运动终板在骨骼肌缺血再灌注损伤中的病理变化

目的 探讨骨骼肌缺血再灌注（ischemia—reperfusion，IR）过程中运动终板（neuromuscular junction，NMJ）损伤的病理改变进程。方法 将Wistar大鼠48只制备仅神经血管蒂及两端腱性相连的腓肠肌内侧头肌瓣模型。分为6组（n=8），A组：正常对照组，不经缺血处理取材；B组：肌肉缺血3h取材；C组：缺血4．5h取材；D组：缺血4．5h，通血1．5h取材；E组：缺血4．5h，通血24h取材；F组：缺血4．5h，通血2周取材。观察肌肉大体变化及肌肉标本行氯化金压染后NMJ的形态学变化，标本经乙酰胆碱酯酶（acetylcholinesterase，AChE）及结合琥珀酸脱氢酶（succinate dehydrogenase，SDH）染色后行半定量分析。另取5只大鼠分别制成如A、C、E组模型，取材后标本分别行HE染色、氯化金（AuCl3）染色连续切片等辅助实验。结果 肌肉大体观察：B组肌肉呈紫色、肿胀；C组呈紫黑色、肿胀加重、无弹性和收缩反应并有较多暗红色血性渗出；D组较C组颜色浅，肿胀减轻，弹性差，无收缩，质脆硬，渗出少且清亮；E组与D组相似、渗出更少；F组呈红灰相间，并有黄色坏死组织外露，肌肉挛缩、无收缩及弹性，与周围广泛粘连。氯化金染色：缺血和再灌注期肌纤维坏死发生最早，随后是坏死肌纤维上NMJ的溃变，最后神经纤维也发生崩解，如同树叶凋落。AChE结合SDH染色：B、C组的AChE含量与A组比较差异无统计学意义（P〉0．05）；D、E、F各组AChE及SDH的含量均急剧下降，各组AChE含量与A、C组比较差异有统计学意义（P〈0．01）。结论 肌肉内神经纤维至NMJ的分布极象树状结构。缺血期NMJ比肌纤维更能耐受缺血损伤，NMJ的存在依赖于肌纤维的存活。应对坏死肌肉实施进一步清创，IR损伤才可得到有效控制。

PATHOLOGICAL CHANGES IN NEUROMUSCULAR JUNCTION DURING ISCHEMIA-REPERFUSION IN RAT SKELETAL MUSCLE

Objective To investigate the pathological changes in the neuromuscular junction during ischemia-reperfusion(IR) in the skeletal muscle. Methods Forty-eight healthy adult Wistar rats (24 male, 24 female) were equally randomised into the following 6 groups: Group A (control group): no ischemia-reperfusion; Group B: ischemia by clamping the blood vessels of the right hindlimb for 3 hours; Group C: ischemia by clamping for 4.5 hours; Group D: ischemia by the clamping for 4.5 hours followed by reperfusion for 1.5 hours; Group E: ischemia for 4.5 hours followed by reperfusion for 24 hours; and Group F: ischemia for 4.5 hours followed by reperfusion for 2 weeks. Then, the medial head of the gastrocnemius muscle flap model was applied to the right hindlimb of each rat. The medial head of the gastrocnemius muscle was isolated completely,leaving only the major vascular pedicle, nerve and tendons intact.The proximal and distal ends (tendons) were ligated while the vessel pedicle was clamped. And then, Parameters of the muscle (performance,contraction index,colour,edema,bleeding) were observed. The muscle harvested was stained with gold chloride (AuCl_3) and the enzymhistochemistry assay (succinate dehydrogenase combined with acetylcholine esterase) was performed. Morphology and configuration of the neuromuscular junction were observed during the ischemia-reperfusion injury by means of the AuCl_3 staining. The result of the enzymhistochemical reactions was quantitatively analyzed with the computer image-analysis system. And then, additional 5 rats were prepared for 3 different models identical with those in Groups A, C and E separately. The specimens were harvested from each rat and were stained with HE and AuCl_3, and they were examined under the light microscope. Results During the period of ischemia, the skeletal muscle of Group B showed the colour of purple and edema.The colour and edema became worse in Group C,while dysfunction of elasticity and contraction appeared obviously with plenty of dark red hemorrhagic effusion at the same time.After reperfusion,the color and edema of muscle in Group D became improved while the elasticity and function of contraction was not improved. Hemorrhagic effusion of Group D turned clearer and less than Group C.Group E was similar to Group D in these aspects of muscle except for much less hemorrhagic effusion. Skeletal muscle in Group F showed colour of red alternating with white, adhesion, contracture of muscle, exposure of necrotic yellow tissue and almost lost all its functions. The AuCl_3 staining showed that during IR, necrosis of the myocytes was followed by degeneration of their neuromuscular junctions, and finally the nerve fibers attached to these neuromuscular junctions were disrupted like the withering of leaves. The enzymhistochemistry assay showed that there was no significant difference in the level of acetylcholine esterase between the ischemic group (Groups B and C) and the control group (Group A) (P>0.05). However, the level of acetylcholine esterase in all the reperfused groups (Groups D, E and F) decreased significantly when compared with the control group(Group A)and the ischemic groups (Groups B and C) (P<0.01). Conclusion The distribution of the nerve fibers and the neuromuscular junctions in the mass of the muscles is almost like the shape of a tree. The neuromuscular junction seems to be more tolerant for ischemia than the myocyte. Survival of the neuromuscular junction depends on its myocytes alive. Therefore, an ischemia-reperfusion injury will not be controlled unless an extensive debridement of the necrotic muscle is performed.