Abstract
Purpose: :
To investigate wound healing and biochemical responses of porcine RPE after SRT for irradiations below and above ED50 threshold radiant exposure for RPE cell death.
Methods: :
Fresh porcine RPE-Bruch-Choroid explants maintained in a culture medium were irradiated with a pulsed Nd:YLF laser (wave length 527 nm, pulse duration 1.7 µs, repetition rate 100 Hz, spot size 200µm) with energies ranged from 100 to 200 mJ/cm2 (n=4-6 for each energy) and cultured in a static culture medium for 4 days. Threshold radiant exposure for cell damage was determined by Calcein-AM cell viability test. RPE wound healing was assessed by actin staining with FITC-phalloidin. Following laser irradiation, media samples were obtained every 24 hours for quantification of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay (ELISA).
Results: :
The threshold radiant exposure for cell viability was determined to be between 150 and 160 mJ/cm2. Complete wound closure occurred within 4 days after laser irradiation. Twenty four hours after laser irradiation with energies ≥ 140 mJ/cm2, levels of MMP-2 increased 1.7 to 2.2-fold compared to the control non-treated samples. After 3-4 days, the level of MMP-2 had decreased to the level of the control. No significant increase of the VEGF level was detected.
Conclusions: :
MMP-2 most likely contributes strongly to the RPE healing response after SRT. Even close below the threshold radiant exposure, significant levels of MMP-2 are released, while levels of VEGF seem to remain unchanged.
Keywords: retinal pigment epithelium • laser • retinal culture