Sub-Threshold Q-Switched Laser (Ellex 2RT) - Induced Migration of Human RPE and Concomitant Release of Matrix Metalloproteinases (MMPs)

Abstract

Purpose: : To investigate cellular and biochemical responses (wound healing and release patterns of MMPs) of human retinal pigment epithelium (RPE) associated with Q-switched laser exposure at energy levels below visual threshold.

Methods: : Human RPE-Bruch’s-Choroid explants (12-72 hours post-mortem) were maintained under organ culture conditions for 3-7 days and baseline MMP release patterns established. The explants were then irradiated with an Ellex 2RT nanosecond laser system (Q-switched YAG laser (532nm) delivering 3 nanosecond pulses; 400um spot size, 0.078mJ energy setting) and returned to the incubator for a further 14 days. At selected intervals, confocal laser scanning, conventional microscopy and a cell proliferation assay (BrdU) assessed RPE cellular dynamics. Following the laser treatment, media samples were obtained every 24 hours for quantification of MMPs 2 & 9 by standard gelatine zymography and densitometric analyses.

Results: : Following 24hour laser intervention, nearly 50% of the RPE cells within the treatment zone showed signs of injury. Most of the damaged RPE was characterised by some degree of cell membrane disruption whilst others showed disintegration of structure. By post-treatment days 5-7, most of the injured cells were replaced by migrating RPE cells from regions surrounding the lesion. Differential effects on the components of the MMP system accompanied laser treatment. Thus, released levels of inactive MMP-2 (pro-enzyme) were little altered over the two-week experimental period. However, inactive MMP-9 (pro-enzyme) increased 1.34-fold by day 1 to reach a 2.8-fold level by day 7 (n=5). Changes in the active forms of MMPs 2&9 were much more profound with levels increasing 6- and 5-fold above controls respectively (n=6). These high levels peaked around 5-7 days post-laser returning to baseline by about day 14.

Conclusions: : The 2RT laser treatment procedure resulted in mild RPE injury with the subsequent wound healing process leading to the release of active matrix metalloproteinase enzymes,This transitory elevation of active MMPs may be beneficial in addressing the pathological alterations of Bruch’s membrane and thus potentially useful for intervention in AMD before significant loss of vision has occurred.

Keywords: age-related macular degeneration • retinal pigment epithelium • laser