活血通督汤对大鼠脊髓损伤后血-脊髓屏障的修复作用

Repair effect of Huoxue Tongdu Decoction on blood-spinal cord barrier in rats after spinal cord injury

目的:通过对大鼠脊髓损伤(SCI)后的行为学评分、脊髓组织病理分析以及血-脊髓屏障(BSCB)功能的评测,观察活血通督汤对大鼠SCI后BSCB的影响。方法:将24只雌性SD大鼠随机均分为假手术组、模型组及活血通督汤组,每组8只。假手术组仅咬除椎板不损伤脊髓,模型组和活血通督汤组采用改良Allen’s法建立SCI模型。分别于第1、3、5、7天行BBB评分,7 d后取材。采用HE染色观察大鼠脊髓组织受损程度,尼氏染色观察神经元形态,透射电镜观察BSCB紧密连接超微结构,EB染色检测BSCB通透性。结果:与假手术组比较,模型组和活血通督汤组相同时点BBB评分显著降低(P<0.01),在干预第7天时,活血通督汤组大鼠BBB评分显著高于模型组(P<0.05)。HE染色结果显示:与模型组比较,活血通督汤组炎症细胞浸润有所减少,神经细胞形态结构较完整且数量增多,但仍存在脊髓空洞样病变;尼氏染色结果显示:与模型组比较,活血通督汤组神经细胞形态较好且数量增多,尼氏体数量较多,空泡样改变减少,瘀血面积较小或消失;透射电镜结果显示:与模型组比较,活血通督汤组大鼠脊髓血管内皮细胞紧密连接较完整,血管管腔连续性好。激光共聚焦定性观察显示:模型组血管周围组织EB渗出明显,弥散范围较大,且固有的血管形态被弥散渗出的EB所遮盖,混为一片,较难分辨。活血通督汤组较模型组EB渗出减少,灰质区血管形态也更清晰,甚至可见少量条管状血管形态;与模型组比较,活血通督汤组脊髓组织EB含量显著降低(P<0.01),与镜下观察结果一致。结论:活血通督汤可促进血管内皮细胞间紧密连接的修复,降低BSCB的通透性,抑制血管中的炎症因子和趋化因子进入脊髓组织,为脊髓微环境的稳态提供有效保护,有利于大鼠SCI后神经细胞的再生以及运动功能的恢复。

Objective:The effect of Huoxue Tongdu Decoction on the blood-spinal cord barrier(BSCB)in rats after spinal cord injury(SCI)was observed through the behavioral score,histopathological analysis of spinal cord,and the evaluation of BSCB function after SCI in rats.Methods:Twenty-four female SD rats were randomly divided into sham operation group,model group,and Huoxue Tongdu Decoction group,8 rats in each group.In the sham operation group,the spinal cord was immediately sutured after laminectomy.The improved Allen's method was adopted to establish the SCI models in the model group and Huoxue Tongdu Decoction group.BBB hind limb motor function score was performed on the first,third,fth,and seventh days,and the samples were taken afer seven days of intervention.HE staining was used to observe the degree of tissue damage.Niss staining was used to observe the morphology of neurons.A transmission electron microscope was used to observe the ultrastructure of the tight junction of the blood spinal cord barrier.Finally,EB staining was used to detect the permeability of the blood spinal cord barrier.Results:Compared with the sham operation group,the BBB scores of the model group and Huoxue Tongdu Decoction group at the same time point were significantly lower(P<0.01),on the 7th day after the operation,the BBB score of rats in the Huoxue Tongdu Decoction group was significantly higher than that of the model group(P<0.05).HE staining:Compared with the model group,the inflammatory cells in the Huoxue Tongdu Decoction group decreased,the morphological structure of nerve cells was complete,and the number increased,but there were still syringomyelia-like lesions.Nissl staining:Compared with the model group,the nerve cells in the Huoxue Tongdu Decoction group had better morphology and increased number,more Nissl bodies,less vacuolar changes,and minor or disappeared blood stasis area.Transmission electron microscope:Compared with the model group,the tight junctions of spinal cord vascular endothelial cells in the Huoxue Tongdu Decoction group was complete,and the continuity of vascular lumen was good.Laser confocal observation showed that the morphology of spinal cord blood vessels in the model group,EB exudation in the perivascular tissue was evident,and the diffusion range was large.The inherent vascular morphology was covered by EB exudation,which was mixed and difficult to distinguish.Compared with the model group,the EB exudation in the Huoxue Tongdu Decoction group was reduced.The vascular morphology in the gray matter area was also clearer,and even a small number of tubular vessels could be seen.Compared with the model group,the content of EB in spinal cord tissue in the Huoxue Tongdu Decoction group decreased significantly(P<0.01),which was consistent with the observation under the microscope.Conclusion:Huoxue Tongdu Decoction can promote the repair of tight junctions between vascular endothelial cells,reduce the permeability of the BSCB,inhibit the entry of inflammatory factors and chemokines in the blood vessels into the spinal cord tissue,and provide effective protection for the homeostasis of the spinal cord microenvironment.It is beneficial to the regeneration of nerve cells and the recovery of motor function after SCI in rats.