| Spinal cord injury (SCI) may be defined as an injury resulting from an insult inflicted on the spinal cord that compromises, either completely or incompletely, its major functions (motor, sensory, autonomic, and reflex). Spinal cord injury remains an important cause of morbidity and mortality in modern society. SCI has profound psychosocial effects that are devastating for patients, families, and friends. A basic understanding of the pathophysiological mechanisms underlying spinal cord injury is of paramount importance to facilitate comprehension of pharmacological interventions...Acute spinal cord injury (SCI) is characterized by its progressive nature, in fact secondary injuries is an active regulatory processes occurring at cellular and molecular levels, which can be regulated by many factors and the process is reversible and can be controlled. In the past decade, it has been shown that the changes of number of genes play a important role in the pathological process of spinal cord injury (SCI).In Gene chip (gene chip) is also called DNA microarray (DNA microarray), is one of the advanced biological technology emerging with the implementation of human genome project. The characteristics of gene chip is high integration, high throughput, miniaturization, diversification and automation. especially the development of recent 10 years, at present already in gene chip have play an important role in analysis of genes expression, gene diagnosis of disease, drug genome science and other fields, which has great application prospect in biotechnology. Collection of a variety of mRNA gene expression microarrays can screening differentially expressed genes in spinal cord in rats after spinal cord injury in.using a combination of several mRNA expression profiles from rats can analyzed the overlapping differentially expressed genes (DEGs) after SCI. By filtering out the relevant genes can be understanding the occurrence and mechanism of the disease quickly, provide a strong guarantee for the diagnosis and treatment of disease.There is a variety of type of lesions that have been used as a model of SCI such as balloon inflation, compression, crush with forceps, lateral hemisection, full transection, dorsal column transection, contusion, and removal of spinal cord segment. For simulation of the primary and secondary spinal cord injury process, our established an easy, cheap, and highly reproducible model of compressive SCI in rat.For that lesion we use a vascular clip, with a known pressure force, that provides a compression in the spinal cord, keeping the dura mater intact. This lesion model presents some advantages such as high reproducibility, low cost, and no need for previous training to use it.Among all of secondary injuries, ischemia has been demonstrated as a focus of postinjury pathophysiological changes of acute SCI since it was believed to aggravate other secondary injuries and is parallel to the neurological dysfunction.Hypoxia-inducible factora (HIFa) is the most direct, and possibly, the only regulatory factor that plays a key role in cell differentiation and survival under hypoxic conditions. Bioinformatics research findings Hypoxia-inducible factor a (HIFa) that plays a key role in spinal cord injury.In the present study, A Highly Reproducible Mouse Model of Compression Spinal Cord Injury was established. The expression patterns of HIF-1a in injured spinal cord were studied at mRNA and protein level, and some important target genes of HIF-lathat participated in hypoxia tolerance and vascularity of tissues were also studied to explore the relationship between expressional network of HIF-1a and development of SCI. At the same time, combined with the detection of VEG, EPO in peripheral blood of patients with SCI, discuss HIF-1 and its target gene VEG, EPO in the developing process of secondary spinal cord, thus providing a theoretical for clinical treatment of spinal cord injury.Methods:1.1 The mRNA microarray data of GSE2770 was downloaded from the Gene Expression Omnibus (GEO) database in NCBI (http://www.ncbi.nlm.nih.gov/geo/).1.2 DEGs in SCI were identified using the limma package in R compared with the corresponding controls.1.3 Enrichment analysis of GO terms was performed using the Cytoscape and its plugin.1.4 Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed for the common DEGs by using the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tool.1.5 The DEGs which enriched in pathways were analyzed by using the DiRE database to look for the regulatory elements of each pathway in cell.2. Mouse Model of Compression Spinal Cord Injury was scored by the means of the BBB open-field locomotor scale. HE staining and Nissl staining observed dynamically pathological changes in spinal cord injury.3.1 Detecting the expression of HIF-1 alpha mRNA by RT-PCR after spinal cord injury at different time points.3.2 Analysis the expression of HIF-1 alpha mRNA and protein by in situ hybridization and immunohistochemistry in the tissue of spinal cord after injured at different time points.3.3 Analysis the expression level of AIF and Caspase-3 protein by Western-blot and observated the apoptosis nerve cell by TUNEL staining in the tissue of spinal cord after injuryed at different time points.3.4 Detecting the expression of protein of VEGF and EPO in peripheral blood with the patient of spinal cord injury by method of ELISA.Results:1. Totally,173 overlapping DEGs were identified. These DEGs were enriched in different GO terms, such as response to wounding and inflammatory response. Pathway enrichment analysis revealed that DEGs were mainly related to inflammation and immunity, such as extracellular matrix (ECM)-receptor interaction pathway. Moreover, the regulatory network showed that HIF1, ras-related C3 botulinum toxin substrate 2 (RAC2),CD44 molecule (CD44), and actin related protein 2/3 complex (ARPC1B)were hub genes.2.Compared with the control group, BBB score was increased in the Mouse Model of Compression Spinal Cord Injury, the difference between the groups were statistically significant (p<0.05). In control group, HE staining and Nissl staining was basically normal, however spinal cord pathological changes aggravated gradually, anterior horn neurons of the spinal cord gray matter showed more obvious, Nissl body showed staining fade or disappear in SCI group.3.1. HIF-1a was remained at a relatively low level in normal spinal cord; in 6-72 h after SCI, the expression of HIF-la significantly increased; after 1 week, its expression began to decrease even though the amount was still above normal. It was also noticed that the expression of HIF-1a was parallel to the degree of ischemic injure of spinal cord:especially after 2-3 days, the peak of clinical ischemic symptoms appeared, coincidence the expression of HIF-la reached its maximum as assayed by RT-PCR.3.2 The IHC of transcriptional factor HIF-la showed that:almost all of the damage area neurons express HIF-alpha mRNA, however almost no obvious signals were detected in normal spinal cord.3.3 Immunohistochemistry showed that:the expression of HIF-1 protein in the spinal cord injury was significantly increased, the trends of protein almost the same as the mRNA, but the expression of the protein earlier than the its mRNA. The expression of VEGF, EPO in normal spinal cord tissue showed a low level, after injured 24 hours the expression began to increase, and reached the peak in 48-72 hours and began to decreased gradually.3.4 Western blot analysis showed that the protein of AIF and caspase-3 after injured 6 hours the expression began to increase, and reached the peak in 3 days and began to decreased gradually at 7 days in the group of SCI compared with the control.TUNEL staining was used to detect the apoptosis of spinal cord nerve cells. TUNEL positive cells was remained at a relatively low level in normal spinal cord, however, after injured 6,12 and 24 hours observed with high fluorescence signal, and reached the peak in 3 days and began to decreased gradually at 7 days.3.5 ELISA analysis showed that the target genes of HIF-1 i.e., VEGF and EPO were significantly increased in peripheral blood with SCI compared with normal.Conclusion:1. SCI is mainly related with inflammation and immunity by the study of gene ontology enrichment and pathway discovery, Our study suggested that RAC2,CD44, and ARPC1B that involved in different pathways including ECM-receptor interaction pathway may associate with the SCI progression.RAC2 and CD44 may affect the inflammatory and immune response, and play crucial roles in SCI progression.2. Improved spinal cord ischemia-reperfusion model can simulate different degrees of ischemia-reperfusion injury, and ischemic broader in scope than the traditional method, and the model is economical and reliable, pathological changes stable.3. The findings suggested that the most important hypoxic regulatory factor HIF-1 a was upregulated in involved cells by activating the transcription and increasing protein stability, and subsequently activated the expression of HIF-la target genes, including EPO and VEGF in SCI. Combined with the pathologic observation, it suggested that overexpression of HIF-1a and its target genes might take part in hypoxia tolerance and vascularity of the injured spinal cord.4. Our study shows that in acute injury of spinal cord may cause cell damaged through apoptosis, and its may be relevant with post-traumatic cord syndrome. But the spinal cord might be through the expressed the HIF-1 alpha and its target gene to overcome the damaged condition. |
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