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標題: | 正中神經損傷對楔狀神經核及背根神經節中甘胺酸、伽傌-胺基丁酸及其受體的影響 The Effects of Median Nerve Injury on Glycine, GABA and Corresponding Receptor Expressions in the Cuneate Nucleus and Dorsal Root Ganglion |
作者: | Seu-Hwa Chen 陳淑華 |
指導教授: | 呂俊宏 |
關鍵字: | 螢光金,楔狀丘腦投射神經元,甘胺酸,普樂芬,神經胜肽,Y,c-Fos蛋白,步態行為分析, Fluorogold,Cuneothalamic projection neuron,Glycine,baclofen,neuropeptide Y,c-Fos,CatWalk, |
出版年 : | 2012 |
學位: | 博士 |
摘要: | 一般正常狀況下,楔狀神經核僅接收頸部、前肢及軀幹上部的初級傳入神經所傳遞的觸覺、震動覺及本體感覺等非傷害性訊息,爾後將訊息再向上投射至丘腦腹基底核。已知正中神經截斷會引起損傷側楔狀神經核內出現神經胜肽Y (neuropeptide Y, NPY)及c-Fos免疫反應,但缺乏探討楔狀神經核的抑制性系統在正中神經損傷後所產生的改變,以及其在神經胜肽Y和c-Fos免疫表現、神經病變疼痛產生方面扮演的角色之相關研究。而甘胺酸及伽傌-胺基丁酸為楔狀神經核內的兩個重要抑制性神經傳遞物質,主要以突觸前或突觸後抑制作用參與訊息的調控。因此本研究期望提供正中神經截斷後,甘胺酸和伽傌-胺基丁酸神經元及其受體數量上的變化,以及它們與神經病變疼痛行為表現之間的關連性。我們將利用免疫組織化學法來觀察正中神經截斷後,甘胺酸、伽傌-胺基丁酸、甘胺酸受體、伽傌-胺基丁酸A和B受體免疫反應神經元在楔狀神經核和背根神經節內的變化。並藉由神經胜肽Y和c-Fos蛋白表現的變化情形,作為評估生理食鹽水 (控制組)、甘胺酸或伽傌-胺基丁酸B受體促效劑 (甘胺酸, glycine或普樂芬, baclofen),以及拮抗劑 (番木虌鹼, strychnine或法克羅芬, phaclofen)處理後對於神經病變疼痛訊息傳遞的影響,進一步透過CatWalk步態分析方式確認受體促效劑是否可以緩解大白鼠正中神經引發之神經病變疼痛症狀。
首先證實在楔狀神經核內甘胺酸受體免疫反應神經元約有半數是具有螢光金 (fluorogold)標誌的楔狀丘腦投射神經元。而在正中神經截斷二和四週後,受傷側楔狀神經核內甘胺酸受體免疫反應神經元數量明顯地遞減,但是甘胺酸免疫反應神經元卻沒有變化。相較於控制組,正中神經截斷4週後在電刺激前給予番木虌鹼處理可顯著地降低神經胜肽Y釋放的程度 (NPY release level);並且甘胺酸和番木虌鹼處理組的楔狀神經核內c-Fos免疫反應神經元數量也會顯著地減少。但是在CatWalk的行為分析時,所測量的前腳掌印寬度和面積參數卻顯示出甘胺酸只輕微地恢復其接近正常值。 另一方面,在單側正中神經截斷後4週,受傷側楔狀神經核內伽傌-胺基丁酸免疫反應神經元和背根神經節內伽傌-胺基丁酸B受體免疫反應神經元的比例下降至最低點,但不影響楔狀神經核內伽傌-胺基丁酸B受體,以及楔狀神經核和背根神經節內伽傌-胺基丁酸A受體的免疫反應。於雙側正中神經截斷4週時,在電刺激單側受傷的正中神經前,分別於腹腔給予生理食鹽水、普樂芬或法克羅芬,我們發現普樂芬處理組的楔狀神經核內神經胜肽Y釋放程度和c-Fos免疫反應神經元數量有明顯地降低。但是法克羅芬處理後的情形卻是完全相反的,兩者皆比生理食鹽水處理組的數值高。而且,利用CatWalk步態分析方式,我們更確認正中神經截斷後所引起的感覺異常可因普樂芬的治療而趨於正常。 總結來說,我們的研究結果顯示正中神經截斷後,會降低楔狀神經核內伽傌-胺基丁酸、甘胺酸受體和伽傌-胺基丁酸B受體的表現量,進而阻礙甘胺酸和伽傌-胺基丁酸的抑制調節作用,而促使神經傷害誘發產生的神經胜肽Y釋放,因此楔狀神經核神經元被誘發而產生c-Fos蛋白。但是甘胺酸處理不僅增加對楔狀神經核神經元的突觸後抑制作用,也壓制部分含有甘胺酸受體的伽傌-胺基丁酸神經元的活性,使得其對初級傳入神經終末的突觸前抑制作用受到影響而導致神經胜肽Y緩慢的釋放。由於以上兩種相互抵觸的作用才會呈現甘胺酸處理後,c-Fos蛋白略微減少而神經病變疼痛行為也只有輕微改善的現象。相對地,普樂芬直接阻斷神經胜肽Y釋放,使得楔狀神經核內神經元的c-Fos蛋白表現減少,以致傳遞到丘腦的神經病變疼痛訊息因此減弱。所以我們推測在楔狀神經核內,伽傌-胺基丁酸B受體可能在止痛方面扮演著比甘胺酸受體更重要的角色,而且希望這些研究結果和CatWalk分析參數,能有效於提供未來研究正中神經傷害所造成的神經病變疼痛的機制和治療方針。 Under normal conditions, the cuneate nucleus (CN) transmits the information related to innocuous tactile, vibratile and proprioceptive sensations from forelimb through primary afferent fibers and relays the information to the contralateral ventrobasal nucleus of thalamus. The upregulation of expressions of neuropeptide Y (NPY) and c-Fos in the ipsilateral CN following median nerve transection (MNT) is well known, but the effect of MNT on the alterations of the inhibitory modulation in the CN associated with the contribution to the expressions of NPY and c-Fos, and neuropathic pain still needs to be investigated. Glycine and γ-aminobutyric acid (GABA) are considered to be the two major components in the inhibitory modulation and integration, which are caused by pre- and postsynaptic inhibition to regulate the information processing. This study aimed to investigate the relationship between temporal changes in glycine and GABA transmission in the CN and behavioral abnormalities after MNT. Immunohistochemistry methods were used to appraise changes of glycine-, GABA-, glycine receptor (GlyR)-, GABAAR- and GABABR-like immunoreactive (LI) neurons in the CN and dorsal root ganglion after MNT. The alterations in NPY and c-Fos expressions were used to assess the effects of saline, receptor agonist (glycine or baclofen) or antagonist (strychnine or phaclofen) treatment. The CatWalk method was used to assess the efficiency of glycine or baclofen treatment on the neuropathic signs of rats with MNT. At first, approximately half of GlyR-LI neurons were fluorogold-labeled cuneothalamic projection neurons in the CN. Following MNT, the number of GlyR-LI neurons significantly decreased in the injured side of CN at 2 and 4 weeks, but the number of glycine-LI neurons remained unchanged. Four weeks after bilateral MNT given with electrical stimulation, strychnine significantly decreased the NPY release level in the stimulated side CN compared to that of the saline group. However, numbers of c-Fos-LI neurons in the glycine and strychnine groups were both significantly less than that in the saline group. But the paw print width and area in CatWalk analysis showed only a moderate recovery. On the other hand, following unilateral MNT, the percentage of GABA-LI neurons in the CN and that of GABABR-LI in the dorsal root ganglion in the injured side decreased and reached a nadir at 4 weeks after median nerve transection. Four weeks after bilateral median nerve transection and intraperitoneal application with saline, baclofen, or phaclofen before unilateral electrical stimulation of the injured median nerve, we found that the neuropeptide Y release level and the number of c-Fos-like immunoreactive neurons in the baclofen group were significantly attenuated, whereas those in the phaclofen group had increased compared to the saline group. Furthermore, using the CatWalk method, we confirmed that the behavioral abnormalities in rats following median nerve transection were reversed by baclofen treatment. Overall, the results indicate that median nerve transection reduces glycine and GABA transmission by decreasing the expression of GABA, glycine and GABAB receptors, promoting injury-induced neuropeptide Y release and consequently evoking c-Fos expression in cuneate nucleus neurons. But glycine increases glycine-mediated postsynaptic inhibition of cuneate neurons, and also blocks GABAergic neurons containing GlyRs which mediate presynaptic inhibition causing temperate NPY release. Consequently, the compromise results showed a weak reduction in c-Fos expression and a slight amelioration of neuropathic behaviors. In the contrast, baclofen treatment direct block neuropeptide Y release, subsequently lessening c-Fos expression in cuneate neurons and consequently attenuating neuropathic signal transmission to the thalamus. Therefore the results suggest the GABABR in the CN may serve as a major potential target for analgesia than GlyR, and we expect that the experimental results and CatWalk analysis parameters presented here will be useful in studying mechanisms and treatment options of neuropathic pain resulting from median nerve injury. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66132 |
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顯示於系所單位: | 解剖學暨細胞生物學科所 |
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