Experimental Airway Hyperresponsiveness Animal Model Of Intrapulmonary Neuropeptides And Their Receptors In Spatial And Temporal Distribution

Part 1 The creation and detection of animal model with airway hyperresponsiveness.Objective: To establish a new type of animal model with AHR by ozone-stressing bronchial epithelial cells.Method: 12 of New Zealand White rabbits were randomly divided into two groups of 6 each: (1) O3-exposed group. Animals were exposed to 2.0ppm O3 for 3 days, Ih/day. (2) Control. All animals were challenged with methacholine at 12h after the last exposure. We detected airway resistance, total cell counts, total protein, the level of IL-1 in BALF and pathologic changes in lung to certify the existence of AHR. Results: (1) After challenge airway resistance increased , especially expiratory resistance;(2)total protein, the level of IL-1, total cell counts in BALF increased significantly in O3-exposed group. (3) A significant inflammation in lung were observed in O3-exposed group, including neutrophils, eosinophils infiltration; mucus exudation; BECs shedding.Part 2 The time and spacial distribution of calcitonin gene-related peptid and receptor in the development of airway hyperresponsivenessObjective: To explore the effect of calcitonin gene-related peptide in the development of airway hyperresponsiveness.Methods: 25 of new Zealand White rabbits were randomly divided into five groups of 5 each, I, II, III, IV, V, and exposed to 2.0ppm ozone 1h/day for 0,l,2,4,8days,respectively.We detected the level of CGRP and the mRNA expression of CGRPR1 in lung. Using in situ hybridisation, we demonstrated the distribution of the mRNAs of CGRPR1 in lung. Results: CD Radioimmunoassay showed the concentration of CGRP in lung began to rise within 24 hours and peak on day 2,then decreased to baseline on day 8.(2)RT-PCR results showed the mRNA expression ofCGRPR1 increased with the elongation of ozone-stressing and peaked on day 4,then decreased slowly.(3)In group I (day 0), CGRPR1 mRNA hybridization signal was detected mainly in pulmonary interstitial with less expression in smooth muscle cells. On day 2 hybridization staining deepen and the majority of VIPR1-positive cells were located in perivascular and peribronchiolar. Up to day 8,only a diffuse weak VIPR1 mRNA signal was detected in lung.Part 3 The time and spacial distribution of vasoactive intestinal polypeptide and receptor in the development of airway hyperresponsivenessObjective: To determine the possible involvement of endogenous VIP in the development of AHR.Methods: Five groups of 5 animals were exposed to 2.0ppm ozone Ih/day for 0,1,2,4,8 days, respectively. We detected the changes of VIP level and the mRNA expression of VIPR1 in lung at various ozone-stressing time points. In situ hybridization was performed to examine the distribution of VIPR1 in lung.Results: (1)the concentration of VIP in lung increased slowly and were maximal at day 4,then reverted to normal.(2)the mRNA expression of VIPR1 in lung were similar to those observed for VIP .Increase in VIPR1 mRNA contents were detectable by 1 days and maximal by 2-4 days, then decreased slowly. (3) On day 0,VIPR1 were expressed on airway epithelium, in pulmonary interstitial and focal areas of airway and vascular smooth muscle. On day 1,the distribution of VIPR1-positive cells had no significant change. By days 2 to 4, hybridization staining deepen and the majority of VIPR1-positive cells were located in perivascular , peribronchiolar with a few in alveolar. On day 8,very few positive cells were seen in lung.