The Study Of Conditions And Mechanism Of Airway Hyperresponsiveness

Background:Airway hyperresponsiveness (AHR) is a very important factor in the pathogenesis of bronchial asthma. Many different factors have been suggested to be involved in causing the airway hyperresponsiveness seen in asthma patients. It is believed that the increased number of eosinophils and other inflammatory cells in asthmatic airways produce many of the tissue changes seen in the disease, including epithelial damage, the release of mediators with the capacity to cause bronchial smooth muscle contraction and exudation of plasma, and other mechanisms interact to produce airway hyperresponsiveness. Nasal allergen challenge has been shown to cause increases both in sputum eosinophils and BHR in patients with allergic rhinitis and asthma. Eosinophilic bronchitis (EB) patients showed that the airway inflammation was similar to that seen in asthma. However, it remains open to question why no functional abnormalities associated with asthma was observed in EB patients. We have found that the intensity of airway inflammation in EB is milder, and the extent was more limited, than that in asthma. Different nebulisers produce aerosol droplets of various sizes. Pari LC star is a small particle nebulizer designed to improve aerosol delivery to the deep airways (peripheral aera) of the lung. It is suitable for asthma model. And Pari TIA nebulizer produces larger particles. It is suitable for establishing animal model which has similar airways inflammation character to EB. These models were very important for study the mechanism of airway hyperresponsiveness.A lot of data indicate a close relationship between the upper and lower airways in airway allergic diseases. Nasal allergen challenge has been shown to cause increases both in sputum eosinophils and BHR in patients with allergic rhinitis and asthma. The data suggest that allergen stimulation of the upper airway can induce physiologic effects in distal airway in the absence of direct allergen challenge. Contrary to large amount of data about the widespread allergic responses involving downstream organ, there are few reports of signs of allergic inflammation in a remote and"upstream"organ after local lower airway allergen challenge. Because study the influence of isolated lower airway inflammation on nose in mice and human requires a sophisticated methodology.Objective:1. We try to use Pari TIA nebulizer to establish mice model that have airway eosinophilic inflammation without airway hyperresponsiveness.2. Distal airway inflammation and responsiveness after antigen challenge in different part of airway in sensitizations mice was studied.Methods:Part I: Establishment of Eosinophilic airway inflammation Mouse Model and Comparison in Airway Inflammation with Asthma ModelSegment 1: Female BALB/c mice were obtained and divided randomly into 3 groups: eosinophilic airway inflammation group (experimental group), asthma group and normal control group. Mice were immunized as before. The experiment group was challenged with an aerosol of 1% w/v OVA in saline using a PARI LC STAR nebuliser(mass median diameter 2.9μm) and asthma group was challenged with an aerosol of 1% w/v OVA in saline using an PARI TIA(mass median diameter 8.6μm) nebuliser on day 28, 29, 30. The control mice received saline sensitization and challenge. Twenty four, 48, 72 and 96 hours after the last challenge, the measurement of airway responsiveness in every group was carried out using the invasive measurement system (RC system, Buxco, USA) and cell different counts in bronchial alveolus lavage fluid (BALF) of every groups were performed and a pathologist blinded to the treatment of the groups performed histopathological evaluation of the trachea and lung.Segment 2: Asthma mice model(asthma group) and eosinophilic airway inflammation mice model without hyper responsiveness(the experimental group) were constructed. Total protein extracted from the lung tissue was separated using two-dimensional electrophoresis (2-DE) with immobilized pH gradient (IPG). After coomassie brilliant blue staining, the gel image analysis was carried out using Image Master 2D Elite 5.0 analysis software to identify the proteins differentially expressed. The differential expression proteins were identified by peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).Part II: Study of the distal airway inflammation and responsiveness after antigen challenge in different part of airways in mice modelMethods: Segment 1: Mice allergic rhinitis (AR) model was established. Mouse was anesthetized after challenge. A cannula was inserted into trachea. A blunt needle connected with a T tube was inserted into nasopharynx. One end of the T tube was connected to a syringe pump which provides consistent air flow. Operating in infuse/withdraw mode, the flow rate was adjusted to 1.0ml/s to mimic the expiration/inspiration cycle. Another end was connected to a pressure transducer to measure pressure difference (ΔP) across nasal passage.ΔP were divided by 1.0ml/s and then deducted catheter resistance to give values of RNA. Histamine was nebulized into nasal cavity to measure early-phase response.Segment 2: Six to 8-week-old BALB/c mice were sensitized by means of OVA systemic intraperitoneal injection. They were then challenged with intranasal OVA, and allergic response and inflammations of the total airways was assessed.Segment 3: BALB/c mice were sensitized by systemic intraperitoneal injection of ovalbumin/saline and then challenged with intratracheal ovalbumin/saline. Inflammation in nose and lung was assessed by nasal and broncheoalveolar lavage cytology and histology at different time points. A new method of nasal airway resistance was created to measure nasal function and response.Results:1. We established an eosinophilic airway inflammation mouse model without hyperresponsiveness using Pari TIA nebulizer. The intensity of airway inflammation in this model was milder than that in the asthma model. By comparative proteomics technique, we have succeeded in the identification of 20 proteins such as GSTM1 and HSPB1.2. RNA comprises at least one half of the total airway resistance.3. Using exclusive local challenge of mouse nares, we found inflammatory changes and airway hyperresponsiveness in both the upper and lower airways, even though distribution of allergen particles appeared to be only in the nares of these animals. Intratracheal application of allergen in anaesthetized mice resulted in exclusive deposition in lower airway. However, histology of nose in intratracheal challenge mice didn't show increased eosinophils infiltration, nasal lavage fluid didn't show increased eosinophils and IL-5 when compared with the control group.Conclusions:1. We established an eosinophilic airway inflammation mouse model without hyperresponsiveness successfully. The character of airway inflammation in this model was similar to that of EB. Our model established a foundation for the further research in airway hyperresponsiveness.2. GSTM1 and HSPB1 are relative to airway hyperresponsiveness.3. We established a relatively simple method for measuring physiological change in the nasal airway in mice.4. We were able to demonstrate inflammatory changes and airway hyper responsiveness in both the upper and lower airways, even though distribution of allergen particles appeared to be only in the nares of mice.5. Exclusive allergen challenge of lower airway elicits a systemic allergic response, but isn't able to promote upper airway inflammatory response in mice model. The nasal mucosa may be more resistant to allergen compared with that of the bronchi..