【作者】 李宁；

【导师】 崔希云；

【作者基本信息】 山东师范大学 ， 动物学， 2014， 硕士

【摘要】 磁场的生物效应一直受到广泛关注。前人对其在分子、细胞、组织、系统和整体等多个层面的生物效应都做了研究，其中神经系统对磁刺激反应尤为敏感。关于磁场对家鸽的影响，前人主要聚焦于其对地磁导航的影响，晚近的研究者以Fos蛋白为指标，研究了磁场变化对家鸽视网膜、前庭神经核中神经元活动的影响，还未见对其他部位的研究报道。本研究在筛选出显示家鸽脊髓、大脑神经元最佳的染色方法和对家鸽脊髓的一般结构特征进行验证性实验的基础上，通过给予家鸽不同强度永磁刺激，利用免疫组织化学技术，以Fos蛋白的表达为指标，探索磁刺激对家鸽脊髓、大脑神经元活动的影响及其作用途径，为家鸽导航生理机制的研究提供实验依据。本文的实验研究分为四部分：一、不同染色方法显示的家鸽海马区神经元形态比较采用中性红、亚甲基蓝、焦油紫、HE、和Cajal’s五种染色方法对正常成年家鸽大脑冰冻切片进行染色，对其显示的家鸽海马区神经元形态进行比较。结果提示，五种染色方法所显示的家鸽海马区神经元形态有差异，以焦油紫染色效果最佳。二、家鸽脊髓的一般结构特征对市场购买的正常成年家鸽（鸽形目，鸠鸽科、鸽属），雌雄不拘，过量麻醉剂处死、心脏灌流固定后分为大体解剖肉眼观察，脊柱横切解剖镜观察，取脊髓冷冻切片、尼氏染色、光镜观察3组（n=6/组）。结果显示，脊髓有38个节段组成，依其位置和结构可分为颈、胸、腰、骶、尾5部分；腰骶部脊髓的后正中沟扩大为第二菱形窝；该窝的底部有糖原体；在颈、腰骶部脊髓的白质中有神经元胞体聚集形成的神经核。三、不同强度永磁刺激对家鸽脊髓神经元Fos蛋白表达的影响对正常成年家鸽（体重300~400g），随机分为对照组（不作处理）和实验组。其中实验组又分为五组，分别为：组一：背磁铁A（重6.98g，磁场强度220mT）；组二：背重物a（玉米粒，重6.98g）；组三：背磁铁A+麻醉；组四：背重物a+麻醉；组五：背磁铁B（重4.31g，磁场强度157mT）；组六：背磁铁C（重3.91g，磁场强度192mT）。按分组处理后，使其自由活动1h，其中组三、组四在实验1min时和30min时，在背负部位及其周围（0.5cm）的皮下和肌肉中注射盐酸普鲁卡因（一种阻断神经冲动传导的局部麻醉剂）。实验结束之后过量麻醉牺牲动物，心脏灌流取脊髓，冰冻切片，免疫组织化学技术处理，光镜观察拍照，利用Imagepro-Plus6.0图像分析软件对各组Fos蛋白免疫反应（Fos-IR）阳性神经元进行观察计数。经SPSS18.0统计软件单因素方差分析，结果显示：实验组的Fos-IR阳性神经元数量在脊髓各个节段中均显著多于对照组（P<0.05）；实验组三的Fos-IR阳性神经元数量在脊髓各个节段中均显著多于组四（P<0.05）。结果表明，3.91g~6.98g的负重刺激、157mT~220mT强度的磁刺激均可诱发家鸽脊髓灰质神经元Fos蛋白的表达加强；胸背部（永磁铁接触部位）皮下、肌组织注射的普鲁卡因（一种神经纤维传导局部阻断剂）可有效阻断负重诱导的Fos蛋白表达，而对磁诱导的Fos蛋白表达的影响却不明显。提示，157mT～220mT强度的磁刺激可诱发家鸽脊髓灰质神经元活动加强；这种刺激作用可能是磁力线直接作用于脊髓灰质神经元所致。四、不同强度永磁刺激对家鸽大脑海马区、海马旁区神经元Fos蛋白表达的影响实验动物分组、免疫组化处理和观察分析方法同第三部分，只是研究目标为家鸽大脑的海马区和海马旁区。本研究结果显示：家鸽大脑海马区、海马旁区的Fos-IR阳性神经元数量，在实验组均分别显著多于对照组（P<0.01）；实验组三均显著多于组四（P<0.01）。结果表明，3.91g、6.98g的负重刺激、157mT、220mT强度的磁刺激均可诱发家鸽大脑海马区、海马旁区神经元Fos蛋白的表达加强；胸背部（永磁铁接触部位）皮下、肌组织注射的普鲁卡因可有效减少负重诱导的Fos蛋白表达，而对磁诱导的Fos蛋白表达的影响却不显著。综上所述，本实验研究证明，胸背部给予157mT~220mT强度磁刺激、3.91g~6.98g的负重刺激均可以诱导家鸽脊髓、大脑的海马部位神经元Fos蛋白表达加强。外周注射局部神经麻醉剂，仅能有效抑制负重诱导的脊髓灰质、海马区和海马旁区神经元Fos蛋白表达，对磁刺激诱导的作用不明显。结果提示，脊髓灰质神经元可能直接接受磁力线的作用，使其活动加强；胸部背磁诱发的家鸽大脑海马区、海马旁区的神经元的活动加强可能与脊髓上传的信息调制有关。关于磁力线诱导神经元活动加强的分子机制还需进一步探究。更多还原

【Abstract】 The biological effects of magnetic field has been widespread concerned. It had been studiedin multiple levels of molecules, cells, tissues, and individuals. The nervous system is especiallysensitive to magnetic stimulation reaction. On the influence of magnetic field on the pigeon,people mainly focused on its effect on the geomagnetic navigation. Researchers recently usedFos protein as index to explore the effects of magnetic field on the neuronal activity.But theresearches were only focused on the pigeon’s retina and vestibular nucleus.It had not beenreported in other parts of pigeon’s.The first part of this study is to screen out the best stainingmethod of neurons in spinal cord and brain.Then,the verified experiment on the general structureof the pigeon’s spinal cord was done.The main part of this study is to explore the effect ofmagnetic stimulation on neuronal activity of the spinal cord and brain by giving differentpermanent magnetic stimulation to pigeons, using molecular immunohistochemical techniquewhich used Fos protein as an index. The purpose of this research is to provide experimental basisfor the study of physiological mechanism of pigeon navigation.The research was divided into four parts:1.The comparison of different method to display the morphology of hippocampalneurons: The frozen sections from pigeons’ brains were stained with neutral red,methyleneblue,cresyl violet,haematoxylin-eosin(HE), and Cajal’s staining method respectively,thencompared the morphology of hippocampal neurons.Results indicate that there are differencesamong the five kinds of staining methods and the cresyl violet staining method is the best one ofthem.2. The general structure of the spinal cord: The pigeons(Columbiformes, Columbidae,Columba,n=18) buying from market were killed by excessive anesthetic and perfused forfixation through heart. They were divided into the group of gross anatomy macroscopicobservation, the group of the spinal transection observed by binocular dissecting microscopeand the group of the spinal cord made into frozen transection, stained with cresyl violet, observed by optical microscope.The result displays that there are38segments of the spinal cord.According to these locations and structures,they can be divided into five parts:the cervical,thoracic, lumbar, sacral and tail. The dorsal median suleus of the lumbosacral spinal cord isbroadened, It is called second rhomboid fossa., and has the glycogen body at the bottom. Thecell bodies of the neurons in white matter of the cervical and lumbosacral spinal cord gather intonerve nucleus.3. The effects of different intensity permanent magnetic stimulation on the expressionof Fos protein in neurons of the spinal cord:The normal adult pigeons (300~400g bodyweight)were randomly divided into control group (no treatment) and experimental group. Theexperimental group was divided into five groups with5pigeons in each group, respectively:group1: the members were carried magnet A (weight6.98g, magnetic field strength of220mT)on their back; group two: the pigeons were carried a bag of corn on their back(weight6.98g); Group Three: the pigeons were carried magnet A and be anesthetized; group four: themembers were carried a bag of corn(weight6.98g) and be anesthetized; group five:they werecarried magnet B (weight4.31g, the magnetic field strength of157mT)on their back; group six:they ere carried magnet C (weight3.91g, the magnetic field strength of192mT)on their back.Then, all animals were allowed to survive for1hour in the waking state and could behavior atease.The group3and4were injectioned with anesthetic(procaine hydrochloride) twice(1minand30min).Then the animals were killed by overdose anesthesia and perfused for fixationthrough heart.The spinal cords of them were made into frozen sections and then were handledwith Immunohistochemical technique.We observed and photographed using lightmicroscope,couted the number of Fos immunoreactive neurons in each group. Results showedThe number of Fos protein immunoreactive (Fos-IR)positive neurons in the spinal cord of eachsegment which were in the experimental group were significantly more than those of the controlgroup (P<0.05); the number of the Fos-IR positive neurons in the spinal cord of each segment inthe experimental group three were significantly more than those of the group four (P<0.05).Itindicates that the weight-bearing stimulation(3.91~6.98g)and magnetic stimulation(157mT~220mT) both can strengthen the expression of Fos protein in spinal cord neurons; Chest and back(permanent magnet contact position) subcutaneous, muscle injection of Procaine(a localanesthetic) can effectively block the expression of Fos protein which was induced by load, while the expression of Fos protein induced by magnetic stimulation can’t be blocked.Therefore,themagnetic stimulation of157mT~220mT can strengthen the neuronal activity; the stimulationmay be a direct effect on neurons induced by magnetic field.4. The effects of different intensity permanent magnetic stimulation on the expressionof Fos protein in neurons of the hippocampus and area parahippocampalis:The methods ofanimal grouping, immunohistochemistry and observation were all the same as section3,exceptthat the experimental object was pigeon’s brain. Results indicated that the number of the Fos-IRpositive neurons in the hippocampus(HP) and area parahippocampalis(APH) which were in theexperimental group were significantly more than those of the control group (P<0.05); there weremore the number of the Fos-IR positive neurons in the experimental group three than those of thegroup four (P<0.05).It indicates that the weight-bearing stimulation(3.91~6.98g)and magneticstimulation(157mT~220mT) both can strengthen the expression of Fos protein in HP and APH;Chest and back (permanent magnet contact position) subcutaneous, muscle injection of Procainecan effectively reduce the expression of Fos protein which was induced by load, while is lesseffect is of the expression of Fos protein induced by magnetic stimulation.In summary, thisexperimental study demonstrated that, the weight-bearing stimulation (3.91~6.98g)and magneticstimulation(157mT~220mT) both can enhanced the expression of Fos protein in the spinal cord,HP and APH. Peripheral nerve local anaesthesia only can effectively inhibit the neuronal activityinduced by loading, not obvious on magnetic stimulation induced effect. The results suggest thatthe magnetic lines of force maybe directly act on the gray matter of the spinal cord neurons toreinforce its activity; the enhance of activity in the HP and APH may be associated with theinformation uploaded by the spinal cord. The molecular mechanism which the magnetic field caninduce the neuronal activity should be further explored.更多还原