不同磁刺激对家鸽脊髓、大脑Fos蛋白表达的影响

发布时间：2018-07-10 15:17

本文选题：磁刺激 + 脊髓　；参考：《山东师范大学》2014年硕士论文

【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（P0.05）；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（P0.05）。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（P0.01）；实验组三均显著多于组四（P0.01）。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。
[Abstract]:The biological effects of magnetic field have been widely paid attention to. Previous studies have been done on the biological effects in molecules, cells, tissues, systems, and the whole. The nervous system is particularly sensitive to magnetic stimulation. The influence of magnetic field on homing pigeons is mainly focused on the effects of the magnetic field on the geomagnetic navigation, and the researchers of the later years have Fos eggs. The effect of magnetic field changes on the activity of neurons in the retina of pigeons and the vestibule nucleus was studied, and no reports on other parts were not reported. This study was based on the screening of the best staining methods for displaying the spinal cord of the pigeon, the best staining method of the brain neurons and the general structural characteristics of the pigeon spinal cord. The effects of magnetic stimulation on the spinal cord of pigeon and the activity of brain neurons and the ways of its action are explored by using the immunohistochemical technique and the expression of Fos protein as the index. The experimental basis is provided for the study of the physiological mechanism of the pigeon navigation.
The experimental research in this paper is divided into four parts:
First, the morphology of the hippocampal neurons of pigeons in different staining methods was compared with neutral red, methylene blue, tar purple, HE, and Cajal 's staining methods to stain the frozen sections of normal adult pigeon brain, and compared the neurons in the hippocampus of the pigeons. The results suggested that the pigeons displayed by five kinds of dyeing methods were shown. The morphology of neurons in the hippocampus is different, with the best effect of tar violet staining. Two, the general structural characteristics of the spinal cord of the pigeon are on the normal adult pigeons (pigeons, doves, doves, pigeons) purchased by the market, the female and male are not restricted, the excessive anesthetic is executed. After the heart perfusion is fixed, the naked eye is divided into gross anatomy, the spinal transverse dissection is observed and the spinal cord is frozen. Section, Nissl staining, and light microscopy 3 groups (n=6/ group). The results showed that the spinal cord was composed of 38 segments, which were divided into 5 parts of the neck, chest, waist, sacrum and tail, and the posterior median groove of the lumbosacral spinal cord was second rhombic; the bottom of the fossa had glycogen; there were neurons in the white matter of the neck and the lumbosacral spinal cord formed in the neck and the lumbosacral spinal cord. The nucleus of the nerve.
Three, the effects of different intensity permanent magnetic stimulation on the expression of Fos protein in the spinal cord neurons of the pigeon were randomly divided into the control group (no treatment) and the experimental group. The experimental group was divided into five groups: the first group: the back magnet A (heavy 6.98g, magnetic field intensity 220mT), and the group two: the back weight a (corn grain, heavy 6.98g); Group Three: anaesthesia with A+ back magnet; group four: back heavy substance a+ anesthesia; Group Five: B of the back magnet (heavy 4.31g, magnetic field intensity 157mT); group six: the back magnet C (heavy 3.91g, magnetic intensity 192mT). After grouping treatment, the free activity 1H is made in group three, group four in 1min and 30min in the experiment 1min and 30min, in the subcutaneous and muscle of the negative parts and around (0.5cm). Injection of procaine hydrochloride (a local anesthetic to block the conduction of nerve impulses). After the experiment, the animals were sacrificed at the end of the experiment. The spinal cord was injected into the heart, the frozen section, the immunohistochemical technique treatment, the light microscope observation and photo taking, and the analysis of the Fos protein immunoreactive (Fos-IR) positive neurons in each group by the Imagepro-Plus6.0 image analysis. The single factor variance analysis of SPSS18.0 statistical software showed that the number of Fos-IR positive neurons in the experimental group was significantly more than that in the control group (P0.05) in all segments of the spinal cord, and the number of Fos-IR positive neurons in three of the experimental group was significantly more than that of the group four (P0.05) in each segment of the spinal cord (P0.05). The results showed that the negative number of 3.91g~6.98g was negative. Heavy stimulation, 157mT~220mT intensity of magnetic stimulation can induce the expression of Fos protein in the gray matter neurons of the pigeon; the hypodermic of the chest back (permanent magnet contact) and the injection of procaine (a nerve fiber conduction local blocker) can effectively block the expression of the Fos protein induced by the weight negative, and the expression of the magnetic induced Fos protein can be effectively blocked. The effect is not obvious. It is suggested that the magnetic stimulation of 157mT ~ 220mT intensity can induce the enhancement of the activity of the gray matter neurons in the spinal cord of the pigeon, which may be caused by the magnetic force line directly acting on the gray matter neurons of the spinal cord.
Four, the effect of different intensity permanent magnetic stimulation on the expression of Fos protein in the hippocampus and parahippocampal neurons of the pigeon, the experimental animal group, the immunohistochemistry and the observation and analysis method are the same as third parts, only the study target is the hippocampus and the parahippocampal region of the pigeon brain. The results of this study show the Fos-I of the hippocampus and the parahippocampal region of the pigeon. The number of R positive neurons in the experimental group was significantly more than that in the control group (P0.01), and the three in the experimental group was significantly more than that of the group four (P0.01). The results showed that the magnetic stimulation of 3.91g, 6.98g, 157mT and 220mT intensity could induce the hippocampus of the pigeon, and the expression of the deity Fos protein in the parahippocampal region of the hippocampus was enhanced; the chest back (permanent magnet contact area) Subcutaneous injection of procaine in muscle tissue can effectively reduce the expression of Fos protein induced by loading, but has no significant effect on the expression of magnetic induced Fos protein.
To sum up, the experimental study showed that the 157mT~220mT intensity magnetic stimulation on the back of the chest was given, and the weight stimulation of 3.91g~6.98g could induce the spinal cord of the pigeon and the expression of Fos protein in the hippocampal neurons of the brain. The peripheral injection of local nerve anesthetic was only effective in inhibiting the gray matter of the spinal cord, and the neuron Fos in the hippocampus and the parahippocampal region. The effect of protein expression on the induction of magnetic stimulation is not obvious. The results suggest that the neurons of the spinal gray matter may directly accept the action of the magnetic line of force to enhance their activity. The enhancement of the activity of the neurons in the hippocampus of the pigeon, the hippocampal region of the pigeon, may be related to the information modulation of the spinal cord. The molecular mechanism of activity strengthening needs to be further explored.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S836

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