一种简便可控的高氧急性肺损伤模型的建立方法

A simple and controllable method for reproduction of a model of high oxygen tension induced acute lung injury

目的建立高氧急性肺损伤（HALI）大鼠模型，寻找一种更简便、价格更低、性能更稳定的高氧模型建立方法。方法自行设计一种可控性的高氧动物饲养箱。将100只SD大鼠按随机数字表法分为正常对照组和HALI组，每组再按高氧暴露时间随机分为0、24、48、72和96h5个亚组，每个亚组10只。正常对照组直接在空气中饲养；HALI组置于高氧箱中饲养，箱内氧浓度≥体积分数90％，温度维持在25～27℃，湿度保持在50％～70％，CO2浓度〈0．5％，每日持续给氧23．5h。分别在各时间点取大鼠动脉血进行血气分析，并计算氧合指数（OI）及呼吸指数（RI）。处死大鼠取右肺组织行苏木素一伊红（HE）染色，光镜下观察肺组织病理改变并进行病理学评分；取左肺测量湿／干质量（W／D）比值。结果随着高氧暴露时间的延长，大鼠肺组织病理学损伤程度逐渐加重，肺泡结构逐渐紊乱，肺泡壁断裂破坏，肺泡间隔逐渐增宽、水肿、炎性细胞浸润，部分大鼠可见少量红细胞渗出，在48～72h达高峰。HALI组随着高氧暴露时间（0、24、48、72、96h）的延长，OI（mmHg，1mmHg=0．133kPa）逐渐降低（446．67±29．93、306．19±37．23、269．70±29．00、253．81±43．40、245．58±35．25），RI（0．25±0．04、0．31±0．06、0．38±0．06、0．46±0．07、0．44±0．03）、肺组织病理学评分（分：0．00±0．00、0．90±0．74、2．90±1．20、4．70±1．57、4．80±1．23）和肺W／D比值（3．84±0．61、4．14±0．46、4．56±0．34、5．32±0．27、5．18±0．25）呈进行性升高，72h内各指标与前一时间点比较差异均有统计学意义（均P〈0．05），而96h与72h比较差异均无统计学意义（均P〉0．05）；24h开始，上述各指标与正常对照组比较差异有统计学意义，并持续至96h[OI（mmHg）：24h为306．19±37．23比435．65±25．34，96h为245．58±35．25比465．42±24．75；RI：24h为0．31±0．06比0．24±0．04，96h为0．44±0．03比0．24±0．06；肺组织病理学评分（分）：24h为0．90±0．74比0．00±0．00，96h为4．80±1．23比0．00±0．00；肺W／D比值：24h为4．14±0．46比3．79±0．44，96h为5．18±0．25比4．12±0．91；均P〈0．05]。结论自行设计的高氧箱操作简便、性能稳定，在持续给予高浓度氧（≥90％）24h后即可成功复制HALI模型，并于48～72h损伤达高峰。

Objective To explore a simpler, more economic and reproducible method to reproduce a model of high oxygen induced acute lung injury （HALl） in rats. Methods An animal feeding box equipped with a controllable high oxygen was designed. 100 Sprague-Dawley （SD） rats were divided into normal control group and HALI group by random number table method, with 50 rats in each group. Each group was randomly subdivided into five subgroups according to the duration of exposure to high oxygen, namely 0, 24, 48, 72 and 96-hour subgroups, with 10 rats in each subgroup. The rats in normal control group were kept in cages with ambient air, and the rats in HALl group were kept in an oxygen tank in which the oxygen concentration was higher than 90% volume ratio, with the temperature maintained at 25-27 ℃ , humidity of 50%-70%, and CO2 concentration 〈 0.5% for 23.5 hours every day. The arterial blood of rats was collected for analysis of blood gas at all time points, and the oxygenation index （OI） and respiratory index （RI） were calculated. Then the rats were sacrificed and the right lung was harvested, which was sectioned and stained with hematoxylin and eosin （HE）. The changes in histopathology were observed with light microscopy, and pathological score was recorded. The left lung was harvested for the measurement of the wet/dry weight ratio （W/D）. Results With the prolongation of high oxygen exposure time, the degree of lung injury in HALI group was gradually increased, and the degree of derangement of alveolar structure appeared in an increasing degree, with destruction of the alveolar wall, widening of alveolar space, and appearance of edema, and inflammatory cell infiltration. A small quantity of red blood cells exudation could be found in some rats. The pathologic changes were most obvious at 48-72 hours after exposure. With the prolongation of high oxygen exposure time （0, 24, 48, 72, 96 hours）, the OI （mmHg, 1 mmHg = 0.133 kPa） in HALl group was gradually decreased （446.67±29.93, 306.19±37.23, 269.70±29.00, 253.81 ±43.40 and 245.58± 35.25）, RI, pathological score of lung tissue and W/D ratio were gradually increased [Rh 0.25 ± 0.04, 0.31±0.06, 0.38±0.06, 0.46±0.07 and 0.44±0.03; pathological score of lung tissue： 0.00±0.00, 0.90±0.74, 2.90±1.20, 4.70±1.57 and 4.80±1.23; lung W/D ratio： 3.84±0.61, 4.14±0.46, 4.56±0.34, 5.32±0.27 and 5.18±0.25]. Statistically significant differences were found in 72-hour group as compared with that of other groups （all P 〈 0.05）, while no significant difference was found between 96 hours and 72 hours groups （all P 〉 0.05）. There were significant differences in changes between 24, 48, 72, and 96 hours as compared with those of the normal control group： OI （mmHg）： 24 h 306.19 ± 37.23 vs. 435.65± 25.34 and 96 h 245.58 ± 35.25 vs. 465.42 ± 24.75; RI： 24 h 0.31±0.06 vs. 0.24 ± 0.04 and 96 h 0.44 ± 0.03 vs. 0.24 ± 0.06. The same as true in pathological scores of lung tissue： 24 h 0.90±0.74 vs. 0.00±0.00 and 96 h 4.80±1.23 vs. 0.00±0.00; lung W/D ratio： 24 h 4.14±0.46 vs. 3.79±0.44 and 96 h 5.18± 0.25 vs. 4.12± 0.91, all P 〈 0.05. Conclusions A self-designed high oxygen box is simple, easy to operate and reproduction of HALl model can be attained. Sustained exposure to high concentrations of oxygen （ ≥ 90%） for 24 hours can replicate the HALl model successfully, and the most serious injury appears at 48-72 hours after exposure.