麝香酮对急性脑缺血损伤大鼠神经元谷氨酸转运体的影响
|
摘要
大量动物实验表明，急性脑缺血状态下，神经元谷氨酸转运体（EAAC1）发生逆向转运，导致细胞外谷氨酸升高。谷氨酸激活突触后兴奋性氨基酸受体介导兴奋性氨基酸毒性，级联反应以此为始触发钙超载、炎症、程序性细胞死亡等下游一系列反应事件的发生,导致神经元死亡。虽然各种有效降低兴奋毒性的方法，均能有效保护神经元，但兴奋性氨基酸受体拮抗剂由于有严重毒副作用，临床使用受到限制。中西医结合治疗急性缺血性中风具有广泛前景。早期选用芳香开窍方案治疗，已证实有较好的疗效。本研究从EAAC1角度，探讨了芳香开窍药物麝香的主要成份—麝香酮对急性脑缺血损伤的神经保护作用及可能作用机理。
目的 论证EAAC1对急性脑缺血损伤大鼠脑损伤的促进作用，探讨麝香酮对急性脑缺血损伤大鼠的神经保护作用并阐明其可能机理。
方法 采用脑内微量注射技术向大鼠注射EAAC1反义寡核苷酸（Antisense），建立大脑中动脉栓塞（MCAO）模型，运用Western blot法和TTC染色观察缺血区EAAC1表达和梗塞体积，探讨EAAC1对急性脑缺血损伤大鼠的影响；在此基础上，运用TTC染色和透射电镜，观察麝香酮对MCAO大鼠梗塞体积和皮层超微结构的作用；为探讨麝香酮作用机理，运用RT-PCR法和Western blot法观察麝香酮对MCAO大鼠海马和大脑皮层EAAC1基因表达、蛋白表达的影响；同时，通过DNA重组技术建立了反映EAAC1功能的细胞模型，观察不同浓度麝香酮对EAAC1逆转运活性的影响。
结果
反义寡核苷酸(Antisense)组EAAC1表达低，梗塞体积为105.67±8.7mm3，而正义寡核苷酸（Sense）组和生理盐水（NS）组EAAC1表达高，梗塞体积分别为180.83±15.7 mm3和182.67±13.7 mm3。Antisense组梗塞体积明显小于各对照组（P<0.01）。提示EAAC1可促进急性缺血性中风脑损伤。
MCAO模型组脑梗塞体积为182.7±18.5mm3，麝香酮组脑梗塞体积为131.2±19.6mm3,差别有显著性意义（P<0.01）。电镜显示 MCAO组核染色质丢失，胞质溶解破坏。麝香酮组见线粒体，内质网扩张，线粒体肿涨，部分有嵴丢失，核染色质正常,较模型组病变明显为轻。提示麝香酮对MCAO大鼠具有神经保护作用。
与假手术组相比，MCAO大鼠海马EAAC1mRNA表达在缺血6小时、24小时持续增高（p<0.01）。大脑皮层EAAC1mRNA表达在缺血24小时表达升高（p<0.01）。在大鼠海马和大脑皮层，EAAC1蛋白在缺血24小时表达升高(p<0.01)。麝香酮可下调海马和大脑皮层EAAC1mRNA表达和蛋白表达的升高（p<0.05或p<0.01）。与EAAC1逆转运模型组相比，麝香酮浓度为0.01%和0.02%时，对EAAC1逆转运活性无影响（p>0.05），浓度为0.04%和0.08%时可抑制EAAC1逆转运活性（p<0.01），至浓度为0.16%时抑制作用开始下降（p<0.01）。提示麝香酮可下调EAAC1表达和抑制EAAC1逆向转运。
结论：(1) EAAC1可促进急性缺血性中风脑损伤。（2）麝香酮可减少MCAO大鼠梗塞体积，改善缺血皮层超微结构变化。（3）麝香酮的作用可能与下调EAAC1表达和抑制EAAC1逆向转运有关。
Neuronal death after cerebral is determined by damage cascade including exitotoxicity, calcium over- loading, oxygen free radical expression, inflammation and so on. During cerebral ischemia, there is a rise in extracelluar cerebral glutamate content that activate glutamate- mediated channels to neurotoxic levels. Recent studies demonstrate that reversal of neural excitatory amino acid transporter(EAAC1) instead of uptake to be a principal cause for elevated extracellular glutamate levels that induce excitotoxity and inniate damage cascade. Therefore, anti-excitotoxity possesses the action of neuro -protection. Xingnaokaiqiao formula was wildly used in the early stage of cerebral ischemia. In this study , we observed whether Muscone could attenuate EAAC1 expression and reverse uptake during cerebral ischemia and participate in neuprotection .Further, the research would suggest a new clew for anti-excitotoxity.
Methods Brain microinjection of neural excitatory amino acid transporter(EAAC1) antisense oligodeoxyneucleotide（EAAC1 antisense）, TTC staining and western blot analysis were adopted for probing the effects of EAAC1 on the brain injury in rat model with MCAO. In order to study its neuroprotection,Muscone was given(1mg/kg b.w) by gavage before middle cerebral artery occlusion(MCAO) operation. At 24h after MCAO, brain tissues were taken by decapitation and infarction volumes were qualified by TTC staining. The change of ultrastructure with cerebral cortex were observed by electron microscope.
Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of different group. In order to study Muscone's neuroprotection mechanism, Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of
different group. At the same time,the effect of muscone (0.01% to 0.16%) on reversed uptake glutamate was examined.
Results: After microinjection of EAAC1 antisense into the central area of cerebral ischemia , the expression of EAAC1 was nearly depressed （P<0.01）and the volume of brain ischemic infarct was reduced（P<0.01）.Treatment with Muscone significantly reduced the infarction volume and ameliorate the damage of ultrastructure. RT-PCR and western blot analysis showed that EAAC1 mRNA expression began to increase gradually at 6hr（p<0.01） while protein expression was higher at 24hr only in hippocampus after MCAO（p<0.01） .Both EAAC1 mRNA in cerebral cortex and protein expression in hippocampus and cerebral were higher at 24hr only after MCAO（p<0.01）. In the Muscone-treated rats , a marked reduction was shown in higher levels of EAAC1 mRNA levels and protein levels at different time after MCAO（p<0.05or p<0.01）.Muscone at concentration 0.04% and 0.08%began to attenuate EAAC1 reverse uptake（p<0.01）while at concentration 0.16%, its action began to decreased（p<0.05）.
Conclution our data indicated that EAAC1 is related to brain damage during cerebral ischemia. Muscone could attenuate infarct volume and ameliorate the damage of ultrastructure with cerebral cortex, so it possesses the action of neuprotection. Moreover, the neuroprotection is possibly related to attenuate EAAC1 expression and reverse uptake during cerebral ischemia
目的 论证EAAC1对急性脑缺血损伤大鼠脑损伤的促进作用，探讨麝香酮对急性脑缺血损伤大鼠的神经保护作用并阐明其可能机理。
方法 采用脑内微量注射技术向大鼠注射EAAC1反义寡核苷酸（Antisense），建立大脑中动脉栓塞（MCAO）模型，运用Western blot法和TTC染色观察缺血区EAAC1表达和梗塞体积，探讨EAAC1对急性脑缺血损伤大鼠的影响；在此基础上，运用TTC染色和透射电镜，观察麝香酮对MCAO大鼠梗塞体积和皮层超微结构的作用；为探讨麝香酮作用机理，运用RT-PCR法和Western blot法观察麝香酮对MCAO大鼠海马和大脑皮层EAAC1基因表达、蛋白表达的影响；同时，通过DNA重组技术建立了反映EAAC1功能的细胞模型，观察不同浓度麝香酮对EAAC1逆转运活性的影响。
结果
反义寡核苷酸(Antisense)组EAAC1表达低，梗塞体积为105.67±8.7mm3，而正义寡核苷酸（Sense）组和生理盐水（NS）组EAAC1表达高，梗塞体积分别为180.83±15.7 mm3和182.67±13.7 mm3。Antisense组梗塞体积明显小于各对照组（P<0.01）。提示EAAC1可促进急性缺血性中风脑损伤。
MCAO模型组脑梗塞体积为182.7±18.5mm3，麝香酮组脑梗塞体积为131.2±19.6mm3,差别有显著性意义（P<0.01）。电镜显示 MCAO组核染色质丢失，胞质溶解破坏。麝香酮组见线粒体，内质网扩张，线粒体肿涨，部分有嵴丢失，核染色质正常,较模型组病变明显为轻。提示麝香酮对MCAO大鼠具有神经保护作用。
与假手术组相比，MCAO大鼠海马EAAC1mRNA表达在缺血6小时、24小时持续增高（p<0.01）。大脑皮层EAAC1mRNA表达在缺血24小时表达升高（p<0.01）。在大鼠海马和大脑皮层，EAAC1蛋白在缺血24小时表达升高(p<0.01)。麝香酮可下调海马和大脑皮层EAAC1mRNA表达和蛋白表达的升高（p<0.05或p<0.01）。与EAAC1逆转运模型组相比，麝香酮浓度为0.01%和0.02%时，对EAAC1逆转运活性无影响（p>0.05），浓度为0.04%和0.08%时可抑制EAAC1逆转运活性（p<0.01），至浓度为0.16%时抑制作用开始下降（p<0.01）。提示麝香酮可下调EAAC1表达和抑制EAAC1逆向转运。
结论：(1) EAAC1可促进急性缺血性中风脑损伤。（2）麝香酮可减少MCAO大鼠梗塞体积，改善缺血皮层超微结构变化。（3）麝香酮的作用可能与下调EAAC1表达和抑制EAAC1逆向转运有关。
Neuronal death after cerebral is determined by damage cascade including exitotoxicity, calcium over- loading, oxygen free radical expression, inflammation and so on. During cerebral ischemia, there is a rise in extracelluar cerebral glutamate content that activate glutamate- mediated channels to neurotoxic levels. Recent studies demonstrate that reversal of neural excitatory amino acid transporter(EAAC1) instead of uptake to be a principal cause for elevated extracellular glutamate levels that induce excitotoxity and inniate damage cascade. Therefore, anti-excitotoxity possesses the action of neuro -protection. Xingnaokaiqiao formula was wildly used in the early stage of cerebral ischemia. In this study , we observed whether Muscone could attenuate EAAC1 expression and reverse uptake during cerebral ischemia and participate in neuprotection .Further, the research would suggest a new clew for anti-excitotoxity.
Methods Brain microinjection of neural excitatory amino acid transporter(EAAC1) antisense oligodeoxyneucleotide（EAAC1 antisense）, TTC staining and western blot analysis were adopted for probing the effects of EAAC1 on the brain injury in rat model with MCAO. In order to study its neuroprotection,Muscone was given(1mg/kg b.w) by gavage before middle cerebral artery occlusion(MCAO) operation. At 24h after MCAO, brain tissues were taken by decapitation and infarction volumes were qualified by TTC staining. The change of ultrastructure with cerebral cortex were observed by electron microscope.
Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of different group. In order to study Muscone's neuroprotection mechanism, Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of
different group. At the same time,the effect of muscone (0.01% to 0.16%) on reversed uptake glutamate was examined.
Results: After microinjection of EAAC1 antisense into the central area of cerebral ischemia , the expression of EAAC1 was nearly depressed （P<0.01）and the volume of brain ischemic infarct was reduced（P<0.01）.Treatment with Muscone significantly reduced the infarction volume and ameliorate the damage of ultrastructure. RT-PCR and western blot analysis showed that EAAC1 mRNA expression began to increase gradually at 6hr（p<0.01） while protein expression was higher at 24hr only in hippocampus after MCAO（p<0.01） .Both EAAC1 mRNA in cerebral cortex and protein expression in hippocampus and cerebral were higher at 24hr only after MCAO（p<0.01）. In the Muscone-treated rats , a marked reduction was shown in higher levels of EAAC1 mRNA levels and protein levels at different time after MCAO（p<0.05or p<0.01）.Muscone at concentration 0.04% and 0.08%began to attenuate EAAC1 reverse uptake（p<0.01）while at concentration 0.16%, its action began to decreased（p<0.05）.
Conclution our data indicated that EAAC1 is related to brain damage during cerebral ischemia. Muscone could attenuate infarct volume and ameliorate the damage of ultrastructure with cerebral cortex, so it possesses the action of neuprotection. Moreover, the neuroprotection is possibly related to attenuate EAAC1 expression and reverse uptake during cerebral ischemia
引文
1. Fisher M. Characterizing the target of acute stroke therapy. Stroke 1997 Apr;28(4):866-72
2. Hakim AM. The cerebral ischemic penumbra. Can J Neurol Sci 1987 Nov;14(4):557-9
3. Fisher M,Schaebitz W.An overview of acute stroke therapy: past, present, and future.Arch Intern Med,2000,160(21):3196-3206
4. Knai,Y.,Trotti,D.,Nussberger,S.,etc. The high-affinity glutamate transporter family.In:Reith,M.E.A,NeurotransmitterTranspoeters:Structure,Function,and Human Press[M].New Jercy: 1997.pp.171-213
5. David J.Rossi,Takeo Oshima, David Attwell. Glutamate in severe brain ischemia is mainly by reversed uptake[J]. Nature,2000,403(6767):316-321
6. 刘亚敏,张赐安,彭胜权。开窍法治疗中风的临床再认识. 新中医，2002，34（8）：7—8
7. 蒋振亚，李常度，周东等.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技 ,2001；8(2)：96-97
8. Zea-longa E,Weinstein PR,Carlson, et al. Reversible middle artery artery occlusion without craniectomy in rats.Stroke,1989,20(1): 84-91
9. Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996 ，16(3):675-86
10. 李瑾翡,王宁生,陆惠文等.麝香酮药代动力学研究.中药新药与临床药理.2000,11(4)：208—210
11. 包新民，舒斯云. 大鼠脑立体定位图谱. 北京，人民卫生出版社. 1991; 20—40
12. F Tao, SD Lu, LM Zhang, etal . Role of excitatory amino acid transporterⅠin neonatal rat neuronal damage induced by hypoxia-ischemia. Neuroscience,2001, 102(3):503-513
13. Zhang YG,Liu TP,Qian ZY, et al.Influence of total saponins of Panax ginseng on infarct size and polyamine contents in rat brain after middle cerebral artery occlusion.Chinese Journal Pharmacology and Toxicology,1994,8(4):250-255
14. 汤耀法,晏义平.脑内缺血区域体积的定量估测.上海医科大学学报,1996,23: 422
Swanson RA, Morton MT, Tsao-Wu G, etal. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab,
15. 1990 ,10(2):290-2903
16. 徐运.实验性脑缺血的氨基酸水平及超微结构变化.临床神经学杂志，1997，10（5）：278—280
17. Zhao JW, Yang XL. Glutamate transporter EAAC1 is expressed on Muller cells of lower vertebrate retinas. J Neurosci Res, 2001 ,66(1):89-95
18. Nishizawa Y. Glutamate release and neuronal damage in ischemia. Life Sci, 2001,69(4):369-81
19. 沈思钰. 醒脑开窍方药对急性缺血性中风兴奋性氨基酸受体及基因表达的影响.复旦大学博士学位论文，2002：49
20. Cain ME,Ambos HD,Witkowski FX,etal.Fast-Fourier transform analysis of signal-averaged electrocardiograms for identification of patients prone to sustained ventricular tachycardia.circulation,1984, 69(4):711-720
21. Haberl R,Jilge G,Pulter R,et al.Comparison of frequency and time domain analysis of the signal-averaged electrocardiogram in patients with ventricular tachycardia and coronary artery disease: methodologic validation and clinical relevance.J Am Coll Cardiol,1988, 12(1):150-158
22. Buckingham TA, Thessen CM, Hertweck D,et al.Signal-averaged electrocardiography in the time and frequency domains.Am J Cardiol,1989,63(12):820-825
23. Nagata K,Yunoki K,Araki G,et al.Topographic electroencephalographic study of
transient ischemic attacks.Electroencephalogr Clin Neurophysiol,1984,58(4):
291-301
24. Kanai Y, Hediger MA. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 1992 ,360(6403):467-471
25. Arriza JL, Eliasof S, Kavanaugh MP, et al. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A ,94(8):4155-60
26. Danbolt NC, Storm-Mathisen J, Kanner BI. An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience,51(2):295-310
27. Storck T, Schulte S, Hofmann K, et al. Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci U S A 1992 Nov 15;89(22):10955-9
Fairman WA, Vandenberg RJ, Arriza JL,et al.An excitatory amino-acid
28. transporter with properties of a ligand-gated chloride channel. Nature ,375(6532):599-603
29. Torp R, Danbolt NC, Babaie E,et al. Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study. Eur J Neurosci 1994 Jun 1;6(6):936-42
30. Rothstein JD, Martin L, Levey AI, et al.Localization of neuronal and glial glutamate transporters. Neuron,13(3):713-25
31. 晏义平，孙凤艳. 谷氨酸转运体的结构和功能.生命科学, 1999, 11(增刊):20-22
32. 吕辉，徐天乐. 兴奋性氨基酸转运体研究进展. 中国药理学报, 2000,16(1):22-25
33. Mennerick S, Zorumski CF. Glial contributions to excitatory neurotransmission in cultured hippocampal cells. Nature,368(6466):59-62
34. Ottersen OP, Laake JH, Reichelt W, et al. Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses. J Chem Neuroanat, 1996 ,12(1):1-14
35. Vemuganti L,Raghavendra Rao, Adibhatla Muralikrishna Rao, et al. Glial glutamate transporter GLT-1 down regul-ation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia.Neurochemistry International, 2000, 36(6): 531-537
36. Marek Szakowski,Boris Barbour,David Attwell. Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature, 1990,348(6300):443-446.
37. David J.Rossi,Takeo Oshima, David Attwell. Glutamate in severe brain ischemia is mainly by reversed uptake. Nature,2000,403(6767):316 -321
38. David Attwell. Brain uptake of glutamate:food for thought. Journal of nutrition, 2000,130(4S,Suppl):1023-1025.
39. Glauser M, Michetti P, Blum AL, et al. Carbon-14-urea breath test as a noninvasive method to monitor Helicobacter felis colonization in mice. Digestion, 1996,57(1):30-34.
40. Fox JG, Correa P, Taylor NS, et al. Helicobacter mustelae-associated gastritis in ferrets. An animal model of Helicobacter pylori gastritis in humans. Gastroenterology 1990 ,99(2):352-361
Handt LK, Fox JG, Stalis IH, et al. Characterization of feline Helicobacter pylori strains and associated gastritis in a colony of domestic cats. J Clin Microbiol
41. 1995 ,33(9):2280-2289
42. Hirayama F, Takagi S, Yokoyama Y, et al. Establishment of gastric Helicobacter pylori infection in Mongolian gerbils. J Gastroenterol ,1996 ,31 Suppl 9:24-28
43. Matsumoto S, Washizuka Y, Matsumoto Y, et al. Induction of ulceration and severe gastritis in Mongolian gerbil by Helicobacter pylori infection. J Med Microbiol ,1997 , 46(5):391-397
44. 董劲松，赵宏，陈伯英.c-fos 反义寡聚核苷酸对脑缺血性损伤和电针抗损伤作用的影响. 中国神经科学杂志,2001，17（2）：194-197
45. Kobayashi S, Millhorn DE. Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells. J Neurochem 2001 Mar;76(6):1935-1948
46. Danbolt NC. Glutamate uptake. Progress in Neurology 2001；65(1): 1-105.
47. Back T. Pathophysiology of the ischemic penumbra--revision of a concept. Cell Mol Neurobiol ,18(6):621-638
48. Rafalowska J. Experimental and human ischaemia: is the penumbra present in human ischaemic stroke? Folia Neuropathol, 40(4):211-217
49. Yepes MS. Thrombolytics and neuroprotective agents in the treatment of the patient with an acute cerebrovascular ischemic accident. Rev Neurol 2001 ,32(3):259-66
50. Fisher M,Schaebitz W.An overview of acute stroke therapy: past, present, and future.Arch Intern Med,2000,160(21):3196-3206
51. 安祯祥.治疗缺血性中风病的中药药理研究现状及前景展望. 贵阳中医学院学报，24（1）：60-62
52. 吴贻谷主编.中国医学百科全书中药学分册.上海：上海科学技术出版社,1991:358－359
53. 蒋振亚，李常度，周东等.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技 ,2001；8(2)：96-97
54. 李仪奎 等. 麝香对中枢耐缺氧能力的影响. 中草药，1985，16（3）：19-21
55. 罗海燕,万文成,陈洁文等.醒脑静对脑缺血大鼠大脑皮质超微结构的保护作用.中医药研究,2000,16(4):37-38
56. 傅强,崔华富,孙中吉等.醒脑静注射液对脑缺血-再灌注诱导的脑神经细胞凋亡防治作用的实验研究.中国中西医结合急救杂志,2000,7(3):144-146
57. 董万超，赵伟刚，刘春华.特产研究.2001，2:48-58
韩国柱 主编.中草药药代动力学. 北京：中国医药科技出版
58. 社,1999:403-406
59. Koizumi J,Yoshida Y,Nakazawa T,et al.Experiment studies of ischemic brain edema: a new experimental model of cerebroembolism in rats in which recirculation can be introduced in the ischemic area.Jpn J stroke,1986,8:1-8
60. Duverger D, MacKenzie ET. The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age. J Cereb Blood Flow Metab 1988 ;8(4):449-61
61. Nedergaard M. Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res 1987, 7,408(1-2):79-85
62. 曹秉振. 血糖水平对大鼠急性脑缺血电镜细胞化学和超微结构的影响. 中国精神神经杂志,1992,18(4):193
63. Schmid-Elsaesser R, Hungerhuber E, Zausinger S, et al .Combination drug therapy and mild hypothermia: a promising treatment strategy for reversible, focal cerebral ischemia. Stroke 1999 Sep;30(9):1891-9
64. Andersen CS, Andersen AB, Finger S. Neurological correlates of unilateral and bilateral "strokes" of the middle cerebral artery in the rat. Physiol Behav 1991 ,50(2):263-9
65. Corbett D, Nurse S. The problem of assessing effective neuroprotection in experimental cerebral ischemia. Prog Neurobiol, 1998;54:531-548.
66. Hunter AJ, Mackay KB, Roger DC. To what extent have functional studies of ischemia in animals been useful in the assessment of potential neuroprotective agents? Trends Pharmacol Sci, 1998;19:59-66.
67. 沈思钰,蔡定芳,刘静,等.局灶性脑缺血神经行为及其相关性分析.中国行为医学科学,2002,11(1):44-45
68. Bederson JB, Pitts LH, Tsuij M, et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke, 1986;17:472-476.
69. Roger DC, Campbell CA, Stretton JL, et al. Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke, 1997;28:2060-2066.
70. Zausinger S,Hungerhuber E,Baethmann A,et al.Neurological impairment in rats after transient middle cerebral artery occlusion:a comparative study under various treatment paradigms.Brain Res,2000, 863:94-105
刘亚敏,张赐安,徐秋英,彭胜权. 麝香、冰片对全脑缺血再灌注大鼠脑组织氨
71. 基酸类神经递质的影响. 中药新药与临床，2002（4）：
72. Torp R, Danbolt NC, Babaie E,et al. Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study. Eur J Neurosci 1994 Jun 1;6(6):936-42
73. Rothstein JD, Martin L, Levey AI, et al.Localization of neuronal and glial glutamate transporters. Neuron,13(3):713-25
74. 张国瑾，赵增荣 主编. 国外医学脑血管疾病研究进展. 北京：中国医药科技出版社，2000：11-12
75. 莫启忠，宫斌，茅惠民等[J]. 3H-麝香酮在体内的吸收、分布和排泄.中成药研究 1984；（7）：1-4
76. Gottlieb M, Domercq M, Matute C. Altered expression of the glutamate transporter EAAC1 in neurons and immature oligodendrocytes after transient forebrain ischemia. J Cereb Blood Flow Metab, 2000 ,20(4):678-687
77. 高天祥,田竟生 主编. 医学分子生物学. 北京：科学出版社,1999：61-80
78. Palmada M, Bohmer C, Centelles JJ, etal . Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1. J Neurochem 1999,73(6):2389-2396
79. Kanai Y, Nussberger S, Romero MF, et al. Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter. J Biol Chem 1995 Jul 14;270(28):16561-165688
2. Hakim AM. The cerebral ischemic penumbra. Can J Neurol Sci 1987 Nov;14(4):557-9
3. Fisher M,Schaebitz W.An overview of acute stroke therapy: past, present, and future.Arch Intern Med,2000,160(21):3196-3206
4. Knai,Y.,Trotti,D.,Nussberger,S.,etc. The high-affinity glutamate transporter family.In:Reith,M.E.A,NeurotransmitterTranspoeters:Structure,Function,and Human Press[M].New Jercy: 1997.pp.171-213
5. David J.Rossi,Takeo Oshima, David Attwell. Glutamate in severe brain ischemia is mainly by reversed uptake[J]. Nature,2000,403(6767):316-321
6. 刘亚敏,张赐安,彭胜权。开窍法治疗中风的临床再认识. 新中医，2002，34（8）：7—8
7. 蒋振亚，李常度，周东等.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技 ,2001；8(2)：96-97
8. Zea-longa E,Weinstein PR,Carlson, et al. Reversible middle artery artery occlusion without craniectomy in rats.Stroke,1989,20(1): 84-91
9. Rothstein JD, Dykes-Hoberg M, Pardo CA, et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 1996 ，16(3):675-86
10. 李瑾翡,王宁生,陆惠文等.麝香酮药代动力学研究.中药新药与临床药理.2000,11(4)：208—210
11. 包新民，舒斯云. 大鼠脑立体定位图谱. 北京，人民卫生出版社. 1991; 20—40
12. F Tao, SD Lu, LM Zhang, etal . Role of excitatory amino acid transporterⅠin neonatal rat neuronal damage induced by hypoxia-ischemia. Neuroscience,2001, 102(3):503-513
13. Zhang YG,Liu TP,Qian ZY, et al.Influence of total saponins of Panax ginseng on infarct size and polyamine contents in rat brain after middle cerebral artery occlusion.Chinese Journal Pharmacology and Toxicology,1994,8(4):250-255
14. 汤耀法,晏义平.脑内缺血区域体积的定量估测.上海医科大学学报,1996,23: 422
Swanson RA, Morton MT, Tsao-Wu G, etal. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab,
15. 1990 ,10(2):290-2903
16. 徐运.实验性脑缺血的氨基酸水平及超微结构变化.临床神经学杂志，1997，10（5）：278—280
17. Zhao JW, Yang XL. Glutamate transporter EAAC1 is expressed on Muller cells of lower vertebrate retinas. J Neurosci Res, 2001 ,66(1):89-95
18. Nishizawa Y. Glutamate release and neuronal damage in ischemia. Life Sci, 2001,69(4):369-81
19. 沈思钰. 醒脑开窍方药对急性缺血性中风兴奋性氨基酸受体及基因表达的影响.复旦大学博士学位论文，2002：49
20. Cain ME,Ambos HD,Witkowski FX,etal.Fast-Fourier transform analysis of signal-averaged electrocardiograms for identification of patients prone to sustained ventricular tachycardia.circulation,1984, 69(4):711-720
21. Haberl R,Jilge G,Pulter R,et al.Comparison of frequency and time domain analysis of the signal-averaged electrocardiogram in patients with ventricular tachycardia and coronary artery disease: methodologic validation and clinical relevance.J Am Coll Cardiol,1988, 12(1):150-158
22. Buckingham TA, Thessen CM, Hertweck D,et al.Signal-averaged electrocardiography in the time and frequency domains.Am J Cardiol,1989,63(12):820-825
23. Nagata K,Yunoki K,Araki G,et al.Topographic electroencephalographic study of
transient ischemic attacks.Electroencephalogr Clin Neurophysiol,1984,58(4):
291-301
24. Kanai Y, Hediger MA. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 1992 ,360(6403):467-471
25. Arriza JL, Eliasof S, Kavanaugh MP, et al. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc Natl Acad Sci U S A ,94(8):4155-60
26. Danbolt NC, Storm-Mathisen J, Kanner BI. An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience,51(2):295-310
27. Storck T, Schulte S, Hofmann K, et al. Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci U S A 1992 Nov 15;89(22):10955-9
Fairman WA, Vandenberg RJ, Arriza JL,et al.An excitatory amino-acid
28. transporter with properties of a ligand-gated chloride channel. Nature ,375(6532):599-603
29. Torp R, Danbolt NC, Babaie E,et al. Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study. Eur J Neurosci 1994 Jun 1;6(6):936-42
30. Rothstein JD, Martin L, Levey AI, et al.Localization of neuronal and glial glutamate transporters. Neuron,13(3):713-25
31. 晏义平，孙凤艳. 谷氨酸转运体的结构和功能.生命科学, 1999, 11(增刊):20-22
32. 吕辉，徐天乐. 兴奋性氨基酸转运体研究进展. 中国药理学报, 2000,16(1):22-25
33. Mennerick S, Zorumski CF. Glial contributions to excitatory neurotransmission in cultured hippocampal cells. Nature,368(6466):59-62
34. Ottersen OP, Laake JH, Reichelt W, et al. Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses. J Chem Neuroanat, 1996 ,12(1):1-14
35. Vemuganti L,Raghavendra Rao, Adibhatla Muralikrishna Rao, et al. Glial glutamate transporter GLT-1 down regul-ation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia.Neurochemistry International, 2000, 36(6): 531-537
36. Marek Szakowski,Boris Barbour,David Attwell. Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature, 1990,348(6300):443-446.
37. David J.Rossi,Takeo Oshima, David Attwell. Glutamate in severe brain ischemia is mainly by reversed uptake. Nature,2000,403(6767):316 -321
38. David Attwell. Brain uptake of glutamate:food for thought. Journal of nutrition, 2000,130(4S,Suppl):1023-1025.
39. Glauser M, Michetti P, Blum AL, et al. Carbon-14-urea breath test as a noninvasive method to monitor Helicobacter felis colonization in mice. Digestion, 1996,57(1):30-34.
40. Fox JG, Correa P, Taylor NS, et al. Helicobacter mustelae-associated gastritis in ferrets. An animal model of Helicobacter pylori gastritis in humans. Gastroenterology 1990 ,99(2):352-361
Handt LK, Fox JG, Stalis IH, et al. Characterization of feline Helicobacter pylori strains and associated gastritis in a colony of domestic cats. J Clin Microbiol
41. 1995 ,33(9):2280-2289
42. Hirayama F, Takagi S, Yokoyama Y, et al. Establishment of gastric Helicobacter pylori infection in Mongolian gerbils. J Gastroenterol ,1996 ,31 Suppl 9:24-28
43. Matsumoto S, Washizuka Y, Matsumoto Y, et al. Induction of ulceration and severe gastritis in Mongolian gerbil by Helicobacter pylori infection. J Med Microbiol ,1997 , 46(5):391-397
44. 董劲松，赵宏，陈伯英.c-fos 反义寡聚核苷酸对脑缺血性损伤和电针抗损伤作用的影响. 中国神经科学杂志,2001，17（2）：194-197
45. Kobayashi S, Millhorn DE. Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells. J Neurochem 2001 Mar;76(6):1935-1948
46. Danbolt NC. Glutamate uptake. Progress in Neurology 2001；65(1): 1-105.
47. Back T. Pathophysiology of the ischemic penumbra--revision of a concept. Cell Mol Neurobiol ,18(6):621-638
48. Rafalowska J. Experimental and human ischaemia: is the penumbra present in human ischaemic stroke? Folia Neuropathol, 40(4):211-217
49. Yepes MS. Thrombolytics and neuroprotective agents in the treatment of the patient with an acute cerebrovascular ischemic accident. Rev Neurol 2001 ,32(3):259-66
50. Fisher M,Schaebitz W.An overview of acute stroke therapy: past, present, and future.Arch Intern Med,2000,160(21):3196-3206
51. 安祯祥.治疗缺血性中风病的中药药理研究现状及前景展望. 贵阳中医学院学报，24（1）：60-62
52. 吴贻谷主编.中国医学百科全书中药学分册.上海：上海科学技术出版社,1991:358－359
53. 蒋振亚，李常度，周东等.麝香对大鼠实验性脑缺血神经元损伤的保护作用[J].中国中医药科技 ,2001；8(2)：96-97
54. 李仪奎 等. 麝香对中枢耐缺氧能力的影响. 中草药，1985，16（3）：19-21
55. 罗海燕,万文成,陈洁文等.醒脑静对脑缺血大鼠大脑皮质超微结构的保护作用.中医药研究,2000,16(4):37-38
56. 傅强,崔华富,孙中吉等.醒脑静注射液对脑缺血-再灌注诱导的脑神经细胞凋亡防治作用的实验研究.中国中西医结合急救杂志,2000,7(3):144-146
57. 董万超，赵伟刚，刘春华.特产研究.2001，2:48-58
韩国柱 主编.中草药药代动力学. 北京：中国医药科技出版
58. 社,1999:403-406
59. Koizumi J,Yoshida Y,Nakazawa T,et al.Experiment studies of ischemic brain edema: a new experimental model of cerebroembolism in rats in which recirculation can be introduced in the ischemic area.Jpn J stroke,1986,8:1-8
60. Duverger D, MacKenzie ET. The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration, and age. J Cereb Blood Flow Metab 1988 ;8(4):449-61
61. Nedergaard M. Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res 1987, 7,408(1-2):79-85
62. 曹秉振. 血糖水平对大鼠急性脑缺血电镜细胞化学和超微结构的影响. 中国精神神经杂志,1992,18(4):193
63. Schmid-Elsaesser R, Hungerhuber E, Zausinger S, et al .Combination drug therapy and mild hypothermia: a promising treatment strategy for reversible, focal cerebral ischemia. Stroke 1999 Sep;30(9):1891-9
64. Andersen CS, Andersen AB, Finger S. Neurological correlates of unilateral and bilateral "strokes" of the middle cerebral artery in the rat. Physiol Behav 1991 ,50(2):263-9
65. Corbett D, Nurse S. The problem of assessing effective neuroprotection in experimental cerebral ischemia. Prog Neurobiol, 1998;54:531-548.
66. Hunter AJ, Mackay KB, Roger DC. To what extent have functional studies of ischemia in animals been useful in the assessment of potential neuroprotective agents? Trends Pharmacol Sci, 1998;19:59-66.
67. 沈思钰,蔡定芳,刘静,等.局灶性脑缺血神经行为及其相关性分析.中国行为医学科学,2002,11(1):44-45
68. Bederson JB, Pitts LH, Tsuij M, et al. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke, 1986;17:472-476.
69. Roger DC, Campbell CA, Stretton JL, et al. Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke, 1997;28:2060-2066.
70. Zausinger S,Hungerhuber E,Baethmann A,et al.Neurological impairment in rats after transient middle cerebral artery occlusion:a comparative study under various treatment paradigms.Brain Res,2000, 863:94-105
刘亚敏,张赐安,徐秋英,彭胜权. 麝香、冰片对全脑缺血再灌注大鼠脑组织氨
71. 基酸类神经递质的影响. 中药新药与临床，2002（4）：
72. Torp R, Danbolt NC, Babaie E,et al. Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study. Eur J Neurosci 1994 Jun 1;6(6):936-42
73. Rothstein JD, Martin L, Levey AI, et al.Localization of neuronal and glial glutamate transporters. Neuron,13(3):713-25
74. 张国瑾，赵增荣 主编. 国外医学脑血管疾病研究进展. 北京：中国医药科技出版社，2000：11-12
75. 莫启忠，宫斌，茅惠民等[J]. 3H-麝香酮在体内的吸收、分布和排泄.中成药研究 1984；（7）：1-4
76. Gottlieb M, Domercq M, Matute C. Altered expression of the glutamate transporter EAAC1 in neurons and immature oligodendrocytes after transient forebrain ischemia. J Cereb Blood Flow Metab, 2000 ,20(4):678-687
77. 高天祥,田竟生 主编. 医学分子生物学. 北京：科学出版社,1999：61-80
78. Palmada M, Bohmer C, Centelles JJ, etal . Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1. J Neurochem 1999,73(6):2389-2396
79. Kanai Y, Nussberger S, Romero MF, et al. Electrogenic properties of the epithelial and neuronal high affinity glutamate transporter. J Biol Chem 1995 Jul 14;270(28):16561-165688