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Effect of Mild Hypothermia on the Apoptosis-related Factors in Rats after Resuscitation
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摘要:
目的 研究亚低温对心肺复苏后大鼠神经细胞凋亡及凋亡基因表达的影响。 方法 60只健康雄性SD大鼠随机分为常温组(T=37℃±0.5℃)和亚低温组(T=33℃±1.0℃), 每组30只; 窒息法建立心肺复苏模型, 于自主循环恢复后12、24 h通过神经功能缺损评分评价神经功能, 使用TUNEL染色观察脑细胞凋亡情况, 并于自主循环恢复后0、2、6、12、24 h采用RT-PCR检测脑组织中凋亡诱导因子(apoptosis-inducing factor, AIF)、caspase-3、Fas基因mRNA表达。 结果 亚低温组大鼠12及24 h神经功能缺损评分明显高于常温组(P < 0.05), 6、12、24 h神经细胞凋亡数量明显少于常温组(P < 0.05);与常温组相比, 自主循环恢复后各时间点AIF、caspase- 3及6、12、24 h Fas基因mRNA表达显著下调(P < 0.05)。 结论 亚低温可能通过抑制脑组织中AIF、caspase-3、Fas凋亡基因表达, 减少神经元凋亡, 改善神经功能。 Abstract:Objective To study the effect of mild hypothermia after resuscitation on rat neuronal apoptosis and its gene expression. Methods Sixty healthy male SD rats were equally randomized into two groups:normal temperature group(T=37℃±0.5℃) and mild hypothermia group (T=33℃±1.0℃).A rat model of asphyxial cardiac arrest and cardiopulmonary resuscitation was established. The neurological functions were assessed using neurological deficit score (NDS) 12 and 24 hours after the restoration of spontaneous circulation. Also, the expressions of the apoptosis-inducing factor (AIF), caspase-3, and Fas mRNA were detected using reverse transcription-polymerase chain reaction (RT-PCR) at 0, 2, 6, 12, and 24 hours, respectively. Results Compared with the normal temperature group, the mild hypothermia group had significantly higher NDS at 12 and 24 hours(P < 0.05) and lower number of apoptotic brain cells at 6, 12, and 24 hours(P < 0.05). The mRNA expressions of AIF and caspase-3 at each time point and Fas at 6, 12, 24 hours were significantly reduced after resuscitation in mild hypothermia group(P < 0.05). Conclusion Therapeutic hypothermia after resuscitation may improve NDS and thus protect brain by inhibiting expressions of apoptotic genes including AIF, caspase-3, and Fas. -
Key words:
- therapeutic hypothermia /
- cerebral protection /
- cardiopulmonary resuscitation /
- apoptosis
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表 1 大鼠神经功能缺损评分标准
分类 标准 整体行为(总分19分) 意识 正常10分，木僵5分，昏迷0分 唤醒 自主睁眼3分，疼痛睁眼1分，不睁眼0分 呼吸 正常6分，异常3分，缺失0分 脑干功能(总分21分) 嗅觉 存在3分，缺失0分 视觉 存在3分，缺失0分 瞳孔对光反射 存在3分，缺失0分 角膜反射 存在3分，缺失0分 恫吓反射 存在3分，缺失0分 触须反射 存在3分，缺失0分 吞咽反射 存在3分，缺失0分 肌张力评分(总分6分) 肌张力(左) 正常3分，僵硬/减低l分，无运动/瘫痪0分 (右) 正常3分，僵硬/减低l分，无运动/瘫痪0分 感觉评分(总分6分) 疼痛(左) 敏锐退缩3分，迟钝/异常1分，缺失0分 (右) 敏锐退缩3分，迟钝/异常1分，缺失0分 运动评分(总分6分) 步态协调 正常3分，异常1分，缺失0分 横梁平衡 正常3分，异常1分，缺失0分 行为评分(总分12分) 反正反射 正常3分，异常1分，缺失0分 趋地实验 正常3分，异常1分，缺失0分 视觉定位 正常3分，异常1分，缺失0分 胡同转弯实验 正常3分，异常1分，缺失0分 癫痫发作(总分10分) 无抽搐10分，局部抽搐5分，全身抽搐0分 总分80分为正常，0分为脑死亡 
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表 2 亚低温组和常温组大鼠不同时间点脑细胞凋亡检测结果(%，x ±s)
组别 0 h 2 h 6 h 12 h 24 h 常温组 0.51±0.48 17.22±1.45 32.61±1.26 42.29±4.03 49.96±4.01 亚低温组 0.49±0.67 15.66±1.73 24.28±0.73* 28.97±1.20* 34.07±3.48* 与常温组比较，*P＜0.05
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表 3 亚低温组和常温组大鼠脑组织中不同时间点AIF、caspase- 3、Fas基因mRNA表达(%，x ±s)
指标 组别 0 h 2 h 6 h 12 h 24 h AIF/GAPDH 常温组 0.46±0.05 1.11±0.37 1.24±0.15 1.95±0.27 2.82±0.51 亚低温组 0.45±0.06 0.71±0.20* 0.88±0.18** 1.43±0.28** 2.11±0.43* Capase-3/GAPDH 常温组 0.51±0.06 1.10±0.36 1.18±0.25 1.65±0.20 1.70±0.24 亚低温组 0.49±0.05 0.69±0.19* 0.87±0.21* 1.36±0.23* 1.42±0.16* Fas/GAPDH 常温组 0.51±0.10 0.56±0.18 2.08±0.30 3.55±0.38 4.21±0.38 亚低温组 0.51±0.08 0.54±0.13 1.37±0.35** 3.12±0.26* 3.44±0.33** AIF：凋亡诱导因子；与常温组比较，*P＜0.05，* *P＜0.01
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