| According to the data of global burden of disease in 2019,atmospheric particulate pollution is the fourth most important risk factor of premature death in the world and China.PM2.5 refers to particulate matter with a diameter of 2.5um or less in the atmosphere,which can be used as a carrier to carry many harmful substances(carcinogens,bacteria,etc)directly spread to small airway and alveoli.Long-term exposure to PM2.5 leads to human lung tissue damage and reduced lung function.In severe cases,it leads to COPD and even lung cancer.Fine particulate matter pollution is one of the main risk factors for lung cancer and COPD,and is closely related to the incidence rate and mortality rate increasing year by year.With the development of imaging technology,the changes of local structure of lung can be clearly displayed by conventional CT,micro CT and phase contrast imaging of synchrotron radiation source.As a new method,quantitative CT is more and more used in early diagnosis and to evaluate the severity of chronic lung disease.At present,which lung structure changes will be caused by PM2.5 exposure is still a question worthy of discussion.In this study,three methods were used to establish PM2.5 exposure rat models: porcine pancreatic elastase(PPE)combined with PM2.5 instillation,simple PM2.5 instillation and PM2.5 aerosol inhalation.The degree of lung injury in the model rats was evaluated by histopathology.Micro CT and synchrotron radiation light source imaging were used to evaluate the dynamic changes of lung parenchyma,airway and blood vessels.The aim is to explore the details and regularities of the variation of the local structure after ambient PM2.5 exposure,so as to provide a theoretical basis for early clinical intervention and treatment of PM2.5 associated lung diseases.Part I Establishment of exposure to ambient air fine particles induce lung injury model in ratsObjective: Different concentrations of atmospheric fine particulate matter(PM2.5)with different exposure methods caused different degrees of lung injury in rats with underlying diseases and healthy rats.Therefore,in this study,three methods were used to establish PM2.5 exposure rat models: porcine pancreatic elastase(PPE)combined with PM2.5 instillation,simple PM2.5 instillation and PM2.5 aerosol inhalation.The degree of lung injury in the model rats was evaluated by histopathology.Methods: Fifty male Wistar rats were divided into 12 PM2.5 group,16 PPE combined with PM2.5 group,7 aerosol inhalation group and 15 control group.The PM2.5 suspensions at different dosages(0.8,1.6 and 3.2 mg/rat for 4,8 and 12 weeks)were respectively given to rats by the intratracheal instillation.Intratracheal instillation of 50 IU trypsin in rats,the rat model of early emphysema induced by elastase was established after three weeks.The PM2.5 suspension was prepared by dissolving 1.5mg PM2.5 particles in 3ml normal saline in the self-made PM2.5 exposure box.The PM2.5 suspension was exposed by aerosol inhalation twice a day,lasting for 1 hour each time,with an interval of 6 hours.The cumulative exposure was 12 weeks and the cumulative use was 252 mg.Weight measurement,histopathological examination and quantitative analysis were performed every 4 weeks.SPSS 25.0 software was used to analyze the data.Results: The weight gain of the rats in the protease combined with PM2.5 instillation group and the aerosol inhalation exposure group were different from that of the control group from the 8th week(P<0.05).The pathological changes of instillation group were slight in 4 weeks,and the wall epithelium of small airway and the wall of small vessels were thickened,and slightly trace carbon deposition in the interstitial lung.The pathological changes of instillation group in 8 weeks group,the alveolar septum was thickened,and the wall epithelium of the small airway was more obvious,and there was a large amount of carbon deposition in the interstitial lung.The pathological changes of instillation group in 12 weeks group showed enlargement of the pulmonary cavity,thickening of alveolar septum,and the largest amount of carbon deposition in interstitial lung.Compared with the PM2.5 group,the epithelial hyperplasia of small airway wall and the thickening of adjacent small vessel wall were more obvious in the protease combined with PM2.5 group,and the non phagocytized fine particles were more easily seen in the alveolar cavity of rats.In PM2.5 group,the quantitative parameters of bronchial wall thickness and mean alveolar area gradually increased with time,but there was no significant difference among the groups(P>0.05).The inhalation exposure group was mainly characterized by chronic inflammatory changes in the lungs,including lymphatic infiltration of the tracheal wall,thickening of the pulmonary septum and inflammatory cell infiltration of the alveolar wall,thickening of the vascular wall.Conclusion: The severity of lung pathology in PM2.5 exposure models of different ways has a dose-dependent feature.In the animal model with underlying diseases(emphysema model induced by protease instillation),there were more small lung nodules and small airway wall inflammation and lumen dilatation.Part II Evaluation of exposure to ambient air fine particles induce lung injury in rats using micro computed tomographyObjective: Micro-CT allows for longitudinal and noninvasive image-based measurements in animal models of lung diseases.In this study,in vivo and in vitro micro CT were used to evaluate exposure to ambient air fine particles induce the dynamic changes of local lung structure in rats exposed to atmospheric fine particles.Methods: Rat models of PM2.5-induced Lung Injury were established with PM2.5 inhalation,PM2.5 instillation,protease and PM2.5 instillation methods.There were 7 rats in the PM2.5 inhalation group,9 rats in the PM2.5 instillation group and 9 rats in the protease and PM2.5 group.In vivo micro CT imaging was performed before modeling,4 weeks,8 weeks and 12 weeks after modeling in rats of aerosol inhalation group and instillation group.In the PM2.5 instillation group,3 rats were executed respectively at the 4th,8th and 12 th week.In the PM2.5 inhalation group,all rats were executed after 12 weeks for pathological examination.The executed rats were respectively subjected to ex vivo micro CT examination and pathological examination.The vivo Micro CT image resolution is 144 ?m.The quantitative parameters included mean gray value,total lung volume and the percentage of low attenuation area volume in total lung volume of both lung tissues(% LAA).The image resolution of conventional ex vivo micro CT is 18?m and 5 ?m.Lung specimens were examine by line HE staining,then observeed by pathological changes.Univariate analysis of variance or Kruskal Wallis h test was used to compare the differences between the groups.Results:(1)All instillation groups showed that both lung were not seen obvious abnormalities.There were significant differences in mean total lung volume(P = 0.001),mean low attenuation area(P = 0.001)and mean low attenuation area as a percentage of total lung volume(LAA)(P = 0.016)between 4-week and 12 week groups.After 4 weeks of PM2.5 inhalation exposure,only one rat had a little inflammation in the right upper lung field.After 8 weeks of inhalation exposure,localized inflammation appeared in the lungs of 4 rats.After 12 weeks of inhalation exposure,the lungs of rats showed consolidation in varying degrees,and the inflammation involved at least two lung fields.For inhalation group,the CT findings were different degrees of consolidation.(2)Ex vivo conventional micro CT whole lung scanning can achieve anatomical segmentation and volume reconstruction image of lung lobe level.Ex vivo conventional high-resolution imaging can only show the internal structure of alveoli,but the local structure of alveolar septum is not well displayed.Conclusion: Conventional in vivo micro CT scanning can quickly and dynamically evaluate the lung changes of PM2.5 inhalation group.Chronic exposure to low dose PM2.5 may cause emphysema.Exposure to high-dose PM2.5 aerosol inhalation resulted in progressive pulmonary inflammation.Conventional ex vivo micro CT can be used to image the whole lung and local lung area,and the image resolution has been further improved.But for the early local slight abnormality,the difference between normal tissue and abnormal tissue is small.Part III Evaluation of exposure to ambient air fine particles induce lung injury in rats using synchrotron radiation imagingObjective: In line phase contrast imaging based on synchrotron radiation can record phase change information that cannot be recorded by conventional X-ray imaging.The study was to evaluate the PM2.5-induced lung local changes in rats using synchrotron radiation source,with histopathology as the gold standard,and to define the early imaging features and the corresponding relationship between pathological histology.Methods: Twenty Samples for synchrotron radiation imaging were resected from right lower lobe of lung specimen.It including three rats in single PM2.5 4-week group,3 PM2.5 8-week group,3 PM2.5 12 week group,3 protease combined PM2.5 12 week group,3 protease combined with normal saline12 week group,one PM2.5 aerosol inhalation 12 week group and one blank control 12 week group.The energy of synchrotron X-ray in this study was 16 Ke V.The distance from the experimental sample and the X-ray source was 25 cm.The pixel size of the detector was 5.2?m,and the field of view was 45mm(H)× 5 mm(V).Seven lung specimens were examined by line HE staining,then observeed by pathological changes.Reconstructed tomographic images were compared with images from histologic slides obtained from the same samples.Results: In the 4-week PM 2.5 instillation group,micro CT showed the larger airway wall was thickened.In the 12-week PM2.5 instillation group,ex vivo phase contrast imaging showed thickening of the small airway wall and a few small patchy exudates in the distal bronchus.In the 12-week protease and PM2.5 instillation group,phase contrast imaging showed that subpleural small patchy ground glass opacity(alveolar septal thickening),solid nodules(PM2.5 deposition in the lung interstitium),small airway dilation(the distal lumen of small airway dilates more proximal).In the 12-week PM 2.5 inhalation group,PC showed strip like high density shadow of alveolar septum,thickening of airway wall and vascular wall.The average value of grade 4-6 bronchial wall in PM2.5 instillation group at 12 weeks was thicker than that in control group at 12 weeks.The average% CSA value of the 8-week protease combined PM2.5 group was smaller than that of the 8-week single PM2.5 group.The average% CSA value of the 12 week protease combined PM2.5 group was smaller than that of the protease combined saline group,but there was no significant difference among the groups.Conclusion: Phase contrast imaging based on synchrotron radiation is a noninvasive method for evaluating PM2.5 exposure in rats,and it has the ability to detect subtle structural changes in lung injury in PM2.5 exposed rats. |
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