Neurogenic Inflammatory In The Lower Respiratory Tract And Viscerosensory Afferent Sites In The External Oesphageal Perfusion Guinea Pig Model For Reflux-associated Respiratory Hyperresponsiveness

AIMGastro-oesophageal reflux disease(GERD) is believed to lead to extra-oesophageal symptoms and complications,primarily in the respiratory tract,such as asthma.With appearance of the drug of effective acid-inhibitor and the 24h PH-monitoring,that GER might be an excitation factor of asthma is recognized gradually.To discuss the relationship between the GER and asthma,appropriate animal model is necessary.The main reason of GER in respiratory tract is the oesophageal-bronchial reflux,that is the acid sensitive receptors in the oesophagus are stimulated by acid which prime the vagus nerve,the airway hyperresponsiveness is caused by this reflux.Sensory nerve in the airway is stimulated because of the oesophageal-bronchial reflux,releases neuropeptide through axon-reflux,causes neurogenic inflammation.On the other hand,impulse conducts to the CNS,forms synaptic with nTS and correspondent spinal dorsal horn,causes respiratory reflux which induces by vagus nerve partly.The receptors of the reflex arc innervate in the airway epithelium and its bady locates in the vagal nodose ganglia and the vagal jugular ganglia.Substance P acts as an important role of neuropeptide family,the diverse actions of SP are often fulfilled by binding to its high-affinity specific receptor,the NK-1.SP is confirmed to participate in the pathogenesis of asthma and acid perfusion of the esophagus,but whether SP also takes part in the process of GER inducing hyperresponsiveness and whether it expresses in the spinal ganglia and corresponding spinal dorsal horn remain open questions.Nerve growth factor,(NGF) is an important medium in pathogenesis of asthma,but whether it take part in the pathogenesis of GER inducing hyperresponsiveness is unknowed.In this study,we detected the expression of SP and NGF in the airway and viscerosensory afferent sites in the external repetitive acid-perfusion oesphageal guinea pig model to test the oesophagus - branchus reflex and neuroginic inflammation.Material and methods1,AnimalsSpecific pathogen-free albino guinea pigs weighing 350-450 g of both sexes were provided by the Laboratory Animal Center,College of Basic Medicine,China Medical University.Thirty guinea pigs were randomized into 3 groups:A group:namely the phosphate buffer solution(PBS)control(n=10);B group:experimental group(n=10);C group:SR140333 group(n=10).In the experimental group the oesophagi were challenged with a solution of hydrochloric acid(HCI) containing 1 g/L pepsin(HCl-P; pH 1.0).In the control group phosphate buffered saline(PBS)(pH 7.0) was used to treat the oesophagi.HCl was used in a 0.1 N solution,the same concentration found in the human stomach.Pepsin was added to simulate the gastric contents during the digestive process.In the HCl-P group,on the day of experimentation,guinea pigs were maintained under ketamine anesthesia delivered intraperitoneally at 50 mg/kg,with additional injections given as necessary.Animals were placed in supine position,and a 5-Fr catheter was inserted through the mouth and into the lumen of the middle and lower oesophagus.The oesophagus of each animal was perfused with HCl-P for 20 min/d for 14 d at a flow rate of 0.3 ml/min via a recirculating system.In the control group,animals received an oesophageal perfusion of PBS.During perfusion,all animals were in a 30°anti-Trendelenburg position.The guinea pigs in C group were injected pertioneally with ST 140333 30min before acid perfusion.2,Determination of airway hypersensitivityThe determination of airway responsiveness to acetylcholine(ACh) was measured 24 hr after the last perfusion,pentobarbital anesthesia.Measure the airway hypersensitivity with AniRes 2005 animal lungs function analysis(Beijing Beilanbo Technology Co.Ltd.)3,Vascular perfusion,sacrifice of guinea pigs,sample preparationCell numbers in BALF were detected;pathological changes of the lower oesophagus,bronchial and lung tissues of different groups were detected by HE staining.4,Hematoxylin-Eosin(HE)staining of the lower oesophagus, bronchial and lung tissues pathologyThe lower oesophagus,right main bronchus,and right lung tissues were removed and fixed in 4%polyformaldehyde for subsequent frozen section HE staining to observe the pathological changes using light microscopy(Olympus BX51,Japan).5,Detecting the expression of SP by means of immunohistochemistry technique in lower respiratory tract and viscerosensory afferent sites of the guinea pigs among different groups.SP protein was observed in groups of A B C,referencing to two-step immunohistochemistrical kits.6,The mRNA levels of SP in the lung and spinal dorsal horn were analyzed by RT-PCR,withβ-actin as an internal control..7,Western blot method to detect protein level of NGF in the lung.After extraction and quantification of protein from the lung,the samples were electrophoresed with SDS-PAGE.The samples were culture with primary and secondary antibody and detected.8,Statistical AnalysisThe data were analyzed with SPSS 13.0 software.The results are expressed as mean values±standard deviation.Differences between the groups were analyzed by t-test and one-way analysis of variance(ANOVA) followed by Fisher's LSD test.A P value of＜0.05 was considered statistically significant.Results1.Pathological changes of the lower oesophagus,bronchial and lung tissuesNo pathological changes of the oesophagus were observed in the PBS control animals.In the oesophagi of HCl-P-perfused animals,we observed infiltration of the mucosa by inflammatory cells,basal cell and spinous cell hyperproliferation,papillae hypertrophy,epithelia hyperkeratinization,squamous cell expansion,and increases in the number of fibrin cells.These results were consistent with the typical histological findings associated with low-grade reflux oesophagitis.Guinea pigs in the PBS control group presented with normal tracheal structures.In the HCl-P-treated model group, obvious hyperplasia,duplicated layer array,and nucleolus darkening of the ciliated columnar epithelium were observed.We observed large numbers of inflammatory cells, namely lymphocytes and eosinophils,infiltrating the submucosa.Airway epithelial desquamation was also evident in experimental animals.We also observed goblet cell metaplasia,ciliated columnar epithelium necrosis and degeneration,alveolus epithelium hyperproliferation,as well as in capillary vessels.Mucus plugs were seen in some of the bronchioles.Constitution of SR140333 group is releaser than that of the HCl-P-perfused group.2.The measurement of airway hyperresponsivenessIn response to increasing doses of intravenously administered ACh,all experimental guinea pigs showed dose-dependent increases in Re.However,when the dose of ACh was increased to 25 g/kg,the airway responsiveness increased significantly in HCl-P model animals when compared with PBS control animals. Airway resistance is more lessened in SR140333 group than the HCl-P-perfused.3.Cell numbers and types observed in guinea pig BALFThe total cell number,as well as lymphocyte and eosinophil counts were significantly greater in the BALF of HCl-P model animals than that of the PBS control animals(P＜0.01).The total cell number and eosinophil is more lessened in SR140333 group than the HCl-P-perfused.4.Immunohistochemistry analysis for SPSP in the lung tissues,C₇-T₅ spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups(P＜0.01).5.The mRNA expression of SPThe ratios of mRNA/β-actin mRNA in HCL-P model animals were increased significantly compared with control group and SR140333 group.6.Detectiong the expression of NGF in lung with Western blot experimentWestern blotting revealed significantly more intense expression of NGF in the lung from model guinea pigs than in the control(P＜0.01).Conclusion1.A novel external oesophageal perfusion model to induce oesophageal,tracheal and pneumonic histological injury similar to that associated with gastroesophageal reflux(GER) was evaluated successfully.2.SP in the lung tissues,C₇-T₅ spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups,which suggested that the neurognic inflammation of airway participated in the pathogenesis in the centre nerve of GER.3.NK-1 receptor antagonist may reduce the airway resistance in external oesophageal perfusion guinea pigs for reflux-associated respiratory symptoms.NK-1 receptor antagonist may also reduce the SP expression in the in lower respiratory tract and viscerosensory afferent sites of HCl-P-perfused group,which suggested that it restrained the neurognic inflammation of airway.4.NGF expression in the lung of control and model groups,and found increased NGF expression in the lung of model,suggest that NGF participates in the pathogenesis of GER inducing asthma.