定量口鼻吸入法建立大鼠慢性阻塞性肺病模型

Quantitative inhalation method to establish a model of chronic obstructive pulmonary disease in rats

目的 比较烟雾经口鼻吸入+脂多糖(lipopolysaccharide, LPS)气管滴注和烟雾全身暴露+脂多糖气管滴注诱导的大鼠慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)模型的差异,为COPD模型的构建提供新的造模方法。方法 将90只雄性SD大鼠随机分为正常对照组、烟雾全身暴露组、烟雾口鼻吸入组,每组30只。烟雾全身暴露组采用“自制熏烟箱”进行烟雾的全身暴露,烟雾口鼻吸入组采用“定量吸烟装置”进行口鼻吸入烟雾,两组动物均每天进行烟雾暴露1次,每次60 min,连续8周,同时分别于造模第1、7、15、21天经气管注入LPS(1 mg/kg),以诱导建立COPD模型。分别对定量吸烟装置和自制烟熏箱生成的烟雾进行质量控制,包括烟雾颗粒的浓度稳定性和均一性验证,烟雾颗粒的粒径分布检测,并分别于造模4、6、8周通过肺功能检查、肺泡灌洗液炎症因子白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量的检测、组织病理学检查来比较两种造模方式的差异。结果 定量吸烟装置能生成浓度稳定的烟雾(浓度分别为1.1 mg/L(以颗粒计)和0.1 mg/L(以尼古丁计)),且其质量中值粒径(median mass aerodynamic diameter, MMAD)(以尼古丁计)为0.86μm,几何标准差(geometric standard deviation, GSD)为2.12,自制烟熏箱所生成的烟雾浓度稳定性和均一性偏差均明显大于定量吸烟装置;烟雾口鼻吸入组大鼠肺功能FEV_(0.2)/FVC、肺顺应性(Cdyn)指标均较烟雾全身暴露组下降更为明显,气道阻力(penh)增加更明显;烟雾口鼻吸入组大鼠肺泡灌洗液IL-6、TNF-α水平在造模后6周即可见显著增加,烟雾全身暴露组大鼠需至造模8周。烟雾口鼻吸入组和全身暴露组大鼠造模后支气管炎症病变程度基本相当,但口鼻吸入组的肺气肿病变程度更严重,且与全身暴露组(造模8周)相比,口鼻吸入组(造模6周出现)肺气肿病变出现统计学差异的时间更早;烟雾口鼻吸入组平均内衬间隔(mean linear intercept, MLI)在造模4~8周均显著增大,平均肺泡数(mean alveolar number, MAN)在造模6~8周均显著减少;烟雾全身暴露组仅造模8周可见MLI显著增大和MAN显著减少。烟雾口鼻吸入组肺功能指标(FEV_(0.2)/FVC、Cdyn、Penh)、肺泡灌洗液细胞因子水平(IL-6、TNF-α)、肺泡组织病理学变化(支气管严重和肺气肿病理评分、MLI、MAN)在造模后均可见显著的异常改变,但各指标变化的变异系数(CV%)明显小于烟雾全身暴露组的相应指标。结论 LPS(1.0 mg/kg)气管滴注联合烟雾全身暴露或烟雾口鼻吸入均能构建典型的大鼠慢性阻塞性肺病模型,其中烟雾口鼻吸入能缩短模型构建的造模周期,连续造模6周即可成模,表现为典型的慢性阻塞性肺病症状(肺通气功能障碍,支气管-肺脏慢性炎症浸润,并同时伴有肺气肿),且模型动物个体间的差异更小(烟雾口鼻暴露vs烟雾全身暴露,CV%值更小)。

Objective To compare differences in chronic obstructive pulmonary disease(COPD)models induced by smoke inhalation through the nose-mouth plus lipopolysaccharide(LPS)or smoke exposure through the whole body plus LPS in rats,providing a new model for COPD.Methods Ninety male SD rats were randomly divided into normal control,whole body exposure,and smoke inhalation groups with 30 rats per group.The whole body exposure group was placed in a smoke box,whereby smoke contacted the whole body of the rats,whereas the smoke inhalation group inhaled smoke via the nose-mouth in a quantitative smoking device.Animals in both groups were exposed to smoke once a day for 60 min over 8 weeks.LPS(1mg/kg)was injected through the trachea on days 1,7,15,and 21 to induce the COPD model.Quality control of the smoke generated by the quantitative smoking device and smoking box included verification of the concentration stability and uniformity of the smoke particles and the particle size distribution of the smoke.At 4,6,and 8 weeks of modeling,pulmonary function examination,the content of proinflammatory factors interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in alveolar lavage fluid,and histopathological examination were performed to compare the two modeling methods.Results Quantitative smoking devices produced smoke with a stable concentration of 1.1 mg/L(total particles)and 0.1 mg/L(nicotine),with a median mass particle size of 0.86μm(nicotine)and a GSD of 2.12.The deviation in stability and uniformity of the smoke concentration produced by the smoking box was obviously greater than that of the quantitative smoking device.Compared with the whole body exposure group,pulmonary function indexes FEVo.2/FVC and pulmonary compliance(Cdyn)in the smoke inhalation group were decreased significantly,and airway resistance(Penh)was increased significantly.IL-6 and TNF-αlevels in alveolar lavage fluid of the smoke inhalation group were significantly increased at 6 weeks after modeling,and those in the whole body exposure group were increased at 8 weeks after modeling.The lesion severity of bronchial inflammation after modeling was similar in smoke inhalation and whole body exposure groups,but the degree of emphysema lesions in the nose-mouth inhalation group was more severe,and statistically significant differences in emphysema lesions appeared earlier in the nose-mouth inhalation group(after 6 weeks of modeling)than in the whole body exposure group(after 8 weeks of modeling).The mean linear intercept(MLI)in the smoke inhalation group was increased significantly at 4~8 weeks of modeling,and the mean alveolar number(MAN)was decreased significantly at 6~8 weeks of modeling.MLI was increased significantly and MAN was decreased significantly in the whole body exposure group after 8 weeks of modeling.In the smoke inhalation group,significant abnormal changes were observed in pulmonary function indexes(FEVo.2/FVC,Cdyn,and Penh),cytokine levels in bronchoalveolar lavage fluid(IL-6 and TNF-α),and alveolar histopathological changes(bronchial severity and emphysema pathological score,MLI,and MAN)after modeling.However,the coefficient of variation(CV%)of each index was significantly lower than that in the whole body exposure group.Conclusion A 1.O mg/kg LPS endotracheal drip combined with whole body smoke exposure or nose-mouth smoke inhalation both establish a typical rat COPD model.Inhaled smoke via the nose-mouth route shortened the modeling period.The model was established after 6 weeks of continuous smoke exposure,presenting typical symptoms of COPD(pulmonary ventilation dysfunction and broncho-lung chronic inflammatory infiltration accompanied by emphysema),and the difference between individual model animals was small(nose-mouth smoke inhalation vs smoke exposure,CV%values were smaller).