Beyond free radical scavenging: Beneficial effects of edaravone (Radicut) in various diseases (Review).

Free radicals play an important role in the pathogenesis of a variety of diseases; thus, they are an attractive target for therapeutic intervention in these diseases. Compounds capable of scavenging free radicals have been developed for this purpose and some, developed for the treatment of cerebral ischemic stroke, have progressed to clinical trials. One such scavenger, edaravone, is used to treat patients within 24 h of stroke. Edaravone, which can diffuse into many disease-affected organs, also shows protective effects in the heart, lung, intestine, liver, pancreas, kidney, bladder and testis. As well as scavenging free radicals, edaravone has anti-apoptotic, anti-necrotic and anti-cytokine effects in various diseases. Here, we critically review the literature on its clinical efficacy and examine whether edaravone should be considered a candidate for worldwide development, focusing on its effects on diseases other than cerebral infarction. Edaravone has been safely used as a free radical scavenger for more than 10 years; we propose that edaravone may offer a novel treatment option for several diseases.

Related papers:

title: The lipophilic multifunctional antioxidant edaravone (radicut) improves behavior following embolic strokes in rabbits: A combination therapy study with tissue plasminogen activator
abstract: Edaravone is a lipophilic drug with multiple mechanisms of action. Because edaravone is a promising drug candidate for the treatment of stroke, we tested the hypothesis that edaravone would be neuroprotective following cerebral ischemia using a rabbit embolic stroke model with a well-defined behavioral endpoint. Using the rabbit small clot embolic stroke model (RSCEM), a drug or drug combination is considered beneficial if it significantly increases the amount of microclots (mg) measured in brain that produce neurologic dysfunction in 50% of a group of animals (P(50)) compared to the control group. Edaravone (100 mg/kg, s.c.), increased the P(50) value to 1.80+/-0.24 mg (p<0.05) when administered 5 min following embolization and increased P(50) values by 195% and 161% (compared to control) when administered 1 and 3 h following embolization, respectively, but was inactive when applied 6 h following embolization, compared to the cumulative control group (P(50)=0.93+/-0.16 mg). To simulate the design of current clinical trials, edaravone was also given following a standard tPA regimen, which by itself increased the P(50) value to 2.72+/-0.28 mg. When tPA was infused 1 h following embolization and edaravone was given 3 h following embolization, the P(50) was 2.68+/-0.56 mg. This study indicates that edaravone may have substantial therapeutic benefit for the treatment of AIS since it had a therapeutic widow of at least 3 h in rabbits. Edaravone can also be administered with a thrombolytic to improve behavior.

title: Neuroprotective Effects of Free Radical Scavengers in Stroke
abstract: Acute ischaemic stroke is a leading cause of death in the majority of industrialised countries and also in many developing countries. Free radicals are generated in the brain during ischaemic injury and these radicals are involved in the secondary injury processes. Several free radical scavengers have been developed and some of them have progressed into clinical trials. One of them, edaravone, has been approved by the regulatory authority in Japan for the treatment of stroke patients. Another scavenger, disodium 4-[(tert-butylimino)methyl] benzene-1,3-disulfonate N-oxide (NXY-059; disufenton), has demonstrated efficacy in a phase III clinical trial (SAINT [Stroke Acute Ischaemic NXY-059 Treatment study]-I) involving a large number of stroke patients. Unfortunately, SAINT II did not show efficacy in the treatment of stroke patients. The purpose of this article is to review the current development of antioxidant strategies, update recent findings for NXY-059 in the treatment of stroke patients, and discuss the future development of neuroprotective agents. Although the development of neuroprotective strategies for the treatment of stroke is challenging, progress in molecular and cellular neuroscience will uncover new information about stroke mechanisms, which should result in the realisation of neuroprotective therapy for this disease.

title: Therapeutic strategies for the treatment of stroke.
abstract: Acute ischaemic stroke is a major health problem with no effective treatments apart from the thrombolytic recombinant tissue plasminogen activator (rt-PA), which must be given within 3h of stroke onset. However, rt-PA increases the risk of symptomatic intracranial haemorrhage and is administered to <5% of stroke patients. New perfusion-enhancing compounds are in development but the risk:benefit ratio remains to be determined. Many neuroprotective drugs have been studied but all those that reached clinical development have failed to demonstrate efficacy. However, adherence to recently published guidelines on preclinical development has resulted in one novel compound (NXY-059) demonstrating efficacy in a Phase III trial, providing encouragement for the validity of the concept of neuroprotection. There are a variety of new neuroprotective compounds in the early stages of investigation and some could prove clinically effective, provided appropriate preclinical development guidelines are observed.

title: The effect of tirilazad mesylate on infarct volume of patients with acute ischemic stroke
abstract: The authors investigated whether the lack of effect of tirilazad on clinical outcome in patients with acute ischemic stroke is explained by failure of tirilazad to reduce infarct volume. Overall, tirilazad had no significant effect on infarct volume. In the subgroups of male patients and of those with a cortical infarct, tirilazad significantly reduced infarct volume. These effects were reduced to nonsignificant trends after adjustment for imbalances in baseline characteristics. In conclusion, early treatment of patients with tirilazad has no effect on infarct volume.

title: NXY-059 for the treatment of acute ischemic stroke.
abstract: The free-radical-trapping agent NXY-059 showed promise as a neuroprotectant in the Stroke-Acute Ischemic NXY Treatment I (SAINT I) trial, reducing disability when given to patients who had acute ischemic stroke. We sought confirmation of efficacy in a second, larger trial.



We enrolled 3306 patients with acute ischemic stroke in a randomized, double-blind trial to receive a 72-hour infusion of intravenous NXY-059 or placebo within 6 hours after the onset of stroke symptoms. Our primary end point was the distribution of disability scores on the modified Rankin scale at 90 days. We examined scores on neurologic and activities-of-daily-living scales as secondary end points. We also tested the hypothesis that NXY-059 would reduce alteplase-related intracranial hemorrhages.



The efficacy analysis was based on 3195 patients. Prognostic factors were well balanced between the treatment groups. Mortality was equal in the two groups, and adverse-event rates were similar. The distribution of scores on the modified Rankin scale did not differ between the group treated with NXY-059 (1588 patients) and the placebo group (1607 patients; P=0.33 by the Cochran-Mantel-Haenszel test; odds ratio for limiting disability, 0.94; 95% confidence interval [CI], 0.83 to 1.06). Analysis of categorized scores on the modified Rankin scale confirmed the lack of benefit: the odds ratio for trichotomization into modified Rankin scale scores of 0 to 1 versus 2 to 3 versus 4 to 6 was 0.92 (95% CI, 0.80 to 1.06). There was no evidence of efficacy for any of the secondary end points. Among patients treated with alteplase, there was no difference between the NXY-059 group and the placebo group in the frequency of symptomatic or asymptomatic hemorrhage.



NXY-059 is ineffective for the treatment of acute ischemic stroke within 6 hours after the onset of symptoms.

title: Hospital-Based Study of the Care and Cost of Acute Ischemic Stroke in Japan
abstract: To evaluate the current status of care and cost of acute ischemic stroke in Japan, we performed a hospital-based analysis at a tertiary emergency hospital with a 24-hour neurology-neurosurgery team and care unit.  During the 12-month period of October 2000 to September 2001, we collected data on 179 patients consecutively hospitalized with acute ischemic stroke within 7 days of onset. We examined demographic data, in-hospital care, length of hospital stay, outcome at discharge, and hospital costs. The medical cost data were collected from official hospital medical cost charts, which calculated direct medical costs for beds, staff, examinations, medications, and rehabilitation.  The mean age was 70 years, and 69% were male. Hospital arrival was within 3 hours of onset in 30% of the patients. A history of stroke was present in 37%. The mean initial National Institutes of Health Stroke Scale score was 8.3 points (median, 6 points). Using the Trial of Org 10172 in Acute Stroke Treatment classification, 25% were lacunar, 27% were atherothrombotic, 33% were cardioembolic, and 15% were of unknown origin. All patients underwent neuroimaging studies during hospitalization; 96% and 92% underwent CT and MRI with MR angiography, respectively. Antithrombotic medications were given in 94%, none of whom received thrombolysis. A newly licensed neuroprotective agent, edaravone, was given in 16%. More than half of the patients (55%) were initially admitted to the neurological intensive care unit. Overall, 64% received in-hospital rehabilitation. Mean length of stay was 33 days. In-hospital mortality rate was 3%. On the modified Rankin Scale (mRS), 63% were independent (mRS, 0 to 2) and 34% were dependent (mRS, 3 to 5) at discharge. Two thirds of the patients (65%) went directly back home. The mean hospital cost per patient was $6887 ($209/d), of which 69% was attributable to the costs for beds and staff, 12% for medications, 7% for rehabilitation, 6% for imaging studies, 5% for laboratory examinations, and 1% for other costs.  Despite the single hospital-based analysis, this study provided current, precise data on short-term inpatient care and costs of acute ischemic stroke in Japan. Because stroke often carries a permanent dependence, long-term cost-effective stroke care should be established.

title: Delayed neuronal death prevented by inhibition of increased hydroxyl radical formation in a transient cerebral ischemia
abstract: The salicylate-trapping method was used to detect hydroxyl radicals by measurement of stable adduct dihydroxybenzoic acid (DHBA). Ten minutes of forebrain ischemia followed by reperfusion induced the increase in DHBA in rat hippocampal perfusates. Postischemic treatment with a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), significantly reduced the increase in DHBA and suppressed delayed neuronal death in the hippocampal CA1 region.

title: Development of the nitrone-based spin trap agent NXY-059 to treat acute ischemic stroke.
abstract: The only current FDA-approved treatment for acute ischemic stroke is thrombolysis with tissue plasminogen activator (tPA). However, there are numerous shortcomings to tPA treatment including an increased incidence of intracerebral hemorrhage (ICH) and a short therapeutic window (3-6 h). In recent years, studies have attempted to identify new therapeutics that might be neuroprotective following ischemic strokes. Free radical scavenging spin trap agents have been proposed as potential candidates for stroke therapy because of the hypothesized role of free radicals in the progression of stroke and ischemia-induced neurodegeneration. Novel spin trap agents like (disodium-[(tert-butylimino) methyl] benzene-1,3-disulfonate N-oxide (NXY-059) are of particular interest, not only because they are broad-spectrum nitrone-based free radical scavengers, but also because of their safety profile in humans. Moreover, the rationale for developing NXY-059 for the treatment of acute ischemic stroke is further supported by the drug's reported neuroprotective effects. In addition, NXY-059 may represent a useful adjunct stroke therapy to tPA, since preclinical studies have demonstrated that NXY-059 increases the therapeutic window for tPA and lowers the occurrence of tPA-induced ICH.

title: Glutathione Peroxidase Overexpression Inhibits Cytochrome c Release and Proapoptotic Mediators to Protect Neurons From Experimental Stroke
abstract: Ischemic injury and reperfusion increases superoxide (O2−) production and reduces the ability of neurons to scavenge free radicals, leading to the release of cytochrome c and apoptosis. Here we test whether overexpression with the use of gene therapy of the antioxidant glutathione peroxidase (Gpx), delivered before or after experimental stroke, is protective against ischemic injury.  Sixty-two rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral vectors expressing Gpx/lacZ or lacZ alone (control) were delivered into each striatum 12 hours before or 2 or 5 hours after ischemia onset.  Striatal neuron survival at 2 days was improved by 36% when Gpx was delivered 12 hours before ischemia onset, 26% with a 2-hour delay, and 25% when delayed 5 hours. After ischemia, Gpx overexpression significantly reduced cytosolic translocation of cytochrome c and increased the proportion of Bcl-2–positive cells compared with cells transfected with control vector. Bax and activated caspase-3, while present in control-transfected neurons after ischemia, were rarely noted in Gpx-transfected cells.  Expression from these herpes simplex viral vectors begins 4 to 6 hours after injection, which suggests a 9- to 11-hour temporal therapeutic window for Gpx. This is the first study to show that overexpression of Gpx with the use of gene therapy protects against experimental stroke, even with postischemic transfection, and the neuroprotective mechanism involves attenuation of apoptosis-related events.

title: Melatonin increases activities of glutathione peroxidase and superoxide dismutase in fetal rat brain
abstract: Melatonin is a powerful scavenger of oxygen free radicals. In humans, melatonin is rapidly transferred from the maternal to the fetal circulation. To investigate whether or not maternal melatonin administration can protect the fetal rat brain from radical‐induced damage by increasing the activities of antioxidant enzymes, we administered melatonin to pregnant rats on day 20 of gestation. Melatonin (10 mg/kg) was injected intraperitoneally at daytime (14:00 hr) and, to remove the fetuses, a laparotomy was performed at 1, 2, or 3 hr after its administration. We measured the melatonin concentration in the maternal serum and in fetal brain homogenates and determined the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) in fetal brain homogenates. Melatonin administration markedly increased melatonin concentrations in the maternal serum and fetal brain homogenates, with peak levels achieved 1 hr after melatonin administration (serum: 538.2±160.7 pM/mL; brain homogenates: 13.8±2.8 pM/mg protein). Between 1 and 3 hr after melatonin administration, GSH‐Px activity in fetal brain homogenates increased significantly (P<0.01). Similarly, SOD activity increased significantly between 1 and 2 hr after melatonin administration (P<0.01). These results indicate that melatonin administration to the mother increases antioxidant enzyme activities in the fetal brain and may thereby provide indirect protection against free radical injury. Thus, melatonin may potentially be useful in the treatment of neurodegenerative conditions that may involve excessive free radical production, such as fetal hypoxia and preeclampsia.

title: Antioxidant MCI-186 inhibits mitochondrial permeability transition pore and upregulates Bcl-2 expression.
abstract: Reperfusion after a period of ischemia is associated with the formation of reactive oxygen species (ROS) and Ca2+ overload resulting in the opening of a nonspecific pore in the inner membrane of the mitochondria, called the mitochondrial permeability transition pore (PTP), leading to cell damage. Although endogenous antioxidants are activated because of oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury. Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia. Here we demonstrated the effects of the antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in preventing reperfusion injury of the heart by inhibition of PTP opening. Ischemia (30 min) by left coronary artery (LCA) occlusion and reperfusion (120 min) in Wistar rats after pretreatment with MCI-186 (10 mg/kg iv) infusion starting from 30 min before LCA occlusion resulted in 1) less area of myocardial infarction (19.2% vs. 61.6%), 2) well-maintained myocardial ATP content (P < 0.03 vs. control), 3) decreased mitochondrial swelling and reduced cytochrome c release, 4) increased expression of BCl-2, 5) lower prevalence of apoptotic cells (14.3% vs. 2.9%), and 6) reduced DNA fragmentation in the MCI-186-treated group. These cytoprotective effects of MCI-186 were inhibited on opening PTP before MCI-186 treatment with the PTP activators lonidamine (10 mg/kg iv) or atractyloside (5 mg/kg iv) but failed to inhibit the protective effects exerted by another antioxidant, allopurinol, suggesting that the PTP inhibiting property is specific for MCI-186. These results demonstrate that the radical scavenger MCI-186, by inhibiting the opening of the PTP, prevents necrosis and cytochrome c release and hence pathological apoptosis.

title: The antioxidant edaravone attenuates ER-stress-mediated cardiac apoptosis and dysfunction in rats with autoimmune myocarditis
abstract: Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T-cells secreting inflammatory cytokines. In this study, rat models of EAM were prepared by injection with porcine cardiac myosin. One week after immunization, edaravone was administered intraperitoneally at 3 or 10 mg/kg/day to rats for 2 weeks. Cardiac function was measured by haemodynamic and echocardiographic studies and TUNEL assay was performed. Left ventricular (LV) expression of NADPH oxidase sub-units (p47phox and p67phox), pro-inflammatory cytokines (TNF-α), endoplasmic reticulum (ER) stress signalling proteins (GRP78, caspase-12 and GADD153) and mitogen-activated protein kinase (MAPK) family proteins (phospho-p38 MAPK and phospho-JNK) were measured by western blotting. Edaravone improved LV function in a dose-dependent manner. Central venous pressure was significantly low and LV ejection fraction and fractional shortening was significantly high in edaravone groups compared with those in the vehicle group. In addition, edaravone treatment down-regulated LV expressions of p47phox, TNF-α, GADD153, phospho-p38 MAPK and phospho-JNK. Furthermore, the LV expressions of p67phox, GRP78, caspase-12 and TUNEL-positive cells of rats with EAM treated with edaravone were significantly low compared with those of the vehicle group. These findings suggest that edaravone ameliorated the progression of EAM by inhibiting oxidative and ER stress and, subsequently, cardiac apoptosis.

title: THE SPECIFIC FREE RADICAL SCAVENGER EDARAVONE SUPPRESSES BLEOMYCIN‐INDUCED ACUTE PULMONARY INJURY IN RABBITS
abstract: 1 Intratracheal instillation of bleomycin induces a condition in rabbits that serves as a useful model of human pulmonary fibrosis. Bleomycin‐induced production of reactive oxygen species leads to acute lung inflammation and induction of apoptosis, which is followed by pulmonary fibrosis at a later chronic stage. In the present study, we tested whether edaravone, a free radical scavenger, would suppress bleomycin‐induced acute pulmonary inflammation. 2 Rabbits were divided into three groups (n = 10 in each): (i) a bleomycin‐treated group, which received intratracheal instillation of 2 mg/kg bleomycin; (ii) a bleomycin + edaravone group, which received a 10 day regimen of daily intravenous injections of edaravone (3 mg/kg per day) beginning 3 days before bleomycin instillation; and (iii) a saline control group. Rabbits were killed for analysis 7 days after bleomycin administration. 3 In lung tissues from the bleomycin‐treated group, marked infiltration of inflammatory cells, consisting mainly of lymphocytes, neutrophils and eosinophils, was observed. In addition, significantly increased numbers of TUNEL‐positive (apoptotic) and transforming growth factor‐β‐positive cells were seen. All these effects were significantly attenuated by treatment with edaravone. 4 The findings of the present study suggest that edaravone may be useful in the prevention of acute lung injury resulting from the production of reactive oxygen species.

title: Beneficial effect of MCI-186 on hepatic warm ischemia-reperfusion in the rat.
abstract: Despite growing evidence that reactive oxygen species are responsible for deleterious effects of ischemia-reperfusion (I/R) injury of the liver, there exists, until now, no reliable antioxidant therapeutics applicable in the clinical setting. We investigated the effects of free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), using an isolated liver perfusion model to elucidate its possible therapeutic effects on hepatic warm I/R injury. Isolated livers from Wistar rats were reperfused for 120 min with an oxygenated Krebs-Henseleit bicarbonate buffer after 1 hr of warm ischemia. Addition of MCI-186 (1 mg/L) into the perfusate significantly improved portal flow, hepatic enzyme release into the perfusate, total bile production, histologic alteration, and malondialdehyde concentration but not sinusoidal endothelial cell function as assessed by the clearance of hyaluronic acid. MCI-186 seems to have protective effects against hepatic warm I/R injury by attenuating the damage of the hepatocyte, which is the major target of oxidative damage in this model.

title: A Free Radical Scavenger, Edaravone, Attenuates Steatosis and Cell Death via Reducing Inflammatory Cytokine Production in Rat Acute Liver Injury
abstract: Reactive oxygen radicals play an important role in various forms of liver injury. In this study, we evaluated the efficacy of edaravone, a newly synthesized free radical scavenger, in its clinical dosage on an experimental model of acute liver injury in rats.  The clinical dose of edaravone (3 mg/kg) was intravenously administered immediately and 3 h after intraperitoneal administration of carbon tetrachloride (CCl4) in rats. Histological evaluation including apoptosis and cytokine profiles were examined.  Fatty degeneration and necrosis with marked elevation of serum alanine aminotransferase and lactate dehydrogenase levels developed after CCl4 administration were significantly reduced by edaravone. In addition, the apoptotic index assessed by TUNEL method was significantly lowered in the edaravone treated group. Serum and liver transcription levels of interleukin-6, tumor necrosis factor-α, interleukin-4, and interleukin-10 were increased following CCl4 administration, and they were attenuated by edaravone treatment. The formation of malondialdehyde, 4-hydroxynonenal adduct and one of the markers for oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, was also inhibited by edaravone treatment.  Edaravone has a remarkable protective effect on acute liver injury caused by oxygen radicals through not only attenuating the membrane lipid peroxidation, but also inhibiting the production of inflammatory cytokines. We theorize that edaravone may have a clinical benefit in the treatment of various liver injuries.

title: Clinical usefulness of edaravone for acute liver injury
abstract: Edaravone, a newly synthesized radical scavenger, has shown an excellent effect on treating stroke patients. The effect of edaravone on carbon tetrachloride (CCl4)‐induced acute liver injury was examined.

title: EDARAVONE PREVENTS FAS-INDUCED FULMINANT HEPATIC FAILURE IN MICE BY REGULATING MITOCHONDRIAL BCL-XL AND BAX
abstract: Fulminant hepatic failure is a serious disease that has a poor cure rate unless liver transplantation is performed. Edaravone, a free radical scavenger, has been approved for the treatment of acute cerebral infarction, and its mechanism of action involves scavenging free radicals generated in ischemic tissues. We assessed the ability of 3-methyl-1-phenyl-2-pyrazolim-5-one (edaravone) to prevent Fas-induced acute liver failure in mice and examined the mechanisms underlying the observed effects. BALB/c mice were administered 0.25 &mgr;g/g (i.v.) body weight of a purified hamster agonist anti-Fas monoclonal antibody (clone Jo2). The mice also received either edaravone or isotonic sodium chloride solution before or after Jo2 treatment. Edaravone improved the survival rate of the mice markedly. Histopathological findings and serum aspartate aminotransferase levels showed that edaravone reduced the degree of liver injury caused by Jo2. Terminal deoxynucleotidyl transferase-mediated 2&vprime;-deoxyuridine 5&vprime;-triphosphate nick end labeling staining showed that edaravone reduced the number of apoptotic hepatocytes. Edaravone also prevented cytochrome c release and caspase 3 activity, recognized as markers of apoptosis after mitochondrial disruption. Therefore, we considered that the antiapoptotic activity of edaravone involved blocking signals in the mitochondria-dependent pathway of Fas-induced apoptosis. Mitochondrial Bcl-xL and Bax, which form a channel in the mitochondrial membrane and, by their balance, regulate its permeability, are involved in mitochondrial disruption. Western blotting showed that the Bcl-xL-Bax ratio of the edaravone group was much higher than that of the control group. In conclusion, edaravone might protect hepatocytes from Fas-induced mitochondria-dependent apoptosis by regulating mitochondrial Bcl-xL and Bax.

title: A Novel Free Radical Scavenger, Edarabone, Protects Against Cisplatin-Induced Acute Renal Damage in Vitro and in Vivo
abstract: Accumulating evidence suggests that enhanced peroxidative damage caused by reactive oxygen species (ROS) may contribute to the pathogenesis of cisplatin-induced acute renal failure. Nevertheless, little is known about the involvement of oxygen radicals in cisplatin nephropathy. In this study, we investigated the effects of a novel free radical scavenger, 3-methyl-1-phenyl-pyrazolin-5-one (MCI-186; edarabone), on murine proximal tubular cell (PTC) damage induced by exposure to cisplatin in vitro and on renal function in an in vivo model of cisplatin-induced acute renal failure. Edarabone inhibited cisplatin-induced (40 μM, 24 h) cytotoxicity in a concentration-dependent manner (10-5 to 10-3 M). Edarabone also attenuated cisplatin-induced mitochondrial transmembrane potential loss and ROS production of PTCs. In the in vivo study, male Wistar rats were cotreated with cisplatin (5 mg/kg, i.p.) and edarabone (1 or 5 mg/kg, i.v.). Effects of edarabone on the kidney were examined 5 days after treatment. Cisplatin resulted in renal dysfunction, renal tubular damage, mitochondrial damage (assayed by histochemical staining for respiratory chain complex IV), renal protein oxidation (examined by Western blot analysis using a specific antibody for carbonyl group-containing proteins), and tubular apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining). The above changes were attenuated by edarabone treatment. Thus, edarabone exhibited cytoprotective effects in PTCs and renoprotective effects against cisplatin. Our findings suggest that ROS, in particular hydroxyl radicals, are involved in cisplatin nephropathy and that edarabone may be potentially useful in protecting the kidneys and prevention of acute renal failure.

title: Edaravone, a known free radical scavenger, enhances X-ray-induced apoptosis at low concentrations.
abstract: Edaravone has been reported to have a radioprotective effect at high concentrations. We now report that a lower dose of edaravone enhanced X-ray-induced apoptosis of some cell lines harboring p53 wild-type status, such as MOLT-4, Nalm-6, and HepG2. The knock-down of p53 using siRNA in MOLT-4 cells abolished the radiosensitizing effect of edaravone. Enhanced phosphorylations of p53 at Ser 15 and Ser 20 and up-regulation of PUMA, a p53 target protein, were observed after X-irradiation in the presence of edaravone. We conclude that the low dose of edaravone sensitized cells to X-irradiation by promoting the p53-dependent apoptotic signaling pathway.

title: Free radical scavenger edaravone suppresses x-ray-induced apoptosis through p53 inhibition in MOLT-4 cells.
abstract: Edaravone, a clinical drug used widely for the treatment of acute cerebral infarction, is reported to scavenge free radicals. In the present study, we investigated the radioprotective effect of edaravone on X-ray-induced apoptosis in MOLT-4 cells. Apoptosis was determined by the dye exclusion test, Annexin V binding assay, cleavage of caspase, and DNA fragmentation. We found that edaravone significantly suppressed the X-ray-induced apoptosis. The amount of intracellular ROS production was determined by the chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate system. We found that the intracellular ROS production by X-irradiation was completely suppressed by the addition of edaravone. The accumulation and phosphorylation of p53 and the expression of p21(WAF1), a target protein of p53, which were induced by X-irradiation, were also suppressed by adding edaravone. We conclude that the free radical scavenger edaravone suppresses X-ray-induced apoptosis in MOLT-4 cells by inhibiting p53.

title: Effect of MCI-186 on postischemic reperfusion injury in isolated rat heart.
abstract: MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a newly developed antioxidant which has been shown to reduce brain edema in cerebral ischemia through inhibition of the lipoxygenase pathway of arachidonic acid. However, its effect on myocardial reperfusion injury after prolonged ischemia has not yet been demonstrated. We compared the mode of the effect of MIC-186 and recombinant human CuZn superoxide dismutase (rh-SOD) in isolated perfused rat hearts subjected to 60-min ischemia followed by 60-min reperfusion. Left ventricular developed pressure (LVDP), necrotic area and the release of creatine phosphokinase (CPK) and endogenous CuZn superoxide dismutase (endoge-SOD) were measured to evaluate myocardial damage. The decrease in left coronary flow (CBF) was measured as an index of the damage of left coronary circulation. MCI-186 (14.5 mg/L) was perfused for 10 min in the MCI group and rh-SOD (70 mg/L) was perfused during the reperfusion period in the SOD group starting 5 min prior to reperfusion. The release patterns of CPK and endoge-SOD were analyzed to elucidate the difference in the mode of protection of MCI-186 and rh-SOD. The LVDP remained higher in both MCI and SOD groups than that of control (76 +/- 1, 77 +/- 2 and 69 +/- 1% of preischemic value, respectively). The necrotic area was significantly attenuated in both MCI and SOD groups compared with that in the control group (16 +/- 1, 14 +/- 1 and 32 +/- 1%, respectively, p < 0.05). Total CPK release was lower in both MCI and SOD groups than in the control (78 +/- 7, 100 +/- 2 and 116 +/- 4 x 10(3) units/g myocardium respectively). The decrease in CPK release was more marked in the MCI group than that in the SOD group (p < 0.05). The reduction in CBF was significantly attenuated by the treatment with rh-SOD or MCI-186, but the effect was much higher in the SOD group than in the MCI group (69 +/- 5, 58 +/- 2, and 48 +/- 2% in SOD, MCI and control groups, respectively). The release pattern of endoge-SOD was identical to that of CPK and thus this did not distinguish the mode of effect of MCI-186 from that of rh-SOD. These results indicate that MCI-186 reduces reperfusion injury in isolated perfused hearts with prolonged ischemia and the effect is more closely related to the reduction of myocyte damage than the preservation of the coronary circulation.

title: Myocardial protection of MCI-186 in rabbit ischemia-reperfusion.
abstract: We observed that 3-methyl-1-1phenyl-2-pyrazolin-5-one (MCI-186), a newly-developed free radical scavenger, attenuated necrosis in the in vivo rabbit hearts upon reperfusion after prolonged ischemia. In rabbits undergoing 1 hour ligation of the anterior ventricular coronary artery, a single bolus injection of MCI-186 (1.5 mg/kg) was introduced into the post-ischemic heart immediately before 4 hour reperfusion. Compared to negligible necrosis in sham-operated control animals and 33.81 +/- 13.50% necrosis in the area at risk for the saline control group (n = 8), the MCI-186 - treated group (n = 8) had a necrosis of 13.27 +/- 4.60% (p < 0.05 vs saline control group). The pressure-rate index had a slight decrease in MCI-186 treated group compared to the control group (p > 0.05). However, the blood levels of malondialdehyde (MDA) in MCI-186 treated group (2.08 +/- 0.23 microM) was significantly smaller than that of 2.65 +/- 0.31 microM in control animals (p < 0.01), while sham control had an average MDA level of 1.91 +/- 0.40 microM, with p > 0.05 relative to that in the MCI-186 treated group. These data support our contention that MCI-186 reduces reperfusion injury in perfused hearts with prolonged ischemia and the mechanism for the in vivo efficacy of MCI-186 is predominantly related to its antioxidant activities.

title: The hydroxyl radical scavenger MCI‐186 protects the liver from experimental cold ischaemia–reperfusion injury
abstract: Oxidative stress contributes to hepatic ischaemia–reperfusion (IR) injury in a biphasic pattern. In addition to direct cytotoxic effects, oxidative stress also initiates the signal transduction processes that promote second‐phase liver injury. The present study investigated the effects of the hydroxyl radical scavenger MCI‐186 on the biphasic process of hepatic cold IR injury.

title: MCI‐186 (edaravone), a free radical scavenger, attenuates hepatic warm ischemia–reperfusion injury in rats
abstract: Hepatic warm ischemia–reperfusion injury (IRI) during hepatectomy and liver transplantation is a major cause of liver dysfunction in which the pathologic role of free radicals is a major concern. To assess the effect of MCI‐186 (edaravone) on hepatic IRI, male Wistar rats were subjected to partial hepatic ischemia for 60 min after pretreatment with vehicle (group C) or MCI‐186 (group M), or after both MCI‐186 pretreatment and additional administration of MCI‐186 12 h after reperfusion (group MX). Groups M and MX showed significantly lower levels of serum alanine aminotransferase and hepatic lipid peroxidation than group C, and also significantly lower expression levels of mRNA for cytokines, chemokines and intercellular adhesion molecule‐1. There were fewer tissue monocytes and neutrophils in groups M and MX than in group C. These effects were more marked in group MX than in group M. Our findings suggest that treatment with MCI‐186 attenuates hepatic IRI in this rat in vivo model.

title: Protective effects of the free radical scavenger edaravone on acute pancreatitis-associated lung injury.
abstract: Impaired lung function is the primary contributor to most deaths associated with severe acute pancreatitis. It is widely accepted that oxidative stress plays a central role in the pathogenesis of pancreatitis and associated complications. Therefore, in the present study, we investigated whether therapeutic treatment with the free radical scavenger edaravone could protect rats against acute pancreatitis and the associated lung injury. Acute pancreatitis was induced by infusion of 1ml/kg of sodium taurocholate (3% solution) into the biliopancreatic duct. Edaravone (8mg/kg) was administered 1h and 13h after inducing pancreatitis, the severity of pancreatic and pulmonary injuries was evaluated 24h after inducing pancreatitis. Edaravone treatment significantly reduced the elevated malondialdehyde levels in rat lungs after acute pancreatitis, suggesting an important role for free radicals in acute pancreatitis-associated lung injury. In addition, edaravone showed significant protective effects against neutrophil infiltration and tissue injury in both pancreas and lung, as demonstrated by serum amylase levels, myeloperoxidase activity and histopathological analysis. Edaravone treatment also attenuated the elevated mRNA levels of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) in rat lungs after acute pancreatitis. In conclusion, edaravone protects rats against acute pancreatitis-associated lung injury, probably through its antioxidant and anti-inflammatory effects. Thus, edaravone shows promise as a treatment for lung injury in patients with acute pancreatitis.

title: Protection by a radical scavenger edaravone against cisplatin-induced nephrotoxicity in rats.
abstract: Acute renal failure is a dose-limiting factor of cisplatin chemotherapy. Here, we show the protective effect of edaravone, a recently developed radical scavenger for clinical use, against cisplatin-induced renal dysfunction in rats. A marked increase in blood urea nitrogen and creatinine in serum, and histological changes including vacuolation, necrosis and protein casts were observed in proximal renal tubules at the fourth day after cisplatin injection (5-10 mg/kg). Repeated injection of edaravone (1-10 mg/kg, i.v. twice a day for 3 days) reversed the cisplatin-induced elevation of blood urea nitrogen and creatinine, and morphological changes in a dose-dependent manner. In particular, the protective effect of edaravone was almost complete at 10 mg/kg. Moreover, the compound was still fully effective, when it was administered only at the second day after cisplatin injection. On the other hand, the glutathione content in renal tissues lowered at the fourth day after cisplatin injection, which was reversed by the late treatment with edaravone. These findings suggest that the clinically available radical scavenger edaravone is potentially useful for the prevention of cisplatin-induced renal toxicity.

title: Radical scavenger edaravone developed for clinical use ameliorates ischemia/reperfusion injury in rat kidney.
abstract: Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent scavenger of free radicals and has the antioxidant ability to inhibit lipid peroxidation. Its protective effect on brain ischemia has been shown. This study aimed to elucidate its possible therapeutic effects on renal oxidative stress in a rat ischemia/reperfusion model.



Ischemic acute renal failure was induced by 45-minute clamp of the left renal artery followed by administration of edaravone. Renal function and pathologic findings were examined. The generation of free radicals was observed by the fluorogenic probe, dichlorodihydrofluorescein (CM-H(2)DCFDA), and lipid peroxidation was observed by immunochemical staining and Western analysis of 4-hydroxy-2-hexenal (HHE). The ability of edaravone to reduce in vitro oxidative stress was investigated using cultured human renal tubular cells (HKC-8) and the cell viability assay with Alamar blue.



Edaravone attenuated renal function and pathologic findings significantly. The protective effect of edaravone showed the dose-response was at the same level as that of dimethylthiourea. In addition, edaravone significantly reduced the generation of free radicals in the tubular cells indicated by CM-H(2)DCFDA. Immunochemical staining and Western analysis of HHE showed significant suppression of lipid peroxidation in edaravone-treated kidney. In HKC-8 cells loaded with CM-H(2)DCFDA, a marked elevation of fluorescence was observed after exposure to hydrogen peroxide (0.5 mmol/L, for 1 hour), which was reduced by edaravone. The cell viability assay with Alamar blue also showed the protective effect of edaravone.



Edaravone ameliorates renal ischemia/reperfusion injury by scavenging free radicals produced in renal tubular cells and inhibiting lipid peroxidation.

title: Protective effect of edaravone, a free‐radical scavenger, on ischaemia‐reperfusion injury in the rat testis
abstract: To investigate the effect of edaravone, a radical scavenger, on ischaemia‐reperfusion (I‐R) injury in the testes.

title: Effects of edaravone on reperfusion injury in patients with acute myocardial infarction.
abstract: This clinical pilot study was a randomized, controlled, open-label study in 80 patients with acute myocardial infarction and was designed to examine the effects of a novel free radical scavenger, edaravone. Administration of edaravone before myocardial reperfusion was associated with smaller enzymatic infarcts and better clinical outcome.

title: Long-term efficacy of edaravone in patients with acute myocardial infarction.
abstract: The effect of edaravone, a free radical scavenger, on long-term prognosis and its efficacy with regards to scavenging injurious free radicals in patients with acute myocardial infarction (AMI) was examined.



One hundred and one initial AMI patients were randomly assigned to receive 30 mg edaravone (n = 50) or a placebo (n = 51) intravenously just before reperfusion. The infarct size, using serum biomarkers and Q-wave formations, and the incidence of reperfusion arrhythmia between the groups were compared. Cardiovascular event-free curves were estimated by using the Kaplan - Meier method. In addition, the serum thioredoxin levels, an oxidative stress marker, to assess the antioxidant effect of edaravone was determined. In all cases, successful reperfusion was obtained within 6 h after the onset of symptoms. Infarct size and reperfusion arrhythmia were significantly attenuated in the edaravone group compared with the placebo group (p = 0.035 and p = 0.031). The cumulative event-free rate was significantly higher in the edaravone group than in the placebo group (p = 0.045). Serum thioredoxin levels were significantly lower in the edaravone group than in the placebo group throughout the acute phase.



The present study suggests that the edaravone administration just prior to reperfusion might reduce oxidative stress and improve the long-term clinical outcomes of AMI patients.

title: Edaravone preserves coronary microvascular endothelial function after ischemia/reperfusion on the beating canine heart in vivo.
abstract: We examined whether edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, exerts its protective effect on coronary microvessels after ischemia/reperfusion (I/R) in vivo. Ninety-minute coronary occlusion followed by reperfusion was performed in 16 open-chest dogs with and without edaravone administration. Coronary small artery (> or = 100 microm in size) and arteriolar (< 100 microm) vasodilation, in the presence of endothelium-dependent (acetylcholine) or -independent (papaverine) vasodilators, was directly observed using intravital microscopy before and after I/R. I/R impaired microvascular vasodilation in response to acetylcholine, whereas administration of edaravone preserved the response in microvessels of both sizes, but to a greater extent in the coronary small arteries. No significant changes were noted with papaverine administration. In the edaravone group, the fluorescent intensity from reactive oxygen species (ROS) was lower, whereas nitric oxide (NO) intensity was higher relative to controls in the microvessels of the ischemic area. In conclusion, edaravone preserves coronary microvascular endothelial function after I/R in vivo. These effects, which were NO-mediated, were attributed to the ROS scavenging properties of edaravone.

title: EDARAVONE REDUCES MYOCARDIAL INFARCT SIZE AND IMPROVES CARDIAC FUNCTION AND REMODELLING IN RABBITS
abstract: 1 In the present study, we investigated the effect of 3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one (edaravone), a free radical scavenger, on myocardial infarct (MI) size and cardiac function in an in vivo model of MI in rabbits. We further investigated the contribution of hydroxyl radicals, superoxide and nitric oxide (NO) to its effects. 2 Anaesthetized open‐chest Japanese white male rabbits were subjected to 30 min coronary occlusion and 48 h reperfusion. The control group (n = 10) was injected with saline 10 min before reperfusion. The edaravone group (n = 10) was injected with a bolus of 3 mg/kg edaravone 10 min before reperfusion. The edaravone + NG‐nitro‐l‐arginine methyl ester (l‐NAME) group (n = 5) was given 10 mg/kg, i.v., l‐NAME 10 min before the administration of 3 mg/kg edaravone. The l‐NAME group (n = 5) was given 10 mg/kg, i.v., l‐NAME 20 min before reperfusion. Infarct size was measured using the triphenyl tetrazolium chloride method and is expressed as a percentage of area at risk. Cardiac function was assessed by echocardiography 14 days after infarction. 3 In another series of experiments, rabbits were subjected to 30 min coronary occlusion and 30 min reperfusion and myocardial interstitial 2,3‐dihydroxybenzoic acid (DHBA) and 2,5‐DHBA levels, indicators of hydroxyl radical, were measured using a microdialysis technique. 4 Infarct size in the edaravone group was significantly reduced compared with that in the control group (27.4 ± 6.8 vs 43.4 ± 6.8%, respectively; P < 0.05). The edaravone‐induced reduction of infarct size was abolished by pretreatment with l‐NAME. Myocardial interstitial levels of 2,3‐DHBA and 2,5‐DHBA increased 20 and 30 min after ischaemia and peaked at 10 min reperfusion in the control group. Edaravone significantly inhibited the increase in 2,3‐DHBA and 2,5‐DHBA levels seen during reperfusion. Dihydroethidium staining showing in situ detectoion of superoxide was less intense in ischaemic myocardium in the edaravone‐treated group compared with the control group. Edaravone improved cardiac function and left ventricular remodelling 14 days after infarction. 5 In conclusion, edaravone significantly reduces MI size and improves cardiac function and LV remodelling by decreasing hydroxyl radicals and superoxide in the myocardium and increasing the production of NO during reperfusion in rabbits.

title: Edaravone Prevented Deteriorated Cardiac Function After Myocardial Ischemia-Reperfusion via Inhibiting Lipid Peroxidation in Rat
abstract: Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has potent effects in the brain as a free radical scavenger in ischemia-reperfusion as well as in ischemic injuries. However, whether this free radical scavenger prevents deterioration of cardiac function and lethal ventricular arrhythmias after ischemia-reperfusion in rat heart is not clear. We aimed to assess whether free radical scavenging by edaravone maintains cardiac function and suppresses life-threatening ventricular tachyarrhythmia after myocardial ischemia-reperfusion. Twenty-nine 7-week-old male Sprague-Dawley rats had acute myocardial ischemia induced by ligation of the left coronary artery for 5 minutes followed by reperfusion. Eleven were treated by intravenous injection of edaravone at 3 mg/kg 2 minutes after coronary ligation, and 18 were left untreated. The index of systolic function (contractility; end-systolic elastance, Ees) and hemodynamics were measured by pressure-volume relationships every 5 minutes before ligation to 25 minutes after reperfusion. Blood levels of malondialdehyde (MDA) and the ischemic areas were also measured 25 minutes after reperfusion. There were no differences in the ischemic areas between the groups. Lethal reperfusion tachyarrhythmia was observed in 5 untreated rats but not in those having edaravone treatment. Ees was significantly greater in the edaravone-treated than in untreated rats from 5 to 25 minutes after reperfusion (1789 ± 866 in untreated versus 2809 ± 273 mm Hg/mL in edaravone-treated rats at 25 minutes, P < 0.001). MDA level was significantly lower in edaravone-treated than in untreated rats (1.44 ± 0.29 nmol/L in edaravone-treated versus 1.90 ± 0.28 nmol/L in untreated group, P < 0.05). The results suggest that edaravone treatment before reperfusion prevented lethal reperfusion ventricular tachyarrhythmias and deteriorated cardiac function with ischemia and ischemia-reperfusion injuries through inhibiting lipid peroxidation in terms of scavenging for free radicals.

title: Effect of edaravone, a novel free radical scavenger, supplemented to cardioplegia on myocardial function after cardioplegic arrest: in vitro study of isolated rat heart
abstract: Cardioplegic arrest has been the main mechanism of myocardial protection during open-heart surgery; however, it causes myocardial injury during ischemia-reperfusion. Free radical scavengers are widely known to attenuate ischemia-reperfusion injury in various settings. We investigated the effects of edaravone, a novel free radical scavenger that was originally used for cerebral protection, on myocardial function during ischemia-reperfusion after cardioplegic arrest. Rat hearts were excised and perfused using Langendorff apparatus. The hearts were cardioplegically arrested for 90 min using St. Thomas’ Hospital cardioplegic solution (ST solution) at 4°C every 45 min and then reperfused for 20 min. The hearts were divided into 4 groups (n = 13 in each group). In Group ST, the hearts were arrested using the ST solution alone. In Groups L, M, and H, the hearts were arrested using the ST solution supplemented with a low-dose (1 μM), moderate dose (10 μM), and high dose (100 μM) of edaravone, respectively. Left ventricular function (+dp/dtmax) and the levels of the cardiac enzymes released were measured before and after cardioplegic arrest. At the end of the study, the water content and the tissue oxidative stress (8-hydroxy-2′-deoxyguanosine) of the heart were measured. During reperfusion, the edaravone-treated groups showed a greater functional recovery with regard to the +dp/dtmax (P < 0.05). The lactate level was the lowest (P < 0.01) in Group M. The water content of the hearts in the edaravone-treated groups was significantly lower (P < 0.05) than that in Group ST. Oxidative stress was significantly lower (P < 0.01) in the edaravone-treated hearts than in Group ST, and it was the lowest in Group M. The addition of edaravone to the cardioplegic solution ameliorates the impairment in myocardial function by reducing the oxidative stress after cardioplegic arrest. In this study, the maximum improvement in the myocardial function was achieved by addition of a moderate dose (10 μM) of edaravone.

title: MCI-186 (edaravone), a novel free radical scavenger, protects against acute autoimmune myocarditis in rats.
abstract: In this study, we tested the hypothesis that MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one; edaravone), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats by the radical scavenging action associated with the suppression of cytotoxic myocardial injury. Recent evidence suggests that oxidative stress may play a role in myocarditis. We administered MCI-186 intraperitoneally at 1, 3, and 10 mg.kg(-1).day(-1) to rats with EAM for 3 wk. The results were compared with untreated rats with EAM. MCI-186 treatment did not affect hemodynamics. MCI-186 treatment (3 and 10 mg.kg(-1).day(-1)) reduced the severity of myocarditis as assessed by comparing the heart-to-body weight ratio and pathological scores. Myocardial interleukin-1beta (IL-1beta)-positive cells and myocardial oxidative stress overload with DNA damage in rats with EAM given MCI-186 treatment were significantly less compared with those of the untreated rats with EAM. In addition, MCI-186 treatment decreased not only the myocardial protein carbonyl contents but also the myocardial thiobarbituric acid reactive substance products in rats with EAM. The formation of hydroxyl radicals in MCI-186-treated heart homogenates was decreased compared with untreated heart homogenates. Furthermore, cytotoxic activities of lymphocytes of rats with EAM treated with MCI-186 were significantly lower compared with those of the untreated rats with EAM. Hydroxyl radicals may be involved in the development of myocarditis. MCI-186 protects against acute EAM in rats associated with scavenging hydroxyl free radicals, resulting in the suppression of autoimmune-mediated myocardial damage associated with reduced oxidative stress state.

title: Efficacy of MCI-186, a free-radical scavenger and antioxidant, for resuscitation of nonbeating donor hearts.
abstract: Oxygen-derived free radicals are responsible in part for reperfusion injury in globally ischemic myocardium. In this study, the efficacy for resuscitation of nonbeating donor hearts of MCI-186, a free-radical scavenger and antioxidant, was investigated in a pig transplantation model.



Cardiac arrest was induced by asphyxiation. After 30 minutes of global ischemia, the hearts were excised and immediately reperfused from the aortic root with normoxemic blood cardioplegia (PO2 100 mm Hg) for 20 minutes, followed by perfusion with hyperoxemic blood (PO2 300 mm Hg). MCI-186 (3 mg/kg) was administered into the aortic root for the first 30 minutes of reperfusion in the treated group (n = 6), and untreated hearts were used as a control group (n = 6). Transplantation was performed with the heart beating.



Posttransplantation recovery of cardiac output, end-systolic pressure-volume ratio, and first derivative of pressure of the left ventricle in the treated group were significantly better than those in the control group. The coronary sinus-aortic root difference in malondialdehyde levels remained low throughout reperfusion in the treated group but abruptly increased after initiation of oxygenated blood perfusion in the control group. The MCI-186-treated hearts showed low degree of edema and well-preserved ultrastructure with normal-appearing organelles, whereas the untreated hearts had marked swelling of mitochondria and scant glycogen granules.



MCI-186 exerts a cardioprotective action at least partly by inhibition of lipid peroxidation. Antioxidant therapy at the initial reperfusion is essential to successful resuscitation of nonbeating hearts by continuous myocardial perfusion.

title: Protective effect of edaravone against hypoxia–reoxygenation injury in rabbit cardiomyocytes
abstract: We examined whether edaravone (Eda), a clinically available radical scavenger, directly protects cardiomyocytes from ischemia/reperfusion (I/R) injury, and whether the timing of its application is critical for protection. Cardioprotective effects of edaravone were tested in the modified cell‐pelleting model of ischemia and under exogenous oxidative stress (hydrogen peroxide: H2O2) in isolated adult rabbit ventricular cells. Cell death and reactive oxygen species (ROS) generation were detected using propidium iodide (PI) and DCFH‐DA, respectively. These parameters were evaluated objectively using flow cytometory. Hypoxia and reoxygenation aggravated the proportion of dead cells from 32.2±1.8% (Baseline) to 51.3±2.7% (Control). When 15 μM edaravone was applied either throughout the entire experiment (Through) or only at reoxygenation (Reox), cell death was significantly reduced to 39.9±1.8% (P<0.01 vs Control) and 43.3±2.5% (P<0.05 vs Control), respectively. In contrast, when edaravone was applied 10 min after reoxygenation, its protective effect disappeared. Cardioprotection by edaravone was more remarkable than that afforded by other free radical scavengers, such as ascorbate and superoxide dismutase (SOD). There is a positive correlation between the cardioprotective effect of edaravone and the extent of ROS reduction. Edaravone blunted the H2O2‐induced changes in electrical properties, and significantly prolonged the time to contracture induced by H2O2 in single ventricular myocytes. Taken together, edaravone directly protects cardiomyocytes from I/R injury by attenuating ROS production, even when applied at the time of reoxygenation, suggesting that edaravone could be a potent cardioprotective therapeutic agent against hypoxia–reoxygenation injury.

title: Effect of the free radical scavenger MCI-186 on pulmonary ischemia-reperfusion injury in dogs.
abstract: Free radical scavengers and superoxide dismutase have been found to protect against cerebral ischemic damage, and it was suggested that oxygen free radicals contribute to ischemia-reperfusion injury induced by cerebral ischemic damage. MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) is a potent scavenger and inhibitor of hydroxyl radicals and protective agent of peroxidative injury. The purpose of this study was to evaluate the effects of MCI-186 on pulmonary ischemia-reperfusion injury in a simulated transplanted lung model.



Fourteen dogs were divided into two groups (n = 7 each). In the MCI group, MCI-186 was continuously administered at 3 mg/kg/hour intravenously (IV) from 30 minutes before reperfusion until 30 minutes after reperfusion (total administration time 1 hour). Vehicle was administered in the control group. Warm ischemia was induced for 3 hours by clamping the left pulmonary artery and veins. Simultaneously, the left stem bronchus was bisected and then anastomosed before reperfusion. The right pulmonary artery was ligated 15 minutes after reperfusion, and the right stem bronchus was then bisected.



The respiratory gas exchange, hemodynamic changes, wet-to-dry weight ratio (WDR) and malondialdehyde (MDA) concentration in the tissue were significantly improved (p < 0.05) in the MCI group. The histologic damage was more severe in the control group and polymorphonuclear neutrophil (PMN) infiltration was reduced in the MCI group.



MCI-186 has a protective effect on pulmonary ischemia-reperfusion injury through the inhibition of lipid peroxidation.

title: Pretreatment with edaravone reduces lung mitochondrial damage in an infant rabbit ischemia-reperfusion model.
abstract: Free radical scavenger edaravone has been approved as a new drug for treatment of stroke patients. The purpose of this study was to examine whether pretreatment with edaravone could attenuate ischemia-reperfusion (IR)-induced lung damage in infant rabbits.



New Zealand White rabbits (Experimental Animal Center, Nanjing Medical University, Nanjing, China) at age from 15 to 21 days were subjected to sham operation, IR, or edaravone plus IR. Ischemia/reperfusion was induced by clamping the right pulmonary hilum for 1 hour and then removal of the clamp for 4 hours. Edaravone (1 mg/kg, intravenous) was given 5 minutes before ischemia. Concentrations of reactive oxygen species-hydroxyl radical (ROS-HR) and malondialdehyde (MDA), and activities of glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) in the lung tissue were measured. Mitochondrial membrane potential, swelling rate, and ultrastructure of the lung were analyzed, and histologic condition of the lung was evaluated.



Edaravone pretreatment reduced markedly the productions of ROS-HR and MDA and increased the activities of GSH-PX and SOD. It attenuated both IR-induced decrease in mitochondrial membrane potential from 60% to 14% and IR-induced increase in mitochondrial swelling. As results, the mitochondrial and lung tissue damages were less, leading to an improved survival rate in IR rabbits pretreated with edaravone compared with IR rabbits without the treatment.



Edaravone pretreatment reduces the IR-induced lung mitochondrial damage in infant rabbits.

title: MCI-186 (edaravone), a free radical scavenger, attenuates ischemia-reperfusion injury and activation of phospholipase A(2) in an isolated rat lung model after 18 h of cold preservation.
abstract: Increased microvascular permeability and extravasation of inflammatory cells are key events in ischemia-reperfusion (IR) injury. We hypothesized that edaravone, a free radical scavenger, is able to attenuate IR lung injury by decreasing oxidative stress and phospholipase A(2) (PLA(2)) activation, which otherwise may lead to lung injury through PAF receptor (PAF-R) activation.



We used an isolated rat lung model. Five groups were defined (n=7, each): in the sham and vehicle group, lungs were immediately washed after thoracotomy or perfused for 2h without an ischemic period, respectively. In the ischemic groups, 10mg/kg of MCI-186 (edavorane group), 1mg/kg of PAF-R inhibitor (ABT-491 group) or saline (control group) were i.v. administered 20 min before harvest. Lungs were flushed with LPD solution, stored at 4 degrees C for 18 h, and reperfused for 2h.



Compared to vehicle group, IR significantly decreased the PO(2) level and increased the wet-to-dry ratio, proteins in bronchoalveolar lavage (BAL), and myeloperoxidase (MPO) activity in the control group, while edaravone treatment maintained the PO(2) similar to the vehicle group and significantly reduced edema formation and neutrophil extravasation. Consistently, IR significantly increased lipid peroxidation, cytosolic-PLA(2) activity mainly via alveolar macrophages, soluble-PLA(2) activity, leukotriene B(4), and PAF-R expression in control lungs, together with a decreased PAF acetylhydrolase (PAF-AH) activity. Edaravone significantly reduced all of these, but increased PAF-AH activity. Furthermore, pharmacological inhibition of the PAF-R attenuated IR injury resembling edaravone action.



Edaravone attenuates lung IR injury by suppressing oxidative damage and PLA(2) activation, which otherwise partially mediates edema formation and neutrophil extravasation through PAF-R activation.

title: Effects of edaravone, a free-radical scavenger, on bleomycin-induced lung injury in mice
abstract: Reactive oxygen species play an important role in the pathogenesis of acute lung injury and pulmonary fibrosis. The present authors hypothesise that edaravone, a free-radical scavenger, is able to attenuate bleomycin (BLM)-induced lung injury in mice by decreasing oxidative stress. Lung injury was induced in female ICR mice by intratracheal instillation of 5 mg·kg−1 of BLM. Edaravone (300 mg·kg−1) was administered by intraperitoneal administration 1 h before BLM challenge. Edaravone significantly improved the survival rate of mice treated with BLM from 25 to 90%, reduced the number of total cells and neutrophils in bronchoalveolar lavage fluid (BALF) on day 7, and attenuated the concentrations of lipid hydroperoxide in BALF and serum on day 2. The fibrotic change in the lung on day 28 was ameliorated by edaravone, as evaluated by histological examination and measurement of hydroxyproline contents. In addition, edaravone significantly increased the prostaglandin E2 concentration in BALF on day 2. In summary, edaravone was shown to inhibit lung injury and fibrosis via the repression of lipid hydroperoxide production and the elevation of prostaglandin E2 production in the present experimental murine system.

title: Edaravone, a newly developed radical scavenger, protects against ischemia-reperfusion injury of the small intestine in rats.
abstract: Although edaravone (3-methyl-1-phenyl-pyrazolin-5-one), a newly developed radical scavenging agent, has been widely used for protection against ischemia-reperfusion (I-R) injury in patients with cerebral infarction, its effects on gastrointestinal I-R injury have not been evaluated. In the present study, we examined the effects of edaravone on experimental intestinal I-R damage in rats. In male Wistar rats with and without edaravone treatment, intestinal damage was induced by clamping the superior mesenteric artery for 30 min, followed by reperfusion. Edaravone was administered via intravenous infusion at 5 min before reperfusion was achieved by removal of the clamp. The rats were sacrificed after 60 min of reperfusion. Luminal protein and hemoglobin concentrations were measured as an index of mucosal injury and histological examination of hematoxylin and eosin-stained sections was performed. Thiobarbituric acid (TBA)-reactive substances and tissue-associated myeloperoxidase (MPO) activity were measured in the mucosa as indicators of lipid peroxidation and neutrophil infiltration, respectively. The mucosal concentration of cytokine-induced neutrophil chemoattractant (CINC)-1 (a member of the IL-8 family) was determined by enzyme-linked immunosorbent assay (ELISA). Additionally, CINC-1 messenger RNA (mRNA) was measured by the reverse-transcription polymerase chain reaction (RT-PCR). As a result, the levels of luminal protein and hemoglobin, TBA-reactive substances, and MPO activity were all increased significantly by I-R injury, and these increases were significantly inhibited by treatment with edaravone. Multiple erosions and bleeding were observed macroscopically after the small intestine was exposed to I-R injury, and these changes were inhibited by administration of edaravone. Microscopic I-R damage was also reduced by treatment with edaravone. CINC-1 protein and CINC-1 mRNA were both increased by I-R injury, while edaravone markedly reduced the levels of both protein and mRNA. In summary, these results suggest that edaravone can protect the small intestine against I-R injury by scavenging oxygen-derived free radicals.

title: Microcirculatory Changes in Experimental Mesenteric Longitudinal Ulcers of the Small Intestine in Rats
abstract: Rats receiving intracolonic administration of indomethacin develop longitudinal ulcers on the mesenteric side of the small intestine that are similar to those seen in the acute phase of Crohn's disease. To investigate the causative role of microcirculatory disturbances and to elucidate the therapeutic effect of antioxidants on this enteropathy in rats, we serially evaluated changes in regional blood flow of the small intestine using laser Doppler perfusion imaging and the colored microsphere injection method. Both methods disclosed stepwise hyperperfusion limited to the mesenteric side of the small intestine following transient ischemia during the initial 30–60 minutes. In addition, both a radical scavenger and a radical production inhibitor significantly ameliorated the mesenteric longitudinal ulcers. We concluded that ischemia–reperfusion on the mesenteric side accompanying excessive production of radicals might be strongly involved in indomethacin-induced longitudinal ulcers of the small intestine in rats.

title: Edaravone, a Novel Free Radical Scavenger, Prevents Liver Injury and Mortality in Rats Administered Endotoxin
abstract: We postulated that a novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone; EDA), would attenuate inflammatory cytokine and chemokine expression in the liver after lipopolysaccharide (LPS) challenge through its antioxidant effect. Rats were administered EDA (0.3, 1.5, 3.0, 6.0, and 12.0 mg/kg) or the same volume of saline intravenously just after LPS (10 mg/kg) injection and then was continued intermittently every 2 h (five administrations in total). Survival was assessed for the next 24 h. In separate experiments, rats were sacrificed at 60 min, 90 min, 6 h, and 9 h after LPS injection. Serum and liver sections were collected for further analysis. Survival was improved by EDA in a dose-dependent manner up to 3 mg/kg, and maximum effects were observed at a dose of 3 mg/kg. After LPS injection, alanine aminotransferase levels increased significantly to about 1,250 IU/l in the vehicle-treated group, whereas values were blunted by about 80% by EDA. Furthermore, increases in 4-hydroxynonenal-modified proteins were also blunted in the liver by EDA. Moreover, mRNA expressions of macrophage infiltrating protein-2, monocyte chemoattractant protein (MCP)-1 and MCP-5 were attenuated by EDA. As a result, increases in the number of infiltrating inflammatory cells and mRNA expression of inflammatory cytokines such as tumor necrosis factor-α and interleukin-6 were significantly blunted in the liver by EDA. This reduction was accompanied by a significant reduction of their serum levels. In conclusion, EDA prevented liver injury by both inhibition of recruitments of inflammatory cells and expression of inflammatory cytokine levels in the liver.

title: Amelioration of hepatic ischemia/reperfusion injury in the remnant liver after partial hepatectomy in rats
abstract: Reactive oxygen species have been implicated in the development of hepatic ischemia/reperfusion (I/R) injury. I/R injury remains an important problem in massive hepatectomy and organ transplantation. The aim of this study was to examine the effect of edaravone, a newly synthesized free radical scavenger, on I/R injury in the remnant liver after partial hepatectomy in rats.

title: Electron spin resonance and spin trapping technique provide direct evidence that edaravone prevents acute ischemia–reperfusion injury of the liver by limiting free radical-mediated tissue damage
abstract: A novel free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), is used for the treatment of acute ischemic stroke and is protective in several animal models of organ injury. We tested whether edaravone is protective against acute liver warm ischemia/reperfusion injury in the rat by acting as a radical scavenger. When edaravone was administered prior to ischemia and at the time of initiation of the reperfusion, liver injury was markedly reduced. Production of oxidants in the liver in this model was assessed in vivo by spin-trapping/electron spin resonance (ESR) spectroscopy. Ischemia/reperfusion caused an increase in free radical adducts rapidly, an effect markedly blocked by edaravone. Furthermore, edaravone treatment blunted ischemia/reperfusion-induced elevation in pro-inflammatory cytokines, infiltration of leukocytes and lipid peroxidation in the liver. These results demonstrate that edaravone is an effective blocker of free radicals in vivo in the liver after ischemia/reperfusion, leading to prevention of organ injury by limiting the deleterious effects of free radicals.

title: A free radical scavenger, edaravone, prevents ischemia-reperfusion injury in liver grafts from non-heart-beating donors.
abstract: Due to the increase in liver transplantation, the donor shortage has become a serious problem, requiring marginal, non-heart-beating donors (NHBDs). The aims of this study were to evaluate the cytoprotective effect of edaravone, a free radical scavenger, on warm ischemia-reperfusion (I/R) injury of liver grafts from NHBDs.



Rat livers were harvested from heart-beating donors (HB group) or from NHBDs undergoing cardiac arrest for 30 minutes led by thoracotomy (NHB group), and reperfused for 60 minutes with Krebs-Henseleit bicarbonate buffer after cold preservation for 6 hours. In another group (ED group), warm ischemic livers from NHBDs were reperfused with buffer containing edaravone (1 mg/L) after cold preservation.



In the ED group, portal flow volume, bile production, and energy charge were significantly ameliorated. Lipid peroxidation, elevation of hepatic enzymes, and release of tumor necrosis factor-alpha and interleukin-1 beta were significantly alleviated, compared with the NHB group.



These results suggested that edaravone has suppressive effects on warm I/R injury in liver grafts from NHBDs.

title: Edaravone protects against ischemia/reperfusion-induced oxidative damage to mitochondria in rat liver.
abstract: This study investigated the effects of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, MCI-186), a potent free radical scavenger, on the prevention of mitochondrial injury induced by hepatic ischemia and reperfusion. Mature male rats were subjected to 70 min of hepatic ischemia and 2 h of reperfusion. The rats received vehicle or edaravone (10 mg/kg body weight) intravenously prior to ischemia, before reperfusion and 1 h after reperfusion. In the vehicle-treated animals, the respiratory control index, ADP/O, State 3 respiration and dinitrophenol-induced uncoupled respiration decreased markedly after ischemia/reperfusion and were restored by edaravone administration. Mitochondrial lipid peroxidation was elevated in the vehicle-treated group, which was attenuated by edaravone, while mitochondrial glutathione peroxidase activity decreased in the vehicle-treated group, which was similarly abrogated by edaravone treatment. Electron microscopic observation demonstrated that treatment with edaravone restored the ischemia/reperfusion-induced disorganization of mitochondrial structures. Edaravone protects against mitochondrial injury, which prevents mitochondrial oxidative stress and improves ischemia/reperfusion-induced hepatic energy metabolism.

title: Edaravone reduces ischemia-reperfusion injury mediators in rat liver.
abstract: In hepatic ischemia-reperfusion (I/R) injury, oxidative stress both directly injures the liver and promotes an inflammatory reaction by up-regulating various inflammatory mediators. We investigated whether edaravone, a new hydroxy radical scavenger, could reduce hepatic I/R injury including expression of inflammatory mediators such as cytokines and adhesion molecules.



Male Sprague-Dawley rats were subjected to 30 min of partial hepatic pedicle clamping (70%) followed by reperfusion. Just after initiation of reperfusion and again 1 h later, edaravone was administered intraportally. After reperfusion hepatic lipid peroxidation was measured by thiobarbituric acid assay, and hepatic injury was quantified by measuring hepatic enzymes in plasma. We serially quantified hepatic expression of mRNAs for tumor necrosis factor (TNF)-alpha and E-selectin, and histologically examined E-selectin expression and neutrophil accumulation.



In the edaravone group, hepatic lipid peroxidation and hepatic enzyme leakage were significantly less than in the saline group. Hepatic expression of TNF-alpha and E-selectin mRNAs was significantly lower in the edaravone than the saline group, at 2 h after initiation of reperfusion. Histologically, E-selectin immunoreactivity and neutrophil accumulation were less evident in hepatic sections from the edaravone group.



Edaravone reduced hepatic I/R injury by minimizing oxidative stress, and inhibited subsequent injurious inflammation by reducing expression of inflammatory cytokines and adhesion molecules.

title: Free radical scavenger (edaravone) prevents endotoxin-induced liver injury after partial hepatectomy in rats.
abstract: Infection after major surgery, such as massive hepatectomy, induces liver dysfunction, occasionally leading to multiple organ failure and death. We demonstrated the anti-inflammatory effects and functional mechanisms of 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone), a newly synthesized free radical scavenger, on an experimental model of endotoxemia after partial hepatectomy in rats.



Rats were treated with lipopolysaccharide (LPS) 48h after 70% hepatectomy. Edaravone was administered intravenously before LPS-treatment.



Edaravone markedly improved the survival rate of LPS-treated rats after hepatectomy and inhibited increases in serum levels of AST and LDH. Histopathological analysis demonstrated that edaravone prevented inflammatory changes in the liver, kidney and spleen. Edaravone inhibited the formation of one of the markers of oxidative damage, malondialdehyde. Increases in inflammatory cytokines and cytokine-induced neutrophil chemoattractant (CINC) in serum and liver tissue were inhibited in the edaravone-treated group. An electrophoretic mobility shift assay revealed that edaravone inhibited the activation of the transcription factor, nuclear factor-kappa B (NF-kappaB). Edaravone also reduced the induction of inducible nitric oxide synthase (iNOS).



Edaravone prevents endotoxin-induced liver injury after partial hepatectomy not only by attenuating oxidative damage, but also by reducing the production of inflammatory cytokines, CINC and iNOS, in part through the inhibition of NF-kappaB activation.

title: The free radical scavenger edaravone suppresses experimental closed duodenal loop-induced acute pancreatitis in rats.
abstract: Recent studies suggest that the enhanced release of reactive oxygen species (ROS) plays an important role in the pathogenesis of clinical acute pancreatitis. In the present study, we investigated the effects of the free radical scavenger edaravone, which is used clinically as an anti-stroke agent, in the development of experimental closed duodenal loop (CDL)-induced acute pancreatitis. In the CDL-pancreatitis model, after edaravone and vehicle saline were injected intravenously, pancreatitis was induced for 7 h by the CDL technique. The subsequent ascites volume, wet pancreatic weight, serum amylase levels, and pancreatic tissue lipid peroxide levels were evaluated. Pancreatic tissue damage was also evaluated histologically. In this CDL-induced pancreatitis model, edaravone treatment tended to reduce the ascites volume and inhibit the increases in the wet pancreatic weight. Edaravone also tended to reduced the microscopic mucosal damage scores and pancreatic tissue lipid peroxide levels. In particular, the serum amylase levels in the edaravone-treated rats (1-20 mg/kg i.v.) were significantly reduced as compared to the vehicle-treated rats. These results strongly support the involvement of ROS in the pathogenesis of CDL-induced acute pancreatitis and cytoprotective effects of free radical scavender against pancreatic acinar cells. A clinical effect for edaravone against acute pancreatitis is strongly expected.

title: A Radical Scavenger, Edaravone, Protects Canine Kidneys from Ischemia-Reperfusion Injury after 72 Hours of Cold Preservation and Autotransplantation
abstract: Cold ischemia-reperfusion (I/R) injury is a prominent cause of delayed graft function after kidney transplantation. Reactive oxygen species play a crucial role in I/R injury. Edaravone is a synthetic radical scavenger that has been used in acute stroke. Some animal experiments have revealed its beneficial effects against I/R injury, but its effects after cold preservation and transplantation of canine kidneys are unknown.  Female hybrid dogs weighing 11 to 13 kg were used. Under anesthesia, the left kidney was harvested. After 72 hr of preservation in cold histidine-tryptophan-ketoglutarate solution, autotransplantation was performed in the right iliac fossa, with contralateral nephrectomy. Animals were divided into control and treatment groups (n=6 per group). In the treatment group, edaravone was administered intravenously at harvest and at reperfusion (3 mg/kg) and in addition was added to the preservation solution (50 &mgr;M).  Animal survival at 2 weeks was four of six in the control group and six of six in the treatment group. Compared with controls, treated animals had higher mean urine output, higher mean glomerular filtration rate, improved tubular cell function, lower mean serum creatinine, and lower renal vascular resistance. Biopsy specimens from treated animals showed less tubular cell damage and decreased P-selectin expression in endothelial cells. Lipid peroxidation of renal tissue and urinary excretion of 8-hydroxy-2′-deoxyguanosine were suppressed by the treatment.  Edaravone reduced cold I/R injury in canine renal transplantation. The agent has the potential to ameliorate preservation injury in clinical transplantation.

title: Effect of a novel free radical scavenger, edaravone, on puromycin aminonucleoside induced nephrosis in rats
abstract: Recent studies indicate that excessive production of oxidants plays a role in the pathogenesis of glomerular injury leading to proteinuria in patients with minimal-change nephrotic syndrome (MCNS). The novel free radical scavenger, edaravone (EDA), which was recently developed in Japan, is currently used in patients with stroke. We studied whether this new agent would be beneficial in patients with MCNS by its antioxidant activity and examined its effect on proteinuria in nephrosis induced by puromycin-aminonucleoside (PAN) in rats. Nineteen Wistar-Kyoto rats injected with PAN were assigned to four groups: group 1, without EDA (n=4); group 2, concomitant EDA injection from 1 day prior to PAN administration (n=5); group 3, concomitant EDA injection from 1 day after PAN administration (n=5); group 4, concomitant EDA injection from 3 days after PAN administration (n=5). Daily urinary excretions of protein and 8-hydroxy-2’-deoxyguanosine (8-OHdG), a new sensitive marker of oxidative DNA damage in vivo, were measured in each group from the 1st to the 30th day after PAN injection. In group 1 proteinuria developed from the 5th day and reached the peak level on the 9th day. In groups 2, 3, and 4 proteinuria did not appear until the 6th day. The excretions in urinary protein and 8-OHdG were significantly lower in groups 2, 3, and 4 than group 1 on days 5, 9, and 25. In conclusion, EDA could delay and ameliorate the urinary protein excretion in accordance with the urinary 8-OHdG excretion in PAN-induced nephrosis.

title: Protective effect of radical scavenger edaravone against puromycin nephrosis.
abstract: Recent studies have indicated that reactive oxygen species (ROS) play a role in the pathogenesis of glomerular injury leading to proteinuria in nephrotic syndrome. In the present investigation, we examined the effects of the radical scavenger edaravone administered at various time points to rats with puromycin nephrosis.



35 Wistar rats were divided into five groups: treatment with puromycin aminonucleoside (PAN) alone, treatment with PAN followed by edaravone in the early period, treatment with PAN followed by edaravone administration in the late period, treatment with PAN and administration of edaravone for the whole experimental period, and untreated controls. On Days 3, 6 and 9, urinary protein excretion was measured. The levels of glomerular thiobarbituric acid-reactive substance (TBArs) were determined in all animals on Day 10.



On Day 9, rats that had been administered edaravone showed reduced urinary protein excretion and reduced glomerular TBArs. In particular, edaravone administration in the late period, during which proteinuria was most acute, had the effect of reducing the severity of proteinuria. Glomerular TBArs were suppressed to the control level. Our results indicate that edaravone exerts a protective effect in the acute phase of PAN nephrosis when administered as antioxidant therapy at the onset of proteinuria.



Edaravone can ameliorate urinary protein excretion after the onset of proteinuria in nephrotic syndrome.

title: Edaravone protects against ischemia/reperfusion-induced functional and biochemical changes in rat urinary bladder.
abstract: To investigate the effects of edaravone on ischemia/reperfusion (I/R) injury in the rat bladder. Increasing evidence has shown that I/R are major etiologic factors in the progression of bladder dysfunction induced by partial outlet obstruction, and that part of the damage is due to the generation of free radicals. Edaravone is a newly developed radical scavenging agent that has been used for protection against I/R injury in patients with cerebral infarction.



Thirty-five adult male rats were divided into five groups. Groups 1 to 4 underwent 1 hour of ischemia followed by 1 hour of reperfusion. Groups 1 to 3 were treated with edaravone at 1, 3, or 10 mg/kg body weight and group 4 with saline. Group 5 consisted of age-matched control rats. In vivo ischemia was created by clamping the vesical arteries for 1 hour. Reperfusion was accomplished by removing the clips and also lasted for 1 hour. Edaravone or saline was administered into the femoral artery after reperfusion for 30 minutes. After reperfusion, the bladder was excised and separated. The responses to electrical field stimulation, carbachol, and KCl were recorded. Other materials were analyzed for malondialdehyde as a measure of lipid peroxidation.



Edaravone administration resulted in protection of the contractile responses to both field stimulation and carbachol, although the responses to KCl were not affected. I/R resulted in an increase in malondialdehyde, which was reduced to control levels by edaravone.



These results suggest that edaravone has a potential protective effect against I/R-induced damage in the rat bladder.

title: Acute urinary retention and subsequent catheterization cause lipid peroxidation and oxidative DNA damage in the bladder: preventive effect of edaravone, a free‐radical scavenger
abstract: To investigate the effect of a free‐radical scavenger, edaravone, on the changes occurring with acute urinary retention (AUR) and subsequent catheterization in the rat bladder.

title: EDARAVONE, A NOVEL FREE RADICAL SCAVENGER, REDUCES HIGH-MOBILITY GROUP BOX 1 AND PROLONGS SURVIVAL IN A NEONATAL SEPSIS MODEL
abstract: Free radicals play an important role in the inflammatory process of sepsis. We hypothesized that edaravone, a novel free radical scavenger, can suppress pathophysiological events and prolong survival in a neonatal sepsis cecal ligation and perforation (CLP) model. Of 32 3-day-old anesthetized and mechanically ventilated piglets, 11 received CLP only, 10 received CLP and edaravone treatment starting 30 min after CLP, and 11 constituted a sham (control) group. Mean arterial pressure (MAP), heart rate, cardiac output, arterial blood gas, serum total hydroperoxide, nitrite and nitrate, TNF-&agr;, and high-mobility group box 1 (HMGB1) were measured before CLP and at 1, 3, and 6 h after CLP. Compared with the CLP group, the edaravone group showed higher MAP at 6 h, lower heart rate at 1 and 3 h, lower total hydroperoxide at 1 h, lower nitrite and nitrate at 3 and 6 h, and higher (although not significantly so) mean cardiac output at 1, 3, and 6 h. TNF-&agr; elevation was delayed from 1 h in the CLP group to 3 h in the edaravone group. In the edaravone group, HMGB1 did not change significantly at any time, whereas in the CLP group, it increased at 6 h. Survival times were longer in the edaravone group than in the CLP group (15.4 ± 1.4 vs. 10.2 ± 1 h; P < 0.005). In addition, each of the serial dilutions of edaravone had a higher biological antioxidant potential than tempol does. In conclusion, edaravone suppressed free radicals, delayed the TNF-&agr; surge, and prevented HMGB1 elevation, thereby maintaining MAP and prolonging survival time in a neonatal sepsis CLP model.

title: The therapeutic efficacy of edaravone in extensively burned rats.
abstract: Extensive burn injury leads to production of free radicals subsequent to massive fluid resuscitation, which in turn increases the risk of acute lung injury. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a novel free radical scavenger, is clinically effective in improving the prognosis after cerebral infarction. However, the effect of edaravone against extensive burn injury has not been tested. Objected To evaluate whether edaravone can reduce free radical precursors in a 30% burn model in rats.



Prospective, randomized controlled experiment.



Animal basic science laboratory.



Male Wistar rats weighing 200 to 220 g.



All rats (n = 10) were given a 30% full-thickness burn according to the Walker and Mason method. Immediately after the burn, edaravone was injected into the rats (n = 5) intraperitoneally at a dose of 9 mg/kg. One hour after burn injury, blood and tissue samples were collected to analyze free radical changes of serum and tissue malondialdehyde (MDA) and xanthine oxidase (XOD) and lung white blood cells.



Statistical significance was found between nontreatment and edaravone treatment relative to serum MDA (mean +/- SD, 2.50 +/- 0.54 vs 1.74 +/- 0.29 nmol/mL), serum XOD (mean +/- SD, 5.04 +/- 1.67 vs 2.26 +/- 0.83 U/L), tissue MDA (mean +/- SD, 1268.7 +/- 289.9 vs 569.1 +/- 135.9 nmol/mg protein), tissue XOD (mean +/- SD, 256.3 +/- 58.1 vs 50.96 +/- 19.60 mU/g tissue), lung white blood cells (mean +/- SD, 3088 +/- 1144 vs 1542 +/- 575 mU/g tissue), and lung XOD (mean +/- SD, 428.3 +/- 210.5 vs 81.8 +/- 36.0 nmol/mg protein).



Edaravone treatment induces significant reduction of free radical precursors and their metabolites compared with controls in burn rats. This suggests that edaravone could be helpful in the clinical treatment of large burns.

title: A free radical scavenger, edaravone (MCI-186), diminishes intestinal neutrophil lipid peroxidation and bacterial translocation in a rat hemorrhagic shock model*
abstract: To investigate the effects of edaravone, a novel free radical scavenger, on bacterial translocation induced by hemorrhagic shock. Prospective, randomized, unblinded animal study. Surgical research laboratories of Shiga University of Medical Science. Male specific-pathogen-free Sprague-Dawley rats. The rats were randomly divided into three groups: conventional saline treatment, edaravone treatment, and sham shock induction. The saline and edaravone groups were subjected to hemorrhagic shock (mean arterial pressure of 30 mm Hg, for 30 or 60 mins). Rats were killed 30 or 60 mins after shock induction. Mesenteric lymph nodes were cultured for determination of bacterial translocation. Systemic plasma silkworm larvae plasma test, which can detect peptidoglycan and &bgr;-glucan, and endotoxin tests were performed. Immunohistochemistry for 4-hydroxy-2-nonenal (4-HNE) was used to assess lipid peroxidation after shock. The incidence and magnitude of hemorrhagic-shock-induced bacterial translocation to mesenteric lymph nodes were reduced by edaravone. Hemorrhagic-shock-induced increase of plasma silkworm larvae plasma test was also reduced by edaravone. Immunohistochemistry for 4-HNE showed many 4-HNE-positive cells in the lamina propria of the ileum 60 mins after hemorrhagic shock. Double immunohistochemistry revealed that many of these 4-HNE-positive cells were also myeloperoxidase positive. Moreover, the percentage of double-labeled cells with 4-HNE and myeloperoxidase in myeloperoxidase-positive cells was significantly lower in the edaravone group than in the saline group. The present findings suggest that lipid peroxidation of intestinal neutrophils is involved in bacterial translocation during hemorrhagic shock and that edaravone is potentially useful in diminishing bacterial translocation after hemorrhagic shock.

title: Edaravone improves the survival of rats subjected to hemorrhagic shock without resuscitation
abstract: Our aim in this study was to find out whether edaravone (3-methyl-1-phenyl-pyrazolin-5-one, MCI-186), a novel free radical scavenger, improved the survival rate in a rat hemorrhagic shock (HS) model. Fifty male Sprague-Dawley rats were divided randomly into an edaravone group and a saline group. Both groups were subjected to HS by inducing a mean arterial pressure of 30 mmHg for 60 min without resuscitation. The edaravone group was divided into four subgroups based on when edaravone was given: 0, 15, 30, or 60 min after HS. The saline group was given saline immediately after HS. We evaluated the 24-h survival rate in each group. The survival rate of the edaravone subgroup given edaravone immediately after HS was significantly better than that of the saline group. Edaravone improved the survival rate in a rat HS without resuscitation model. Edaravone was most effective when given immediately after HS.

title: Protective effect of the radical scavenger edaravone against methamphetamine-induced dopaminergic neurotoxicity in mouse striatum.
abstract: The administration of high doses of methamphetamine causes the degeneration of striatal dopaminergic fibers in the brains of rodents, and oxidative stress appears to be one of the main factors of neurotoxicity. This study examined whether edaravone, a radical scavenger, protects against methamphetamine-induced neurotoxicity in mice. Methamphetamine treatment (4 mg/kg, s.c. x 4 with 2 h intervals) showed striatal dopaminergic degeneration as observed by decreases in dopamine levels and tyrosine hydroxylase immunoreactivity in the striatum. The neurotoxicity was reduced by edaravone (3 mg/kg, i.p.), when it was administered four times 30 min before methamphetamine at 2 h intervals and additionally four times after methamphetamine at 12 h intervals. An immunohistochemical study showed that methamphetamine increased 3-nitrotyrosine immunoreactivity, an in vivo marker of peroxynitrite production, and activated microglia and astrocytes in the striatum. Edaravone blocked the increase in 3-nitrotyrosine immunoreactivity and the activation of astrocytes, but it did not affect the activation of microglia. Edaravone did not affect methamphetamine-induced hyperthermia and striatal dopamine release. These results suggest that edaravone protects against methamphetamine-induced neurotoxicity in the striatum by blocking peroxynitrite production. This study also suggests that methamphetamine activates microglia in a radical-independent mechanism.

title: Inhibitory effects of edaravone on the production of tumor necrosis factor-α in the isolated heart undergoing ischemia and reperfusion
abstract: We evaluated the effects of edaravone, a hydroxyl radical scavenging agent, on the production of tumor necrosis factor-α (TNF-α) in myocardium, and the release of TNF-α and P-selectin from myocardium after ischemia–reperfusion injury in isolated Langendorff-perfused rat hearts. Cardiodynamic function at stable points during perfusion and 5, 15, 30, and 60 min after the initiation of reperfusion was evaluated by left ventricular developed pressure, rate of increase in left ventricular pressure and rate of decrease in ventricular pressure, coronary flow, and heart rate. At 60 min after the initiation of reperfusion, myocardial infarct size was estimated microscopically using triphenyltetrazolium chloride staining, and expression of TNF-α in myocardium was detected by Western blot and immunohistochemistry. At the same time points as the measurement of cardiodynamic function, TNF-α and the soluble form of P-selectin in coronary effluent were measured by enzyme immunoassay. At all time points during reperfusion, edaravone markedly improved cardiodynamic function and reduced myocardial infarct size in comparison to the control. In myocardium in the control, TNF-α was detected in the endothelial cells and other cells bearing some resemblance to interstitial cells and monocyte cells. Edaravone suppressed this cytokine expression in the corresponding sites. P-selectin as well as TNF-α was found in the coronary effluent of the control, and edaravone significantly decreased soluble P-selectin levels in comparison to the control (P < 0.01). Edaravone might have protective effects on cardiac function through reduction of infarct size via decrease of production of TNF-α in myocardium induced by ischemia–reperfusion injury and through reduction of the release of adhesion molecules such as P-selectin from vascular endothelial cells.

title: Cardioprotective effects of 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), a novel free radical scavenger, on acute autoimmune myocarditis in rats.
abstract: Recent evidence suggests that oxidative stress may play a role in myocarditis.



To test the hypothesis that 3-methyl-1-phenyl-2-pyrazolin-5-one (edaravone or MCI-186), a novel free radical scavenger, protects against acute experimental autoimmune myocarditis (EAM) in rats.



MCI-186 was administered intraperitoneally (1 mg/kg/day, 3 mg/kg/day or 10 mg/kg/day) in rats with EAM for three weeks. The results were compared with those from untreated rats with EAM.



MCI-186 treatment did not affect the hemodynamics of the rats, but did reduce the severity of myocarditis when the heart weight:body weight ratio and the pathological scores were compared. There were significantly fewer myocardial interleukin-1beta-positive cells in rats with EAM treated with MCI-186 (at 3 mg/kg/day and 10 mg/kg/day) than in the untreated rats with EAM.



MCI-186 ameliorated acute EAM in rats, suggesting that free radicals may be involved in the development of myocarditis.

title: Preventive effects of edaravone, a free radical scavenger, on lipopolysaccharide‐induced lung injury in mice
abstract: Reactive oxygen species (ROS) play an important role in the pathogenesis of acute lung injury (ALI) and pulmonary fibrosis. It was hypothesized that edaravone, a free radical scavenger, would be able to attenuate LPS‐induced lung injury in mice by decreasing oxidative stress.