protein 2, and Nrf2 transcription factor that promotes the expression of several antioxidant proteins (Della-Morte et al., 2009; Morris-Blanco et al., 2014; Koronowski et al., 2015; Narayanan et al., 2015). Resveratrol PC has also been associated with activation of estrogen and NMDA receptors (Saleh et al., 2010). Treatment with resveratrol has been shown to induce miR-21 in the ischemic heart, which is beneficial as miR-21 is anti-apoptotic and anti-inflammatory (Mukhopadhyay et al., 2010). However, a recent study showed that, although resveratrol PC protects the brain and decreases infarct volume after transient MCAO, it does not have a significant effect on cerebral miRNA expression profiles (Lopez et al., 2016).

Remote ischemic PC of a limb was also shown to promote significant tolerance to subsequent stroke in both humans and rodents by promoting release of the pro-survival factors into the blood stream from the preconditioned organ (Dave et al., 2006; Wilson et al., 2007; Zhang et al., 2012b; Connolly et al., 2013). In rodents, the effects of remote ischemic PC are linked to neuroprotective pathways and alterations in peripheral inflammation and immune responses (Malhotra et al., 2011; Wei et al., 2012; Cheng et al., 2014; Liu et al., 2016). Importantly, miRNAs that are encapsulated in exosomes can be released and transported to organs of need, inducing ischemic tolerance (Giricz et al., 2014; Chopp and Zhang, 2015). Circulating miR-144 has been shown to be increased in the serum of both humans and mice subjected to remote ischemic PC, though its neuroprotective significance was not yet investigated (Li et al., 2014a).

7. microRNAs as ischemic stroke biomarkers

As they are detectable in many bodily fluids, including blood and cerebrospinal fluid (CSF), miRNAs can serve as diagnostic stroke biomarkers in humans (Tan et al., 2009, 2013; Li et al., 2015b; Vilar-Bergua et al., 2016). Several miRNAs were shown to be altered in serum and CSF of stroke patients in a time-dependent manner. A study of plasma samples of ischemic stroke patients had identified upregulation of miR-125b-2*, miR-27a*, miR-422a, miR-488, and miR-627 in stroke patients at acute time points (1, 2 and 7 days after stroke onset) but not during the recovery phase (6 months and 2 years after the stroke) (Sepramaniam et al., 2014). Another study showed that miR-106b-5p and miR-4306 were upregulated and miR-320e and miR-320d were downregulated in the blood of stroke patients collected at 3, 6, 12, and 24 h after stroke onset (Wang et al., 2014c). A comparison of CSF and plasma miRNA profiles showed increased levels of miR-151a-3p and miR-140-5p in plasma and increased levels of let-7c and miR-221-3p and decreased levels of miR-18-5p in CSF of patients at 3 days after stroke onset compared to patients with other neurological diseases (Sorensen et al., 2014). Interestingly, some miRNAs, including miR-523-3p, were found exclusively in the CSF (Sorensen et al., 2014). At 1 day after stroke onset in humans, miR-32-3p, miR-106-5p, and miR-1246 levels were upregulated and miR-532-5p levels were downregulated in plasma compared to those of healthy controls (Li et al., 2015b). In a cohort of younger patients (18–49 years), miR-145 levels were significantly increased during the acute phase after stroke and returned to basal levels after several months compared to those of healthy controls (Gan et al., 2012). A recent study showed increased miR-145 levels in a cohort of patients at 1 day after stroke, which was correlated with the infarct volume, National Institute of Health Stroke Scale (NIHSS) score, plasma IL-6 and hs-CRP (Jia et al., 2015). At 1 day after stroke in humans, elevated levels of miR-16 and decreased levels of miR-21 and miR-24 compared to those of healthy controls were demonstrated (Leung et al., 2014; Zhou and Zhang, 2014). In addition, levels of brain-specific miRNAs miR-107, miR-128b, and miR-153 were shown to be upregulated in plasma of stroke patients and positively correlated with NIHSS score (Yang et al., 2016b). Furthermore, circulating levels of two other brain-specific miRNAs, miR-124 and miR-9, were observed to be decreased in the plasma of stroke patients and was associated with higher infarct volume, hs-CRP and MMP9 (Liu et al., 2015b). The miR-29b levels were observed to be downregulated in the plasma of patients collected at 3 days after stroke, and this might be related to BBB disruption and edema as aquaporin-4 is a target of miR-29b (Wang et al., 2015). Studies also showed that miR-30a and miR-126 were downregulated and let-7b was upregulated in patient plasma during acute phase after stroke, which returned to basal levels by 15 to 48 days (Long et al., 2013; Peng et al., 2015). Plasma from acute stroke patients also showed that miR-99a was significantly decreased compared to that of healthy subjects (Tao et al., 2015b). In addition to stroke, ischemia of peripheral organs was also shown to be associated with altered blood microRNA levels. Limb ischemia was shown to be associated with increased expression levels of miR-15a and miR-16in circulating proangiogenic cells (Spinetti et al., 2013), and myocardial ischemia and/or coronary artery disease was shown to increase circulating levels of miR-15a and miR-17-5p (Liu et al., 2012a; Chen et al., 2015c).

8. Role of miRNAs in brain damage in hemorrhagic stroke

Hemorrhagic strokes, which account for ~15% of all stroke cases, are comprised of intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH) (American Heart Association) (Passos et al., 2016). Although the precise cause of hemorrhagic strokes is not known, they are more prevalent in patients with certain co-morbidities, such as diabetes and hypertension. Recent studies showed that both types of hemorrhagic stroke are associated with altered cerebral and blood miRNA profiles.

In adult rats subjected to ICH induced by injecting autologous blood into the brain, several miRNAs were shown to be altered in both brain and blood (Liu et al., 2010). Particularly, 29 miRNAs were altered (17 up- and 12 downregulated by >2-fold) in the brain after ICH. Interestingly, miR-542-3p was upregulated and miR-155, miR-362-3p, miR-122, and miR-450a-5p were downregulated in ICH as well as ischemic stroke (Liu et al., 2010). In the same rats, 41 miRNAs were also altered in blood (21 up- and 20 downregulated). Also in the blood, miR-96, miR-152, miR-298, miR-333, and miR-505 were upregulated and miR-125a-5p, miR-130b, miR-142-3p, miR-330, miR-342-5p, miR-685, and miR-347 were downregulated in ICH as well as in ischemic stroke, indicating that they might be common stroke biomarkers (Liu et al., 2010). Changes in circulating miRNAs were shown to serve as biomarkers to predict secondary hematoma enlargement in patients with ICH (Zheng et al., 2012). Thirty miRNAs associated with inflammation were observed to be upregulated in the blood in both male and female patients with ICH (Guo et al., 2013). Furthermore, a high level of miR-130a was shown to be a marker of edema and a worse outcome after ICH in rats (Wang et al., 2016). In ICH patients, perihematoma edema was also shown to be associated with alteration of serum miRNAs that include miR-126, miR-146, Let-7a and miR-26a (Zhu et al., 2015). Levels of miR-132-3p and miR-324-3p were also shown to be increased significantly in the blood of patients with SAH (Su et al., 2015). Many miRNAs were also shown to be altered in the CSF of patients with SAH (Powers et al., 2016; Stylli et al., 2016).

miRNAs were also shown to modulate secondary brain damage after ICH. Inhibition of Let7c, which was induced after ICH, was shown to decrease edema, apoptotic cell death and inflammation in adult rats (Kim et al., 2014). In adult mice, miR-223 was shown to target and silence the inflammasome NLRP3 and thus prevents the post-ICH edema leading to improved neurological outcome (Yang et al., 2015b). Microglial activation following ICH and the resulting inflammation is known to be modulated by interleukin-1 receptor-associated kinase 4 (IRAK4) upregulated after ICH, which is an essential part of the MyD88-dependent pathway (Yuan et al., 2015). Interestingly, IRAK4 is a target of miR-367, and ICH leads to decreased levels of miR-367 in microglia (Yuan et al., 2015). Treatment of adult mice subjected to ICH with miR-367 mimic inhibited IRAK4, NF-kB, p65, IL-6, IL-1β and TNF-α and thus resulted in decreased inflammation leading to curtailed secondary brain damage and cerebral water content (Yuan et al., 2015).

9. Application of miRNA-based therapies

As described above, miRNAs likely represent both new biomarkers and new therapeutic targets for stroke. As targets for stroke therapy, miR-based strategies provide the merit of rapid onset of action, an essential factor in developing valid clinical treatments for stroke. Several companies are developing miR-based therapeutics. A successful phase 2 trial of the first miR-targeted drug, a locked nucleic acid (LNA) targeting miR-122 to treat hepatitis C, which was completed, demonstrating that translation of miR-based therapies to clinic may be possible if only candidate targets are identified (Janssen et al., 2013). However, no consensus seems to have emerged at the time-points of treatment, administrative routes, chemical modifications and dose of miRNAs in stroke therapy. Ongoing research reveals that both pre- and post-treatment with miRNAs could affect ischemia injury, the time-point of treatment might depend on the temporal changes of miRNAs during the process of stroke.

9.1. Current delivery strategies for microRNAs

Administrative routes of miRNAs in animals include transgene or gene knockout, or exogenous injection into the lateral ventricle, the infarct area, the vein, or intranasal administrations. But targeted delivery of miRNAs, particularly into the CNS of stroke patients remains the major barrier. Direct administration of miRNAs into the ventricles, is a potential method (Yin et al., 2010b; Ouyang et al., 2012b), but the resulting immune-system activation and tissue damage present some defects. Therefore, delivery methods that afford passage across the BBB have been explored. Using adeno-associated virus (AAV) vector is a way to cross the BBB, and recombinant AAV9 capsid effectively passes through the BBB (Foust et al., 2009). The miRNA in the vectors can be continually expressed, resulting in strong substitution expression of miRNAs downregulated following stroke. Furthermore, AAV is currently used in a number of clinical trials for gene therapy, and the safety profiles have looked quite well. Another method involves the use of intranasal administrations can gain access to the brain rapidly along the olfactory nerve pathway from the nasal cavity directly to the brain, bypassing the BBB. This noninvasive method has been proved effectively with a miR-206-neutralizing antagomir (Lee et al., 2012). In contrast to exogenously administered cells delivered systemically, exosomes, given their nano dimension may easily pass through the BBB and readily enter the brain and deliver miRNA molecules (Schorey and Bhatnagar, 2008; Alvarez-Erviti et al., 2011; Katakowski et al., 2013). However, as a result of their ability to target numerous genes, systemic administration of miRNAs may also have unexpected effects on other organs, which may limit the clinical utilization of a candidate miRNA.

9.2. Chemical modification and design of microRNAs

The therapeutic application of miRNAs can be carried out by two approaches, either by inhibition of miRNAs using multifarious chemically modified oligonucleotides complementary with the sequence of the miRNA (Hutvagner et al., 2004) or by mimicking the function of miRNAs by the use of so-called miRNA mimics (van Rooij et al., 2008). Another drawback of RNA-based treatment paradigms is miR degradation by endogenous RNAases, limiting their pharmacological efficacy. However, miRs can be chemically modified to enhance both stability and transfer across cell membranes. For in vivo use, some distinct chemical modifications have been exploited to improve their pharmacokinetics (Port and Sucharov, 2010; Latronico and Condorelli, 2011). These modifications include the initial antagomiRs in which anti-miR oligonucleotides are modified by incorporations of a methyl group (2′-O-methyl) together with the partial phosphorothioate linkage and cholesterol conjugation at the 3′ end of the strand (which improves tissue distribution and cellular uptake), alterations to the sugar moiety with 2′-O-methoxyethyl phosphorothioate and the use of LNAs. Santaris Pharmaceuticals has launched a phase II clinical trial using liver-specific anti-miR-122 LNA for the treatment of hepatitis C. Besides, modified double-stranded miRNA mimics with one strand containing the same sequence as the mature miRNA contain uridine bases and uridine-quanin nucleotide pairs that induce a non-specific immune reaction by activating Toll-like receptors (Judge et al., 2005). As the sequence of miRNA mimic cannot be changed without its specificity or efficacy being concessive, chemical modifications of these nucleotide-based are necessary to minimize the immunological response. Exploring more clinically applicable approaches to alter endogenous miRNA production, such as intravenous or intraperitoneal administration of chemically-stabilized miRNA inhibitors and mimics, are pivotal next steps for efficient clinical translation.

In addition, the dose of miRNAs treatment in stroke animals was determined by miRNAs types, administrative routes, animal weight, etc. All studies by now have showed that miRNAs intervention can affect the prognosis of stroke, but there is a lack of direct studies on the dose, type of miRNAs and administrative routes. So, further work is needed to explore the stroke-related function of miRNAs in dose, administrative routes and more time points of brain ischemia injury.

10. Conclusions

Emerging research has identified miRNAs as gene regulators that are essential for controlling various functions in both normal and diseased brain. miRNAs can target hundreds of proteins in multiple regulatory networks in the cell in normal and diseased states. Normal expression of miRNAs is essential for appropriate brain development and function, whereas dysregulation of miRNAs in brain cells or the neurovasculature increases susceptibility to stroke and other neurological disorders. These changes have been shown to play a critical role in stroke pathophysiology.

Studies from experimental animal models have suggested that promoting or suppressing expression of miRNAs involved in various pathogenic mechanisms by specific pharmaceutical and non-pharmaceutical therapies may be beneficial for post-stroke recovery. However, several challenges may stand in the way of using miRNAs effectively in post-stroke therapy. A major hurdle in the stroke field is the limited understanding of the multiphasic nature and time-dependent interactions of the molecular and cellular changes after ischemia and the role of various miRNAs in these processes. At present, miRNA stroke research has focused on the relationship between a single miRNA and its target genes. However, investigations on co-regulated networks of miRNAs are needed to understand the role of integrated mechanisms of complex post-transcriptional regulations. Introducing agents that facilitate the transport of miRNAs across the BBB and deliver them to the affected tissue is another major challenge. Hundreds of miRNAs have been observed to undergo changes in peripheral blood samples of patients with acute stroke as well as chronic time points, and they might provide new avenues to serve as biomarkers for rapid diagnosis and treatment efficacies in various stroke subtypes.

Abbreviations:

AAV

adeno-associated virus

ABCA1

ATP-binding cassette transporter 1

AGO

argonaute

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazole propionate

AT1R

angiotensin II type 1 receptor

AQPs

aquaporins

BBB

blood–brain barrier

Bcl-6

B-cell lymphoma-6

BMSCs

bone-marrow stromal cells

CDC25A

cell division cycle 25A

CDK6

cell division protein kinase 6

CNS

central nervous system

CSF

cerebrospinal fluid

DG

dentate gyrus

ERK

extracellular signal-related kinase

GLT-1

glutamate transporter 1

GluR2

glutamate receptor 2

GRP78

glucose-regulated protein 78

HIF1-α

hypoxia-inducible factor 1 alpha

HMGB1

high mobility group box-1

hs-CRP

high-sensitivity C-reactive protein

ICH

intracerebral hemorrhage

IL-1R

interleukin-1 receptor

IL-6

interleukin 6

IRAK4

interleukin-1 receptor-associated kinase 4

LNAs

locked nucleic acids

miRNAs

microRNAs

Mcl-1

myeloid cell leukemia-1

MCAO

middle cerebral artery occlusion

MMP-9

matrix metalloproteinase-9

Mn-SOD

manganese superoxide dismutase

MyD88

myeloid differentiation factor 88

NCOA1

nuclear receptor coactivator 1

NF-κB

nuclear factor kappa B

NIHSS

National Institute of Health stroke scale

NMDA

N-methyl-d-aspartate

NO

nitric oxide

NR2B

NMDA receptor subunit 2B

Nrf2

nuclear factor erythroid 2-related factor

3-NT

3-nitrotyrosine

OGD

oxygen and glucose deprivation

PC

preconditioning

PPAR-δ

peroxisome proliferator-activated receptors-δ

pre-miRNAs

precursor miRNAs

pri-miRNA

primary miRNAs

REST

RE1-silencing transcription factor

RISC

RNA-induced silencing complex

ROS

active oxygen species

SAH

subarachnoid hemorrhage

shh

sonic hedgehog protein

SHRs

spontaneously hypertensive rats

SVZ

subventricular zone

TNFα

tumor necrosis factor alpha

VEGF

vascular endothelial growth factor

VNS

vagus nerve stimulation

ZO-1

zonula occludens-1