行政院國家科學委員會專題研究計畫 期中進度報告
賴激迷走分支與交感神經節對於大鼠呼吸道血管滲透性與
泩汰神經元免疫活性影響及機轉之研究(3/3)
計畫類別： 整合型計畫 計畫編號： NSC91-2320-B-110-012-執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立中山大學生物科學系(所) 計畫主持人： 黃宏圖 報告類型： 完整報告 處理方式： 本計畫涉及專利或其他智慧財產權，1 年後可公開查詢中
華
民
國 92 年 6 月 16 日
行政院國家科學委員會專題研究計畫期中報告
* 第三神經系統對肺及呼吸道之調整— 刺激迷走分支與交感神經節
*
* 對於大鼠呼吸道血管滲透性與泩汰神經元免疫活性影響及機轉之
*
*
研
究
(3/3)
*
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計畫類別: 整合計畫
子計畫編號: NSC 91-2320-B-110-012
執行期間: 91年8月1日~ 92年7月31日
主持人: 黃宏圖
執行單位: 國立中山大學生物科學系
中華民國 92 年 5 月 30 日
第三年研究進度報告
神經性發炎為經由感覺神經的軸突反射而引發的發炎反應，其間牽涉發炎 仲介物快太自 C 型神經元的神經纖維末梢釋放，並作用於微小血管壁。神經性 發炎發生在呼吸道，除造成微小血管的血漿外滲、並形成黏膜層水腫外，尚會引 起黏膜層上皮杯狀細胞的去顆粒作用。辣椒素為一感覺神經刺激物，能活化 C 型纖維進而引起發炎反應。本研究利用辣椒素(90 ug/kg）作為發炎誘發劑，長期 探討經性發炎引發後，大鼠在自然存活狀況下，在 5 min ~ 7 d之後,呼吸道黏膜 層水腫消退及黏膜上皮杯狀細胞黏液分泌及生成作用之變化情形。藉影像處理系 統之助，從氣管切片計量其水腫率，發現大鼠在辣椒素注射後迅速引發的黏膜水腫，經過 1 日即有顯著的消退情況，但消退至與未誘發發炎之動物(對照組)相 近的程度，則約需 5 至 7 日。透過經 CAE reagent 反應與 Alcian blue（AB） 染色之氣管整體封埋標本，可清楚觀察到含有黏液顆粒的杯狀細胞。在以辣椒素 溶劑處理的實驗組中，AB染色正反應的杯狀細胞密度為每mm2 有2,200 ±230 （mean ±S E, n＝6）個細胞，其平均直徑為 4.3 ±0.3 μm ，投予辣椒素後 5 分 鐘，Alcian blue-positive goblet cells 的細胞密度驟減至約一半，但平均直徑無 明顯差異。辣椒素注射誘發後 3 天，細胞密度回復至與控制組者無異但平均直 徑明顯變小，推測為黏液生成作用加強之故。透過切片觀察並以半計量形態計量 法評估杯狀細胞分泌作用，發現於發炎誘發後 5 分鐘迅即提高的分泌比率，在 第 1 天即有顯著降低，並在第 3 至 5 天降至與控制組無顯著差異的程度。
Abstr act
Previous studies investigate the short-term effect of capsaicin on edema formation and goblet cell secretion in the trachea. The present study sought to investigate the
long-term effect of a high dose of capsaicin (90 μg/ml/kg), administered intravenously, on changes in formation of endothelial gaps between venular endothelial cells, mucosal tissue edema, and secretory activity of goblet cells
including the number and size of goblet cells, and mucus score and secretory ratio of goblet cell mucus secretion in the trachea of rats. The tracheal whole mounts with silver staining, and those stained with chloroacetate esterase reagent and Alcian blue, and tracheal tissue sections stained with Alcian blue and periodic acid-Schiff reagent were used for evaluation. Formation of endothelial gaps occurred in a few min after administration of capsaicin, and gaps almost closed within 12 min after capsaicin injection. Five min after capsaicin, the leaky blood vessels were numerous and subepithelial edema ratio (% of length of edema along the inner circumference of tracheal cross section) was found to be 57.8 ± 3.0% (N=6). The number of Alcian blue-positive goblet cells (1,090 ±220 per mm2 of mucosal surface) was reduced to
half the number of goblet cells in the vehicle-treated rats
(2,200 ±330). Mucus score of goblet cell secretion was not changed. Secretory ratio was greatly increased. One day after capsaicin, edema ratio remained large and the number of Alcian blue-positive goblet cells was also small. Mucus score was also not changed. Secretory
ratio was still large. On day 3, the edema ratio remained large, but the number of Alcian blue-positive goblet cells was increased to the level of controls. Mucus score and secretory ratio returned to the control level. On day 5, edema ratio was greatly decreased, but it was still significantly larger than controls. Mucus score and secretory ratio remained at the baseline level. Seven days after capsaicin, the edema ratio was similar to the controls. The number of goblet cells was even larger than controls. It is concluded that capsaicin-induced acute inflammation in the rat trachea involves formation of endothelial gaps, extensive plasma extravasation and edema formation, and depletion of goblet cell secretory granules. Spontaneous gradual remission of edema was accompanied by regranulation of goblet cells
with gradual mucogenesis for several days.
Key words: Neurogenic inflammation, Respiratory tract, Plasma exudation, Cessation of edema, Goblet cell degranulation, Goblet cell regranulation
本研究論文發表於:
Jing-Jang Guo, Di-Seng Wang, Hung-Tu Huang (2003) Spontaneous remission of edema and regranulation of goblet cells in rat tracheae after capsaicin-induced acute inflammation. Anatomy and Embryology 206: 301-309.
論文全文如下:
Revised Ms submitted to Anatomy and Embryology, Oct. 18, 2002
tr achea after capsaicin-induced acute inflammation
Jin-Jang Guo, Di-Seng Wang, and Hung-Tu Huang*
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan
Content: 1 Text, 6 Figures, and 2 Tables
Running title: Tracheal edema and goblet cell secretion
*Correspondence: Hung-Tu Huang, Department of Biological Sciences, National Sun Yat-Sen
University, Kaohsiung, Taiwan. Tel: (07)5252000 x3611; Fax: (07)5253609; e-mail: [email protected]
Abstr act
Previous studies investigate the short-term effect of capsaicin on edema formation and goblet cell secretion in the trachea. The present study sought to investigate the
long-term effect of a high dose of capsaicin (90 μg/ml/kg), administered intravenously, on changes in formation of endothelial gaps between venular endothelial cells, mucosal tissue edema, and secretory activity of goblet cells
including the number and size of goblet cells, and mucus score and secretory ratio of goblet cell mucus secretion in the trachea of rats. The tracheal whole mounts with silver staining, and those stained with chloroacetate esterase reagent and Alcian blue, and tracheal tissue sections stained with Alcian blue and periodic acid-Schiff reagent were used for evaluation. Formation of endothelial gaps occurred in a few min after administration of capsaicin, and gaps almost closed within 12 min after capsaicin injection. Five min after capsaicin, the leaky blood vessels were numerous and subepithelial edema ratio (% of length of edema along the inner circumference of tracheal cross section) was found to be 57.8 ±3.0% (N=6). The number of Alcian blue-positive goblet cells (1,090 ±220 per mm2 of mucosal surface) was reduced to half the number of goblet cells in the vehicle-treated rats
(2,200 ±330). Mucus score of goblet cell secretion was not changed. Secretory ratio was greatly increased. One day after capsaicin, edema ratio remained large and the number of Alcian blue-positive goblet cells was also small. Mucus score was also not changed. Secretory
ratio was still large. On day 3, the edema ratio remained large, but the number of Alcian blue-positive goblet cells was increased to the level of controls. Mucus score and secretory ratio returned to the control level. On day 5, edema ratio was greatly decreased, but it was still significantly larger than controls. Mucus score and secretory ratio remained at the baseline level. Seven days after capsaicin, the edema ratio was similar to the controls. The number of goblet cells was even larger than controls. It is concluded that capsaicin-induced acute inflammation in the rat trachea involves formation of endothelial gaps, extensive plasma extravasation and edema formation,
and depletion of goblet cell secretory granules. Spontaneous gradual remission of edema was accompanied by regranulation of goblet cells
with gradual mucogenesis for several days.
Key words: Neurogenic inflammation, Respiratory tract, Plasma exudation, Cessation of edema, Goblet cell degranulation, Goblet cell regranulation
Intr oduction
Goblet cells are mucus-secreting cell in the mucosal surface epithelium lining the respiratory tract and the intestine. This cell type is characterized by the presence of numerous large glycoprotein-containing secretory granules. The supranuclear
cytoplasm of goblet cell is filled with secretory granules, and the nucleus is located at the cell base (Adler et al., 1982; Basbaum et al., 1988; Furuya et al., 1998).
Goblet cell secretion in mammals is under neural control. Vagus nerve
stimulation causes discharge of mucus granules and is blocked by either atropine or by capsaicin pre-treatment, indicating that both cholinergic nerves (Tokuyama et al., 1990) and capsaicin-sensitive sensory nerves (McDonald, 1988a; Huang et al., 1989; Tokuyama et al., 1990; Kuo et al., Huang, 1993; Huang et al., 1995) contribute to control of goblet cell secretion. Capsaicin produces inflammation in the airways by releasing and ultimately depleting substance P from the sensory nerve endings, which acts on the NK-1 receptors. Endothelial gaps form in venules (Bowden et al., 1994; McDonald, 1994) where the plasma leaks to the perivascular tissue. Associated with the acute neurogenic inflammation and edema formation in the airway mucosa induced by either capsaicin or substance P (McDonald, 1988b; Brokaw et al., 1990), airway goblet cells discharge many mucus granules from the supranuclear cytoplasm. It is suggested that substance P also acts directly on the goblet cells to cause
discharge of mucus granules into the airway lumen.
Previous studies investigate the short-term effect of capsaicin on plasma leakage and goblet cell secretion in the airways from 1 min up to a few hours. Goblet cell secretion is assessed in histologic sections of the tracheal mucosa stained with Alcian blue and periodic acid-Schiff reagent by a semiquantitative morphometric analysis
(mucus score). Mucus score is inversely related to the magnitude of mucus discharge (Kuo et al., 1990; Tokuyama et al., 1990; Takeyama et al., 1996). When goblet cell content of mucus is partially depleted by a low dose of capsaicin (Kuo et al., 1990), sensory neuropeptides (Kuo et al., 1990), or histamine (Takeyama et al., 1996), mucus score is useful. This method may be not applicable to a high dose of capsaicin,
substance P, or histamine which can produce acute inflammation and may cause complete depletion of secretory granules in many tracheal goblet cells.
Therefore, the total number of mucus granule-containing goblet cells may be largely reduced. The purpose of the present study was to characterize the formation and closure of endothelial gaps, plasma
leakage, cessation of mucosal edema, and changes in the secretory activity of goblet cells including the number and size of goblet cells, and the mucus score and the secretory ratio of goblet cell secretion. Whole mounts and plastic sections of rat trachea were studied 3, 5, and 12 min, and 1, 3, 5, and 7 days after acute inflammation was induced by a high dose of capsaicin administered intravenously.
Mater ials and Methods
Animal exper iments and pr epar ation of micr oscopic specimens
Induction of plasma leakage and secretory activity of goblet cells.
After anesthesia with an intraperitoneal injection of sodium pentobarbital (50 mg/kg, Koch-Light Laboratories, Colnbrok, Buckinghamshire, UK), 36 Sprague-Dawley rats received an intravenous injection of capsaicin (Sigma, St. Louis, Mo.) at the dosage of 90μg/ml/kg, or injection of vehicle (1% ethanol and 1% Tween 80) at the dosage of 1 ml/kg, over 2 min via the femoral vein. At different time points after the onset of injection, the magnitude of plasma extravasation, the edematous status of tracheal mucosa, the cell number and cell size of goblet cells, as well as the mucus score and secretory ratio of goblet cell secretion were
determined and evaluated.
For evaluation of the permeability-producing ability of capsaicin, India ink was used as a tracer dye to label the leaky blood vessels in the mucosal tissue (Lo and Huang, 1997; Hwang et al., 1999). One group of 6 rats received an intravenous injection of India ink (1 ml/kg, over 5 s) followed by an intravenous injection of capsaicin. Another group of 6 rats received an injection of India ink followed by a
dose of vehicle.Five min after capsaicin or vehicle injection, the rat chest was opened and a cannula was inserted through a cut in the left ventricle of the heart into the aorta to perfuse the circulation with 0.05 M phosphate buffer, pH 7.4 for 2 min, and then with 0.05 M phosphate-buffered 1% paraformaldehyde and 0.5% glutaraldehyde, pH 7.4, for 2 min at a pressure of 120-140 mm Hg. Afterwards, fixative was
retrogradely perfused through a cut in the mainstem bronchus into the tracheal lumen to wash off the covering mucus film and further fix the tracheal mucosal tissue. In the remaining groups each with 6 rats, injection of capsaicin was followed by suturing the wound skin with metal clips at the groin where the femoral vein was exposed. They were allowe d to survive for 1, 3, 5, or 7 days before perfusion fixation to study the time -course of changes in mucosal edema and secretory activity of goblet cells. Injection of India ink was not carried out.
Goblet cells and globule leukocytes in tracheal sections and tracheal whole mount The rostralmost portion of trachea with the 1st to 6th cartilage rings was removed for preparation of glycol methacrylate sections for the measurement of edema ratio in the mucosa and the measurement of mucus score and secretory ratio of goblet cell
secretion. The remaining middle and distal portion of trachea was used for the
prepartion of whole mount for the measurement of the size of goblet cells, and for the count of number of goblet cells.
Globule leukocytes and goblet cells in tracheal epithelium of whole mounts were differentiated. Whole mount with the 7th to 25th cartilage rings was pinned on a piece of Sylgard (Dow Corning, Midland, MI, USA) using stainless steel pins and
incubated with chloroacetate esterase medium containing naphthol AS-D chloroacetate (Sigma) as the
substrate and hexazotized pararosanilin (Sigma) as the chromogen (Tam et al., 1989) for 14-18 hours at 4 °C, followed by a wash in distilled water for 2 hours. Globule granules of variable sizes in globule leukocytes were stained orange to red color. Whole mounts were
further stained with 0.5% Alcian blue (Sigma) in 3% acetic acid, pH 2.5, for 30 minutes. At this time, globule leukocytes appeared dark red, because cytoplasmic granules were also stained with Alcian blue. Goblet cells that contained a large number of cytoplasmic mucus
granules were only stained with Alcian blue, so they appeared brilliantly blue. Tracheal whole mounts were dehydrated with ethanol, cleared with toluene, and
mounted with Permount (Fisher Scientific, New Jersey, USA) with the mucosal surface upward.
The rostral part of trachea was dehydrated with a graded series of acetone, and embedded in glycol methacrylate (Merck). Cross sections of trachea 3 μm in thickness were stained with 0.5% Alcian blue in 3% acetic acid, pH 2.5, for 30 min, treated with 1% periodic acid (Merck) for 5 min, and stained again with the Schiff reagent for 5-10 min (Huang et al., 1989).
F ormation and count of endothelial gaps in tracheal venules
Three groups of rats, each with 4 animals, were used. After anesthesia with sodium pentobarbital, rats received an intravenous injection of capsaicin 90 μg/ml/kg over 2 min. Three, 5, or 12 min after the onset of capsaicin injection, rats were perfused with the fixative containing 0.5% glutaraldehyde and 1% paraformaldehyde in 0.075 M cacodylate buffer, pH 7.4, for 5 min at a pressure of 120-140 mm Hg. Then, 80 ml of 0.9% NaCl were perfused over 2 min at a pressure of 120-140 mm Hg, 25 ml of 5% glucose in water over 10 s, 20 ml of 0.2% AgNO3 in water over 7 s, 25 ml of 5%
glucose in water over 10 s, and finally 50 ml of fixative were perfused over 1 min at a pressure of 80-100 mm Hg. The trachea was cut open along the ventral midline, excised, and pinned to a layer of Sylgard on a Petri dish. The silver halide was developed by a 15-min exposure of light from a fiber optic illuminator as in a previous study (McDonald, 1994). Whole mounts of tracheas were prepared by the above-mentioned method. Silver dots observed between endothelial cells of venules were identified as endothelial gaps. For each rat, 40 to 60 endothelial cells from 4 to 6 postcapillary venules were examined and the total number of endothelial gaps between cells sampled in the middle portion of trachea was recorded.The data were expressed as the number of gaps per endothelial cells, which was calculated by dividing the total number of gaps by the total number of observed endothelial cells.
Measur ement of plasma leakage
The magnitude of plasma leakage was expressed by the area density of India ink-labeled leaky blood vessels in the mucosal tissue of tracheal whole mounts determined by a point counting method (Lo and Huang, 1997; Hwang
et al., 1999).
Meassur ement of mucosal tissue edema
For each rat, a single crossly sectioned tracheal tissue was used. The subepithelial edema pushed the tracheal surface epithelium away from its underlying lamina propria. The perimeter of the tracheal surface epithelium was recorded by tracing along the bases of epithelial cells and the length of the border between the subepithelial edema and its covering epithelium recorded by tracing along the basement membrane with a computer based image analysis system and ImageTool software (University of Texas Health Science Center at San Antonio, USA). The magnitude nof mucosal edema was represented by the subepithelial edema ratio (% of length of edema along the inner circumference of tracheal cross section).
Measur ement of the number and size of goblet cells in the tr acheal whole mounts
The tracheal whole mounts stained with chloroacetate esterase reagent and Alcian blue were used. Goblet cells were sampled from the mucosal surface epithelium in the areas between cartilage rings near to the posterior membrane. Twelve visual fields, 6 from the right side and
6 from the left side, were magnified to x400 under a conventional light microscope. An ocular micrometer with a 100-square grid was placed on the visual field of tissue. The number of goblet cells was counted and their diameters were measured with an ocular micrometer in 60 squares for each visual field. The area of each square measured 773 µm2. For each rat trachea, the total number of squares sampled was 720, and the total area of which was 555,560 µm2. Only goblet cells with a round or oval mass of accumulation of mucus granules that appeared brilliantly blue were sampled. Over 500 Alcian blue-positive goblet cells from each trachea were studied. For each trachea, the average size of goblet cells was calculated as the average of diameters of sampled goblet cells.
Semiquantative assessment of goblet cell secr etion in tr acheal sections
from two serial, but not consecutive, tracheal sections. Mucus score (in ar bitr ar y units) was calculated as: (1 x n1 + 2 x n2)
where 1 and 2 were the gradings, and n1 and n2 the total numbers of cells in each
grade, respectively (Kuo et al., 1990; Tokuyama et al., 1990). Grade 1 was that the vertical distance of the stained area of goblet cell was within one-third of the
epithelial layer, measured from basement membrane to cell apices. Grade 2 was that the vertical distance of the stained area of goblet cell exceeded one-third of the epithelial layer. Stained cells that were agranular or did not have a well-defined boundaries were not included in the counts.
The secretor y r atio was calculated as the ratio of the number of grade 1 cells to the number of grade 2 cells in each animal (Kuo et al., 1990) .
Statistical analysis
Experimental data were expressed as means ± SEM. Data were analyzed with
one-way ANOVA followed by Newman-Keuls method. Probability (P) values of less than 0.05 were considered significantly different.
Results
Magnitude of inflammation
Five min after the onset of capsaicin injection, plasma leakage was extensive in the trachea as evidenced by the numerous India ink-labeled leaky blood vessels in the mucosa. The area density of India ink-labeled blood vessels was 15.7 ± 2.1% (N=6). The vehicle of capsaicin produced only a little extravasation, with an area density of India ink-labeled blood vessels of 0.2 ± 0.1% (N=6), significantly less than after capsaicin (P < 0.01).
For mation and closur e of endothelial gaps
Three min after the onset of intravenous injection of capsaicin, many endothelial gaps (4.80±0.11 per endothelial cell, N=4) were observed along the cell borders between endothelial cells of postcapillary venules.The number of gaps per
endothelial cell was greatly reduced by 52% at 5th min after capsaicin injection (P < 0.01, Table 1).
Twelve min after capsaicin, the number of gaps per endothelial cell was reduced by 98%. They had almost disappeared from the venules.
Edema r atio
Mild local subepithelial edema was found in the tracheal section of control rats receiving injection of vehicle. Extensive edema in the lamina propria was observed 5 min after the onset of capsaicin injection (Fig. 1). Prominent subepithelial edema lasted for 3 days (Fig. 2). It was interesting to find that the subepithelial edema area was continuous, but not limited to the right, left, ventral, or dorsal side of the tracheal mucosa. Edema formation was not present beneath the stratified squamous
epithelium that covered the mass of lymphoid tissue. The edema ratio was 57.8±3.0 (N=6) 5 min after capsaicin (Fig. 3). It remained large on days 1 and 3, was
diminished strikingly on day 5, and on day 7 was restored to the baseline level of vehicle-treated rats.
Cell number and cell size of goblet cells
Goblet cells were clearly differentiated from globule leukocytes in the surface epithelium of the tracheal whole mounts stained with chloroacetate esterase reagent and Alcian blue (Figs.1 and 2). Control rat trachea exhibited numerous Alcian blue-stained goblet cells (Fig. 1a), 2200 ±230 per mm2 of mucosal surface (N=6) (Fig. 4), with an average diameter of 4.26 ±μm (Table 2). Five min after capsaicin, goblet cell number was reduced to 1090 ±22 (P < 0.01) about half the number present in vehicle controls (Figs. 1a, 1c, and 4). One day after capsaicin, the cell number was also smaller than in controls (P < 0.05). On days 3 and 5, the cell
number was increased to the level of the controls (P > 0.05; Figs. 2a, 2c, and 4). Cell size of goblet cells was small on day 3 (Table 2). On day 7 after capsaicin, the cell number was even larger than in the controls (P < 0.01).
There were numerous globule leukocytes in the tracheal epithelium (Figs. 1 and 2). Although the present study did not pay attention to these cells, capsaicin seemed not to have depleted the cytoplasmic granules in globule leukocytes or reduced the cell number.
Histological str uctur e of mucosal sur face epithelium and goblet cell str uctur e The tracheal epithelium was consisted of goblet cells, columnar ciliated cells, globule leukocytes, and basal cells. In control rats, goblet cells contained a large
accumulation of purple mucus granules in the supranuclear cytoplasm that made the cells goblet-shaped or dumb bell-shaped (Fig. 1b). Five min after capsaicin, many goblet cells were partially or completely depleted of mucus granules (Fig. 1d). These cells were no longer goblet-shaped or dumb bell-shaped. Now, they assumed a thin, rod-like shape. Partially depleted goblet cells
had a small aggregate of mucus granules in apical cytoplasm. The diameter of these cells was obviously decreased. Three days and 5 days after capsaicin, goblet cells appeared to store more mucus granules in the cytoplasm (Figs. 2b and 2d). Seven days after capsaicin, many
goblet cells were hypertrophied due to refilling of mucus granules in the cytoplasm (Fig. 2f).
Mucus scor e
The mucus score between groups was not significantly different when analyzed with one-way ANOVA (P > 0.05). This was due to a large variation in the number of mucus granule-storing goblet cells (Fig. 5) during the process of degranulation and regranulation.
Secr etor y r atio
The secretory ratio of goblet cell secretion was significantly different (P < 0.01) between the control rats and rats 5 min or 1 day after capsaicin (Fig. 6). Three, 5, or 7 days after capsaicin, the secretory ratio was not statistically different (P > 0.05) from that of controls, but significantly smaller (P < 0.01) than that of 5 min after capsaicin.
Discussion
The present study was potentially applicable for the study of the effects of neural and non-neural inflammatory agents on the time course of edema formation and cessation, and discharge and reappearance of mucous cell secretion, and for the elucidation of the
underlying mechanism. Most previous studies on experimentally-induced inflammation in the trachea focus on the magnitude of plasma leakage by determination of the amount of extravasated tracer dye, e.g. Evans blue, in the tissue with a spectrophotometer (Saria and Lundberg, 1983; Lundberg and Saria, 1983; Brokaw et al., 1990), measurement of the relative number of leaky venules labeled by the colloidal dye particles with a
morphometric method (Huang et al., 1989; Luo and Huang, 1997; Baluk et al., 1997), and measurement of the optical density of colloidal dye that labels the leaky venules with a microscope photometer (McDonald, 1988a, 1988b). After an intravenous injection of capsaicin (McDonald, 1988b; Huang et al., 1989; Huang, 1993) or substance P (McDonald, 1988b; Huang et al., 1995), the exudate that leaks out from the venules accumulates largely
underneath the mucosal surface epithelium and forms a subepithelial edema. The degree of edema formation and how long it is retained are largely neglected.
Previous studies have noted that the tracheal edema correlates with the extensive plasma leakage, which is associated with the formation of venular endothelial gaps that are resulted from the contraction of endothelial cells after the binding of substance P to the NK -1 receptors of cells (McDonald, 1994; Thurston et al., 1996; Baluk et al., 1997). The present study demonstrated that prominent edema produced by capsaicin persisted for 3 days, a duration that was much longer than expected. Edema ratio was near 60% at 5 min after
capsaicin, and was about 40% on day 1 or 3 after capsaicin. Although the
time-course of change in the edema ratio was only based on single cross sections of the rostral part of the trachea, serial sections exhibited that the edematous region was usually continuous. The
edema pushed the pseudostratified columnar epithelium away from the lamina propria. In our observations, the India ink-labeled leaky microvessels were evenly distributed in the rostral, middle, and distal parts of the trachea.
Venular endothelial gaps are short-lived. The number of endothelial gaps (14 – 16 per endothelial cell) in postcapillary venules peaks at 1 min after a high dose of
substance P, 5 μg/kg, is intravenously injected (McDonald, 1994; Baluk et al., 1997). The number declines
rapidly with a half-life of 1.9 min. Endothelial gaps almost disappear at 10 min after substance P. The time-course of change in plasma leakage is correlated well with the formation and closure of gaps (McDonald, 1994; Baluk et al., 1997). Formation of the number of gaps and the magnitude of plasma leakge are dose-dependent. In the present study, the magnitude of plasma leakage produced by the dose of capsaicin 90 μg/kg was equivalent to the dose of substance P 3μg/kg (Brokaw et al., 1990; Huang et al., 1995). The present study presented a time-course of the formation and closure of endothelial gaps after
capsaicin, that was similar to the previous studies using substance P (McDonald, 1994; Baluk et al., 1997). It is reasonable for the present study to find that the number of gaps at each time point was smaller than the previous results.
Capsaicin-induced acute inflammation was associated with total depletion of mucus secretion from many goblet cells. Five min and 1 day after capsaicin injection, the number of Alcian blue-positive goblet cells in the tracheal whole mounts was greatly reduced. Secretory ratio of goblet cell secretion was greatly increased. In addition to its action on the endothelial cells of venules, substance P might simultaneously act on the goblet cells to stimulate their secretory function. Goblet cells probably contracted and discharged mucus granules into the tracheal lumen to clean the harmful irritant and the exudate present in the tracheal lumen, and to protect the mucosal surface. The affected C-fiber sensory neurons develop desensitization to capsaicin after the release of tachykinins (Holzer, 1991). It is not known if goblet cells developed desensitization to capsaicin. They soon began to synthesize and store mucosubstance to prepare to meet the coming challenges. Goblet cells that were partially or
completely depleted of their secretory granules were thin, rod-shaped. Mucogenesis in goblet cells resulted in an increase in the size and number of Alcian blue-positive cells. Accumulation of mucus granules that caused thickening of cells started at the basal portion of the cytoplasm, and then extended to the apical portion.
In conclusion, the secretory activity of goblet cells during the acute hypersecretory phase, and the subsequent restoration and granular refilling was related to the mucosal edema in rat trachea following capsaicin-induced neurogenic inflammation. The results suggest that degranulation of many goblet cells is associated with the
occurrence of extensive plasma extravasation and formation of mucosal edema in the rat trachea. Regranulation by mucogenesis in goblet cells was associated with the cessation of inflammation.
Acknowledgment
This study was supported by the grant NSC 90-2314-B-110-001, from the National Science Council, Republic of China.
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