Abstract
Purpose: :
Radiation therapy is a possible modality for treatment of choroidal neovacularisation based on the principle that radiation will selectively affect the choroidal vascular endothelial cells, which are undergoing active proliferation with less damage to the surrounding tissue. However, conventional mode of radiation does not have the resolution to deliver the radiation to the eye without damaging the surrounding structures. Proton beam radiation is able to focus the energy in a smaller area with minimal radiation to the surrounding tissue. The purpose of this study is to evaluate the ability of choroidal endothelial cell and retinal pigment epithelial cells to form colonies after exposure to various doses of proton beam radiation.
Methods: :
RF/6A and ARPE-19 cells were grown in culture flasks until they reached log rhythmic scale. Prior to identifing the survival rate, efficiency of each of these cells to replicate and form a colony, within 12-15 days, without any exposure to radiation was identified by plating at low density of 250 cells and counting the number of colonies after 13 days to determine their plating efficiency (PE).The semi-confluent RF/6A and ARPE-19 cells were irradiated using proton beam at the doses of 0,2,4,8 and 12 Cobalt Gray Equivalent (CGE). Following radiation, the cells were detached from the flask and serially diluted to replate them at a sparse density based on PE. The ability of the cells to repair and replicate to form colonies were analysed 13 days after radiation with crystal violet stain. The survival rate was calculated as the ratio of number of colonies observed to that of number of cells plated multiplied by plating efficiency for each radiation dose for both types of cells separately.
Results: :
The PE of RF/6Aand ARPE-19 were of 12.96±0.29% and 8.46±0.24% respectively. The survival ratio of irradiated cells were normalized against the unexposed cells and interpreted in percentage. Survival rate of RF/6A to escalating radiation doses of 2 and 4CGE were of 64.18% and 50.22%; which significant reduction to 10.25% at 8CGE and 1.2% at 12CGE (p<0.001). ARPE-19 cells showed 33.61% and 8.6% of survival at 2 and 4CGE (p<0.001) whereas at 8 and 12CGE they were not able to form colonies.
Conclusions: :
Although the proliferation rates of ARPE-19 cells were affected at 2 and 4 CGE, these low doses may offer possible therapeutic window because in human eyes RPE cells do not proliferate in non-disease setting.
Keywords: radiation therapy • neovascularization • retinal culture