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Elisabeth Richert, Alexa Karina Klettner, Stefan Otto Johannes Koinzer, Johann Roider, Jost Hillenkamp; Release of different cell mediators during retinal pigment epithelium (RPE) wound healing after Selective Retina Therapy (SRT) for prevention of early age-related macular degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):198.
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SRT is a micropulse laser technique which stimulates the RPE-regeneration but avoids thermal damage of the retina. SRT has been shown to effectively reduce soft drusen. The aim of this study was to investigate the effect of SRT on the release of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) during RPE wound healing using different laser spot sizes.
Porcine RPE-choroid explants were treated with a pulsed 532 nm Nd:YLF laser (duration 250 ns, repetition rate 100 Hz, spot sizes 100/200 µm, energies 100-200 mJ/cm²). Threshold titration of cell death and wound healing were investigated by calcein staining. Explants were cultivated in modified Ussing chambers which allowed the differentiation of basal and apical compartments. Release of MMPs was analyzed by zymography. Expression of MMP-2 was observed by immunofluorescence applied to RPE-choroid flat mounts. Secretion of VEGF and PEDF were analyzed by ELISA. Statistical analyses were conducted with Student's t-test.
Laser energies of 140 mJ/cm² for 200 µm and 200 mJ/cm² for 100 µm spot size resulted in 100% cell death after 24 hours. Four days after laser exposure irregularly shaped cells with prominent nuclei covered the RPE wound. For 200 µm spots we observed a significant increase of basal release of pro-MMP-2 after 3 days to 1,15 fold of control (SD±0,3, p<0,05) and a significant increase of active MMP-2 after 4 days to 1,74 (SD±0,85, p<0,05). These effects were not observed at 100 µm spots. In immunofluorescence irradiated explants showed a localized expression of MMP-2 within the healing lesions. The content of PEDF increased significantly to 125% (SD±32,2, p<0,05) while VEGF decreased to 88% (SD±19,2, p<0,05) in treated explants in comparison to controls.
Selective laser therapy has been proposed as a treatment of degenerative changes in early AMD to improve flux across Bruch's membrane. SRT triggers the basaolateral release of MMPs during RPE wound healing which could slow the macular ageing process while VEGF expression was decreased and antagonistic PEDF expression was increased. The results suggest that SRT does not stimulate angiogenesis. SRT laser spots need to be of a minimal size (200 µm spots) to trigger the desired protein release.
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