脊髓爆震伤后神经细胞的形态学变化及凋亡细胞的时空分布特点

Morphological changes and spatial and temporal distribution of apoptotic neural cells after blast injury to spinal cord

目的:研究脊髓爆震伤后神经细胞的形态学变化及凋亡细胞的时空分布特点。方法:90只家兔随机分为正常组(A组,n=6)、Allen′s撞击组(B组,n=42)和爆震损伤组(C组,n=42)。用改良Allen′s法造成B组不完全性急性脊髓损伤模型;而用0.7g单质金属炸药黑索金(cyclotrimethylene trinitramine)在垂直距离动物T9～T10节段4cm处引爆造成C组脊髓爆震伤模型,5h后B、C组动物均用诱发电位仪刺激下肢的MEP以筛选造模成功的动物。B、C两组分别于损伤后8h、1d、3d、7d、14d和30d取材(损伤段脊髓2cm),每组每个时间点取材7只,而A组动物不作任何处理,统一于30d取材。通过HE染色、透射电镜检测、DNA电泳分析和凋亡细胞原位末端标记法(TUNEL),观察各组脊髓中神经细胞的形态学变化及凋亡细胞的时空分布。结果:HE染色显示,A组为正常组织学表现;B组损伤后8h即出现大片的出血坏死灶,白质呈严重的脱髓鞘样改变,1、3d时前角运动神经元大量减少,7d时白质中空洞形成,而且空洞逐渐融合,到14、30d时,可见部分巨大空洞;而C组爆震后8h仅出现散在的小型出血点,但病变进展迅速,1d时前角运动神经元即明显减少并伴有大量微型空洞形成,3、7d时整个白质结构松散,可见液化坏死灶及广泛的脱髓鞘样改变,而且透射电镜证实这种髓鞘损伤还具有方向性特征,到14、30d时,脊髓可见巨大空洞及散在的小空洞,但面积明显小于B组。B组的前角运动神经元数目在8h时的降低幅度比C组要大(P<0.05),而到损伤7d后,两组却无明显差异。DNA电泳显示B、C两组损伤后3d均出现明显的DNA梯形条带和Smearing现象,而且这种现象逐渐向邻近节段扩散。TUNEL检测显示,B、C两组于损伤后的灰质和白质中均能检测到阳性细胞,而且分布于灰质内的阳性细胞数均于1d时达到高峰(P<0.001),但是在白质内的阳性细胞数量,B组于3d时达到高峰(P<0.001),C组却于7d时才达到高峰(P<0.001)。结论:与脊髓Allen′s撞击模型相比,脊髓爆震伤后同样存在神经细胞的凋亡现象,而且有其时间和空间分布特点。

Objective：To investigate morphological changes and spatial and temporal distribution of apoptotic neural cells after blast injury to spinal cord.Method：Ninety rabbits were randomly assigned into normal group （group A,n=6）,Allen＇s group（group B,n=42） and spinal cord blast injury group（group C,n=42）.Incomplete ASCI model was made based on the modified method of Allen＇s in group B.0.7g eyclotrimethylene trinitramine was placed at the site 4cm vertically to the dorsal of T9-T10 level and detonated in group C to make the blast injury model to spinal cord.5h later,MEP of all rabbits were examined to confirm whether the model was successful.After that,all rabbits in group B and C were sacrificed to harvested the injured spinal cord （2cm） at 8 hours and 1,3,7,14,30 days（n=7） respectively.While in control group,animals were left intact. All injured spinal cords were taken for morphological studies,including hematoxylin and eosin staining,TEM （transmission electron microscope） examination,electrophoretic analysis of DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL） methods.Result：Under HE stain,the specimens in group A displayed normal histology,however in group B,severe hemorrhage,necrosis and demyelination were observed at 8h,the amount of motor neurons decreased significantly at 1 or 3d,invanity were observed until 7d and inosculated gradually,and some huge cavities were evidenced at 14d or 30d.Compared with group B, there were only some hemorrahage spots in group C at 8h,which deteriorated rapidly with time processing.A significant reduction of motor neurons and a large number of micro-cavities were observed in group C at ld, necrosis and extensive demyelination were evidenced at 3d or 7d after blast injury.However,the transmission electron microscopy confirmed that the myelin sheath also had orientation from injury.Huge cavities and scattered small cavities were observed in group C at 14d or 30d,but areas of these cavities was significantly smaller than those in group B.For quantitative analysis,the extent of neuron reduction in group B were greater than those in group C at 8h（P〈0.05）,and there was no significant difference between this two groups 7d after the injury.DNA gel electrophoresis revealed DNA ladderring and apparent with smearing phenomenon at 72h after injury in both group B and C.Moreover,the gross lesion area expanded over time.After TUNEL assay,the TUNEL-positive cells were found in the gray matter and white matter in group B and C,with a maximum presence at ld in the grey matter of injured segment（P〈0.001） ,however the labeled cells in white matter reached peak at 3d in group B（P〈0.001） ） and 7d in group C（P〈0.001）.Conclusion：Compared with Allen＇s model,neuronal apoptosis are also presented in the blast injury to spinal cord,and have characteristics of spatial and temporal distribution.