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.