Effect Of Intra-esophageal Perfusion With Different Physical-chemical Solutions On The Guinea Pig Airways And Its Mechanism

Intra-esophageal acid infusion can induce the airway neurogenic inflammation, which may be mediated by esophageal-tracheobronchial reflex. There are no reports whether intra-esophageal perfusion with cold, hot or other different physical-chemical solutions could induce inflammation in the airways. Transient receptor potential (TRP) ion channel family has been recognized to sense a vast of chemical and physical stimuli and distribute widely in different tissues and organs. The members of TRP family: transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanillic-1(TRPV1) can be activated directly by cold (<17℃)or hot stimulation (>43℃) respectively, and other chemical stimuli, working as polymodel sensors. This study is to investigate the effect of intra-esophageal perfusion with different physical-chemical solutions on the airway inflamationin in guinea pig model.PartⅠEffect of intra-esophageal perfusion with different physical–chemical solutions on the airway plasma extravasationSegment 1Objective: To observe the the plasma extravasation of airway when intra-esophagus was perfused with different physical–chemical solutions, role of TRPV1, TRPA1 and neuropeptide in this process.Method: Guinea pigs were perfused for 20mins with 37℃normal saline (NS) (control group), cold NS(4℃) group, hot NS (45℃) (hot group), hydrochloric acid (0.1M)(HCL group), 50% glucose solution ( hypertonic group) and ethanol (ethanol group), in the presence or absence of neutral endopeptidase inhibitor phosphoramidon, TRPV1 antagonist capsazepine, TRPA1 antagonist HC-030031(only in cold group), or atropine and propranolol. The plasma extravasation of the trachea, bronchi and bronchiole was measured by Evans blue dye, the total number and cell differiential in BALF were calculated.Results1. The plasma extravasation in trachea, main bronchi and bronchiole was increased significantly [(31.2±10.61)pg/mg, (18.82±5.11)pg/mg, (12.24±1.94)pg/mg in cold group, P<0.05; (54.02±13.51)pg/mg, (40.59±11.57)pg/mg, (28.31±10.03)pg/mg in hot group, P<0.001 in trachea and P<0.05 in main bronchi and bronchiole] compared with control group [(16.69±6.02)pg/mg,(11.59±2.45)pg/mg,(7.55±2.40)pg/mg ].2. Intra-esophageal perfusion with hydrochloric acid and ethanol significantly increased the trachea, main bronchi and bronchiole plasma extravasation [(74.41±12.03)pg/mg,(73.37±13.69)pg/mg,(53.84±17.18)pg/mg in ethanol group, P <0.001; (42.80±13.41)pg/mg,(29.74±11.60)pg/mg,(16.77±5.58)pg/mg in acid group, P <0.05 ]. No significantly difference was found in hypertonic group.3. Phosphoramidon significantly potentiated the trachea, main bronchi and bronchiole plasma extravasation induced by perfusion with cold, hot, acid and ethanol solutions (, P<0.001 in trachea and main bronchi, P<0.05 in bronchiole for both cold and hot group; P<0.05; ethanol: P<0.05 in trachea and main bronchi for acid group).4. TRPA1 antagonist HC-030031 inhibited the airway plasma extravasation induced by perfusion with cold solution (trachea and main bronchi: P<0.01); Capsazepine inhibited the airway plasma extravasation induced by perfusion with hot solution and acid (P<0.05 in trachea and bronchi). Plasma extravasation didn't change in ethanol group pretreated with capsazepine (P>0.05).5. When animals were pretreated with atropine and propranolol group before cold stimulation, the plasma extravasation is not different compared to single cold stimulation group (P>0.05).6. The total number and cell differential in the bronchia-alveolar lavage fluid is not changed significanyly in all groups (P>0.05).Conclusions: Intra-esophageal perfusion with different physical-chemical solutions induce the airway plasma extravasation. which may be related with the release of neuropeptid in airway and TRPA1 and TRPV1 activation.Segment 2Objective: To measure the concentrations of substance P (SP) in main bronchi when the esophagus was perfused by different solutions discriptioned above.Methods: Guinea pigs were divided as segment 1. Concentrations of SP in bronchi were measured by ELISA.Results1. The concentrations of SP in main bronchi in cold, hot, acid and ethanol groups were significantly higher than control group [(6.79±1.11)pg/mg, (12.71±3.03)pg/mg, (10.76±3.61)pg/mg, (19.98±7.84)pg/mg vs (4.12±1.15)pg/mg, respectively, P<0.05)]. No significant difference was found between hypertonic group and control group (P>0.05).2. Being pretreated with phosphoramidon in heat and cold, acid and ethanol groups significantly increased the concentration of SP in bronchi (P<0.05). 3. HC-030031 significantly decreased the concentration of SP in bronchi in cold stimulus group (P<0.05); Capsazepine significantly decreased the concentration of SP in hot group (P<0.01) and acid group (P<0.05). The concentration of SP didn't change in ethanol group pretreated with capsazepine(P>0.05).Conclusions: Intra-esophageal perfusion with cold and hot saline, acid and ethanol increaded the concentration of SP in bronchi, which could be inhibited by TRPA1 antagonist and TRPV1 antagonist. The activation of TRPV1 and TRPA1 may contribute to the release of SP in the airway.PartⅡThe expression of temperature-chemical receptors TRPV1, TRPA1 and C fibers in esophagus and ganglionsObjective: To study the expression of temperature-chemical receptors TRPV1, TRPA1 and C fibers in guinea pigs esophagus and ganglions.Methods: Six Hatlay guinea pigs were anaesthetized and then the esophagus, nodose ganglion and dorsal root ganglion were removed and sectioned on a cryostat. The expression and distribution of TRPV1, TRPA1 and C fibers on esophagus was examined with fluorescence immunostaining and observed with laser scanning confocal microscope.Results:TRPA1-immunoreactivity fibers were found on the mucous epithelial, mucous layer and vessel walls of the esophagus, and TRPA1 immonoreactivity neurons on doral root ganglion (DRG) and nodose ganglion. SP-immunoreactivity fibers existed on the mucous layer and muscul layer, SP-immunoreactivity neurons were observed on the DRG and nodose ganglion, and TRPV1-like immunoreactivity fibers on mucous layer and the vessel wall of the esophagus, TRPV1- immunoreactivity neurons also were found on DRG and nodose ganglion.Conclusions: There are TRPV1, TRPA1 and C fibers positive nerve expressed on guinea pigs esophagus and ganglions, which suggests that TRPV1 and TRPA1 may be activated by intra-esophageal different physical-chemical materials.