氯碘羟喹对沙土鼠全脑缺血再灌注后锌离子致神经元死亡的保护作用及其机制
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摘要
前言
     金属锌是人体内含量第二位的过渡金属元素,也是正常生长发育、基因表达、蛋白质代谢、免疫功能等过程中所必需的元素。在啮齿类动物中,短暂的全脑缺血可以使海马CA1区的锥体细胞出现延迟性神经元死亡。有证据显示大脑局部缺血中风的大鼠皮层中出现了锌离子从突触前神经元“涌入”突触后神经元的现象。在短暂全脑缺血后神经元轴突终末释放的过量锌离子可能是导致脑选择性延迟神经元死亡的重要原因之一。许多研究表明在缺血动物模型体内注射锌离子螯合剂对缺血所致的神经功能障碍有保护作用。例如,在全脑缺血模型中给予锌离子螯合剂Ca-EDTA可以显著的减少神经元死亡,在蒙古沙土鼠全脑缺血模型给予Ca-EDTA可以减轻CA1区神经元的损伤。氯碘羟喹(Clioquinol,CQ,5-氯-7-碘-8-羟喹,分子量:305.5)是一种具有膜通透性和疏水性的锌离子螯合剂,能穿过血脑屏障。本研究通过制作沙土鼠全脑缺血模型并给予CQ,探讨CQ对海马CA1区锥体细胞的保护作用及其机理。
     材料与方法
     一、实验动物及分组
     健康成年2月龄蒙古沙土鼠随机分为3组:(1)假手术对照组;(2)脑缺血溶剂对照组(1d,3d,7d);(3)脑缺血CQ治疗组(1d,3d,7d)。二甲基亚砜(DMSO)作为CQ的溶剂。
     为了验证CQ有螯合锌离子的作用,正常2月龄沙土鼠随机分为两组:(1)正常组;(2)注射CQ2小时组。用硒化锌金属自显影(autometallography,AMG)法检测CQ对锌离子的螯合作用。
     二、沙土鼠全脑缺血模型的建立
     沙土鼠用4%戊巴比妥钠麻醉(40mg/kg,ip),颈部正中切口游离迷走神经纤维暴露双侧颈总动脉,用无创动脉夹阻断双侧颈总动脉(假手术对照组不夹闭),造成全脑缺血,夹持10min后松开动脉夹,见双侧颈总动脉血液充盈,双侧颈总动脉重新恢复供血。颈部切口用4号尼龙线缝合,酒精消毒。术后沙土鼠在加热灯照射下保温2h直至复苏。脑缺血溶剂对照组在术后立即腹腔注射溶剂DMSO(10mg/kg/day),脑缺血CQ治疗组在术后立即腹腔注射CQ溶液(10mg/kg/day),按照1d,3d,7d的时间点取材。
     三、检测指标
     应用硒化锌金属自显影AMG法和TSQ锌离子荧光染色检测CQ螯合锌离子的作用。应用Nissl染色检测脑缺血后溶剂对照组和CO治疗组之间海马CA1区锥体细胞的丢失情况。采用TUNEL染色检测脑缺血3d CQ是否有对脑缺血后CA1区锥体细胞的保护作用。采用原位杂交来检测脑缺血3d各组Caspase-3和Caspase-9的mRNA的表达变化。采用Western Blot来检测在脑缺血1d、3d和7d各组之间Caspase-3和凋亡诱导因子(apoptosis inducing factor,AIF)的蛋白含量的变化的情况。
     实验结果
     一、硒化锌AMG染色结果
     在注射锌离子螯合剂CQ的正常沙土鼠标本切片中可在整个海马区域见AMG阳性反应与未注射锌离子螯合剂的沙土鼠标本切片相比染色减弱,在齿状回和苔藓纤维处的阳性反应也与未注射锌离子螯合剂CQ沙土鼠切片相比较减弱。
     二、TSQ锌离子荧光染色结果
     荧光显微镜下观察,在沙土鼠海马苔藓纤维和齿状回部位脑缺血3d后TSQ荧光染色较假手术对照组增强,而注射了锌离子螯合剂CQ的沙土鼠TSQ荧光染色较假手术组和溶剂对照组相比最弱。
     三、海马CA1区Nissl染色结果及CA1区神经元计数
     假手术对照组沙土鼠海马CA1区未见明显神经元损害性改变。脑缺血1d,3d,7d溶剂对照组沙土鼠海马CA1区可见显著的锥体细胞变性、坏死。与溶剂对照组相比,脑缺血1d,3d,7d CQ治疗组海马CA1区锥体细胞死亡数目减少,锥体细胞较排列,形态较完整。
     四、TUNEL染色结果
     假手术对照组沙土鼠在海马CA1区仅见少量TUNEL阳性反应细胞;脑缺血3d CQ治疗组沙土鼠海马CA1区锥体细胞内呈深棕色的TUNEL阳性细胞数目明显较溶剂对照组减少。脑缺血3d溶剂对照组海马CA1区TUNEL阳性细胞数与CO治疗组之间比较有显著性差异(P<0.01)。
     五、Caspase-3和Caspase-9原位杂交染色结果
     光镜下见Caspase-3和Caspase-9原位杂交阳性反应产物均呈深棕黄色。假手术对照组沙土鼠海马CA1区仅见少量Caspase-3和Caspase-9原位杂交阳性反应细胞。脑缺血3d CQ治疗组沙土鼠海马CA1区锥体细胞内呈深棕黄色的Caspase-3和Caspase-9原位杂交阳性细胞数量明显减少。脑缺血3d溶剂对照组海马CA1区Caspase-3和Caspase-9原位杂交阳性细胞数与CQ治疗组之间比较有显著性差异(P<0.01)。
     六、Caspase-3和AIF的Western blot结果
     脑缺血术后1d,3d,7d的溶剂对照组Caspase-3和AIF在动物海马的表达高于CQ治疗组动物。其中Caspase-3(17KD)溶剂对照组和CQ治疗组相比,在1d、3d、7d时间点各组(P<0.01);Procaspase-3(32KD)溶剂对照组和CQ治疗组相比,在1d、7d时间点(P<0.05),在3d时间点(P<0.01);AIF(67KD)溶剂对照组和CQ治疗组相比,在1d时间点(P<0.01),在3d、7d时间点(P<0.05)。
     结论
     1、应用AMG染色和TSQ荧光技术证实CQ对锌离子具有螯合作用。
     2、脑缺血后1d、3d和7d CQ治疗组CA1区锥体细胞数目较溶剂对照组增多。TUNEL染色证实在脑缺血后3d CQ治疗组CA1区锥体细胞TUNEL阳性细胞数目较溶剂对照组减少。
     3、锌螯合剂CQ通过影响凋亡因子Caspase-3,Caspase-9和AIF的表达和活性,对沙土鼠全脑缺血神经元死亡具有保护作用
Preface
     Zinc(Zn~(2+)) is the second most abundant transition metal in the human body.And it's required for normal growth,gene expression,protein metabolism,and immune function.Short time cerebral ischemia gives rise to delayed neuronal death of pyramidal neurons in the CA1 region of the hippocampus in rodents.Evidence for the early Zn~(2+) translocation from presynaptic terminals into postsynaptic neuronal cell bodies has also been demonstrated in rat cortex subjected to focal ischemia,as occurs in stroke.Excessive release of synaptic Zn~(2+) may contribute to the pathogenesis of delayed selective neuronal cell death after transient global ischemia.Many researches have shown that chelation of zinc in vivo puts a protective effect on neurological disorders in animal models.For instance,injection of the zinc chelator,Ca-EDTA, significantly reduced neuronal death resulting from experimentally-induced forebrain ischemia.It has been verified that late injection of Ca-EDTA could rescue CA1 neurons from damaging in global-ischemia Mongolian gerbils.In the recent study,it has been suggested that clioquinol(iodochlorhydroxy-quin,5-chloro-7-iodo-8-hydroxyquinoline, CQ) is a membrane-permeable and hydrophobic zinc chelator.So it can easily cross the blood-brain barrier.In the present study,we examine the protective effects of CQ on the ischemia gerbil brains,to address whether administration of CQ after ischemia can rescue the CA1 pyramidal cells.
     Materials and Methods
     1.Experimental animals and Treatments
     Adult Mongolian gerbils were randomly divided into three groups:(1) sham,(2) vehicle-treated(1d,3 d,7d) ischemia groups,(3) CQ-treated(1d,3d,7d) ischemia groups.DMSO was used as the vehicle for CQ.
     To verify the chelation of CQ,adult gerbils were randomly divided into normal and CQ 2h-normal groups.ZnSe AMG was used for checking the chelation of CQ.
     2.Induction of Global Cerebral Ischemia
     The gerbils were anesthetized with pentobarbital(40mg/kg,i.p.).Ventral neck incision was made;the bilateral common carotid arteries were separated carefully from vagus nerve and were occluded bilaterally for 10 min with non-traumatic aneurysm clips.Ten minutes later,the aneurysm clips were removed and the complete reperfusion of the arteries was verified by direct visual observation.Then the neck incision area was sutured by 4-0 nylon and ethanol for disinfection was applied.The gerbils were kept under heating lamp for 2 h until recovery.The sham-operated non-ischemia gerbils underwent the same surgical procedures,except bilateral common carotid arteries were not occluded.CQ and the vehicle were administered immediately after ischemia and then were given everyday(i.p.,10mg/kg) till sacrificed(1d,3d,7d).
     3.Morphological and molecular biological methods
     The chelation of CQ was evaluated by the ZnSe autometallography(AMG).TSQ fluorescence was used to detect the chelation of CQ in CQ-treated animals.We used Nissl staining to detect the loss of pyramidal neurons in the CA1 region of the hippocampus between vehicle-treated groups and CQ-treated groups after the surgery. TUNEL staining was used to examine whether CQ administered after ischemia can rescue the CA1 neurons.We also used in situ hybridization to detect the expression of Caspase-3 and Caspase-9 mRNA between vehicle-treated 3d groups and CQ-treated 3d groups.Furthermore,Western blot was used to detect the expression of Caspase-3 and AIF between vehicle-treated groups and CQ-treated after the surgery(1d,3d,7d).
     Results
     1.ZnSe AMG result
     Under light microscope,compared with non-CQ group,the CQ group ZnSe AMG staining intensity was decreased seriously,and the stain was lower in the region of DG and mossy fiber in hippocampus.
     2.TSQ fluorescence
     TSQ fluorescence results showed that the fluorescence in the CQ-treated group was lower than sham and vehicle-treated group after 3d of surgery.And vehicle-treated group was higher than the sham group.
     3.Nissl staining and hippocampal CA1 cell count
     Under light microscope,there was no any cell damage in sham group after 1d,3d, 7d of surgery.Cell degeneration,cell death and cellular swelling were observed in CA1 pyramidal neurons in the vehicle-treated groups.Cell number of CA1 in the CQ-treated groups was raised than that of vehicle-treated groups,the arrangement was more regularity and the shape was more integrity.
     4.TUNEL assay
     Under light microscope,there were few TUNEL positive cells in sham group after 3d of surgery.TUNEL positive cell count of CA1 pyramidal neurons in the CQ-treated groups was decreased than that of vehicle-treated groups;the TUNEL positive cells were brown.The vehicle-treated 3d group was statistically different from the CQ-treated 3d group:P<0.01.
     5.Caspase-3 and Caspase-9 in situ hybridization assay
     Under light microscope,in situ hybridization results revealed that Caspase-3 and Caspase-9 mRNA positive cells were brown.There were few Caspase-3 and Caspase-9 mRNA positive cells in sham group after 3d of surgery.Caspase-3 and Caspase-9 mRNA positive cell number of CA1 pyramidal neurons in the CQ-treated a groups was decreased than that of vehicle-treated groups.The vehicle-treated 3d group was statistically different from the CQ-treated 3d group:P<0.01.
     6.Western blot
     The expression levels of Caspase-3 and AIF protein were measured using Western blot analysis.The semiquantitative analysis of immuno-blots showed statistically significant elevations of Caspase-3 and AIF in the hippocampi of vehicle-treated groups(1d,3d,7d) compared with CQ-treated groups(1d,3d,7d).Caspase-3(17KD): the vehicle-treated groups were statistically different from the CQ-treated groups: P<0.01.Procaspase-3(32KD):the vehicle-treated groups were statistically different from the CQ-treated groups:P<0.05(1d,7d);P<0.01(3d).AIF(67KD):the vehicle-treated groups were statistically different from the CQ-treated groups:P<0.05 (3d,7d);P<0.01(1d).
     Conclusion
     1.AMG staining and TSQ fluorescence results showed that CQ had the effect of chelation of zinc.
     2.Nissl staining showed that the cell number of CA1 pyramidal neurons in the CQ-treated groups was raised than that of vehicle-treated groups.TUNEL staining showed that the TUNEL positive cell number of CA1 pyramidal neurons in the CQ-treated groups was decreased than that of vehicle-treated groups.
     3.The detection of apoptosis factors Caspase-3/Caspase-9 and AIF showed the CQ could protect the CA1 pyramidal neurons death after global ischemia in the gerbil.
引文
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