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The Effects of Hemodilution on Changes of the Pathology and Concentration of Amino Acids at Different Brain Areas after Deep Hypothermia Circulation Arrest of Rats

苏殿三 王祥瑞 郑拥军 赵延华 张挺杰

SU dian-san,WANG xiang-rui,ZHENG yongjun,et al.

Institute Of <?xml:namespace prefix = st1 ns = "urn:schemas-microsoft-com:office:smarttags" />Anesthesiology Renji Hospital,Shanghai Second Medical University,Shanghai (200001),China.

Abstract

　　Objective：To develop a rat model of deep hypothermia circulation arrest(DHCA) and to observe the effects of hemodilution on Changes of the Pathology and Concentration of Amino Acids in Different Brain Areas after DHCA.

　　Methods：48 rats were distributed randomly into four groups:Hematocrit(Hct)10% group,Hct20% group,Hct30% group and control group,12 rats in each group. All experiment groups underwent 90 minutes of DHCA at 18℃. After then we measured the changes of the pathology,and evaluated the concentration of Glutamate(Glu),Aspartate(Asp),Glycine(Gly),γ-Aminobutyric Acid(GABA) and Taurine(Tau) with the high performance liquid chromatograpy.

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　　Results：The numbers of injury neurons in the hippocampus and parietal cortex but not in the thalamus of the Hct 30% group were significantly greater than those of other groups (p<0.05). Although the concentration of the said five amino acid all increased after DHCA,the concentration of the Glu,Asp and Gly in the hippocampus and parietal cortex but not in the thalamus of the Hct 30% group were significantly less than those of other groups (p<0.05). However,the concentration of the GABA and Tau had no significant difference among the three experiment groups.

　　Conclusions：Maintaining Hct of 30% would attenuate the brain injury after DHCA,and the mechanism of this neuroprotective effects may contribute to the inhibition of the concentration of the excitatory amino acids in the brain.

　　Key words：Hemodilution;Deep Hypothermia Circulation Arrest;Amino Acid;Brain;Rats

　　深低温停循环（DHCA）是临床大血管手术、巨大脑动脉瘤手术时常用的技术，但是术中的最佳血液稀释程度一直没有定论。因此，本研究观察了不同程度血液稀释对大鼠DHCA模型不同脑区病理学和氨基酸浓度变化的影响，以探讨不同血液稀释程度与DHCA后脑损伤的关系。

材料和方法

　　1.动物及分组

　　成年健康雄性SD大鼠48只，体重400～450g(中科院上海动物中心提供)，随机均分为四组，每组12只动物：Hct10%组；Hct 20%组；Hct30%组和对照组。前三组为实验组，控制体外循环预充液的构成，将转流后动物Hct分别控制在相应的水平，对照组动物只作假手术，不行体外循环，麻醉时间与实验组相同。

　　2. 制作大鼠DHCA模型

　　（1）CPB的构成和监测

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　　CPB环路由20＃压力注射管连接储血器（10ml注射器）、血泵（兰格蠕动泵BT00-100M/YZ1515）和氧合变温器（中空纤维膜式氧合器，氧合面积为0.1m2，变温面积0.03m2，预充量为8ml，上海复旦大学生物材料有限公司提供）组成。预充液总量为20ml。Hct30%组的预充液为全血10ml、乳酸林格氏液4ml、20%甘露醇1ml、肝素1ml（100UI）、6%贺斯4ml；Hct20%组预充液为乳酸林格氏液10ml、20%甘露醇1ml、肝素1ml（100UI）、6%贺斯8ml；Hct10％组预充液成分与20%组相同，但是在转机开始前抽血10ml并同时补充乳酸林格氏液和贺斯（1:1）混合液10ml。连续测量动物直肠温度、心电图（ECG）、心率（HR）、平均动脉压（MAP），血气分析仪测定股动脉血氧分压（PaO₂）、pH值、红细胞压积（Hct）、颈静脉血氧饱和度（SjvO₂）和血乳酸（Lac）。

　　（2）动物模型的制作过程

　　术前20min肌肉注射阿托品0.03mg/kg。腹腔注射混合麻醉药2.4ml/kg（芬太尼0.005%，氯胺酮5%，氟哌利多0.25%）诱导并行气管插管，麻醉仍以该复合液维持。插管后的大鼠实行机械通气，呼吸频率60次/min，潮气量为8ml/kg。

　　分离并穿刺双侧股动脉，一侧连接动脉测压系统，另一侧用于动脉灌注。分离并穿刺右侧颈内静脉，以监测颈内静脉的血气。分离右侧颈静脉，以末端带有侧孔的14G静脉留置针穿刺（深度为3.5cm左右），使头端到达右心房水平。穿刺成功后立即开始体外循环转流（Hct10%组在转流前抽血10ml同时补充乳酸林格氏液和贺斯（1:1）混合液10ml）。转流开始时灌注流量为10ml.min^-1，逐渐将灌注流量逐渐加大到约50ml.min^-1。转流开始后停止机械通气。

　　调节变温水箱的温度，在20min内将大鼠的体温降到18℃，停止体外循环90min，依靠体表降温维持深低温状态，然后在30min内将温度复到(36~37)℃，停体外循环，恢复机械通气，保持循环呼吸稳定60min视为成功。假手术对照组仅作各种插管，不进行体外转流。

　　模型制作成功后，一半动物经灌注固定后取脑组织待测，另外一半动物不灌注直接断头取脑置入液氮保存待测。

　　3.反相高效液相色谱法检测氨基酸的浓度

　　分离液氮保存的鼠脑额顶叶皮层、海马和丘脑三部分的脑区，称重后加入两倍体积0.1M高氯酸（Hclo4）按文献1中介绍的方法检测谷氨酸（Glu）、天冬氨酸（Asp）、甘氨酸（Gly）、γ氨基丁酸（GABA）和牛磺酸（Tau）5种氨基酸的浓度^[1]。

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　　GABA和Tau是抑制性氨基酸。GABA的主要生理功能是突触前抑制兴奋性递质的释放。脑缺血后GABA和Tau升高认为是一种自我保护作用^[11-12]。本实验发现，DHCA后3个脑区GABA和Tau浓度都明显升高，但是3个实验组之间没有显著性差异，其原因还有待于进一步研究。

　　总之，在DHCA过程中将Hct值保持在30%左右具有减轻脑损伤的作用，并且这种保护作用的机制与抑制兴奋性氨基酸升高有关。

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