先天性眼球震颤眼外肌栅栏状终末结构的研究

Palisade endings of extraocular muscles in eyes with congenital nystagmus

目的研究人眼外肌栅栏状神经终末（PE）结构的形态、分布和功能，观察其在先天性眼球震颤（CN）中发生的改变，探讨CN的病因和发病机制，并为指导临床治疗提供理论依据。方法眼肌手术时留取眼外肌远端的肌一腱连接区部分，标本分为CN组和对照组（共同性斜视患者），两组再按相近年龄大小（差别〈3岁）、相同取材部位配对分组并进行下述各项检测。（1）超微结构观察：标本经4％戊二醛前固定后，自附着点处向后连续每1mm切取1或2块横断的1mm×2mm×3mm组织，常规电子显微镜标本处理，透射电子显微镜下观察两组眼外肌及PE的超微结构。（2）眼外肌肌一腱连接区整体经4％多聚甲醛固定、胆碱乙酰转移酶抗体标记后，行25Ixm厚的纵、横断面冰冻切片，激光扫描共聚焦显微镜下观察PE的胆碱乙酰转移酶表达情况、形态及分布。（3）两组选取相同部位的肌一腱连接区组织，提取组织蛋白，进行胆碱乙酰转移酶的免疫印迹检测。采用两个独立样本t检验，对CN组与对照组内直肌横断面PE的数量、肌一腱连接区胆碱乙酰转移酶阳性表达量进行比较。结果与对照组相比，透射电子显微镜下CN组PE的结构水肿，电子密度增高呈明显的嗜锇酸样改变；肌细胞内线粒体肿胀，肌浆网扩张。人眼外肌PE的胆碱乙酰转移酶呈阳性表达。眼外肌纵断面切片可见供应PE的神经纤维起自肌肉，延伸人腱后返回，发出栅栏状分支，分布于肌纤维末端。横断面切片可见内直肌在附着点后约3～4mm处PE的分布最为集中，对照组和CN组栅栏状终末分布数量分别为26．800±3．421、39．200±2-3．564，差异有统计学意义（t=-5．613，P〈0．05）。CN组的ChAT表达条带的灰度值（1．503±0．203）高于对照组（1．000±0．113），差异有统计学意义（t=-3．730，P〈0．05）。结论人眼外肌PE为胆碱能神经，分布于眼外肌远端的肌一腱连接处，可能与眼外肌的收缩有关。CN患者的眼外肌PE结构发生了明显改变，可能导致异常的眼球运动。

Objective To evaluate the morphology, distribution and function of palisade endings （PE） in human extraocular muscles （EOM） , and observe the alterations in eyes with congenital nystagmus （CN）. The etiology and pathogenesis of CN were also investigated. Methods It was a experimental study. The distal myotendinous junctions of the EOM were obtained during operation for CN （ CN group ） and concomitant strabismus （control group ）. The samples from patients with similar age and same extraction sites in the two groups were compared. The muscles cut during operation were immediately put into 4% glutaraldehyde fixative solution. And 1 - 2 transverse bands of tissue were cut every 1 mm from tendon insertion for specimens processing. The ultrastructure of EOM and PE in the two groups was observed by transmission electron microscopy. The distal parts of EOM cut during operation were put into 4% paraformaldehyde promptly. Myotendinous junction region whole mounts were labeled with antibodies against choline aeetyltransferase （CHAT）. Muscle fibers were counterstained with phalloidin. And longitudinal and transverse eryostat serial sections were cut at 25μm and analyzed by confocal laser scanning microscopy. The ChAT expression, morphology and distribution of PE were observed. The same fragment of myotendinous junction in the two groups was selected. After the total protein was extracted, ChAT was detected by western blot. The expression level of ChAT was analyzed. Results Compared with the controls, the uhrastructure in the CN group had considerable variations. The axon of PE was swelled and deformed partly. The electron density was increased and presented as addicted to osmic acid. In the muscle cells, mitochondria was swelled, and sarcoplasmic reticulum was dilated. All PE exhibited ChAT immunoreactivity in human EOM. In the longitudinal section, nerve fibers extended from the muscle into the tendon, looped back and divided into several terminal arborizations （palisade endings） around the muscle fiber tip. The PE of medial rectus were richest at the location 3 - 4 mm from tendon insertion. In the cross section, the amount of PE in the CN group was higher than the control group （ t = - 5. 613, P 〈 0.05 ）. The expression level of ChAT in the CN group was higher than the control group （ t = - 3. 730, P 〈 0.05 ）. Conclusions Palisade endings in myotendinous junction of human EOM are cholinergic nerves, which might innervate the contraction of EOM. Significant changes of palisade endings in the EOM of the CN subjects may affect eye movement.