隐性脊椎裂胎鼠支配肛提肌脊髓运动神经元定量研究

Quantitive analysis of motor neurons innervating the levator ani muscle in fetuses with spina bifida occulta

目的采用神经解剖学常用的荧光金(FG)标记逆向神经示踪法,结合胎儿外科显微手术和显微注射技术,探讨脊椎裂胎鼠支配肛提肌的脊髓运动神经元的发育特点。方法在大鼠孕20d用10%水合氯醛麻醉,应用胎儿外科显微手术和显微注射技术,用纤细玻璃针将5%FG注入雄性胎鼠直肠两侧肛提肌;胎鼠子宫内存活24h后,4%多聚甲醛进行灌注固定;取脊柱标本阿尔新蓝软骨染色后实体显微镜下数码相机采集脊柱软骨染色图像,经计算机软件测量出每个椎体椎弓两个软骨端距离并判定隐性脊椎裂;采集图像后取腰骶段脊髓标本置入20%蔗糖溶液内,24h后连续冰冻横切片,脊髓冰冻切片荧光显微镜下观察并计数FG标记的运动神经元数目。结果正常组脊髓前角FG标记的运动神经元呈团簇状分布,左右两侧对称,主要位于脊髓前角的背内侧群和背外侧群;畸形组脊髓前角FG标记的运动神经元数目少,而且在脊髓前角的背内侧群和背外侧群之间也有少量散在分布,分布团簇状、对称性差,畸形组FG标记的运动神经元数量(244±41)个,与正常组(426±36)个和用药无畸形组(397±20)个差异显著,P<0.05。结论隐性脊椎裂胎鼠支配肛提肌的脊髓运动神经元发育异常,运动神经元数目明显减少,分布异常。

Objective Combining the microsurgery and microinjection technique, we investigated the motor neuron development of the spinal cord in fetal rats with spina bifida occulta by injecting the retrograde tracer fluorogold （FG） into the muscle levator ani. Methods On day 20 of gestation, pregnancy rat was anesthetized with 10% intraperitoneal chloral hydrate （300 mg/kg, b. t. ）. Fetal microsurgery and microinjection were performed to expose the muscle levator ani. 5% FG was injected with microinjector. 24 hours later, the operated fetus was perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline （PBS）. After the spine sample was stained with Alcian blue GX, the distance parameter of the two cartilaginous ends of the vertebra arch was measured with image-analyser. The lumbosacral spinal cord was cryoprotected in 20% sucrose in PBS and for serial transverse cryosection after 24 hours. The FG-labelled motor neurons were visualized with a wideband ultraviolet-fluorescence filter and the number of the FG-labelled motor neurons was recorded . Results The FG-lablled motor neurons in the ventral horn of normal spinal cord occupied the dorsolateral （DL） corner and dorsomedial （DM） corner of the ventral horn in cluster. The FG-lablled motoneurons in the ventral horn of malformation spinal cord were less than that of normal spinal cord and the motoneurons situated at the dorsolateral corner and dorsomedial corner and were scattered, the number of FG-labelled motoneurons in malformation spinal cord was 244±41 and that in normal spinal cord 426±36, 397±20 in the spinal cord of fetuses administered by RA, but no malformation. The data were significantly different （P〈0. 05）. Conclusions The motor neurons innervating the muscle levator ani in fetal rats with spina bifida occulta are fewer than that in the normal fetal rats. Its distribution is abnormal.