Evaluation of choroidal endothelial cell proliferation after exposure to varying doses of proton beam radiation.
OBJECTIVE
Focal epiretinal radiation has emerged as a promising tool in the management of choroidal neovascularization associated with age-related macular degeneration. However, the dosages tested are not backed by cell culture studies used in the clinical setting empirically.


METHODS
Choroidal endothelial cells (RF6A) were maintained in a log scale and exposed to a single fraction of 2, 4, 8, and 12 cobalt gray-equivalent of proton radiation with an internal control. Cell viability was quantified using Vi-cell XR and neutral red assay at days 5, 9, and 12 after radiation. Mitochondrial viability using WST-1 and reactive oxygen species levels using dihydrorhodamine 123 were measured at similar intervals.


RESULTS
By using neutral red assay, on day 12, the percentages of viable cells compared with control were 100.1 ± 5.7%, 96.7 ± 23.3%, 27.6 ± 6.6%, and 19.5 ± 3% at radiation doses of 2, 4, 8, and 12 cobalt gray-equivalent, respectively (P < 0.001). Increase in reactive oxygen species levels correlated with the number of dead cells implicating reactive oxygen species as an intermediary molecule (r = 0.85-0.96).


CONCLUSIONS
Our study shows sensitivity of cultured choroidal endothelial cells to proton beam radiation at doses of 8 and 12 cobalt gray-equivalent in an in vitro model.
Chalam
Kakarla V
KV
Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USA. kchalam@jax.ufl.edu
Balaiya
Sankarathi
S
Malyappa
Robert S
RS
Hsi
Wen
W
Brar
Vikram S
VS
Murthy
Ravi K
RK
D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects; D002829:Choroid / Q000166:cytology* / Q000528:radiation effects*; D004396:Coloring Agents / Q000493:pharmacokinetics; D004307:Dose-Response Relationship, Radiation; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology / Q000528:radiation effects*; D008928:Mitochondria / Q000201:enzymology / Q000528:radiation effects; D009499:Neutral Red / Q000493:pharmacokinetics; D011522:Protons; D017382:Reactive Oxygen Species / Q000378:metabolism; D013997:Time Factors
D004396:Coloring Agents; D011522:Protons; D017382:Reactive Oxygen Species; D009499:Neutral Red