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吸入性一氧化氮对全猪体外循环模型肺表面活性物质的影响

时间:2010-08-24 11:35:07  来源:  作者:

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Influence of inhaled nitric oxide on surfactant in the total cardiopulmonary bypass pig model

 

王天龙1△,杨拔贤2 ,Gilbert A Blaise1

1. 加拿大蒙特利尔大学Notre-Dame 医院麻醉实验室;2. 北京大学人民医院麻醉科)

WANG Tianlong1△,YANGBaxian2,Gilbert A Blaise1

1 Labarotory of Anesthesia,Notre-Dame Hospital,University of Montreal,Canada;2 Department of Anesthesia,Peking University People’s Hospital)

 

SUMMARY

  Objective:To study whether or not inhaled nitric oxide(INO)can improve surfactant production and pulmonary mechanics after CPB in pigs,and to seek the mechanism of lung injury during mechanical ventilation.

  Methods:Thirty pigs were randomized into 6 groups:Sham(n=5),Sham + INO(n=5),CPB(n=5),CPB+INO(n=5),CPB + lipopolysaccharide(L PS)(n=5),and CPB + L PS + INO(n=5). After anesthesia induction,INO (mass fraction,20×10-6),added to the gas mixture,was given to the animals throughout the procedure. After 2 hours of INO treatment,broncho-alveolar lavage(BAL)fluid was taken for surfactant assay and cytology analysis. Pulmonary hemodynamics parameters and lung compliance were measured as well. CPB was performed for 90 minutes after the first BAL sampling at T0. Four hours(T4)and 24 hours(T24)following CPB,BAL and other measurements were repeated. After CPB,LPS (4μg/kg-1)was infused to specific groups of pigs within 90 minutes in order to stimulate the inflammation process.

  Results:Pulmonary hemodynamics parameters(pulmonary artery pressure,pulmonary vascular resistance and oxygenation)in all INO groups were much better than those of the control groups at T4 and T24. However,lung compliance of pigs in all groups declined with time,and showed statistically significant differences at T24 compared with T0. At T0,the active sub-fraction of surfactant(large aggregate,LA)was increased in animals given INO treatment compared with the controls,but decreased with time,and at T24 significantly reduced in all groups. In LPS groups,this decrease LA after T4 was very obvious. Total cell counts and the percent differentials of neutrophils in BAL increased with time,being lower in the INO groups than in other groups.

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  Conclusion:INO exposure exerted time-related effects on the lung surfactant (LA). Initially,INO resulted in short-term increase of surfactant production at T0. However,with time passing by,INO exposure following CPB did not prevent long-term decrease of lung surfactant and lung compliance although INO was beneficial for pulmonary hemodynamics and oxygenation. In summary,despite the use of INO,synergetic effect s of long-term hyperoxia,mechanical ventilation and inflammation following CPB may exacerbate pulmonary mechanics and result in surfactant dysfunction.

  Key word:Cardiopulmonary bypass;Nitric oxide;Pulmonary surfactants;Swine

 

  吸入性一氧化氮(inhaled nitric oxide,INO)具有降低肺动脉压,减少肺内分流和改善氧合的作用,同时它还具有抑制激活白细胞在肺血管内皮表面黏附并减少氧自由基产生的抗炎效应。然而,一旦炎性反应启动,超氧离子(O2-)产生,一氧化氮(NO)会具有促炎和促氧化的作用,这些负面效应使NO在肺部炎症反应病人的应用及作为心肺转流术(cardiopulmonary bypass,CPB)后病人的常规治疗方面引起争议。肺表面活性物质(surfactant,S)是维持肺功能稳定的重要因素之一,其合成减少和功能障碍都会导致肺泡萎陷(肺不张)和肺顺应性下降。INO在有益于肺血流动力学和肺氧合的基础上,是否也对肺顺应性和S具有保护作用值得探索。

  本研究使用猪模型行CPB,尽可能地接近临床过程以获得可用于人的实验数据。通过CPB 后给予小剂量内毒素[1,2],以增强CPB激发的炎性反应,并观察炎性反应增强状态下,与高氧、机械通气协同对S和肺功能的损伤效应及与时间的关联;通过支气管-肺泡灌洗液(broncho-alveolar lavage,BAL)中细胞学分析,研究该条件下中性粒细胞(polymor-phonuclear neut rophil,PMN)在肺损伤中的作用。

 

  1. 材料与方法

  1.1 实验分组

  30头杂种健康无肺疾病的阉割雄性猪(魁北克省规定所有3个月大的雄性猪均需阉割),体重30~35kg,随机分为6组,分别为假手术(Sham,n=5),Sham + INO(n=5),CPB(n=5),CPB+INO(n=5),CPB+脂多糖(LPS,n=5)和CPB+L PS+INO(n=5)。

  1.2 麻醉

  麻醉前禁食12h。为建立静脉通路,诱导前用药采用肌注阿托品0.04mg/kg、azaperone4mg/kg、氯胺酮25mg/kg;麻醉诱导采用硫喷妥钠5mg/kg、芬太尼5μg/kg、潘库溴胺0.2mg/kg行气管内插管(8mm)。麻醉维持采用芬太尼20μg/kg/h,硫喷妥钠5mg/kg/h连续静脉输注,间断推注潘库溴胺0.1mg/kg以维持良好的外科条件。

  1.3 实验程序

  麻醉诱导后即刻,INO治疗组开始吸入NO(质量分数为20×10-6)直至实验结束。插入S-G导管以监测肺和全身血流动力学;行气管切开,重新放置气管插管导管,以利支气管镜的置入。初始吸入INO后90min,下纤维支气管镜,行BAL,同时测量肺顺应性和血流动力学指标(T0)。初始数据采集完毕后劈胸骨,Sham和Sham + INO组劈胸骨后未行任何外科操作,3h后关胸。其余组则行CPB 90min,停机后关胸。CPB+L PS和CPB+L PS+INO组关胸后连续输注L PS 4μg/kg共90min。CPB后4h(T4)和24h(T24)重复所有测量和BAL。

  1.4 实验监测

  所有动物监测如前所述。血流动力学监测使用M1166ATM型66S监测仪。动静脉血气和电解质由iSTAT血气检测仪监测。呼吸数据由CapnomagUltima监测系统搜集。

  通气方式和INO吸入采用7200AETM Puritan Bennett呼吸机,间歇正压通气,呼气末正压(PEEP)5cm H2O(1cm H2O=0.098 kPa),VT 8ml kg,调节呼吸频率(RR)使呼气末二氧化碳分压(PETCO2)维持于40~50mm Hg(1mmHg=0.133kPa)。使用UltimaTM O2检测仪监测FiO2,并维持在100%,关胸后减至50%。CPB期间潮气量VT减至3ml/kg,RR减至8次 min。INO组通过NO喷射器持续喷入NO(质量分数为20×10-6)。NO/氮气输入浓度由电化学器监测。CPB期间,INO被直接加入氧合器的混合气体中。

  1.5 肺泡灌洗和表面活性物质分析

  每次通过纤维支气管镜向肺上叶内灌入20ml灭菌生理盐水3次,灌洗液滤过、离心,上清浓缩10倍,于-80℃保存,并行细胞计数及分析。使用原始肺泡提取液、150g上清液、150g小体(pellet)分别测量小聚体(SA)、总表面物质(TS)、大聚体(LA)含量。磷脂水平由Duck-Chong生化法测量。S含量表示为mg/L/kg,LA百分比为[LA/(LA+SA)]。

  1.6 灌洗液总细胞计数和形态学分类

  细胞用20ml RPMI-1640介质和1%(体积分数)FCS悬浮,于4℃1500 r/min离心8min,再用10mlRPMI-1640介质和1%FCS重新悬浮。用100倍显微镜行全细胞计数和分类计数。

  1.7 统计分析

  所有结果表示为<?xml:namespace prefix = v ns = "urn:schemas-microsoft-com:vml" /> ,组间配对采用单因素方差分析,组内采用配对t检验(T4和T24结果分别与T0比较),P<0.05为差异有显著性。

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  2 结果

  2.1 呼吸功能

  所用动物在试验过程中均存活。组间动物体重及体长差异均无显著性。配对组间猪肺每分钟通气量、PETCO2差异无显著性。呼吸系统总顺应性采用VT/呼气末平台压计算。与T0相比,各组猪肺顺应性在T24均明显降低(P<0.05,CPB+LPS和CPB+LPS+INO组在T4后,已显著下降(P<0.05,表1)。

 

 

 

  2.2 肺血流动力学和氧合指数

  肺血流动力学指标(肺动脉压,肺血管阻力)和氧合指数(PaO2/FiO2)在INO组较对照组明显改善(P<0.05,表2)。

 

  2.3 表面活性物质

  肺灌洗回收量组间差异无显著性。实验过程中,LA含量随时间而降低(P<0.0)总S呈现相同的下降趋势,但配对组间差异无显著性;SA随时间有轻度下降,但差异亦无显著性(表3)。

  2.4 INO对LA的总效应

  比较INO组(n=15)和未用INO组(n=15)猪在INO后90min(T0)LA含量的变化,INO组LA含量明显增加(P<0.05),但这种增加效应在T4后消失(图1)。

 

  2.5 灌洗液细胞学

  灌洗液总细胞计数(中性粒细胞、巨噬细胞、嗜酸性粒细胞和淋巴细胞)随时间延长而增加,各组在T24中性粒细胞分类计数明显增加,但增加幅度略低于非配对INO组(表4,5)。

 

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