Blood microRNAs as potential diagnostic markers for hemorrhagic stroke.

Proper medical treatment of a stroke victim relies on accurate and rapid differentiation between ischemic and hemorrhagic stroke, which in current practice is performed by computerized tomography (CT) or magnetic resonance imaging (MRI) scans. A panel of microRNAs could be an extremely useful clinical tool for distinguishing between hemorrhagic and ischemic stroke. This review has shown that blood miRNA profile can distinguish hemorrhagic from ischemic stroke in patients and in experimental animal models. It also seems likely they can differentiate between intracerebral and subarachnoid hemorrhage stroke. The miRNA profile in cerebrospinal fluid could be a useful diagnostic tool for subarachnoid hemorrhagic stroke. Decreased or increased miRNA levels may be needed either as prevention or treatment of stroke. Administration in vivo of miR-130a inhibitor or miRNA mimic (miR-367, miR-223) in an intracerebral hemorrhage animal model improved neurological outcomes.

Introduction

Strokes can be broadly classified as ischemic or hemorrhagic. Hemorrhagic strokes account for about 20% of all strokes and are divided into categories depending on the site and cause of bleeding. In intracerebral hemorrhage (ICH), bleeding occurs from a ruptured blood vessel within the brain. Hypertension, excessive alcohol intake, and advanced age are all important risk factors. Ischemic strokes can convert to an ICH (Berger et al., 2001), and may be associated with infective endocarditis (Morris et al., 2014). A subarachnoid hemorrhage (SAH) involves bleeding from a damaged blood vessel causing blood to accumulate at the surface of the brain. Most often, a SAH happens because of a leaking saccular aneurysm. Hemorrhagic stroke is life threatening with up to 50% of all people with ICH dying, many within the first two days. Surgical removal of the hematoma as an early-stage treatment for ICH may improve long-term prognosis (Morgenstern et al., 1998), but no effective targeted therapy for hemorrhagic stroke exists yet. ICH is more likely to result in death or major disability than ischemic stroke or SAH. The sudden buildup of pressure outside the brain in SAH may cause loss of consciousness or death.

Proper medical treatment of a stroke victim relies on accurate and rapid differentiation between ischemic and hemorrhagic stroke. Not only do ischemic and hemorrhagic stroke have completely divergent therapeutic options, the treatment itself can convert ischemic stroke to hemorrhagic stroke (Zhang et al., 2014). Clinically, it is therefore crucial to monitor and distinguish ischemia versus hemorrhage stroke within the first week of symptom onset to prevent adverse outcome. Also it is important to distinguish between ICH and SAH as this will influence possible treatment. In current practice, diagnosis of hemorrhage versus ischemia stroke is performed by computerized tomography (CT) or magnetic resonance imaging (MRI) scans. There is a need for a reliable, relatively inexpensive method for differentiating between ischemic and hemorrhagic stroke in patients - potentially a point-of-care assay that can be performed on a daily basis within the first week of stroke onset. Most biomarkers associated with stroke and proposed as diagnostics in the emergency room for acute stroke are blood-borne proteins of tissue injury such as C-reactive protein, matrix metallopeptidase 9, D-dimer, S100β protein, and B-type natriuric peptide (Lopez et al., 2012).

MicroRNAs are small non-coding RNAs of approximately 22 nucleotides long, involved in the regulation of gene expression, thus controlling a range of physiological and pathological functions such as development, differentiation, apoptosis and metabolism (Ambros, 2004). It has been shown that serum or plasma miRNAs are stable and indicative of the disease state (Chen et al., 2008). Recently much interest has developed in the use of circulating cell-free miRNAs as novel markers in the clinical diagnosis of disease especially in cancer (Ho et al., 2010). This article reviews recent human and animal studies of miRNAs as biomarkers of hemorrhagic stroke, and whether specific miRNAs, or a combination, can be used to distinguish between ischemic and hemorrhagic stroke.

MicroRNAs in Hemorrhagic Stroke

Leung et al. (2014) compared miR-124-3p and miR-16 plasma levels in 93 stroke patients, median age 72 years, 51% male. Ischemic stroke was diagnosed in 74 patients and 19 patients were diagnosed with hemorrhagic stroke, with blood samples being collected within 24 hours of symptom onset. Twenty-three age- and sex-matched healthy individuals were recruited as controls. Hypertension was a major stroke risk factor in the patients. Hemorrhagic stroke patients had higher median plasma miR-124-3p levels than ischemic stroke patients and healthy controls (1.94 and 2.55 fold change, respectively). The median plasma level of miR-16 was increased in ischemic stroke patients compared with hemorrhagic stroke patients and healthy controls (1.24 and 1.35 fold change, respectively). This study did not indicate the number of hemorrhagic stroke patients diagnosed with ICH or SAH, and therefore it is not known whether plasma levels of miR-124-3p and miR-16 can differentiate between these two categories. The findings from ICH and SAH studies are summarized in Tables and , respectively.

Intracerebral hemorrhage

Human studies

Four clinical studies were found. They indicated that serum miR-130a, or a panel of blood specific miRNAs, could distinguish ICH patients from controls. Additionally, plasma miR-29c and miR-122 could distinguish between hematoma enlargement group and non-hematoma enlargement group of ICH patients.

Animal studies

Three studies had been performed with ICH rats and one study with ICH mice. Inhibition of miR-130a or enhancement of miR-367 or miR-223 had positive outcome by inhibiting inflammation ().

Subarachnoid hemorrhage

Four clinical studies were found. Blood miR-132 and miR-324 could differentiate SAH patients with delayed cerebral infarction and SAH patients with non-delayed cerebral infarction from controls. Also a panel of specific miRNAs in cerebrospinal fluid could distinguish SAH patients from controls, and SAH patients with no vasospasm from SAH patients with vasospasm.

One study in SAH rats showed miR-30a and miR-143 might be useful biomarkers for SAH ().

Targeting MicroRNAs as a Novel Therapeutic Approach

Both increased and decreased miRNA levels may be needed either as prevention or treatment of hemorrhagic stroke. Using an experimental model of ICH, injection of miR-130a inhibitor into the right lateral ventricle before ICH induction in male rats significantly reduced endogenous expression of miR-130a, decreased brain edema, and alleviated brain-blood barrier disruption at 1 day after ICH. Neurological function was significantly improved (Wang et al., 2016). Also in a mouse model of ICH, intracerebroventricular injection of miR-367 mimic significantly increased the miR-367 level in vivo, and significantly inhibited interleukin-1 receptor-associated kinase 4 (IRAK4), nuclear factor-κB (NF-κB), p65, interleukin 6 (IL-6), IL-1β and tumor necrosis factor-alpha (TNF-α) expression in brain tissues after ICH, indicating that miR-367 could inhibit inflammatory response in vivo. A miR-367 mimic significantly decreased brain edema and neurological injury (Yuan et al., 2015; ). Overexpression of mir-223 following intracerebroventricular injection of miR-223 mimic in ICH mice resulted in reduced brain edema, and improved neurological functions. MiR-223 significantly inhibited caspase-1 p20, NLRP3, TNF-α, IL-1β, and IL-6 expression in brain tissues after ICH, showing that miR-223 could inhibit inflammatory response in vivo (Yang et al., 2015).

Possible molecular mechanism of miR-367 mimic-mediated support of post-stroke recovery in intracerebral hemorrhage (ICH) mice.

Upregulation of brain tissue miR-367 results in decreased brain edema and brain injury, and improved neurological function in treated animals. Possible molecular mechanisms mediating these processes could be associated with inhibition of brain tissue interleukin (IL)-1 receptor-associated kinase 4 (IRAK4) expression and decreased brain tissue nuclear factor-κB (NF-κB) activation by miR-367 mimic, leading to a decrease in proinflammatory mediators interleukin 6 (IL-6), IL-1β, and tumor necrosis factor-alpha (TNF-α).

Future Perspectives

Numerous circulating miRNAs have been reported to have a potential value in diagnosis of hemorrhagic stroke, with considerable variation in findings. Most of the studies had performed RT-PCR to validate changes in miRNAs detected by microarray analysis. In several studies, changes in specific miRNAs were confirmed in experimental hemorrhagic stroke in healthy rats or mice. The findings of previous clinical studies need to be repeated in other hospital centers and include both male and female patients. Also experimental animal studies should be performed with hemorrhagic stroke rats or mice of both sexes, and to use antagomirs or mimics to decrease or increase miRNAs, respectively. As most hemorrhagic stroke patients have existing comorbidities and are aged ≥ 50 years, confirmation studies should involve animal models with hypertension, hyperlipidemia, diabetes mellitus, and aging. The clinical study by Leung et al. (2014) and the experimental animal study by Liu et al. (2010) have shown that miRNA profile can distinguish hemorrhagic stroke from ischemic stroke. The reported miRNA profiles in the studies reviewed would suggest that they can differentiate between ICH and SAH, and clinical studies should be performed to confirm this.

Table 1

MicroRNAs in intracerebral hemorrhage (ICH) patients and experimental animal models

Table 2

MicroRNAs in subarachnoid hemorrhage (SAH) patients and experimental animal models