| BackgroundsIschemic stroke is the second of the main causes of death and disability in the world. It seriously affects the health of the human as well as brings heavy burden to society and patient’s families. It has a serious impact on the patient’s family and social life during the late period. Therefore, looking for the safe and effective new method that can relieve symptoms and promote early functional recovery in the acute phase of cerebral infarction is one of the highlights of the current research in the field of neurology.The previous basic research hints that ozone treatment may play a role in the case of tissue ischemia. First, ozone not only can provide sufficient energy to ischemic tissue directly, but also can reduce the damage of biological macromolecules through reconstructing the redox equilibrium of the cell. Second, many studies also suggests that ozone can improve the liveness of tissue, to improve oxygen supply to the hypoxia tissue, restore the cell function, change the oxygen metabolism of hypoxia tissue effectively. Major ozonated autoheamotherapy(MOAH) can maintain the ATP and energy metabolism of brain tissue in the condition of ischemia and hypoxia,to reduce apoptosis. Third, the ability of ozone recovery antioxidant system is the key to treat a typical chronic inflammatory injury, and correct the oxidative stress. Such as cardiovascular disease, multiple sclerosis and chronic obstructive pulmonary are all in use. Studies have found that the encephaledema and tissue damage may be caused by a large number of products produced by oxygen free radicals after cerebral infarction, especially after cerebral ischemia-reperfusion. Ozone can activate the antioxidant enzymes and free radicals and reduce necrosis stage of nerve cell damage after acute cerebral infarction.In clinical application, many studies suggest that MOAH can relieve chronic oxidative stress, delay of serious complications, improve the quality of life of diabetic patients and improve cerebral blood flow. Thus to support the ozone treatment is applied to ischemic and metabolic diseases. Some studies also suggests ozone combined with hemoglobin quickly after it enters the body to make O2 turn into O3, to increase the blood oxygen saturation, activate the red blood cell metabolism, make the brain tissue oxygen condition improved, improve blood circulation of the brain and increase the activity of brain cells. That is, to improve the anoxic condition of ischemic penumbra, promote the formation of collateral circulation and restore nerve cell function.The mechanism of MOAH for the treatment of acute cerebral infarction is unclear at present. If the experiment can further define its clinical effect and the impact of nerve cell apoptosis and the oxygen free radical damage, it can be further applied in clinic. And provide reliable evidence of evidence-based medicine for MOAH treatment in the acute phase of cerebral infarction.Study contents:We proposed to build the model of middle cerebral artery occlusion (MCAO) of SD rat. To observe indicators like the neural functional recovery, cortex neuron apoptosis and oxygen free radical change and so on with clinical functional score, Hoechst staining, immunohistochemistry, Western Blot and biochemical detection. To prove that MOAH has the improvement and protection effect on nerve function, apoptosis and oxygen free radical damage after acute ischemia of middle cerebral artery. Lay a further theoretical foundation for the widely use of MOAH, and provide new evidence for early treatment of cerebral infarction to find an effective treatment.Next, we performed MOAH in patients with acute cerebral infarction, and assessed outcomes according to the U.S. National Institutes of Health Stroke Score, biochemical detection of oxygen free radicals and transcranial magnetic stimulation motor evoked potential(TMS MEP). Study furtherly on the effect of MOAH in the treatment of the patients with acute cerebral infarction on the recovery of motor function and the role of oxidative stress after cerebral ischemia, so as to assess the clinical validity of MOAH.Objectives:1.Primary discussion for the influence of MOAH on the recovery of motor functions, the apoptosis of cerebral ischemia and oxygen free radical injury in the rats during the period of acute cerebral ischemia with the method of clinical function scoring, Hoechst staining, immunohistochemistry, western blot and biochemical detection of oxygen free radicals.2.Assessing outcomes according to NIHSS and MEP to clear the influence of MOAH to the recovery of clinical motor function in the patients with acute cerebral infarction.Methods1. Experiment of the animals1.1 Establish MACO models and groups of the rats treated with MOAH.Select male SD rats weigh 250-280-g in 3 months old. Using the line switch method to establish the model of MACO and divided into three groups randomly:the sham operated group, the control group and the treatment group with 18 rats in each group. Put 2 ml femoral venous blood of rats into the special syringe in the treatment group after surgery 12 hours later and blended with ozone (47 μg/ml) at a scale of 1:1 for 5-10min. Then put the blood back to femoral vein, once every 12 hours for 6 times.1.2 The neurological scoreScore the neurobehavior by Longa method at 12 hours later of the operation and the 6th time of MOAH.1.3 Apoptosis DetectionThe rats were executed to detect their indicators after the neurobehavioral scoring.Test their apoptosis of ischemic cortex, and apoptosis-related protein expression Bcl-2、Bax、Caspase-2 in the ischemic brain tissue through the method of Hoechst staining, Tunel staining, immunohistochemistry and western blot.1.4 The detection of oxygen free radicals The rats were executed to detect their indicators after the neurobehavioral scoring. Test the content of superoxide dismutase and malonaldehyde in use of relevant kit.2. Clinical trails2.1 Study object:A total of 86 patients with acute cerebral infarction(male58, female 28) were divided randomly into the control group (n=43) and the ozone treatment group (n=43). The basic medicines of the two groups are Bayaspirin (Aspirin Enteric Coated Tablets) 0.3 g 1/night+atorvastatin 20mg,1/night, 1/day. The ozone treatment group were added MOAH at baseline,1/day, a treatment of 10 days. The control group was treated only with the basic medicines and rehabitation.2.2 Cases selection:2.2.1 Grouping criteria:①age from 30～80;Exclude the patients with strokes caused by hemorrhage, TIA, brain tumor, brain trauma, cerebral parasitic disease and metabolic disorders, to confirm the patients with acute cerebral infarction.③ Scored by NIHSS in hospital, scoring between 4 to 20.④The onset of acute cerebral infarction is less than 72h and more than 6h. Besides, the patients can’t be performed intravenous thrombolysis and artery embolectomy.2.2.2 Exclusion criteria:①Severe stroke patients, such as more cerebral lobe infarction (CT low density greater than 1/3 of the brain hemispheres);② The patients with thrombolysis therapy; ③The patients with shock、vital signs are unstable、severe cardiopulmonary complications, hepatic and renal dysfunction and life expectancy is not more than 1 month;④The patients in the case of blood coagulation dysfunction, the abnormal volume and function of platelet, thalassemia, drepanocytemia and 6PGD(glucose-6-phoshate dehydrogenase deficiency), that is favism.⑤uncontrolled hyperthyroidism;⑥allergic constitution⑦in use of kinases and free-radical preparation;⑧Women during pregnancy or lactation;⑨Not suitable for performing MEP:patients with implanted peacemaker and epilepsy。⑩patients with poor compliance can’t perform relevant examination, treatment and records.2.3 Detection method:2.3.1 Clinical function score:Assess the clinical features change of the patients in the two groups according to NIHSS before the treatment and 10 days after.2.3.2 The detection of oxygen free radical:Perform blood drawing and the use of biochemical kit to the patients with testing NO, SOD and MDA before the treatment and 10 days later.2.3.3 The detection of MEP:Adopt KeypointR.net and MagPro to perform the detection of motor evoked potential and stimulate coils by circular stimulator. Use magnetic stimulator to stimulate functional cortex area, cervical7 nerve root and Lumbar5 --scrall nerve root of the patients in the supine position and seat position respectively. Surface electrodes records abductor pollicis brevis of upper limbs and anterior tibial muscle of lower limbs.3. Statistical analysis:SPSS 16.0 software was used for statistical analysis. Normally distributed data were recorded as mean± SD, and the M (QR) was used for recording skewed distribution data. 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 two 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.Results1. Animal experimental results1.1.The scores of Neurological behavior.The cerebral ischemia of rats of MCAO was given the score of neurological behavior, and the scores are during the value of 1-3 points. After 3 days, the scorce of the ischemia control group and the treatment group decreased significantly than 12h later of the operation (P> 0.05). While the score of the treatment group decreased significantly than that in the control group (P< 0.05).1.2 The detection of apoptosis.1.2.1 Hoechst staining:To observate the results of these three cortex ischemic groups with Hoechst staining after 3 days. We can see a lot of nucleus were densely stained, and many were chunky, dense, hyperchromatic, and whitish in colour. Apoptosis rates of three groups were calculated. Apoptosis rates of the treatment group decreased than the control group significantly. [(39.20±5.12)% vs (45.82±4.89)%] (P>0.05).1.2.2 Tunnel stain:To observate the results of these three cortex ischemic groups groups with Tunnel staining after 3 days. We can see a lot of brown apoptotic fine, which volume reduced, nucleus pycnosised, and were triangular or fan-shaped under fluorescence microscope. Through the analysis of the IPP, the area positive for staining in the treatment group is less than that in the control group. [(3.01±0.92)% vs (3.91±0.30)%]. The Immunohistochemisty staining intensity of the treatment group is also obvious less than that in the control group. (28083.33±7846.55 vs 37754.75±7028.85). It had statistical difference(P>0.05).1.2.3 The detection of apoptosis related proteins:1) The staining of immunohistochemistry. Through microscope observation, it showed that positive cells staining of Bcl-2 Ab, Bax Ab, caspase-2 Ab of rat brain tissue in all three groups were in the cytoplasm with brownish yellow substance deposition, there were also part of the cell membrane and nuclear membrane coloring. The relative area of staining positive region of Bcl-2 antibody staining in ischemia treatment group was significantly higher compared with the ischemia control group[(2.36±0.34)% vs (1.66±0.26)%] (P<0.01), The staining intensity of immunohistochemistry increased greatly [(4741.40±408.76) vs (7464.90±2441.29)] (P<0.05); The relative area of Bcl-2 positive staining in the treatment group was obviously decreased than that in the control group,[(2.41±0.58)% vs (3.59±1.10)%] (P<0.05). The detection results of the intensity of immunohistochemical staining had no significant difference[(20526.69±5473.95) vs (23916.83±7291.13)] (P>0.05). The immunohistochemical staining analysis of capase-2 antibody staining of the control group and the treatment group had no significant difference. [(1.57±0.38)% vs (1.90±0.30)%][(10017.86±2221.08) vs (10222.43±1257.99)] (P>0.05)2) The detection of Western Blot protein electrophoresis and gray value.The gray values of Bcl-2 of the ischemic treated group were significantly higher than ischemia control group (P< 0.01). And the Bax and Caspase-2 gray of the ischemic treated group, was still higher than that of ischemia control group significantly. It had significance difference. (P<0.05). The specific value of Bcl-2/Bax in the treated group decreased obviously than that in the control group. (P<0.01)1.3 The detection of oxygen free radicals1.3.1 Ischemic brain tissue SOD vigor level measurement:After treatment, SOD vitality values of the sham operation, the control group and the ischemia treatment group were respectively (5.10±0.78) U/mg.prot,(1.62±0.53) U/mg.prot, (3.26 ±0.58)U/mg.prot. The control group and the treatment group compared with the sham group decreased, the data is of statistically difference(P<0.01)And the ischemia treatment group was obviously higher than the ischemic control group(P< 0.01).1.3.2 Ischemic brain tissue MDA vigor level measurement:After treatment, plasma MDA value of the sham operation group, the ischemia control group, the treatment group, were respectively:(0.45±0.11) nmol/mg.prot, (1.55±0.29) nmol/mg.prot, (0.79±0.16) nmol/mg.prot, The ischemia treatment group and the ischemia control group were increased compared with the sham operation group, the data is considered statistically difference (P<0.01). the ischemic control group was obviously higher than the ischemic treatmentgroup (P<0.01)。2.Clinical trial results2.1 Clinical function scoring of the acute cerebral infarction patientsBefore the treatment,there was no significant difference in NIHSS score between both groups(P> 0.05) Treatment 10 days later, NIHSS score was significantly lower,which was not only in the treatment group, but also in the control group (P< 0.05). Compared with the control group,the treatment group decreased obviously (P< 0.05); The clinical total effective rate of patients of the treatment group was obviously higher than that of the control group (P<0.05)2.2 The detection of the relevant indicators of oxygen free radical:2.2.1 Detection of serum NO level:Before the treatment, the treatment group NO and the control group NO was respectively (67.30±10.71)μmol/1/, (65.74± 11.97)μmol/1. The treatment of 10 days later, the treatment group NO was(48.12± 10.59)μmol/1, and the control group was(53.95±11.55)μmol/1. Before the treatment, the treatment group and the control group has no significant statistical difference(P>0.05).SOD content was significantly increased in the two groups after the treatment P<0.01).But SOD content was significantly higher in treatment group, the difference between the two groups have statistical significance(P<0.05)2.2.2 Detection of serum SOD level:Before the treatment, serum SOD level of the treatment group and the control group were respectively(67.30±20.71)μmol/l, (68.74±21.97)μmol/1. The treatment of 10 days later, the content of the treatment group and the control group were respectively (96.80±8.73)U/ml,(90.45±9.54) U/ml.Before the treatment, there was no significant statistical difference between the treatment group and the control group. (P>0.05); After the treatment, SOD content of the treatment group and the control group increased clearly (P<0.01). But the treatment group SOD content was significantly higher than that of the control group. The difference between the two groups has statistical significance (P<0.05)。2.2.3 Detection of serum MDA level:Before the treatment, the content of plasma MDA of the treatment group was (9.63±3.98) nmol/ml and the control group was (9.58±3.55) nmol/ml. After treatment for 10 days, the MDA content in the treatment group was (7.32±3.78) nmol/ml and in the control group was (6.11±3.60) nmol/ml. The difference between the two groups has no statistical significancebefore the treatment (P>0.05). After treatment, the MDA content of the two groups were both significantly increased (P<0.01). But compared with the control group, MDA content of treatment group significantly decreased. The difference between the two groups has statistical significance (P<0.01)2.3 Related research of neurological function and motor evoked potential in patients of acute cerebral infarction treated with MOAH.2.3.1 The total effective rate in patients of acute cerebral infarction treated with MOAH:Before treatment, there was no significant difference in the National Institutes of Health Stroke Scale between the groups (P> 0.05). However, the score for the National Institutes of Health Stroke Scale was significantly reduced after treatment for 10 days (P< 0.05); the reduction in the control group was significantly less than in the ozone group (P< 0.05). The clinical total effective rate in the ozone group was significantly higher than that in control group.2.3.2 Comparison of cortical potential rise rate before and after treatment:Motor evoked potential showed there was no significant difference in the cortical potential rise rate in the treatment group and in the control group before treatment (P> 0.05). After ozone treatment for 10 days, the number of cases of MEP on the upper and lower limbs increased in both of the two groups, but the increasement of number of cases of MEP in the treatment group had no significant difference compared with that in the control group (P> 0.05); the cortical potential rise rate of the lower limbs in the treatment group had no significant increasement compared with the control group (P>0.05).3.3 Comparison of central motor conduction time before and after treatment.By comparison, CMCT of the upper and lower limbs reduced significantly in the two groups after treatment(P< 0.05). There was no significant difference of upper limbs and lower limbs between the treated group and the control groups (P> 0.05). After treatment, CMCT of the upper limbs in the treated group was significantly shorter than that in the control group (P< 0.05). CMCT of the lower limbs in the treated group weren’t shorter clearly than in the control group (P> 0.05).After treatment, the central motor conduction times of the upper and lower limbs in both groups were significantly shorter than those before treatment (P< 0.05).3.4 Comparison of MEP amplitude before and after treatmentMEP amplitude of the upper and lower limbs increased significantly in the two groups after treatment (P<0.05). Before treatment, there were no differences in the cortical MEP amplitudes of the upper and lower limbs between the treated and the control groups (P> 0.05). After treatment, the cortical MEP amplitudes of the upper limbs were significantly higher in the treated group than in the control group (P< 0.05), while there was no difference in cortical MEP amplitudes of the lower limbs between the groups (P> 0.05). The cortical MEP amplitudes of the upper and lower limbs were significantly greater after treatment than those before treatment in both groups (P< 0.05).3.5 Correlation analysis of National Institutes of Health Stroke Scale to central motor conduction time and MEP amplitude:The National Institutes of Health Stroke Scale improvement rate was positively correlated with the central motor conduction time improvement rate of the upper limb before and after treatment (r= 0.782, P< 0.05), as well with the improvement rate of MEP amplitude of the upper limb(r= 0.847, P< 0.05). However, there was no correlation with the central motor conduction time and MEP amplitude of the lower limb (P> 0.05).Conclusions:1. MAOH in the treatment of rat cerebral ischemia caused by middle cerebral artery occlusion can improve the neurological function scores of ischemic rats. MAOH in the treatment of rat cerebral ischemia caused by middle cerebral artery occlusion has certain effect on apoptin. It may adjust apoptosis-related protein Bcl-2, Bax and increase the ratio of bcl-2 to Bax of the ischemic cerebral tissue to improve the clinical neural function. The apoptosis inhibition of ischemia by MOAH may be related to the Caspase-2 inhibition of cortical.2. MOAH in the treatment of acute cerebral ischemia may adjust the level of SOD. NO and MDA to reach the role of resistance to free radical damage, to maintain mitochondrial function and metabolism. MOAH could inhibit apoptosis potentially through the mitochondrial protection.3. MOAH can obviously improve motor function in patients with acute cerebral infarction. It is the effective means to promote early functional recovery of cerebral infarction patients. Through correlation analysis, we also think the upper limbs motor evoked potential central conduction time and amplitude is relatively lower limb clinical motor function in patients with more direct response, showing that motor evoked potential can be the effective method to evaluate clinical motor function in patients with stroke. |
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