| Objective: The animal model of bronchial stenosis was established by APC through bronchoscope,and then cryotherapy was performed with a new cryoprobe,to evaluate the therapeutic effect and mechanism,and to provide the theoretical basis for the clinical human experiment with the new cryoprobe in the later stage.Methods:The new cryoprobe used in this study was different from the conventional cryoprobe.The front end of the probe had a certain bending angle,hyperelasticity,memorability and automaticity,and animal experiment had been carried out to verify the self-bending angle of the new cryoprobe.The purpose of this study was to verify the effect of cryotherapy and study the mechanism of cryotherapy so as to lay a foundation for clinical human experiment with new cryoprobe.A total of 24 healthy beagle dogs,twelve beagle dogs were randomly selected to establish the animal model of bronchial stenosis.On the premise that the animal model had been established successfully,the model of bronchial stenosis was repeatly established in 12 remaining beagle dogs,and then cryotherapy was carried out.1.Establishment of animal model(1)12 beagle dogs were randomly divided into model group A(n = 6),and model group B(n = 6);(2)Bronchial mucosa in left and right lobes of 12 beagle dogs were cauteried by APC through bronchoscope;(3)On the 7th day and on the 14 th day after APC,bronchoscope was performed,and the diameter of bronchus was measured,and statistical analysis was carried out;(4)Model group A were taken on the 7th day after APC,and model group B were taken on the 14 th day after APC,and the normal bronchi of the same branch were obtained.HE staining,collagen fiber staining,and the histopathological changes were observed under the light microscope,and the percentage of the collagen fiber area was detected,and the statistical analysis was carried out;2.Study on the therapeutic effect and mechanism of a new cryoprobe(1)According to the principle of self-control,the remaining 12 beagle dogs were used to repeatly establish the animal model of bronchial stenosis;(2)The right bronchi of 12 beagle dogs were treated with cryotherapy on the 7th day after APC,and the left bronchi were used as a control group.The bronchial diameter was measured under bronchoscope and analyzed statistically;on the 14 th day after APC(on the 7th day after cryotherapy),bronchoscope was performed,and the diameters of bronchi were measured and analyzed statistically.(3)On the 14 th day after APC(on the 7th day after cryotherapy),12 beagle dogs were sacrified.HE staining,collagen fiber staining and the histopathological changes were observed under light microscope,and the percentage of collagen fiber area was measured for statistical analysis;(4)Immunohistochemical method was used to detect the expression of CD34 in the microvessels of bronchial tissue.The microvessel density(MVD)was counted and analyzed statistically;(5)Immunohistochemical method was used to detect the expression of IL-17,VEGF-A and TGF-β 1 in bronchial tissues.The mean optical density(MOD)was counted and analyzed statistically.Results:1.Establishment of animal model(1)Bronchoscope observation: The mucosal surface became scabby and whitened immediately after APC;on the 7th day after APC,the bronchial lumen was blocked by granulation tissue,accompanied by swelling of peribronchial tissue;on the 14 th day after APC,scar formation was observed in the bronchial lumen.(2)Measurement of bronchial diameter: The diameters of bronchi before APC,on the 7th day after APC and on the 14 th day after APC were significantly different(P<0.05);there was no significant difference in diameter between left and right bronchi at the same time point(P>0.05).(3)Observation of bronchial gross specimen: On the 7th day after APC,granulation tissue proliferation,tissue edema and uneven surface were observed at the bronchus;on the 14 th day after APC,the scar formation was observed around the bronchus and the surface was not smooth.(4)Histological observation:(1)Light microscope pathology: On the 7th day after APC,necrotic tissues,granulation tissue proliferation,edema,lymphocytes,neutrophils could be seen in the submucosal tissue,and injury reached the smooth muscle layer,cartilage layer appeared to be normal;on the 14 th day after APC,mucosal epithelial hyperplasia,granulation tissue decreased,lymphocytes,neutrophils could be seen in the submucosal tissue.(2)Collagen fiber hyperplastic results: Collagen fibers on the 14 th day after APC were more abundant than those on the 7th day after APC.The percentage of collagen fiber area before APC,on the 7th day and on the 14 th day after APC were significantly different(P<0.05).2.Study on the therapeutic effect and mechanism of a new cryoprobe(1)Bronchoscope observation: The effect of cryotherapy on granulation tissue was significant,and the bronchial lumen became larger;on the 14 th day after APC(on the 7th day after cryotherapy),the granulation tissue in the treatment side decreased and scar tissue was seen in the peripheral bronchus of the control side.(2)Measurement of bronchial diameter: The diameters of bronchi before APC,on the 7th day after APC and on the 14 th day after APC(on the 7th day after cryotherapy)were significantly different(P<0.05);there was no significant difference in bronchial diameter between treatment side and control side at the same time point(P>0.05).(3)Observation of bronchial gross specimen: On the 14 th day after APC(on the 7th day after cryotherapy),the granulation tissue in the bronchial lumen of the treated side decreased and the surface of the treated side was relatively flat,and scar formation around the left bronchus was observed in the control side.(4)Histological observation:(1)Light microscope pathology: In the treated side,capillaries,edema,lymphocytes,neutrophils could be seen in the submucosal tissue;in the control side,capillaries,lymphocytes,neutrophils could be seen in the submucosal tissue.(2)Collagen fiber hyperplastic results: The collagenous fibers in the control side were more abundant and dense than those in the treatment side.The percentage of collagen fiber area in the treated side was lower than that in the control side(t=7.89,P<0.05).(3)Measurement of MVD: The MVDs in the bronchial tissue of the treated side were lower than those of the control side(t=6.64,P<0.05).(4)Measurement of MOD: The levels of IL-17,VEGF-A and TGF-β1 MODs in the bronchial tissue of the treated side were lower than those of the control side(P<0.05).Conclusion:1.The establishment of animal model of bronchial stenosis by APC through bronchoscope was a simple,rapid and safe technique,which can provide a stable experimental animal model for the exploration of therapeutic methods for bronchial stenosis.2.Although there were no significant differences in the diameters of bronchus before and after cryotherapy,the results showed that cryotherapy was effective for granulation tissue hyperplasia from the aspects of bronchoscope,gross specimens of bronchus and pathological observation.In addition,the results of IL-17,VEGF-A,TGF-β 1 in bronchial tissue showed that cryotherapy could reduce inflammation and granulation tissue proliferation.In the process of cryotherapy,whether there were other factors involved in the reaction needed to be further studied.3.The new cryoprobe had better freezing effect and no adverse reaction,which provided experimental data for later clinical human experiment. |
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