Summary: | 碩士 === 國立中山大學 === 生物科學系研究所 === 94 === Stimulation of C-fiber sensory neurons innervating the respiratory tract with electricity or capsaicin leads to the liberation of substance P, CGRP and other neuropeptides from the nerve terminals. Substance P (SP) binds to the NK-receptors on the membrane of vascular endothelial cells and elicits neurogenic inflammatory responses. These inflammatory responses include plasma leakage and the subsequent edema formation (Lundberg and Saria, 1983;McDonald et al., 1988). Evans blue is a hydrophilic dye and is often used as a tracer of plasma leakage due to its’ high affinity to the plasma proteins. Plasma leakage causes Evans blue extravasates from the blood vessels and remains in the tissues. The more plasma leaks from the blood vessels, the more Evans blue will extravasate into the tissues. Measuring extravasated Evans blue dye that is extracted from tissues, is useful for evaluation of the amount of plasma leakage. Potassium channel openers can inhibit neurogenic plasma leakage in the airways and urinary bladder (Hollywood et al., 1998). Evans blue directly stimulates large-conductance Ca2+-activated K+ channels in cultured endothelial cells of human umbilical vein (Wu et al., 1999). This suggests that it may influence the permeability of the microvessels in vivo. A previous study shows that Evans blue dye blocks capsaicin-induced cough and bronchospasm in the guinea pig (Bolaer et al., 1995). We postulated that pretreatment with Evans blue may influence the extent of neurogenic inflammation in the rat airways induced by the application of either SP or capsaicin. India ink was used as a colloidal tracer dye to label the leaky vessels. The present study investigated whether different concentration of Evans blue (0, 3, 15 and 30 mg/ml/kg) pretreatment could affect the plasma leakage and edema formation in rat lower airways in response to intravenous injection of either SP or capsaicin. The amount of plasma leakage was expressed by the area density of India ink-labeled leaky blood vessels. We also investigated whether Evans blue influenced the ultra-structural change in tracheal serous cells induced by intravenous injection of SP. Our results showed that pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea caused by SP application (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), compared to the control group that received saline prior to SP. However, no statistical significance was observed in edema ratio between any two groups (P>0.05). In the neurogenic inflammation of the airways caused by injection of capsaicin, pretreatment with high concentration of Evans blue reduced more than seven tenths of the area density of plasma leakage in the trachea (P<0.01); reduced more than seven tenths in the left main bronchus (P<0.01) and reduced about seven tenths in the right main bronchus (P<0.01), respectively, compared to the control group that received saline prior to capsaicin. Pretreatment with high concentration of Evans blue prior to capsaicin also reduced more than eight-tenth in edema ratio (P<0.01). In the ultra-structure change of serous cells and the stastical analysis of the number of active serous per 1000μm2 of tracheal epithelium, Evans blue pretreatment prior to SP significantly reduced the number of active serous cells by seven tenths (P<0.01) as compared to control group that received saline prior to SP. Therefore, we concluded that pretreatment with high concentration of Evans blue exerted its’ effect by opening large-conductance Ca2+-activated K+ channels and inhibited the plasma leakage induced by SP or capsaicin. But no significant inhibition was observed in edema formation induced by SP application. Low concentration of Evans blue might enhance the neurogenic inflammation of the airway. Under the observation with SEM, we found that SP activated serous cells in airway epithelium, and high concentration of Evans blue pretreatment lowered the secretory activity of serous cells. Therefore Evans blue might inhibit the activation of serous cells.
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