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Objective: To evaluate the protective effect of adenovirus mediated vascular endothelial growth factor 165 (VEGF165) gene transfer on dopaminergic neurons in Parkinson disease (PD).
Methods: Adenovirus vector coding VEGF165 (Ad-VEGF165) was injected into the striate bodies of 16 SD rats, and adenovirus Ad-LacZ was injected into 25 rats and phosphate-buffered saline (PBS) was injected into 16 rats as controls. Then 6-hydroxydopamine (6-OHDA) was injected to establish PD model. X-gal staining was used to detect the expression of the report gene LacZ in the brain of the Ad-LacZ group 3 d, 2 w, and 6 w after injection, 3 rats in each time point. RT-PCR was used to detect the VEGF165 mRNA expression in the striate body of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. Western blotting was performed to check the protein expression of VEGF165 in the striate bodies of the rats of the 3 groups 2 weeks after injection, 3 rats for each group. A certain numbers of rats in each group underwent rotational behavior analysis 1, 2, and 6 weeks after the 6-OHDA injection. Immunohistochemistry was used to examine the number of tyrosine hydroxylase (TH) positive neuron, density of TH-positive fiber in striate body and black substance, laminin-positive vessel density, and glial fibrillary acidic protein (GFAP) positive glial cells. High performance liquid chromatography-electric-chemical discharge (HPLC-ECD) was performed to detect the contents of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum.
Results: Beta-gal was expressed in the striate bodies of all Ad-LacZ transfected rats, showing the successful transfection of LacZ gene. The mRNA expression and protein expression of VEGF165 in the striate body were significantly higher in the Ad-VEGF165 group than in the other 2 groups. The apomorphine-induced rotation number in the Ad-VEGF165 group was 8.3 turns/min +/- 8.7 turns/min 1 week after the transfection, then gradually decreased, and became 5.0 turns/min +/- 4.4 turns/min 6 weeks after. The rotation numbers of the Ad-LacZ and PBS group were 14.7 turns/min +/- 11.2 turns/min and 13.9 turns/min +/- 8.3 turns/min respectively 1 week after the injection, then increased gradually, and became 20.2 turns/min +/- 13.6 turns/min and 21.8 turns/min +/- 11.8 turns/min respectively 6 weeks later, all significantly higher than those of the Ad-LacZ group (all P < 0.01). The ratios of TH-positive cells in the black substance in the transfected side was 0.42 +/- 0.11, and the density of fibers in the striate body of the transfected side to that of the contralateral side was 0.56 +/- 0.10 in the Ad-VEGF165 group, both significantly higher than those of the Ad-LacZ group (0.20 +/- 0.10 and 0.28 +/- 0.09) and PBS group (0.22 +/- 0.13 and 0.24 +/- 0.08), (all P < 0.01). The ratio of laminin-positive vessel density of the transfected side to that of the contralateral side in the Ad-VEGF165 group was 2.09 +/- 0.42, and the ratio of GFAP-positive glial cells of the striate body of the transfected side to that of the contralateral side was 2.77 +/- 1.21 in the Ad-VEGF165 group, both significantly higher than those in the Ad-LacZ group (1.01 +/- 0.16 and 1.64 +/- 0.28) and the PBS group (1.04 +/- 0.09 and 1.56 +/- 0.62) (P < 0.01 and 0.05). HPLC-ECD showed that the contents of DA, HAV, and DOPAC of the striate body at the destroyed side in the Ad-VEGF165 group were all significantly higher than those in the other 2 groups (all P < 0.01). The ratios to the DA, DOPAC, and HVA of the destroyed side striate body to those of the contralateral side in the Ad-VEGF165 group was 0.35 +/- 0.11, 0.46 +/- 0.09, and 0.38 +/- 0.09 respectively, all significantly higher than those in the Ad-LacZ group (0.17 +/- 0.15, 0.21 +/- 0.07, and 0.16 +/- 0.05) and PBS group (0.19 +/- 0.06, 0.20 +/- 0.09, 0.14 +/- 0.03) (all P < 0.01).
Conclusion: Gene transfer of Ad-VEGF165 has a protective effect on the dopaminergic neurons of PD. The proliferation of vessels and glial cells induced by VEGF may involve in the process of neuroprotection to the dopaminergic neurons of PD.
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