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John Peter Wood, Glyn Chidlow, Marzieh Tahmasebi, Malcolm Plunkett, Robert James Casson; RESPONSE OF THE RETINAL PIGMENTED EPITHELIUM TO RETINAL REGENERATION THERAPY (2RT) LASER, IN VITRO AND IN VIVO. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5670.
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© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to delineate the effects of the retinal regeneration therapy (2RT) laser to the retinal pigmented eiithelium (RPE), both in vitro and in vivo, concentrating on levels of ablation, and induction of proliferation and potential protective factors from surviving cells.
For in vitro studies, primary cultures of rat RPE cells grown on glass coverslips were subjected to laser irradiation at a range of energy settings from 0.3mJ to 1.8mJ. Some cells were fixed immediately and some returned to culture for varying times up to 7 days before being fixed, for immunocytochemical analysis of normal cell markers (α-tubulin, RPE65, ZO-1), markers of cell stress (nestin, αB-crystallin, heat shock proteins), indicators of proliferation (cyclin D1, PCNA) or growth factor expression (FGF-2, CNTF, VEGF). In other cases cells were subjected to time-lapse videomicroscopy (VDM) for up to a week, to assess their physical reaction. For in vivo studies, anaesthetised adult Dark Agouti rats were exposed to retinal laser irradiation at the same energy range as used for in vitro studies. At different times after treatment RPE whole-mounts were analysed by immunohistochemistry as outlined for the in vitro studies.
RPE cells in vitro and in vivo were ablated in a manner linearly-dependent upon laser energy delivered; at the extremes, 1.8mJ killed 100% of cells within the lased zone and 0.3mJ only killed approximately 25% of cells. Cells surrounding the kill zones produced significant levels of stress proteins (nestin, αB-crystallin, heat shock proteins) and growth factors (FGF-2, CNTF, VEGF) within 6-24 hours of treatment; cellular proliferation markers were also detected, from 24-72 hours post-laser. Effects were dependent upon energy levels used: the greater the energy level, the greater the relative amount of detectable factor produced by surrounding cells. VDM demonstrated cells proliferating back into the ablated zones within 1-3 days post-laser, in vitro.
The findings demonstrate that RPE cells surrounding 2RT laser ablation sites actively respond to injury by producing a range of protein factors which likely have autocrine and paracrine effects. Thus, the 2RT laser not only avoids causing significant collateral damage to photoreceptors, but it can also induce expression of potentially protective factors.
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