| Objective Sympathetic denervation model of adult rat was established by superior cervical ganglionectomy(SCGx).The histological structure as well as the protein and m RNA expression of following factors: matrix metalloproteinase 9(MMP9), aquaporin 9(AQP9), vascular endothelial growth factor(VEGF), Bcl-2-assaciated X protein(Bax) and B cell lymphoma/lewkmia-2(Bcl-2) were detected in adult rats’ hippocampus after SCGx 1d, 3d, 7d and 15 d.Subsequently, this research discussed the mechanism of injury and recovery of rat’s hippocampus involved in MMP9, AQP9, VEGF, Bax and Bcl-2 after SCGx. Further, the domination of sympathetic ganglion on rat’s hippocampus were explored.Method 1. Modelling SCGx of adult rats and grouping: dividing 125 adult male Wistar rats into SCG was exposed in the sham operation(Control group); surviving 1d after bilateral SCGx(SCGx-1d); surviving 3d after bilateral SCGx(SCGx-3d); surviving 7d after bilateral SCGx(SCGx-7d); surviving 15 d after bilateral SCGx(SCGx-15d). 2. Histological observation: Hematoxylin—Eosin(HE) staining was used to observe neuron amount and morphological changes of CA1, CA3 and DG in rat hippocampus of each group. 3. Location of positive immunoreactivity: Immunohistochemistry(IHC) was used to observe the location, amount and dynamic changes of MMP9, AQP9, VEGF, Bax and Bcl-2 positive immunoreactivity in rat hippocampal CA1, CA3 and DG of each group. 4. Quantification of protein expression: Western-blotting(WB) was used to detect the changes of MMP9, AQP9, VEGF, Bax and Bcl-2 protein expression in rat hippocampus of each group. 5. Quantification of m RNA expression: Quantitative Real-time PCR(RT-PCR) was used to detect the changes of MMP9, AQP9, VEGF, Bax and Bcl-2 m RNA expression in rat hippocampus of each group. 6. Correlation analysis: SPSS Pearson correlation was used to analysis the correlation of rat hippocampal MMP9, AQP9, VEGF, Bax and Bcl-2 between protein expression and m RNA expression; and the correlation of dynamic changing discipline of rat hippocampal MMP9, AQP9, VEGF, Bax and Bcl-2 at 1d, 3d, 7d and 15 d after SCGx.Result 1. The research modelling sympathetic denervation of adult rats by modified SCGx, rat of SCGx bilateral miosis, eyeballs were hollow, palpebrae got constricted and pupillary reaction to light was absent, naming ”Horner syndrome”. 2. Result of HE staining: there was no significant difference between SCGx-1d group and Control group in rat hippocampal neuron amount and morphology; SCGx-3d group hippocampal neuron irregular arranged, part of the cells were lost, neuronal degeneration and lots of inflammatory cell infiltration; compared with SCGx-3d group, the arrangement of hippocampal neuron, the degree of neuronal degeneration and the amount of inflammatory cells in SCGx-7d group were improved; the amount of SCGx-15 d group hippocampal neuron is increased significantly, the arrangement of neuron was tidier than SCGx-3d group, and inflammatory cells are disappeared. 3. Result of IHC: there was a few weakly positive immunoreactivity of MMP9, AQP9, VEGF, Bax and Bcl-2 in rat hippocampus of Control group; MMP9, AQP9, VEGF and Bax positive neurons in hippocampal CA1, CA3 especially DG of SCGx-1d group are increased and stained deeper than Control group, however, positive immunoreactivity of Bcl-2 had trended down; there were lots of MMP9, AQP9, VEGF and Bax strongly positive immunoreactivity in hippocampal CA1, CA3 and DG of SCGx-3d group, capillary endothelial cell, microglia, neutrophil, choroid plexus and ependymal cell also showed MMP9, AQP9 and VEGF positive immunoreactivity, however, positive immunoreactivity of Bcl-2 was minimized; positive immunoreactivity of MMP9, AQP9 and Bax in hippocampus of SCGx-7d group and SCGx-15 d group had trended down, but they were still higher than Control group, furthermore, there were still lots of VEGF positive neurons and positive immunoreactivity of Bcl-2 was picking up. 4. Result of WB: MMP9, AQP9, VEGF and Bax protein expression in rat hippocampus of Operation group were increased than Control group, Bcl-2 protein expression was decreased, the results in line with ICH. MMP9 protein expression of rat hippocampus were as follows: SCGx-3d group(compared with Control group, P<0.01) >SCGx-7d group(P<0.01) >SCGx-15 d group(P<0.01) >SCGx-1d group(P<0.05) >Control group; AQP9 protein expression of rat hippocampus were as follows: SCGx-3d group(P<0.01) >SCGx-7d group(P<0.01) >SCGx-15 d group(P<0.01) >SCGx-1d group(P>0.05) >Control group;VEGF protein expression of rat hippocampus were as follows: SCGx-3d group(P<0.01) >SCGx-7d group(P<0.01) =SCGx-15 d group(P<0.01) >SCGx-1d group(P<0.01) >Control group; Bax protein expression of rat hippocampus were as follows: SCGx-3d group(P<0.01) >SCGx-1d group(P<0.05) >SCGx-7d group(P<0.05) >SCGx-15 d group(P>0.05) >Control group; Bcl-2 protein expression of rat hippocampus were as follows: Control group>SCGx-15 d group(P>0.05) >SCGx-7d group(P>0.05) >SCGx-1d group(P<0.05) >SCGx-3d group(P<0.01). 5. Result of RT-PCR: MMP9, AQP9, VEGF and Bax m RNA expression in rat hippocampus of Operation group were increased than Control group, Bcl-2 m RNA expression was decreased, the results in line with WB. MMP9 m RNA expression of rat hippocampus were as follows: SCGx-3d group(compared with Control group, P<0.01) >SCGx-7d group(P<0.01) >SCGx-15 d group(P<0.01) >SCGx-1d group(P<0.05) >Control group; AQP9 m RNA expression of rat hippocampus were as follows: SCGx-3d group(P<0.01) >SCGx-7d group(P<0.05) >SCGx-15 d group(P<0.05) >SCGx-1d group(P>0.05) >Control group; VEGF m RNA expression of rat hippocampus were as follows: SCGx-15 d group(P<0.01) >SCGx-7d group(P<0.01) >SCGx-3d group(P<0.01) >SCGx-1d group(P>0.05) >Control group; Bax m RNA expression of rat hippocampus were as follows: SCGx-3d group(P<0.01) >SCGx-1d group(P<0.05) >SCGx-7d group(P<0.01) >SCGx-15 d group(P<0.05) >Control group; Bcl-2 m RNA expression of rat hippocampus were as follows: SCGx-15 d group(P>0.05) >Control group >SCGx-1d group(P>0.05) >SCGx-7d group(P>0.05) >SCGx-3d group(P<0.01). 6. Result of correlation analysis: the correlation of rat hippocampal MMP9, AQP9, VEGF, Bax and Bcl-2 between protein expression and m RNA expression were positive; results of pairwise comparison indicated that the correlation of dynamic changing discipline of rat hippocampal MMP9, AQP9 and Bax was positive at 1d, 3d, 7d and15 d after SCGx; the correlation of dynamic changing discipline of rat hippocampal Bax and Bcl-2 was negative at 1d, 3d, 7d and15 d after SCGx.Conclusion 1. Sympathetic denervation model of adult rat was established by SCGx was stable and reliable. 2. In sympathetic denervation model of adult rat, the protein expression and m RNAexpression of MMP9 and AQP9 all showed upgrade firstly then descending latter tendency, indicating that MMP9 and AQP9 involved in the degradation of extracelluar matrix, the damage of blood-brain barrier and brain edema. 3. In sympathetic denervation model of adult rat, the protein expression and m RNA expression of VEGF kept at a high level, indicating that VEGF involved in the protective effect of neuron and angiogenesis after SCGx. 4. In sympathetic denervation model of adult rat, the protein expression and m RNA expression of Bcl-2/Bax showed descending firstly then upgrade latter tendency, indicating that Bax and Bcl-2 involved in the anoikis of hippocampal neurons by SCGx. |
