DISCUSSION

OME is a major public health problem in paediatric ENT practice (8) and understanding the mechanism of tympanic complications is fundamental in reducing the sequelae of this disease (5). Proteolytic enzymes can play an important role in the development of TM atrophy associated with OME, and then into cholesteatoma. Many authors refer to the role of proteases in the pathogenesis of OME complications (9–11,15,20). Our study suggests a non-significant link between the level of MMP-2 in OME fluid and the existence of tympanic atrophy. A higher concentration of MMP-2 in the effusions of patients with atrophic tympanic membranes has been found. Its specific inhibitor TIMP-2, assessed in order to estimate the active MMP-2 level without the inactive pro-enzymes (14), was also lower in the effusions of patients with atrophic tympanic membranes. The MMP-9 level tended to be lower in children with atrophy, while the MMP-7 rate was quite similar in both groups. So, the MMP-2 enzyme activity tended to be higher in cases of tympanic atrophy, even if the difference was not significant.
The ELISA technique was used for the assays. Gelatin or casein zymography is an electrophoresis examination frequently performed in other studies to measure MMP activity, because it dissociates MMPs from TIMPs. However, ELISA, which is also commonly used for this type of assay, is a validated technique. The MMP concentrations measured in this study in ng/µl were consistent with MMP levels found in previous publications, except for the MMP-9 concentrations which were higher than in other studies. A South Korean study (21) studying MMP-9 levels in OME with and without allergy found also high concentrations ranging from 100 to 920 pg/mg total protein.
Destruction of fibrous layer in OME may be related to the duration of the OME’s evolution (5), but this cannot be demonstrated in the general population because OME is often asymptomatic. In the literature, the duration of OME is often estimated from the number of VT insertions, and the age of the patients. In our study, MMP2 and TIMP-2 concentrations tended to decrease with age (figure 2), as reported in a 1994 American study (16).However, the ration MMP-2/TIMP-2 did not vary among age.
In our study, the number of previous VT was not correlated with the MMP level, but the MMP-7 rate which was significantly higher in children who had no prior history of VT placement. The study by Jennings and al. (20) found an increase in MMP-2 levels with the number of VT operations, while a Swedish study by Juhn and al. (16) found no relationship between the number of VT insertions and MMP-2 and 9 concentrations.
In our study, a significantly higher concentration of MMP was found in the thicker mucous effusions, the difference being significative for MMP-9 and TIMP-2 (p=0.0017 – p=0,013 respectively). This result has been demonstrated in other studies (9,10,15,20). Interleukins such as TNFα and IL-1 are present in higher concentrations in mucous effusions (16). The role of these molecules in inducing the transcription of MMPs is consistent with the results of our research. On the other hand, our results for TIMP-2 concentrations are at odds with the results from Moon’s work, since we found a significant link between elevated TIMP-2 concentrations and mucous effusions (9).
One of the limitations of our study is assessment of the degree of tympanic atrophy by examination under a microscope, even with an examination under microscope performed by two different examiners. The gold standard would be to perform biopsies of the eardrum (4). However, this could lead to perforations and is questionable in children. In further studies it could be possible to analyse an animal model to quantify the relationship between the degree of eardrum atrophy and MMP concentrations in OME effusion.
The second limitation was the number of children enrolled: inclusion of more subjects would increase the strength of our study. In addition, it would be interesting to compare intra-individual MMP-2 levels in children with unilateral tympanic atrophy, the child being his own control.