Oxidative stress is one of the mechanisms involved in ischemic brain injury induced by ischemia-reperfusion [1,2]. In addition to the direct cytotoxic effects of lipid peroxidation, oxidative DNA damage occurs immediately after ischemia-reperfusion and leads to ischemic brain injury, as reflected by neural death and brain edema [3-7]. Thus, the burst of free radicals is thought to be associated with stroke severity. However, it remains uncertain how to evaluate the association between increased oxidative stress and functional outcomes of patients. In the presence of oxidative stress, reactive oxygen species, including superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals, can cause oxidative damage to lipids, proteins, and nucleic acids [8]. During oxidative stress, DNA is constantly being damaged and oxidatively modified. Nuclear and mitochondrial DNA in tissues and blood lymphocytes are typical sites of oxidation damage [8].

The oxidatively modified product 8-hydroxy-2-deoxyguanosine (8-OHdG) is one of the predominant forms of free radical-induced lesions of DNA. 8-OHdG can be detected in human tissues or blood samples. Because oxidative DNA lesions, i.e., oxidized nucleosides and bases, are reasonably water soluble and excreted into the urine without being further metabolized, urinary 8-OHdG is considered an important biomarker of generalized and cellular oxidative stress [9]. In fact, urinary 8-OHdG levels have been most frequently measured to indicate the extent of oxidative damage, since it is a non-invasive and technically less involved procedure [10].

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, Mitsubishi Pharma Corp), a novel radical scavenger, has been shown to inhibit lipid peroxidation and vascular endothelial cell injury and ameliorate brain edema, tissue injury, delayed neuronal death, and neurological deficits [11-13]. Edaravone is the first neuroprotective agent to be approved for clinical use in Japan [14]. The effect of edaravone on ischemic processes has been investigated experimentally, but no data is available on its effects on oxidative stress markers in the human ischemic brain.

In the present study, we examined oxidative stress in patients with acute ischemic stroke by determining the changes in urinary 8-OHdG, a maker of oxidative damage. We evaluated the relationships between 8-OHdG levels, stroke subtypes, and clinical outcomes based on the National Institutes of Health Stroke Scale (NIHSS) and the modified Rankin Scale (mRS) at discharge. The effect of edaravone on oxidative damage was also examined.

In order to evaluate ischemic brain damage, numerous biomarkers related to oxidative stress, such as nitric oxide, matrix metalloproteinase-9, S100β, and oxidized LDL, have been investigated [19-22]. The main finding of our study is the demonstration of an association between urinary 8-OHdG and ischemic stroke. The present study provides evidence that Δ 8-OHdG, specifically the rate of change of urinary 8-OHdG, may be considered a marker of tissue injury in brain ischemia. In fact, mRS scores (i.e., clinical outcomes at discharge) showed a significant correlation with Δ 8-OHdG (Fig. 3). These findings suggest that Δ 8-OHdG may act as a mediator during the evolution of ischemic damage.

In all 3 subtypes of ischemic stroke, lacunar, atherothrombotic, and cardioembolic infarctions, urinary 8-OHdG levels increased on Day 7. When we compared the rate of change of urinary 8-OHdG (Δ 8-OHdG), Δ 8-OHdG was significantly higher in patients in the atherothrombotic infarction group than that of patients in the lacunar infarction group (Fig. 1). Moreover, Δ 8-OHdG of patients in the lacunar infarction group tended to be lower than that of patients in the cardioembolic infarction group, suggesting that a smaller infarction volume can result in lesser amounts of oxidative stress. However, the questions arise as to why Δ 8-OHdG increased more in the atherothrombotic infarction than in the cardioembolic infarction group.

Numerous studies have shown that urinary 8-OHdG is not only a biomarker of oxidative stress but also a possible risk factor for cancer, coronary heart disease, diabetes, and atherosclerosis [23,24]. For example, elevated levels of urinary 8-OHdG have been detected in patients with diabetes mellitus or hypertension [25]. In human atherosclerotic plaques, increased amounts of oxidatively modified DNA and 8-OHdG are present [26]. Thus, DNA oxidative stress may readily occur in patients with these conditions. In this study, the atherothrombotic infarction group had more number of risk factors for arteriosclerosis, such as hypertension, diabetes mellitus, and hyperlipidemia, and the cardioembolic group had less number of risk factors than the other groups (Table 1). In addition, the risk factors for arteriosclerosis such as diabetes mellitus, hyperlipidemia, and BMI influence on a production of the oxidative stress (Fig. 2). Thus, patients with atherothrombotic infarctions may have some background conditions for which 8-OHdG is easily generated, and more increased oxidative damage may cause poor outcome comparable to that of patients with cardioembolic infarctions, despite having the less infarction volume.

There is increasing evidence for the contributions of oxidative stress to ischemic brain damage in primary and secondary pathophysiological mechanisms [1,2]. In addition to the direct cytotoxic effects of lipid peroxidation and oxidative DNA damage that occurs immediately after ischemic injury, the burst of free radicals also induces formation of inflammatory mediators through redox-mediated signaling pathways, leading to post-ischemia/reperfusion inflammatory injury [4,6]. Therefore, the control of these 2 impacts of oxidative damage is important to achieve neuroprotection.

Recent studies demonstrated that edaravone provided neuroprotection in the integrated processes of oxidative stress-induced injury in ischemic stroke [27,28]. Edaravone has antioxidant properties that result in suppressing lipid peroxidation products and oxidative DNA damage, as well as anti-inflammatory effects that result in reduced iNOS production [29-33]. Further studies by using animal model suggested that a combination therapy with edaravon and tissue plasminogen activator (rt-PA) could provide important therapeutic benefits for acute stroke patients, not only in reducing the infarct size but also in minimizing the catastrophic hemorrhagic transformation. In these studies, edaravone suppressed matrix metalloproteinase-9 (MMP-9) upreguration and hemorrhagic transformation by scavenging rt-PA-induced reactive oxidative stress derived from ischemia/reperfusion injury [34,35].

As the present study was not a randomized trial, there could have been a bias in the selection of patients for edaravone use. However, although patients who received edaravone tended to be classified as more severe based on NIHSS scores at admission than those who did not receive edaravone, both groups showed equivalent mRS scores at discharge (Table 3). Furthermore, Δ 8-OHdG levels in patients who received edaravone tended to be reduced compared to those who did not receive edaravone within the atherothrombotic infarction group (Fig. 4). Thus, edaravone treatment might provide a neuroprotective effect by reducing oxidative damage and it may contribute to limiting brain damage.

The present study demonstrated a significant association between elevated urinary 8-OHdG levels and acute ischemic stroke, especially for those with atherothrombotic infarctions. Because Δ 8-OHdG, the rate of change of urinary 8-OHdG, showed a significant relationship to mRS scores at discharge, Δ 8-OHdG may be a useful predictor of poor outcomes after ischemic stroke. Edaravone is useful for reducing oxidative damage, especially in patients with atherothrombotic infarctions, and thus, it may contribute to limiting brain damage in patients with ischemic stroke.

The authors declare that they have no competing interests.

HN performed analyses, collected data and wrote the manuscript. KU helped to draft the manuscript and collected data. TI, HK, and FK helped to draft the manuscript. All authors read and approved the final manuscript.