International Journal of Pediatric Otorhinolaryngology (2008) 72, 1845—1848
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Does exogenous nitric oxide influence tympanosclerosis? An experimental study Sinan Atmaca a,*, Yesim Erol a, Ahmet Yilmaz Coban b, Levent Yildiz c, Senem Cengel a, Mehmet Koyuncu a a
Department of Otolaryngology and Head and Neck Surgery, Ondokuz Mayis University School of Medicine, Kurupelit 55139, Samsun, Turkey b Department of Microbiology and Clinical Microbiology, Ondokuz Mayis University School of Medicine, 55139 Kurupelit, Samsun, Turkey c Department of Pathology, Ondokuz Mayis University School of Medicine, 55139 Kurupelit, Samsun, Turkey Received 16 July 2008; received in revised form 2 September 2008; accepted 4 September 2008
KEYWORDS Nitric oxide; Tympanosclerosis
Summary Objective: We aimed to elucidate the effects of exogenous nitric oxide application via myrigotomized rat tympanic membranes on tympanosclerosis. Materials and methods: Forty male rats were included in the study. After myringotomy, nitric oxide was given to the left tympanic cavities and saline was given to the right as the control group. The procedure was repeated 3 times with weekly intervals. Three months after the first procedure, otomicroscopic examination was made and the rats were sacrificed for histologic examination. Results: Neither otomicroscopic examination, nor histologic examination of middle ears and tympanic membranes revealed any significant difference between the right and left sides. Tympanic membrane thicknesses were also similar on both sides without showing any significant difference. Conclusions: Data from this study indicate that exogenous nitric oxide application did not change the occurence of tympanosclerosis in the rat model. Other cytokine interactions are needed to start the reaction sequence leading to tympanosclerosis. # 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
* Corresponding author at: Ondokuz Mayis Universitesi, Tip Fak. KBB Anabilim Dali, Kurupelit 55139, Samsun, Turkey. Tel.: +90 362 3121919x2749; fax: +90 362 4576041. E-mail address:
[email protected] (S. Atmaca).
TS is a degenerative process in the connective tissue layer affecting both the tympanic membrane (TM) and the middle ear mucosa, characterized by hyalin degeneration in the lamina propria and increase in collagenous fibers with accumulation of calcium and
0165-5876/$ — see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2008.09.008
1846 phosphorous, leading to crystallization and sclerosis [1—8]. Tympanosclerosis (TS) is a frequent sequel of acute and chronic otitis media and is also seen after ventilation tube insertion [1—9]. The TM is the most common localization of the process and when solely engaged, the disease is defined as myringosclerosis (MS) [1,5]. Although several hypotheses have been put forward, no single cause was identified [3,4]. Free oxygen radicals were blamed to exacerbate the inflammatory process [6,7]. Nitric oxide (NO) and the other free oxygen radicals can be produced through intracellular and extracellular effects of cytokines and inflammatory mediators [6]. Several studies have focused on the role of nitric oxide in the development of TS and MS in traumatized TMs [8— 10]. The aim of this study was to elucidate the effects of exogenous NO application on tympanosclerosis in experimental rat model.
2. Materials and methods 2.1. Chemical for nitric oxide NO-nucleophile complexes termed diazeniumdiolates including DETA-NO, MAHMA-NO, DEA-NO are compounds with such specifications like the capability of releasing NO in aqueous or low pH environments without the need of a redox reaction or electron transfer. NO donor DETA-NO was purchased from Sigma. Stock solution of DETA-NO as 1 M was freshly prepared in sterile distilled water.
S. Atmaca et al. After intraperitoneal pentobarbital (80 mg/kg) injection, an ear—nose—throat specialist performed otomicroscopic evaluation blindly. Sclerotic lesions were scored by a 4-point scale: 0 (no visible myringosclerosis), + (occasional myringosclerosis), ++ (moderate myringosclerosis), +++ (severe myringosclerosis). After otomicroscopic examination, rats were decapitated and tympanic bullae were opened. The specimens were fixed in 10% buffered neutral formalin solution for 24 h. For histologic examination, the specimens were decalcified in 10% formic acid solution (Gooding—Stewart solution) and tympanic bullae were cut into 2 pieces with a transverse section of TM bisecting through the short process of malleus. Formalin-fixed biopsies were dehydrated in a graded alcohol series using an over-night tissue processing schedule (Leica TP1020, Leica Microsystem). The tissues were embedded in paraffin and sectioned at a thickness of 4—6 mm (Leica 2125 rotary microtome, Leica Microsystem). The sections were stained with hematoxylin—eosin (HE), (Varistain 24-4, Shandon Inc.) and examined under light microscopy (Olympus BX51, Japan). Histopathologically; TM and middle ear inflammations were scored as ‘‘mild’’ (presence of inflammatory cells in perivascular areas, Fig. 1), ‘‘moderate’’ (presence of inflammatory cells in perivascular areas and subepithelial fields) or ‘‘severe’’ (presence of diffuse inflammatory cell infiltration, Fig. 2) by a blinded pathologist and TM thickness was measured by light microscope by Samba image analysis system, morphometry module (Samba Technologies, France). Statistical analysis was performed by Wilcoxon signed ranks and Chi-square tests.
2.2. Animals Forty healthy male Sprague—Dawley rats with intact TMs weighing 250—300 g were included in the study. Experiments for this study was approved by the Animal Use Committee at Ondokuz Mayis University Medical Center. Sample size calculated to acquire 80% power of test and 95% confidence interval for estimated 30% difference between NO and saline cohorts was 40.
3. Results The rats were healthy before the sacrifice. All TM perforations of the right (saline) and left (NO) sides
2.3. Experimental design All animals were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg). Under otomicroscopic examination, 3 mm bilateral perforations were created at posterior quadrant of TMs with a pick. After myringotomy, 25 ml saline (0.9% NaCl) solution was given into right tympanic cavities and 25 ml, 5 mM NO solution was given into left tympanic cavities using a syringe. The same procedure was repeated for 3 times with weekly intervals. The rats were sacrificed 3 months after the first procedure.
Fig. 1 Mild middle ear inflammation involving the perivascular areas.
Exogenous nitric oxide and tympanosclerosis
1847 Table 3 Histopathologic evaluation of tympanic membranes and middle ear mucosas.
R TM inf. (S) L TM inf. (NO) R ME inf. (S) L ME inf. (NO)
Number of ears
Mild
Moderate
Severe
40 40 40 40
16 17 21 20
18 19 14 16
6 4 5 4
R, Right; L, left; TM, tympanic membrane; Inf., inflammation; S, saline; NO, nitric oxide; ME, middle ear; Chi-square test, for all variables listed, p > 0.05.
Fig. 2 Severe middle ear inflammation with diffuse inflammatory cell infiltration.
had healed and closed. Neither otomicroscopic evaluation nor TM thicknesses revealed significant difference between right (saline) and left (NO) ears (Tables 1 and 2). There was also no significant difference between right and left ears with regards to TM and middle ear inflammation (Table 3).
4. Discussion NO is a transmitter, secondary messenger and regulatory molecule that exists in immune, gastrointestinal, cardiovascular and urogenital systems. Besides its physiologic functions, it is also involved in septic shock, hypertension, cerebrovascular events, epilepsy and neurodegenerative diseases. NO with other oxygen radicals were shown to induce histopathologic changes in the tympanic cavity and tympanosclerosis by several authors [3—10]. Russell and Giles [2] showed that sequela leading to TS started within 1 month of Eustachian tube Table 1 Otomicroscopic evaluation of tympanic membranes.
Right ear (S) Left ear (NO)
Number of ears
0
+
++
+++
40 40
3 2
14 15
18 19
5 4
0 (no visible myringosclerosis); + (occasional myringosclerosis); ++ (moderate myringosclerosis); +++ (severe myringosclerosis). S, Saline; NO, nitric oxide; Chi-square test, p > 0.05.
obstruction and calcium deposition started after 3 months. Ozcan et al. [3] demonstrated that topical application of N-acetylcysteine (NAC) solution to myringotomized TMs reduced the occurence of myringosclerosis in rats. They also demonstrated that the levels of lipid peroxides and NO products decreased more in NAC treated groups than the control groups suggesting that NAC application prevents myringosclerosis by acting as a free radical scavenger. Exposure of myringotomized rat TMs to different oxygen concentrations was studied by Mattsson et al. [4]. In this study, all hyperoxic animals had more myringosclerotic lesions supporting the role of oxygen radicals in myringosclerosis formation. In a similar setting, Mattsson et al. [7] showed that treatment with copper zinc-superoxide dismutase plus catalase and deferoxamine reduced the development of myringosclerosis in rats with perforated TMs in an atmosphere containing 40% oxygen. Forseni-Flodin and Hultcrantz [5] proposed a hypothetical reaction sequence leading to tympanosclerosis. In this rat otitis media model, cytokine induced macrophages produce NO and differentiate into osteoclasts whereas osteoblasts are induced by IL-6 and interact with bone remodelling. Hukkanen et al. [11] showed NO production by osteoblasts in response to cytokine production. In this study, topical application of exogenous NO to middle ear cavities of rats via myringotomies did not increase the development of TS as expected. NO mediates cell-to-cell signaling among osteoblasts, osteoclasts and macrophages in addition to its role as an intracellular signaling molecule involved in mediation of calcium and cytokine effects in osteoblasts and osteoclasts [5,11]. In our design, exogenous NO application might have by-passed the previous steps of cytokine interactions and not
Table 2 Evaluation of tympanic membrane thicknesses TM thickness
Right (saline) ear (n = 40)
Left (NO) ear (n = 40)
p
74.9 23.3 mm
75.6 15.8 mm
>0.05
Wilcoxon signed ranks test. Values are expressed as mean standard error of means.
1848
S. Atmaca et al.
started the reaction sequence leading to TS properly or NO in liquid form might have leaked to nasopharynx through Eustachian tubes (ET) without spending enough time inside the tympanic cavities to interact with the surrounding structures. We believe the presence of moderate to severe middle ear inflammation after 3 months in the control (saline) ears is due to the repeated trauma caused by weekly myringotomies for 3 weeks. Histopathologic changes in the middle ear after ET blockage was studied by Vicente et al. [12]. Besides TM and mucoperiosteal changes, fibrous tissue hypertrophy with immature collagen fibers and osteogenic activity were observed at 15 days. After 90 days of ET blockage, increased number of goblet cells and submucosal secretory glands with epithelial metaplasia and edema was observed. They concluded that these pathologic changes was the result of unspecific repairing processes instead of the original cause of the acute inflammation. Collagen fiber deposition is an indispensable step in the formation of TS supporting the 15 day findings of the previous study [1]. Our further experiments will be designed to solve the complex mediator effects of cytokines and NO in experimental ET blockage model.
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