International Journal of Pediatric Otorhinolaryngology 77 (2013) 792–795

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International Journal of Pediatric Otorhinolaryngology

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Levels of reactive oxygen species in rat tympanic membranes after incisionalversus radiofrequency myringotomy

Erdal Sakalli a,*, Serdar Baylancicek a, Meral Yuksel b, Selcuk Cem Erdurak a, Burhan Dadas a

a Sisli Etfal Training and Research Hospital, Department of Otorhinolaryngology, Istanbul, Turkeyb Medical Laboratory, Vocational School of Health Professions, Marmara University, Istanbul, Turkey

A R T I C L E I N F O

Article history:

Received 18 October 2012

Received in revised form 10 February 2013

Accepted 12 February 2013

Available online 9 March 2013

Keywords:

Myringotomy

Reactive oxygen species

Radiofrequency

Myringosclerosis

A B S T R A C T

Objective: A close relationship between reactive oxygen species (ROS) and myringosclerosis, which is a

common complication of myringotomy, was recently reported. The objective of this study was to

measure ROS levels directly in rat tympanic membranes using luminol-aided chemiluminescence (CL) in

order to compare the levels of ROS after incisional and radiofrequency (RF) myringotomy.

Methods: Fifteen Sprague-Dawley rats were separated into three groups of five animals each. Bilateral

myringotomies were made using an appropriate myringotomy lancet in Group 1 and RF in Group 2.

Group 3 served as the control group with no myringotomy. Twenty-four hours after the procedure, all

tympanic membranes were inspected with an otomicroscope and then excised for the measurement of

ROS using luminol-aided CL.

Results: The mean ROS level in Group 1 was significantly higher than that in Groups 2 and 3 (p < 0.05 for

both). The difference in mean ROS level between Groups 2 and 3 was not significant (p > 0.05).

Otomicroscopy revealed increased vascularity and vessel dilation in all tympanic membranes that

underwent myringotomy. Vascular dilation was observed in the annular region in the vessels that passed

along the long arm of the malleus, in addition to the vessels feeding the anterior and posterior

tympanomalleolar folds.

Conclusions: Although the relationship between ROS and the development of myringosclerosis after

myringotomy has been demonstrated, the present study is the first to compare incisional and RF

myringotomy based on the measurement of ROS levels. Our results indicate that the increase in ROS due

to myringotomy was greater following incisional myringotomy than RF myringotomy.

� 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Myringotomy with the concomitant application of a ventilationtube (VT) is the most effective surgical treatment for otitis mediawith effusion (OME) [1]. Tympanosclerosis is the most commonlong-term complication of this procedure, which is performedfrequently in children. In myringosclerosis, the tympanoscleroticplates are limited to the ear membrane [2].

Myringosclerosis of the tympanic membrane is characterizedby hyaline degeneration and an increased number of collagenfibers in the lamina propria. The accumulation of calcium andphosphorus in this structure results in crystallization and sclerosis[3]. Although the etiology of myringosclerosis is unclear, recentstudies have suggested that reactive oxygen species (ROS) areformed as a result of the increased oxygen content in this area, and

* Corresponding author. Tel.: +90 5334417710.

E-mail address: erdalkbb1979@hotmail.com (E. Sakalli).

0165-5876/$ – see front matter � 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 ofmyringosclerosis [4–6]. The oxygen concentration in the middleear ranges between 5% and 10%; however, it increases to 21% aftermyringotomy, contributing to the formation of ROS [7]. Theproduction of ROS under high oxygen concentrations is caused byinflammatory cells that infiltrate into the region, resulting frommyringotomy-induced trauma, and the level increases as thenumber of inflammatory cells in the area increases [8].

Several myringotomy methods have been developed. Incisionalmyringotomy is the most frequently performed method; however,radiofrequency (RF) myringotomy has recently increased inpopularity [9–12]. Although numerous studies have comparedvarious changes in rat tympanic membrane after incisional versusRF myringotomy, there are no studies comparing RF with incisionalmyringotomy in terms of increased levels of ROS.

In this study, a quantitative analysis of ROS using luminol-amplified chemiluminescence (CL) was conducted, and the effectsof incisional versus RF myringotomy on the development ofmyringosclerosis were considered.

[(Fig._1)TD$FIG]

Fig. 1. Rat tympanic membrane before myringotomy (arrow; malleus).

E. Sakalli et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792–795 793

2. Materials and methods

This study was approved by the Animal Research EthicsCommittee of Marmara University (Istanbul, Turkey). All animalswere treated according to the Helsinki Universal Declaration ofAnimal Rights.

Fifteen adult Sprague-Dawley rats weighting between 250 and350 g each obtained from the Marmara University ExperimentalResearch and Animal Laboratory were used in this study. Theanimals were housed in steel cages and were fed ad libitum.

The animals were examined after 1 week in quarantine; thosewith pathologies in the tympanic membrane or middle ear wereexcluded from the study. The animals were anesthetized with asingle intraperitoneal injection of 50 mg/kg ketamine hydrochlo-ride (Ketalar; Eczacibasi, Warner Lambert, Istanbul, Turkey). Underan otomicroscope (S1 with a 300-mm objective lens; Zeiss, Jena,Germany), an appropriately sized speculum was inserted into theexternal ear of the rat, and the external ear canal and tympanicmembrane were examined (Fig. 1). The animals were randomlyseparated into three equal groups, consisting of two study groupsand one control group, with five animals in each group. In Group 1,an incision was made bilaterally in the tympanic membrane usingan appropriate myringotomy lancet (Fig. 2). In Group 2, bilateralmyringotomies in the same location in each tympanic membranewere made with an RF probe using a CelonENT (Celon MedicalInstruments) RF device at power level 6, creating a perforation thatwas 1.5 mm in diameter (Fig. 2). During the procedure, care wastaken to avoid contact between the electrode and the external earcanal and promontory. No procedures were performed on the earmembranes of the five animals in the control group.

The animals were sacrificed using a high dose of ketamine,injected intraperitoneally 24 h after myringotomy, and theirtympanic membranes were evaluated by otomicroscopy. Allanimals were decapitated and the temporal bullae were removed.Both tympanic membranes were dissected carefully under amicroscope, washed in ice-cold saline, and analyzed as describedbelow. After 10 min, the specimens were examined by CL to detectthe ROS levels.

CL was measured at room temperature using a Mini Lumat LB9506 luminometer (EG&G Berthold, Bad Wildbad, Germany) in thepresence of 0.2 mM luminol. Counts were obtained at 5-s intervals;the results are given as the area under curve (AUC) for luminol CLwith a 5-min counting period. Afterwards, the tissues wereremoved from the tubes, the liquids were absorbed using filterpaper, and the dry weights were determined. The results areexpressed as AUC rlu/mg of tissue.

The data were transferred to a computer for statistical analysis.Group differences were analyzed by one-way Anova. The limit forstatistical significance was set at a p-value of 0.05.

[(Fig._2)TD$FIG]

Fig. 2. Rat tympanic membrane after RF myringotomy (A) (arrows show bloodless and sm

(arrows show hemorrhagic and rough perforation borders).

3. Results

3.1. Otomicroscopic results

In all tympanic membranes that underwent myringotomy,vessel dilation was observed in the annular area, in the vesselspassing along the long arm of the malleus, and in the vesselsfeeding the anterior and posterior tympanomalleolar folds.

3.2. CL measurement (Tables 1 and 2)

The luminol-amplified CL levels in the rat tympanic membranesof all groups are shown in Table 1; the results of the statisticalanalysis are summarized in Table 2. In those animals undergoingRF and incisional myringotomy, the ROS levels as measured byluminol CL were 46.15 � 8.79 and 55.91 � 9.00 rlu/mg of tissue,respectively. In the incisional myringotomy group, trauma to thetympanic membrane caused a significant increase in ROS as measuredby luminol CL compared to the RF myringotomy group (p < 0.05)(Fig. 3). The mean ROS level in the control group was 43.63 � 7.66 rlu/mg of tissue, which is significantly lower than that in the incisionalmyringotomy group (p < 0.05), but not significantly different fromthat in the RF myringotomy group (p > 0.05).

4. Discussion

The results of our study show that incisional and RFmyringotomy resulted in an increase in ROS levels, withsignificantly higher levels reported in animals undergoing the

ooth perforation borders), Rat tympanic membrane after Incisional myringotomy (B)

Table 1Luminol-amplified chemiluminescence values in rats’ tympanic membranes (rlu

[relative light unit] per milligram Tissue).

Ear number Incisional myringotomy RF myringotomy Control

1 70.0 52.2 30.1

2 69.4 48.3 47.8

3 63.7 43.7 38.5

4 51.1 40.7 41.2

5 59.2 68.2 36.1

6 52.8 37.8 40.9

7 48.3 40.5 44.6

8 46.2 44.2 53.1

9 50.4 41.0 51.1

10 48.0 44.9 52.9

RF, radiofrequency.

[(Fig._3)TD$FIG]

Fig. 3. Comparison of mean � SD values for free radical amount. Chemiluminescence

levels in tympanic membranes are shown to be significantly higher in incisional

myringotomy group compared with radiofrequency myringotomy and control groups.

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E. Sakalli et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792–795794

incisional method. This study is original because it compares thetwo techniques based on the quantitative measurement of ROSlevels using luminol-aided CL.

The etiopathogenesis of myringosclerosis is unclear; suggestedcauses include infection, trauma, allergy, hypersensitivity, inflam-mation, bacterial enzymes, Eustachian tube disorders, geneticpropensity, and cholesteatoma [3]. In addition, it is well-knownthat myringotomy or VT insertion increases the risk of myringo-sclerosis [13]. Mattsson et al. [14] conducted an experimentalstudy in which dexamethasone and RU486 (a glucocorticoidreceptor antagonist) were applied to bilaterally myringotomizedrats. They discovered a lesser degree of inflammation in bothgroups as compared to the control group, which did not receive anytreatment. An examination of the rat ears revealed that thesubmucosal tissues were more edematous and included moresclerotic lesions than those in the control group. The authorsconcluded that an inflammatory reaction could be involved in thedevelopment of myringosclerosis. Dogan et al. [15] investigatedthe anti-inflammatory and antifibrotic effects of N-nitro-L-argininemethyl ester in experimentally induced myringosclerosis. Theyshowed that the use of these agents decreased the formation ofmyringosclerosis in myringotomized rat tympanic membranes.Furthermore, Park et al. [16] showed the preventive effect ofsodium thiosulfate, which is the antidote for cyanide poisoning, inthe development of myringosclerosis in an experimental animalmodel.

Alternatively, recent studies have demonstrated the role of ROSin the development of myringosclerosis after myringotomy [4–6].Normally, most of the oxygen in the human body is reduced towater via the cytochrome oxidase complex, located insidemitochondria. Exposure of cells to hyperoxic conditions, followedby reperfusion or the activation of phagocytic cells (e.g.,macrophages) during inflammation produces significant amountsof superoxide radicals that cause tissue injury [8]. The oxygenconcentration in the middle ear is between 5% and 10%, andincreases up to 21% with myringotomy, creating a relativelyhyperoxic condition [7]. The increased oxygen content in themiddle ear triggers the formation of ROS, causing tissue injury,followed by fibrosis and hyaline degeneration, and the initiation ofcalcium and phosphorus accumulation in the tissues [4–6].

Table 2Descriptive statistical summary of middle ear tympanic membranes ROS levels.

Incisional myringotomy

Median (min–max) 51.950 (46.200–70.000)

Mean� SD 55.910�9.006

Standard error mean (SEM) 2.848

95% Confidence interval 49.468–62.352

Size 10

ROS, reactive oxygen species; RF, radiofrequency.

Mattsson et al. [6] conducted a study in which they separatedmyringotomized rats into four groups, and followed the animalsfor 1 week at oxygen concentrations of 10%, 15%, and 40% underambient air conditions. Otomicroscopic and histopathologicalexaminations of the rat tympanic membranes revealed higherrates of myringosclerosis in the hyperoxic groups, and lowerrates in the hypoxic animals. Yildirim et al. [17] found thatantioxidant agents such as zinc aspartate reduced the develop-ment of myringosclerosis in myringotomized rats. In anotherstudy, Kazikdas et al. [18] gave rats intramuscular alpha-tocopherol injections after myringotomy and noted that theformation of myringosclerosis was reduced in the alpha-tocopherol-treated group; they concluded that the antioxidantalpha-tocopherol reduced the occurrence of myringosclerosis.Spratley et al. [19] applied topical ascorbic acid to rat ears aftermyringotomy. Using otomicroscopic and histopathologicalexaminations, they noted that the extent of sclerotic lesionswas significantly less in the ascorbic acid-treated group. Thus,they concluded that the antioxidant ascorbic acid reduced theoccurrence of myringosclerosis. In the same study, a higherconcentration of myringosclerotic deposits around the annulusand malleus was linked to higher concentrations of solubleoxygen in the more highly vascularized areas and to theresultant increase in ROS production.

Polat et al. [4] and Uneri et al. [5] investigated the protectiveeffects of topical vitamin E on the myringotomized ears of animalsand measured the ROS levels using a luminol-aided CL technique.These studies support the hypothesis that ROS formation contrib-utes significantly to the development of myringosclerosis.However, neither of these studies involved quantitative measure-ments, and therefore did not show the presence of ROS, which arebelieved to originate from the tympanic membrane and middle earmucosa.

RF myringotomy Control

43.950 (37.800–68.200) 42.900 (30.100–53.100)

46.150�8.798 43.630�7.668

2.782 2.425

39.857–52.443 38.145–49.115

10 10

E. Sakalli et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 792–795 795

The measurement of ROS under biological conditions isdifficult, due to their short half-life. Spin trapping techniques,calorimetric determinations, CL, and electron spin resonancespectroscopy are used to show ROS presence. CL is used as adirect method for measuring ROS [20].

In our study, we measured the ROS level directly in thetympanic membranes of rats using a luminol-aided CL technique24 h after myringotomy, when inflammation is known to reachmaximum levels [21]. The ROS levels measured in the controlgroup indicate basal values after dissection of the tympanicmembrane, and reflect the amount of ROS released due todissection trauma. A comparison with the control group showedsignificant elevation in the ROS level with incisional myringotomy,while this difference was insignificant with RF myringotomy. TheROS levels were also significantly different between incisional andRF myringotomy.

RF has been used to create myringotomies, demonstrating adelay in closure time in recent years. Cinar et al. [9] compared RFmyringotomy with incisional myringotomy in terms of themyringotomy patency time. The authors suggested that RFmyringotomy is a safe, simple procedure that can be used insteadof incisional myringotomy. Cakir et al. [10] created myringotomiesusing an RF device at power level 4–5. They were able to control thedimensions of the myringotomy easily. This was suggested to be anadvantage over classic surgical myringotomy. Lachanas [11,12]emphasized that RF waves passing through the tissues created alocalized thermal injury. These effects were confined only to thearea of application, and the procedure did not cause hemorrhage;this is the greatest advantage of the technique.

During surgery, we observed that the perforation induced byRF myringotomy was bloodless, straightforward, and hadsmooth borders. For these reasons, we believe that less tissueinjury and inflammation around the site of myringotomydecreases ROS production by phagocytic cell activation. Therelative increase in bleeding in incisional myringotomies mayincrease the number of inflammatory cells and ROS, resulting inmore sclerotic changes. However, the present study wasperformed using animals with healthy tympanic membranesand middle ears. In the treatment of OME in humans, pre-existing inflammation may further increase ROS levels and thedevelopment of myringosclerosis.

5. Conclusions

Our results show that incisional myringotomy caused signifi-cantly elevated ROS levels, and that the ROS levels were notsignificantly different between the RF and control groups.Increased use of RF myringotomy may decrease the rate ofmyringosclerosis, which can occur after myringotomy. However, itshould be emphasized that this study was conducted in rats. Ourmodel might produce different findings if done in humans.Additional clinical studies on this topic will be helpful indetermining the outcome.

Conflict of interest statement

There is not any financial and personal relationships with otherpeople or organizations. Also there is not any funding source.

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