| Research BackgroundWhat hypoxic ischemic encephalopathy(HIE) means is that various factors in the perinatal period, especially asphyxia, cause dysfunctions of cerebral energy metabolism in fetus or newborns, who can appear serious clinical symptoms in severe cases, and even died in infancy, which is the leading cause of disable in pediatric. In spite of perinatal medicine in constant progress, and perinatal asphyxia are gradually reduced, HIE is still a threat to the infants. There is no specific treatment. Current evidence supports the use of therapeutic hypothermia on neonatal brain damage. But a Meta analysis with a number of clinical trials by Tagin MA et al. indecated that therapeutic hypothermia therapy was significant on moderate neonatal encephalopathy, but no obvious effect on severe cases. In foreign countries, hypothermia is widely used in clinic, but is not yes comprehensive promotion. And as clinicians, it’s inportant to recognize the contraindications and complications when choose hypothermia for treatment. The clinical application of therapeutic hypothermia is based on the possibility to inhibit or lessen a myriad of destructive processes that take place in the injured tissue following ischemia-reperfusion. When the children exist severe infection, blood coagulation dysfunction or multiple organ failure, it should be reservation to choose the treatment. Meanwhile, the clinicians should pay attention to the therapeutic time window which is within 6 hours after birth, there is no neuroprotective effect when the time delays to 8.5 hours. Another therapy considered an effective treatment to improve the prognosis of HIE is hyperbaric oxygen(HBO) therapy, which can improve nervous system symptoms and reduce sequelae and mortality. Domestic numerous clinical evidences confirm the short term effects of HBO, whereas lacking of multi center randomized studies and difficult to assess the long term outcomes. Foreign researchers believe that HBO treating acute focal cerebral ishcemia has shown predominantly benefits in neurobehavioral testing or infarct volume. However, further preclinical research is needed to explore detailed mechanism and outcomes of oxygen application at higher pressures following focal cerebral ischemia. Other therapy such as oxygen free radical inhibitor and scavenger, excitatory amino acid antagonists, neuroprotective agents, stem cell transplatation still need more animal studies and preclinical verification.Medical ozone, used to disinfect and treat disease, has been around for over 150 years. In 1840, the German chemist C.F.Schonbein found a odorful gas and named it as ozone when passed electrical discharge through water. During the first world war, ozone was used to treat gas gangrene caused by anaerobic bacteria and discoved ozone not only remedied infection, but also had hemodynamic and antiinflamatory properties. Since two German physicists developed the first ozone generator in 1857, ozone therapy started for medical use. In 1929,a book called ozone and its medical effect was published in USA, and illuminated that ozone could be used in 114 diseases. In the 1950s, ozone befinicial effects was questioned by clinicans because of lacking of basic and clinical research. Later, with general progress in medical research, the mechanisms and effects of mefical ozone was elucidated gradually and was recognized by clinicians. In 1999, Italy clinicians initinated and established the International Medical Ozone Association (IMOS), which aimed to promote the research and application of medical ozone and made usage specification. Currently, ozone is used mainly for the treatment of lumbar disc herniation, joint pain, scapulohumeral periarthritis and diabetic ulcer. Ozone has excellent health benefits in decreasing blood cholesterol,stimulation of antioxidative responses and modifying oxygenation in resting muscle and is used in complementary treatment of hypoxic and ischemic sydromes.Animals, such as rat, mouse, rabbit, monkey, have maken a great contribution to huamn biological research. Apart from those animals mentioned above, the vertebrates-zebrafish has gradually become an attractive and potential animal model. Advantageous features of zebrafish include saving cost, their ease of breeding, their small size, rapid growth and embryo transparent easily to observe the ontogenetic process. Zebrafish have been used successfully to understand the biological activity of genes orthologous to human disease-related genes.69% of zebrafish genes have at least one human orthologue. Among the orthologous genes,47% of human genes have a one-to-one relationship with a zebrafish orthologue.82% can be related to at least one zebrafish orthologue amonge 3176 human morbid genes. Therefore, zebrafish can be used for the study of human diseases. Several disease models have been established including hypoxic ishcemia brain damage to research the pathogenesis and therapy.Obejective and significanceBased on the research background above, this experiment is aimed to use the efficient and convenient zebrafish models to make a hypoxic reperfusion brain injury models to imitate neonatal hypoxic ischemic brain damage and explore the mechanisms of hypoxia injury and curative effect with application of medical ozone in neonatal brain injury. We observed the zebrafish larvae brain oxidative stress indications(SOD, MDA, total oxidation level) and the expression level of SOD mRNA, combined with the change of larvae behavior and histopathology to investigate whether medical ozone has protective effects on hypoxic brain damage in zebrafish larvae and what its mechanisms are. We explored the influence of different concentration ozone to provide related animal level research data for futher clinical trials.Methods1. Animal model and Grouping5 days post fertilization (dpf) wild-type zebrafish larvae by the same batch were randomly divided into three groups before hypoxia experiment:normal control group, experimental group(hypoxia-system water group) and treatment group(hypoxia-ozone water group). According to different ozone concentrations, the treatment groups were divided into three subgroups. High pure nitrogen was filled into a closed water tank and oxygen content was monitored by dissolved oxygen meter. As the dissolved oxygen content was less than 0.5mg/L, put the experimental larvae into the hypoxic device to establish the model of hypoxic brain damage. Larvae were subjected to hypoxia treatment until it became motionless, lying on its side on the bottom of the chamber for one minute, followed by transferring into a prepared recovery beaker. Samples were extracted in 3 hours after hypoxia. The dissolved oxygen content in control group maintained at 7.5mg/L.2. Measuring methods of the indicators2.1 General conditions in zebrafish larvaeRecorded recovery time of each group and observed the rate and swimming ability under the microscope to assess the effect of hypoxia on larvae survial state.2.2 Pathological changes of brain tissueTermina deoxiynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of zebrafish larvae brain was observed under optical microscope.2.3 Oxidative stress indicators of zebrafish brain tissue Activity of superoxide dismutase (SOD) was determined by xanthine oxidative technique; contet of malonadehyde (MDA) was determined by thiobarbituric acid (TBA) method; the total oxdative level was determined by T-AOC method.2.4 The expression level of SOD mRNAThe gene expression of SOD was measured by real-time quantitative RCP (qRT-PCR).3. Statistical analysisAll data were statistical described and normal distribution measurement data were presented as mean±standare deviation. The data were analyzed by SPSS 20.0. Between the two groups were compared using independent samples t test. Differences between groups were determined by a one-way ANOVA. LSD method was used for homogeneity of variance and Dunnett’s post hoc test for heterogeneity of variance. A p value of less than 0.05 was considered to be sigificant. Kaplan-Meier was used to analysed survial time. Graphpad Prism 5.0 was used to make figures.Results1.General state of zebrafish larvaeThe larvae of normal group were in good condition with smooth heart rate and normal athletic ability. Compared with normal group, the larvae of experimental group appeared slowing heart rate, changes of behavior spontaneous movements decreased, swimming distance shortend, convulsions and motionless at 3 hours after hypoxic-reoxygen. Recovery time in 3 hours after hypoxia/reoxygen in each group (hypoxia-systerm water reoxygen,0.05ppm-ozone water reoxygen, O.lppm-ozone water reoxygen, 0.1ppm-ozone water reoxygen) was 37.5±6.1 min,33.0±3.8min, 32.5±5.0min,32.6±5.6min, respectively. Recovery time in hypoxia-systerm water reoxygen group was longest. Compared with control group at the same time point, heart rates of larvae’s decreased in experimental group and 0.1ppm-reoxygen group, while accelerated in both 0.05ppm and 0.2ppm groups, but there were no significant difference. Within 14 days after hypoxia/reoxygen, the median survial time of control group, experi-mental group and treatment groups were 12.87,7.67,9.96,10.35,8.96 days.The overall survial rate of survial curves analysed by Log-Rank test was different(P<0.001).2.Effect on TUNEL staining of zebrafish larvae brainTUNEL staining was employed to sutdy the effect of hypoxia on the level of apoptosis. The cells were scored as apoptotic when they were TUNEL-positive staining. TUNEL-positive cells nucleus in experimental and treatment groups were speckled and brown deep dyeing with irregular shape and sizes, distributed in perinuclear or concentrated into a group. While a minority of the nuclei were uniformly yellow indifferent. TUNEL-positive cells scattered in normal cells, more obvious around the center region. TUNEL-positive cells significantly(p<0.05) increased in experimental group as compared with the normal group, where TUNEL staining was barely evident. There were no significant difference in experimental, 0.05ppm group and 0.2ppm groups. The numbers of TUNEL-positive cells in the group of O.lppm significantly(p<0.05) decrease as compared with experimental group.3.Oxidative stress indicators in zebrafish larvae brain tissue3.1 The level of total antioxidant capacityCompared with control group, the level of total antioxidant capacity in experimental group significantly (p<0.05) decreased, while that of the three treatment groups all increased(p<0.05).3.2 The level of SOD activityCompared with control group, the level of SOD activity of experimental group larvae brain significantly (p<0.05) decreased, while that of the three treatment groups significantly (p<0.05) increased..3.3 The level of MDA contentCompared with control group, the level of MDA content of experimental group and the three treatment groups larvae brain significantly (p<0.05) increased.. However, the MDA content of experimental group was significant (p<0.05) higher than that of treatment groups.4.SOD gene experssion of zebrafish larvae brainCompared with control group, the expression level of SOD in experimental group decreased, while increased in treatment, there were significant difference among the groups (p<0.05).Conclusion1. The model of neonatal hypoxic brain damage could be established through the method that zebrafish larvae were placed in very low concentrations.2. Hypoxia could cause damage to zebrafish brain tissue by oxidative stress. Oxidative stress is one of the mechanisms of zebrafish hypoxic brain injury.3. Medical ozone inhibited the oxidative stress induced by hypoxia of zebrafish larvae,improved the antioxidant levels and reduced apoptosis, suggesting that the protective effect of medical ozone on zebrafish hypoxic brain injury. |
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