Role Of Endogenous Pro-survival Protein Iduna In Neonatal Hypoxic-ischemic Brain Injury

Part 1: Expression and cellular localization of endogenouspro-survival protein Iduna in neonatal rat brainObjective To clear the expression and cellular localization of endogenous pro-survival protein Iduna in neonatal rat brain. Methods Taken each organ organization of neonatal SD rats and evaluated the tissue specificity of endogenous Iduna protein by Western blot. Embedded paraffin and frozen sections of neonatal SD rats brain, both the expression level and localization of Iduna protein were observed using immunohisto-chemical staining. The localization of Iduna in neurons and astrocytes was confirmed using immunofluorescence staining in frozen sections. In primary neurons and astrocytes, the m RNA levels of Iduna was compared by Real-time PCR.Used double immuno--fluorescence staining to test Neuronal nuclei(Neu N) and glial fibers acid protein(GFAP) expression,conform the cellular localization of Iduna in primary neurons and astrocytes. Results The highest expression level of Iduna is in the lung tissue of neonatal rats, followed by the brain and spinal cord. Immunohistochemical staining showed Iduna mainly located in the cortex and hippocampus. Immunofluorescence staining indicated that Iduna mainly expresses in the cytoplasm of neurons(79.85%), the result is also verified in primary cultured neurons(80.6%). The expression of Iduna in neurons is far higher than it in astrocytes in primary neural cell culture(P<0.001).Conclusion The expression of endogenous Iduna protein has a tissue specificity in normal neonatal rats organs, especially has a high expression in the brain. Iduna mainly distributes in the hippocampus and cortex of the brain.Iduna mainly localises in the cytoplasm of neurons in cortex and hippocampus, the dates implies that Iduna may be play an important role in the neurons.Part 2 : Expression of Iduna and AIF in neonatal brain afterhypoxia-ischemiaObjective To determine the relationship of Iduna expression with nuclear translocation of AIF in cerebral cortex of postnatal day 7 rats after HI. Method Ninety-six rats were divided into three groups: sham, hypoxia 1h and hypoxia 2h. The HI insult included permanent ligation of left common carotid artery. Brain damage was evaluated using hematoxylin and eosin staining, Nissl staining, transmission electron microscopy, TUNEL staining and immunofluorescence. Immunohistochemistry and Western blot analysis were used to assess expression of Iduna and AIF. Learning and memory ability were analyzed based upon Morris water maze test. Results Compared with sham animals, the number of surviving neurons in cerebral cortex was decreased, and cell damage and DNA breakage were severe in rats with HI, with damage greatest in the 2-h group. Moreover, the expression of Iduna was significantly decreased, whereas the expression of nuclear AIF were increased. Furthermore, down-regulation of Iduna negatively correlated with nuclear AIF abundance in the 2-h HI group(r = –0.950; P < 0.0001). In addition, learning and memory ability were decreased. Conclusion 1. In the process of neonatal Hypoxic-Ischemic brain injury,cerebral cortex damage seriously, the number of survival neurons decrease, nerve cells DNA have a seriously damage. 2. With the increases of hypoxia ischemia, Iduna content in cortical tissue declines significantly, and the level of AIF increases obviously in nuclei. 3.The long-term learning and memory ability of rats can be affected significantly by early hypoxia ischemic injury.Part 3: Effects of over-expression Idunaon oxygen glucose deprivation(OGD) induced neuron injury invitroObjective To construct the rat Iduna c DNA lentiviral vector plasmid(PCDH- Iduna) and improve its expression in primary neurons, then observe the influence of Iduna on cell activity after oxygen glucose deprivation. Method Extract SD rats total RNA, using RT-PCR method to get Iduna c DNA sequence,then insert it to PCDH plasmid. The recombinant plasmid PCDH-Iduna was verified by enzyme digestion and sequencing. Put PCDH-Iduna(PCDH-EGFP as negative control) and packaging plasmid into HEK293 T cells, packaging Iduna overexpression slow virus, observe HEK293 T cell morphological change microscope, to understand the packing of the virus.The original generation of neurons was infected by lentivirus,Western blot detected the expression efficiency of Iduna.After Iduna was improved,taken oxygen glucose deprivation experiment then detected cell activity by MTT, LDH and comet assay. Results The agarose gel electrophoresis showed Iduna PCR fragment is about 1068 bp, enzyme identification of recombinant plasmid about 1000 bp fragment release, The results of sequencing data were all inconcordance with that expected. After the recombinant lentivirus plasmid of PCDH-Iduna and packaging plasmid transfection introduced into HEK293 T cells together, a peculiar cytopathic effect of cell under confocal microscopy.When Iduna over expression lentivirus and its control virus infected neurons, immune fluorescence and Western blot show that the expression of Iduna in infection neurons was improved. Conclusion 1.The recombinant lentivirus plasmid of PCDH-Iduna is successfully constructed. 2. Packaging and obtaining a good slow virus infection efficiency of Iduna, then improve the expression of Iduna in primary neurons. 3.Improcing the expression of Iduna can significantly inhibit neuron injury caused by OGD(e.g., improve cell state, resistance to neuronal death, protect cell integrity and reduce DNA rupture).