| BackgroundMany factors, especially perinatal asphyxia, can lead to varying degrees of hypoxic-ischemic brain injury in fetus or newborns within perinatal period. Hypoxic injury to the developing human brain causes life-long intellectual and behavioral deficits. So far, the mechanisms of neonatal cerebral damage caused by hypoxia during the perinatal period have not been clearly demonstrated, and there have no effective drugs or therapeutic methods to improve hypoxia-induced cerebral damage. So far, the mechanisms of neonatal cerebral damage caused by hypoxia during the perinatal period have not been clearly demonstrated, and there have no effective drugs or therapeutic methods to improve hypoxia-induced cerebral damage. With the development of medicine, although great progress in neonatal brain injury after hypoxic has been acquired, the pathogenesis and effective therapeutic purposes have not been clear, and need more studies for a long period. Therefore further study on the pathogenesis and effective therapeutic methods of neonatal hypoxic-ischemic brain damage is an important topic in the field of pediatrics for domestic and overseas scholars.The effective establishment of animal disease models is beneficial to the deep study of many disease, as well as hypoxic brain damage. In recent years, zebrafish has quickly become a popular model organisms in the development of the nervous system and related diseases, and its role in this field could not substitutes. Compared with the classical model organism--nematodes and Drosophila, zebrafish are vertebrates, the genome, proteome, embryo development and the pathogenesis are remarkably similar to that in humans. Compared to rodents, with the advantages of its small size, high reproducibility, short reproductive cycle, rapid growth, in vitro fertilization and transparent embryo, it is helpful for the research on the pathogenesis of neurological disease and high-throughput neurotrophic drug screen. Researching of nervous system development and making anoxic brain injury model by zebrafish could provide a new theoretical basis of new paths and discovery new drug for hypoxic brain damage.Objective1By simulating the environment of asphyxia, making a hypoxic brain injury model of zebrafish larvae which provides an economic, efficient and convenient experimental animal models for neonatal hypoxic brain injury;2Compared the change of acute/chronic hypoxia zebrafish larvae brain mitochondrial oxidative stress items and the gene expression changes of HIF-la, EPO, and p53, analysis the mechanisms of zebrafish larvae brain injury after acute and chronic hypoxia, and try to provide a new ideas for zebrafish larvae hypoxic brain injury mechanisms on gene level;3Compared the differential gene expression of nervous related genes nestin, mbp and syn2a between hypoxia group and recovery group ofzebrafish larvae,explore the spontaneous recovery mechanism after hypoxia, in order to provide a new therapeutic target for neonatal hypoxic ischemic encephalopathy.Methods6dpf zebrafish larvae by the same batch were randomly divided into three groups: control group, chronic hypoxia group and acute hypoxia group. First, the chronic group were put into the ypoxic devices in advance, and then filled the high-pure nitrogen gas into water, until reach the endpoint of the experiment, took all the chronic hypoxia group larvae out after hypoxia; When the dissolved oxygen content of the water was less than0.5mg/L, put the acute hypoxic hypoxia group into the device, making acute hypoxic brain injury model of zebrafish larvae. Observe the heart rate(HR), neurobehavioral changes and the pathological changes by HE brain staining to evaluate the hypoxia model of zebrafish larvae. The survival, morphology, HR and swimming ability of larvae was observed by vivo imaging technique; The zebrafish larvae sample were observed by optical microscopy histopathological examination; The concentration of nitric oxide (NO), the content of malonaldehyde (MDA) and the activity superoxide dismutase (SOD) were measured in each group of zebrafish larval brain by the corresponding kit; Apoptosis was analyzed by acridine orange staining; The gene expression of HIF-1α, EPO and p53were measured by real-time quantitative PCR (qRT-PCR) to explore the different mechanisms of brain injury after hypoxia between chronic hypoxia group and acute hypoxia group,and also compared the differential gene expression of nervous related genes nestin, mbp and syn2a between hypoxia group and recovery group of zebrafish larvae.Result1. Evaluation of hypoxic model of zebrafish larvae and brain injury:HR of zebrafish larvae after hypoxia were significantly difference as compared with normal group (p<0.05), and appeared changes of behavior spontaneous movements decreased, swimming distance shortened and motionless; Larvae brain tissue morphology of control group was not changed abnormally, but there were pathological changes in hypoxia group, such as apoptotic bodies were found on hypoxia group of zebrafish in acridine orange staining, ventricular contraction, swelling of part of neurons, cells in hypoxia group shrinked and round, cytoplasmic concentration in HE staining.2. Comparison the damage degree of acute hypoxia and chronic hypoxia groupa) The population mean of HR of acute hypoxia group and chronic hypoxia group compared with control group all have significant difference(p<0.05).HR of acute hypoxia group was faster than control group and chronic hypoxia group was more slowly;b) There were statistical significance of recovery time after hypoxia among the chronic hypoxia group and acute hypoxia group (p<0.05), the recovery time of acute hypoxia group was shorter than the other group.c) Within14days, the median survival time of normal control group, chronic hypoxia group and acute hypoxia group were13.00,1.00,7.00days after hypoxia, Overall Comparisons survival curves analyzes by Log-Rank test showed the overall survival rate of survival curves between each group level was differences, the difference between the two groups was statistically significant(p<0.05)..3. Items of zebrafish larvae brain tissue oxidative stressa) The level of NO of chronic hypoxia and acute hypoxia group larvae brain were significantly higher as compared with control group (p<0.05).b) The level of MDA of chronic hypoxia and acute hypoxia group larvae brain were significantly higher as compared with control group (p<0.05).c) The level of SOD activity of chronic hypoxia and acute hypoxia group larvae brain was significantly higher as compared with control group (p<0.05).The SOD activity of chronic hypoxia group was significantly lower than the acute hypoxia group.4. Hypoxia-related gene expression of control group, chronic hypoxia group and acute hypoxia injurya) The relative expression of chronic hypoxia group HIF-la mRNA was0.674±0.0476, and acute hypoxia group was1.422±0.136, the expression level of gene HIF-1αof chronic hypoxia group decreased, while increased1.422-fold in acute hypoxia group, there was a significant difference (p<0.05).b) The relative expression of chronic hypoxia group EPO mRNA was0.698±0.079, and acute hypoxia group was3.539±0.811, the expression level of gene EPO of chronic hypoxia group decreased, while increased3.539-fold in acute hypoxia group, there was a significant difference (p<0.05).c) The relative expression of chronic hypoxia group p53mRNA was0.724±0.097, and acute hypoxia group was1.651±0.184, the expression level of gene p53of chronic hypoxia group decreased, while increased1.651-fold in acute hypoxia group, there was a significant difference (p<0.05).5. Nerve repair-related gene expression of control group, chronic hypoxia group and recovery groupa) The relative expression of chronic hypoxia group nestin mRNA was0.790±0.055, and recovery group was2.622±0.617the expression level of gene nestin of chronic hypoxia group decreased, while increased2.622-fold in recovery group, there was a significant difference (p<0.05).b) The relative expression of chronic hypoxia group mbp mRNA was0.710±0.060, and recovery group was1.555±0.0665, the expression level of gene mbp of chronic hypoxia group decreased, while increased1.555-fold in recovery group, there was a significant difference (p<0.05).c) The relative expression of chronic hypoxia group syn2a mRNA was0.57±0.126, and recovery group was0.623±0.037, the expression level of gene syn2a decreased in both chronic hypoxia and recovery group, and syn2a expression level of the recovery group was higher than control group, there was a significant difference(P <0.05).Conclusion1. The method by filling high-purity nitrogen into the water, decreased dissolved oxygen in the water to simulate the environment of asphyxia, could be making the model of neonatal hypoxic brain damage.2. Oxidative stress is an important causation of hypoxia zebrafish’s brain injury.NO, MDA and SOD play crucial roles in brain of zebrafish larvae oxidative stress injury.3. The damage of chronic hypoxic brain is more serious than acute hypoxia; Meanwhile, the expression of hypoxic-related genes (HIF-la, EPO and p53) are significant difference, indicated that the brain injury mechanisms of hypoxia between acute and chronic hypoxia may be different.4. Zebrafish larvae could be spontaneous recovery after hypoxic, it possibly repairs by regulating the expression of nerve repair-related gene (nestin, mbp and syn2a)... |
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