Building Rats Model Of Radiation Brain Injury And Study The Expression Changes Of P35 And P25 In Hippocampal Neurons

【Background】Radiation injury of brain is a serious complication of carcinoma of head and neck after radiation therapy.Besides iatrogenic post-radiation brain injury,radiation accident such as nuclear disembosom unexpected,nuclear weapon injury in war can also result in radiation injury of brain.Brain and spinal after overdose therapy can be lead to dropsy partly,to block or to necrosis,and influence the survivorship of patients seriously.Clinical observation results showed that the severity of recognition functional lesion concerned with the dose of the temporal lobe accepted.Because neurons in hippocampus are very sensitive to radioactive ray,recognition function is related to the damage of hippocampus.Radiation brain injury has become the restrictive factor of the therapeutic dose of the head and neck neoplasm.Radiotherapy experts and neurologist experts have paid close attention to how to prevent and lessen the injury of brain after radiation therapy.It is of great social significance and clinical value to elevate the life quality of patients.Up to now,it is considered that radiation-induced brain injury results from comprehensive function of multiple factors.Radioactive-ray damaged nerve cells and lead to degenerative disease of brain tissues via both direct physical effect and indirect mechanisms induced by free radical.In central nervous system,hippocampus and subependymal region are the most sensitive districts to radiation.The destroyed nerve cells in these districts can effect the damage level and the process of recovery.Injury of blood vessel is one of the most important pathological basis at late stage in radiation-induced brain injury.Oligodendroglial cells and their enzymatic system can produce autologous antigen,guide autoimmune response,lead to demyelination and cerebral edema,and aggravate the destroy of nerve cells.Apoptosis induced by radiation results from biochemical cascade in the guidance of many genes such as damage of DNA,change of signal transduction system,enzyme activation and cell envelope structural change.Radiobiologists have long recognized that the most critical target of ionizing radiation passing throuth living tissues is the DNA that is present in the nucleus and mitochondria of most cells.Exposure of cells to ionizing radiation results in immediate and widespread oxidative damages to DNA by both direct and indirect mechanisms.Recently many research concentrated on the effect of Cdk5(cyclin-dependent kinase 5) on the process of neuron apoptosis induced by toxic substance.It has been discovered that Cdk5 plays an important role in the occurrence and development in nerve degenerative disease.But its contribution in radiation injury has not been elucidated.Cyclin-dependent kinase 5 is a member of the small proline-directed serine/threonine kinase family,and it is a special member of the cyclin-dependent kinase family(Cdks).Cdk5 is critical to the CNS(central nervous system) development.Since the discovery of Cdk5,several important functions have been assigned to this unique kinase,including synaptic plasticity,cellular motility and adhesion,drug addiction and neurodegeneration.Although Cdk5 is expressed in most tissues,its activity is found predominantly in CNS.The reason for the localized activity of Cdk5 is that its activators,P35 and P39,are expressed almost exclusively in the CNS.SiRNA-mediated Cdk5 knockdown resulted in a reduction in primary sensory neurons of the trigeminal ganglia of the peripheral nervous system, suggesting that Cdk5 also plays a crucial role in the development of the peripheral nervous system.Mouse embryos lacking Cdk5 or both activators display severe neuronal migration defects throughout the brain,including the neocortex, hippocampus,cerebellum,olfactory bulb,thalamus,and brain stem.Cdk5 knockout mice are embryonic lethal with inverted cortical layering defects.Although the expression and activity of Cdk5 in the CNS have been reported,the precise molecular functions of this kinase have not been characterized.Cdk5 plays an important role in human degenerative disease such as alzheimer's disease, amyotrophic lateral sclerosis,Parkinson's disease,progressive supranuclear palsy and Pick disease.It has been recognized that DNA is the most critical target of ionizing radiation passing through living tissues.Fistly,toxicity caused DNA damage.Specific activation of calpain,a calcium-dependent cysteine protease,directly cleaves P35 to P25 and P10.Conversion of P35 to P25 causes prolonged activation and mislocalization of Cdk5.Consequently,the P25/Cdk5 kinase hyperphosphorylates tan, disrupts the cytoskeleton and promotes the apoptosis of primary neurons.In cultured primary cortical neurons,excitotoxins,hypoxic stress and calcium influx can induce the production of P25.If we confirm that Cdk5 and its neuron-specific activator P35 and P25 can contribute to apoptosis,we can protect brain tissues from radiation injury and elevate the curative effect by regulate these kinases.【Objective】1) To observe the changes of hippocampus neurons after radiation by building rat model of post-radiation brain injury,and to provide a theoretical basis to clinical therapy.2) To observe the expression changes of P35 and P25 in hippocampal neurons after radiation-induced brain injury and to explore the mechanism,and to provide a new idea to therapy and evaluation of prognosis.【Methodes】 1) A model of whole brain radiation injury was established in the male Sprague-Dawley rat.72 rats were randomly divided into 4 groups:0Gy,10Gy,20Gy and 30Gy by radiation.2) The neurons of CA1 were detected by nissl's staining at 3,6,12h after radiation.3) After the model of whole brain had been established,12 rats were randomly divided into 3 groups:0Gy,10Gy,20Gy and 30Gy groups.Western blot was used to observe the expression of P35 and P25 in hippocampus.4)Another 9 rats were applied to test the protein expression of P35 and P25 in different time(3h,6h,12h after radiation) after irradiation with a dose of 30Gy.【Results】1)Estabished radiaton induced brain injury in vivo successfully and neuronal opoptisis could be observed after radiation.2)Compared with 0Gy group,the number of neurons in 10Gy group showed no obvious change,there was no statistically difference between the above groups(P＞0.05).However,the number of neurons in 20Gy and 30Gy groups decreased obviously,with statistical significance as compared with 0Gy group(P＜0.05).And it reached the apoptisis peak at 6h after radiation(P＜0.05).3)Compared with 0Gy group,the expression changes of P35 and P25 in 10Gy group was not obvious(P＞0.05).The P35 expression decreased while P25 expression increased in 20Gy and 30Gy groups,the differences were significant(P＜0.05).4)The protein expression of P35 and P25 after radiation in different time:The expression of P35 and P25 has no significant difference between 3h and 12h groups(P＞0.05).The expression of P35 decreased while the expression of P25 increased at 6h after radiation compared with other time(P＜0.05).【Conclusion】 1) The neuron apoptisis in CA1 of hippocampus was related with irradiation dose and duration after irradiation.2) The neural injury after radiation brain injury in hippocampus might be related with the expression changes of P35 and P25.