天麻钩藤饮对脑灌注不足大鼠VEGF的表达和神经发生的影响
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摘要
背景与目的：临床以及实验研究证实了慢性脑灌注不足与认知功能障碍之间的关系，慢性脑灌注不足是导致认知功能障碍的一个重要因素，是血管性认知障碍发展过程中的一个重要的病理过程。通过永久性双侧颈总动脉结扎(permanent occlusion of bilateral common carotid arteries，2-vessel occlusion，2VO)建立的2VO模型导致了慢性中度脑血流的减少，构建了一个慢性脑灌注不足的模型。2VO所导致的脑组织的主要病理改变为：白质疏松，胶质增生，皮质、海马CA1区神经元的固缩、缺失；在行为学上表现为长期的学习记忆能力障碍，因此是研究轻度认知功能障碍的一个良好的模型。天麻钩藤饮这一古老的中药组方显示了较好的临床应用效果，可以改善患者的认知，但是基础研究甚少，作用机制不甚清楚。韩国学者进行的体外研究表明，钩藤可以促进人脐静脉内皮细胞VEGF的基因表达和蛋白分泌，那么天麻钩藤饮这一中药复方组分是否也发挥了这一作用呢?是否还有其它的作用机制参与了其神经保护功能呢?因此，本研究拟通过建立大鼠脑灌注不足模型，观察天麻钩藤饮早期干预对脑灌注不足大鼠学习记忆能力的影响，以及药物干预对脑缺血后血管内皮生长因子(vascular endothelial growth factor，VEGF)的表达和海马齿状回神经发生的影响，探讨其可能的神经保护机制。
材料与方法：健康SD大鼠，144只，雌雄不限，体重342±16g。实验动物随机分为正常对照组、假手术组、模型组和治疗组，共4组。每组又分为2周、4周、8周3个时间点进行观察。采用永久性双侧颈总动脉结扎制备脑灌注不足模型。在造模前、后对动物进行Morris水迷宫评价，以筛选合格动物入组。治疗组于造模后次日开始给予天麻钩藤饮(5g／kg·d)灌胃，分别灌胃2周，4周，8周。假手术组，模型组给予相同体积蒸馏水灌胃；正常对照组不予灌胃处理。在各个时间点结束后行水迷宫，对大鼠的学习记忆能力进行评价。BrdU标记S期细胞，观察海马齿状回细胞增殖。动物于处死前12h内，腹腔注射5-溴-2′-脱氧尿嘧啶核苷(bromodeoxyuridine，BrdU)(50mg／kg)，1次／4h，共3次，末次注射4h后处死。免疫组化观察3个时间点各组大鼠的VEGF及VEGFR-2／Flk-1的表达变化，以及BrdU标记的海马齿状回新生细胞数量的变化。免疫荧光检测神经元特异性烯醇化酶(neuron-specific enolase，NSE)(标记神经元)与VEGF的共表达，即观察脑缺血是否诱导了神经元表达VEGF。
结果：(1)行为学结果显示，模型组的潜伏期均明显延长，与假手术组大鼠比较具有显著性差异(P＜0.05)，表明大鼠学习记忆能力明显受损；治疗组大鼠与模型组比较，潜伏期明显缩短(P＜0.05)，表明药物干预改善了脑缺血后大鼠的学习记忆能力。(2)免疫组化结果显示，模型组VEGF的表达较假手术组明显增多(P＜0.05)，随后呈现递减的趋势。治疗组VEGF的表达较模型组明显增多(P＜0.05或P＜0.01)。2周治疗组的VEGF的表达较相应模型组明显增多，不仅表达的细胞数量多，且胞浆丰富，染色浓密，部分呈颗粒状，并且这种高表达可持续到4周，8周治疗组仍呈中度表达。模型组Flk-1的表达较假手术组明显增多(P＜0.05)；治疗组Flk-1的表达较模型组明显增多(P＜0.05或P＜0.01)。(3)模型组的BrdU阳性细胞较假手术组明显增多(P＜0.05)，治疗组较模型组明显增多(P＜0.05或P＜0.01)，增生的细胞主要分布于海马齿状回颗粒细胞层亚颗粒增生带中。模型组的BrdU阳性细胞随着时间的推移呈现减少的趋势，而治疗组的BrdU阳性细胞却未随着时间的延长则呈现减少的趋势。(4)免疫荧光观察到模型组神经元VEGF表达增加。
结论：脑缺血可以诱导神经元VEGF表达增加，可能是脑缺血后内源性的神经保护机制之一；天麻钩藤饮可以改善脑灌注不足后大鼠的学习记忆能力；天麻钩藤饮可以促进脑缺血后VEGF及Flk-1的表达增加，可能通过该途径发挥脑保护功能；天麻钩藤饮可以促进海马齿状回神经发生。
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.
材料与方法：健康SD大鼠，144只，雌雄不限，体重342±16g。实验动物随机分为正常对照组、假手术组、模型组和治疗组，共4组。每组又分为2周、4周、8周3个时间点进行观察。采用永久性双侧颈总动脉结扎制备脑灌注不足模型。在造模前、后对动物进行Morris水迷宫评价，以筛选合格动物入组。治疗组于造模后次日开始给予天麻钩藤饮(5g／kg·d)灌胃，分别灌胃2周，4周，8周。假手术组，模型组给予相同体积蒸馏水灌胃；正常对照组不予灌胃处理。在各个时间点结束后行水迷宫，对大鼠的学习记忆能力进行评价。BrdU标记S期细胞，观察海马齿状回细胞增殖。动物于处死前12h内，腹腔注射5-溴-2′-脱氧尿嘧啶核苷(bromodeoxyuridine，BrdU)(50mg／kg)，1次／4h，共3次，末次注射4h后处死。免疫组化观察3个时间点各组大鼠的VEGF及VEGFR-2／Flk-1的表达变化，以及BrdU标记的海马齿状回新生细胞数量的变化。免疫荧光检测神经元特异性烯醇化酶(neuron-specific enolase，NSE)(标记神经元)与VEGF的共表达，即观察脑缺血是否诱导了神经元表达VEGF。
结果：(1)行为学结果显示，模型组的潜伏期均明显延长，与假手术组大鼠比较具有显著性差异(P＜0.05)，表明大鼠学习记忆能力明显受损；治疗组大鼠与模型组比较，潜伏期明显缩短(P＜0.05)，表明药物干预改善了脑缺血后大鼠的学习记忆能力。(2)免疫组化结果显示，模型组VEGF的表达较假手术组明显增多(P＜0.05)，随后呈现递减的趋势。治疗组VEGF的表达较模型组明显增多(P＜0.05或P＜0.01)。2周治疗组的VEGF的表达较相应模型组明显增多，不仅表达的细胞数量多，且胞浆丰富，染色浓密，部分呈颗粒状，并且这种高表达可持续到4周，8周治疗组仍呈中度表达。模型组Flk-1的表达较假手术组明显增多(P＜0.05)；治疗组Flk-1的表达较模型组明显增多(P＜0.05或P＜0.01)。(3)模型组的BrdU阳性细胞较假手术组明显增多(P＜0.05)，治疗组较模型组明显增多(P＜0.05或P＜0.01)，增生的细胞主要分布于海马齿状回颗粒细胞层亚颗粒增生带中。模型组的BrdU阳性细胞随着时间的推移呈现减少的趋势，而治疗组的BrdU阳性细胞却未随着时间的延长则呈现减少的趋势。(4)免疫荧光观察到模型组神经元VEGF表达增加。
结论：脑缺血可以诱导神经元VEGF表达增加，可能是脑缺血后内源性的神经保护机制之一；天麻钩藤饮可以改善脑灌注不足后大鼠的学习记忆能力；天麻钩藤饮可以促进脑缺血后VEGF及Flk-1的表达增加，可能通过该途径发挥脑保护功能；天麻钩藤饮可以促进海马齿状回神经发生。
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.
引文
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39. Kumagai AK, Kang YS, Boado RJ, et al. Upregulation of blood-brain barrier GLUTI glucose transporter protein and mRNA in experimental chronic hypoglycemia. Diabetes. 1995; 44(12): 1399-1404.
40. Shepherd PR, Kahn BB. Glucose transporters and insulin action--implications for insulin resistance and diabetes mellitus. N Engl J Med. 1999; 341(4): 248-257.
41. Dantz D, Bewersdorf J, Fruehwald-Schultes B, et al. Vascular endothelial growth factor: a novel endocrine defensive response to hypoglycemia. J Clin Endocrinol Metab. 2002; 87(2): 835-840.
42. Pekala P, Marlow M, Heuvelman D, et al. Regulation of hexose transport in aortic endothelial cells by vascular permeability factor and tumor necrosis factor-alpha, but not by insulin. J Biol Chem. 1990; 265(30): 18051-18054.
43. Sone H, Deo BK, Kumagai AK. Enhancement of glucose transport by vascular endothelial growth factor in retinal endothelial cells. Invest Ophthalmol Vis Sci. 2000; 41 (7): 1876-1884.
44．朱晓峰 主编．神经干细胞基础及应用．第1版．北京：科学出版社，2005．5
45. Kawai T, Takagi N, Mochizuki N, et al. Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat. Neuroscience. 2006; 141(3): 1209-1216.
46. Mateo I, Llorca J, Infante J, et al. Case-control study of vascular endothelial growth factor (VEGF) genetic variability in Alzheimer's disease. Neurosci Lett. 2006; 401(1-2): 171-173.
47. Hashimoto T, Zhang XM, Chen BY, et al. VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation. Development. 2006; 133(11): 2201-2210.
48. Maurer MH, Tripps WK, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett. 2003; 344(3): 165-168.
49. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature. 2005: 438(7070): 954-959.
50. Zhu Y, Jin K, Mao XO, et al. Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression. FASEB J. 2003; 17(2): 186-193.
51. Wang YQ, Guo X, Qiu MH, et al. VEGF overexpression enhances striatal neurogenesis in brain of adult rat after a transient middle cerebral artery occlusion. J Neurosci Res. 2007; 85(1): 73-82.
52. Sun FY, Guo X. Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factor. J Neurosci Res. 2005; 79(1-2): 180-184.
53．孙晓芳，王巍，王丹巧，等．天麻及其制剂神经保护机制的研究进展．中国中药杂志．2004；29(4)：292-295．
54. Terasawa K, Shimada Y, Kita T, et al. Choto-san in the treatment of vascular dementia: a double-blind, placebo-controlled study. Phytomedicine. 1997; 4(1): 15-22.
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3. Hayashi T, Abe K, Itoyama Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J Cereb Blood Flow Metab. 1998;18(8):887-895.
4. Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol. 2000;156(3):965-976.
5. Maurer MH, Tripps WK, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett. 2003; 344(3): 165-168.
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7. Banai S, Jaklitsch MT, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994;89(5):2183-2189.
8. Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1 alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis. 2003;14(13):524-534.
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10. Yang ZJ, Bao WL, Qiu MH, et al. Role of vascular endothelial growth factor in neuronal DNA damage and repair in rat brain following a transient cerebral ischemia. J Neurosci Res. 2002;70(2):140-149.
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26．朱晓峰 主编．神经干细胞基础及应用．北京：科学出版社．2005．5
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31．罗春霞，李露斯，迟路湘．脑灌注不足大鼠血管内皮生长因子的表达及其意义．中国临床康复．2004；8(34)：7694-7695．
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35. Graham SH, Chen J. Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab. 2001; 21(2): 99-109.
36. Volm M, Mattern J, Koomagi R. Inverse correlation between apoptotic (Fas ligand, caspase-3) and angiogenic factors (VEGF, microvessel density) in squamous cell lung carcinomas. Anticancer Res. 1999; 19(3A): 1669-1671.
37. Chen J, Nagayama T, Jin K, et al. Induction of caspase-3-1ike protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J Neurosci. 1998; 18(13): 4914-4928.
38. Jin K, Nagayama T, Mao X, et al. Two caspase-2 transcripts are expressed in rat hippocampus after global cerebral ischemia. J Neurochem. 2002; 81(1): 25-35.
39. Kumagai AK, Kang YS, Boado RJ, et al. Upregulation of blood-brain barrier GLUTI glucose transporter protein and mRNA in experimental chronic hypoglycemia. Diabetes. 1995; 44(12): 1399-1404.
40. Shepherd PR, Kahn BB. Glucose transporters and insulin action--implications for insulin resistance and diabetes mellitus. N Engl J Med. 1999; 341(4): 248-257.
41. Dantz D, Bewersdorf J, Fruehwald-Schultes B, et al. Vascular endothelial growth factor: a novel endocrine defensive response to hypoglycemia. J Clin Endocrinol Metab. 2002; 87(2): 835-840.
42. Pekala P, Marlow M, Heuvelman D, et al. Regulation of hexose transport in aortic endothelial cells by vascular permeability factor and tumor necrosis factor-alpha, but not by insulin. J Biol Chem. 1990; 265(30): 18051-18054.
43. Sone H, Deo BK, Kumagai AK. Enhancement of glucose transport by vascular endothelial growth factor in retinal endothelial cells. Invest Ophthalmol Vis Sci. 2000; 41 (7): 1876-1884.
44．朱晓峰 主编．神经干细胞基础及应用．第1版．北京：科学出版社，2005．5
45. Kawai T, Takagi N, Mochizuki N, et al. Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat. Neuroscience. 2006; 141(3): 1209-1216.
46. Mateo I, Llorca J, Infante J, et al. Case-control study of vascular endothelial growth factor (VEGF) genetic variability in Alzheimer's disease. Neurosci Lett. 2006; 401(1-2): 171-173.
47. Hashimoto T, Zhang XM, Chen BY, et al. VEGF activates divergent intracellular signaling components to regulate retinal progenitor cell proliferation and neuronal differentiation. Development. 2006; 133(11): 2201-2210.
48. Maurer MH, Tripps WK, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett. 2003; 344(3): 165-168.
49. Greenberg DA, Jin K. From angiogenesis to neuropathology. Nature. 2005: 438(7070): 954-959.
50. Zhu Y, Jin K, Mao XO, et al. Vascular endothelial growth factor promotes proliferation of cortical neuron precursors by regulating E2F expression. FASEB J. 2003; 17(2): 186-193.
51. Wang YQ, Guo X, Qiu MH, et al. VEGF overexpression enhances striatal neurogenesis in brain of adult rat after a transient middle cerebral artery occlusion. J Neurosci Res. 2007; 85(1): 73-82.
52. Sun FY, Guo X. Molecular and cellular mechanisms of neuroprotection by vascular endothelial growth factor. J Neurosci Res. 2005; 79(1-2): 180-184.
53．孙晓芳，王巍，王丹巧，等．天麻及其制剂神经保护机制的研究进展．中国中药杂志．2004；29(4)：292-295．
54. Terasawa K, Shimada Y, Kita T, et al. Choto-san in the treatment of vascular dementia: a double-blind, placebo-controlled study. Phytomedicine. 1997; 4(1): 15-22.
1. Marti HH, Risau W. Systemic hypoxia changes the organ-specific distribution of vascular endothelial growth factor and its receptors. Proc Natl Acad Sci USA. 1998;95(26):15809-15814.
2. Shweiki D, Itin A, Soffer D, et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992; 359(6398):843-845.
3. Hayashi T, Abe K, Itoyama Y. Reduction of ischemic damage by application of vascular endothelial growth factor in rat brain after transient ischemia. J Cereb Blood Flow Metab. 1998;18(8):887-895.
4. Marti HJ, Bernaudin M, Bellail A, et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am J Pathol. 2000;156(3):965-976.
5. Maurer MH, Tripps WK, Feldmann RE, et al. Expression of vascular endothelial growth factor and its receptors in rat neural stem cells. Neurosci Lett. 2003; 344(3): 165-168.
6. Hayashi T, Abe K, Suzuki H, et al. Rapid induction of vascular endothelial growth factor gene expression after transient middle cerebral artery occlusion in rats. Stroke. 1997; 28(10):2039-2044.
7. Banai S, Jaklitsch MT, Shou M, et al. Angiogenic-induced enhancement of collateral blood flow to ischemic myocardium by vascular endothelial growth factor in dogs. Circulation. 1994;89(5):2183-2189.
8. Mu D, Jiang X, Sheldon RA, et al. Regulation of hypoxia-inducible factor 1 alpha and induction of vascular endothelial growth factor in a rat neonatal stroke model. Neurobiol Dis. 2003;14(13):524-534.
9. Jin KL, Mao XO, Nagayama T, et al. Induction of vascular endothelial growth factor and hypoxia-inducible factor-1 alpha by global ischemia in rat brain. Neuroscience. 2000;99(3):577-585.
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