Abstract
Purpose: :
To investigate the dose-response relationship for laser (Ellex 2RT) induced changes in the MMP release profiles of human RPE cells and their effects on the diffusional status of underlying Bruch’s membrane.
Methods: :
Human RPE-Bruch’s-Choroid (RPE-BCM) explants were maintained under organ culture conditions for 3 days and were then irradiated with the Ellex 2RT nanosecond laser system, delivering energies of 0.3mJ and 0.6 mJ/pulse (total energy 120mJ/cm2) and returned to the incubator for a further 14 days. Following laser treatment, media samples were obtained every 24 hours for quantification of MMPs 2&9 by standard gelatine zymography and densitometric analyses. Permeability studies of the BCM preparation were performed in Ussing chambers by quantifying the rate of diffusion of a dye across the preparation using standard colorimetric instrumentation. Having determined basal diffusion levels, primary RPE cells were plated on BCM. At various time intervals, RPE were brushed off and diffusion rates measured.
Results: :
Varying laser energy was without effect on levels of Pro-MMP2 but the higher energies considerably elevated levels of Pro-MMP9. Active levels of both MMPs 2&9 showed a time dependent change with increasing levels peaking around post-laser day 7 with a subsequent decline that had not reached baseline by day 14. At their peak, active MMP 2&9 levels were 6.7 ± 2.6 and 4.37 ± 1.1 (Mean± SEM) fold higher than control. Transport studies performed after the seeded RPE cells had achieved confluence demonstrated much improved diffusional status of Bruch’s membrane.
Conclusions: :
The Ellex 2RT laser system provides a facility for enhancing the synthesis and release of active forms of MMPs 2&9 from RPE cells. Entry of such active species into Bruch’s allows for improvement in the transport characteristics of the membrane.
Keywords: retinal pigment epithelium • Bruch's membrane • laser