| Background and objective: Clinical practice and experimental research have confirmed that chronic cerebral hypoperfusion is associated with cognitive decline. Chronic cerebral hypoperfusion is a key factor which induced cognitive impairment. And chronic cerebral hypoperfusion is an important pathological change in the process of vascular cognitive impairment. Permanent occlusion of the bilateral common carotid arteries (2VO) which caused chronic moderate cerebral blood flow reduction is a suitable model for the research of chronic cerebral hypoperfusion. The neuropathological changes that followed 2VO exhibited rarefaction in the white matter, gliosis, shrinkage of neurons and neuronal loss in the cerebral cortex and hippocampus CA1. And it was observed persistent learning and memory impairments in this model. Therefore 2VO rats appear to be a useful model for research of mild cognitive impairment. As a traditional medicine, Tianma Gouteng Decoction shows good clinical efficacy, it could improve patients' cognition. But the basic researchs are rare, and the mechanism of its effect is unknown. Experts in Korea demonstrated that Uncaria increased VEGF gene expression and protein secretion of HUVECs(human umbilical vein endothelial cells). Does Tianma Gouteng Decoction have this effect? And are there any other effects participated in its neuroprotection effect? Therefore a cerebral hypoperfusion model which was produced by permanent occlusion of bilateral common carotid arteries (2VO) in rats was established. Rats were treated with Tianma Gouteng Decoction after the 2VO operation. The purpose of this study intended to investigate the effect of Tianma Gouteng Decoction on memory deficits of rats induced by cerebral hypoperfusion and elucidated whether it could increase vascular endothelial growth factor (VEGF) expression, and enhance neurogenesis in the hippocampal dentate gyrus in order to clarify its mechanisms of neuroprotection.Materials and methods: 144 healthy SD rats(median weight: 342±16g, either sex) were divided into normal control group, sham-operated group, model group and treatment group at random. The experimental groups were divided into four subgroups that were kept for 2 weeks, 4 weeks and 8 weeks respectively after 2VO operation. Before and after the operation, the performance of rats in Morris water maze was investigated in order to screen qualified rats. The next day of 2VO operation treatment group rats were administered Tianma Gouteng Decoction (5g/kg.d) for 2 weeks, 4 weeks and 8 weeks respectively. Sham-operated groups and model groups were treated with same volume distilled water, and normal control group with no administration. At the end of the 3 time points, the performance of rats was investigated by Morris water maze in order to evaluate the changes of learning and memory abilities. Cells in S phase can be labeled by Bromodeoxyuridine (BrdU). 12 hours before sacrificed rats were injected with BrdU for 3 times, 4 hours once. The neurogenesis in the hippocampal dentate gyrus was evaluated by BrdU and the presence of VEGF and its receptor Flk-1 were investigated by immunohistochemistry. And in this experiment we also investigated whether rats following cerebral hypoperfusion could express VEGF in neurons by immunofluorescence. The neurons were labeled by neuron-specific enolase (NSE).Results: (1) The model rats showed a significantly increased latency of the rats to find the hidden platform in the Morris water maze task compared with sham-operated rats (P<0.05). It was indicated that the spatial learning and memory abilities of 2VO rats were impaired significantly. 2VO rats administered Tianma Gouteng Decoction showed significantly improved latency in Morris water maze compared to model groups (P<0.05) . It was suggested that Tianma Gouteng Decoction ameliorated the memory deficits of 2VO-rats. (2) The results of immunohistochemistry study showed that VEGF expression in model group rats increased compared to sham-operated groups (P<0.05) , and it decreased gradually over time. The expression of VEGF in treatment groups increased significantly compared to model groups (P<0.05 or P< 0.01) . Drug treatment prolonged the immunoreactivity for VEGF till 8 weeks. Flk-1 expression in model group rats increased compared to sham-operated groups (P<0.05) , and the expression in treatment groups increased compared to model rats (P<0.05 or P< 0.01) . (3) The number of BrdU in model rats significantly increased compared with sham-operated rats (P<0.05) , and the drug treatment groups increased positive cells of BrdU more than model groups (P<0.05 or P<0.01) . The new-born cells were located in hippocampal dentate gyrus subgranular layer. The number of BrdU positive cells was declined in models over time, but in drug treatment it didn't decreased. (4) Increased VEGF expression was observed in neurons on 2VO-rats by immunofluorescence.Conclusions: Cerebral hypoperfusion could induce VEGF expression in neurons, and it may be one of the endogenous neuronal protection effects for cerebral ischemia. Tianma Gouteng Decoction has an ameliorating effect on rats with spatial memory deficits caused by cerebral hypoperfusion. The drug may induce VEGF and Flk-1 expression, and it may exert neuroprotective effects via VEGF/Flk-1 pathway. Tianma Gouteng Decoction also promotes neurogenesis in hippocampal dentate gyrus.
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