title: Evaluation of choroidal endothelial cell proliferation after exposure to varying doses of proton beam radiation.
abstract: 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.
authors.affiliation: Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USA. kchalam@jax.ufl.edu
authors.forename: Kakarla V
authors.initials: KV
authors.lastname: Chalam
authors.forename: Sankarathi
authors.initials: S
authors.lastname: Balaiya
authors.forename: Robert S
authors.initials: RS
authors.lastname: Malyappa
authors.forename: Wen
authors.initials: W
authors.lastname: Hsi
authors.forename: Vikram S
authors.initials: VS
authors.lastname: Brar
authors.forename: Ravi K
authors.initials: RK
authors.lastname: Murthy
mesh_terms: 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
chemical_list: D004396:Coloring Agents; D011522:Protons; D017382:Reactive Oxygen Species; D009499:Neutral Red