The effect of melatonin and vitamin C treatment on the experimentally induced tympanosclerosis: study in rats / Efeito do tratamento com melatonina e vitamina C na timpanoesclerose induzida experimentalmente: estudo em ratos

Abstract Introduction: The ethiopathogenesis of tympanosclerosis has not been completely under- stood yet. Recent studies have shown that free oxygen radicals are important in the formation of tympanosclerosis. Melatonin and Vitamin C are known to be a powerful antioxidant, interacts directly with Reactive Oxygen Species and controls free radical-mediated tissue damage. Objective: To demonstrate the possible preventative effects of melatonin and Vitamin C on tympanosclerosis in rats by using histopathology and determination of total antioxidant status total antioxidant status. Methods: Standard myringotomy and standard injury were performed in the middle ear of 24 rats. The animals were divided into three groups: Group 1 received melatonin, Group 2 received vitamin C, and Group 3 received saline solution. Results: The mean values of total antioxidant status were similar in the all study groups before the treatment period. The mean values of total antioxidant status were significantly higher in the melatonin and vitamin C groups compared to control group but vitamin C with melatonin groups were similar after the treatment period (p < 0.001). Minimum and maximum wall thicknesses were lower in the melatonin and vitamin C groups compared to the control group but the differences were insignificant. Conclusion: Melatonin increases total antioxidant status level and might have some effect on tympanosclerosis that develops after myringotomy.

Resumo Introdução: A etiopatogênese da timpanoesclerose (TE) não foi ainda totalmente esclarecida. Estudos recentes têm demonstrado que os radicais livres de oxigênio são importantes na formação de TE. Melatonina e vitamina C são conhecidas por serem poderosos antioxidantes, interagir diretamente com espécies reativas de oxigênio (ROS) e controlar danos em tecidos mediados por radicais livres. Objetivo: Demonstrar os possíveis efeitos preventivos da melatonina e da vitamina C na TE em ratos com histopatologia e determinação da capacidade antioxidante total (CAT). Método: Miringotomias padronizadas foram feitas na orelha média de 24 ratos. Os animais foram divididos em três grupos: o Grupo 1 recebeu melatonina, o Grupo 2 vitamina C e o grupo 3 solução salina. Resultados: Os valores médios de CAT foram semelhantes em todos os grupos de estudo antes do período de tratamento. Os valores médios de CAT foram significativamente maiores nos grupos que receberam melatonina e vitamina C em comparação com o grupo de controle, mas os grupos vitamina C e melatonina foram semelhantes após o período de tratamento (p < 0,001). As espessuras mínimas e máximas de parede foram menores nos grupos melatonina e vitamina C, em comparação com o grupo controle, mas as diferenças não foram significativas. Conclusão: A melatonina aumenta os níveis de CAT e pode ter algum efeito sobre a TE que se desenvolve após a miringotomia.

The effect of caffeic acid phenethyl ester (CAPE) on tympanosclerosis.

OBJECTIVE: To investigate the effects of caffeic acid phenethyl ester (CAPE) on tympanosclerosis. MATERIALS AND METHODS: Thirty-two male Sprague Dawley rats were separated into 4 groups as CAPE (n = 10), alcohol (n = 10), control (n = 8) and normal (n = 4) groups. All tympanic membranes except normal group were myringotomised and type 3 Streptococcus pneumoniae strains was injected into their middle ears. Myringotomies were repeated for 5 weeks. Intraperitoneal (i.p) CAPE were administrated to the CAPE group at 10 µmol/kg/day and 10% ethyl alcohol administrated to the alcohol group for 5 weeks. The control group were left untreated. Findings of myringosclerosis were recorded by otomicroscope at sixth week. Then, all rats were sacrificed and tympanic membrane thickness and severity of middle ear mucosal inflammation evaluated histopathalogically. RESULTS: Severity of myringosclerosis was significantly higher in the alcohol and control groups compared to the CAPE group (p < 0.001), but was not significant when alcohol and control groups were compared (p = 0.17). The tympanic membrane thickness measured in the alcohol and control groups were significantly higher compared to the CAPE group (p < 0.001), but was not significant when alcohol and control groups were compared (p = 0.17). The severity of inflammation in the middle ear mucosa was significantly higher in the alcohol and control groups compared to the CAPE group (respectively, p < 0.001, p = 0.03). The severity of inflammation in the middle ear mucosa was not significant between alcohol and control groups (p = 0.30). CONCLUSION: CAPE has anti-inflammatory and antioxidant effects on the development of MS in myringotomized rats, so reduces the severity of tympanosclerosis.

The effect of melatonin and vitamin C treatment on the experimentally induced tympanosclerosis: study in rats.

INTRODUCTION: The ethiopathogenesis of tympanosclerosis has not been completely under- stood yet. Recent studies have shown that free oxygen radicals are important in the formation of tympanosclerosis. Melatonin and Vitamin C are known to be a powerful antioxidant, interacts directly with Reactive Oxygen Species and controls free radical-mediated tissue damage. OBJECTIVE: To demonstrate the possible preventative effects of melatonin and Vitamin C on tympanosclerosis in rats by using histopathology and determination of total antioxidant status total antioxidant status. METHODS: Standard myringotomy and standard injury were performed in the middle ear of 24 rats. The animals were divided into three groups: Group 1 received melatonin, Group 2 received vitamin C, and Group 3 received saline solution. RESULTS: The mean values of total antioxidant status were similar in the all study groups before the treatment period. The mean values of total antioxidant status were significantly higher in the melatonin and vitamin C groups compared to control group but vitamin C with melatonin groups were similar after the treatment period (p<0.001). Minimum and maximum wall thicknesses were lower in the melatonin and vitamin C groups compared to the control group but the differences were insignificant. CONCLUSION: Melatonin increases total antioxidant status level and might have some effect on tympanosclerosis that develops after myringotomy.

The impact of topical and systemic enoxaparin sodium use on traumatic tympanic membrane perforation and myringosclerosis.

The objective of this study is to investigate the effect of topical and systemic enoxaparin sodium on the healing pattern of experimentally induced tympanic membrane perforation and formation of myringosclerosis. A total of 24 Wistar-Albino strain rats were included in the study. Standard myringotomies were performed on each rat. In the first group, isotonic serum physiologic was dropped on external ear canal (control group). Topical enoxaparin was dropped on external ear canal and daily topical doses of enoxaparin were dropped on external ear canal of the rats for 14 days (topical treatment group). Third group received subcutaneous injections of enoxaparin for 14 days (systemic treatment group). Five micrometer thick sections of the bullae of the rats were stained with H&E. Inflammation, edema and sclerotic lesions and neovascularization observed in the lamina propria layer of the tympanic membrane, and total thickness of the tympanic membrane were evaluated. In intergroup comparisons, significant difference in the distribution pattern of severity of inflammation in all three groups was not observed (p = 0.784, p > 0.05). Total TM thickness differed among all three groups (p = 0.028, p < 0.05). A statistically significant difference was observed between the systemic enoxaparin and the control groups (p = 0.022, p < 0.05). A statistically significant difference was observed between the topical enoxaparin and the control groups (p = 0.037, p < 0.05). However, comparison between the topical and systemic treatment groups could not reveal any statistically significant intergroup difference (p = 0.682, p > 0.05). A significant difference was not observed among three groups as for the distribution of myringosclerotic plaques, severity of edema and neovascularization in the lamina propria (p = 0.539, p > 0.05), (p = 0.063, p > 0.05), (p = 0.152, p > 0.05). Topical and systemic enoxaparin treatment did not prevent formation of sclerotic plaques; however, it decreased TM thickness significantly in comparison with the control group.

The Impact of Systemic and Local Administration of Ascorbic Acid on Traumatic Perforation of Tympanic Membrane and Myringosclerosis.

OBJECTIVE: In the present study, tympanic membranes (TM) harvested from myringotomized rats were analyzed histopathologically to compare the systemic and local effects of ascorbic acid on the development of myringosclerosis. MATERIALS AND METHODS: Forty male Wistar-Albino rats weighing between 350-400 g were included in this study. Under otomicroscopic examination, a standard 2-mm myringotomy incision was made on the posteroinferior quadrant of the TM of both ears. Rats were randomized into five groups as control, topical ascorbic acid 50 mg/kg, systemic ascorbic acid 50, 100, and 200 mg/kg groups, each group containing eight rats. On the 15th day of the study, the rats were decapitated, and bullas of the rats were extracted. Sections were stained with hematoxylin-eosin and examined through light microscopy. Inflammation, distribution width of plaques, edema, and neovascularization were observed on the lamina propria. Thickness of the TM was evaluated under the microscope and scored semiquantitatively. RESULTS: When intergroup comparisons of parameters related to total TM thickness were performed, differences between the control group and topical AA (ascorbic acid) or systemic treatment groups were found to be statistically significant (p<0.005). A statistically significant difference was detected among control, topical and systemic 200 mg/kg ascorbic acid groups for the edematous lamina propria (p=0.003 and p<0.05, respectively). CONCLUSION: For the total TM thickness, systemic and topical ascorbic acid use was effective when compared with the control group. It has been concluded that systemic use of higher doses of (200 mg/kg) ascorbic acid is beneficial in the resolution of the edematous lamina propria.

Future opportunities in preventing ototoxicity: Caffeic acid phenethyl ester may be a candidate (Review).

Caffeic acid phenethyl ester (CAPE) is an important active component of propolis, which is derived from honeybee hives. It has received increasing attention in a variety of medical and pharmaceutical research, due to its anti­oxidant, antiproliferative, anti­inflammatory, antiviral and antifungal activity, in addition to its antineoplastic properties. Besides the use of CAPE as an antioxidant and anti­inflammatory agent in a number of in vivo studies of ear disease, its beneficial effects have been reported in the treatment of cancer, arthritis, allergies, heart disease, diabetes, kidney disease, liver disease and neurological disease. CAPE influences a number of biochemical pathways, as well as several targets involved in ear diseases, in particular, in ototoxicity. The protective effects of CAPE in ototoxicity, which may be induced by a number factors, including lipopolysaccharides, hydrogen peroxide and streptomycin, are evaluated and discussed in the present review.

Does systemic clarithromycin therapy have an inhibitory effect on tympanosclerosis? An experimental animal study.

OBJECTIVE: To demonstrate the inhibitory effects of clarithromycin on in vitro tympanosclerosis. METHOD: Twenty-eight rats were divided into three groups: a clarithromycin group, a non-clarithromycin group and a negative control group. Those in the first two groups were injected with Streptococcus pneumoniae following a myringotomy, and tympanosclerosis was experimentally induced. Oral clarithromycin therapy was administered in the clarithromycin group. The other groups received no medical treatment. RESULTS: All eardrums in the clarithromycin and non-clarithromycin groups developed myringosclerosis, but there was only one eardrum, in the clarithromycin group, with very severe myringosclerosis. In the clarithromycin group, 11 ears showed no inflammation and there were no ears with severe inflammation. In the non-clarithromycin group, there were 11 ears with severe inflammation. The mean eardrum thickness in the clarithromycin group was 20.93 µm and in the non-clarithromycin group it was 42.71 µm. CONCLUSION: Acute otitis media and myringotomies induced tympanosclerosis, but clarithromycin reduced the severity of tympanosclerosis.

The effect of lycopene on experimental myringosclerosis.

OBJECTIVES: The aim of this study was to investigate the effect of lycopene on myringosclerosis development using histopathological and immunohistochemical analyses. METHODS: Fifty-six intact tympanic membranes of 28 guinea pigs were included in the study. Subjects were randomly divided into four groups (n=7/group). Group I (control group) did not receive any treatment after myringotomy. Group II (lycopene treatment after myringotomy) received oral lycopene (once daily at the same time, 10mg/kg, dissolved in water, administered with a catheter). The treatment lasted seven days. Group III (lycopene treatment before and after myringotomy), received lycopene treatment (same dose and route of administration) for seven days. Myringotomy was performed on day 8, and lycopene treatment was initiated immediately, and continued for seven days (same dose and route of administration). Group IV (lycopene treatment before myringotomy) received lycopene treatment one week before myringotomy using the same method and dose for seven days. Myringotomy was performed on day 8. Lycopene was not administered after myringotomy. Fourteen days after myringotomy, myringosclerosis was evaluated automicroscopically and scored. Following decapitation, bulla were removed and immersed in a 10% formaldehyde solution. Sections were cut for histopathological and immunohistochemical examination, and thickness, sclerosis, inflammation, and collagen-IV accumulation were scored semi-quantitatively. RESULTS: In the present study, the level of myringosclerosis was significantly lower in lycopene-treated groups compared to the control group (p<0.05). In addition, thickness, inflammation, sclerosis, and collagen-IV accumulation were significantly lower in the lycopene-treated groups compared to the control group (p<0.05). The timing of lycopene administration - i.e. before and/or after surgery - did not cause any difference with respect to myringosclerosis development. CONCLUSION: Lycopene, a strong antioxidant, may represent a good alternative treatment to prevent the development of myringosclerosis.

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

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.

Combining use of captopril and losartan attenuates the progress of Streptococcus pneumoniae-induced tympanosclerosis through the suppression of TGF-ß1 expression.

OBJECTIVES: In this study, using an Streptococcus pneumoniae-induced tympanosclerosis (TS) model, we explored the effects of captopril and losartan in the treatment of TS and the possible mechanisms. STUDY DESIGN: A prospective experimental animal study. METHODS: We set up the TS models in both guinea pig and wistar rat by inoculation of type-3 Streptococcus pneumoniae microorganisms and then treated the animals with the combining use of captopril and losartan. Otomicroscopy was employed to observe the development of TS. Auditory brainstem response was used to test the hearing function of animals. Hematoxylin-eosin and von Kossa staining were performed to determine the morphological changes and calcium depositions. The protein expressions of transforming growth factor ß1 (TGF-ß1) were assessed by western blot and immunohistochemistry staining, and the mRNA level of TGF-ß1 was measured by quantitative reverse transcription- polymerase chain reaction. RESULTS: The combining use of captopril and losartan attenuated TS responses in terms of a decrease in the TS incidence and the ABR threshold, a reduction of hyalinization and calcification in the middle ear mucosa and the thickness of the mucosa. In addition, the TGF-ß1 expression was decreased at both protein and mRNA levels. CONCLUSION: Our data indicate, for the first time, that the combining use of captopril and losartan obviously attenuates TS progress through inhibiting the overexpressing of TGF-ß1.