電針對擬血管性癡呆小鼠海馬單胺類神經(jīng)遞質(zhì)和氧自由基影響的研究
發(fā)布時(shí)間：2018-07-14 18:18
【摘要】：目的：觀察電針對腦缺血再灌注擬血管性癡呆（Vascular Dementia，VD）小鼠的行為學(xué)、腦組織海馬CA1區(qū)病理形態(tài)學(xué)、單胺類神經(jīng)遞質(zhì)與氧自由基代謝的影響，并進(jìn)行不同干預(yù)時(shí)間的比較，探討電針治療VD的效應(yīng)及作用機(jī)制。 方法： 1模型復(fù)制 將60只昆明小鼠隨機(jī)分為第1批和第2批，每批各設(shè)假手術(shù)組、模型組、電針組，每組各10只。參照文獻(xiàn)，，各批電針組和模型組小鼠采用雙側(cè)頸總動(dòng)脈反復(fù)缺血再灌注的方法復(fù)制VD模型，術(shù)前12h禁食水，用10%水合氯醛0.35g/kg腹腔注射，在麻醉狀態(tài)下，常規(guī)消毒，行頸正中切口，將雙側(cè)頸總動(dòng)脈（Common CarotidArtery，CCA）逐層鈍性分離，穿線備用，拉緊絲扣阻斷血流20min，同時(shí)剪斷距尾尖1cm處，放血大約0.3ml，采用熱凝止血。將絲扣松開使血液灌注10min后，再次阻斷血流20min，然后觀察30min，縫合傷口。假手術(shù)組小鼠只分離雙側(cè)頸總動(dòng)脈，穿線但不結(jié)扎，不進(jìn)行尾部放血，觀察時(shí)間與電針組和模型組相同。造模后每日肌注青霉素0.2萬U，連續(xù)3d。為了防止低溫對缺血缺氧后腦損傷的保護(hù)作用，造模過程中維持動(dòng)物肛溫（36±0.5）℃。 2治療方法 第1批于造模后蘇醒當(dāng)日即開始進(jìn)行治療，第2批于造模第3d開始進(jìn)行治療。各批電針組選取：大椎、百會、膈俞（雙）、后三里（雙），用0.35mm×13mm毫針刺入，連接韓氏電針儀，施以疏密波，頻率為2/80Hz，強(qiáng)度以小鼠肢體輕輕顫動(dòng)，不嘶叫掙扎為度，每次針刺10min，1次/d，連續(xù)15d。各批假手術(shù)組和模型組均采用相同體位固定10min。3檢測方法 于治療結(jié)束后，采用跳臺實(shí)驗(yàn)對各組小鼠進(jìn)行行為學(xué)檢測，觀察學(xué)習(xí)記憶能力。行為學(xué)檢測結(jié)束后，斷頭處死小鼠，在冰盤上快速剖取腦組織，棄去嗅球和小腦，將左側(cè)腦組織固定于4%多聚甲醛溶液，用石蠟包埋，冠狀切片，常規(guī)HE染色，光鏡下觀察各組小鼠海馬區(qū)的病理形態(tài)學(xué)改變。取右側(cè)海馬組織，于電子天平上稱重，加入預(yù)冷的生理鹽水，用電動(dòng)勻漿器研磨，以鏡檢無完整細(xì)胞為標(biāo)準(zhǔn)，制成10%的海馬組織勻漿，4℃，3600r/min，離心5min，提取上清液備用。海馬單胺類神經(jīng)遞質(zhì)NE、DA和5-HT含量測定采用熒光分光光度法；海馬SOD活性測定采用黃嘌呤氧化酶法；海馬MDA含量測定采用硫代巴比妥酸（TBA）比色分析法。 結(jié)果：1行為學(xué)檢測 經(jīng)跳臺實(shí)驗(yàn)檢測，兩批模型組小鼠的反應(yīng)時(shí)間均延長，潛伏期均縮短，錯(cuò)誤次數(shù)均增加，與同批假手術(shù)組相比，差異有統(tǒng)計(jì)學(xué)意義(P0.01)；兩批電針組小鼠反應(yīng)時(shí)間均縮短，潛伏期均延長，錯(cuò)誤次數(shù)均明顯降低，與同批模型組相比，有顯著差異（P0.01）；兩批電針組之間比較，學(xué)習(xí)成績無明顯差異（P0.05），第2批電針組記憶成績優(yōu)于第1批電針組（P0.05或P0.01）。 2海馬CA1區(qū)病理形態(tài)學(xué)檢測 在光鏡下觀察海馬CA1區(qū)錐體細(xì)胞顯示：兩批假手術(shù)組海馬CA1區(qū)錐體細(xì)胞均排列規(guī)整緊密，細(xì)胞呈圓形，細(xì)胞核大而圓，細(xì)胞核仁清晰，核質(zhì)均勻。兩批模型組CA1區(qū)錐體細(xì)胞排列紊亂、疏松，細(xì)胞脫失嚴(yán)重，部分細(xì)胞結(jié)構(gòu)不完整，形態(tài)欠規(guī)則，胞漿與胞核界限模糊，胞核體積固縮變小，核仁呈三角型或多角型，核質(zhì)不均勻，視野中可見大量壞死細(xì)胞。第1批電針組海馬CA1區(qū)錐體細(xì)胞少量脫失，細(xì)胞結(jié)構(gòu)比較清晰，細(xì)胞排列比較緊密，可見少數(shù)細(xì)胞核固縮為三角形或多邊形；第2批電針組海馬CA1區(qū)錐體細(xì)胞僅有少量脫失，細(xì)胞結(jié)構(gòu)比較清晰，細(xì)胞排列比較緊密，細(xì)胞核大而圓，核仁較清晰，有極少數(shù)細(xì)胞核固縮為三角形或多邊形。 3海馬單胺類神經(jīng)遞質(zhì)的檢測 兩批模型組小鼠海馬NE、DA、5-HT含量均明顯降低，與同批假手術(shù)組比較，差異有統(tǒng)計(jì)學(xué)意義（P0.01）；兩批電針組NE、DA、5-HT含量均明顯升高，與同批模型組比較有顯著性差異（P0.01）；且兩批電針組之間比較有差異，第2批電針組DA、5-HT含量高于第1批電針組（P0.05或P0.01）。 4海馬氧自由基的檢測 與假手術(shù)組比較，各批模型組小鼠海馬SOD活性均明顯下降，MDA含量均顯著升高，差異具有統(tǒng)計(jì)學(xué)意義（P0.01）；與模型組比較，各批電針組海馬SOD活性均明顯增高，MDA含量均顯著降低，有顯著性差異（P0.01），且兩批電針組之間比較，第2批電針組SOD活性高于第1批電針組(P0.05)。 結(jié)論：采用電針刺激“大椎”、“百會”、“膈俞”、“后三里”穴可以改善因缺血再灌注導(dǎo)致VD小鼠的學(xué)習(xí)記憶能力；其作用機(jī)制可能與電針提高VD模型小鼠海馬單胺類神經(jīng)遞質(zhì)NE、DA、5-HT含量，增強(qiáng)SOD活性，降低MDA含量，改善缺血損傷后海馬單胺類神經(jīng)遞質(zhì)代謝紊亂，提高機(jī)體清除自由基的能力，減輕自由基對神經(jīng)系統(tǒng)的損傷有關(guān)。并初步發(fā)現(xiàn)，電針干預(yù)時(shí)間不同，其療效存在一定差異。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R245
本文編號：2122557
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R245
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