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Ling Zhi Heng, Ali Hussain, Sobha Sivaprasad, Robin Hamilton, John Marshall; Vectorial release of matrix metalloproteinase enzymes from porcine RPE in explant culture following nanosecond laser exposure. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1382.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously shown the release of active matrix metalloproteinase(MMP) enzymes from RPE cells in response to nanosecond pulse laser irradiation (Q-switched, frequency doubled YAG, 532nm). The present work has examined the vectorial nature, time-course, and dose-response characteristics of the MMP response to the laser.
Explant cultures of porcine RPE were maintained in modified using chambers. After an equilibration period of 48 hours, explants were irradiated with 0.10 and 0.15mJ per exposure incorporating 25 and 50, 400um spots. Incubation was continued for a further 7 days with apical and basal medium being replaced every 24 hours. The collected medium samples were subjected to gelatin zymography for quantification of released MMPs.
Pro-MMP9 levels declined continuously in culture and were not affected by the laser regime. Active MMP 9 was not detectable in these studies. In control explants, levels of pro and active-MMP2 showed a transient increase around days 3-4, returning to baseline within 2 days. Laser exposures of 0.1 and 0.15mJ increased pro-MMP 2 within 24 hours, peaking at 4-5 days and returning to baseline by day 10 of incubation. Peak levels of pro-MMP 2 for 0.1 and 0.15mJ exposures were elevated 1.6±0.1 and 2.9±0.75 fold (Mean±SD) compared to basal, (both p<0.05). Levels ofa ctive MMP2 showed similar changes to pro-MMP2 but recovery to baseline did not occur by day 10 for the higher 0.15mJ exposure. Peak levels for active MMP2 for the 0.15mJ exposure were 3-fold higher compared to the 0.1mJ level(p<0.05).
The nanosecond laser system evoked the release of pro and active MMP2 from botha pical and basal surfaces of porcine RPE in a dose dependent manner without effect on the level of MMP9 species. Response was discernable within 24 hours, peaking at around 3-4days post laser, and returning to baseline by about 10 days. Further work is required to assess beneficial effects(if any) of this laser-mediated enzyme release on the structural and functional aspects of underlying Bruch's membrane. These results are essential for designing treatment protocols for future clinical studies.
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