大鼠皮质脊髓束LuxolFastBlue染色

大鼠皮质脊髓束Luxol Fast Blue染色

作者：吕广明，吴辉群，栗卓，刘苏，韩笑，季达峰

【摘要】目的：探讨显示大鼠皮质脊髓束的特殊染色方法。方法：选用正常成年SD大鼠延髓和脊髓冰冻切片，应用Luxol Fast Blue 染色法进行染色。结果：Luxol Fast Blue染色后，在延髓和脊髓切片中可见灰质呈淡红色，白质呈蓝色，延髓锥体染成深蓝色。锥体中Luxol Fast Blue标记的深蓝色阳性纤维，经锥体交叉后至脊髓灰质后连合背侧，沿脊髓后索腹侧深层下行，至荐段后逐渐消失。在延髓锥体和脊髓颈、胸、腰段后索中，深蓝色的Luxol Fast Blue阳性纤维边界清晰，与周围结构区分明显。结论：运用Luxol Fast Blue 染色可清楚显示大鼠皮质脊髓束在脊髓内的定位，是一种简便可靠的皮质脊髓束形态学研究方法。

【关键词】皮质脊髓束；Luxol Fast Blue；髓鞘染色；大鼠

[Abstract] Objective: To explore the special staining method for revealing the localization of corticospinal tract in rats. Methods: Luxol Fast Blue staining method was utilized to stain the sections of medulla oblongta and spinal cord of the normal adult Sprague-Dawley rats. Results: In Luxol Fast Blue

staining sections, the gray matter of medulla oblongta and spinal cord was stained in light red, while the white matter around the gray matter was dyed in blue. The dark blue stained Luxol Fast Blue labeled fibers in the pyramid passed through the pyramidal decussation to the contralateral side, descended at the ventral part of posterior funiculus just dorsal to the gray commissure throughout most of the length of the spinal cord and progressively diminished to end at the sacral spinal segments. The Luxol Fast Blue positive fibers in the pyramid and the ventral part of posterior funiculus at cervical, thoracic, lumbar spinal segments were easily discriminated from the surrounding structures. Conclusion: Using Luxol Fast Blue staining method may demonstrate the localization and distribution of corticospinal tract in the spinal cord of rats, which is an easy and effective method in morphological study of corticospinal tract.

[Key Words] Corticospinal tract；Luxol Fast Blue；Myelin stain；Rat

长期以来，脊髓损伤后皮质脊髓束的再生修复研究一直是神经科学研究的热点。最近有人尝试运用微电子芯片技术重建皮质脊髓束功

能[1]。此项研究中一个关键指标就是皮质脊髓束在脊髓内定位和走行情况。常用的组织学染色方法不能显示皮质脊髓束，本实验采用Luxol Fast Blue染色法对大鼠皮质脊髓束进行形态学研究，取得良好效果。

1 材料和方法

1.1 材料选用健康成年SD大鼠6只，清洁级，体重250~300g,雌雄不拘，由南通大学实验动物中心提供。0.1 % Luxol Fast Blue 液（Sigma公司），Leica CM1900冰冻切片机，显微镜为Leica DMR 显微镜（Leica公司，德国）。

1.2 方法切片制备：给予大鼠复合麻醉剂（0.2 ml/100g体重），腹腔注射麻醉后开胸，穿心脏经升主动脉灌注，先用温（37℃）生理盐水200 ml冲净血液，再用预冷（4℃）的含4%多聚甲醛的磷酸盐缓冲液500 ml快速灌注固定，1 h后取脑和脊髓放入含4%多聚甲醛的PB溶液中后固定4~6 h,再移入30%蔗糖的PB溶液中4℃过夜，待组织沉底后取脑（延髓）和脊髓颈、胸、腰、荐段行连续横切面冰冻切片，切片厚度为30 μm，直接贴于预先涂有明胶的载玻片上，置室温下晾干待用。

Luxol Fast Blue 染色：（1）将切片放入乙醇∶氯仿溶液（无水乙醇和氯仿的体积比为1∶1）中5 min；（2）将切片置入 95%乙醇溶