| Objective:To investigate the expression of nerve growth factor(NGF)in neonatal rats with hypoxic-ischemic brain damage(HIBD)and the effect of NGF on damaged cortical neurons in vitro,then lay the theoretical foundation for the clinical development of new therapeutic strategies.Methods:To explore the molecule mechanism of HIBD,the Hypoxic-ischemic encephalopathy(HIE)model of neonatal Sprague-Dawley(SD)rats and the cell model of oxygen glucose deprivation(OGD)were established,respectively.The healthy neonatal SD rats were randomly divided into two groups:sham operation group(Sham group)and hypoxia-ischemia(HI)group.TTC staining and Zea-longa score were separately performed to determine the cerebral ischemia situation and neurologic function in rats after operation.Gene chip technology was employed to detect the expression changes of genes in right cortex,hippocampus,lung tissue and serum.The String database was utilized to analyze the interrelation of different expression gene in right cortex of rats undergoing HI.To determine the target gene,the downregulation genes of cortex,hippocampus,lung tissue and serum were analyzed by using Venny 2.1.0 online software and searching literature databases.Western blot(WB)and immunohistochemical staining were performed to test the expression of NGF in cortex and hippocampus,respectively.Immunofluorescence assay was used to detect the expression of NeuN/NGF and GFAP/NGF in neurocytes.Then,then the NGF overexpression virus was constructed and transfection efficiency was determined by transfecting into the neurons.Meanwhile,the cultured neurons were randomly divided into Normal group,OGD group,OGD+GFP group and OGD+NGF-ORF group,and the Polymerase Chain Reaction(PCR)assay was utilized to test the expression of NGF in different groups.Moreover,the immunofluorescence staining was employed to determined the expression of Tuj1,and the growth condition of neurons was analyzed consisting of area,number and neurite length.Results:To investigate the related pathological mechanism of HIBD,the HIE model of neonatal rats was established.The results of TTC staining suggested that there was significant white ischemic infarction in right cerebral hemisphere of HI group,while the Sham ones were no obviously different.Zea-longa score revealed that there was overt neurologic impairment in HI group,but which of in Sham group was no abnormal.To study the effect of HI on neonatal rats,the gene expressional changes were separately determined in right cortex,hippocampus,lung tissue and serum.The results of gene microarray showed that 37 genes were downregulated in right cortex of HI group compared to that of in Sham group;and 37 genes were downregulated in right hippocampus of HI group when compared with Sham group;and 33 genes were downregulated in right lung tissue of HI group compared to Sham group.String database was employed to analyze the differential expression genes(DEGs)of right cortex in rats following HI.The results showed that the biological process(BP)of DEGs was involved in 220 categories;cellular component(CC)included 20 kinds;and MF(molecular function)comprised 14 categories.The KEGG(Kyoto Encyclopedia of Genes and Genomes)analysis suggested that the significantly enriched signal pathways of downregulated DEGs mainly included 32 kinds.The network of Protein-Protein interaction(PPI)revealed that there were 22 nodes and 48 edges among downregulated DEGs.Then,Venny 2.1.0 online software and literature databases were used to analyze the downregulated DEGs of right cortex,hippocampus,lung tissue and serum,and ultimately NGF gene was selected as the study subject.The results indicated by WB assay showed that the NGF protein level in right cortex is significantly decreased at 24h after operation compared to that of in Sham' group(P<0.05),and which is lower in right hippocampus at 12h and 24h post operation(P<0.05).Meanwhile,the results of immunohistochemical test suggested that the NGF staining was obviously lighter in right cortex and hippocampus,while which in the left side of cortex and hippocampus was only slightly lighter.To further study the NGF expression in neurons following HI,the immunofluorescence assay(NeuN/NGF)was performed to detect the expression of NGF in cortex and hippocampus,respectively.The results showed that the staining of NeuN and NGF in right cortex and hippocampus was significantly lighter and the number of pigmenting cell is overtly decreased in HI group than that of in Sham group,but which in left side of cortex and hippocampus was no significantly different.The co-location results showed that the staining of NeuN and NGF was overlapped in the cell bodies of cortical neurons and hippocampal neurons.Similarly,the immunofluorescence experiment(GFAP/NGF)was carried out to detect the expression of NGF in astrocytes in Sham group and HI group after HI.The results showed that the staining of GFAP and NGF in right cortex and hippocampus was overtly lighter and the number of pigmenting cell was significantly decreased in HI group than Sham group,while which in left side of cortex and hippocampus was no significantly different.The co-location results indicated that the staining of GFAP and NGF was overlapped in the cell bodies of cortical and hippocampal astrocytes.Moreover,to further investigate the role of NGF on the growth of cortical neurons,the recombinant of herpes simplex virus(HSV)carrying NGF was constructed and transfected into the neurons,and the polymerase chain reaction(PCR)was utilized to assess the NGF mRNA level.The results showed that the cells was well transfected.Meanwhile,the results of PCR assay showed that NGF mRNA level was decreased in OGD group compared with Normal group(P<0.05),but which was increased in OGD+NGF-ORF group compared to OGD+GFP group(P<0.05).Furthermore,to explore the effect of NGF overexpression on cortical neuron growth,HSV-NGF overexpression virus was transfected into cortical neuron under normal and oxygen-glucose deprivation(OGD)condition.The results of immunofluorescence test showed that the dyeing of Tuj1 was lighter and the number of positive staining cell was decreased in OGD group than that of in Normal group,and the coloring intensity was higher and the number of positive staining cell was increased in OGD+NGF-ORF group when compared to OGD+GFP group.And the quantitative analysis indicated that the area and number of neurons and the neurite length were decreased in OGD group compared to Normal group(P<0.05),and that of in OGD+NGF-ORF was obviously increased when compared with OGD+GFP group(P<0.05).Conclusion:The expression of NGF was decreased in neonatal rats with hypoxic-ischemic brain damage(HIBD),and the NGF overexpression by HSV vector carrying can promote the growth of cortical neurons following hypoxic-ischemic injury,then it therefore may facilitae the recovery of HIBD. |
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