European Archives of Oto-Rhino-Laryngology

, Volume 272, Issue 1, pp 29–34

Effect of caffeic acid phenethyl ester on myringosclerosis development in the tympanic membrane of rat

Authors

    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Musa Ozbay
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Ulas Alabalik
    • Department of PathologyDicle University Medical College
  • Aylin Gul
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Beyhan Yilmaz
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Fazi Emre Ozkurt
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Engin Sengul
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
  • Ismail Topcu
    • Department of ENT & Head and Neck SurgeryDicle University Medical College
Otology

DOI: 10.1007/s00405-013-2832-2

Cite this article as:
Kinis, V., Ozbay, M., Alabalik, U. et al. Eur Arch Otorhinolaryngol (2015) 272: 29. doi:10.1007/s00405-013-2832-2

Abstract

Myringosclerosis is hyalinization and calcification of certain areas of the tympanic membrane, especially the fibrous lamina propria layer and appears as white sclerotic lesions. Ventilation tube insertion is one of the most performed operations in the pediatric otorhinolaryngology practice to treat chronic otitis media with effusion. Myringosclerosis is a very common sequela of ventilation tube insertion. In this experimental study, our aim was to show the histopathological effects of caffeic acid phenethyl ester on myringosclerosis development in rat tympanic membrane after myringotomy. The rats were randomly categorized into four experimental groups including the comparison group (n = 4), non-treated group (n = 7), the saline (control) group (n = 7), the caffeic acid phenethyl ester group (n = 7). Non-treated group did not receive any treatment for 15 days. Saline (2.5 mL/kg, intraperitoneal) was administered to the third group once a day for 15 days. Fourth group received caffeic acid phenethyl ester intraperitoneally once a day at a dose of 10 μmol/kg for 15 days. Myringotomy was performed on the right tympanic membrane of all rats except comparison group using a sterile pick with the help of an operating microscope. Histopathological examination of myringosclerosis formation was done by a pathologist under light microscope. In histopathological analysis of groups, the severity of inflammation was milder in caffeic acid phenethyl ester group compared to non-treated and saline groups (p < 0.05). There was less myringosclerotic plaques in caffeic acid phenethyl ester group than in non-treated and saline groups (p < 0.05). TM thickness measurements were very close to each other in non-treated and saline groups. The tympanic membrane thickness of caffeic acid phenethyl ester group was much thinner than the other two groups (p < 0.05). Caffeic acid phenethyl ester decreases inflammation severity and the formation of myringosclerotic plaques. These two effects resulted in thinner tympanic membranes of rats which were treated with caffeic acid phenethyl ester. As a result, caffeic acid phenethyl ester has potential preventive effects on myringosclerosis development after myringotomy and ventilation tube insertion.

Keywords

Caffeic acid phenethyl esterMyringosclerosisVentilation tubeTympanic membraneInflammationRat

Introduction

Tympanic membrane (TM) is composed of three layers: a lateral keratinized squamous epithelium layer continuous with external auditory meatus epithelium, the middle lamina propria layer and medial mucosal layer continuous with mucosa of tympanic cavity. The rat TM structure is very similar to human [1]. Myringosclerosis (MS) is hyalinization and calcification of certain areas of TM, especially the fibrous lamina propria layer and appears as white sclerotic lesions. Histologically, there is an increase in collagen fibers, degeneration and calcification in the lamina propria of TM seen after irreversible tissue damage [24]. MS is usually considered a healed inflammation or a particular form of scar tissue. The etiology and pathogenesis of MS are not still well known. Production of free oxygen-derived radicals also known as reactive oxygen species (ROS), mechanical injury and inflammation may contribute to the formation of MS [58].

Myringotomy, ventilation tube (VT) insertion, recurrent otitis media, physical trauma, middle ear infections, various chemical agents, genetic tendency, immunity, and local metabolic changes are the reasons which have been suggested for MS formation [6, 9]. VT insertion is among the most frequently performed operations in the pediatric otorhinolaryngology practice to treat chronic otitis media with effusion and recurrent acute otitis media (AOM). There are many frequently seen complications of this procedure such as chronic otorrhea, permanent perforations, MS, tympanosclerosis, iatrogenic cholesteatoma and formation of granulation tissue. MS is a very common sequela of VT insertion [3, 4, 10]. Most of the cases are asymptomatic or cause mild hearing loss [5]. However, MS may cause marked hearing loss, if the plaques involve large areas of the TM or limit the movement of ossicles.

Caffeic acid phenyl ester (CAPE) is a biologically active ingredient of propolis from honey hives. CAPE has several biological properties including anti-oxidative, anti-metastatic, anti-inflammatory, antiviral, immunomodulatory, anti-invasive, and carcinostatic effects on different organs and tissues [1113]. In this experimental study, our aim was to demonstrate the histopathological effects of CAPE on the development of MS in rat TM after myringotomy.

Materials and methods

The study was performed in accordance with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health, Commission on Life Sciences, and National Research Council [14]. The experimental protocol was approved by the Ethical Committee of our institution with the document no. 2012/50.

Twenty-five male adult Wistar albino rats, weighing between 220 and 331 g, were used in this study. The rats were kept in suitable cages under standard environmental conditions with the standard guidelines (room temperature between 22 and 24 °C, 50 % relative humidity and 12-hourly light and dark cycles). The animals had free access to water and were fed with conventional laboratory diet until they were euthanized. The rats were randomly categorized into four groups including the comparison group (n = 4), non-treated group (n = 7), saline (control) group (n = 7), and CAPE group (n = 7). We used comparison group to find the normal TM thickness of healthy rats without any treatment and procedure. The third group was administered saline intraperitoneally (2.5 mL/kg, IM) once a day for 15 days. At a concentration of 10 μmol, CAPE completely blocks the production of ROS in human neutrophils and xanthine/xanthine oxidase system [13, 15]. Therefore, we injected CAPE (Sigma-Aldrich Co LLC, St Louis, MO) intraperitoneally once a day at a dose of 10 μmol/kg for 15 days. Intraperitoneal injection of ketamine hydrochloride 60 mg/kg (Ketalar, Pfizer, Istanbul, Turkey) and 10 mg/kg of 2 % xylazine hydrochloride (Rompun, Bayer, Istanbul, Turkey) were used for anesthesia. Otomicroscopic evaluation of all rats was done to exclude any external and middle ear disorder. All rats included in study had normal tympanic membrane. Myringotomy was performed on the right TM of all rats except comparison group under aseptic conditions using a sterile pick with the help of an operating microscope. At day 15, otomicroscopic examination of all rats was done under anesthesia. Then the rats were decapitated and tympanic bulla of each rat was removed by microdissection. Otomicroscopic evaluation of MS is a very subjective examination. It may change easily from person to person. So, the degree of MS severity was not evaluated with otomicroscope in our study as in previous studies. We just performed a general otomicroscopic evaluation. Histopathological examination of MS formation was done by a pathologist which is much more objective method.

Tissue preparation and histopathological examination

TM and the surrounding bony annulus were fixed in 10 % formaldehyde solution for 24 h and decalcified in a 10 % ethylenediamine tetraacetic acid (EDTA) solution for 3 weeks. After fixation and decalcification processes, the specimens were rinsed in tap water for 24 h, dehydrated through a reaction with graded alcohol series, rendered transparent and blocked after the infiltration with paraffin. The samples embedded in paraffin were sectioned with the help of a microtome (Microm HM 360) into a thickness of 5 μm and the obtained cross sections were stained with hematoxylin & eosin (H&E) solution before the examination using a microscope (Nikon ECLIPSE 80i). Stained specimens were evaluated by the same pathologist who was unaware of study groups. Histopathologically, the degree of fibroblastic proliferation and the intensity of the inflammation in the lamina propria were evaluated semiquantitatively and scored as follows: (0) no inflammation, (+) mild inflammation, (++) moderate inflammation and (+++) severe inflammation. The thickness of the myringotomized area of pars tensa was measured by a photomicroscope. The histopathological evaluation of myringosclerotic lesions was performed by a 4-point scale: 0 (no visible myringosclerotic plaque); 1, occasional MS (few single lesions in the lamina propria); 2, moderate MS (confluent lesions in lamina propria); and 3, severe MS (extensive lesions in the lamina propria).

Statistical analysis

Statistical analyses were performed using the SPSS 15.0 software package for Windows (SPSS inc., Chicago, IL, USA). We used Kruskal–Wallis test for the comparison of continuous variables among the three groups. Mann–Whitney U test was used in the binary comparisons when there was a significant difference among the groups. Pearson correlation was used to test the correlation between the grade of inflammation and TM thickness. Statistical significance was based on a p value below 0.05 (p < 0.05).

Results

H&E staining showed moderate to severe inflammation in non-treated and saline groups and there was no statistically significant difference between them (Figs. 1, 2). But the degree of inflammation in CAPE group was milder when compared to these two groups (Fig. 3; p < 0.05; Table 1). Confluent or extensive myringosclerotic plaques were present in non-treated and saline groups (Figs. 1, 2). This result was not statistically significant when we compared non-treated and saline groups. There was less myringosclerotic plaques in CAPE group (Fig. 3), (p < 0.05) (Table 2). In the comparison group, the mean TM thickness of healthy rats was 27.21 μm (Fig. 4). TM thickness measurements were very close to each other in non-treated and saline groups (Figs. 1, 2). This caused no significant importance statistically. But the TM thickness of CAPE group was much thinner than the other two groups (Fig. 3; p < 0.05; Table 3). There was a statistically important correlation between degree of inflammation and TM thickness.
https://static-content.springer.com/image/art%3A10.1007%2Fs00405-013-2832-2/MediaObjects/405_2013_2832_Fig1_HTML.jpg
Fig. 1

Sclerotic changes, increased fibroblastic proliferation and inflammation in the lamina propria of comparatively thickened TM (asterisk) (H&E, ×20)

https://static-content.springer.com/image/art%3A10.1007%2Fs00405-013-2832-2/MediaObjects/405_2013_2832_Fig2_HTML.jpg
Fig. 2

Sclerotic changes, increased fibroblastic proliferation and inflammation in the lamina propria of comparatively thickened TM (asterisk) (H&E, ×20)

https://static-content.springer.com/image/art%3A10.1007%2Fs00405-013-2832-2/MediaObjects/405_2013_2832_Fig3_HTML.jpg
Fig. 3

Comparatively thin TM, mild fibroblast proliferation and inflammation in the lamina propria (arrows) (H&E, ×20)

Table 1

Severity of inflammation in groups

 

Non-treated

Saline

CAPE

N (%)

N (%)

N (%)

None (0)

0

0

0

0

4

57.14

Mild (+)

2

28.57

2

28.57

3

42.85

Moderate (++)

3

42.85

4

57.14

0

0

Severe (+++)

2

28.57

1

14.28

0

0

Total

7

100

7

100

7

100

p*: 0.08, (p†: P1; 0.7, P2; 0.08, P3; 0.08)

p*: Significance of Kruskal–Wallis Test, p†: Significance of Mann–Whitney U Test

1Comparison of the group 1 and 2

2Comparison of the group 1 and 3

3Comparison of the group 2 and 3

Table 2

Histopathological evaluation of myringosclerotic plaques

 

Non-treated

Saline

CAPE

N-Percentage (%)

None (0)

0

0

0

0

4

57.14

Occasional (+)

2

28.57

3

42.85

2

28.57

Confluent (++)

2

28.57

2

28.57

1

14.28

Extensive (+++)

3

42.85

2

28.57

0

0

Total

7

100

7

100

7

100

p*: 0.015, (p†: P1; 0.6, P2; 0.01, P3; 0.02)

https://static-content.springer.com/image/art%3A10.1007%2Fs00405-013-2832-2/MediaObjects/405_2013_2832_Fig4_HTML.jpg
Fig. 4

Normal appearance and thickness of healthy non-myringotomized rat TM (H&E, ×20)

Table 3

Analysis of TM thickness

 

Non-treated (n = 7) Mean ± SD

Saline (n = 7) Mean ± SD

CAPE (n = 7) Mean ± SD

p*

p

TM thickness (μm)

98.36 ± 1.47

99.71 ± 1.84

64.69 ± 6.74

0.011

0.61, 0.0152, 0.0073

Refer Table 1 footnote

TM tympanic membrane, μm micrometer, SD standard deviation

Discussion

MS is an irreversible, nonspecific end result of chronic inflammation of the TM [16]. Histopathologically, MS is characterized with the increase of collagen tissue, hyalinization, and calcification in the lamina propria of TM [3, 4, 6, 17]. Although the exact cause and pathogenesis of MS is not well known, several hypotheses have been blamed concerning the etiology and pathophysiology of MS.

Tos et al. [5] stated that reduced mobility of the TM in the ear with inflammatory fibrous hyperplasia promotes hyalinization and calcification in the collagen layer. In another study, it was emphasized that excessive inflammatory response and tissue damage in the fibrous lamina propria layer of the TM contribute to the formation of MS [8, 17, 18]. Similarly, Dawes et al. [19] hypothesized that the risk of development of MS increased when there is traumatic VT insertion, hemorrhage, or excessive aspiration of middle ear fluid. Also mechanical injury and metabolic disturbance were blamed as the reasons of MS [2, 20]. Most probably, the main factors which trigger the formation of MS after myringotomy or VT insertion are severe inflammation of TM and tissue damage by mechanical trauma. The most accepted and popular theory of MS formation after myringotomy or VT insertion is ROS. The oxygen concentration in the middle ear cavity of human and animals is 5–10 %, which is much lower than in ambient air [21]. Ambient air containing 21 % O2 easily passes into the middle ear cavity after VT insertion by myringotomy, resulting in a relatively hyperoxic condition [22]. This increase of partial pressure of oxygen in the middle ear cavity after VT insertion may cause an excessive formation of ROS, which probably initiates the process that results in the development of calcification and sclerotic plaques [68, 16, 17, 23].

In a study by Mattsson et al. [6], myringotomized rats were exposed to different concentrations of oxygen. More extensive sclerotic lesions were seen in TM at higher oxygen concentrations. ROS may come from both the hyperoxic environment and the inflammation induced by mechanical trauma of tympanic membrane. Inflammatory reaction consists of inflammatory cells around sclerotic aggregates. Inflammatory reaction causes the migration of polymorphonuclear cells and macrophages. These cells produce high amount of ROS in response to inflammation. Also, during phagocytosis and metabolism of arachidonic acid, superoxide radicals are produced in increased quantities, which may contribute to the development of MS [6, 23]. Increase in production of ROS can be the first stage in the accumulation and aggregation of calcium and phosphorus that form sclerotic deposits and result in MS [6].

(CAPE) is a biologically active major component of propolis from honey hives and it has been used in traditional medicines for many years for its anti-oxidative and anti-inflammatory effects. CAPE has many preventive and protective effects against toxic properties of several agents in various tissues such as the cochlea, tympanic membrane, kidney, heart, red blood cell, brain, and spinal cord [12, 18, 2426]. The possible beneficial effects of CAPE on MS formation after myringotomy were demonstrated in this experimental study.

In many studies performed on rats and humans, many substances with anti-oxidant and anti-inflammatory properties including l-carnitine, vitamin E, ascorbic acid superoxide dismutase, N-acetylcysteine (NAC), selenium, N-nitro l-arginine methyl ester (l-NAME), ginkgo biloba extract (GBE), CAPE have been used to show their effects on MS development. Sclerotic plaque formation was either prevented or diminished by these agents. They probably achieved this task by decreasing inflammation in TM after myringotomy and production of ROS by neutrophils in response to inflammation [24, 6, 7, 17, 18, 2730].

TM thickness, inflammation grade and myringosclerotic plaques are the parameters that were measured to show the presence of MS and the effects of the antioxidants and anti-inflammatory agents in these studies. Emir et al. [30] reported that sclerotic lesions were much lesser in the group administered with GBE when compared with the saline group. In another study, NAC significantly decreased the formation of sclerotic plaques when compared with saline and non-treated groups [4]. Similarly, Akbas et al. [28] found that sclerotic plaques were less in carnitine group than in saline group. In present study, the sclerotic plaques were significantly less in CAPE group than the non-treated group and saline group. CAPE significantly decreased the sclerotic lesions.

However, inflammation severity is probably one of the most important factors that lead to MS formation. It has not been a frequently evaluated parameter in experimental studies. In a study done by Dogan et al. [29], the inflammation severity was significantly milder in topically versus intraperitoneally treated rats with l-NAME groups when compared with non-treated and saline groups. In our study, we found that inflammation severity significantly was much lesser in CAPE group than in non-treated and saline groups.

In previous studies, TM thickness was a another important parameter to evaluate the MS formation. Song et al. [18] reported that TM thickness was 0.75 ± 0.22 mm in CAPE group, whereas it was found to be 1.17 ± 0.49 and 1.19 ± 0.39 mm, respectively, in non-treated and saline groups. This difference in TM thickness was statistically significant. In another study, TM thickness was 1.55 mm in l-carnitine group and 3.35 mm in control group [28]. Emir et al. [30] reported that TM thickness was 0.055 and 0.050 mm in two groups of GBE with different doses. TM thickness was found to be 0.1 mm in the saline group. The thinner TM in GBE groups was statistically important. In another study, TM thickness was 28.0 μm in topical l-NAME group, 25.5 μm in IP l-NAME group, 77.9 μm in non-treated group and 62.8 μm in saline group [29]. They reported that both forms of l-NAME significantly decreased the TM thickness compared to non-treated and control groups. In the present study, the mean TM thickness of healthy rat was measured as 27.21 μm. TM was significantly thinner in CAPE group compared to non-treated and saline groups. But the mean TM of CAPE group was thicker than that of healthy rat TM. So we should investigate the long-term effect of CAPE on TM thickness after myringotomy. This is a weakness of our study.

Song et al. [18] investigated the effect of CAPE on MS after myringotomy on rat and also evaluated TM thickness and performed otomicroscopic examination of sclerotic lesions. We evaluated the sclerotic lesions histopathologically instead of otomicroscopic evaluation. We also evaluated the grade of inflammation in TM after myringotomy. The examination of these two measurements is an important difference from the study of Song et al. [18]. Since inflammation is one of the major factor that leads to MS formation, we also found that there was an important correlation between TM thickness and inflammation.

In conclusion, as a result of long-term and comprehensive studies, CAPE may become a promising treatment option for MS which is the most important problem of the VT insertion.

Conflict of interest

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Copyright information

© Springer-Verlag Berlin Heidelberg 2013