Establishment of A Hypoxia Model with Apnea in Anesthetized Rats

金海龙^#　　博士研究生
徐　雪^#　　博士研究生
王保国^*　　教授
孙　异^△　　研究员
袁　芳^△　　研究员
^#首都医科大学2002级博士研究生
^*中国医学科学院首都医科大学附属北京天坛医院麻醉科，北京100050
^△北京市神经外科研究所，北京100050<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

Hai-long Jin^*, Xue Xu^*,Bao-guo Wang^*, Yi-lin Sun^△ and Fang Yuan^△
*Department of Anesthesiology,Beijing Tiantan Hospitalm, Capital University of Medicai Sciences, Chinese Academy of Medical Sciences, Beijing 100050
^ΔBeijing Neurosurgical Institute, Beijing 100050

ABSTRACT

　　Objective：To establish a hypoxia model with apnea in anesthetized rats.
　　Methods：144 male Wistar rats, 260～290g in body weight, were randomly allocated to 8 groups：group I, II, III, IV, V, VI, VII and sham, with 18 rats in each group. The rats were anesthetized, paralyzed and mechanically ventilated. Apnea was induced by disconnecting the tracheal tube with ventilator for1,2,3,4,5,6and7min, respectively, then the ventilator was reconnected and cardiopulmonary resuscitation began if cardiac arrest happened. The sham group received anesthesia, mechanical ventilation without apnea. Blood pressure, heart rate, mortality, time of cardiac arrest (T_CA) and return of spontaneous circulation (T_ROSC) were recorded. Neural deficit scores(NDS)were evaluated at 24,48and 72h after resuscitation.CA1 region of left hippocampus were removed 3days later for electron microscope examination.
　　Results: No cardiac arrest occurred in sham, I and II groups; all the rats underwent cardiac arrest in group III, IV, V, VI and VII, and TCA were 142.8±14.7sec,144.1±13.6sec,146.3±13.7sec, 148.3±12.4sec, and 146.6±13.7sec, respectively, without significant differences among groups（P＞0.05）.All the rats in sham, I,II and III groups survived, while the mortality in group IV,V and VI were 33.3%、61.1% and 83.3%,respectively, and no rats survived in group VII.T_ROSC in group III, IV,V and VI　were　4±14.8sec,　56.9±17.0sec,　56.9±17.　0sec and 91.0±13.5sec, respectively, T_ROSC were significantly longer in group IV,V,VI than in group III（P＜0.05).NDS in sham, I,II and III groups remained unaffected while the scores were significantly lower in IV,V and VI group than in control group（P＜0.01).Electron microscope examination of hippocampus CA1 region showed graded injury of neuron , organelle and astrocyte with the prolongation of apnea.
　　Conclustion: An hypoxia animal model was established with 4minutes apnea, which provides a stable and repeatable tool for investigating the related mechanisms.
　　Key words: Anesthesia; Hypoxia model; Apnea; Rat
　　Corresponding author: Bao-guo Wang, MD; E-mail:wbgttyy@sina.com

　　约80%与麻醉有关的死亡或脑损害与麻醉中呼吸管理不当有关^[1]，且尤以各种原因造成的呼吸停止危害最大，目前尚未见到代表这种损伤特征的实验模型。为此我们拟建立一种麻醉后大鼠不同时间停通气缺氧损伤模型，通过观察基本生命体征、死亡率、神经功能及脑组织细胞形态学的变化确定其稳定性，以期为临床上正确认识和防治这种缺氧损伤提供研究工具。<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />

资料与方法

1. 实验动物准备
  　雄性清洁级Wistar大鼠144只，体重260～290g(由首都医科大学动物中心提供，许可证号：SCXK11-00-0012)，随机分为8组：停通气1分钟组(I组)、停通气2分钟组(II组)、停通气3分钟组(III组)、停通气4分钟组(IV组)、停通气5分钟组(V组)、停通气6分钟组(VI组)、停通气7分钟组(VII组)和假手术组，每组18只。实验室由北京神经外科研究所提供，许可证号：SYXK(京)2003-0001。饲养室恒温22℃，12小时间隔照明，标准鼠料饲养。
  　10%水合氯醛(30mg／kg)腹腔麻醉后在腹股沟中点沿下肢纵轴在右侧股部切开约1cm切口，分离股血管，从股静脉置入20号套管针连接微量输液泵以2ml／h的速度输入乳酸钠林格氏液，从股动脉置入24号套管针连接压力转换器，使用Spacelabs多功能生理监护仪(美国)监测有创动脉压和心电图。经口插入气管导管(14G套管针外套管)后应用动物呼吸机行纯氧机械通气(潮气量1ml／100g，呼吸频率60次/分，吸：呼=1：2)，静脉注射维库溴铵(2mg／kg)消除大鼠自主呼吸。应用灯照法维持直肠温度37.5±0.3℃。
2 .模型的建立
  　各实验组均在机械通气10分钟后关闭和呼吸机并断开呼吸机接头，I、II、III、IV、V、VI和VII组停止通气时间分别为1、2、3、4、5、6和7分钟，每组到达相应预定时间后立即重新连接呼吸机接头与气管导管恢复机械通气，呼吸模式同前，发生循环停止者(MAP＜25mmHg)在恢复通气的同时静脉注射肾上腺素0.01mg／kg和碳酸氢钠1mmol／kg，并施以200次/分的胸外按压，SBP＞60mmHg认为自主循环恢复并停止按压，如按压3分钟仍未恢复自主循环则放弃抢救，视为死亡。复苏成功者继续呼吸机支持60分钟，若自主呼吸恢复满意即可拔除气管导管，结扎股动静脉后缝合皮肤，在吸氧箱中观察30分钟后送回笼。假手术组除未停通气外其它处理同实验组。记录循环停止时间(time of cadiac arrest, T_CA)：从开始停通气到MAP＞25mmHg的时间和自主循环恢复时间(time of recovery of spontaneous circulation, T_ROSC)：从开始复苏到SBP＞60mmHg的时间。

3. 神经功能评定
  　参照Geodadra的神经功能评分(neural deficit scores, NDS)方法并加以改良，于实验后24、48和72小时从意识、基本反射、运动、感觉、行为学等方面对存活大鼠进行神经功能评定，评分范围0～80分，0分为脑死亡，80分为完全正常^[2]，具体评分方法见表1
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4 .海马电镜检查
  　每组选取3只存活动物于实验后72小时10%水合氯醛（90mg／kg）腹腔麻醉后开胸，迅速自心尖部插入灌注针至主动脉并固定，剪开右耳，首先灌注4℃生理盐水约150ml至右心耳流出淡粉色液体时续灌4℃4%多聚甲醛约200ml，灌注结束后断头取脑，视交叉后4mm冠状切开脑组织，切取约1mm×1mm×1mm大小左侧海马CA1区标本3～4块，置于2%多聚甲醛-2.5%戊二醛中初固定2小时，二甲砷酸钠缓冲液冲洗，1%锇酸后固定2小时，梯度乙醇脱水，环氧丙烷置换，环氧树脂Epon812包埋，修块后制备1um半薄切片，天青-美兰染色，光学显微镜观察定位后用超薄切片机制备40nm超薄切片，最后经醋酸双氧铀枸橼酸铅染液染色，在Philips EM208s透射电镜下观察。
5. 统计分析
  　采用SPSS 10.0 for Windows统计软件，计量资料数据以X±SD表示，组内、组间比较采用多个均数之间两两比较的q检验，计数资料的比较采用Fisher精确概率法，P＜0.05为差异有显著性。

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