Abstract
Purpose: :
We have shown that optic nerve head (ONH) cells like trabecular meshwork (TM) cells have a higher incidence of Cross-linked Actin Networks (CLANs) when exposed to dexamethasone (DEX). CLAN induction in TM cells is more pronounced with transforming growth factor beta (TGF-β2) than with DEX. This study investigated whether there is also TGF-β2 CLAN induction in primary ONH cells from normal and glaucomatous donors.
Methods: :
A range of 11 primary cultures of human ONH cells from donors aged 26-91, were grown to confluence and treated with media containing 1% foetal calf serum (FCS) or media containing 1% FCS with 4ng/ml TGF-β2, (concentration based on dose response curves). The cells were fixed at 7 days in 10% neutral buffered formalin, stained with Alexa-fluor 488 phalloidin and propidium iodide. Masked immunofluorescent counts were carried out in the standardised manner used in our laboratory and imaged using a conventional fluorescence microscope and confocal microscopy.
Results: :
In 1% FCS control conditions the percentage of ONH cells containing CLANs after 7 days was extremely variable and ranged from 2-50%, compared to 0-12% in TM cells. TGF-β2 was shown to be a potent inducer of CLANs in ONH cells and in every primary culture there was some level of induction above baseline levels. The response was hugely varied ranging from a 50% induction above FCS levels to a 30 and a 40 fold induction in the ONH cells of 2 donors. CLAN incidence in one glaucoma donor reached 80% of the cells by the combined effects of TGF-β2 and 1% FCS but if the FCS effect was subtracted then the TGF-β2 effect was far more modest and averaged 17% (range 33.1 to 2.3%) and compared favourably with the TGF-β2 response TM cells in the same conditions (average was 15.1%).There was no correlation between donor age and CLAN frequency either in 1% FCS alone or FCS plus TGF-β2. Whereas TGF-β2 CLAN induction was far more pronounced in TM cells from elderly donors.
Conclusions: :
TGF-β2 was shown to be an effective inducer of CLANs in ONH cells as it was for TM cells. There was variability in response that did not relate to age of donor or to the donor having glaucoma. We were interested to find that our 1% FCS maintenance medium contained as yet unknown CLAN inducers which we wish to identify in future studies.
Keywords: cytoskeleton • optic nerve • trabecular meshwork