An open-label, prospective, non-randomized study of intravenous H2-administration was performed at 3 institutions in Japan (the National Defense Medical College, the Kuki General Hospital, and the Ken-o-Tokorozawa Hospital) from July 2011 through December 2012. The Clinical Research Ethics Committee of these institutions approved this study. Prior informed consent was obtained from all study participants or their relatives.
Patients were eligible for enrollment if they were 18 years or older and had a clinical diagnosis of acute ischemic stroke within 72 h of symptom onset. All patients underwent examination using computed tomography (CT) or magnetic resonance imaging (MRI) immediately after admission. A diagnosis of stroke was based on clinical findings. Baseline data (age, sex), conventional vascular risk factors (hypertension, diabetes mellitus, hyperlipidemia), and past history of smoking, atrial fibrillation, ischemic stroke and hemorrhagic stroke were recorded. Patients whose pertinent data could not be evaluated at the time of stroke onset were excluded from this study. Based on the location of the ischemic lesions, the patients were divided into 2 groups . In Group I (GI) patients, the infarct was located in cortical regions within the cerebral hemisphere and involved the frontal, parietal, and temporal lobe or the occipital lobe and cerebellum. In Group II (GII) patients, the infarct involved basal ganglia regions in the anterior circulation (putamen, caudate head), corona radiate, or brain stem and thalamus. The stroke subtypes were defined according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification system . Of the 38 patients, 12 (31.6%) had cardioembolic infarcts, 14 (36.8%) had atherothrombotic infarcts, 11 (28.9%) had lacunar infarcts, and 1 (2.6%) had a stroke of undetermined etiology. The atherothrombotic infarct group included patients with clinical and imaging findings of either significant stenosis or occlusion of a major artery or branch of the cortical artery, due to atherosclerosis. The cardioembolic infarct group included patients with arterial occlusion due to an embolus arising from the heart. The lacunar infarction group included patients with one of the traditional clinical lacunar syndromes and no evidence of cerebral cortical dysfunction, and patients whose MRI did not show any lesions exceeding 1.5 cm in diameter.
There was no control group in this study. All patients received intravenous H2-enriched glucose-electrolyte solution in addition to edaravone immediately after the diagnosis of acute ischemic stroke. For all patients, intravenous edaravone (a 30 mg Edaravone Kit) was given twice a day (every 12 h), and H2-enriched intravenous solutions (200 ml) were added at the speed of 200 ml/h twice a day (every 12 h). All patients received appropriate routine stroke care. Acute stroke patients within 3 h of onset received intravenous t-PA treatment (0.6 mg/kg), and those patients receiving t-PA had to commence the administration of H2-enriched intravenous solution and edaravone before or at the same time that the t-PA was infused.
Production of an H2-enriched glucose-electrolyte solution
An H2-enriched glucose-electrolyte solution was produced using a non-destructive hydrogen adding apparatus (Miz Co., Fujisawa, Japan; Patent No.4486157, Patent Gazette of Japan 2010) as has been reported elsewhere . Bags of glucose-electrolyte solution (Soldem 1, 200 ml/bag, Terumo, Tokyo, Japan) were immersed, without opening or altering the bag, in a water tank in which water is electrolyzed periodically to produce water with hydrogen concentrations of up to 1.6 ppm. The concentration of hydrogen in the bag reached saturation, increasing to more than 1.0 ppm because of diffusion through the wall of the bag. Additional information describing this process can be found at: http://www.e-miz.co.jp/english/technology.html#non_destructive.
Clinical diagnosis and evaluation
An NIHSS score was assigned upon admission to the hospital, and 7, 30, and 90 d after admission to evaluate neurological deficits. Patients were also evaluated using a modified Rankin scale (mRS) (7, 30, and 90 d after admission) and the Barthel Index (7, 30, and 90 d after admission). Vital signs were recorded at enrollment and at specified times throughout the infusion and follow-up periods. Venous blood sampling for the malondialdehyde (MDA)-modified low-density lipoprotein (LDL) was taken as a serum-based indicator of oxidative stress . Other biochemical analyses, urine sampling, 12-lead electrocardiography (ECG), and plain chest X-rays were performed at the time of enrollment, and on the 7th day after admission, and were analyzed centrally.
Prior to administration of t-PA, 3D-CT angiography (3DCTA) or MR angiography (MRA) were performed to identify the occluded arteries. Follow-up 3DCTA or MRA was performed 24 h after t-PA treatment to identify the presence or absence of early recanalization in the occluded arteries. Recanalization was graded as complete, partial, or no recanalization according to a previous report , as follows: 1) complete recanalization: reappearance of the entire occluded artery and the distal branches of vessels; 2) partial recanalization: restoration of part of the distal vessel supplied by the occluded artery; and 3) no recanalization: persistent occlusion. To assess any effect of H2 on hemorrhagic transformation after t-PA administration, brain imaging was repeated after 24 h in patients who were receiving concomitant treatment with t-PA. Symptomatic hemorrhagic transformation was defined as an increase of or more 4 points according to the NIHSS score that occurred within 24 h, and evidence of any blood on neuroimaging performed 24 h after treatment with t-PA.
All data are presented as the mean ± SD. All analyses were performed using Student’s t test for paired data. A value of P < 0.05 was considered statistically significant. The GraphPad Prism 4.0 software program (San Diego, CA, USA) was used for all statistical tests.