| Objective:To observe the variation of expression and distribution of IhhmRNA and GlilmRNA in the lesion areas after acute injury of spinal cord and to analyze the regulation of Ihh/Glil signal pathways on endogenous neural stem cells to ensure the molecule treatment mechanism of Ligustrazine for spinal cord.Methods:The SD (Sprague Dawley)rats were randomly divided into gap group (group A), pseudo surgery group (group B), model group (group C), Methylprednisolone control group (group D) and Ligustrazine treatment group (group E). The rat models of acute spinal cord injury were established by modified Allen’s method in the experiment. The rats of group E after molding were given abdominal Ligustrazine Hydrochloride Injection for400mg/kg per day, and the first injection was given after30min after the operation. Adapting Methylprednisolone high dose impulsive therapy, the rats in group D were given187.5mg/kg Methylprednisolone injection in abdomen after30min surgery. After one hour, the total amount of23hours with33.75mg/kg per hour was worked out to averagely inject four times in23hours for the rats. The group A, B and C were given the same amount of physiological saline in abdomen at the same time for ten days while group D was given the same mount with the former three groups in the following nine days after molding on the second day. Oblique board test and BBB evaluation on nerve function were applied to test the postoperative results on the8th hour,1st day,7th day,14th day, and28th day respectively.6rats in each group were sacrificed on the8th hour,1st day,7th day,14th day, and28th day after spinal cord injury. Then the injuried spinal cord about the5mm right and left of it was cut out to be detected,3of them for the HE, Nissl staining, immunohistochemistry and in situ hybridization, and3of them only for Real-time PCR detection. Meanwhile, the experiment adapted HE and Nissl staining methods to observe the morphological changes of spinal cord. Immunohistochemistry was applied to test the expression of Brdu+and Nestin+cells. Besides, in-situ hybridization and Real-time PCR were used to test the distribution and expression of IhhmRNA and GlilmRNA in the spinal cord. All of the test results were statistically analysis, and the correlation analysis were made among the expression of Brdu+and Nestin+cells, IhhmRNA and GlilmRNA in the spinal cord.Results:Firstly, motor function of hind limbs and the function of spinal cord of the rats decreased significantly after spinal cord injury; The maximum angles the rats maintained on oblique board in the Ligustrazine treatment group were on the uprising trend and there was significant difference compared with model control group on the14th day and28th day(p<0.05); There was significant difference between Methylprednisolone control group and Ligustrazine treatment group (P<0.01), and Methylprednisolone control group is better than Ligustrazine group from3th to28th after spinal cord injury. There was significant difference compared with model control group on the3th day,14th day and28th day according to the BBB score(P<0.05or P<0.01). However, the impact was declined compared with Methylprednisolone control group (P<0.05or P<0.01)Secondly, Ligustrazine could remarkably reduce edema, inflammatory cell infiltration and blooding and promote the repair of spinal cord tissues after injury.Thirdly, there was significant difference (P<0.01) on the7th day,14th day and28th day after spinal cord injury since Ligustrazine enabled to increase the proliferation of Brdu+cells (compared with group C P<0.05or P<0.01) while Methylprednisolone restrained its proliferation(compared with group C P<0.05or P<0.01). Ligustrazine could conspicuously enhance the expression of Nestin+cells and reach its summit on the7th day and there was significant difference compared with group C and group D on the7th day,14th day and28th day after spinal cord injury(P<0.05or P<0.01) while Methylprednisolone control group revealed minor expression (compared with group C P<0.05or P<0.01)Fourthly, there were IhlimRNA and GlilmRNA in the spinal cord of normal adult rats. IhhmRNA mainly distribute in the whiter matter as well as minor expression in ependymocytes. While GlilmRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. In the experiment, the expression of IhhmRNA and GlilmRNA decreased to the lowest on the3th day and7th day, and then gradually increased, but still lower than the normal value. There is no obvious difference between among Ligustrazine treatment group, Methylprednisolone control group and model control group at each time point (P>0.05). Fifthly, IhhmRNA is positively correlated with the expression of GlilmRNA and is negatively related to the Nestin+cells according to the partial correlation analysis.Finally, The correlation analysis was performed on the expression of IhhmRNA, GlilmRNA, Nestin+cells and Brdu+cells in the spinal cord. IhhmRNA positively correlated with the expression of GlilmRNA, and is negatively related to Nestin+expression.Conclusion:Firstly, Ihh distributes in the spinal cord of normal adult rats with main distribution in the white matter and minor in the ventricular zone. Gli1mRNA are mainly expressed in the cytoplasm of glial cells and the cell nucleolus of gray matter neurons in addition to outside the plasmosome. Secondly, Ihh/glil signal transduction pathway negatively regulates the proliferation and differentiation of endogenous neural stem cells. Thirdly, Ligustrazine could promote the proliferation and differentiation of endogenous neural stem cells after spinal cord injury. Fourthly, the influence Ligustrazine exerted on proliferation and differentiation of endogenous neural stem cells may not realize by the intervention of Ihh/Glil signal pathways. |
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