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Experimental otitis media with effusion induced by leukotriene D4
Auris Nasus Larynx 29 (2002) 127– 132 www.elsevier.com/locate/anl
Experimental otitis media with effusion induced by leukotriene D4 Naoki Tada a,*, Masayuki Furukawa a, Manabu Ogura a, Shoji Arai a, Yasushi Adachi b, Susumu Ikehara b, Toshio Yamashita a a
Department of Otorhinolaryngology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi 570 -8506, Japan b Department of Pathology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi 570 -8506, Japan Received 27 June 2001; received in revised form 26 October 2001; accepted 16 November 2001
Abstract Objecti6e: inflammatory mediators such as prostaglandins (PGs), leukotrienes (LTs), and platelet-activating factor (PAF) have been identified in human middle ear effusions (MEEs), as well as in the experimentally induced MEEs of animals. However, the exact function of LTs in the middle ear cavity is still unknown. In this study, the role of LTs was investigated using an experimental model in which OME was induced by LTD4. Methods: to examine whether leukotrienes (LTs) induce otitis media with effusion (OME), 2 × 10 − 6, 2× 10 − 5, 1× 10 − 4 and 2 × 10 − 4 M of LTD4 was injected into the rat ear. The severity of OME was assessed based on the histological findings. The concentrations of IL1-b, TNF-a, and GRO/CINC-1 in MEE were also measured. Additionally the therapeutic efficacy of a specific LTs antagonist, pranlukast on experimental OME was investigated. Results: all ears developed middle ear effusion (MEE) within 24 h and about 50% of the animals continued to demonstrate MEE for 14 days in the 1 ×10 − 4 and 2×10 − 4 M groups. The cytokine levels seemed to correspond well with the persistence of OME. The oral administration of a specific LTs antagonist, pranlukast, was found to alleviate the experimental OME. Conclusion: these findings suggest that LTs appear to plays an important role in the pathogenesis of OME. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Otitis media with effusion; Leukotrienes; Cytokine
1. Introduction The mechanism of otitis media with effusion (OME) is still not clearly understood. Inflammatory mediators such as prostaglandins (PGs), leukotrienes (LTs) and platelet-activating factors (PAF) have been identified in human middle ear effusions (MEEs) [1,2], as well as in the experimentally induced MEEs of animals [3,4]. These inflammatory mediators released in the middle ear, induced by either viral or bacterial infection, eustachian tube dysfunction, or allergies, seem to play a major role in the pathogenesis of OME. However, the exact function of LTs in the middle ear cavity is still unknown. Smooth muscle contraction and increased blood vessel permeability have all been shown to be pharmacological effects of LTs [5]. In addition, mu* Corresponding author. Tel.: + 81-6-6992-1001x3334; fax: + 8172-832-5924. E-mail address: [email protected] (N. Tada).
cociliary clearance is one of the important functions of the eustachian tube. If this clearance system fails to function, properly then fluid will accumulate in the tympanic cavity, when the middle ear mucosa becomes inflamed. LTD4 has been demonstrated to suppress the entire mucociliary transport function [6]. Both LTD4 and LTC4 induce a dose-related increase in the glycoprotein secretion by human bronchial mucosa [7]. In this study, the role of LT was investigated using an experimental model in which OME was induced by LTD4. Thereafter we examined the middle ear and eustachian tube mucosa in rats. The cytokines levels were quantified and the pathogenetic role of cytokines was investigated in the experimental OME. The LTs antagonist, pranlukast, has been extensively studied and proven to clearly have an inhibitory effect against LTs actions. However, its inhibitory action against OME has yet to be elucidated. As a result, the therapeutic efficacy of pranlukast on experimental OME was therefore investigated.
0385-8146/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 8 5 - 8 1 4 6 ( 0 1 ) 0 0 1 4 3 - 2
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2. Materials and methods
2.1. Induction of otitis media with effusion Healthy male Wister rats, which were free of any middle ear infection and weighing 200– 300 g were used. Under ketamine anesthesia, the tympanic membranes were visualized under a microscopic examination and the external canals were cleaned with alcohol. Using a 25-gauge needle, 2×10 − 6, 2 ×10 − 5, 1 ×10 − 4, 2 × 10 − 4 M of LTD4 in 0.5 ml with 1/15 M of phosphate-buffer saline (PBS, pH 7.4) was injected transtympanically in the superior anterior pars flaccida area. The left ear of these groups serve as controls, and thus were only injected with 0.5 ml of PBS. The animals were thereafter killed at 1, 3, 5, 7, 10 and 14 days, at which time either the middle ear or the tubal mucosa and MEE were obtained. The presence of MEEs was determined for identification of fluids by dissection of bullae.
commercially available enzyme-linked immunosorbent assay kits (BioSource International, Camarillo, CA). These assays employed the immunoenzymetric ‘sandwich’ technique. The optical density was measured with an automated double-beam spectrometer (EAR 400, SLT Labinstrument, Salzburg, Austria) at an optical density of 450 nm.
2.5. Administration of pranlukast To examine the therapeutic efficacy of LT antagonist pranlukast, 24 rats were given 100 mg/kg of pranlukast by the gastric intubation daily starting 1 day before injection of 2× 10 − 4 M LTD4 into the tympanic cavity and then were killed on days 1, 3 and 5 in order to undergo a cytokines analysis and histological examination.
3. Results
2.2. Monitoring of otitis media with effusion
3.1. Presence of middle ear effusion
Each rat was monitored by a microscopic observation of the ear drum after LTD4 instillation into the tympanic cavity. The severity of OME as determined by microscopic observation was classified as follows: grade 1: the presence of MEE below the mid-point to the top of tympanic membrance (TM). grade 2: the presence of OME above the mid-point to the top of TM. grade 3: the presence of MEE up to the top of TM.
Table 1 shows the occurrence of OME according to the number of days after LTD4 instillation. In the 2×10 − 6 and 2× 10 − 5 M groups, MEE was present in each of the three LTD4-injected ears (100%) on the 1st day (grade 2) after injection, however, no MEE was present on day 3. All animals of the 1× 10 − 4 and 2× 10 − 4 M groups developed MEE to the top of the tympanic membrane (grade 3) on day 1 and about 50% of animals continued to demonstrate MEE for 14 days. On classification of severity of OME by microscopic observation, almost all cases of OME were grade 2 from day 3 to day 7 and on day 14, all affected ears were grade 1 in the two groups. No remaining fluids were observed on day 1 in any control ear.
2.3. Histological examination of the middle ear and eustachian tube mucosa For the histological examination, the bullae were dissected, fixed with 10% neutral-buffered formaline solution, decalcified with 10% EDTA– Tris buffer, embedded in paraffin wax, cut at 6 mm thickness and stained with hematoxin– eosin (HE).
2.4. Measurement of cytokines
Table 1 Occurrence of OME days after LTD4 installation
In order to examine the possible role of cytokines in LTD4-induced OME, levels of IL-1b, TNF-a, and GRO/CINC-1 were measured in MEEs. The MEEs were aspirated through the ear drum with a 26-gauge needle, next a 1-ml syringe filled with saline was inserted into the cavity, and then the saline was instilled and thereafter immediately withdrawn. Each MEE and recovered fluid sample was combined and centrifuged at 1500 rpm for 10 min, the supernatant was then collected for a cytokine assay. IL1-b, TNF-a (BioSource International, Camarillo, CA) and GRO/CINC1 (IBL, Fujioka, Gunnma, Japan) were examined with
Data shows the number of affected ears studied in each group. In parenthese are number of ears affected per number of ears studied.
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Fig. 1. The IL1-b, TNF-a and GRO/CINC-1 levels from rat middle ear samples. The ears which received (A) 2 ×10 − 5 M, (B) 2×10 − 6 M injection demonstrated significantly higher cytokines levels than did the ears which received PBS injection. The results are given as the mean9 S.D. of each four experimental ears.
3.2. Cytokine le6els in MEE The levels of IL1-b, TNF-a and GRO/CINC-1 in MEEs were measured in the 2×10 − 5 (A) and 2× 10 − 6 M (B) group animals and are presented in Fig. 1. The levels of the cytokines in the 2×10 − 5 M group were higher than the 2× 10 − 6 M group. Almost all cytokines showed high levels on day 3, especially IL1-b. For the 1 ×10 − 4 and 2 × 10 − 4 M groups, the GRO/CINC1 levels were examined and the high levels of GRO/ CINC-1 were detected for 14 days (Fig. 2). In contrast, the washes of control ears consistently had low to nondetectable levels of cytokines.
3.3. Histologic findings Since aerosolized crysteinyl LTD4 has been reported to elicits a migration of inflammatory cells, especially eosinophils, into guinea pig lungs [10], we examined the histologic changes of the LTD4-injected middle ears of rats. The histologic findings of the mucosa near the tympanic orifice of the tube in 2× 10 − 4 M LTD4 injected rat are showed in Fig. 3. On day 1, various inflammatory cells, such as lymphocytes, macrophages and polynuclear leukocytes (PNL), infiltrated in the middle ear, while eosinophils were not conspicuous. Subsequently, the infiltration of PNL worsened in a time dependent manner. Especially, prominent PNL infiltration was observed on day 7, and this severe infiltration continued until day 14. In contrast, the middle ears of the PBS-injected rats remained unchanged from 1 to 14 days after injection, as well as those of the non-treated rats (data not shown). In the case 2× 10 − 5 M LTD4 injection, the histological changes were similar to the case of 2×10 − 4 M LTD4 injection, but the degree of inflammation was milder and the duration of inflammation was shorter (data not shown).
Table 2 shows the mucosal thickness near the tympanic orifice of the tube (A) and the peripheral portion of the tympanic bulla (B) of each group over time. A significant increase was seen in the thickness of mucosa and the increase was dose dependent.
3.4. Effect of pranlukast on LTD4 -induced OME As for the animals that pretreated with pranlukast, no MEE was observed up to 5 days. In the recovered fluid in the middle ear, the GRO/CINC-1 levels were significantly lower than in the 2×10 − 4 M LTD4 injected animals (Fig. 4). Histological observations revealed that the degree of mucosal edema was minimal and the level of cellular infiltration decreased (data not shown). The histological finding might thus reflect the antagonistic effect of pranlukast on LTD4. Both the thickness of the mucosa of tube (A) and the bulla (B) are shown in Table 2. The mucosal thicknesses of both parts were reduced significantly compared with those of LTD4-injected ears (PB 0.01).
Fig. 2. The GRO/CINC-1 levels from rat middle ear samples of the 1 ×10 − 4 M and 2 ×10 − 4 M LTD4 injected ears. The results are given as the mean 9S.D. of each seven experimental ears.
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Fig. 3. The histologic findings of the middle ear mucosa near the tympanic orfice of the tube in the 2 × 10 − 4 M LTD4 injected rats.
4. Discussion Although MEE contains various beneficial substances, such as enzymes antibodies, lysozyme, there are also many inflammation-induced substances; in particular, high concentrations of histamine, leukotrienes, prostaglandins, platelet-activating factor, activated complement, and others have been reported [1,2,11– 13]. LTC4 has also been identified in human MEE [1] and is considered to be the most persistent eicosanoid in the infected chinchilla middle ear [14]. However, the exact function of LTs in the middle ear cavity is still unknown. This study demonstrated that the injection of LTD4 into the middle ear of rats without any obstructed eustachian tubes can cause inflammation and effusion. All of the LTD4 injected ears developed MEE on 1 day and about 50% of the LTD4 injected ears in the 1 ×10 − 4 and 2 × 10 − 4 M groups continued for 14 days. Jung et al. [3] investigated the effect of several arachidonic acid metabolites including the LTC4 chinchilla model and reported that LTC4 inoculation produced the greatest degree of inflammation. However, it was necessary to give daily inoculations to induced OME. Our present study demonstrated that LTD4 can induce OME by a single inoculation and high levels of LTD4 can last over 14 days, as LTD4 stimulates the release of cytokines. This finding could thus explain, in part, that the effect of LTD4 is ten-fold greater than that of LTC4 on the permeability of blood vessels [5]. The initial LTD4-induced MEE is probably due to increased vascular permeability in the middle ear mucosa. Further production of MEE and progression of OME appeared to be caused by the interaction of various inflammatory mediators including cytokines produced by the initial effect of LTD4. Cytokines are
known to be released from inflammatory cells as well as epithelial cells stimulated by various mediators [15,16], and are known to regulate both inflammation and the immune response. Recent studies have demonstrated the presence of a variety of cytokines in human MEEs, including IL1-b, TNF-a and most recently IL-8 [17– 19]. These cytokines in MEE might form a network that is responsible for inflammatory cell migration and activation, and mucosal changes in the enzyme activities. Rat GRO/CINC-1 has a structural and functional homology to human IL-8 [20]. Since high levels of a highly chemotactic chemokine, GRO/CINC-1 were detected in MEE samples from 2× 10 − 4 M LTD4 injected ears, it was not surprising to find dense leukocyte infiltration. The cytokines levels seem to correspond well with both the degree of inflammation in the middle ear cavity and the persistence of OME. A specific LTs antagonist, pranlukast was developed in Japan and found to have high and specific affinity for LT receptors and to antagonize the binding LTD4 and LTE4 to these receptors 2000–3000 times more strongly than the standard antagonist did [21]. It has been demonstrated to be a useful agent for the treatment of bronchial asthma [8] and allergic rhinitis [9]. However, basic and clinical trials have not yet been performed for the treatment of OME. Pranlukast has been demonstrated to supress the production of cytokines by peripheral blood mononuclear cells under stimulation with specific antigens in patients with bronchial asthma [22]. Although leukotriene receptor (LT-R) has not yet been demonstrated in middle ear mucosa, our results suggested that pranlukast acts directly on the middle ear and that blockade of LT-R in middle ear mucosa or inflammatory cells by pranlukast inhibits release of
N. Tada et al. / Auris Nasus Larynx 29 (2002) 127–132 Table 2 Thickness of the mucosa (mm) of the tube (A) and bulla (B)
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The findings of this study suggest that LT thus appear to play an important role in the pathogenesis of OME and, as a result, we recommend that a clinical trial for use of pranlukast to treat OME should be conducted. References
Values are 9 S.D. Four ears of rats were studied in each group. LTD4-leukotrien D4 showed significant difference between PBS injected ears and various concentrations of LTD4 injected ears (*PB 0.01, **PB0.05).
inflammatory cytokines, as a consequence of which local inflammation may be attenuated. This may be one mechanism by which pranlukast inhibits the occurrence of OME.
Fig. 4. The effect of pranlukast on the GRO/CINC-1 production in the rat middle ear sample. The production of GRO/CINC-1 in middle ear fluid was significantly reduced by pretreatment with pranlukast (*PB0.01). The results are given as the mean 9 S.D. for four experimental ears each.
[1] Jung TTK. Prostaglandins. Leukotrienes, and other arachidonic acid metabolites in the pathogenesis of otitis media. Laryngoscope 1988;98:980 – 93. [2] Furukawa M, Kubo N, Yamashita T. Biochemical evidence of platelet-activating factor (PAF) in human middle ear effusions. Laryngoscope 1995;105:118 – 21. [3] Jung TTK, Park YM, Schlnnd D, Weeks D, Miller S, Wong O, Jung SK. Effect of prostaglandin, leukotriene, and arachidonic acid on experimental otitis media with effusion in chinchillas. Ann Otol Rhinol Laryogol 1990;99:28 – 32. [4] Rhee C-K, Jung TTK, Miller S, Weeks D. Experiment otitis media with effusion induced by platelet activating factor. Ann Otol Rinol Laryngol 1993;102:660 – 5. [5] Samuelsson B. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 1983;220:568 –75. [6] Yamaki K, Noda Y, Takagi K, Satake T. Effect of adrenergic and cholinergic agents and leucotrienes on mucociliary transport force measured by using frog palate. Nagoya J Med Sci 1987;49:21 – 30. [7] Coles SJ, Neill KH, Reid LM, Austen KF, Nii Y, Corey EJ, Lewis RA. Effect of leukotrienes C4 and D4 on glycoprotein and lysozyme secration by hman bronchial mucosa. Prostaglandins 1983;25:155 – 70. [8] Fujimura M, Sakamoto S, Kamino Y, Matsuda T. Effects of a leukotriene antagonist, ONO-1078, on bronchial hyper-responsiveness in patients with asthma. Respir Med 1993;87:133 –8. [9] Fujita M, Nakagawa N, Yonetomi Y, Takeda H, Kawabata K, Ohno H. Cysteinyl leukotrienes induce nasal symptoms of allergic rhinitis via a receptor-mediated mechanism in guinea pigs. Jpn J Pharmacol 1997;75:355 – 62. [10] Underwood DC, Osborrn RR, Newsholme SJ, Torphy TJ, Hay DWP. Persistent airway eosinophilia after leucotriene (LT) D4 administration in the guinea pig. Am J Respir Crit Care Med 1996;154:850 –7. [11] Tsuji H, Furukawa M, Ikeda H, Asako M, Yamashita T. The presence of platelet-activating factor acetylhydrolase (PAF-AH) in human middle ear effusions. ORL 1998;60:25 – 9. [12] Mogi G, Bernstein JM. Immune mechanisms in otitis media with effusion. In: Immunology of the ear. New York: Raven Press, 1987:289 – 95. [13] Berger G, Hawke M, Proops DW, Ranadive NS, Wong D. Histamine levels in middle ear effusions. Acta Otolaryngol (Stockh) 1984;98:385 – 90. [14] Swarts JD, Westcott JY, Chan KH. Eicosanoid synthesis and inactivation in healthy and infected chinchilla middle ears. Acta Otolaryngol (Stockh) 1997;117:845 – 50. [15] Peplow PV. Action of cytokines in relation to arachidonic acid metabolism and eicosanoid production. Prostaglandins Leucok Essent Fatty Acids 1996;54(5):303 – 17. [16] Strober W, James SP. The interleukins. Pediatr Res 1998;24:549 – 57. [17] Maxwell KS, Fitzgerald JE, Burleson JA, Leonard G, Carpenter R, Kreutzer DL. Interleukin-8 expression in otitis media. Laryngoscope 1994;104:989 – 95. [18] Himi T, Suzuki T, Kodama H, Takezawa H, Kataura A. Immunologic characteristics of cytokines in otitis media with effusion. Ann Otol Rhinol Laryngol 1992;101:21 – 5.
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[19] Hotomi M, Samukawa T, Yamanaka N. Interleukin-8 in otitis media with effusion. Acta Otolaryngol (Stockh) 1994;114:406 – 9. [20] Yoshioka I, Himi T, Kataura A. In vivo induction and regulation of interleukin-8-like chemokine GRO/CINC-1 in rat middle ear. Acta Otolaryngol (Stockh) 1997;117:719 –23. [21] Nakagawa N, Obata T, Kobayashi T. In vivo parmacologic
profile of ONO-1078: a potent, selective and orally active peptide leukotriene (LT) antagonist. Jpn J Pharmacol 1992;60:217 –25. [22] Tohda Y, Nakahara H, Kubo H, Hamaguchi R, Fukuoka M, Nakajima S. Effects of ONO-1078 (pranlukast) on cytokine production in peripheral blood mononuclear cell of patients with bronchial asthma. Clin Exp Allergy 1999;29:1532 – 6.
Experimental otitis media with effusion induced by leukotriene D4 Naoki Tada a,*, Masayuki Furukawa a, Manabu Ogura a, Shoji Arai a, Yasushi Adachi b, Susumu Ikehara b, Toshio Yamashita a a
Department of Otorhinolaryngology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi 570 -8506, Japan b Department of Pathology, Kansai Medical Uni6ersity, 10 -15 Fumizono, Moriguchi 570 -8506, Japan Received 27 June 2001; received in revised form 26 October 2001; accepted 16 November 2001
Abstract Objecti6e: inflammatory mediators such as prostaglandins (PGs), leukotrienes (LTs), and platelet-activating factor (PAF) have been identified in human middle ear effusions (MEEs), as well as in the experimentally induced MEEs of animals. However, the exact function of LTs in the middle ear cavity is still unknown. In this study, the role of LTs was investigated using an experimental model in which OME was induced by LTD4. Methods: to examine whether leukotrienes (LTs) induce otitis media with effusion (OME), 2 × 10 − 6, 2× 10 − 5, 1× 10 − 4 and 2 × 10 − 4 M of LTD4 was injected into the rat ear. The severity of OME was assessed based on the histological findings. The concentrations of IL1-b, TNF-a, and GRO/CINC-1 in MEE were also measured. Additionally the therapeutic efficacy of a specific LTs antagonist, pranlukast on experimental OME was investigated. Results: all ears developed middle ear effusion (MEE) within 24 h and about 50% of the animals continued to demonstrate MEE for 14 days in the 1 ×10 − 4 and 2×10 − 4 M groups. The cytokine levels seemed to correspond well with the persistence of OME. The oral administration of a specific LTs antagonist, pranlukast, was found to alleviate the experimental OME. Conclusion: these findings suggest that LTs appear to plays an important role in the pathogenesis of OME. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Otitis media with effusion; Leukotrienes; Cytokine
1. Introduction The mechanism of otitis media with effusion (OME) is still not clearly understood. Inflammatory mediators such as prostaglandins (PGs), leukotrienes (LTs) and platelet-activating factors (PAF) have been identified in human middle ear effusions (MEEs) [1,2], as well as in the experimentally induced MEEs of animals [3,4]. These inflammatory mediators released in the middle ear, induced by either viral or bacterial infection, eustachian tube dysfunction, or allergies, seem to play a major role in the pathogenesis of OME. However, the exact function of LTs in the middle ear cavity is still unknown. Smooth muscle contraction and increased blood vessel permeability have all been shown to be pharmacological effects of LTs [5]. In addition, mu* Corresponding author. Tel.: + 81-6-6992-1001x3334; fax: + 8172-832-5924. E-mail address: [email protected] (N. Tada).
cociliary clearance is one of the important functions of the eustachian tube. If this clearance system fails to function, properly then fluid will accumulate in the tympanic cavity, when the middle ear mucosa becomes inflamed. LTD4 has been demonstrated to suppress the entire mucociliary transport function [6]. Both LTD4 and LTC4 induce a dose-related increase in the glycoprotein secretion by human bronchial mucosa [7]. In this study, the role of LT was investigated using an experimental model in which OME was induced by LTD4. Thereafter we examined the middle ear and eustachian tube mucosa in rats. The cytokines levels were quantified and the pathogenetic role of cytokines was investigated in the experimental OME. The LTs antagonist, pranlukast, has been extensively studied and proven to clearly have an inhibitory effect against LTs actions. However, its inhibitory action against OME has yet to be elucidated. As a result, the therapeutic efficacy of pranlukast on experimental OME was therefore investigated.
0385-8146/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 8 5 - 8 1 4 6 ( 0 1 ) 0 0 1 4 3 - 2
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2. Materials and methods
2.1. Induction of otitis media with effusion Healthy male Wister rats, which were free of any middle ear infection and weighing 200– 300 g were used. Under ketamine anesthesia, the tympanic membranes were visualized under a microscopic examination and the external canals were cleaned with alcohol. Using a 25-gauge needle, 2×10 − 6, 2 ×10 − 5, 1 ×10 − 4, 2 × 10 − 4 M of LTD4 in 0.5 ml with 1/15 M of phosphate-buffer saline (PBS, pH 7.4) was injected transtympanically in the superior anterior pars flaccida area. The left ear of these groups serve as controls, and thus were only injected with 0.5 ml of PBS. The animals were thereafter killed at 1, 3, 5, 7, 10 and 14 days, at which time either the middle ear or the tubal mucosa and MEE were obtained. The presence of MEEs was determined for identification of fluids by dissection of bullae.
commercially available enzyme-linked immunosorbent assay kits (BioSource International, Camarillo, CA). These assays employed the immunoenzymetric ‘sandwich’ technique. The optical density was measured with an automated double-beam spectrometer (EAR 400, SLT Labinstrument, Salzburg, Austria) at an optical density of 450 nm.
2.5. Administration of pranlukast To examine the therapeutic efficacy of LT antagonist pranlukast, 24 rats were given 100 mg/kg of pranlukast by the gastric intubation daily starting 1 day before injection of 2× 10 − 4 M LTD4 into the tympanic cavity and then were killed on days 1, 3 and 5 in order to undergo a cytokines analysis and histological examination.
3. Results
2.2. Monitoring of otitis media with effusion
3.1. Presence of middle ear effusion
Each rat was monitored by a microscopic observation of the ear drum after LTD4 instillation into the tympanic cavity. The severity of OME as determined by microscopic observation was classified as follows: grade 1: the presence of MEE below the mid-point to the top of tympanic membrance (TM). grade 2: the presence of OME above the mid-point to the top of TM. grade 3: the presence of MEE up to the top of TM.
Table 1 shows the occurrence of OME according to the number of days after LTD4 instillation. In the 2×10 − 6 and 2× 10 − 5 M groups, MEE was present in each of the three LTD4-injected ears (100%) on the 1st day (grade 2) after injection, however, no MEE was present on day 3. All animals of the 1× 10 − 4 and 2× 10 − 4 M groups developed MEE to the top of the tympanic membrane (grade 3) on day 1 and about 50% of animals continued to demonstrate MEE for 14 days. On classification of severity of OME by microscopic observation, almost all cases of OME were grade 2 from day 3 to day 7 and on day 14, all affected ears were grade 1 in the two groups. No remaining fluids were observed on day 1 in any control ear.
2.3. Histological examination of the middle ear and eustachian tube mucosa For the histological examination, the bullae were dissected, fixed with 10% neutral-buffered formaline solution, decalcified with 10% EDTA– Tris buffer, embedded in paraffin wax, cut at 6 mm thickness and stained with hematoxin– eosin (HE).
2.4. Measurement of cytokines
Table 1 Occurrence of OME days after LTD4 installation
In order to examine the possible role of cytokines in LTD4-induced OME, levels of IL-1b, TNF-a, and GRO/CINC-1 were measured in MEEs. The MEEs were aspirated through the ear drum with a 26-gauge needle, next a 1-ml syringe filled with saline was inserted into the cavity, and then the saline was instilled and thereafter immediately withdrawn. Each MEE and recovered fluid sample was combined and centrifuged at 1500 rpm for 10 min, the supernatant was then collected for a cytokine assay. IL1-b, TNF-a (BioSource International, Camarillo, CA) and GRO/CINC1 (IBL, Fujioka, Gunnma, Japan) were examined with
Data shows the number of affected ears studied in each group. In parenthese are number of ears affected per number of ears studied.
N. Tada et al. / Auris Nasus Larynx 29 (2002) 127–132
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Fig. 1. The IL1-b, TNF-a and GRO/CINC-1 levels from rat middle ear samples. The ears which received (A) 2 ×10 − 5 M, (B) 2×10 − 6 M injection demonstrated significantly higher cytokines levels than did the ears which received PBS injection. The results are given as the mean9 S.D. of each four experimental ears.
3.2. Cytokine le6els in MEE The levels of IL1-b, TNF-a and GRO/CINC-1 in MEEs were measured in the 2×10 − 5 (A) and 2× 10 − 6 M (B) group animals and are presented in Fig. 1. The levels of the cytokines in the 2×10 − 5 M group were higher than the 2× 10 − 6 M group. Almost all cytokines showed high levels on day 3, especially IL1-b. For the 1 ×10 − 4 and 2 × 10 − 4 M groups, the GRO/CINC1 levels were examined and the high levels of GRO/ CINC-1 were detected for 14 days (Fig. 2). In contrast, the washes of control ears consistently had low to nondetectable levels of cytokines.
3.3. Histologic findings Since aerosolized crysteinyl LTD4 has been reported to elicits a migration of inflammatory cells, especially eosinophils, into guinea pig lungs [10], we examined the histologic changes of the LTD4-injected middle ears of rats. The histologic findings of the mucosa near the tympanic orifice of the tube in 2× 10 − 4 M LTD4 injected rat are showed in Fig. 3. On day 1, various inflammatory cells, such as lymphocytes, macrophages and polynuclear leukocytes (PNL), infiltrated in the middle ear, while eosinophils were not conspicuous. Subsequently, the infiltration of PNL worsened in a time dependent manner. Especially, prominent PNL infiltration was observed on day 7, and this severe infiltration continued until day 14. In contrast, the middle ears of the PBS-injected rats remained unchanged from 1 to 14 days after injection, as well as those of the non-treated rats (data not shown). In the case 2× 10 − 5 M LTD4 injection, the histological changes were similar to the case of 2×10 − 4 M LTD4 injection, but the degree of inflammation was milder and the duration of inflammation was shorter (data not shown).
Table 2 shows the mucosal thickness near the tympanic orifice of the tube (A) and the peripheral portion of the tympanic bulla (B) of each group over time. A significant increase was seen in the thickness of mucosa and the increase was dose dependent.
3.4. Effect of pranlukast on LTD4 -induced OME As for the animals that pretreated with pranlukast, no MEE was observed up to 5 days. In the recovered fluid in the middle ear, the GRO/CINC-1 levels were significantly lower than in the 2×10 − 4 M LTD4 injected animals (Fig. 4). Histological observations revealed that the degree of mucosal edema was minimal and the level of cellular infiltration decreased (data not shown). The histological finding might thus reflect the antagonistic effect of pranlukast on LTD4. Both the thickness of the mucosa of tube (A) and the bulla (B) are shown in Table 2. The mucosal thicknesses of both parts were reduced significantly compared with those of LTD4-injected ears (PB 0.01).
Fig. 2. The GRO/CINC-1 levels from rat middle ear samples of the 1 ×10 − 4 M and 2 ×10 − 4 M LTD4 injected ears. The results are given as the mean 9S.D. of each seven experimental ears.
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Fig. 3. The histologic findings of the middle ear mucosa near the tympanic orfice of the tube in the 2 × 10 − 4 M LTD4 injected rats.
4. Discussion Although MEE contains various beneficial substances, such as enzymes antibodies, lysozyme, there are also many inflammation-induced substances; in particular, high concentrations of histamine, leukotrienes, prostaglandins, platelet-activating factor, activated complement, and others have been reported [1,2,11– 13]. LTC4 has also been identified in human MEE [1] and is considered to be the most persistent eicosanoid in the infected chinchilla middle ear [14]. However, the exact function of LTs in the middle ear cavity is still unknown. This study demonstrated that the injection of LTD4 into the middle ear of rats without any obstructed eustachian tubes can cause inflammation and effusion. All of the LTD4 injected ears developed MEE on 1 day and about 50% of the LTD4 injected ears in the 1 ×10 − 4 and 2 × 10 − 4 M groups continued for 14 days. Jung et al. [3] investigated the effect of several arachidonic acid metabolites including the LTC4 chinchilla model and reported that LTC4 inoculation produced the greatest degree of inflammation. However, it was necessary to give daily inoculations to induced OME. Our present study demonstrated that LTD4 can induce OME by a single inoculation and high levels of LTD4 can last over 14 days, as LTD4 stimulates the release of cytokines. This finding could thus explain, in part, that the effect of LTD4 is ten-fold greater than that of LTC4 on the permeability of blood vessels [5]. The initial LTD4-induced MEE is probably due to increased vascular permeability in the middle ear mucosa. Further production of MEE and progression of OME appeared to be caused by the interaction of various inflammatory mediators including cytokines produced by the initial effect of LTD4. Cytokines are
known to be released from inflammatory cells as well as epithelial cells stimulated by various mediators [15,16], and are known to regulate both inflammation and the immune response. Recent studies have demonstrated the presence of a variety of cytokines in human MEEs, including IL1-b, TNF-a and most recently IL-8 [17– 19]. These cytokines in MEE might form a network that is responsible for inflammatory cell migration and activation, and mucosal changes in the enzyme activities. Rat GRO/CINC-1 has a structural and functional homology to human IL-8 [20]. Since high levels of a highly chemotactic chemokine, GRO/CINC-1 were detected in MEE samples from 2× 10 − 4 M LTD4 injected ears, it was not surprising to find dense leukocyte infiltration. The cytokines levels seem to correspond well with both the degree of inflammation in the middle ear cavity and the persistence of OME. A specific LTs antagonist, pranlukast was developed in Japan and found to have high and specific affinity for LT receptors and to antagonize the binding LTD4 and LTE4 to these receptors 2000–3000 times more strongly than the standard antagonist did [21]. It has been demonstrated to be a useful agent for the treatment of bronchial asthma [8] and allergic rhinitis [9]. However, basic and clinical trials have not yet been performed for the treatment of OME. Pranlukast has been demonstrated to supress the production of cytokines by peripheral blood mononuclear cells under stimulation with specific antigens in patients with bronchial asthma [22]. Although leukotriene receptor (LT-R) has not yet been demonstrated in middle ear mucosa, our results suggested that pranlukast acts directly on the middle ear and that blockade of LT-R in middle ear mucosa or inflammatory cells by pranlukast inhibits release of
N. Tada et al. / Auris Nasus Larynx 29 (2002) 127–132 Table 2 Thickness of the mucosa (mm) of the tube (A) and bulla (B)
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The findings of this study suggest that LT thus appear to play an important role in the pathogenesis of OME and, as a result, we recommend that a clinical trial for use of pranlukast to treat OME should be conducted. References
Values are 9 S.D. Four ears of rats were studied in each group. LTD4-leukotrien D4 showed significant difference between PBS injected ears and various concentrations of LTD4 injected ears (*PB 0.01, **PB0.05).
inflammatory cytokines, as a consequence of which local inflammation may be attenuated. This may be one mechanism by which pranlukast inhibits the occurrence of OME.
Fig. 4. The effect of pranlukast on the GRO/CINC-1 production in the rat middle ear sample. The production of GRO/CINC-1 in middle ear fluid was significantly reduced by pretreatment with pranlukast (*PB0.01). The results are given as the mean 9 S.D. for four experimental ears each.
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