Investigation The Expression Of Insulin And NeuroD1 In Embryo And Pup Brain Of SD Rats Through Different Level Transient Hypoxia In Utero

Objective At 17d(E17) SD rats continued low tensile anoxia, fetal intrauterine anoxia animal model is set up. By testing the offspring rats in Neurogenic differentiation factor1(Neuro D1) and Insulin instant expression, discusses intrauterine hypoxia of rat behavior, the influence of different expression level Neuro D1 and Insulin, their mutual relations and change of neurons in the role of repair, for the treatment of fetal anoxic encephalopathy(HIE) to provide basic experiment basis.Methods1. On the 17 th embryonic day(E17) SD rats were continued low anoxia, to set up the fetal intrauterine hypoxia ischemia model. By the abnormal behavior identify, frozen section HE stain and neurobehavioral test(Muscle test, Ramp test, Elevated plus maze, Open field test, Morris water maze) to found the change of the offspring rats at different degree of hypoxia ischemia(1h, 2h,3h).2. Using real-time PCR analyzed the relative expression of Neuro D1 and Insulin on the different degree of hypoxia ischemia group of fetal rats; Using protein immunoblot(Western blot) detected the relative expression of Neuro D1 and Insulin on the different degree of hypoxia ischemia group of fetal rats;Using immunofluorescence double labeled staining(MAP-2 and DAPI, Neu N and DAPI), and three labeled staining(Neuro D1, MAP-2 and DAPI, Neuro D1,Neu N and DAPI, Neuro D1, Insulin and DAPI), to observation whether Neuro D1 and Insulin have co-localization and the fluorescence intensity.Results1. With the intrauterine hypoxia, four groups of neonatal rat had no different inskin color, weight, screaming, limbs activity. The behavior experiment(Muscle test, Ramp test, Elevated plus maze, Open field test, Morris water maze) results showed that compared with the sham group, have no effect on motor function and psychological anxiety were not obvious, the learning and memory ability has declined on hypoxia ischemic 1h group; however hypoxia ischemic 2h group increased the offspring rats spontaneous activities, and not obvious declined on learning ability; but the lack of oxygen to 3h, the ability of learning and memory has improved and closed to the sham group.2. The real-time PCR results showed that the expression of Insulin up to the highest on the 3h group, followed by 1h group; the expression of Neuro D1 on sham group was the highest. Western blot results showed that the protein expression of Insulin and Neuro D1 up to the highest on the 1h group, Neuro D1 and Insulin correlation index reached statistical significance.3. Immunofluorescence labeling MAP-2, Neu N, Neuro D1, Insulin and laser confocal results show different degree intrauterine hypoxia ischemia affected the expression of Neuro D1 and Insulin on offspring rats. The expression of Neuro D1 and Insulin had a rising trend after hypoxia ischemia rats, offspring rats caused Neuro D1 and Insulin co-localization, 2h group was the most obvious, Neu N also appeared a similar trend.Conclusions Intrauterine short of oxygen have not affect the motion system and psychological anxiety, but influenced offspring rats learning and memory ability.Intrauterine hypoxia help stimulate the gene of Neuro D1 and Insulin transcription and high expression in offspring rats, in order to induce NSCs to differentiate into neurons, participated in the reconstruction of the damaged nerve tissue repair,function and brain protection. Studies have shown that Neuro D1 has close relationship with Insulin, for cerebral protective effect of Insulin still need further study.