<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Effects of lidocaine on brain damage induced by transient global cerebral ischemia in mice of different apolipoprotein E genotypes

宋萍萍  王东信  王沛钰

SONG Ping－ping，WANG Dong－xin，WANG Pei－yu. Department of Anesthesiology，Peking University First Hospital，Beijing 100034，China Corresponding author：WANG Dong－xin

Abstract

　　0bjective：To evaluate the effects of lidocaine on changes of neuropathological outcome as well as the learning and memory abilities induced by transient global cerebral ischemia in mice of different apolipoprotein E genotypes.

　　Methods：Transient global ischemia was induced by bilateral common carotid arteries occlusion（BCCAO）for a period of 17 minutes. Healthy male C57BI/6J wild－type mice（C57mice）and apolipoprotein E knockout mice（apoE mice）were randomly divided into six groups：C57 control group（n＝15，undergoing sham operation，neither BCCAO was performed nor pharmacologic intervention was given），C57 ischemia group（n＝21，BCCAO for 17 minutes was performed and normal saline was given intraperitoneally），C57 lidocaine group（n＝22，BCCAO for 17 minutes was performed and lidocaine was given intraperitoneally），apoE control group（n＝15，undergoing the same procedure as that of the C57 control group），apoE ischemia group（n＝19，undergoing the same procedure as that of the C57 ischemia group），apoE lidocaine group（n＝16，undergoing the same procedure as that of the C57 lidocaine group）. The mice were allowed to recover for 7 days. Twenty－eight mice were randomly selected from these 6 groups for neuropathological studies on the 7th postoperative day. The percentage of ischemic neurons in the CA1 region of hippocampus was calculated. Morris water maze tasks were performed for the rest mice from the 8th postoperative day. Mice were tested four times daily for 5 consecutive days. The latency period was recorded and the percentage of effective search strategies were calculated.

 <?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

　　Results：（1）The percentage of ischemic neurons in the CA1 region of hippocampus was 0.3％±0.1％ in the C57 control group，19.3％±4.5％ in the C57 ischemia group，36.9％±2.5％ in the C57 lidocaine group，0.6％±0.3％ in the apoE control group，65.5％±2.2％ in the apoE ischemia group，and 39.4％±6.5％ in the apoE lidocaine group，significantly higher in the ischemia and lidocaine groups than in the corresponding control groups（all P<0.01），significantly higher in the C57 lidocaine and apoE ischemia groups than in the C57 ischemia group（both P<0.01），however，significantly lower in the apoE lidocaine group than in the apoE ischemia group（both P<0.01）.（2）The latency period decreased significantly along with the test days in all groups except in the apoE ischemia group（P<0.05 or 0.01），significantly longer in the ischemia and lidocaine groups than in the corresponding control groups（P <0.05 or 0.01），significantly longer in the C57 lidocaine group than in the C57 ischemia group on the 3 rd day of test[73.1（22.1－120.1）s vs.40.2 （28.4－91.1）s]，and in the apoE ischemia group than in the C57 ischemia group on the 3 rd and 4 th days of test[88.2（41.0－120.1）s vs.40.2（28.4－91.1）s，and 78.2（32.9－120.1）s vs.46.3（11.6－ 81.9）s]（P<0.05 or 0.01），and，however，significantly shorter in the apoE lidocaine group than in the C57 lidocaine group on the 3rd day of test[39.0（15.5－103.5）s vs.73.1（22.1－120.1）s]，and in the apoE lidocaine group than in the apoE ischemia group from the 3 rd to the 5 th days of test [39.0（15.5. 103.5）s vs.88.2（41.0－120.1）s，24.9（11.8－68.0）s vs.78.2（32.9－120.1）s，and 29.1（6.6－ 57.2）s vs.66.3（14.2－97.0）s respectively]（P<0.05 or 0.01）.（3）The percentage of effective search strategy increased significantly along with the test day in all groups except in the C57 lidocaine and apoE ischemia groups（P<0.05 or 0.01），significantly lower in the ischemia and lidocaine groups than in the corresponding control groups（P<0.05 or 0.01），significantly lower in the C57 lidocaine group than in the C57 ischemia group on the 4 th and 5 th days of test[25（0－50）％ vs.50（25－75）％ and 37.5（0－75）％ vs.50（50－100）％），and in the apoE ischemia group than in the C57 ischemia group from the 3 rd to the 5th days of test[25（0－25）％ vs.50（25－75）％，25（0－25）％ vs.50（25－75）％，and 25（0－5O）％ vs.50（50－100）％ respectively]（P<0.05 or 0.O1），and，however，significantly higher in the apoE lidocaine group than in the C57 lidocaine group[50（0－75）％ vs.25（0－50）％ and 50（25－100）％ vs.37.5.（0－75）％]，and in the apoE lidocaine group than in apoE ischemia group[50（0－75）％ vs.25 （0－25）％ and 50 （25－100）％ vs.25 （0－50）％ ]on the 4 th and 5 th days of test（all P<0.05）.

　　Conclusion：Transient global cerebral ischemia causess significant brain damage，which is more severe in the apoE mice than in the C57 mice.Lidocaine significantly worsens the ischemic brain damage in the C57 mice，and，however，significantly alleviates the ischemic cerebral result in the apoE mice.

　　Key words：Apolipoproteins E；Brain ischemia；Lidocaine；Brain injuries；Mice，knockout

　　轻度脑缺血损害后运动功能障碍可逐渐恢复，但患者常遗留学习记忆能力减退^[1]。载脂蛋白E（apoE）基因型与神经损伤和学习记忆功能减退关系密切。动物研究显示，与apoE基因正常的野生型小鼠相比，apoE基因缺陷的小鼠会在一定月龄后出现学习记忆功能减退^[2]，且对脑缺血损害更为敏感^[3]。临床研究发现，apoE.84基因携带者易于在老年期出现认知功能减退^[4]。以往的研究表明利多卡因对于缺血性脑损害具有保护作用^[5，6]。但利多卡因对不同apoE基因型动物的神经保护作用有无差异尚不明确。本研究的目的是观察利多卡因对不同apoE基因型小鼠短暂全脑缺血后神经病理结果和学习记忆功能变化的影响。

 <?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

材料与方法

　　一、实验动物

　　健康的雄性C57BL/6J野生型小鼠（C57小鼠）和apoE敲除型小鼠（apoE小鼠），周龄8～10周，由北京大学医学部实验动物中心提供。实验前饲养1周以适应环境。饲养室室温22～24℃，相对湿度50％～60％，12 h明暗交替光照。

　　二、动物模型和实验步骤

　　1.小鼠短暂全脑缺血模型：以3％异氟醚、60％氧化亚氮和氧的混合气进行吸入麻醉诱导。小鼠翻正反射消失后保持自主呼吸，将混合气中的异氟醚浓度降至1％用面罩吸入维持麻醉。术中保持肛温为37.5～38.5℃。经腹腔穿刺置管接微量泵持续腹腔内给药。颈部正中切口，暴露双侧颈总动脉，以微动脉夹夹闭17 min，之后松开恢复血流，缝合伤口。

　　2.动物分组：116只小鼠（其中C57小鼠66只，apoE小鼠50只）。C57小鼠中随机抽取8只进行利多卡因血药浓度检测。剩余108只小鼠随机分为6组：（1）C57对照组（15只），进行全部手术操作，但不夹闭双侧颈总动脉，不给药物干预；（2）C57缺血组（21只）：进行手术并夹闭双侧颈总动脉17min，经腹腔给予生理盐水；（3）C57利多卡因组（22只）：进行手术并夹闭双侧颈总动脉17 min，经腹腔给予利多卡因；（4）apoE对照组（15只），进行全部手术操作，但不夹闭双侧颈总动脉，不给药物干预：（5）apoE缺血组（19只）：进行手术并夹闭双侧颈总动脉17 min，经腹腔给予生理盐水；（6）apoE利多卡因组（16只）：进行手术并夹闭双侧颈总动脉17min，经腹腔给予利多卡因。

　　3.利多卡因给药：利多卡因组小鼠在缺血开始前5 rain经腹腔注射利多卡因（溶于生理盐水，浓度1mg/m1）1.5ms/kg，继以2 mg/kg^－1/h^－1 持续输注直至再灌注后10 min。缺血组小鼠在同样时间以相同的容量和速度给予生理盐水。

　　三、麻醉和手术操作对动物模型的影响观察

　　随机选取C57对照组小鼠2只、C57缺血组小鼠4只、C57利多卡因组小鼠4只、apoE对照组小鼠2只、apoE缺血组小鼠4只和apoE利多卡因组小鼠4只。除完成上述手术操作外，还在右股动脉穿刺置管，连续监测整个手术过程中的平均动脉压，并在开放颈总动脉10 min后抽取动脉血进行血气分析和血糖测定。这些动物不再参加以后的实验。

 <?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

6. Wang D，Wu X，Zhou Y，et a1. Lidocaine improving cerebral protection provided by retrograde cerebral perfusion. Chin Med J （Eng1），1998，l1l：880－890.

7. B1ock F. Global isehemia and behavioural deficits. Prog Neurobi0l. 1999，58：279－295.

8. Duchen LW. General pathology of neurons and neuroglia. In：Adams JH，Duchen LW. eds. Greenfield's neuropathology. 5th ed. London：Edward Amold.1992.1－68.

9. D'Hooge R. De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev，2001，36：60－9O.

10. Yang G，Kitagawa K，Matsushita K，et a1.C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains：selective neuronal death in the murine transient forebrain ischemia. Brain Res，1997，752：209－2l8.

11. Wellons JC 3rd，Sheng H，Laskowitz DT.et a1.A comparison of strain－related susceptibility in two murine recovery models of global cerebral ischemia. Brain Res，2000，868：14－21.

12. Horsburgh K，Kelly S，McCulloch J，et al. Increased neuronal damage in apolipopmtein E－deficient mice following global ischemia. Neuroreport. 1999，l0：837.841.

13. Hodges H，Nelson A，Virley D，et al. Cognitive deficits induced by global cerebral ischaemia： prospects for transplant therapy Pharmacol Biochem Behav，1997，56：763－780.

14. Laskowitz DT，Sheng H，Bart R，et al. Apolipoprotein E－deficient mice have increased susceptibility to focal cerebral ischemia. J Cereb Blood Flow Metab，1997，17：753－758.

15. Horsburgh K. McCulloch J，Nilsen M，et al. Intraventricular infusion of apolipoprotein E ameliorates acute neuronal damage after global cerebral ischemia in mice. J Cereb Blood Flow Metab，2000，20：458－462.

16. Wang D，Wu X，Li J，et al. Effect of lidocaine on early postoperative cognitive dysfunction after coronary artery bypass surgery. Anesth Analg，2002，95：1134－1141.

17. Song RP.Wu XM. Effect of lidocaine on hippocampus neuronal apoptosis after transient global cerebral ischemia. Chin J Anesthesio1.2O00.20：546－548.

18. 宋瑞平，吴新民.利多卡因对短暂脑缺血后海马区细胞凋亡的影响.中华麻醉学杂志，2000，20：546－548.

19. Franklin JL. Johnson EM Jr. Suppression of programmed neuronal death by sustained elevation of cytoplasmic calcium. Trends Neurosci，1992，15：501－508.

20. Koike T，Martin DP，Johnson EM Jr.Role of Ca₂⁺ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic－factor deprivation：evidence that levels of internal Ca₂⁺ determine nerve growth factor dependence of sympathetic ganglion cells. Proc Natl Acad Sci USA.1989.86：6421－6425.

21. Cao DO，Wu XM，Du MY. Effect of lidocaine on calcium currents mediated by NMDA in rat hippocam pal neurons.Chin J Anesthesiol. 2004.24：447－449. 21.

22. 曹殿青，吴新民，杜敏逸. 利多卡因对大鼠海马锥体神经元 NMDA介导钙电流的影响. 中华麻醉学杂志，2004，24：447－449.