A Systematic Review and Meta-analysis of the Efficacy of Edaravone in Animal Models of Focal Ischaemic Stroke Background Acute ischaemic stroke is a major cause of mortality and morbidity worldwide, the majority of which is the focal ischaemia due to the blockage of certain cerebral arteries [1, 2]. Though thousands of candidate drugs have been tested and more than five hundred neuroprotive agents are proved to be effective in animal models, few of them has shown the efficacy in clinical practice [3]. Thus the development of anti-stroke strategies is still in urgent demand. Oxidative stress is responsible for several neuropathological conditions. Edaravone, an oxygen free radical scavenger, has demonstrated neuroprotective effects by protecting cell membranes against oxidative stress [4-7]. Therefore, it shows ability to suppress neuronal damage and neurovascular injury in such conditions in studies in vitro and in vivo. Edaravone further proves to have beneficial effects in animal models of several neurodegenerative diseases, such as neonatal hypoxic-ischemic encephalopathy [8], acute intracerebral haemorrhage (ICH) [9], subarachnoid haemorrhage (SAH) [10], traumatic brain injury [11], amyotrophic lateral sclerosis (ALS) [12] , and Parkinson disease (PD) [13]. Further, it has already been applied in clinical practice for some of them [14, 15]. Based on these perspectives, there is significant implication for the edaravone management of human ischaemic stroke and its free radical scavenging activity has been evaluated experimentally in focal ischaemia models. Edaravone was first reported in Japan to be beneficial in animal models with focal cerebral ischaemia [16-18], and was then tested clinically [19-20]. However, though it has been marketed as a neuroprotective agent for acute ischaemic stroke in Japan for a decade [19], and, as well, has been widely used in China [21, 22], very limited clinical data on edaravone has been reported in other countries [23]. For those drugs tested, the animal data seemed not to be robust enough as a useful guide to clinical practice. We are interested in carrying out a systematic review and meta-analysis to investigate the safety and efficacy of edaravone in animal models with focal ischaemia, trying to explore sufficient fidelity of animal studies to support the transition of its efficacy from bench to bedside. The recombinant tissue plasminogen activator (rt-PA) is currently the only licensed acute treatment for ischaemic stroke and is recommended as a first-line drug [28-30] worldwide . However, only a few patients receive the treatment [27, 31, 32], as it is [24-27] strictly conditioned to patients within 3 hours of onset and bears a great risk of Page 1 of 7 [33, 34] haemorrhage transformation . Thus, we would also like to explore the potential benefits of the combination therapy of edaravone and rt-PA over the rt-PA therapy alone. Methods 1. Criteria for selecting studies for the review We will include studies describing the effect of edaravone in animal models of focal ischaemic stroke, compared with control animals receiving vehicle or no treatment, or the combination therapy of edaravone with rt-PA compared with rt-PA alone, regardless of randomisation. We will take studies in which stroke models were established by the occlusion of the middle cerebral or anterior cerebral arteries, or their branches. Either permanent or transient/reperfusion model would be included. All species of animals are eligible, regardless of age and sex. Studies with any route and dose of drug administration are eligible. Any time of delivery will be considered, whether prior or post to the induction of ischaemia, or before/after reperfusion. Outcomes are measured as neurobehavioral scores and/or infarct volume. 2. Search strategy We will search PUBMED (from 1950), EMBASE (from 1980), and ISI Web of Science (from 1981). We develope the Pubmed search strategy* as follows and will adapt it for the other databases. Two investigators will independently assess the titles and abstracts obtained from the searches of the electronic databases, and compare their results of included papers. If there is any discrepancy in the decision, they will discuss to reach a consensus. Then full text of the included studies will be obtained. Articles in foreign languages will be translated, and reference listed in articles will be assessed to identify additional studies not located by the initial search. Two authors will assess each study for inclusion in the review based on our inclusion criteria. Any disagreement will be resolved by discussion and, if necessary, we will consult a third review author. 3. Data extraction Data on characteristics of study design, attributes of study quality, functional outcome and structural outcome will be recorded. Two investigators will extract data from the first 10 papers and compare the data to enhance the inter-rater reliability, and then one of them will do the remaining papers. Data will be recorded in the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Stroke (CAMARADES) Microsoft Access 2003 data manager application. Data will be extracted and recorded separately according to the single edaravone therapy and combined therapy with rt-PA for further individual analyses of therapeutic effects of either therapy. Page 2 of 7 3.1. Design of study Data on study design will be recorded, including: species and sex of animals used; anaesthetics and ventilation; types of ischaemic injury; methods of ischaemia induction and duration of ischaemia; dose, route and time of drug administration; time-point of outcome measured; methods of outcome measurement. In combination therapy of rt-PA, the dose, route and time of rt-PA administration will be recorded. 3.2. Quality of study Study quality will be assessed according to the CAMARADES quality checklist [35] with 10 points in total, and we will calculate the group median scores: (1) peer-reviewed publication; (2) control of animals’ temperature; (3) using of animals with co-morbidities; (4) avoid using anaesthetics with prominent intrinsic neuroprotective abilities; (5) reporting the sample size calculation; (6) randomization of treatment allocation; (7) concealment of treatment allocation; (8) blinded assessment of outcome; (9) statement of compliance with animal welfare regulations; and (10) statement of possible conflicts of interest. 3.3. Outcome extraction We will extract the number of animals, the mean outcome and its standard deviation (SD) or standard error (SE) for each treatment comparison. Our outcomes are neurobehavioural scores, and the infarct volume or size. When neurobehavioral tests were performed at different times, only the final test will be included. Values of graphical data will be requested from the authors, and if not available, we will use digital ruler software to measure data from the graphs. In case full data required for meta-analysis are not available from abstracts or publications, we will request them directly from authors. When data required are not obtainable, we will exclude such studies from the analysis. 4. Statistical analysis Data obtained from single edaravone therapy and edaravone plus rt-PA therapy will be analysed separately. We will use normalised mean difference as effect size (ES), and calculate ES and its SE for each comparison. The 95% confidence interval of the effect size will be calculated as well. Data will be aggregated using a weighted average method in which greater weight is given to more precise studies. For anticipated heterogeneity between studies, the random-effects model of Dersimonian and Laird is going to be used, which is more conservative than fixed-effect model, giving the weighting to individual comparisons depends on the variance within those comparisons and on overall heterogeneity. The heterogeneity between studies will be assessed by using the Chi-square statistc with n-1 degrees of freedom (df). To allow for multiple comparisons we set a significance level using Bonferroni correction taking into the number of comparisons. We will look for publication bias using funnel plotting [36] , Egger regression [37] and “trim and fill” [38]. Page 3 of 7 Subgroup meta-analysis is performed according to characteristics of study design and study quality to assess their impact on efficacy, as following subgroups: species, dose (low or high dose, single or multiple), time of administration (before/after ischaemia, or before/after reperfusion), time of assessment (acute phase or long term), and types of ischaemia (transient or permanent), duration of ischaemia, as well as scores of quality checklist, especially the use of randomisation and blinded assessment of outcome. Appendix References 1) World Health Organization. Preventing Chronic Diseases. A Vital Investment: WHO Global Report. Cited from: http://www.who.int/chp/chronic_disease_report/en/ (2005) 2) Wardlaw, JM. et al. Thrombolysis for acute ischaemic stroke. Cochrane Database of Systematic Reviews. Issue 4 (2009) 3) O’Collins, VE. et al. 1,026 experimental treatments in acute stroke. Ann Neurol. 59, 467–77 (2006) 4) Watanabe, T. et al. Preventive effect of MCI-‐186 on 15-‐HPETE induced vascular endothelial cell injury in vitro. Prostaglandins Leukot Essent Fatty Acids. 33, 81–87 (1988) 5) Yoshida, H. et al. Edaravone, a novel radical scavenger, inhibits oxidative modification of low-‐density lipoprotein (LDL) and reverses oxidized LDL-‐mediated reduction in the expression of endothelial nitric oxide synthase. Atherosclerosis. 179, 97-‐102 (2005) 6) Ishii, J. et al. The free-‐radical scavenger edaravone restores the differentiation of human neural precursor cells after radiation-‐induced oxidative stress. Neuroscience Letters. 423, 225-‐230 (2007) 7) Lee, BJ. et al. Edaravone, a free radical scavenger, protects components of the neurovascular unit against oxidative stress in vitro. Brain research. 1307, 22-‐27 (2010) 8) Ikeda, T. et al. Effect of the free radical scavenger, 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one (MCI-‐186), on hypoxia-‐ischemia-‐induced brain injury in neonatal rats. Neurosci Lett. 329, 33–36 (2002) 9) Nakamura, T. et al. Edaravone attenuates brain edema and neurologic deficits in a rat model of acute intracerebral hemorrhage. Stroke. 39, 463–469 (2008) 10) Nakagomi, T. Effect of edaravone on cerebral vasospasm following experimental subarachnoid hemorrhage. Journal of Stroke and Cerebrovascular Diseases. 12, 17-‐21 (2003) 11) Wang, GH. et al. Free-‐Radical Scavenger Edaravone Treatment Confers Neuroprotection Against Traumatic Brain Injury in Rats. Journal of Neurotrauma. 28, 2123-‐2134 (2011) 12) Ito, H. et al. Treatment with edaravone, initiated at symptom onset, slows motor decline and decreases SOD1 deposition in ALS mice. Exp Neurol. 213, 448–455 (2008) 13) Xiong, N. et al. Edaravone guards dopamine neurons in a rotenone model for Parkinson's disease. PLoS ONE | www.plosone.org 6(6): e20677 (2011) Page 4 of 7 14) Munakata, A. et al. Effect of a free radical scavenger, edaravone, in the treatment of patients with aneurysmal subarachnoid hemorrhage. Neurosurgery. 64, 423-‐428 (2009) 15) Dohi, K. et al. Alkoxyl radical-‐scavenging activity of edaravone in patients with traumatic brain injury. J Neurotrauma. 23, 1591–1599 (2006) 16) Abe, K. et al. Attenuation of ischemic and post-‐ischemic brain edema in rats by a novel free radical scavenger. Stroke. 19, 480-‐485 (1988) 17) Nishi, H. et al. Effect of MCI-‐186 on brain edema in rats. Stroke. 20, 1236-‐1240 (1989) 18) Oishi, R. et al. Effect of MCI-‐186 on ischemia-‐induced changes in monoamine metabolism in rat brain. Stroke. 20, 1557-‐1564 (1989) 19) Edaravone Acute Infarction Study Group. Effect of a novel free radical scavenger, edaravone (MCI-‐186), on acute brain infarction: randomized, placebo-‐controlled, double-‐blind study at multicenters. Cerebrovasc Dis. 15, 222-‐229 (2003) 20) Otomo, E. et al. Clinical efficacy of a free radical scavenger, MCI-‐186, on acute cerebral infarction: Early phase II clinical trial. Ther Res. 19, 1311–1332 (1998) 21) Yang, QW. et al. Edaravone for acute cerebral infarction: a systematic review. Chinese Evidence-‐Based Medicine. 6, 18-‐22 (2006) 22) Feng, S. et al. Edaravone for acute ischaemic stroke. Cochrane Database of Systematic Reviews. Issue 3 (2008) 23) Green, AR. et al. Free radical trapping as a therapeutic approach to neuroprotection in stroke: experimental and clinical studies with NXY-‐059 and free radical scavengers. Current Drug Targets -‐ CNS & Neurological Disorders. 4, 109-‐118 (2005) 24) STARS. Intravenous tissue-‐type plasminogen activator for treatment of acute stroke: the Standard Treatment with Alteplase to Reverse Stroke (STARS) study. JAMA. 283, 1145 (2000) 25) Hill, MD, et al. Thrombolysis for acute ischemic stroke: results of the Canadian Alteplase for Stroke Effectiveness Study. Canadian Medical Association Journal. 172, 1307-‐1312 (2005) 26) Wahlgren, N, et al. Thrombolysis with alteplase for acute ischaemic stroke in the Safe Implementation of Thrombolysis in Stroke-‐Monitoring Study (SITS-‐MOST): an observational study. The Lancet. 369, 275-‐282 (2007) 27) Yamaguchi, T, et al. Alteplase at 0.6 mg/kg for Acute Ischemic Stroke Within 3 Hours of Onset: Japan Alteplase Clinical Trial (J-‐ACT) Supplemental Appendix 2. Stroke. 37, 1810-‐1815 (2006) 28) The European Stroke Initiative Executive Committee and European Stroke Initiative Writing Group. European stroke initiative recommendations for stroke management—update 2003. Cerebrovasc Dis. 16, 311-‐337 (2003) 29) Adams, HP Jr, et al. Guidelines for the early management of patients with ischaemic stroke: a scientifi c statement from the Stroke Council of the American Stroke Association. Stroke. 34, 1056-‐1083 (2003) 30) Guidelines for Management of Acute Ischemic Stroke Writing Committee, Chinese Stroke Society, Chinese Society of Neurology, Chinese Medical Association. Guidelines for Management of Acute Ischemic Stroke in China 2010.Chin J Neurol. 43, 146-‐153 (2010) 31) Samson, K, et al. Ten Years after FDA Approval, US Hospitals Slow to Adopt t-PA for Ischemic Strokes. Neurology Today. May, 32-33 (2007) 32) Albers, G. W. et al. Intravenous alteplase for ischaemic stroke. The Lancet. 369, 249-‐250 Page 5 of 7 (2007) 33) Wardlaw, J. M., et al. Thrombolysis for acute ischaemic stroke. Cochrane Database of Systematic Reviews (4) (2009) 34) Hacke, W, et al. Association of outcome with early stroke treatment: pooled analysis of ATLANTIS, ECASS, and NINDS rt-‐PA stroke trials.Lancet.363, 768-‐774 (2004) 35) Macleod, MR. et al. Pooling of animal experimental data reveals influence of study design and publication bias. Stroke. 35, 1203-‐1208 (2004) 36) Light, RJ, et al. 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J Am Stat Assoc. 95, 89-‐98 (2000) Pubmed search strategy All fields search: #4Search: (#1) AND #2 Limits: Animals 183 #3Search: (#1) AND #2 284 #2Search: edaravone OR "MCI 186" OR MCI-‐186 OR radic t OR 1-‐phenyl-‐3-‐methyl-‐5-‐pyrazolone OR edarabone OR 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one 614 #1Search: stroke OR ischemi* OR ischaemi* OR cerebrovascular OR "middle cerebral artery" OR MCA OR MCAO OR ACA OR ACAO OR "anterior cerebral artery" 467452 Title/Abstract search: 183 #4Search (#1) AND #2 Limits: Animals 183 #3Search (#1) AND #2 284 #2Search edaravone OR "MCI 186" OR MCI-‐186 OR radic t OR 1-‐phenyl-‐3-‐methyl-‐5-‐pyrazolone OR edarabone OR 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one[Title/Abstract] 614 #1Search stroke OR ischemi* OR ischaemi* OR cerebrovascular OR "middle cerebral artery" OR MCA OR MCAO OR ACA OR ACAO OR "anterior cerebral artery"[Title/Abstract] 467376 Comparing these two sets of 183 papers in EndNote, they are all the same. ISI Web of Science: 379 Topic=(stroke OR ischemi* OR ischaemi* OR cerebrovascular OR "middle cerebral artery" OR MCA OR MCAO OR ACA OR ACAO OR "anterior cerebral artery") AND Topic=(edaravone OR "MCI 186" OR MCI-‐186 OR radic t OR 1-‐phenyl-‐3-‐methyl-‐5-‐pyrazolone OR edarabone OR 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one) Page 6 of 7 Databases=SCI-‐EXPANDED, SSCI, A&HCI, CPCI-‐S, CPCI-‐SSH Timespan=All Years Lemmatization=On EMBASE: keywords in all fields 232 stroke OR ischemi* OR ischaemi* OR cerebrovascular OR "middle cerebral artery" OR MCA OR MCAO OR ACA OR ACAO OR "anterior cerebral artery" 588050 edaravone OR "MCI 186" OR MCI-‐186 OR radic t OR 1-‐phenyl-‐3-‐methyl-‐5-‐pyrazolone OR edarabone OR 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one 790 (edaravone or "MCI 186" or MCI-‐186 or radic t or 1-‐phenyl-‐3-‐methyl-‐5-‐pyrazolone or edarabone or 3-‐methyl-‐1-‐phenyl-‐2-‐pyrazolin-‐5-‐one) and (stroke or ischemi* or ischaemi* or cerebrovascular or "middle cerebral artery" or MCA or MCAO or ACA or ACAO or "anterior cerebral artery") 402 Limit to animals: 232 Totally retrieved 183+379+232=794 Find duplicate: 438 Keep: 356 Page 7 of 7