International Journal of Pediatric Otolaryngology 77 (2013) 792-795
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Levels of reactive oxygen species in the rat eardrum after incision versus radiofrequency myringotomy Erdal Sakalli a,*, Serdar Baylancicek a, Meral Yuksel b, Selcuk Cem Erdurak a, Burhan Dadas a a b
Sisli Etfal Training and Research Hospital, ENT Department, Istanbul, Turkey Medical Laboratory, Vocational School for Health Professions, Marmara University, Istanbul, Turkey
ARTIKEL I N F O
A B S T R A C T
Article History: Retrieved October 18, 2012. Retrieved in revised form February 10, 2013. Accepted February 12, 2013. Available online March 9, 2013.
Objective: A close association between reactive oxygen species (ROS) and myringosclerosis, a common complication of myringotomy, has recently been reported. The aim of this study was to measure ROS levels directly in the eardrums of rats using luminol-assisted chemiluminescence (CL) to compare ROS levels after incisional and radiofrequency (RF) myringotomy. Methods: Fifteen Sprague-Dawley rats were divided into three groups of five animals each. Bilateral myringotomies were performed using the right myringotomy lancet in group 1 and RF in group 2. Group 3 served as a control group without myringotomy. Twenty-four hours after the procedure, all eardrums were examined under an otomicroscope and then excised for ROS measurement with CL using luminol. Results: The mean level of ROS in group 1 was significantly higher than in groups 2 and 3 (p < 0.05 for both). The difference in the mean level of ROS between groups 2 and 3 was not significant (p > 0.05). Otomicroscopy revealed increased vascularization and dilation of blood vessels in all tympanic membranes that underwent myringotomy. Vascular dilatation was observed in the annular region in the vessels running along the long arm of the malleus, in addition to those supplying the anterior and posterior tympanomalleolar folds. Conclusions: While the relationship between ROS and the development of myringosclerosis after myringotomy has been demonstrated, this study is the first to compare incisional and RF myringotomy based on measurement of ROS levels. Our results show that the increase in ROS due to myringotomy was greater after incisional myringotomy than after RF myringotomy. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Myringotomy Reactive oxygen species Radiofrequency myringosclerosis
1. Introduction Myringotomy with concomitant use of a breathing tube (VT) is the most effective surgical treatment of otitis media with effusion (OME) . Tympanosclerosis is the most common long-term complication of this procedure, often performed in children. In myringosclerosis, tympanosclerotic plaques are confined to the eardrum . Myringosclerosis of the tympanic membrane is characterized by hyaline degeneration and an increased number of collagen fibers in the lamina propria. Accumulation of calcium and phosphorus in this structure leads to crystallization and sclerosis . Although the etiology of myringosclerosis is unclear, recent studies suggest that reactive oxygen species (ROS) are produced as a result of increased oxygen levels in this area, and
* Corresponding author. Tel.: +90 5334417710. Email address:[emailprotected](E. Sakalli). 0165-5876/$ – see cover ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2013.02.013
that they have a significant effect on the development of myringosclerosis [4-6]. The oxygen concentration in the middle ear varies between 5% and 10%; however, it increases to 21% after myringotomy, contributing to the generation of ROS . ROS production under high oxygen concentrations is caused by inflammatory cells infiltrating the area, which is a consequence of myringotomy-induced trauma, and the level increases as the number of inflammatory cells in the area increases . Several methods of myringotomy have been developed. Incisional myringotomy is the most commonly performed method; however, radiofrequency (RF) myringotomy has become increasingly popular in recent times [9-12]. Although numerous studies have compared different changes in the rat eardrum after incisional versus RF myringotomy, there are no studies comparing RF to incisional myringotomy in terms of increased ROS levels. In this study, a quantitative analysis of ROS was performed using luminoamplified chemiluminescence (CL) and the effects of incisional versus RF myringotomy on the development of myringosclerosis were considered.
E. Sakalli i sur. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792-795
2. Materials and Methods This study was approved by the Animal Research Ethics Committee of Marmara University (Istanbul, Turkey). All animals were treated in accordance with the Helsinki Universal Declaration of Animal Rights. Fifteen adult Sprague-Dawley rats weighing between 250 and 350 g obtained from the Experimental Research and Animal Laboratory of Marmara University were used in this study. The animals were housed in steel cages and fed ad libitum. Animals were examined after 1 week in quarantine; those with pathologies in the eardrum or middle ear were excluded from the study. Animals were anesthetized with a single intraperitoneal injection of 50 mg/kg ketamine hydrochloride (Ketalar; Eczacibasi, Warner Lambert, Istanbul, Turkey). Under an otomicroscope (S1 with 300 mm objective; Zeiss, Jena, Germany), an appropriately sized speculum was inserted into the rat's external ear and the external auditory canal and tympanic membrane were examined (Figure 1). The animals were randomly divided into three equal groups, consisting of two test groups and one control group, with five animals in each group. In group 1, a bilateral incision was made on the tympanic membrane using an appropriate lancet for myringotomy (Figure 2). In group 2, bilateral myringotomies were made at the same site in each eardrum with an RF probe using a CelonENT (Celon Medical Instruments) RF device at power level 6, creating a 1.5 mm diameter perforation (Figure 2 ). Care was taken during the procedure to avoid electrode contact with the external auditory canal and promontory. No procedures were performed on the ear membranes of five animals in the control group. Animals were sacrificed using a high dose of ketamine, injected intraperitoneally 24 hours after myringotomy, and their tympanic membranes were assessed by otomicroscopy. All animals were decapitated and the temporal bullae removed. Both eardrums were carefully dissected under a microscope, washed in ice-cold saline and analyzed as described below. After 10 minutes, samples were probed by CL to detect ROS levels. CL was measured at room temperature using a Mini Lumat LB 9506 luminometer (EG&G Berthold, Bad Wildbad, Germany) in the presence of 0.2 mM luminol. Counts were obtained at 5 second intervals; results are given as area under the curve (AUC) for luminol CL with a count period of 5 minutes. The tissues were then removed from the tubes, the fluids were absorbed using filter paper and the dry weights were determined. Results are expressed as AUCrlu/mg tissue. The data was transferred to a computer for statistical analysis. Group differences were analyzed by one-way analysis. The limit of statistical significance was set at a p-value of 0.05.
Figure 1. Rat eardrum before myringotomy (arrow; malleus).
3. Results 3.1. Otomicroscopic Results In all tympanic membranes that underwent myringotomy, dilation of vessels was observed in the annular region, in the vessels running along the long arm of the malleus, and in the vessels supplying the anterior and posterior tympanomalleolar folds. 3.2. CL measurement (Tables 1 and 2) Luminol-enhanced CL levels in rat eardrums of all groups are shown in Table 1; the results of the statistical analysis are summarized in Table 2. In those animals that underwent RF and incisional myringotomy, ROS levels measured by luminol CL were 46.15 ± 8.79 and 55.91 ± 9.00 rlu/, respectively mg tissue. In the incisional myringotomy group, tympanic membrane trauma caused a significant increase in ROS measured by luminol CL compared to the RF myringotomy group (p < 0.05) (Figure 3). The mean ROS level in the control group was 43.63 ± 7.66 rlu/mg tissue, which was significantly lower than that in the incision myringotomy group (p < 0.05), but not significantly different from that in the group with by RF myringotomy (p > 0.05). 4. Discussion The results of our study show that incisional and RF myringotomy resulted in increased ROS levels, with significantly higher levels reported in animals receiving
Figure 2. Rat eardrum after RF myringotomy (A) (arrows indicate bloodless and smooth perforation boundaries), rat eardrum after incisional myringotomy (B) (arrows indicate hemorrhagic and rough perforation boundaries).
E. Sakalli i sur. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792-795
Table 1 Luminol-enhanced chemiluminescence values in rat eardrums (rlu [relative light unit] per milligram of tissue). ear number
Incision and myringotomies
1 2 3 4 5 6 7 8 9 10
70,0 69,4 63,7 51,1 59,2 52,8 48,3 46,2 50,4 48,0
52,2 48,3 43,7 40,7 68,2 37,8 40,5 44,2 41,0 44,9
30,1 47,8 38,5 41,2 36,1 40,9 44,6 53,1 51,1 52,9
RF, radio frequency.
incisional method. This study is original in that it compares two techniques based on the quantitative measurement of ROS levels using luminol-assisted CL. The etiopathogenesis of myringosclerosis is unclear; suggested causes include infection, trauma, allergy, hypersensitivity, inflammation, bacterial enzymes, Eustachian tube disorders, genetic predisposition, and cholesteatoma . In addition, myringotomy or VT implantation is known to increase the risk of myringosclerosis . Mattson et al.  conducted an experimental study in which dexamethasone and RU486 (a glucocorticoid receptor antagonist) were administered to bilaterally myringotomized rats. They found lower levels of inflammation in both groups compared to the control group, which received no treatment. Examination of rat ears revealed that the submucosal tissues were more edematous and contained more sclerotic lesions than those in the control group. The authors concluded that the inflammatory response could be involved in the development of myringosclerosis. Dogan et al.  investigated the anti-inflammatory and anti-fibrotic effects of N-nitro-L-arginine methyl ester in experimentally induced myringosclerosis. They showed that the use of these agents reduces the formation of myringosclerosis in the eardrums of rats with myringotomy. Furthermore, Park et al.  demonstrated the preventive effect of sodium thiosulfate, which is an antidote for cyanide poisoning, in the development of myringosclerosis in an experimental animal model. Alternatively, recent studies have shown a role for ROS in the development of myringosclerosis after myringotomy [4-6]. Normally, most of the oxygen in the human body is reduced to water by the cytochrome oxidase complex in the mitochondria. Exposure of cells to hyperoxic conditions followed by reperfusion or activation of phagocytic cells (e.g. macrophages) during inflammation produces significant amounts of superoxide radicals that cause tissue damage . The oxygen concentration in the middle ear is between 5% and 10%, and upon myringotomy it increases to 21%, creating a relatively hyperoxic state . Increased oxygen content in the middle ear induces the formation of ROS, which causes tissue damage, followed by fibrosis and hyaline degeneration, and the onset of calcium and phosphorus accumulation in tissues [4-6].
Figure 3. Comparison of mean SD values for the amount of free radicals. Chemiluminescence levels in the eardrums were found to be significantly higher in the incisional myringotomy group compared to the radiofrequency myringotomy and control groups. The English in this document has been reviewed by at least two professional native English editors. For confirmation see: http://www.textcheck.com/certiﬁcate/3Rv7Ub.
Mattson et al.  conducted a study in which they divided myringotomized rats into four groups and followed the animals for 1 week at oxygen concentrations of 10%, 15% and 40% in ambient air. Otomicroscopic and histopathological studies of rat eardrums revealed higher rates of myringosclerosis in hyperoxic groups and lower rates in hypoxic animals. Yildirim et al.  found that antioxidants such as zinc aspartate reduced the development of myringosclerosis in myringotomized rats. In another study, Kazikdas et al.  gave rats intramuscular injections of alpha-tocopherol after myringotomy and found that myringosclerosis formation was reduced in the group treated with alpha-tocopherol; concluded that the antioxidant alpha-tocopherol reduces the occurrence of myringosclerosis. Spratley et al.  applied ascorbic acid topically to rat ears after myringotomy. Using otomicroscopic and histopathological examinations, they determined that the size of sclerotic lesions was significantly smaller in the group treated with ascorbic acid. For example, they concluded that the antioxidant ascorbic acid reduces the occurrence of myringosclerosis. In the same study, a higher concentration of myringosclerotic deposits around the annulus and malleus was associated with higher soluble oxygen concentrations in higher vascularized areas and a resulting increase in ROS production. Polat et al.  and Uneri et al.  investigated the protective effects of topical vitamin E on myringotomized animal ears and measured ROS levels using a luminol-assisted CL technique. These studies support the hypothesis that ROS generation contributes significantly to the development of myringosclerosis. However, none of these studies included quantitative measurements and thus failed to demonstrate the presence of ROS, which are believed to originate from the eardrum and middle ear mucosa.
Table 2. Descriptive statistical summary of middle ear eardrum ROS levels.
Median (min-max) Mean SD Mean Standard Error (SEM) 95% Confidence Interval Size ROS, reactive oxygen species; RF, radio frequency.
Incision and myringotomies
51.950 (46.200–70.000) 55.910 9.006 2.848 49.468–62.352 10
43.950 (37.800–68.200) 46.150 8.798 2.782 39.857–52.443 10
42.900 (30.100–53.100) 43.630 7.668 2.425 38.145–49.115 10
E. Sakalli i sur. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792-795
Measuring ROS under biological conditions is difficult due to their short half-life. Spin trapping techniques, calorimetric determinations, CL and electron spin resonance spectroscopy are used to demonstrate the presence of ROS. CL is used as a direct method to measure ROS . In our study, we measured the ROS level directly in the eardrums of rats using the luminol-assisted CL technique 24 hours after myringotomy, when inflammation is known to reach its maximal level . ROS levels measured in the control group indicate basal values after eardrum dissection and reflect the amount of ROS released as a result of dissection trauma. Comparison with the control group showed a significant increase in ROS levels with incisional myringotomy, while this difference was insignificant with RF myringotomy. ROS levels were also significantly different between incisional and RF myringotomy. RF was used to create a myringotomy, showing a slowdown in closing time in recent years. Cinar et al.  compared RF myringotomy with incisional myringotomy in terms of myringotomy patency time. The authors suggested that RF myringotomy is a safe, simple procedure that can be used in place of incisional myringotomy. Cakir et al.  performed myringotomies using an RF device at a power level of 4-5. They were able to easily determine the dimensions of the myringotomy. It has been suggested that this is an advantage over classical surgical myringotomy. Lachanas [11,12] emphasized that RF waves passing through tissues cause localized thermal injury. These effects were limited to the area of application and the procedure did not cause bleeding; this is the greatest advantage of the technique. During surgery, we noticed that the perforation caused by RF myringotomy had bloodless, simple, and smooth edges. For these reasons, we believe that mild tissue damage and inflammation around the myringotomy site decreases ROS production by activating phagocytic cells. The relative increase in bleeding in incisional myringotomies may increase the number of inflammatory cells and ROS, resulting in a greater number of sclerotic changes. However, this study was conducted in animals with healthy eardrums and middle ears. When treating OME in humans, existing inflammation can further increase ROS levels and the development of myringosclerosis.
5. Conclusions Our results show that incisional myringotomy caused significantly increased ROS levels and that ROS levels did not differ significantly between RF and control groups. Increasing use of RF myringotomy may reduce the rate of myringosclerosis that can occur after myringotomy. However, it should be emphasized that this study was performed on rats. Our model may yield different findings when applied to humans. Additional clinical studies on this topic will help determine the results.
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