电针对拟血管性痴呆小鼠海马单胺类神经递质和氧自由基影响的研究

发布时间：2018-07-14 18:18

【摘要】：目的：观察电针对脑缺血再灌注拟血管性痴呆（Vascular Dementia，VD）小鼠的行为学、脑组织海马CA1区病理形态学、单胺类神经递质与氧自由基代谢的影响，并进行不同干预时间的比较，探讨电针治疗VD的效应及作用机制。方法： 1模型复制将60只昆明小鼠随机分为第1批和第2批，每批各设假手术组、模型组、电针组，每组各10只。参照文献，，各批电针组和模型组小鼠采用双侧颈总动脉反复缺血再灌注的方法复制VD模型，术前12h禁食水，用10%水合氯醛0.35g/kg腹腔注射，在麻醉状态下，常规消毒，行颈正中切口，将双侧颈总动脉（Common CarotidArtery，CCA）逐层钝性分离，穿线备用，拉紧丝扣阻断血流20min，同时剪断距尾尖1cm处，放血大约0.3ml，采用热凝止血。将丝扣松开使血液灌注10min后，再次阻断血流20min，然后观察30min，缝合伤口。假手术组小鼠只分离双侧颈总动脉，穿线但不结扎，不进行尾部放血，观察时间与电针组和模型组相同。造模后每日肌注青霉素0.2万U，连续3d。为了防止低温对缺血缺氧后脑损伤的保护作用，造模过程中维持动物肛温（36±0.5）℃。 2治疗方法第1批于造模后苏醒当日即开始进行治疗，第2批于造模第3d开始进行治疗。各批电针组选取：大椎、百会、膈俞（双）、后三里（双），用0.35mm×13mm毫针刺入，连接韩氏电针仪，施以疏密波，频率为2/80Hz，强度以小鼠肢体轻轻颤动，不嘶叫挣扎为度，每次针刺10min，1次/d，连续15d。各批假手术组和模型组均采用相同体位固定10min。3检测方法于治疗结束后，采用跳台实验对各组小鼠进行行为学检测，观察学习记忆能力。行为学检测结束后，断头处死小鼠，在冰盘上快速剖取脑组织，弃去嗅球和小脑，将左侧脑组织固定于4%多聚甲醛溶液，用石蜡包埋，冠状切片，常规HE染色，光镜下观察各组小鼠海马区的病理形态学改变。取右侧海马组织，于电子天平上称重，加入预冷的生理盐水，用电动匀浆器研磨，以镜检无完整细胞为标准，制成10%的海马组织匀浆，4℃，3600r/min，离心5min，提取上清液备用。海马单胺类神经递质NE、DA和5-HT含量测定采用荧光分光光度法；海马SOD活性测定采用黄嘌呤氧化酶法；海马MDA含量测定采用硫代巴比妥酸（TBA）比色分析法。结果：1行为学检测经跳台实验检测，两批模型组小鼠的反应时间均延长，潜伏期均缩短，错误次数均增加，与同批假手术组相比，差异有统计学意义(P0.01)；两批电针组小鼠反应时间均缩短，潜伏期均延长，错误次数均明显降低，与同批模型组相比，有显著差异（P0.01）；两批电针组之间比较，学习成绩无明显差异（P0.05），第2批电针组记忆成绩优于第1批电针组（P0.05或P0.01）。 2海马CA1区病理形态学检测在光镜下观察海马CA1区锥体细胞显示：两批假手术组海马CA1区锥体细胞均排列规整紧密，细胞呈圆形，细胞核大而圆，细胞核仁清晰，核质均匀。两批模型组CA1区锥体细胞排列紊乱、疏松，细胞脱失严重，部分细胞结构不完整，形态欠规则，胞浆与胞核界限模糊，胞核体积固缩变小，核仁呈三角型或多角型，核质不均匀，视野中可见大量坏死细胞。第1批电针组海马CA1区锥体细胞少量脱失，细胞结构比较清晰，细胞排列比较紧密，可见少数细胞核固缩为三角形或多边形；第2批电针组海马CA1区锥体细胞仅有少量脱失，细胞结构比较清晰，细胞排列比较紧密，细胞核大而圆，核仁较清晰，有极少数细胞核固缩为三角形或多边形。 3海马单胺类神经递质的检测两批模型组小鼠海马NE、DA、5-HT含量均明显降低，与同批假手术组比较，差异有统计学意义（P0.01）；两批电针组NE、DA、5-HT含量均明显升高，与同批模型组比较有显著性差异（P0.01）；且两批电针组之间比较有差异，第2批电针组DA、5-HT含量高于第1批电针组（P0.05或P0.01）。 4海马氧自由基的检测与假手术组比较，各批模型组小鼠海马SOD活性均明显下降，MDA含量均显著升高，差异具有统计学意义（P0.01）；与模型组比较，各批电针组海马SOD活性均明显增高，MDA含量均显著降低，有显著性差异（P0.01），且两批电针组之间比较，第2批电针组SOD活性高于第1批电针组(P0.05)。结论：采用电针刺激“大椎”、“百会”、“膈俞”、“后三里”穴可以改善因缺血再灌注导致VD小鼠的学习记忆能力；其作用机制可能与电针提高VD模型小鼠海马单胺类神经递质NE、DA、5-HT含量，增强SOD活性，降低MDA含量，改善缺血损伤后海马单胺类神经递质代谢紊乱，提高机体清除自由基的能力，减轻自由基对神经系统的损伤有关。并初步发现，电针干预时间不同，其疗效存在一定差异。
[Abstract]:Objective: To observe the effect of Electroacupuncture on the behavior of Vascular Dementia (VD), the pathomorphology of the hippocampus CA1 region, the influence of monoamine neurotransmitters and oxygen free radical metabolism in the hippocampus CA1 region of the brain tissue, and to compare the different intervention time, and to explore the effect and mechanism of electroacupuncture in the treatment of VD.
Method:
1 model replication
60 Kunming mice were randomly divided into first batches and second batches. Each group had sham operation group, model group, electroacupuncture group and 10 rats in each group. The VD model was replicated by repeated ischemia and reperfusion of bilateral common carotid artery in each group of electroacupuncture groups and model groups. The pre operation 12h fasting water was injected with 10% chloral chloral 0.35g/kg in the anesthetic state. Under the routine disinfection, the cervical median incision was performed, the bilateral common carotid artery (Common CarotidArtery, CCA) was separated by blunt layer, the thread was put on the line, the thread was pulled to block the blood flow 20min, and the blood flow was cut from 1cm at the tail tip of the tail, and the bleeding was about 0.3ml. The blood liquid was poured into 10min, and the blood flow 20min was blocked again, and then 30min and suture were observed. In the sham operation group, the mice in the sham operation group were only separated from the bilateral common carotid artery, wearing line but not ligation and no tail bleeding. The time of observation was the same as that in the electroacupuncture group and the model group. After making the model, 2 thousand U of penicillin was injected daily in order to prevent the protective effect of hypothermia on the brain injury after ischemia and hypoxia, and the temperature of the animal anus was maintained (36 + 0.5) during the process of modeling.
2 treatment methods
The first batches of the first groups began to be treated on the day of awakening, and the second batch began to be treated at the model 3D. Each group of electroacupuncture groups was selected: Da Zhui, Baihui, phrenic Shu (double), and then three li (double), with a 0.35mm x 13mm needle, connected with the Han's electroacupuncture instrument, the density wave, the frequency of 2/80Hz, the strength of the mice's limbs, and the degree of no struggle. For the second time acupuncture 10min, 1 /d, continuous 15d. each false leave, the operation group and the model group were all fixed position 10min.3 test method.
After the end of the treatment, the mice were tested and the learning and memory ability was observed by the platform of the platform. After the end of the behavior test, the mice were killed, the brain tissue was quickly dissected, the olfactory bulb and the cerebellum were discarded, the left brain tissue was fixed to 4% poly Formaldehyde Solution, paraffin embedded, coronal section, routine HE staining, and light microscope. Observe the pathological morphological changes in the hippocampus of each group. Take the right hippocampal tissue, weigh it on the electronic balance, add the pre cooled physiological saline, use the electric homogenizer to grind it, and make 10% of the hippocampal homogenate, 4, 3600r/min, 5min, and the supernatant. The hippocampus monoamine neurotransmitter NE, D Fluorescence spectrophotometry was used to determine the content of A and 5-HT, and xanthine oxidase method was used for the determination of SOD activity in the hippocampus, and thiobarbituric acid (TBA) colorimetric assay was used for the determination of MDA content in the hippocampus.
Results: 1 behavioral test
The reaction time of the two batch model group was prolonged, the incubation period was shortened, the number of errors increased, and the difference was statistically significant (P0.01) compared with the same batch operation group. The response time of the two groups of electroacupuncture groups was shortened, the latency was prolonged, and the number of errors decreased significantly. Compared with the same batch model group, there was a significant difference. There was no significant difference between the two groups of electroacupuncture groups (P0.05), and the memory scores of the second batch electroacupuncture group were better than those of the first group of electro acupuncture group (P0.05 or P0.01).
Pathomorphological detection of 2 hippocampal CA1 region
Under the light microscope, the pyramidal cells in the hippocampal CA1 region showed that the pyramidal cells in the hippocampal CA1 area of two batches of the hippocampus were arranged regularly, the cells were round, the nuclei were large and round, the nucleolus of the cells were clear and the nuclear quality was uniform. The two batches of CA1 pyramidal cells in the model group were arranged in disorder, loosely, and the cells were lost seriously, the structure of some cells was incomplete and the morphologic under rules, The nucleus of the cytoplasm and nucleus is blurred, the nucleus volume contraction is smaller, the nucleolus is triangular or multi angle type, the nucleolus is not uniform, and a large number of necrotic cells are visible in the field of vision. The first batches of hippocampal CA1 pyramidal cells are a small amount of loss of cell structure, the cell structure is clear, and the cells are arranged closely. A few nuclei can be condensed into triangles or polygons; the second batch of nuclei can be found to be triangles or polygons. The pyramidal cells in the hippocampal CA1 region of the electroacupuncture group were only a small amount of loss of loss. The cell structure was clear, the cells were arranged closely, the nuclei were large and round, the nucleolus were clearer, and a few nuclei were condensed into triangles or polygons.
3 detection of monoamine neurotransmitters in the hippocampus
The contents of NE, DA and 5-HT in the hippocampus of the two groups of mice were significantly lower than those in the same batch group (P0.01). The contents of NE, DA and 5-HT in the two groups of electroacupuncture groups were significantly higher than those in the same batch model group (P0.01), and the two batches of electroacupuncture groups were different, second batches of electroacupuncture groups were DA and 5-HT content was higher than first Group of electroacupuncture (P0.05 or P0.01).
4 detection of oxygen free radicals in the hippocampus
Compared with the sham operation group, the activity of SOD in the hippocampus of the mice was significantly decreased and the content of MDA increased significantly (P0.01). Compared with the model group, the activity of SOD in the hippocampus was significantly higher in each group of electroacupuncture groups, and the content of MDA decreased significantly (P0.01), and the two batches of electroacupuncture groups were compared, and the second groups of electroacupuncture groups were compared. The activity of SOD was higher than that in the first group of electroacupuncture groups (P0.05).
Conclusion: electroacupuncture stimulation of "Da Zhui", "Baihui", "phrenic Shu" and "Three Li" can improve the learning and memory ability of VD mice caused by ischemia reperfusion. The mechanism of action may improve the content of NE, DA, 5-HT, SOD activity, SOD activity, MDA content and improve the ischemic injury after ischemia reperfusion. The metabolic disorder of monoamine neurotransmitters in the hippocampus improves the ability of the body to clear free radicals and reduces the damage of free radicals to the nervous system. It is found that there are some differences in the therapeutic effect of electroacupuncture at different time.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R245

【参考文献】