Corticosteroid Inhibition of Airway Microvascular Leakage 1- 4
PIERA BOSCHETTO,5 DUNCAN F. ROGERS, LEONARDO M. FABBRI, and PETER J. BARNES
Introduction
Increased permeability of the microvasculature with leakage of plasma into the interstitium is an important component of the acute inflammatory response. In the airways, chronic inflammation may underlie the pathogenesis of a number of bronchial diseases, in particular, asthma, where the contribution of both inflammation (1)and plasma exudation (2) to bronchial hyperresponsiveness(3) have been emphasized. Specifically, edema is a distinguishing characteristic of the airways in patients who have died in status asthmaticus (4). However, there is evidence of edema in comparatively healthy asthmatics, including those in remission (4, 5). In addition, plasma proteins can be detected in the sputum (6-9) and, more importantly, in the bronchoalveolar lavage fluid (10)of asthmatics. Airwayedema leads to bronchial narrowing, but, perhaps more importantly, interstitial plasma will induce activation of biochemical pathways leading to the generation of a number of inflammatory mediators that may amplify the leakage response (11). As well as inducing further leakage, some of these mediators may directly affect airway smooth muscle and its normal control (12). Luminal plasma, in addition to increasing the volume of airway fluid, may induce mucus secretion (13)and increase the viscosity of the mucus (14), both of which would impair mucociliary transport (15). Edema of the airway wall may have important functional consequences and may markedly amplify the airway narrowing induced by bronchoconstriction (16).Thus, indirect evidence for the involvement of plasma exudation in the pathogenesis of asthma is compelling (2, 17), and strategies to reduce leakage may be important in the therapy of chronic asthma. Corticosteroids are veryeffective in the treatment of chronic asthma, although the precise mechanisms underlying their beneficial action are unknown (18). Inhibition of plasma leakage is one possibility because corticosteroids inhibit mediatorinduced edema and plasma leakage in a number of systemic vascular beds (19-
SUMMARY We studied the effect of dexamethasone on microvascular leakage (using Evans blue dye as a marker of plasma exudation) Induced in rat airways by platelet·actlvatlng factor (PAF). Intra· venously administered PAF caused a dose-related Increase In plasma leakage over the range 0.1 to 1 l19/kg. At 500 ng/kg PAF, the response was maximal In the extrapulmonary airways elUlmlned with increases In leakage above those In control animals of 312% In the larynx, 295% In the trachea, and 167% In the main bronchi. A mexlmal response was not achieved In the Intrapulmonary airways at the doses of PAF tested: at 1 l19/kg the increase was 206% above that In control animals. Dexamethasone, given by Intraperitoneal injection 24 hand 4 h before PAF at a dose of 0.2 mg/kg on each occasion, partially Inhibited leakage Induced by PAF (1 l19/kg) In all airway levels studied by 43 to 65%. At each level the tissue concentration of dye was reduced to a value that was significantly (p < 0.05)different from either PAFor control values. Wealso determined whether a high dose (8 mglkg) of dexamethasone given Intraperltoneally would Inhibit plasma leakage of dye Induced by either PAF or antlgen-ehallenge of sensitized rats. When given 4 h before antigen, dexamethasone completely prevented allergen.lnduced leakage in the airways showing significant leakage (larynx, trachea, and Intrapulmonary airways). Similarly, dexamethasone (4 h before) partially Inhibited PAFinduced leakage In the trachea and main bronchi. In summary, In rat airways, both low and high doses of dexamethasone markadly inhibit medlator·lnduced plasma exudation. This effect may contribute to the beneficial action of corticosteroids In allergic disease, Including asthma. AM REV RESPIR DIS 1991; 143:605-609
23), as well as attenuating capsaicin-induced leakage of plasma into the tracheal lumen in guinea pigs (24). In the present study we investigated whether a comparatively low dose of dexamethasone (0.2 mg/kg, twice in 24 h), would inhibit plasma exudation in both extrapulmonary and intrapulmonary airwaysinduced in rats by platelet-activating factor (PAF). We used dexamethasone because it is a potent corticosteroid that is water-soluble and so may be given by intravenous injection. We used rats because a number of studies have demonstrated that steroids are markedly more potent in this species than, for example, in guinea pigs (25). PAF was used to induce plasma leakage because it is potent, its mechanism of action has been well characterized (26, 27), and it exhibits a number of biologic properties consistent with a putative mediator of asthma (28). Wealso determined whether a singledose of 8 mg/kg dexamethasone would inhibit airway leakage induced by either PAF or allergen challenge of sensitized rats. We used Evans blue dye as a marker of plasma leakage since this allows the measurement of leakage at different airway levels. Methods Male Sprague-Dawley rats (250 to 400 g) were
premedicated with diazepam (3 mg/kg) by intraperitoneal injection and anesthetized with 0.5 ml Hypnorm (containing 0.155 mg fentayl citrate and 5 mg fluanisone) by intramuscular injection.
Measurement of Plasma Exudation The jugular veins were exposed, and drugs were administered intravenously by passing the injection needle through the pectoralis major to avoid bleeding on withdrawal. Evans blue dye (30 mg/kg) was injected, followed I min later by PAF, ovalbumin, or their appropriate controls (see below). The magnitude of plasma leakage was determined by measuring the concentration of dye extravasated into the airway tissue after first removing intravascular dye by a method described
(Received in original form May 16, 1990) 1 From the Department of Thoracic Medicine, National Heart & Lung Institute, London, United Kingdom. 2 Supported bythe Asthma ResearchCouncil and Medical Research Council of Great Britain. 3 Presented in part at the Annual Meeting of the American Thoracic Society, Cincinnati, May 1989. • Correspondence and requests for reprints should be addressed to Dr. D. F. Rogers, Department of Thoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY. United Kingdom. S Recipient of Grant No. 87.00266.56 from the Italian Research Council.
605
606 previously(26).Fiveminutes after PAF or ovalbumin, the thorax was opened and a bluntended 13-gauge needle was passed through a left ventriculotomy into the aorta. The heart was clamped, the right atrium was incised to allow outflow of perfusate, and the systemic circulation was perfused of blood with 100 ml saline (pH, 5.5; 21° C) at 100mm Hg pressure. The larynx, trachea, main bronchi, and lungs were removed. The main bronchi were separated from the trachea, and the remaining intrapulmonary airways were stripped of parenchyma by gently scraping with a razor blade. Excess fluid was removed by squeezing between filter papers, and the tissue wet weights were recorded. Evans blue dye was extracted by incubating tissues in formamide at 37° C for 16 h, and its concentration was determined against a formamide blank by light absorbance at a wavelength of 620 nm (SP 1750 spectrophotometer; Pye Unicam, Cambridge, UK) and interpolation on a standard curve of dye concentrations in the range 0.5 to 10 ug/ml in formamide. Airway concentration of dye was expressed as nanograms Evans blue per milligram wet weight tissue.
Experiment 1: Low Dose Dexamethasone The relationship between dose of PAF and magnitude of leakage of Evans blue dye was examined by comparing the response to intravenously administered doses of PAF of 50 (n = 5), 100 (n = 5), 250 (n = 3), and 500 (n = 7) ng/kg and 1 ug/kg (n = 5) with the response to control vehicle (BSA-saline; see below). Because control procedures reduced the response to PAF (see RESULTS), the inhibitory effect of dexamethasone was determined at the highest dose of PAF above. Dexamethasone was administered intraperitoneally at a dose 0.2 mg/kg on each of two occasions, 24 h and 4 h, before 1 ug/kg PAF (n = 6). Baseline leakage was determined in six rats given saline (the diluent for dexamethasone) 24 hand 4 h before BSA-saline. The effect of dexamethasone (0.2 mg/kg, 24 h and 4 h) on baseline leakage to BSA-saline was determined in a further six rats. Finally the effect of the administration procedure was accounted for by injecting animals with saline 24 h and 4 h before 1 ug/kg PAF (n = 5). Experiment 2: High Dose Dexamethasone Dexamethasone was given intraperitoneally atadoseof8mg/kg2h(n = 3)or4h(n = 6) before 500 ng/kg of PAF was given intravenously. Baseline leakage was determined in five rats given saline 4 h before BSA-saline. The effect on leakage of saline given intraperitoneally 4 h before PAF was assessed in a further five rats. The inhibitory effect of the high dose of dexamethasone was also assessed in rats sensitized 3 wk prior to experimentation using a single intraperitoneal injection of 1 ml saline containing 267 mg aluminium hydroxide and 1.33mg ovalbumin (29). Sham-sensitized (control) animals were injected with 1 ml saline containing 267 mg aluminium hydroxide alone. To investigate the dose-response effect
BOSCHETTO, ROGERS, FABBRI, AND BARNES
of antigen challenge on microvascular leakage, sensitized animals were injected with ovalbumin, 50 (n = 3), 100 (n = 3), 250 (n = 3), and 500 (n = 4) mg/kg 5 min before perfusion. Baseline leakage was determined by giving 250 rag/kg ovalbumin (n = 3) to sham-sensitized animals. The time course of the response to 250 mg/kg ovalbumin was determined in sensitized animals perfused after 5 min (n = 3) or 15min (n = 3) and compared with sham-sensitized control animals challenged and perfused at the same time points (n = 3 in each group). Dexamethasone, 8 mg/kg given intraperitoneally, was administered to sensitized rats 4 h before challenge with 250 mg/kg ovalbumin (n = 4). Baseline leakage was assessed in five sham-sensitized animals by injecting saline 4 h before ovalbumin. The effect on leakage in sensitized animals of the pretreatment procedure was determined by injecting saline 4 h before ovalbumin challenge.
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250 500 750 1000 C 0 Drugs and Chemicals [PAF] (ng/kg) Evans blue dye (Sigma Chemical Ltd., Poole, UK) was dissolved at a concentration of 30 Fig. 1. Effect of platelet-activating factor (PAF) on plasmg/ml in saline and filtered to exclude 5-l1m ma exudation in rat airways. Values are mean concenparticles. PAF (Novabiochem, Nottingham, tration Evans blue dye (vertical bars = SEM) for three UK), 1 mg/kg in ethanol, was stored at - SOo C to seven animals per group (n = number; C = control; and diluted on each day of experimentation I.p.a. = intrapulmonary airways). Single asterisks indiin 0.9llJo sodium chloride in distilled water (sa- cate p < 0.05; double asterisks indicate p < 0.Q1 comline) containing 0.35llJo bovine serum albu- pared with C. Solid squares = main bronchi; open min (BSA; Sigma). Dexamethasone sodium squares = trachea; solid circles = I.p.a.; open circles = larynx. phosphate (Merck, Sharp and Dohme Ltd., Hoddesdon, UK) and ovalbumin (for acute challenge) were diluted in saline and, together with PAF, were injected as 1 ml/kg. in the larynx, 295 % in the trachea, and
Statistical Analysis Data for the concentration of Evans blue dye extractable from tissues does not approximate a Gaussian (normal) distribution but shows positive skew (26). Therefore, the MannWhitney U-Test(30) was used to compare median values between groups. Bonferroni's correction (31) was used when appropriate for multiple comparisons. For ease of presentation, data in results are means ± one standard error (SEM). The null hypothesis was rejected at p values less than 0.05. Results
Effect of PAF on Plasma Leakage PAF (50 ng/kg to l ug/kg given intravenously) induced immediate tachypnea in all animals, which subsided after 1 min, and increased the concentration of Evans blue dye in all airway tissues examined in a dose-related manner (figure 1). PAF (100 ng/kg) significantly increased dye content above control values in the most proximal airways studied (larynx and trachea), whereas 250 ng/kg was the minimum effective dose tested that increased leakage in all airways (p < 0.05). Leakage was maximal in the extrapulmonary airways at 500 ug/kg PAF with increases above that in control animals of 312070
167% in the main bronchi. Over the dose range used, the intrapulmonary airways did not demonstrate maximal leakage: at 1 ug/kg there was a 206010 increase above control values.
Inhibitory Effect of Low Dose Dexamethasone Rats pretreated with two intraperitoneal injections of saline (the steroid diluent) 24 hand 4 h before PAF demonstrated significantly less leakage in comparison with the equivalent vehicle-treated control rats in the dose-response study (see above). At 500 ng/kg PAF the reduction was significant in the trachea (p < 0.05), main bronchi (p < 0.01), and intrapulmonary airways (p < 0.05) but not in the larynx, whereas at 1 ug/kg PAF the reduction was significant (p < 0.05) in the trachea. The control (baseline) response was also reduced (p < 0.05) in the main bronchi, although not sufficiently to account significantly for the difference in response to PAR In rats pretreated with the two saline injections, the response to l ug/kg PAP was similar to that with 500 ng/kg PAF with no prior treatment. Dexamethasone, 0.2 mg/kg givenintraperitoneally 24 hand 4 h before 1 ug/kg
607
CORTICOSTEROID INHIBITION OF AIFrNAY MICROVASCULAR LEAKAGE
PAF was given intravenously, partially inhibited PAF-induced dye leakage in all airwaysexamined (figure 2). The concentration of dye in rats treated with dexamethasone plus PAF was significantly different from that in rats treated with either saline plus PAF or saline plus BSAsaline. The dexamethasone-induced reductions in response were 65070 in the larynx, 64% in the trachea, 43% in the main bronchi, and 60% in the intrapulmonaryairways. The inhibition by dexamethasone was not due to a reduction in baseline leakage, which, if anything, was slightly increased by the steroid (although not significantly).
Inhibitory Effect of High Dose Dexamethasone Rats injected with saline intravenously 4 h before PAF (500 ng/kg) had a significantly increased tissue dye concentration in all airways studied in comparison with animals injected with saline 4 h before BSA-saline (figure 3A), with increases of 147% in the larynx, 361% in the trachea,
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Fig. 2. Inhibitory effect of low dose dexamethasone on plasma exudation induced in rat airways by plateletactivating factor (pAF). Dexamethasone (Dex., 0.2 mglkg) or saline was injected intraperitoneally 24 hand 4 h before PAF (1 ~/kg) or saline was given intravenously. Values are mean concentration Evans blue dye (vertical bars = SEM) in each airway tissue (MB = main bronchi; Ipa = intrapulmonary airways). Number of animals in each group are indicated in the first set of columns. Single asterisk indicates p < 0.05; double asterisks indicate p < 0.01 compared with saline + saline; double daggers indicate p < 0.01 compared with saline + PAF.
Fig. 3. Inhibitory effect of high dose dexamethasone on plasma exudation induced in rat airways by either plateletactivating factor (PAF) or antigen challenge of sensitized animals. A. Dexamethasone (Dex., 8 mglkg) or saline was injected intraperitoneally 4 h before PAF (500 ng/kg) or saline was injected intravenously. B. Rats were either sensitized to ovalbumin or given a sham procedure. All animals were challenged acutely with ovalbumin with or without Dex. (8 mgl kg). Values are mean concentration Evans blue dye (vertical bars = SEM) in each airway tissue (MB = main bronchi, Ipa = intrapulmonary airways). Number of animals in each group is indicated in the first set of columns. For each airway, asterisks indicate p < 0.05 compared with open columns; single and double daggers indicate p < 0.05 and p < 0.01, respectively, compared with solid columns.
200
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PIERA BOSCHETTO,5 DUNCAN F. ROGERS, LEONARDO M. FABBRI, and PETER J. BARNES
Introduction
Increased permeability of the microvasculature with leakage of plasma into the interstitium is an important component of the acute inflammatory response. In the airways, chronic inflammation may underlie the pathogenesis of a number of bronchial diseases, in particular, asthma, where the contribution of both inflammation (1)and plasma exudation (2) to bronchial hyperresponsiveness(3) have been emphasized. Specifically, edema is a distinguishing characteristic of the airways in patients who have died in status asthmaticus (4). However, there is evidence of edema in comparatively healthy asthmatics, including those in remission (4, 5). In addition, plasma proteins can be detected in the sputum (6-9) and, more importantly, in the bronchoalveolar lavage fluid (10)of asthmatics. Airwayedema leads to bronchial narrowing, but, perhaps more importantly, interstitial plasma will induce activation of biochemical pathways leading to the generation of a number of inflammatory mediators that may amplify the leakage response (11). As well as inducing further leakage, some of these mediators may directly affect airway smooth muscle and its normal control (12). Luminal plasma, in addition to increasing the volume of airway fluid, may induce mucus secretion (13)and increase the viscosity of the mucus (14), both of which would impair mucociliary transport (15). Edema of the airway wall may have important functional consequences and may markedly amplify the airway narrowing induced by bronchoconstriction (16).Thus, indirect evidence for the involvement of plasma exudation in the pathogenesis of asthma is compelling (2, 17), and strategies to reduce leakage may be important in the therapy of chronic asthma. Corticosteroids are veryeffective in the treatment of chronic asthma, although the precise mechanisms underlying their beneficial action are unknown (18). Inhibition of plasma leakage is one possibility because corticosteroids inhibit mediatorinduced edema and plasma leakage in a number of systemic vascular beds (19-
SUMMARY We studied the effect of dexamethasone on microvascular leakage (using Evans blue dye as a marker of plasma exudation) Induced in rat airways by platelet·actlvatlng factor (PAF). Intra· venously administered PAF caused a dose-related Increase In plasma leakage over the range 0.1 to 1 l19/kg. At 500 ng/kg PAF, the response was maximal In the extrapulmonary airways elUlmlned with increases In leakage above those In control animals of 312% In the larynx, 295% In the trachea, and 167% In the main bronchi. A mexlmal response was not achieved In the Intrapulmonary airways at the doses of PAF tested: at 1 l19/kg the increase was 206% above that In control animals. Dexamethasone, given by Intraperitoneal injection 24 hand 4 h before PAF at a dose of 0.2 mg/kg on each occasion, partially Inhibited leakage Induced by PAF (1 l19/kg) In all airway levels studied by 43 to 65%. At each level the tissue concentration of dye was reduced to a value that was significantly (p < 0.05)different from either PAFor control values. Wealso determined whether a high dose (8 mglkg) of dexamethasone given Intraperltoneally would Inhibit plasma leakage of dye Induced by either PAF or antlgen-ehallenge of sensitized rats. When given 4 h before antigen, dexamethasone completely prevented allergen.lnduced leakage in the airways showing significant leakage (larynx, trachea, and Intrapulmonary airways). Similarly, dexamethasone (4 h before) partially Inhibited PAFinduced leakage In the trachea and main bronchi. In summary, In rat airways, both low and high doses of dexamethasone markadly inhibit medlator·lnduced plasma exudation. This effect may contribute to the beneficial action of corticosteroids In allergic disease, Including asthma. AM REV RESPIR DIS 1991; 143:605-609
23), as well as attenuating capsaicin-induced leakage of plasma into the tracheal lumen in guinea pigs (24). In the present study we investigated whether a comparatively low dose of dexamethasone (0.2 mg/kg, twice in 24 h), would inhibit plasma exudation in both extrapulmonary and intrapulmonary airwaysinduced in rats by platelet-activating factor (PAF). We used dexamethasone because it is a potent corticosteroid that is water-soluble and so may be given by intravenous injection. We used rats because a number of studies have demonstrated that steroids are markedly more potent in this species than, for example, in guinea pigs (25). PAF was used to induce plasma leakage because it is potent, its mechanism of action has been well characterized (26, 27), and it exhibits a number of biologic properties consistent with a putative mediator of asthma (28). Wealso determined whether a singledose of 8 mg/kg dexamethasone would inhibit airway leakage induced by either PAF or allergen challenge of sensitized rats. We used Evans blue dye as a marker of plasma leakage since this allows the measurement of leakage at different airway levels. Methods Male Sprague-Dawley rats (250 to 400 g) were
premedicated with diazepam (3 mg/kg) by intraperitoneal injection and anesthetized with 0.5 ml Hypnorm (containing 0.155 mg fentayl citrate and 5 mg fluanisone) by intramuscular injection.
Measurement of Plasma Exudation The jugular veins were exposed, and drugs were administered intravenously by passing the injection needle through the pectoralis major to avoid bleeding on withdrawal. Evans blue dye (30 mg/kg) was injected, followed I min later by PAF, ovalbumin, or their appropriate controls (see below). The magnitude of plasma leakage was determined by measuring the concentration of dye extravasated into the airway tissue after first removing intravascular dye by a method described
(Received in original form May 16, 1990) 1 From the Department of Thoracic Medicine, National Heart & Lung Institute, London, United Kingdom. 2 Supported bythe Asthma ResearchCouncil and Medical Research Council of Great Britain. 3 Presented in part at the Annual Meeting of the American Thoracic Society, Cincinnati, May 1989. • Correspondence and requests for reprints should be addressed to Dr. D. F. Rogers, Department of Thoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY. United Kingdom. S Recipient of Grant No. 87.00266.56 from the Italian Research Council.
605
606 previously(26).Fiveminutes after PAF or ovalbumin, the thorax was opened and a bluntended 13-gauge needle was passed through a left ventriculotomy into the aorta. The heart was clamped, the right atrium was incised to allow outflow of perfusate, and the systemic circulation was perfused of blood with 100 ml saline (pH, 5.5; 21° C) at 100mm Hg pressure. The larynx, trachea, main bronchi, and lungs were removed. The main bronchi were separated from the trachea, and the remaining intrapulmonary airways were stripped of parenchyma by gently scraping with a razor blade. Excess fluid was removed by squeezing between filter papers, and the tissue wet weights were recorded. Evans blue dye was extracted by incubating tissues in formamide at 37° C for 16 h, and its concentration was determined against a formamide blank by light absorbance at a wavelength of 620 nm (SP 1750 spectrophotometer; Pye Unicam, Cambridge, UK) and interpolation on a standard curve of dye concentrations in the range 0.5 to 10 ug/ml in formamide. Airway concentration of dye was expressed as nanograms Evans blue per milligram wet weight tissue.
Experiment 1: Low Dose Dexamethasone The relationship between dose of PAF and magnitude of leakage of Evans blue dye was examined by comparing the response to intravenously administered doses of PAF of 50 (n = 5), 100 (n = 5), 250 (n = 3), and 500 (n = 7) ng/kg and 1 ug/kg (n = 5) with the response to control vehicle (BSA-saline; see below). Because control procedures reduced the response to PAF (see RESULTS), the inhibitory effect of dexamethasone was determined at the highest dose of PAF above. Dexamethasone was administered intraperitoneally at a dose 0.2 mg/kg on each of two occasions, 24 h and 4 h, before 1 ug/kg PAF (n = 6). Baseline leakage was determined in six rats given saline (the diluent for dexamethasone) 24 hand 4 h before BSA-saline. The effect of dexamethasone (0.2 mg/kg, 24 h and 4 h) on baseline leakage to BSA-saline was determined in a further six rats. Finally the effect of the administration procedure was accounted for by injecting animals with saline 24 h and 4 h before 1 ug/kg PAF (n = 5). Experiment 2: High Dose Dexamethasone Dexamethasone was given intraperitoneally atadoseof8mg/kg2h(n = 3)or4h(n = 6) before 500 ng/kg of PAF was given intravenously. Baseline leakage was determined in five rats given saline 4 h before BSA-saline. The effect on leakage of saline given intraperitoneally 4 h before PAF was assessed in a further five rats. The inhibitory effect of the high dose of dexamethasone was also assessed in rats sensitized 3 wk prior to experimentation using a single intraperitoneal injection of 1 ml saline containing 267 mg aluminium hydroxide and 1.33mg ovalbumin (29). Sham-sensitized (control) animals were injected with 1 ml saline containing 267 mg aluminium hydroxide alone. To investigate the dose-response effect
BOSCHETTO, ROGERS, FABBRI, AND BARNES
of antigen challenge on microvascular leakage, sensitized animals were injected with ovalbumin, 50 (n = 3), 100 (n = 3), 250 (n = 3), and 500 (n = 4) mg/kg 5 min before perfusion. Baseline leakage was determined by giving 250 rag/kg ovalbumin (n = 3) to sham-sensitized animals. The time course of the response to 250 mg/kg ovalbumin was determined in sensitized animals perfused after 5 min (n = 3) or 15min (n = 3) and compared with sham-sensitized control animals challenged and perfused at the same time points (n = 3 in each group). Dexamethasone, 8 mg/kg given intraperitoneally, was administered to sensitized rats 4 h before challenge with 250 mg/kg ovalbumin (n = 4). Baseline leakage was assessed in five sham-sensitized animals by injecting saline 4 h before ovalbumin. The effect on leakage in sensitized animals of the pretreatment procedure was determined by injecting saline 4 h before ovalbumin challenge.
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250 500 750 1000 C 0 Drugs and Chemicals [PAF] (ng/kg) Evans blue dye (Sigma Chemical Ltd., Poole, UK) was dissolved at a concentration of 30 Fig. 1. Effect of platelet-activating factor (PAF) on plasmg/ml in saline and filtered to exclude 5-l1m ma exudation in rat airways. Values are mean concenparticles. PAF (Novabiochem, Nottingham, tration Evans blue dye (vertical bars = SEM) for three UK), 1 mg/kg in ethanol, was stored at - SOo C to seven animals per group (n = number; C = control; and diluted on each day of experimentation I.p.a. = intrapulmonary airways). Single asterisks indiin 0.9llJo sodium chloride in distilled water (sa- cate p < 0.05; double asterisks indicate p < 0.Q1 comline) containing 0.35llJo bovine serum albu- pared with C. Solid squares = main bronchi; open min (BSA; Sigma). Dexamethasone sodium squares = trachea; solid circles = I.p.a.; open circles = larynx. phosphate (Merck, Sharp and Dohme Ltd., Hoddesdon, UK) and ovalbumin (for acute challenge) were diluted in saline and, together with PAF, were injected as 1 ml/kg. in the larynx, 295 % in the trachea, and
Statistical Analysis Data for the concentration of Evans blue dye extractable from tissues does not approximate a Gaussian (normal) distribution but shows positive skew (26). Therefore, the MannWhitney U-Test(30) was used to compare median values between groups. Bonferroni's correction (31) was used when appropriate for multiple comparisons. For ease of presentation, data in results are means ± one standard error (SEM). The null hypothesis was rejected at p values less than 0.05. Results
Effect of PAF on Plasma Leakage PAF (50 ng/kg to l ug/kg given intravenously) induced immediate tachypnea in all animals, which subsided after 1 min, and increased the concentration of Evans blue dye in all airway tissues examined in a dose-related manner (figure 1). PAF (100 ng/kg) significantly increased dye content above control values in the most proximal airways studied (larynx and trachea), whereas 250 ng/kg was the minimum effective dose tested that increased leakage in all airways (p < 0.05). Leakage was maximal in the extrapulmonary airways at 500 ug/kg PAF with increases above that in control animals of 312070
167% in the main bronchi. Over the dose range used, the intrapulmonary airways did not demonstrate maximal leakage: at 1 ug/kg there was a 206010 increase above control values.
Inhibitory Effect of Low Dose Dexamethasone Rats pretreated with two intraperitoneal injections of saline (the steroid diluent) 24 hand 4 h before PAF demonstrated significantly less leakage in comparison with the equivalent vehicle-treated control rats in the dose-response study (see above). At 500 ng/kg PAF the reduction was significant in the trachea (p < 0.05), main bronchi (p < 0.01), and intrapulmonary airways (p < 0.05) but not in the larynx, whereas at 1 ug/kg PAF the reduction was significant (p < 0.05) in the trachea. The control (baseline) response was also reduced (p < 0.05) in the main bronchi, although not sufficiently to account significantly for the difference in response to PAR In rats pretreated with the two saline injections, the response to l ug/kg PAP was similar to that with 500 ng/kg PAF with no prior treatment. Dexamethasone, 0.2 mg/kg givenintraperitoneally 24 hand 4 h before 1 ug/kg
607
CORTICOSTEROID INHIBITION OF AIFrNAY MICROVASCULAR LEAKAGE
PAF was given intravenously, partially inhibited PAF-induced dye leakage in all airwaysexamined (figure 2). The concentration of dye in rats treated with dexamethasone plus PAF was significantly different from that in rats treated with either saline plus PAF or saline plus BSAsaline. The dexamethasone-induced reductions in response were 65070 in the larynx, 64% in the trachea, 43% in the main bronchi, and 60% in the intrapulmonaryairways. The inhibition by dexamethasone was not due to a reduction in baseline leakage, which, if anything, was slightly increased by the steroid (although not significantly).
Inhibitory Effect of High Dose Dexamethasone Rats injected with saline intravenously 4 h before PAF (500 ng/kg) had a significantly increased tissue dye concentration in all airways studied in comparison with animals injected with saline 4 h before BSA-saline (figure 3A), with increases of 147% in the larynx, 361% in the trachea,
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Fig. 2. Inhibitory effect of low dose dexamethasone on plasma exudation induced in rat airways by plateletactivating factor (pAF). Dexamethasone (Dex., 0.2 mglkg) or saline was injected intraperitoneally 24 hand 4 h before PAF (1 ~/kg) or saline was given intravenously. Values are mean concentration Evans blue dye (vertical bars = SEM) in each airway tissue (MB = main bronchi; Ipa = intrapulmonary airways). Number of animals in each group are indicated in the first set of columns. Single asterisk indicates p < 0.05; double asterisks indicate p < 0.01 compared with saline + saline; double daggers indicate p < 0.01 compared with saline + PAF.
Fig. 3. Inhibitory effect of high dose dexamethasone on plasma exudation induced in rat airways by either plateletactivating factor (PAF) or antigen challenge of sensitized animals. A. Dexamethasone (Dex., 8 mglkg) or saline was injected intraperitoneally 4 h before PAF (500 ng/kg) or saline was injected intravenously. B. Rats were either sensitized to ovalbumin or given a sham procedure. All animals were challenged acutely with ovalbumin with or without Dex. (8 mgl kg). Values are mean concentration Evans blue dye (vertical bars = SEM) in each airway tissue (MB = main bronchi, Ipa = intrapulmonary airways). Number of animals in each group is indicated in the first set of columns. For each airway, asterisks indicate p < 0.05 compared with open columns; single and double daggers indicate p < 0.05 and p < 0.01, respectively, compared with solid columns.
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