【作者】 王春霞；

【导师】 孙远征；

【作者基本信息】 黑龙江中医药大学 ， 针灸推拿学， 2012， 博士

【摘要】 目的：通过观察头穴丛刺法对慢性脑缺血损伤大鼠学习记忆能力及HIF-1α、VEGF表达的影响,探讨头穴丛刺治疗慢性脑缺血致学习记忆障碍的作用机制,为临床应用头穴丛刺治疗该病提供理论依据,以更好的指导临床。方法：利用Morris水迷宫筛选出学习记忆能力正常的Wistar大鼠72只,随机分为空白对照组(A)、假手术组(B)、和手术组。手术组采用改良的双侧颈总动脉结扎法(2VO)制备慢性脑缺血致学习记忆障碍大鼠模型,术后4周,再通过Morris水迷宫检测大鼠的学习记忆能力,将造模成功的大鼠分为模型组(C)、尼莫地平组(D)和头穴丛刺组(E)。A组、B组和C组不予治疗。D组给予尼莫地平1mg/kg灌胃,每天1次,治疗4周。E组采用头穴丛刺法,取百会和百会左右旁开2mm三个穴位,留针30min,每日1次,治疗4周。术后8周(治疗后4周)再次采用Morris水迷宫检测大鼠的学习记忆能力；光镜下观察额叶皮层、海马组织病理学变化；免疫组化检测额叶皮层、海马HIF-1α、VEGF蛋白表达；RT-PCR检测额叶皮层、海马HIF-1α、VEGFmRNA表达。结果：1.Morris水迷宫检测结果：术后8周A组和B组相比无显著性差异(P>0.05)；C组、D组和E组大鼠学习记忆能力明显下降,与A组相比差异显著(P<0.05)；D组和E组与C组相比,具有显著性差异(P<0.05),大鼠的学习记忆能力有所提高；D组和E组相比无显著性差异(P>0.05)。2.HE染色结果：A组和B组未见明显病理学变化,C组可见大量神经元细胞固缩,胞体形态不规则,胞浆浓缩深染,核仁变小或消失,细胞周围可见明显的空壳区；D组和E组神经细胞排列层次及细胞形态较清晰,少见固缩细胞,与C组相比脑组织损害程度明显减轻；而D组与E组相比,固缩细胞数量明显多于E组3.HIF-1α、VEGF免疫组化染色结果：A组和B组神经细胞数量形态分布正常,排列紧密有序,细胞核圆而大,染色浅,核仁清晰,偶见HIF-1α、VEGF阳性细胞表达,两者比较无显著性差异(P>0.05)；C组、D组和E组均可见HIF-1α、VEGF表达增高,与A组比较差异显著(P<0.05)；而D组和E组较.C组表达更高,有显著性差异(P<0.05)；D组与E组相比,后者阳性细胞表达率明显升高,差异有统计学意义(P<0.05)。4.HIF-1α、VEGF RT-PCR检测结果：A组和B组HIF-1α、VEGF mRNA表达极弱,两组相比无显著性差异(P>0.05)C组、D组和E组均出现HIF-1α、VEGF mRNA扩增条带,表达丰度明显高于A组,差异显著(P<0.05)；而D组和E组的表达较C组高,差异有统计学意义(P<0.05)；与D组相比,E组表达量更高,差异显著(P<0.05)。结论：1.采用改良的2VO法制作慢性脑缺血致学习记忆障碍大鼠模型,并通过Morris水迷宫从行为学和HE染色从组织学两方面予以评价,模型成功、可靠。2.头穴丛刺可提高慢性脑缺血致学习记忆障碍大鼠的学习记忆成绩,改善额叶皮层、海马组织病理学变化,减少神经元死亡,从而改善大鼠的学习记忆能力3.头穴丛刺可能通过上调HIF-1α和VEGF的表达,促进血管和神经元的再生,从而发挥脑保护功能。4.头穴丛刺可应用于慢性脑缺血致学习记忆障碍的防治,有望成为慢性脑缺血致学习记忆障碍防治新的治疗方法之一。更多还原

【Abstract】 Objective:To discuss the mechanism of scalp cluster-needling on chronic cerebral ischemia big rats, by observing the efficiency of learning and memory ability, HIF-1α and VEGF expression. The objective is to provide theoretical basis of treating chronic cerebral ischemia by scalp cluster-needling on clinical.Methods:72wistar big rats with normal learning and memory ability which were chosen by Morris water maze, were randomly divided into a control group (Group A), a sham operation group (Group B), and an operation group. To establish chronic cerebral ischemia models by modified double common carotid artery permanent ligation for operation group. Determined the big rats’ learning and memory ability by Morris water maze4weeks after operation, and divided successfully modeled rats into a model group(Group C), a Nimodipine (Group D) and a scalp cluster-needling group group (Group E). No treatment for Group A, B and C. Group D were given Nimodipine1mg/kg by gavage, once daily and4weeks treatment.Group E were given scalp cluster-needling, acupunctured Baihui and both points2mm aside, remained the needles for30min, once daily and4weeks treatment. Determined the big rats’learning and memory ability by Morris water maze8weeks after operation (4weeks after treatment), observed form changes of prefrontal cortex and hippocampus cells by light microscope, determined protein expression of prefrontal cortex, hippocampus HIF-1α、VEGF by Immunohistochemistry and expression of prefrontal cortex, hippocampus HIF-1α、VEGFmRNA were determined by RT-PCR.Results:1. Results of Morris water maze:Learning and memory ability were markedly decreased in Group C, D and E8weeks after operation, with significant differences compared with Group A (P<0.05); there were significant differences in Group D and E, compared with Group C (P<0.05), learning and memory ability in Group E had increased; there were no significant differences between Group D and E.2. Results of hematoxylin-eosin staining:There were no marked pathological changes in Group A and B, neuro cells in Group C were markedly decreased with cells sparsely arranged, seriously deformed, karyopyknosis, cytoplasm disappeared, uneven dye color marks and fuzzy neucleolus. Neuro cells in Group A and B were regularly arranged and formed, with little pycnosis pyramidal cell. Brain tissue damages were markedly reduced compared with Group C3. Results of immunohistochemical staining on HIF-1α、VEGF: Neuro cells in Group A and B were regular with a little positive cells express of HIF-1α、VEGF, there were no significant differences btween Group A and B (P>0.05); HIF-1α、VEGF expression in Group C and D were increased with big body, polygon and different stainings with significant differences compared with Group A and B (P<0.05); there were higher expressions in Group D. and E compared with Group C with significant differences (P<0.05). Positive expression rates in Group E were higher than in Group D with significant differences (p<0.05).4. Results of HIF-1α、VEGF determined by RT-PCR:Expression in Group A and B were inhibited with no significant differences compared within the two groups (P>0.05); there were amplified band of HIF-1α、VEGF amRNA in Group C, D and E, with higher expression abundance compared with Group A with significant differences (P<0.05); Expressions in Group D and E were higher compared with Group C with significant differences (P<0.05); expressions in Group E were higher than in group D with significant differences (P<0.05)Conclusion:1.Chronic cerebral ischemia models established by modified double common carotid artery permanent ligation, and commented by behaviouristics (Morris water maze) and histology (HE staining), were successful and reliable.2.Scalp cluster-needling can improve chronic cerebral ischemia big rats’learning and memory ability by improving pathological changes of prefrontal cortex and hippocampus tissue.3.Scalp cluster-needling can up-regulate expression of HIF-la and VEGF, promote regeneration of vesscular and neuron to protect the brain.4.Scalp cluster-needling can be used on pretreatment of learning and memorizing disorder caused by chronic cerebral ischemia, and is expected to be a new therapy.更多还原