A Initial Study Of Regulation Mechanism About Insulin In Hippocampus Of Neonatal Rats After Hypoxia

Objective:A rat model mimicking term newborns with hypoxic ischemic brain damage was established.We aim to study whether hippocampus can generate the insulin and whether hypoxia can influence the expression of insulin in hippocampus,then discuss the upstream regulatory mechanism of insulin in hippocampus from a perspective of neurotrophic factor treatment.Methods:1)8⁹ days old SD rats were put into a Gas box with N₂ and O₂ mixture for2 hours,according to the improved method of Zhao.First,the animal neurobehavioral changes were observed attentively during the modeling.Second,the hippocampus were drawn from the brain and put into fixative immediately after the modeling,histological changes in the hippocampus were observed by Nissl staining at day 1 post-hypoxia,the ultra-structural changes in the hippocampus were observed by transmission electron microscopy.In summary,the animal models were evaluated by the above mentioned technical methods.2)Rats were divided into control groups and hypoxia groups randomly sampled according to the weight.Rats exposed to N2 and O2 mixture for 2hours were sacrificed at day 1,7 and 14 post-hypoxia.RT-qPCR,Western-blot and immunohistochemistry methods were used to investigate the changes of mRNA and protein expression ofβ-catenin,NeuroD1 and insulin.Results:Compared with the control groups,the hypoxia groups showed that（1）Abnormal behavior,such as head&body shaking violently,deep breathing,hind legs movement actively during the hypoxia.（2）Cell morphology showed that Pyramidal cells in CA1 and Granular layer cells in DG arranged in disorder,loose structure,some cells swelling,cells gap became significantly large.（3）Ultra structural results of the neuronal cells showed that light chromatin,swelling endoplasmic reticulum,free ribosomes and swelling mitochondria.Microvascular endothelial cells swelled and cell organelles arranged disorderly.Cells gap among astrocytes,pericytes and endothelial cells became large.Basement membrane continuity was incomplete.The real-time quantitative PCR and Western-blot results（1）The real-time quantitative PCR results showed that the mRNA expression levels ofβ-catenin,NeuroD1 and insulin genes in hypoxia groups were significantly different from those of the control group（P<0.01）at day 1 post-hypoxia.At 7 and 14 days post-hypoxia,the relative expression ofβ-catenin,NeuroD1and insulin genes had no significant difference with that of the control group.（2）The Western-blot results showed that the relative expression levels of active-β-catenin,NeuroD1 and insulin in the hypoxia group were significantly different from those of the control group（P<0.05）at day 1 post-hypoxia.At 7 day post-hypoxia,the relative expression of NeuroD1 was significantly different from those of the control group（P<0.05）.At 14 day post-hypoxia,the relative expression of total-β-catenin,active-β-catenin,NeuroD1 and insulin had no significant difference with that of the control group.Immunofluorescence showed that the brightness of active-β-catenin,NeuroD1 and insulin immunoreactive substance in the hippocampus CA1 and DG areas of hypoxia rats was significantly higher than that of the control group,active-β-catenin distributed both in cytoplasm and nucleus.NeuroD1 and insulin were co-localization with the nucleus,at day 1 post-hypoxia.At 7-day post-hypoxia,the brightness of NeuroD1 immunoreactive substance was different from those of control group,while the other proteins had no difference than that of the control group.At 14-day post-hypoxia,the brightness of the protein was not significantly different from those of control group.Conclusions:1)A model is similar to human term neonates with hypoxic-ischemic brain damage can be established ideally while the 8⁹ days old SD rats were put into a Gas box with N₂ and O₂ mixture.2)Insulin express mainly in the hippocampus pyramidal cell nuclei and granular cell nuclei,transient hypoxia can stimulate the expression of active-β-catenin,NeuroD1,insulin.Most importantly,insulin expression may follow the Wnt3a/β-catenin/NeuroD1 signaling pathway.