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Tympanometric changes in an experimental myringosclerosis model after myringotomy.

HYPOTHESIS: The goal of this experimental study was to investigate the specific effect of myringosclerosis on tympanograms in the tympanic membranes of myringotomized rats by using otomicroscopy, tympanometry, and histopathology. BACKGROUND: Myringosclerosis is a common sequela of ventilation tube treatment of otitis media with effusion. The condition involves the hyalinization and calcification of the collagen layer in certain areas of the tympanic membrane. Previous animal experiments suggest an intimate relationship between the formation of myringosclerosis and an increased oxygen concentration in the environment of the wound after myringotomy. The result of a myringotomy therefore is an increased production of free oxygen radicals, initiating irreversible tissue damage involving fibrosis, hyalin degeneration, and finally apoptosis as observed in myringosclerosis. We propose an experimental model specific for creating sclerotic plaques solely on the tympanic membrane and for performing tympanometric measurements on this pure myringosclerosis model without creating any abnormality in the middle ear to test in what proportion myringosclerosis contributes to decrease of amplitude in tympanograms. METHODS: To assess the normal tympanometric values of Wistar albino rats, the pressure and peak admittance of the left middle ears were measured using a semiquantitative computerized clinical admittance meter using a sound frequency of 226 Hz. Twelve animals were randomly selected for the myringotomy group and perforations in the left ears were created. All tympanic membrane perforations in this group had healed and closed prior to the otomicroscopic examination and no pathologic reaction was observed in the external ear canals of rats. Otomicroscopic and tympanometric measurements were carried out on Day 15 and the degree of myringosclerosis was noted before the animals were killed. Twelve specimens in the myringotomy group were histopathologically examined for the presence of myringosclerotic plaques. RESULTS: Under light microscopy, extensive sclerotic lesions were found in the tympanic membranes of the myringotomy group, and these sclerotic deposits were located in the lamina propria. The myringosclerosis occurred predominantly adjacent to the handle of the malleus, but also near the annular region. In all ears with myringosclerosis, the magnitude of the maximum admittance reduced to approximately 50% of the Day-0 values, and this reduction was statistically significant (Z=-3.061, p=0.002). CONCLUSION: The present findings in this study are consistent with the fact that the movement of the tympanic membrane is hampered by lesions of sclerotic material, resulting in a decrease of amplitude in tympanograms (such as Type As) without any effusion or inflammation in the middle ear.

Acoustic Impedance Tests↗

The effect of Vitamin E treatment on the development of myringosclerosis after ventilation tube insertion.

OBJECTIVES: Recent studies have established the relationship between the reactive oxygen species and myringosclerosis. Furthermore several antioxidants have been known to prevent myringosclerosis. All the previous studies supporting this hypothesis have been performed on animals. The aim of our study is to investigate the possible effect of Vitamin E on the development of tympanosclerosis after VT insertion on human subjects. METHODS: 72 children undergoing myringotomy and VT insertion were included in the study. Vitamin E was applied to the right ear and no treatment was applied to the left ear. Both ears were examined at the end of 9 months with otomicroscopy. Myringosclerosis formation at the end of the study period was noted for each ear. RESULTS: Myringosclerosis was found in 33 of the 144 ears. The overall incidence was 22.9%. While 22 (30.6%) of the 72 left ears showed myringosclerosis otomicroscopically, in only 11 (15.3%) of the 72 right ears that were treated with Vitamin E was myringosclerosis observed at the end of the study period. Of these nine cases were bilateral. CONCLUSION: Animal studies have well documented the development of myringosclerosis after myringotomy and VT insertion and beneficial effects of different antioxidants. Our study has shown similar results in human subjects. Further clinical studies consisting of a larger patient population are needed to bring about routine clinical use of antioxidants in myringotomy and VT insertion.

Antioxidants↗

The inhibitory effect of topical N-acetylcysteine application on myringosclerosis in perforated rat tympanic membrane.

OBJECTIVE: Myringosclerosis often occurs in patients in whom ventilation tube insertion and tympanoplasty procedures are performed. Recent studies have revealed a relationship between the development of myringosclerosis and oxygen-derived free radicals, and some investigations have demonstrated that free radical scavengers prevent the development of myringosclerosis. N-acetylcysteine is a well-known anti-oxidant and anti-inflammatory agent. In this study, we aimed to investigate the preventive effect of N-acetylcysteine on myringosclerosis in myringotomized rat tympanic membranes. METHODS: Twenty Sprague-Dawley rats were bilaterally myringotomized and divided into four groups. Group 1 received no treatment, group 2 was treated with topical saline solution in Spongostan, group 3 received topical 0.6 mg N-acetylcysteine in Spongostan and group 4 received 1.2 mg N-acetylcysteine in Spongostan daily for 12 days. Tympanic membranes were examined by otomicroscopy on day 12. Then, the membranes were harvested and evaluated histologically by light microscopy. RESULTS: The tympanic membranes of groups 1 and 2 (saline and non-treated) showed extensive occurrence of myringosclerosis, whereas groups 3 and 4 (treated with N-acetylcysteine) showed lesser occurrence of myringosclerosis in otomicroscopic evaluation (P<0.01). Under light microscopic examination, lamina propria of pars tensa was found thicker in groups 3 and 4 when compared with groups 1 and 2. There was no significant difference between groups 3 and 4 (P: 0.30). CONCLUSIONS: Topically applied N-acetylcysteine was found to be effective in the prevention of sclerotic lesions in myringotomized rat tympanic membranes.

Acetylcysteine↗

The anti-oxidant effect of alpha-tocopherol in the prevention of experimentally induced myringosclerosis.

HYPOTHESIS: The objective of this study was to investigate the possible effect of alpha-tocopherol on the prevention of experimentally induced myringosclerosis. BACKGROUND: Myringosclerosis is a common sequela of ventilation tube treatment of otitis media with effusion. The relationship between oxygen-derived free radicals and occurrence of myringosclerosis has been proven in experimental models, and it was also shown that the formation of myringosclerosis after experimental myringotomy could be reduced by application of various free radical scavengers. METHODS: Eighteen Wistar albino rats were myringotomized on the left side and randomly separated into two groups: group A consisted of rats which received intramuscular alpha-tocopherol injections 100 mg/kg daily and group B which were injected with physiological serum only. The occurrence of myringosclerotic plaques in the tympanic membranes of the two groups was compared by otomicroscopy, histopathology, and tympanometry, which is a novel method of quantification. Blood samples were collected for biochemical evaluation, and the tympanic membranes were harvested on the 15th day of the experiment. RESULTS: In otomicroscopic evaluation, tympanic membranes in group B revealed varying degrees of myringosclerotic plaques; on the other hand, tympanic membranes in group A showed faint or no existence of myringosclerosis. The mean malondialdehyde levels were 1.33 +/- 0.11 micromol/L in group A and 7.49 +/- 1.37 micromol/L in group B (Z = -1.906, p = 0.057). In all ears from group B, the magnitude of the maximum admittance measured by tympanometry reduced to approximately 40% of the values obtained from group A (Z = -2,160, p = 0.031). The mean magnitude of the maximum admittance from group A was very close to the standardization values of Wistar albino rats, which predicts a functional outcome. CONCLUSION: The formation of myringosclerosis after experimental myringotomy can be diminished by intramuscular alpha-tocopherol injections.

Acoustic Impedance Tests↗

Otomicroscopic and histologic findings of induced myringosclerosis in rats: a critical study of an experimental model.

UNLABELLED: Myringosclerosis is characterized by hyaline changes of the lamina propria of the tympanic membrane. Experimental studies have used otomicroscopy or histology to evaluate myringosclerosis in animals, but they do not correlate precisely these two methods. AIM: The present study evaluates the accuracy of otomicroscopy in the diagnosis of myringosclerosis in rats. STUDY DESIGN: Experimental. MATERIAL AND METHOD: Myringosclerosis was induced by transtympanic inoculation of Streptococcus pneumoniae in 25 Wistar rats, which were examined weekly through otomicroscopy and sacrificed after eight weeks for histologic study of their tympanic membranes. RESULTS: From the comparison of the otomicroscopic data in relation to the histologic findings, we could observe sensibility of 80% and specificity of 75% for the otomicroscopy. CONCLUSION: Considering the results in this study, otomicroscopy did not represent a good method to evaluate myringosclerosis in this experimental model.

Animals↗

Development of myringosclerosis during acute otitis media caused by Streptococcus pneumoniae and non-typeable Haemophilus influenzae: a clinical otomicroscopical study using the rat model.

OBJECTIVE: The present study was performed in order to study development of myringosclerosis during acute otitis media caused by different bacteria in myringotomized and non-myringotomized ears. MATERIAL AND METHODS: A rat model of acute otitis media caused by Streptococcus pneumoniae type 3 and non-typeable Haemophilus influenzae was used. A sample consisted of 42 animals. Four days following middle ear inoculation, a myringotomy was performed in 10 animals from the Streptococcus pneumoniae group and 6 from the non-typeable Haemophilus influenzae group. Another group of 24 animals was inoculated only. On day 4, 7, 14 and 28 after inoculation the status of the drum was inspected under the otomicroscope for vascular reaction, effusion, perforation, myringosclerosis and scarring. RESULTS: On day 4 after inoculation all infected ears had typical signs of acute otitis media. Tympanic membranes healed with scar formation in most cases of myringotomized Streptococcus pneumoniae type 3 infection and deposition of sclerotic plaques was observed by day 14. Otomicroscopically visible myringosclerosis was not found after non-typeable Haemophilus influenzae induced acute otitis media neither in myringotomized nor in non-myringotomized animals. We conclude that Streptococcus pneumoniae type 3 provokes a severe clinical course of acute otitis media that healed with scarring and myringosclerosis formation in the tympanic membrane. Clinically visible myringosclerosis develops after middle ear infection caused by Streptococcus pneumoniae type 3, but not in cases caused by non-typeable Haemophilus influenzae.

Acute Disease↗

The effect of topical adrenaline on the development of myringosclerosis after tympanostomy tube insertion.

OBJECTIVE: To determine the effect of topical adrenaline application after myringotomy and before tympanostomy tube placement on the development of myringosclerosis. STUDY DESIGN: A prospective, randomized, double-blind study, with each patient acting as his or her own control. Ethical approval and full parental consent were obtained. SETTING: Department of Otorhinolaryngology-Head and Neck Surgery in a university teaching hospital. PATIENTS: Fifty children satisfying inclusion and exclusion criteria for first-time tympanostomy tube insertion. THERAPEUTIC INTERVENTION: Myringotomy followed by adrenaline application to incision before tympanostomy tube insertion. Control contralateral ear received saline application after myringotomy. Follow-up examination was done 14 to 21 days after surgery and again after 1 year by a single blinded surgeon. MAIN OUTCOME MEASURE: Comparison of myringosclerosis between adrenaline-treated ears and matched control ears. RESULTS: No difference was found in early morbidity between the two groups of ears. Myringosclerosis after 1 year was not found to have been significantly affected by adrenaline application (p = 0.2) CONCLUSION: The use of adrenaline on the myringotomy site before tympanostomy tube placement was not found to influence early postoperative morbidity or the later development of myringosclerosis.

Administration, Topical↗

Treatment with dexamethasone arrests the development of myringosclerosis after myringotomy.

HYPOTHESIS: To attempt to inhibit the development of myringosclerosis by intraperitoneal injection of dexamethasone. BACKGROUND: The authors' earlier report showed that the development of myringosclerosis after myringotomy was associated with an inflammatory reaction. The present study was performed to secure evidence for this hypothesis. METHODS: Three groups of bilaterally myringotomized rats were treated at 12-hour intervals with intraperitoneal injection of dexamethasone, RU486 (a glucocorticoid receptor antagonist), and saline, respectively. At 6, 12, 24, and 48 hours after the myringotomy, 2 animals were anesthetized on each occasion and examined otomicroscopically. The animals were then killed, and the tympanic membranes were excised and prepared for light microscopic studies. RESULTS: Dexamethasone treatment retarded and diminished the development of sclerotic lesions markedly. Moreover, no inflammatory signs were seen in the flaccida specimens. When the RU486-treated animals were compared with the animals in the control group, there were no evident differences concerning the development of myringosclerosis or the extent of the inflammatory reaction. CONCLUSION: These findings confirm the earlier hypothesis that an inflammatory reaction in collagen tissue is involved in the mechanism that causes the development of myringosclerosis.

Animals↗

The effect of L-carnitine on the prevention of experimentally induced myringosclerosis in rats.

The objective of this study was to investigate the possible effect of L-carnitine on the prevention of experimentally induced myringosclerosis. Twenty Sprague-Dawley rats were bilaterally myringotomized. The rats were divided into two groups randomly: group 1 which were intraperitoneally administered saline and group 2 which were intraperitoneally administered L-carnitine. Blood samples were collected for biochemical evaluation and the tympanic membranes were harvested after 28 days. Histopathological and immunohistochemical evaluation were done under light microscopy. The mean malondialdehyde levels were 3.9+/-0.9 in group 2, and 7.9+/-1.1 in group 1 (P<0.001), nitric oxide levels were 25.6+/-6.4 in group 2 and 30.8+/-8.2 in group 1 (P=0.14) and acetylcholinesterase was 1035+/-60 in group 2 and 678+/-35 in group 1 (P=0.001). Myringosclerosis was more frequent and severe in group 1 than group 2 (P<0.007). Immunoreactivity was seen in 16 of 20 tympanic membranes in group 2 and six of 20 tympanic membranes in group 1 (P=0.005). We conclude that L-carnitine diminishes the occurrence of myringosclerosis in rats after myringotomy possibly by antioxidant activity and decreasing the formation of reactive oxygen species.

Animals↗

Myringotomized mice develop myringosclerosis in the pars flaccida and not in the pars tensa.

The development of myringosclerosis has been correlated with increased production of oxygen-derived free radicals. For the present study, we used a null mutant mouse lacking extracellular superoxide dismutase to test the hypothesis that increased production of free radicals can cause the development of myringosclerosis. Null mutant mice and wild-type, control mice were myringotomized and kept in ambient air for 3 weeks. Both groups developed myringosclerosis in the pars flaccida, but not in the pars tensa. The sclerotic lesions were visible in both the light and the electron microscope but not in the otomicroscope. In particular, the localization of the sclerotic deposits was found beneath both the inner and outer epidermal epithelium. No difference concerning the extent or number of sclerotic lesions between the null mutant and the wild-type mice could be distinguished.

Animals↗

Myringosclerosis develops within 9h of myringotomy.

The aim of the present experimental study was to elucidate the temporal development of myringosclerosis. Twenty-four Sprague-Dawley rats were myringotomized bilaterally. At 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, 72 and 84 h after the myringotomy, 2 animals at each time were examined otomicroscopically and thereafter sacrificed. The pars flaccida and pars tensa were excised and prepared for light- and electron-microscopic studies. Otomicroscopically, myringosclerosis was visible in the pars tensa 24 h after myringotomy, whereas no sclerotic lesions were noted in the pars flaccida. Histologically, sclerotic lesions were present in the pars tensa and pars flaccida 9 and 12 h, respectively, after myringotomy. The pars flaccida responds promptly with an inflammatory reaction characterized by abundant macrophages. Myringosclerosis develops promptly after myringotomy and its establishment is related to an inflammatory reaction.

Animals↗

Topical ascorbic acid reduces myringosclerosis in perforated tympanic membranes. A study in the rat.

Myringosclerosis, a common finding after myringotomy, has been recently associated with an increased production of oxygen free radicals. Ascorbic acid's proposed actions include collagen synthesis, antioxidation, and free radical scavenging. The effects of topical ascorbic acid on healing tympanic membranes were studied. Particular attention was given to detecting the presence of myringosclerosis. Twelve Sprague-Dawley rats were bilaterally myringotomized. Their ears were randomized into group A, which received topical ascorbic acid in Gelfoam, group B, which received topical saline solution in Gelfoam, and group C, which received no treatment. The tympanic membranes were harvested on day 13, after routine otomicroscopy. Under light microscopy, the connective tissue layer of the untouched side of the pars tensa was distinctly thicker in group A than in group B or group C. At this level, the extent of sclerotic lesions was significantly less in the ascorbic acid-treated group. It is inferred that topical ascorbic acid reduces the occurrence of myringosclerosis following tympanic membrane perforations in the rat.

Administration, Topical↗

Application of oxygen free radical scavengers to diminish the occurrence of myringosclerosis.

The present study was designed to establish whether or not an increased production of oxygen-derived free radicals is involved in the causation of myringosclerosis. Sclerotic lesions in the tympanic membrane were experimentally elicited by keeping rats with perforated tympanic membranes in an atmosphere containing roughly 40% oxygen. The animals were treated daily with a solution containing either copper zinc-superoxide dismutase plus catalase, deferoxamine, or copper sulfate plus iron chloride, applied to the traumatized area. After 1 week the extension of myringosclerotic plaques was determined otomicroscopically. The pars tensa and pars flaccida were then dissected free and prepared for light microscopic studies. The results showed that treatment with copper zinc-superoxide dismutase plus catalase and deferoxamine inhibited or reduced the development of myringosclerosis, whereas the ears treated with copper sulfate plus iron chloride appeared unaffected. Consequently, the findings support the hypothesis that the formation of oxygen free radicals contributes significantly to the development of myringosclerosis.

Animals↗

Inhibition of the development of myringosclerosis by local administration of fenspiride, an anti-inflammatory drug.

Earlier studies have revealed a relationship between the development of myringosclerosis and oxygen-derived free radicals. The latter can be blocked by the anti-inflammatory drug fenspiride. The present study was undertaken to test the ability of fenspiride to prevent myringosclerosis from developing during healing of the tympanic membrane. Myringotomized rats were treated with either topical applications or intraperitoneal injections of fenspiride for 12 days, after which the tympanic membranes were examined by otomicroscopy and studied histologically by light microscopy. Topically applied fenspiride was found to inhibit the development of sclerotic lesions, whereas intraperitoneal injections were ineffective.

Administration, Topical↗

Myringotomized tympanic membranes cultured in vitro do not develop myringosclerosis.

The aim of this study was to evaluate the development of myringosclerosis in in vitro-cultured tympanic membranes. Sprague-Dawley rats were myringotomized bilaterally and the tympanic membranes were excised after sacrifice. The explants were placed in inserts in wells filled with a nutrient medium. Every second day the tympanic membranes were photodocumented and after 9 days the explants were prepared for histology. On the 4th day the explants had attached to the bottom of the inserts and the specimens had thickened. From the perforation borders and the dissection edges a thin outgrowth was extending across the surface. By Day 9 the perforation had clearly diminished in size when examined in a stereomicroscope. In a light microscope the keratin layer was seen to protrude towards the centre of the perforation and, at the borders, epithelial cells were bridging the gap. Neither the pars tensa nor the pars flaccida showed any sclerotic lesions. The pars flaccida had thickened and the basal cells of the outer keratinized epithelium had invaded the connective tissue. Inflammatory cells were sparse in both the pars tensa and pars flaccida. The in vitro-cultured myringotomized tympanic membrane therefore shows a similar healing pattern to that in vivo. However, inflammatory reactions are sparse and there is no development of myringosclerosis.

Animals↗

The anti-oxidant and anti-apoptotic activities of selenium in the prevention of myringosclerosis in rats.

The possible effect of selenium on the prevention or reduction in occurrence of myringosclerosis was investigated. Fifteen rats were myringotomized bilaterally and separated into two groups. Nine rats were treated with selenium in the study group. Six rats were administered physiological serum and formed the control group. The occurrence of myringosclerotic lesions and anti-apoptotic activity in the tympanic membranes of the two groups were compared otomicroscopically and histopathologically. The sclerosis was occasional in three, moderate in five and severe in three tympanic membranes in the control group. On the other hand sclerosis was observed in only two of 18 specimens in the study group and sclerosis was seen only occasionally in these two sections. Although Bcl-2 staining, which indicates apoptosis, was not statistically different between the groups, it was observed that apoptosis was slightly more apparent in the study group (eight of 18 tympanic membranes versus two of 12 tympanic membranes in the control group). In conclusion, the formation of myringosclerosis following myringtomoy in rats can be reduced by intraperitoneal selenium administration.

Animals↗

The effect of local administration of N-acetylcysteine in perforated rat tympanic membrane: an experimental study in myringosclerosis.

Myringosclerosis (MyS) is a common sequela of acute and chronic otitis media and ventilation tube treatment of serous otitis media. We aimed to study the effect of topical administration of N -acetylcysteine (NAC) on MyS by assessment of otomicroscopic evaluation, lipid peroxidation and nitric oxide (NO) (nitrite/nitrate) levels in experimental myringotomized rat tympanic membrane. Thirty adult rats were used and the upper posterior quadrant of the tympanic membranes of rats was myringotomized. Thereafter, they were divided into four groups. Group I received no treatment, group II was treated with saline, groups III and IV were treated with topical NAC (0.1 ml of 6 and 12 mg ml(-1), respectively). The levels of nitrite/nitrate and malondialdehyde (MDA) were measured in serum samples. In the otomicroscopic evaluation, non-treated and saline treated ears (controls) showed extensive occurrence of myringosclerotic plaques. Groups III and IV showed fewer occurrences of sclerotic plaques. There was no significant difference between groups III and IV regarding the development of MyS. The development of myringosclerotic lesion was found to be significantly different between NAC treated groups (III and IV) and the control groups (I and II). The levels of nitrite/nitrate of both groups III and IV were significantly lower than the control groups. The levels of MDA of these groups were also significantly lower than the control group. The relationship between groups III and IV was not statistically significant for the levels of nitrite/nitrate and MDA. We conclude that the topical treatment of NAC reduces the levels of MDA and NO products in rats. These results suggest that topical NAC application may be useful for the prevention of MyS.

Acetylcysteine↗

The role of experimental myringosclerosis in interpretation of tympanograms and its possible clinical implications.

Myringosclerosis (MS) is hyalinization and calcification of the collagen layer in certain areas of the tympanic membrane (TM) and appears as white chalky patches in otomicroscopy. One of the most common sequela from the use of grommets is the development of MS and its rate in the tubed ear ranges between 44% and 54% during long term follow-up. Among tympanometric configurations, type As tympanograms may indicate ossicular fixation, tympanosclerosis or otitis media with effusion. In case of multiple pathologies (e.g., MS and otitis media with effusion), it is not possible to evaluate the effect of a specific pathology in the absence of other, with otomicroscopy and tympanometry. We believe that the movement of TM is hampered by lesions of sclerotic material, thus resulting in decrease of amplitude in tympanograms without any effusion in middle ear. Now, we propose an experimental model specific for creating MS solely on TM and for performing tympanometric measurements on this pure MS model without creating any pathology in the middle ear, to test in what proportion this specific pathology contributes to decrease of amplitude in tympanograms. A myringotomy in rat's TM admits ambient air into the middle ear cavity, resulting in a relatively hyperoxic condition. The result of a myringotomy therefore is an increased production of free oxygen radicals, initiating irreversible tissue damage involving fibrosis, hyalin degeneration and finally apopitosis as observed in MS. After the closure of perforations, tympanometric measurements can be made on this pure MS model. When evaluating a child for suspected otitis media with effusion, the results of the experimental model might have far-reaching clinical implications and might provide suitable target for prevention of unnecessary myringotomies especially in the pediatric age-group.

Acoustic Impedance Tests↗