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B. E. Uygun, Q. Zhang, M. Yarmush, M. Latina; Sub-lethal Pulsed Laser Treatment Induces Enhanced Regeneration in Retinal Pigmented Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):679.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the biological response of human fetal retinal pigmented epithelial (RPE) cells to pulsed laser treatment with the purpose of developing a selective and safe laser treatment regimen for improved RPE regeneration.
Human fetal retinal pigmented epithelial (hfRPE) cells were isolated from 19-week old fetal eyes and cultured on laminin coated glass slides for 4 weeks until they become confluent and darkly pigmented. Human fetal RPE cells were treated with a pulsed dye laser (Palomar 3010; 590nm, 1µs, 1mm dia). The cells were irradiated with a single pulse laser at fluences between 50-700 mJ/cm2 and were analyzed using a fluorescent live/dead stain (Invitrogen, Carlsbad, CA) two hours after the treatment. The fluorescent images of stained cells were used to determine the number of live and dead cells within the irradiation field using the METAMORPH software (Molecular Devices, Sunnyvale, CA). The laser energy level that caused 50% cell death within the irradiation area was determined to be the threshold energy level and sub-lethal energy was defined as the energy level where no cell death was observed. The experiments were performed at an energy level that is 70% of the sub-lethal energy to account for inter-pulse variations. The regeneration response of hfRPE cells upon single pulse laser treatment was evaluated in an in vitro scratch wound healing model. The expression of mitogenic growth factors such as platelet derived growth factor (PDGF), fibroblast growth factor (bFGF), and insulin-like growth factor (IGF1 and IGF2) two hours post laser treatment was analyzed via reverse transcriptase polymerase chain reaction (RT-PCR).
Using the 1-µs single pulse dye laser, the threshold energy level was determined to be 496 mJ/cm2 and the sub-lethal energy level was determined to be 285 mJ/cm2 for hfRPE cells. In the scratch type in vitro wound healing model, there was approximately 20-fold increase in proliferation of hfRPE cells following single pulse dye laser irradiation when compared to sham controls. In addition, we have found upregulated expression of bFGF (1.96-fold), IGF1 (4.6-fold), IGF2 (2.2-fold) and PDGF (0.14-fold) following laser treatment as analyzed by RT-PCR and densitometry.
The results indicate that sub-lethal pulsed laser treatment of hfRPE cells stimulate expression of mitogenic factors that lead to enhanced proliferation in an in vitro wound healing model. Focal sub-lethal pulsed laser treatment of the RPE preserves surrounding cells and can be used to improve RPE regeneration in diseases implicated by RPE degeneration.
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