Some characteristics of econazole-induced pleurisy in rats

Gen. Pharmac. Vol. 16, No. 6, pp. 637 640, 1985 Printed in Great Britain. All rights reserved

0306-3623/85 $3.00 + 0.00 Copyright ~" 1985 Pergamon Press Ltd

SOME C H A R A C T E R I S T I C S OF E C O N A Z O L E - I N D U C E D P L E U R I S Y IN RATS SEICO HANADA, SEIZI OGA a n d MARCIA T. HIRANO Department of Pharmacology, Instituto de Ci~ncias Biom~dicas, University of S~o Paulo, 05508, S~o Paulo, Brazil (Received 20 February 1985)

Alrstract--1. Aqueous suspension of econazole nitrate injected into the pleural cavity of rats induces sharp exudate formation and small leucocyte mobilization. 2. The econazole pleurisy development is monophasic and its peak value occurs between 8 and 12 hr after econazole injection. 3. The exudate volume and the leucocyte mobilization are inhibited by chlorpheniramine, cyproheptadine and steroidal and non-steroidal anti-inflammatory drugs.

INTRODUCTION

Carrageenin-induced pleurisy

Pleurisy was induced by injecting 0.25 ml of a 0.2% carrageenin suspension in sterile saline into the pleural cavity, using the same procedure described above for econazole-induced pleurisy.

The characteristics of the acute i n f l a m m a t i o n induced by s u b c u t a n e o u s injection o f aqueous suspension of econazole into rat paws a n d the sensitivity of the i n f l a m m a t i o n to several drugs were previously rep o r t e d by Oga et al. (t983). In a n effort to clarify the m e d i a t o r s o f econazoleinduced inflammation, we have n o w established the rat pleural cavity as an i n f l a m m a t o r y site. Over the past 10 years, this site has been used to determine the cellular response after the i n t r o d u c t i o n of an i n f l a m m a t o r y stimulus (Parente et al., 1979; Vinegar et al., 1973, 1976; Willoughby, 1975). T h e effects of a n t i - i n f l a m m a t o r y drugs on exudate f o r m a t i o n a n d p o l y m o r p h o n u c l e a r a n d m o n o n u c l e a r leucocyte acc u m u l a t i o n have been d e m o n s t r a t e d in a n u m b e r of papers (Almeida et al., 1980; K o h et al., 1979; M i y a s a k a a n d M i k a m i , 1982; Vinegar et al., 1976). We describe here the features of the a c c u m u l a t i o n o f i n f l a m m a t o r y cells a n d exudate, following the intrapleural injection of econazole in n o r m a l a n d drug-treated rats. The t e m p o r a l b e h a v i o r o f this process has been studied a n d c o m p a r e d with that o f carrageenin-induced pleurisy.

Total leucocyte counts and differential counts A 0.05 ml aliquot was used to determine the total leucocyte count. Red blood cells were lysed by the addition of Tuerk's solution (Costacurta, 1969). For differential cell counts, the remaining fluid was centrifuged for 4 min at 900 rpm and the cells resuspended in 0.2 ml of rat plasma. Exudate smears were prepared, air-dried and fixed with Leishmann stain. Differential counts were determined by standard procedures. Mast cell counts

At the 2nd and 12th hr after econazole or saline injection, the rats were decapitated and exsanguinated. Mast cells were obtained by lavage of the rat pleural cavity with Tyrode solution (Foreman and Mongar, 1972). Mast cell counts were determined by mixing one part of 0.1% dimethylotuthionine chloride (toluidine blue) with 19 parts of the pteural fluid (Bray and Van Arsdel, 1961). All cells staining metachromatically with toluidine blue were assumed to be mast cells. Drug effects on econazole pleurisy

MATERIALS AND METHODS

The effects of dexamethasone (0.2 mg/kg), phenylbutazone (100mg/kg), cyproheptadine (10mg/kg) and chlorpheniramine (l.6mg/kg) on exudate volume, total cell number and differential leucocyte count of econazoleinduced pleurisy were examined. All drugs were administered p.o. by gavage 1 hr prior to econazole injection.

Animals"

Male Wistar rats (180 _+ 20 g) were used. The rats were fasted overnight, but had free access to tapwater before each experiment. Econazole-induced pleurisy

Pleurisy was induced in rats by injecting 0.25 ml of a 4% econazole nitrate suspension in sterile saline into the pleural cavity. The intrapleural injection was given between the third and fifth ribs on the right side of the mediastinum using a procedure described by Vinegar et al. (1973). After a fixed period (2, 4, 8, 12 or 24 hr), the rats were carefully bled from the carotid arteries. The chest was opened, the exudate removed by aspiration and the exudate volume determined. The pleural cavity was washed with 0.1% EDTA solution in PBS buffer, adjusted to pH 7.2. Exudate and washes were pooled and their volume adjusted to 5 ml with 0.1% EDTA solution in PBS buffer. Any exudates contaminated with blood were discarded.

Statistical assessment

Statistical evaluation of data was carried out by analysis of variance. Sequential differences among means were tested according to Tukey contrast analysis at P < 0.05 (Sokal and Rolf, 1969). The significance of the difference between two means was evaluated using Student's t-test. Drugs

Econazole nitrate (AchO, phenylbutazone calcium (Boehringer), chlorpheniramine maleate (Schering), dexamethasone phosphate and cyprohepradine hydrochloride (Merck Sharp & Dohme) were used as received.

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Fig. 1. Time course of pleurisy induced by econazole and carrageenin in rats. Exudate formation (A) and leucocytemobilization (B) in response to intrapleural injection of econazole (0) and carrageenin (C)). Each value represents the mean +_SEM of 6 animals. *Significantlydifferent (P < 0.05) from carrageenintreated group.

RESULTS The intrapleural injection of econazole induced a rapid exudate formation and a small leucocyte mobilization. Appreciable exudate was still present after 24hr. The time course of development of exudate and leucocyte migration differed from that of carrageenin-induced pleurisy. The maximum development of the pleural exudate occurred between 8 and 12 hr after econazole injection as compared to 4hr after carrageenin injection (Fig. 1A). Carrageenin provoked an accelerated inflammatory response, the maximum leucocyte migration reaching 55.8 x 106 cells at the 8th hr. In contrast, with econazole this count rose to a modest 15.1 × 10 6 cells at the 12thhr (Fig. 1B). The neutrophil mobilization resulting from the intrapleural injection of carrageenin increased sharply until the 8th hr, decreasing slowly thereafter. The mononuclear leucocyte mobilization, though less than that of neutrophils, was nearly linear with neutrophil mobilization (Fig. 2). Econazole induced small neutrophil mobilization and a somewhat greater mononuclear leucocyte mobilization. The peak value occurred at the 12th hr, i.e. at the moment of maximum pleural exudation (Figs 1 and 2). The effects of several drugs on exudate volume and total leucocyte number of econazole-induced pleurisy were examined at the 2nd and 12th hr after injection of econazole. Pretreatment with dexamethasone (0.2mg/kg) markedly reduced exudate volume and cell number throughout the econazole pleurisy development. These inhibitory effects of dexamethasone were more marked in the late phase than in the early phase (Tables l and 2). Phenylbutazone (100 mg/kg) produced a 67% inhibition of exudate formation at the 2nd hr, but by the 12th hr the inhibition was only 27.4%. At the 12thhr, leucocyte mobilization was only slightly inhibited by phenylbutazone. In all experiments, the inhibition elicited by dexamethasone

was greater than that of phenylbutazone (Tables 1 and 2). Cyproheptadine (10mg/kg) significantly reduced exudate volume and cell number. Chlorpheniramine (1.6mg/kg) significantly inhibited exudate volume and cell number for 2 hr after econazole injection, but had no effect after 12hr (Tables 1 and 2). After intrapleural injection of econazole, no mast cells were found in the pleural exudate (Table 3). DISCUSSION

The injection of econazole into the pleural cavity of rats induced an edema-time curve different from that

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Fig. 2. Time course of neutrophil, mononuclear and eosinophil accumulation in the pleurisiesinduced by econazole and carrageenin in rats. In (A) carrageenin-treated group and in (B) econazole-treated group. Each value represents the mean _+SEM of 6 animals.

639

Econazole pleurisy Table 1. Effects of drugs on exudate volume on econazole-inducedpleurisy 2nd hr Dose (mg/kg)

Treatment Control Cyproheptadine Chlorpheniramine Dexamethasone Phenylbutazone

Volume (ml) 1.00 ± 0.08 ± 0.35 ± 0.25 _ 0.33 ±

10 1.6 0.2 100

0.03 0.01" 0.02* 0.02* 0.02*

12th hr Inhibition (~)

Volume (ml)

Inhibition (~)

92 65 75 67

1.90 ___0.06 1.34 ± 0.04* 1.75 ± 0.05 0.04 ± 0.01" 1.38 ± 0.04*

-29.5 7.9 97.9 27.4

All drugs were given orally 1 hr before econazole. Exudate volumes were measured at 2nd and 12th hr after intrapleural injection of econazole. Each value represents the mean + SEM of 6 rats. *Significantly different (P < 0.05) from the control.

Table 2. Effects of drugs on leucocyte mobilization on econazole-induced pleurisy 2nd hr Treatment Control Cyproheptadine Chlorpheniramine Dexamethasone Phenylbutazone

12th hr

Dose (mg/kg)

Leucocytes ( x 106)

Inhibition (~o)

Leucocytes ( × 106)

Inhibition (~o)

10 1.6 0.2 100

4.13 ± 0.04 0.74 ± 0.04* 1.44 + 0.11 * 0.40 ± 0.02* 1.57 _+0.09*

-82.1 65.1 90.3 62

15.13 + 0.22 13.74 4-_0.49 13.99 ± 0.67 0.65 + 0.04* 12.25 4- 0.35*

9.2 7.5 95.7 19

All drugs were given orally 1 hr before econazole. Total leucocyte counts were determined at 2nd and 12th hr after intrapleural injection of econazole. Each value represents the mean _+ SEM of 6 rats. *Significantly different (P < 0.05) from the control.

Table 3. Mast cell number in econazole-induced pleurisy Mast cells x l06 Treatment

2nd hr

12th hr

Control Econazole

0.64 + 0.09 0

0.55 ± 0.09 0

Mast cells were measured at 2nd and 12th hr after intrapteural injection of saline (control) or econazole. Each value represents the mean _+ SEM of 6 rats. *Significantly different (P < 0.05) from the control.

induced by injection in the rat's paw. The pleural curve was monophasic, while the hindlimb curve was biphasic (Oga et al., 1983). Similar findings have been reported after subplantar and intrapleural injection of carrageenin in the rat (Dohert and Robinson, 1975; Vinegar et al., 1973). As described in our previous paper (Oga et al., 1983), when injected in the rat hindlimb, econazole produces two defined phases of accelerated edema formation. The development of the first phase begins immediately after econazole injection and that of the second phase 6 h r later with its peak value at the 12thhr. Notwithstanding its monophasic development, the pleural exudate accumulation evidences similar etiology to paw edema in both its magnitude and sensitivity to drugs. Both edemas are strongly inhibited by oral doses of phenylbutazone and dexamethasone. The early phase of these edemas is also inhibited by cyproheptadine and chlorpheniramine. The features of inflammatory process elicited by econazole are different from those produced by carrageenin. The present data demonstrate a lack of correlation between neutrophil mobilization and exudate volume in econazole pleurisy. Econazole produced an acute edematous response, but failed to elicit the mobilization of an appreciable number of neutrophils. Pleural exudation showed only moderate interdependence with mononuclear leucocyte mobilization.

The temporal characteristics of neutrophil mobilization in carrageenin pleurisy can readily be correlated with exudate volume. Studies on the development of carrageenin inflammation in granulocytopenic rats led to the conclusion that neutrophils are required for the production of edema (Vinegar et al., 1974). Indeed, drug inhibition studies carried out by Vinegar et al. (1976) revealed that neutrophil mobilization precedes edema formation. They refuted the role of histamine and serotonin per se as mediators of the carrageenin-induced inflammatory process, since the antihistamine triprolidine and the antiserotonin agent cyproheptadine did not affect the development of hindlimb edema and pleural edema. On the other hand, Capasso et al. (1974) found increased levels of histamine and serotonin in the rat pleural cavity 1 hr after intrapleural injection of carrageenin. More recently Caciagli et al. (1977) detected a large quantity of histamine in the acetic acid-induced exudate. Our data show a significant inhibition of econazole-induced pleurisy by cyproheptadine and chlorpheniramine. Thus, histamine and serotonin appear to play an important role in this inflammatory process. Pleural mast cell counts provide additional evidence supporting this hypothesis. After intrapleural injection of econazole no mast cells were found in the pleural exudate, suggesting that amines released from mast cells mediate the econazoleinduced edema. However further biochemical experiments are required to elucidate these mechanisms. SUMMARY

The intrapleural injection of an aqueous suspension of econazole nitrate, a broad spectrum antimycotic agent, induced sharp pleural exudation and small leucocyte mobilization. The temporal characteristics of its development differed from those of

SEICO HANADA et al.

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carrageenin-induced pleurisy. The m a x i m u m exudate a c c u m u l a t i o n occurred between 8 a n d 1 2 h r after econazole injection a n d at the 4th hr after carrageenin injection. C a r r a g e e n i n p r o v o k e d a n accelerated inflammatory response, with a peak value of 55.8 x 106 cells at the 8th hr. In contrast, with econazole the m a x i m u m c o u n t was only 15.1 x 106 cells at the 12th hr. P r e t r e a t m e n t with p h e n y l b u t a z o n e ( 1 0 0 m g / k g ) or d e x a m e t h a s o n e ( 0 . 2 m g / k g ) h a d a p r o n o u n c e d effect o n exudate f o r m a t i o n a n d o n p o l y m o r p h o n u c l e a r a n d m o n o n u c l e a r leucocyte mobilization. C h l o r p h e n i r a m i n e (1.6 mg/kg) a n d cyproheptadine (10 mg/kg) significantly inhibited the pleural exudate, b u t did not affect the total leucocyte mobilization.

Acknowledgements--This work was supported by CNPq

(Proc.30.0622/81-BFD 3), FAPESP (Proc. 84/1239-2).

Fundo

BUNKA/1983

and

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