论文标题:大鼠脑和脊髓星形胶质细胞及神经元对一侧胫、腓骨骨折所致疼痛的反应及其关系的形态学研究
Morphological Study on Response and Relationship Of Neurons and Astrocytes in Rat Brain and Spinal Cord to Pain Induced by the Unilateral Tibia and Fibula Fracture
论文作者 张辉
论文导师 饶志仁,论文学位 博士,论文专业 神经生物学
论文单位 第四军医大学,点击次数 159,论文页数 104页File Size5780k
2002-05-01论文网 http://www.lw23.com/lunwen_1197707/ 星形胶质细胞,神经元,胶质细胞-神经元通讯网络,大鼠疼痛,免疫组化,免疫电镜
Astrocytes;Neuron;Neuron-glia signaling network;pain;Rats;Immunohistochemistry;immuno-electron-microscopy;Neuron-Astrocyte Complex;Central Nervous System;
目的 观察一侧胫、腓骨骨折刺激后，大鼠中枢神经系统(脊髓、全脑)的星形胶质细胞(AS)与神经元在光镜、电镜水平结构的变化，以探明伤害性刺激条件下脊髓和脑内哪些部位或核团的胶质细胞起反应，反应性AS与神经元相互关系如何?从而研究AS在外周疼痛刺激反应中是否对神经元具有主动调控作用，并进一步探讨AS和神经元之间的信息通讯结构在疼痛刺激反应调节中的作用。 方法 ①采用免疫组织化学技术，显示外周疼痛刺激后不同时间点中枢神经系统不同部位即刻早期基因Fos、胶质原纤维酸性蛋白(GFAP)与抗酪氨酸羟化酶(TH)、蛋白激酶C(PKC)及磷脂酶C(PLC)的表达变化、相互关系及分布规律。②利用免疫电镜方法，观察骨折刺激前后大鼠腰髓背角内AS与神经元树突间接触部位超微结构以及缝隙连接蛋白Cx43与Cx32的表达变化。③ 采用鞘内注射缝隙连接特异性阻断剂—甘珀酸，阻断AS与神经元间的信息连接，并测定热刺激疼痛时大鼠缩足潜伏期的变化，以了解缝隙连接在疼痛反应调节中的作用。 结果 ①骨折同侧腰髓背角清晰可见大量激活的AS呈PKC-LI，PLC-LI或GFAP／Fos-LI免疫阳性，而激活的神经元则呈PKC-LI，PLC-LI及Fos-LI免疫阳性。激活的神经元和AS主要分布在背角浅层，且阳性产物随存活时间的延长而逐渐向背角深层推进。②在脑干GFAP-LI星形胶质细胞主要分布于MVZ内的孤束核(NTS)、腹外侧延髓(VLM)以 第四军医大学博士学位论文 及两者之间的中间带（IRt）上。三叉神经脊束核尾侧亚核（VC）、外 侧楔柬核（Ecu）、蓝斑（LC）、臂旁外侧核（LP）、中缝大核 （RMg）、中脑导水管周围灰质腹外侧区（VI PAG）、中缝背核（DR）等部 位也出现一定数量的GFAP阳性细胞。F。S—LI神经元的分布与GFAP阳 性细胞基本一致。③在前脑的外侧僵核内侧区（LMHB）、下丘脑室旁核 （PVN）、视上核（SON）、视交叉上核（SCh）、终纹床核（BST）、和杏仁中 央核（Ce）均可以观察到有Fos飞I神经元和GFAP－＊胶质细胞的分 布，表现出船和神经元对疼痛刺激反应部位的一致性。④从时间点 上，GFAP／Fos｛I或PKC－LI AS在骨折后15min开始出现，45 min 时 达高峰：而FOS十I或PKC1神经元则在骨折后45 min才出现，90 min 时达高峰，即GFAP／FOS－LI或叫C－LI AS开始出现及达到高峰的时间都 LLF“1I或PKC1I神经元要早。⑤首次发现一种一侧为胶质细胞突、 另一侧为神经元树突构成的超微结构，其特点是细胞膜增厚、深 染，在两层细胞膜间各有一狭缝，CX32＊与CX43儿I产物分别出现在 神经元一侧和AS一侧（本文暂称EDA），并发现骨折组脊髓背角的 EDA 数目显著高于对照组。③对大鼠后足热刺激致痛缩足潜伏期的测定 结果说明，鞘内注射缝隙连接特异性阻断剂后潜伏期较对照组明显 延长，提示胶质细胞一神经元间信息通讯被阻断后痛阈升高。同时 与对照组相比，阻断组F。S＊I神经元数量明显减少，两组的GFA卜LI AS表达差别却很小。这表明EDA的信息传导是单方向。 结论①脊髓和脑内AS对非病理性疼痛起反应，且具有明确的机 能定位特点。②在外周组织损伤导致疼痛条件下，AS的生化学可塑 性反应早于神经元。③光、电镜水平发现AS与神经元之间有多种途 径进行信息交流，新发现的EDA有可能是神经元与AS之间信息交流的 结构之一。④阻断AS与神经元间信息交流连接后，前者对外周组织 4 第四军医大学博士学位论文 损伤所致疼痛的反应不受影响，而后者的反应明显受到抑制，提示 在非病理性疼痛反应中，胶质细胞对神经元具有主动调控作用。⑤ 中枢神经系统可能以神经元为中心，周围包绕AS共同构成神经元－胶 质细胞复合体（N－ASC），作为中枢神经系统一个功能单位，共同对 各种刺激做出反应。
Morphological Study on Response and Relationship OfNeurons and Astrocytes in Rat Brain and Spinal Cord to PainInduced by the Unilateral Tibia and Fibula FractureAS constitute nearly half of the total CNS. Recent years, an increasingly important role of the AS has been revealed. More and more evidences have shown that the AS have quite a few founctions neglected before in many aspects: they respond to neurotransmitters, integrate inputs from neurons and other AS, signal over a long range and modulate synaptic activity. There are probably some important communication and material exchange between them. Now, AS have come to the center stage in our efforts to understand the function of the brain.The present study, (D by using immunohistochemical single , double or triple staining method, showed the expression, relationship and distribution pattern of Fos-protein,GFAP or TH in rat CNS, investigated the plastic response and relationship of rat lumbar spinal cord AS and neurons to pain induced by the unilateral tibia and fibula fracture; (2) by using a double immuno-electron-microscopic method , investigated the ultrastructural characters of junction areas between neurons and AS in the dorsal horn of rat lumbar spinal cord following the unilateral tibia andfibula fracture; ? after intrathecal application of the carbenoxolone, a gap junction blocker, recorded the paw withdrawal thermal latency and compared with control.Our results were that ﹖he PKC-LI), PLC-LI, Fos-LI AS and neurons, and GFAP-LI AS were distributed in dorsal horn of rat lumbar spinal cord, predominantly in the superficial layer; ﹖he PKC-LI, PLC-LI, Fos/GFAP-LI AS were detected earlier than that of PKC-LI, PLC-LI and Fos-LI neurons; (3) double immune-electron-microscopic staining studies firstly observed a structure temporarily named electron-dense area (EDA) consisted of the astrocytic process on one side and the neuron (dendrite) on the other side, and the character of EDA was the cytomembrane thickening and dark staining, and there was a narrow cleft between both membranes, meanwhile Cx32-LI and Cx43~LI products appeared in the neuron side or astrocyte side respectively;甶n the experimental group rats 73 of 100 per large dendrites (^lu m) and 19 of 100 per smaller dendrites ( |
