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K V Chalam, S Balaiya, R Malyapa; Proton Beam Radiation Selectively Destroys Proliferating Choroidal Endothelial Cells And Preserve Mature Retinal Ganglion Cells And Retinal Pigment Epithelial Cells: Therapeutic Model For AMD. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5564.
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© ARVO (1962-2015); The Authors (2016-present)
Neovascular form of age related macular degeneration is a leading cause of severe visual loss in the elder population above the age of 65. Conventional radiation suppresses neovascularization 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. However the sensitivity of variety of retinal cells to the effects of proton beam radiation has not been defined. The purpose of this study is to identify the differential sensitivity of retinal cells to escalating doses of proton beam radiation.
To mimic in vivo conditions, we chose staurosphorine differentiated retinal ganglion cells (RGC), serum starved confluent retinal pigment epithelial cells (ARPE-19) and the proliferating choroidal endothelial cells (RF/6A) and irradiated them with single fraction escalating doses of proton radiation. Dosages ranged from 4 CGE (Cobalt grey equivalent) to 8 CGE (4, 5, 6, 7 and 8) with an internal control. Post radiation viability was analyzed on day interval of day 5 and day 9 using neutral red (NR) uptake assay. Cell numbers were quantified by trypan blue dye exclusion assay using Vi-cell XR on day 5 and day 9.
On day 5, NR assay showed 99.8%, 92.9%, 83.4%, 88.5% and 95.01% of differentiated RGC viability at 4,5,6,7 and 8 CGE doses respectively, compared with control (p>0.05). On day 9, cells recovered from radiation effects and showed 111.88%, 130.2%, 108.3%, 101.1 and 98.3% at respective dosages of 4, 5, 6, 7 and 8 CGE. Vi cell XR results showed decrease in cell numbers on day 5 from 85.7% at 4 CGE to 58.5%, 45.7%, 46.4% and 64.9% at 5,6,7 and 8 CGE doses. On day 9, cells recovered and 25% over the control cell rate in all the doses.In ARPE-19, on day 5, they revealed 92.6%, 90.3%, 86.8%, 78.5% and 68.7% of cell viability at 4, 5, 6, 7 and 8 CGE, respectively. By day 9, cell viability in 4,5, 6, 7 and 8 CGE (102%, 99.1% and 98.6%, 94.7% and 91.1%; p<0.01). Vi-cell XR showed a dose dependent decline of cell numbers by day 5 only at dosage of 7 CGE (60.3%) and 8 CGE (61.8%) which further increased by day 9 (101%, 91%) in both doses (p<0.001).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 8 CGE, respectively.
Proton beam radiation at doses 7 and 8 CGE significantly decreased cell viability of choroidal endothelial cells while preserving differentiated retinal ganglion cells as well as retinal pigment epithelial cells. 8 CGE is the optimal starting proton radiation dose in exudative age related macular degeneration
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