The Effect Of Transient Hypoxia On Insulin Receptor In Hippocampus Of 3-day-old Rats

ObjectiveA model of hypoxic brain injury was built by transient hypoxia in a three-day-old rat to simulate hypoxia in preterm infants,followed by detection of insulin receptor levels in hippocampus.Ultimately,to study the effects of transient hypoxia on insulin receptor in hippocampus of three-day-old rats.we are able to figure out the experimental evidence of the clinical treatment for hypoxic encephalopathy from the point of neuroprotective effect.Method 1 To establish a model of hypoxic brain injury,the newborn rats were placed in a nitrogen-oxygen mixed gas for 2h.During the experiment period,the behavioral changes of the newborn rats were observed and recorded.The samples from hippocampus were takenat 24 h after the end of hypoxia,and then,the hypoxia model was examined withthe techniques of Nissl staining and transmission electron microscopy by using the samples.2 The hypoxia model and the control group were successfully established.The sampleswere taken at 2h,4h and 24 h post-hypoxia.Real-time quantitative PCR,Western blotting and immunofluorescence staining were used to investigate the changes of β-catenin、NeuroD1 and insulin receptor at the levels of gene and protein.Result 1 Evaluation of hypoxic model Compared with the control group,the newborn rats in the model group: 1)Behavioral change monitoring:neonatal rats with varying degrees of behavioral changes during hypoxia,such as rapid breathing,body twitching.2)Neuronal morphological changes: the number of neurons was significantly reduced,the cells were arranged loosely,the layers were reduced,the staining was light,and the nucleolus was not obvious.3)Neuronal ultrastructural changes: neuronal nuclear envelop brokedown incompletely,nucleus was swollen,mitochondria,rough endoplasmic reticulum and other organelles were swollen simultaneously,and neurites also showed different degrees of edema.2 Real-time quantitative PCR2 h post-hypoxia,the relative expression of the three genes in the hypoxia group was not significantly different from the control group.4h post-hypoxia,the relative expression of β-catenin gene decreased(P<0.01),The relative expression of NeuroD1(P<0.05)and insulin receptor gene was increased(P<0.05).There was no significant difference in the expression of β-catenin and insulin receptor in hippocampus at 24 h post-hypoxia,but the relative expression of NeuroD1 gene in hippocampus of hypoxia group continuously increased(P <0.05).3 Western blotting2 h post-hypoxia,the expression levels of the three proteins in the hypoxia group were not significantly different from that in control group;however,4h post-hypoxia,the expressionof active-β-catenin(P<0.05)and NeuroD1(P<0.01)increased.The expression levels of insulin receptor barely showed difference.24 h posthypoxia,the expression of active-β-catenin(P<0.05)、NeuroD1(P<0.01)and insulin receptor(P<0.05)was higher than the control group.4 Immunofluorescence staining 2h post-hypoxia,the fluorescence intensity of the target proteins in the hypoxia group was not significantly different from that in the control group;the active-β-catenin and NeuroD1 were mainly located in the cytoplasm,while the insulin receptor was located in the nucleus and cytoplasm.4h post-hypoxia,the fluorescence intensity of active-β-catenin and NeuroD1 increased in hypoxia group;active-β-catenin was mainly distributed in cytoplasm;NeuroD1 was mainly located in cytoplasm in control group,but in the hypoxia group,a large amount was distributed in the nucleus;the fluorescence intensity of the insulin receptor in the hypoxia group was not significantly different from that in the control group,but it was mainly located in the cytoplasm.At 24 h post-hypoxia,the fluorescence intensity of the three target proteins in the hypoxia group was higher than that in the control group;the active-β-catenin was mainly distributed in the cytoplasm;the NeuroD1 and insulin receptor were increased in the nucleus.Conclusion 1 Three-day-old Sprague-Dawley rats were used to as experimental subjects to simulatehypoxic encephalopathy of premature infants,placed in a mixed gas for 2 hours.The model of hypoxic brain injury in neonatal rats was successfully established,which provides a model basis for subsequent experiments.2 Transient hypoxia can stimulate not only the expression of mRNA of active-β-catenin,NeuroD1 and insulin receptor but also the expression of their proteins.Insulin receptor was increased in the nucleus after transient hypoxia,and its expression mechanismbe affected by Wnt/β-catenin /NeuroD1 pathway.