Effect of Hypoxia after Reperfusion on Neuronal Apoptosis and Expression of C-fos Gene in Brain of Zebrafish Embryos
|School||Southern Medical University,|
|Keywords||c-fos gene neuron cell apoptosis hypoxia reperfusion zebrafish|
[Background]The most long-term harm of perinatal hypoxia brain damage is the neurological sequelae, especially occurred in children with moderate and severe hypoxic-ischemic encephalopathy (HIE). The data of World health organization showed that there are still four million newborn deaths associated with suffocation every year in2005. In Europe and the United States and other developed countries, the mortality and severe disability of moderate and severe neonatal HIE up to53%to61%, and the situation of developing countries even more than this. As a result of hypoxic-ischemic encephalopathy among Chinese infants, there are about300,000asphyxial neonates leave over permanent brain damage, such as cerebral palsy, epilepsy, mental retardation and audio-visual disorders, resulting in varying degrees of disability to families and caused great harm to society.HIE is the most common and serious complications of perinatal asphyxia,90%occurred before birth and birth, which accounts for about20%of the factors before birth, such as hypotension after maternal bleeding, pregnancy-induced hypertension, placental abnormalities and intrauterine growth retardation. The birth factors accounted for about70%, such as dystocia, fetal distress, umbilical cord knotted around the neck. The postnatal factors accounted for about10%, for example a serious lung disease, including respiratory distress syndrome, apnea and congenital heart disease.1. Hypoxic-ischemic brain damage following reperfusionThe peak of neonatal asphyxia leading to brain damage does not occur at the time of hypoxia-ischemia, but when blood is restored or collateral vessels is forming to that area of the brain. The brain injury was further worsening because the secondary metabolic chain reaction following reperfusion. The mechanism of this phenomena involves excitatory amino acids and their toxicity; role of oxygen free radicals; calcium balance disorders; toxic effects of nitric oxide; humoral factors (β-endorphin, vasopressin, platelet-activating factor (PAE), heart sodium prime, monoamine neurotransmitters, etc.); cellular signal transduction, mitochondrial damage, apoptosis, immune and inflammatory injury, but also related to p53, bcl-2, bax and c-fos gene expression and regulation.2. Apoptosis of cells in the central nervous systemApoptosis, also known as programmed cell death, is one of the basic characteristics of the cell life. As a new concept of cell death has been caused by a wide range of interest and more and more attention. In recent years, due to the wide application of biotechnology, more and more studies have shown that apoptosis plays an important role in ischemic brain injury.Neonatal asphyxia return to normal soon after cardiopulmonary resuscitation, but delayed brain damage can occur after a few hours, its pathogenesis is not clear. It was found that there are obvious signs of apoptosis in the brain cells when detecting the asphyxial neonates’brain, indicating that this transient cerebral hypoxic-ischemic delayed cell death mechanism may be at least part of the apoptotic program inappropriate activation. Characteristics of apoptosis can be seen in newborn pigs with hypoxic ischemic brain damage, it is showed that a large number of cell apoptosis was happened when delayed hypoxic-ischemic brain damage occurred. Feature of apoptosis can also be seen in the immature and mature rat hypoxia and neuronal hypoxia in vitro experiments. The positive correlation between the proportion of apoptotic cells after hypoxia48hours and the degree of hypoxia-ischemia.The mechanism of Hypoxia-ischemia induced apoptosis is not fully understood. Maybe through a variety of different or overlapping pathways, including DNA damage, increasing intracellular Ca2+concentration, destroyed other cellular structures, disrupt signal transduction pathways, cell secretion function decrease, damage to the membrane receptor or disrupt signal transduction pathways.Neonates were suffered to HIE because of that substances produced by the above pathways, such as glutamate, and NO, under certain conditions, to initiate apoptosis, suggesting that acute necrotic brain cells were failed to induced by the original hypoxic-ischemic time, but induced after several hours. The study proves that necrosis of brain cells can be induced by hypoxic-ischemic, apoptosis cells and c cells in hypoxic ischemic core area ratio is1:9on the time of hypoxic-ischemic, while in hypoxic ischemic penumbra with a ratio of1:6. But there are apoptosis cells based in the period of the delayed cerebral hypoxic-ischemic neuronal death. Apoptosis and necrosis are responding differently to treatment. It can significantly reduce the proportion of apoptotic cells, while brain temperature was reduced3℃within12hours after cardiopulmonary resuscitation, but no effects on cell necrosis.3. c-fos proto-oncogeneThe human c-fos gene is located on human chromosome (14q21-31), formed by four exons and three introns, it is a3.5kb DNA in length. The characteristic of the c-fos gene in a resting cell is that fast transient expression by external stimuli. It is difficult to detect the low level of c-fos gene expression in the central nervous system of normal situation. c-fos gene can be activated to transcribe to a2.2kb mRNA by hypoxia, light stimulation, mechanical stimulation and pain stimulation, resulted in380amino acid and synthesized proteins of molecular weight55KD, called Fos protein. The Fos protein’s functional areas are very conservative in the evolution, the area is the a-helix, every three to four amino acids surround one circle, there is a leucine in every seven amino acids, formed Fos leucine zipper. It is considered that Fos protein plays a role of the nuclear third messenger in external stimuli and transcription-coupled signal transduction process, can be detected by immunohistochemical methods. c-fos gene function is not directly to stimulate the expression of genes through binding with DNA strands, but achieve by encoding Fos nucleoprotein. A variety of extracellular stimuli signal activating transcription factor by second messenger, such as Ca+, cAMP, c-fos gene was induced to transcription and synthesis of Fos protein, then Fos protein was combining to Jun protein, a kind of phosphoprotein synthesis by the other proto-oncogene c-jun. They bonded to form a heterodimer through the "leucine zipper ", binding with the regulatory site-TGACTA of transcription factors activating protein (AP-1) within the target gene to further activate the expression of target genes and respond to the stimulus response finally, regulating many long-term delayed response genes involved in signal transduction. c-fos gene is therefore referred to as the "third messenger".4. The expression of c-fos proto-oncogene and cell apoptosisIt is thought that c-fos gene’s on was a potential mechanism inducing neuronal cell apoptosis after hypoxic-ischemic. There is a close relationship between c-fos gene highly expressed in and neuronal apoptosis after hypoxic-ischemic. The correlation of the c-fos gene expression and the distribution, schedule and number of nerve cells apoptosis confirmed that the higher the degree of c-fos gene expression, the corresponding brain damage more serious. The mechanism may be formed with AP-1after the c-fos gene expression, through regulation of downstream target genes, this process ultimately results cell apoptosis because the proteins maintaining cell survival synthesis were decreased and the killer proteins were increases.However, some studies have reported the increase of the c-fos gene expression by affecting the nerve growth factor and peroxide dismutase, promoting nerve cell survival gene expression and different adaptive changes in the nerve cells to stimuli, enhance the repair capacity of the neurons, which play an anti-apoptotic role. There are other studies proved that apoptosis morphological and biochemical changes later than the time of c-fos gene quickly and abundantly expressed, c-fos gene persistently highly expressed in the nerve cells can induce the irreversibility apoptosis pathological change, so that the early signs of the start flag as apoptosis and neuronal activation.The relationship between c-fos gene and neuronal apoptosis after cerebral hypoxia ischemia-reperfusion is still inadequacies. The current researches have focused on the application of different types of hypoxic-ischemic modefs; observe the c-fos gene expression in aging, dose, and brain distribution. The experiments carried out the new model named zebrafish. In the past decade, a growing number of scientists are using zebrafish to create disease models and study of the pathogenesis in-depth. Researchers found that zebrafish shows a good correlation with rodent in the processes of in-depth studying disease, it is an alternative animal model in research. Zebrafish is in vitro fertilization, its early embryo is transparent, can be clearly observed through an electron microscope changes during embryonic development, and growth and development rapidly. Because the similarity of its genes with human genes is near87%, which means the, it is expected to intervention regulation on c-fos target gene by using zebrafish, and further realizes in-depth study of the c-fos gene expression after hypoxia reperfusion.[Objective]Hypoxic-ischemic reperfusion lead to neurological dysfunction, associated with the death of nerve cells after injury, including necrosis and apoptosis, especially the latter. Apoptosis occurs late and longer duration, the neural function impact is much more noticeable. To further investigate the relationship of c-fos gene and cerebral hypoxic-ischemic neuronal apoptosis after reperfusion. The study provided an experiment basis on the exploring the new cerebral hypoxic-ischemic therapy entry point and the drug effective time window. This experiment was carried out in the new model of zebrafish, the impact of the tested animals brain cell apoptosis and c-fos gene expression levels were observed after hypoxia and reperfusion, the aim to explore the method of neuronal apoptosis inhibitory explore after hypoxia ischemia is expect for providing new ideas to clinical treatment and prevention of neonatal hypoxic-ischemic encephalopathy.[Methods]Zebrafish embryos were divided into three groups at48hpf. Hypoxia and subsequent reperfusion was induced by gradually leading nitrogen (99.999%) into the device lasting for6,12and24hours and followed by reperfusion for6hours. Apoptosis was analyzed by acridine orange staining and image analysis system. The expression of c-fos gene was measured by real-time quantitative PCR.[Result]The number of apoptotic cells of48hpf embryos has increased in the three hypoxia groups, especially a strong induction of apoptosis in the24h hypoxia groups (P<005, respectively). Expression of c-fos gene were lower in the control groups, but improve in the three hypoxia group (P<005, respectively), and the gene in hypoxia6h was increased distinctly. [Conclusion]Hypoxia causes upregulation of c-fos gene in the zebrafish brain cells, which is likely related to the one of the mechanisms leading to increased apoptosis cells in the brain in the later stages of hypoxia. This study is the first time to analyse the expression of c-fos gene on the zebrafish after hypoxia reperfusion, enriched the research of hypoxia brain damage on zebrafish model. According to the study on previous c-fos gene expression in other animals’ model after hypoxia reperfusion, it establishes the foundation for further production zebrafish hypoxia-reperfusion brain injury model and the role of c-fos gene in the response of zebrafish embryos brain to hypoxia and subsequent reperfusion and a role in the formation of apoptosis.