Design
A controlled, prospective study.
Time and setting
The study was performed at Guangzhou General Hospital of Guangzhou Military Command, China from May 2008 to November 2009.
Subjects
Diagnostic criteria of patients with cerebral infarction
Blood supply disturbances in the brain are induced by various causes and often result in brain ischemia or ischemic necrosis, which corresponds with neurological deficits over one to several days. The injury is often confined to the blood supply region of a certain artery. In the present study, cerebral infarction was confirmed by history and physical examination and brain MRI[27].
Inclusion criteria
(1) Patients were included from 30–80 years of age. (2) Acute cerebral infarction that was not induced by tumor, traumatic brain injury, brain parasitic diseases, or metabolic disorder was confirmed through CT or MRI. (3) The National Institutes of Health Stroke Scale score was between 6–21. (4) Diseased time of patients was more than 6 hours, but less than 72 hours, and patients included were those who could not be treated with intravenous thrombolysis.
Exclusion criteria
(1) Patients with severe stroke, such as multi-lobe infarction (low density in CT larger than 1/3 cerebral hemisphere). (2) Patients subjected to thrombolytic therapy. (3) Patients with shock, severe heart and lung complications and liver dysfunction, or life expectancy of less than 1 month. (4) Patients with coagulation dysfunction or abnormal amount of platelet, including thalassemia, sickle cell anemia, and glucose-6- phosphate dehydrogenase deficiency (favism). (5) Hyperthyroidism symptoms are not under control. (6) High-sensitivity and ozone allergy. (7) Using kinase or anti-free radical agents. (8) Pregnant or lactating women. (9) Not suitable for motor-evoked potential, including a pacemaker or with a history of epilepsy. The program was discussed and adopted by Ethics Committee of Guangzhou General Hospital of Guangzhou Military Command, China, and all subjects signed the informed consent.
In total, 86 patients were included in this study, which was performed in accordance with requirements of Declaration of Helsinki and Administrative Regulations on Medical Institution, formulated by the State Council of China[26]. Written informed consent was obtained from all patients.
Methods
Drug treatment and ozone therapy
The conventional standard treatment guidelines were based on the a guide to early treatment issued by the Stoke Affiliated Society of American Heart Association in 2003[27], and included: (1) stroke unit care; (2) monitoring and treatment of complications such as hypertension, arrhythmia, and high blood glucose; and (3) treatment of neurological complications. The basic medicines used by patients in both groups were 450 mg Xueshuantong injection (a traditional Chinese patent medicine; the main component is arasaponin; Lyophilized, Zhunzi Z20025652, Lot No. 11100407; Guangxi Wuzhou Pharmaceutical Co., Ltd., Wuzhou, Guangxi Zhuang Autonomous Region, China) and 0.1 g aspirin (Bayer HealthCare Manufacturing S.r.l, Beijing, China), or 75 mg Clopidogrel bisulfate tablet (Sanofi Winthrop Industries, France), once per day. In the ozone group, the patients were treated with major ozonated autohemotherapy, once per day, for 10 ± 3 days.
An ozone generating device (HUMARES, Bruchsal, Germany) was used for the major ozonated autohemotherapy. The operating procedure was as follows: 100 mL blood was collected from the cubital vein of patients into a sterile closed blood bag with 10 mL of 2.5% sodium citrate. The blood was then mixed with 100 mL ozone (47 μg/mL) for approximately 2 minutes, and then re-transfused fast through the initial intravenous access (< 30 minutes) into the patient.
Evaluation of treatment efficacy
Evaluation of clinical motor function
Neurological deficits before and after a cycle of 10 ± 3 days treatment were evaluated using the National Institutes of Health Stroke Scale and the Modified Rankin Scale score[28]. Basic cure: 91–100% reduction of impairment scores, disability level 0; significant improvement: 46–90% reduction of impairment score, disability level 1–3; some improvement: impairment score decreased by 18–45%; no effect: impairment score decreased by 17% or less; worse: impairment score increased more than 18%; death and the events causing test suspension were all classified as worse situation. The basic cure, significant improvement, and some improvement conditions were considered as effective outcomes.
Analysis of cortical motor function by cortical motor- evoked potential
Motor-evoked potential examination was applied using The KeypointR.net EMG evoked potential equipment (Danndy, Skovlunde, Denmark) and the MagPro magnetic stimulator (Danndy), with a circular 90 mm hand-held coin. The center of the coin was placed at the vertex or slightly lateral toward the stimulated hemisphere. Face ‘A’ (visible face) was used for left hemisphere stimulation and face ‘B’ for right hemisphere stimulation. Slight displacements were made in all directions until the position yielding the lowest threshold was found. Cortical functional areas and the C7 nerve root/L5–S1 nerve root were stimulated by the MagPro magnetic stimulator when the patients were lying in the supine and sitting positions. The cortical stimulation intensity was 85–90% of the maximum output, and the nerve root stimulation intensity was 55%. The magnetic motor-evoked potential latency was defined as the shortest latency from eight responses. The surface electrodes were recorded at the abductor muscle of the thumb of the upper limb and the tibialis anterior muscle of the lower limb. The recording electrode (diameter 0.8 cm) was set at the muscle venter, the reference electrode (diameter 0.8 cm) was at the tendon, and the ground wire was fixed in the contralateral limb. For the patients whose muscle strength was too low to be stimulated, the record was taken through a slight voluntary contraction or a passive activity[29]. When the peripheral nerve root was stimulated, the motor-evoked potential with the longest incubation period was considered as the nerve root motor potential[5]. The shortest period from the beginning of cortical stimulation to production of muscle contraction was recorded as the total motor conduction time. The central motor conduction time was calculated by subtracting the nerve root motor potential from the total motor conduction time.
Statistical analysis
Normally distributed data were recorded as mean ± SD, and the M (QR) was used for recording skewed distribution data. SPSS 16.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Ranked data were tested by the rank sum test. Measurement data within the group were compared with a paired t-test. Percentages were compared using the binomial distribution. Multivariate analysis of variance was used to compare trends before and after treatment between the ozone and control groups, and the paired-sample Wilcoxon rank sum test was used to test the heterogeneity of variance. The Spearman rank correlation analysis was used for correlation analyses. A value of P < 0.05 was considered statistically significant.
