活血通督法改善脊髓缺血再灌注损伤微环境炎性反应的机制

Infulence of microenvironment and inflammatory of spinal cord during ischemia-reperfusion injury by Huo Xue Tong Du..Spinal cord ischemia reperfusion injury is tough clinical problem with high paralyed and death rate. Our previously study has shown that excitation aminoacid-mediated neurotoxicity are reased into the synapticcleft, or the postsynaptic membrance of N-methyl-D-aspartate receptor. Previous research has shown that spinal nerve cells on microcirculation disorder induced by ischemia and hypoxia is very sensitive, more transient spinal cord ischemia can cause the loss of motor neurons in the spinal cord function. Delayed neuronal apoptosis is secondary to spinal cord injury leading to neuronal dysfunction in the final pathway. It is comfirmed that Huo Xue Tong Du estanbished by Prof. Zhang An-Zhen has definite clinial effect, so that we surposed that Huo Xue Tong Du may activate blood circulation to dissipate blood stasis, inhibit blood platelet agglutination, reduce blood lipid concentrations, remove free radicals, and reduce oxygenation and inflammation. Microenvironment inflammatory of spinal cord plays importment role which may induce neuronal damage following ischemia reperfusion injury. .This project is to estanblish rabbit spinal cord ischemia reperfusion injury model, through the behavior assessment of experimental animals models by measurements of Tarlov scores and BBB scores, we further confirm founction of Huo Xue Tong Du..To observe the changes of inflammatory response factor ICAM - 1, NF - B, VCAM-1, IL - 8, IL-1 beta of spinal cord during spinal cord ischemia reperfusion injury to invest its inflammatory mechanism. Finally, to examine BDNF, Slit2 and GFAP of spinal cord during ischemia reperfusion which might indicate nerve regeneration microenvironment improved by Huo Xue Tong Du or not.

脊髓缺血再灌注损伤是临床治疗的世界难题，瘫痪率和死亡率高。应用张安桢教授活血通督法具有一定的临床疗效。课题拟补肝肾，强督脉，生新髓以防治该病，采用代表性方剂活血通督方,探讨其作用与机制。前期的研究已证明：迟发性神经细胞凋亡是继发性脊髓损伤导致神经功能障碍的最终共同通路。病理状态下炎性路径介导的神经元凋亡是导致脊髓缺血再灌注损伤的关键。本研究创建家兔脊髓缺血再灌注损伤的新模型，通过活血通督方脑脊液的药代动力学明确该药的作用途径和靶点；采用行为学评定、脊髓病理生理学技术分析活血通督方对脊髓缺血再灌注损伤的修复作用及对脊髓炎性通路核心因子ICAM-1、NF-κB、VCAM-1、IL -8， IL-1β的作用，探讨其炎性机制；通过检测脊髓微环境神经轴突再生的关键因子BDNF、Slit2和GFAP，探讨活血通督方改善脊髓缺血再灌注损伤微环境的作用和机制。

腰动脉阻滞造模法夹闭20min造成兔脊髓可逆性损伤，无截瘫；夹闭30min和40min均可造成兔脊髓部分不可逆性损伤，致截瘫 ；夹闭30min所建立的SCII模型病理损伤轻于夹闭40min的模型，最理想。. 活血通督汤组1h始见VCAM-1阳性血管, ICAM-1阳性血管4h时开始表达之后逐渐降低(P<0.01，P<0.05)；NF-κB p65各时点显著低于生理盐水组(P<0.05),脊髓神经细胞细胞浆的染色更淡，细胞形态不规则，细胞核呈现蓝色，脊髓见不均匀分布的神经细胞。脊髓TNF-a、IL-1β、IL-8假手术组低表达，模型组及活血通督汤组各时点表达显著增高（P<0.01），提示正常脊髓表达稳定，损伤诱导表达。同时,IL-1β、IL-8增高与TNF-a一致，提示TNF-a促IL-1β、IL-8表达。脊髓TNF-a在各时点显著增高，脊髓前角的神经元胞体染色较明显，提示脊髓神经元TNF-a表达增多与SCII有关。活血通督汤组上述指标各时点明显减少（P<0.01)提示有效 。. BDNF表达增高(P<0.01），7d最高，14d有所下降，推测SCII后BDNF表达存在时间窗。活血通督汤组各时点显著增高，阳性染色细胞多为脊髓前角神经元和胶质细胞，提示可促脊髓组织BDNF分泌，促损伤修复。Slit2的表达1d、3d、7d、14d增高(P<0.05)，提示与脊髓修复有关,活血通督汤促脊髓Slit2分泌，能促轴突的再生修复。 GFAP的表达1d、3d、7d、14d持续增高(P<0.05)，提示 SCII后脊髓星形胶质细胞增生。活血通督汤组在上述时点显著降低，提示该药可抑制脊髓组织GFAP的表达，减少胶质瘢痕形成，有利于再生轴突的连接。.12h和24h Tunnel染色脊髓前角运动神经细胞核呈棕黄色深染，HE染色胞核固缩、深染、结构模糊，提示凋亡，解释了脊柱减压术后部分患者症状反而加重。3d、1w凋亡指数较12h和24h下降，提示坏死程度改善。HE染色见各时点脊髓间隙见不同程度的出血，炎症反应,中性粒细胞渗出，提示SCII坏死和凋亡同时存在，不同时点严重程度不同，治疗时应区分。活血通督汤组脊髓细胞凋亡指数在各时点均低（P<0.01)，存活的脊髓前角神经细胞数目更多，脊髓和神经细胞的形态和结构良好，说明可抑制神经元凋亡，减轻脊髓神经元的损伤，从而改善SCII.