实验性脑缺血大鼠再灌注前后给予藻酸双酯钠对脑组织神经细胞内钙离子浓度及神经细胞凋亡的影响(英文)

Influence of alginic sodium diester on intraneuronal Ca^(2+) content and nerve cell apoptosis before and after reperfusion in experimental ischemic rats

背景:藻酸双酯钠具有选择性钙拮抗作用,从而产生神经保护作用。目的:观察局灶性脑缺血大鼠再灌注前后给予藻酸双酯钠对脑组织神经细胞内钙离子浓度的影响以及对神经细胞凋亡保护作用的差异。设计:随机对照观察。单位:中南大学湘雅医院神经内科和南华大学第一附属医院激光整形外科。材料:实验于2003-11/2004-04在中南大学湘雅医院神经内科实验室完成,选用雄性SD大鼠65只。大鼠随机分为6组,实验过程中脱失17只,剩余48只,每组8只。方法:假手术组仅切开颈部皮肤后缝合切口。缺血组、藻酸双酯钠5mg/kg缺血前组、藻酸双酯钠5mg/kg缺血后组、藻酸双酯钠10mg/kg缺血前组、藻酸双酯钠10mg/kg缺血后组建立右侧大脑中动脉缺血模型后,除缺血组外,其余4组分别在再灌前30min或再灌后5h经腹腔给予不同剂量的藻酸双酯钠或赋形剂。流式细胞术测量受损脑组织神经元细胞内Ca2+浓度及凋亡率,同时测定大鼠的神经功能障碍评分。主要观察指标:①藻酸双酯钠对右侧大脑中动脉缺血大鼠神经功能障碍评分、脑细胞内Ca2+荧光强度及神经元凋亡的影响。②右侧大脑中动脉缺血大鼠行为障碍评分与细胞内Ca2+荧光强度和凋亡相关性。结果:选取大鼠65只,脱失17只,纳入48只进入数据分析。①藻酸双酯钠对右侧大脑中动脉缺血大鼠功能障碍、大脑细胞内Ca2+荧光强度及凋亡率的影响:藻酸双酯钠5mg/kg缺血前组、藻酸双酯钠5mg/kg缺血后组、藻酸双酯钠10mg/kg缺血前组、藻酸双酯钠10mg/kg缺血后组神经功能障碍评分较缺血组明显减轻(1.80±0.21,2.20±0.23,1.20±0.11,2.00±0.22,3.40±0.65),再灌注前给药较再灌注后给药的功能改善更为明显;给予藻酸双酯钠后各给药组的Ca2+浓度较缺血组均有不同程度的下降,再灌前30min给予10mg/kg藻酸双酯钠组的Ca2+浓度下降最为明显,约下降70%;给药后缺血侧神经元凋亡率明显下降。并呈时间依赖性,再灌注前给药较再灌注后给药抑制凋亡作用更明显(5.68%,10.03%;4.00%,9.91%)。②右侧大脑中动脉缺血大鼠行为障碍评分与细胞内Ca2+荧光强度和膜联蛋白/碘化丙啶凋亡相关性:大鼠行为障碍评分与细胞内Ca2+荧光强度和膜联蛋白/碘化丙啶凋亡检出率均呈明显的正相关(r=0.51,0.62,P<0.05);细胞内Ca2+荧光强度与膜联蛋白/碘化丙啶凋亡检出率亦呈正相关(r=0.84,P<0.05)。结论:①藻酸双酯钠能通过抑制胞内钙离子浓度增高,发挥抗凋亡效应,从而起到神经保护作用,最终改善神经功能障碍。②其药效呈时间依赖性,再灌注前给药较再灌注后给药作用更明显。

BACKGROUND： Alginic sodium diester （ASD） possesses neumprotective function because of its selective calcium antagonist effects. OBJECTIVE： To compare the influences of ASD on intraneuronal Ca^2＋ content and nerve cell apoptosis before and after reperfusion in focal cerebral ischemic rats. DESIGN： Randomized controlled observation. SETTING： Neurological Department of Xiangya Hospital Affiliated to South China University; Laser Orthopedic Surgery of the First Hospital Affiliated to Southern China University. MATERIALS： This experiment was carried out between November 2003 and April 2004 at the Neurological Department of Xiangya Hospital Affiliated to South China University. A total of 65 male SD rats were recruited and randomized into 6 groups; 17 got lost during the experiment, and the other 48 rats completed the experiment with 8 rats in each group. METHODS： In sham operation group, an incision was made on rats＇ cervical skin and sutured. Right cerebral middle artery was occluded in rats of ischemic group, ASD 5 mg/kg preischemic group, ASD 5 mg/kg postischemic group, ASD 10 mg/kg preischemic group, and ASD 10 mg/kg postischemic group. After that, rats in all but ischemic group were subjected to intraperitoneal injection of various dosage of ASD or excipient 30 minutes before reperfusion and 5 hours after reperfusion. FCM was used to determine intraneuronal Ca^2＋ content and rate of nerve cell apoptosis; meanwhile, neurological dysfunction was scored. MAIN OUTCOME MEASURES： ① Influence of ASD on the score for neurological dysfunction, intraneumnal Ca^2＋ fluorescence intensity, and neuronal apoptosis in rats with fight cerebral middle artery ischemia. ② Correlation of behavioral obstacle score with intraneumnal Ca^2＋ fluorescence intensity and neuronal apoptosis in rats with right cerebral middle artery ischemia. RESULTS： Totally 65 rats were enrolled in this study, 17 of which got lost and the other 48 rats entered the result analysis. ① Influence of ASD on the score for neurological dysfunction, intraneumnal Ca^2＋ fluorescence intensity, and neuronal apoptosis in rats with right cerebral middle artery ischemia： The score was obviously reduced in ASD 5 mg/kg preischemic group, ASD 5 mg/kg postischemic group, ASD 10 mg/kg preischemic group and ASD 10 mg/kg postischemic group as compared with ischemic group （1.80±0.21, 2.20±0.23, 1.20±0.11, 2.00±0.22, 3.40±0.65）; moreover, functional improvement was more obvious due to pre-reperfusional administration than post-reperfusional administration. Intraneuronal Ca^2＋ concentration was reduced after ASD administration at different degrees and lower than that of ischemic group. Decrement of intraneumnal Ca^2＋ concentration was found most obvious due to 10 mg/kg ASD administration 30 minutes before reperfusion, approximately reduced by 70%; moreover, neuronal apoptosis rate on the ischemic side was obviously suppressed by ASD administration, displaying time-dependent manner, with apoptotic suppression effect more obvious in pre-reperfusional group than in post-reperfusional group （5.68%, 10.03%; 4.00%, 9.91%）. ② Correlation of behavioral obstacle score of right cerebral middle artery ischemic rats with intraneuronal Ca^2＋ fluorescence intensity and membrane associated protein/propidium iodide apoptosis： Obvious positive correlation was found between behavioral obstacle score and intraneuronal Ca^2＋ fluorescence intensity and detection rate of membrane associated protein/propidium iodide apoptosis （r=0.51, 0.62, P〈0.05）; intraneuronal Ca^2＋ fluorescence intensity was also positively correlated with the detection rate of membrane associated protein/propidium iodide apoptosis （r=0.84, P 〈 0.05）. CONCLUSION： ② ASD can exert anti-apoptosis effect by suppressing the increment of intraneuronal Ca^2＋ concentration, thus having neuroprotective function and ultimately improving neurological dysfunction. ② Its effect displays time-dependent manner, and neurological functional improvement is more obvious by pre-reperfusional administration than by post-operational administration.