Abstract
Purpose::
To evaluate NGF levels in aqueous samples of patients with primary open angle glaucoma.
Methods::
Nerve growth factor (NGF) plays a vital role in a number of pathological conditions and the presence of growth factor receptors on human trabecular meshwork make it interesting to investigate changes in NGF levels in aqueous samples.Aqueous samples were collected from 32 consecutive patients undergoing surgery for cataract or POAG. 18 patients had POAG and 14 patients (controls) had cataract. ELISA immunoassay was used to quantify the NGF levels in the aqueous (Promega). 50 micL of undiluted sample was used to run the assay. All samples were run in single, except the standard and control.
Results::
In POAG samples (n=18) the mean age of the subjects was 74.2 yrs and in controls (n=14) the mean age of the subjects was 69 years. Maximum available sample volumes varying from 40 to 50 µl of undiluted samples were assessed. In pilot experiments quantities ranging from 15 to 50 µl and dilutions ranging from 1:1.25-3.3 were used but NGF levels were found to be undetectable. With undiluted samples, the typical average levels of NGF were 0.91pg/ml (standard error 0.52) in aqueous samples of patients with POAG and 0.82pg/ml (standard error 0.47) in aqueous samples of controls. Levels were considered undetectable as these were below the detection sensitivity of the kit (7.8pg/ml). In each experiment external recombinant NGF controls were kept and their values were detected as predicted. However, in both glaucoma and control groups approximately 5.5% and 7.1% of samples had NGF values as high as 75.3pg/ml and 17.9pg/ml respectively. These samples were considered outliers because (1) these values were rare and highly inconsistent with others in the group (2) they were found with equal frequency in control and glaucomatous eyes. Histories of outliers were reviewed to identify any apparent cause but none was identified.
Conclusions::
NGF levels were below undetectable levels in aqueous humor from eyes with both POAG and controls by immunoassay. Our results highlight the difficulties in assessing NGF levels in aqueous. It may be necessary to pool the samples from different patients (but from same group) in order to increase the sample volume and therefore the delectability. However, results also show that outliers may skew the results obtained by pooling the samples. The outliers may be a result of variables at the time of collection of sample (e.g. per operative trauma, bleeding, iris handling etc). Considering these limitations, other technologies such as multiplex assays might be more useful.
Keywords: growth factors/growth factor receptors • aqueous • outflow: trabecular meshwork