短暂性全脑缺血再灌注大鼠经高压氧治疗后脑组织神经节苷脂的变化

背景:神经节苷脂是神经组织中含量丰富并可发挥神经保护作用的含唾液酸的鞘糖脂,在脑缺血或缺氧性疾患时有含量或组分的变化。目的:通过观察全脑缺血再灌注大鼠应用高压氧治疗后脑组织神经节苷脂的变化情况,探讨高压氧对缺血再灌注损伤治疗作用的可能途径。设计:随机-对照观察。单位:首都医科大学附属朝阳医院高压氧科和首都医科大学基础医学院生物化学与分子生物学系。材料:动物模型制作于2002-03/04在首都医科大学附属朝阳医院高压氧科(北京市重点实验室)完成,各项指标检测在首都医科大学基础医学院生物化学与分子生物学系完成。选择雌性SD大鼠54只,将大鼠随机分成9组,即假手术组、缺血再灌注6h,24h,48h,96h组及高压氧6h,24h,48h,96h组,每组6只。方法:除假手术组外,其余8组大鼠均建立脑缺血再灌注动物模型,按四动脉阻断法建立,缺血20min后再通。假手术组同样手术但不阻断动脉。将高压氧组大鼠置于实验舱内,纯氧洗舱5min,升压5min至0.1MPa后稳压,吸纯氧45min,减压10min。高压氧组大鼠在缺血再灌注3h后行高压氧处理1次,24h,48h及96h组在每天同一时间行高压氧处理1次。缺血再灌注组和假手术组处于常压空气环境中。再灌注组和高压氧处理组大鼠分别于再灌注6h,24h,48h及96h麻醉取全脑标本测定总神经节苷脂及各组分百分含量,神经节苷脂各组分以高效薄层层析法测定。主要观察指标:各组大鼠全脑组织神经节苷脂的总含量及其各组分的百分含量。结果:54只大鼠全部进入结果分析,无脱失。①高压氧24h及48h组总神经节苷脂均显著高于假手术组及相应缺血再灌注时相组(F=12.730,122.246,P<0.01),但缺血再灌注各时相组与假手术组相比,差异均无显著性意义(P>0.05)。②对缺血再灌注24h组大鼠GT1b相对含量显著低于假手术组(F=13.575,P<0.01),再灌注48h组GD1b及GM1分别低于假手术组(F=4.015,3.979,P<0.05),GM3于再灌注24h高于假手术组及其他时相组(F=21.450,P<0.01),并于再灌注96h反弹。③高压氧24h组GM1,GM3分别高于假手术组(F=3.970,21.450,P<0.05,<0.01),GD1a、GD1b和GT1b均低于假手术组(F=13.575,5.745,8.783,P<0.05～0.01),但GT1b明显高于缺血再灌注各相应时相组(F=8.783,P<0.05)。结论:大鼠短暂全脑缺血再灌注后可引起神经节苷脂总含量、GT1b,GD1b,GM1百分含量降低及GM3百分含量升高,其中提高脑组织神经节苷脂总量、GM1含量及加速GT1b的恢复可能为高压氧治疗改善缺血性脑损伤的作用途径,而GD1a,GD1b百分含量的下降有何作用尚不清楚。

Changes of brain gangliosides in rats transient global cerebral ischemia/reperfusion models after hyperbaric oxygen treatment

BACKGROUND: Ganglioside (Gls) is a kind of N-acetylneuraminatecontaining glycosphingolipid, which is abundant in neural tissues and exerts neuroprotective roles, and has been found its changes in content and composition pattern after cerebral ischemia or hypoxia diseases.OBJECTIVE: To investigate the effect of hyperbaric oxygen on brain gangliosides after cerebral ischemia/reperfusion and explore the possible mechanism of hyperbaric oxygen treatment to ameliorate cerebral ischemia/reperfusion damage.DESIGN: Randomized-control observation.SETTING: Department of Hyperbaric Oxygen, Chaoyang Hospital Affiliated to Capital University of Medical Sciences and Department of Biochemistry and Molecular Biology, Basic College, Capital University of Medical Sciences.MATERIALS: Animal models were established at Department of Hyperbaric Oxygen, Chaoyang Hospital Affiliated to Capital University of Medical Sciences (Key Laboratory of Beijing) from March to April 2002. All indexes were determined at the Department of Biochemistry and Molecular Biology, Basic College, Capital University of Medical Sciences. A total of 54 SD male rats were selected and randomly divided into 9 groups: shamoperated group; ischemia/reperfusion 6, 24, 48 and 96 hours group; and hyperbaric oxygen 6, 24, 48 and 96 hours group with 6 rats in each group.METHODS: Animal models with cerebral ischemic/reperfusion were established in the other 8 groups, except the sham-operation group. Global cerebral ischemia was induced by a four-vessel occlusion and reperfusion allowed after 20-minute ischemia. The rats in sham-operation group were operated in the same way except of arterial occlusion. The rats in the HBO group were placed in experimental animal chamber. After 5-minute wash by pure oxygen, the pressure of oxygen cabins was increased to 0.1 MPa in 5 minutes and kept stable for 45 minutes, then decreased to ambient level within 10 minutes. The rats in the HBO groups were treated once by hyperbaric oxygen at reperfusion 3, 24, 48 and 96 hours respectively; while the rats in the ischemia/reperfusion group and sham-operation group were placed in normal atmospheric environment. The rats of HBO and ischemia/reperfusion groups were killed by decapitation at the 6th, 24th, 48th and 96th hours respectively, and the brains removed quickly. Gls and its percentage content in each group were detected with high performance thin-layer chromatography plate.MAIN OUTCOME MEASURES: Total content of gangliosde in the whole brain tissue of rats and its percentage content.RESULTS: Totally 54 rats were involved in the result analysis without drop out. ①The contents of Gangliosides in HBO groups at 24 and 48 hours was significantly higher than those in sham-operation group and ischemia/reperfusion group at corresponding time phase (F=12.730,122.246,P ＜ 0.01), but there was no significant difference between ischemia/reperfusion and sham-operation groups (P ＞ 0.05). ② N-acetylneuraminosylgalactosyl-N-acetylgalactosaminyl- (N-acetylneuraminosylα2→8- N-acetylneuraminosyl) -galactosylglucosylceramide (GT1b) proportion in the ischemia/reperfusion 24 hours group was lower markedly than that in the S-O (F=13.575,P ＜ 0.01); Both galactosyl-N-acetylgalactosaminyl- (N-acetylneuraminosyl- α2→8-N-acetylneuraminosyl) - galactosylglucosylceramide (GD1b) and galactosyl- N-acetylgalactosaminyl-(N-acetylneuraminosyl)galactosylglucosylceramide (GM1) in the reperfusion 48 hours group were lower than those in the sham-operation group (F= 4.015,3.979,P ＜ 0.05); (N-acetylneuraminosyl)galactosylglucosylceramide (GM3)in ischemia/reperfusion 24 hours group was much higher than that in sham-operation and any other ischemia/reperfusion groups (F=21.450,P＜ 0.01 ); An unknown increase of GM3 was found again at the 96th hour;③GM1and GM3 proportion in the hyperbaric oxygen group was higher than that of sham-operation group at the 24th hour (F=3.970,21.450,P＜ 0.05, ＜ 0.01), and all of GD1a, GD1b and GT1b were lower than that in the sham-operation group at the same times (F= 13.575,5.745,8.783, P＜ 0.05-0.01), but GT1b was remarkably higher than that in ischemia/reperfusion group (F=8.783 ,P ＜ 0.05).CONCLUSION: The pattern of brain gangliosides changed after transient whole cerebral ischemia/reperfusion. The new mechanism of neuron damage after transient whole cerebral ischemia/reperfusion might be the decreasing of GT1b, GD1b and GM1 with increasing of GM3 proportion. The hyperbaric oxygen treatment could ameliorate cerebral ischemia/reperfusion damage by increasing total gangliosides content and GM1 proportion and accelerating GT1b restoration to normal level. It is unknown that the effect of percentage content of GD1a and GD1b decreased.