Vagal and spinal sensory innervation is responsible for the regulation of neurogenic inflammation in the airways. Neurogenic inflammation is the result of the activation of sensory nerve endings by stimulant and induced through axon reflex to release neuropeptides from sensory nerve endings. These neuropitides are tachykinins, including substance P, neurokinin A and neuronkinin B. Tachykinin-1 (NK-1) receptors are mainly involved in neurogenic inflammation in the airway. It is found that 6-hydroxydopamine (6-OHDA) acts as a stimulant of sensory neurons that produces inflammation in the rat trachea. The magnitude of plasma leakage was expressed by the area density (%) of India ink-labeled blood vessels in tissue whole mounts. The present study found that area density of India ink-labeled blood vessel were 36.5%, 29.5%, 27.7%, 28.2%, 19.2%, 15.5% in the rat larynx, trachea, left bronchus, right bronchus, upper esophagus and distal esophagus after i.v. injection of 6-OHDA (100 mg /Kg), respectively. 6-OHDA could stimulate sensory neurons by free radicals that produced by non-enzymatic oxidation. NK-1 receptor antagonist can inhibit plasma leakage in airways. This study also tested the effect of a free radical scavenger. Rats are pretreated with a full dose (2.25 g/kg, i.v.) or lower doses of dimethylthiourea (DMTU) for a period of 15 min. We found that pretreatment with a full dose of DMTU could inhibit inflammatory plasma leakage induced by 6-OHDA, that was 4.8%, 1.6%, 1.1%, 2.4%, 0.4% and 1.0% in the rat larynx, trachea, left bronchus, right bronchus, upper esophagus and distal esophagus, respectively. It is suggested hydroxyl radicals mediated the inflammatory response in the respiratory tract and esophagus. DMTU dose-dependently decreased 6-OHDA-induced plasma leakage in the rat respiratory tract and esophagus. One sixth dose was effective in inhibition in esophagus. 6-OHDA-induced inflammation in the left and right bronchus could be reduced with 2/3 dose of DMTU. A full dose of DMTU (2.25g/Kg) was needed to inhibit inflammation in the larynx and trachea. It is concluded that sensitivity to 6-OHDA was different in the different part of lower airways and esophagus.

Endotoxin (lipopolysaccharide, LPS) a chemical component of cell wall of gram-negative bacteria, is an important mediator in pathogenesis of sepsis and acute respiratory distress syndrome. It causes production and release of a wide array of mediators including cytokines, chemokines, oxygen free radicals and nitric oxide from neutrophils, macrophages, endothelial cells and epithelial cells through the NF-£eB pathway. LPS increases the permeability of microcirculation, and causes the acute formation of numerous endothelial gaps among venular endothelial cells, resulting in extensive plasma leakage in the inflammatory tissue. Urethan is commonly used as an animal anesthetic for nonrecovery laboratory surgery. It is aslo an £\2-adrenoreceptor antagonist, which can suppress the activation of the cardiovascular system and reduce the angiotensin which increases the blood pressure. Urethan or its metabolites protect animals against LPS, in part, by reducing TNF-£\ release. The aims of the present study to investigate the time-course of vascular permeability in microcirculation of rat trachea, bronchus and esophagus after intravenous application of a high dose of LPS (15 mg/kg), and to reveal the role of urethan (1 g/¢V) and dimethylthiourea (DMTU, 0.375 g/¢V) in inhibition of LPS-induced plasma leakage and edema. India ink was used as a tracer dye to mark leaky microvessels after LPS application. Endothelial gaps were made visible for light microscopy by staining the borders of endothelial cells with silver nitrate. Tracheal sections were stained with toluidine blue to show the subendothelial edema formation. A high dose of LPS was administered intravenously to induce serious plasma leakage and edema and a large number of endothelial gaps formed in postcapillary and collecting venules in the rat trachea and esophagus. The peak values of plasma leakage and edema occurred 5 min after LPS (P<0.01). Urethan anesthesia significantly inhibited LPS-induced plasma leakage by 95 ¡Ó 1.7% in various parts of the respiratory tracts and inhibited edema ratio in the trachea by 57%. Urethan was also found to reduce leukocyte infiltration and the number of endothelial gaps by 46.8 ¡Ó 4.6%. DMTU pretreatment significantly inhibited plasma leakage by 88.5 ¡Ó 2.5% in the respiratory tract and inhibited edema ratio in the trachea by 89% at 5 min after LPS. It is concluded that LPS-induced increase in plasma leakage and edema correlated with the formation of endothelial gaps, and association with activation of alpha 2-adrenergic receptors and hydroxyl free radical production.

Intravenous application of a high dose of endotoxin, such as lipopolysaccharide (LPS), results in endotoxemia and sepsis in experimental animals. LPS induces production of cytokines and free radicals, plasma leakage and systemic inflammation. But the relationship between LPS-induced plasma leakage and endothelial gap formation is still unknown. Under normal physiological and pathological conditions, the mucus of intestine plays an important role in host defense mechanism as a barrier to prevent invasion of bacteria and endotoxin. The integrity of the intestinal epithelium is an important determinant of clinical outcome in septic patients. It is reported that, after LPS application, ileal mucosa is injured consequently. Necrosis of epithelial cells is also prominent feature in the villus epithelium. However, the response of mucin-secreting goblet cells is often ignored. The present study was designed to prove (1) whether LPS application increased plasma leakage by endothelial gap formation in rat intestinal tract, (2) whether LPS application increased goblet cell secretion by compound exocytotic activity in mucosal villi of small intestine; and (3) whether hydroxyl radicals were involved in LPS-induced compound exocytosis in goblet cells and plasma leakage. First, the microcirculation of large intestine in rats was shown by using silver nitrate staining method, and India ink was used to label the leaky microvessels to express the magnitude of plasma leakage. Endothelial gaps formed between endothelial cells in the venules after LPS-induced inflammation were investigated by light and scanning electron microscopy. In saline control, the number of endothelial gaps per 1000 £gm2 endothelium of postcapillary and collecting venules was 0.2 ¡Ó 0.1 ~ 0.4 ¡Ó 0.1 / 1000 £gm2 (n = 5). At 5 minutes after LPS application, the endothelial gap density drastically increased to 12.1 ¡Ó 1.6 ~ 27.5 ¡Ó 2.2 / 1000 £gm2 (n = 5 or 6), about 43-69 times (P < 0.01) as much as control. At the same time, the magnitude of plasma leakage, expressed by area density of India ink-labeled blood vessels, in the cecum and colon of LPS-treated rats increased to 7.8-8.2 times (P < 0.01) as much as control. Unusually high degree of plasma leakage and high number of endothelial gaps persisted for at least 30 minutes after treatment. Then, a significant reduction to the baseline level occurred at 60 minutes after LPS application (P > 0.05). The results evidently indicated that LPS-induced intestinal plasma leakage and the endothelial gap formation of venules were closely related. In the following experiment, in order to obtain an actual number of goblet cells in the mucosal epithelium, an innovative and effective experimental method was developed and adopted to prepare small intestine specimens in this study. Tissue pieces with two rows of mucosal villi were taken under a dissecting microscope. Then, scanning electron microscope was used to observe goblet cells and histochemistry staining was applied to further identify mucosubstance. The degree of goblet cell secretion in the villus epithelium of the duodenum and ileum was expressed by the number of cavitated goblet cells undergoing compound exocytosis. Digital morphometric software SimplePCI was employed to measure the epithelial surface area of sampled villi and to count the number of goblet cells. In addition, hydroxyl radical scavenger ¡V dimethylthiourea (DMTU) was also applied to explore the role of hydroxyl radicals involving in LPS-induced goblet cells secretion and plasma leakage. From scanning electron microscopy study, the numbers of cavitated goblet cells per mm2 of ileal villus epithelium in rats at 5 and 30 minutes after LPS injection were 693 ¡Ó 196 (n = 6) and 547 ¡Ó 213 (n = 6), respectively, which were 5.1 and 8.4 times (P < 0.05) compared with the number of saline control. The percentage of villus cavitated goblet cell numbers, in both duodenum and ileum 5 minutes after LPS and in the ileum 30 minutes after LPS, increased significantly (P < 0.05). When DMTU was given prior to LPS, the number of cavitated goblet cells and the amount of plasma leakage was inhibited and remained at the level as control (P > 0.05). It is concluded that the mechanism of the LPS-induced increase in compound exocytotic activity of goblet cells and increase in plasma leakage during acute phases of inflammatory response in rat small intestine was associated with hydroxyl radicals.