本文選題：磁刺激 + 脊髓�。� 參考：《山東師范大學》2014年碩士論文

【摘要】：磁場的生物效應一直受到廣泛關注。前人對其在分子、細胞、組織、系統(tǒng)和整體等多個層面的生物效應都做了研究，其中神經(jīng)系統(tǒng)對磁刺激反應尤為敏感。關于磁場對家鴿的影響，前人主要聚焦于其對地磁導航的影響，晚近的研究者以Fos蛋白為指標，研究了磁場變化對家鴿視網(wǎng)膜、前庭神經(jīng)核中神經(jīng)元活動的影響，，還未見對其他部位的研究報道。本研究在篩選出顯示家鴿脊髓、大腦神經(jīng)元最佳的染色方法和對家鴿脊髓的一般結構特征進行驗證性實驗的基礎上，通過給予家鴿不同強度永磁刺激，利用免疫組織化學技術，以Fos蛋白的表達為指標，探索磁刺激對家鴿脊髓、大腦神經(jīng)元活動的影響及其作用途徑，為家鴿導航生理機制的研究提供實驗依據(jù)。 本文的實驗研究分為四部分： 一、不同染色方法顯示的家鴿海馬區(qū)神經(jīng)元形態(tài)比較采用中性紅、亞甲基藍、焦油紫、HE、和Cajal’s五種染色方法對正常成年家鴿大腦冰凍切片進行染色，對其顯示的家鴿海馬區(qū)神經(jīng)元形態(tài)進行比較。結果提示，五種染色方法所顯示的家鴿海馬區(qū)神經(jīng)元形態(tài)有差異，以焦油紫染色效果最佳。二、家鴿脊髓的一般結構特征對市場購買的正常成年家鴿（鴿形目，鳩鴿科、鴿屬），雌雄不拘，過量麻醉劑處死、心臟灌流固定后分為大體解剖肉眼觀察，脊柱橫切解剖鏡觀察，取脊髓冷凍切片、尼氏染色、光鏡觀察3組（n=6/組）。結果顯示，脊髓有38個節(jié)段組成，依其位置和結構可分為頸、胸、腰、骶、尾5部分；腰骶部脊髓的后正中溝擴大為第二菱形窩；該窩的底部有糖原體；在頸、腰骶部脊髓的白質中有神經(jīng)元胞體聚集形成的神經(jīng)核。 三、不同強度永磁刺激對家鴿脊髓神經(jīng)元Fos蛋白表達的影響對正常成年家鴿（體重300~400g），隨機分為對照組（不作處理）和實驗組。其中實驗組又分為五組，分別為：組一：背磁鐵A（重6.98g，磁場強度220mT）；組二：背重物a（玉米粒，重6.98g）；組三：背磁鐵A+麻醉；組四：背重物a+麻醉；組五：背磁鐵B（重4.31g，磁場強度157mT）；組六：背磁鐵C（重3.91g，磁場強度192mT）。按分組處理后，使其自由活動1h，其中組三、組四在實驗1min時和30min時，在背負部位及其周圍（0.5cm）的皮下和肌肉中注射鹽酸普魯卡因（一種阻斷神經(jīng)沖動傳導的局部麻醉劑）。實驗結束之后過量麻醉犧牲動物，心臟灌流取脊髓，冰凍切片，免疫組織化學技術處理，光鏡觀察拍照，利用Imagepro-Plus6.0圖像分析軟件對各組Fos蛋白免疫反應（Fos-IR）陽性神經(jīng)元進行觀察計數(shù)。經(jīng)SPSS18.0統(tǒng)計軟件單因素方差分析，結果顯示：實驗組的Fos-IR陽性神經(jīng)元數(shù)量在脊髓各個節(jié)段中均顯著多于對照組（P0.05）；實驗組三的Fos-IR陽性神經(jīng)元數(shù)量在脊髓各個節(jié)段中均顯著多于組四（P0.05）。結果表明，3.91g~6.98g的負重刺激、157mT~220mT強度的磁刺激均可誘發(fā)家鴿脊髓灰質神經(jīng)元Fos蛋白的表達加強；胸背部（永磁鐵接觸部位）皮下、肌組織注射的普魯卡因（一種神經(jīng)纖維傳導局部阻斷劑）可有效阻斷負重誘導的Fos蛋白表達，而對磁誘導的Fos蛋白表達的影響卻不明顯。提示，157mT～220mT強度的磁刺激可誘發(fā)家鴿脊髓灰質神經(jīng)元活動加強；這種刺激作用可能是磁力線直接作用于脊髓灰質神經(jīng)元所致。 四、不同強度永磁刺激對家鴿大腦海馬區(qū)、海馬旁區(qū)神經(jīng)元Fos蛋白表達的影響實驗動物分組、免疫組化處理和觀察分析方法同第三部分，只是研究目標為家鴿大腦的海馬區(qū)和海馬旁區(qū)。本研究結果顯示：家鴿大腦海馬區(qū)、海馬旁區(qū)的Fos-IR陽性神經(jīng)元數(shù)量，在實驗組均分別顯著多于對照組（P0.01）；實驗組三均顯著多于組四（P0.01）。結果表明，3.91g、6.98g的負重刺激、157mT、220mT強度的磁刺激均可誘發(fā)家鴿大腦海馬區(qū)、海馬旁區(qū)神經(jīng)元Fos蛋白的表達加強；胸背部（永磁鐵接觸部位）皮下、肌組織注射的普魯卡因可有效減少負重誘導的Fos蛋白表達，而對磁誘導的Fos蛋白表達的影響卻不顯著。 綜上所述，本實驗研究證明，胸背部給予157mT~220mT強度磁刺激、3.91g~6.98g的負重刺激均可以誘導家鴿脊髓、大腦的海馬部位神經(jīng)元Fos蛋白表達加強。外周注射局部神經(jīng)麻醉劑，僅能有效抑制負重誘導的脊髓灰質、海馬區(qū)和海馬旁區(qū)神經(jīng)元Fos蛋白表達，對磁刺激誘導的作用不明顯。結果提示，脊髓灰質神經(jīng)元可能直接接受磁力線的作用，使其活動加強；胸部背磁誘發(fā)的家鴿大腦海馬區(qū)、海馬旁區(qū)的神經(jīng)元的活動加強可能與脊髓上傳的信息調制有關。關于磁力線誘導神經(jīng)元活動加強的分子機制還需進一步探究。
[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.
【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：S836

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