神经生长因子和血管内皮生长因子联合应用对兔局灶性脑缺血再灌注损伤神经元的保护时间窗

Time window of nerve growth factor and vascular endothelial growth factor in protection of neural function in rabbits with focal cerebral ischemia reperfusion injury

目的:观察神经生长因子和血管内皮生长因子对兔局灶性脑缺血再灌注损伤的神经元的保护作用,以及发挥作用的有效时间窗。方法:实验于2005-05/08在河北医科大学第二医院神经分子影像医学和神经病学实验室进行。34只雄性4.5～5月龄新西兰白兔随机数字表法分为假手术组(n=6)、生理盐水组(n=8),再灌注3h因子治疗组(n=10)和再灌注6h因子治疗组(n=10)。采用兔大脑中动脉阻断局灶性脑缺血再灌注模型,假手术组线栓插入的深度不同。因子治疗组在缺血2h再灌注损伤后3h和6h应用微量进样器将2.5μg/L血管内皮生长因子16550μL和神经生长因子400AU(相当于16μg/L)立体定向导入梗死灶周,生理盐水组则注入同等剂量的生理盐水,于再灌注72h,应用MR影像学、红四氮唑染色和流式细胞术评价各组动物脑梗死体积、灶周缺血半暗带细胞凋亡率、表观弥散系数值比率及半胱氨酸蛋白酶3活性表达。在大脑中动脉阻塞2h再灌注后24,72h,采用Purdy评分标准行神经功能缺损评分。总得分最低为2分,表示无神经功能缺陷;总得分最高为11分,表示动物意识丧失或死亡。结果:在实验过程中,无动物死亡,均进入结果分析。①缺血2h再灌注72h,MR影像测得梗死灶主要限于左侧大脑中动脉供血区的皮质和皮质下白质和部分尾壳核。再灌注3h因子治疗组、再灌注6h因子治疗组脑梗死百分率分别较生理盐水组下降44.0%和33.3%。②缺血2h再灌注72h的再灌注3h因子治疗组、再灌注6h因子治疗组神经功能缺损评分分别较生理盐水组明显减少,差异有显著性意义犤(6.4±0.5),(4.8±0.8),(5.4±0.5),P<0.01);犤(2.8±0.4),(3.2±0.8),(4.6±0.5),P<0.01犦。③再灌注3h因子治疗组、再灌注6h因子治疗组脑组织含水量与生理盐水组相比明显减少,差异均有显著性意义犤(79.2±0.5)%,(79.9±0.6)%,(81.8±0.3)%,0.01犦。④缺血2h再灌注72h的再灌注3h因子治疗组、再灌注6h因子治疗组灶周皮质区梗死灶周区表观弥散系数值与生理盐水组相比明显升高,差异有显著性意义犤(89±3)%,(83±3)%,(74±4)%,P<0.01犦。这两组灶周皮质凋亡率及半胱氨酸蛋白酶3活性表达与生理盐水组相比则明显降低,差异均有显著性犤(10.4±0.7)%(15.5±1.2)%(20.2±1.3)%,P<0.01犦;犤(17.4±1.3),(26.1±1.0),(54.1±6.9),P<0.01犦。结论:联合神经生长因子和血管内皮生长因子治疗脑缺血再灌注损伤具有明显的神经元护作用,有效时间窗最少是再灌注后6h。

AIM： To observe the protective effect of nerve growth factor （NGF） and vascular endothelial growth factor （VEGF） on neural function in rabbits with focal ischemia/reperfusion and effective time window to exert its role. METHODS： The study was carried out at the Laboratory of Molecular Imaging and Neurology, Second Hospital, Hebei Medical University form May 2005 to August 2005.Thirty-four male New Zealand white rabbits , aged 4.5 to 5 months , were randomly divided into sham-operation group （n =6）,physiological saline group （n =8）, factor treatment group of reperfusion for 3 hours （n=10）and factor treatment group of reperfusion for 6 hours （n=10）. Local ischemia and reperfusion models caused by blocking middle cerebral artery of the rabbits were adopted and the inserting depth of sham-operation group was different. 50μL VEGF 165 （2.5μg/L） and 400 AU nerve growth factor （16μg/L） were led into the perifocal region of infarction in the factor treatment groups of ischemia 2 hours and reperfusion 3 hours and 6 hours respectively with microsyringe. Same volume of physiological saline was injected into the rabbits in the physiological saline group. At 72 hours after reperfusion, cerebral infarcted volume, cellular apoptosis rate and apparent dispersion coefficient ratio of perilocal ischemia region and the expression of caspase-3 activity were evaluated in the animals in each group with MR imaging, methylthiazolyl tetrazolium assay and flow cytometry. At middle cerebral artery occlusion 2 hours and reperfusion 24 hours or 72 hours, neurologic impairment scoring was performed according to Purdy scoring standard. The lowest was 2 points, suggesting no neurologic impairment; the highest was 11 points, suggesting animals lost consciousness or died. RESULTS： During the experiment, all the animals entered the stage of result analysis. ① At ischemia 2 hours and reperfusion 72 hours , MR imaging measured that＇the infarcted locals were mainly at the cortex and white matter as well as partial dorsal caudate putamen of blood-supplying region of left middle cerebral artery. The infarcted percentage of was decreased by 44.0% and 33.3% in the factor treatment group of reperfusion for 3 hours and for 6 hours than physiological saline group respectively. ②At ischemia 2 hours reperfusion 72 hours, neurologic impairment was significantly lower in the factor treatment group of reperfusion 3 hours and factor treatment group of reperfusion 6 hours thanin the physiological saline group , with significant difference [（6.4±0.5）, （4.8±0.8）, （5.4±0.5） ,P 〈 0.01]; [（2.8±0.4）, （3.2±0.8）, （4.6±0.5） ,P 〈 0.01]. ③ Water content of cerebral tissue was obviously reduced in the factor treatment group of reperfusion 3 hours and factor treatment group of reperfusion 6 hours as compared with physiological saline group, with significant difference [ （ 79.2±0.5 ） %, （ 79.9±0.6 ） %, （ 81.8±0.3 ） %, P 〈 0.01]. ④At isehemia 2 hours reperfusion 72 hours, apparent dispersion coefficient of perilocal region of infarotion was significantly increased in the factor treatment groups of reperfusion for 3 hours and 6 hours as compared with physiological saline group, with significant difference [（89±3）%, （83±3）%, （74±4）%,P〈 0.01]. Apoptotic rate of cortex around local and caspase-3 activities expression were obviously decreased in the factor treatment groups of reperfusion for 3 hours and 6 hours as compared with physiological saline group , with significant difference [（10.4±0.7）% （15.5±12）% （20.2±13）% ,P 〈 0.01]; [（17.4±1.3 ）, （26.1±1.0）, （54.1±6.9）,P〈 0.01]. CONCLUSION： Combination of NGF and VEGF has obvious neuronal protective role in treatment of cerebral ischemia and reperfusion injury. The effective time window is at 6 hours after reperfusion at least.