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S. Balaiya Mehanathan, R. Malyapa, K. V. Chalam; Proton Beam Radiation Selectively Destroys Proliferating Chorodial Endothelial Cells and Preserves Non-Proliferating Retinal Pigment Epithelial Cells: A Model for Choroidal Neovascular Membrane. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2775.
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© ARVO (1962-2015); The Authors (2016-present)
Subfoveal choroidal neovascular membrane is a leading cause of severe visual loss in the elder population affected with age-related macular degeneration. Conventional radiation suppresses neovascularisation but damage the overlying retina in the path of radiation. Proton beam radiation has three dimensional focal delivery of dosage with minimal entry and exit dose. The purpose of this study is to identify the differential tissue sensitivity of non-proliferating retinal pigment epithelial cells and proliferating choroidal endothelial cells to proton radiation.
Serum starved confluent retinal pigment epithelial cells (ARPE-19) and the proliferating choroidal endothelial cells (RF/6A) were irradiated with protons in a single fraction. The dosages ranged from 4CGE (Cobalt grey equivalent) to 8CGE (4, 5, 6, 7 and 8) with a presence of an internal control. Post radiation viability of serum starved confluent ARPE-19 and proliferating RF6A were analyzed on post incubation day interval of day 5, day 9 and day 12 using neutral red (NR) uptake assay. Cell numbers were quantified by trypan blue dye exclusion assay using Vi-cell XR on day 5, day 9 and day 12.
On day 5, using NR assay, RF/6A showed 85.8%, 80.6%, 73.4%, 74.2% and 49.6% cell viability at 4,5,6,7, and 8CGE, respectively. Similar decrease in cell numbers was noted using Vi-cell XR studies on day 5. Further decline of cell numbers was observed on day 9 to 53.9%, 36.6%, 47.9%, 43.3% and 19.3%. On day 12, the reduction of cell numbers were maintained at all dose ranges studied (52.7%, 45.4%, 51.8%, 52.7% and 36.3% (p<0.001).In ARPE-19, on day 5, NR assay revealed 92.6%, 90.3%, 86.8%, 78.5% and 68.7% of cell viability at 4, 5,6,7 and 8CGE, respectively. By day 9, there was a recovery in cell viability in 4, 5 and 6CGE (102%, 99.1% and 98.6%), with a slight decrease on day 12 (93.6%, 81.3% and 79%). Cells exposed to 7CGE and 8CGE radiation dosage showed 94.7% and 91.1% cell viability on day 9 with further decline to 79.8% and 75% by day 12 (p<0.01). Vi-cell XR showed a dose dependent decline of cell numbers by day 5 at dosage of 7CGE (60.3%) and 8CGE (61.8%) which further increased by day 9 (101%, 91%) and 12 (94.6%) in both doses (p<0.001).
Proton beam radiation at doses 7 and 8 CGE significantly decreases cell viability of proliferating choroidal endothelial cells compared to non-proliferating retinal pigment epithelial cells.
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