Abstract
Context: Proton beam therapy offers the advantage of precise delivery with limited damage to the healthy tissue and is being tested in the management of exudative age-related macular degeneration (AMD). However, the dosages tested are empirical and not based on preclinical studies.
Objective: In this study we evaluated the effects of varying doses of proton beam radiation on choroidal endothelial cells (CECs) and retinal ganglion cells (RGCs) using clonogenic assay to determine differential sensitivity.
Materials and methods: Each cell type has different efficiency to replicate (plating efficiency (PE)). PE of CEC (RF/6A) and RGC (RGC-5) grown in culture flasks was determined by plating 250 cells each (without any treatment) and counting the number of colonies after 13 days. Radiation induced sensitivity was determined by exposing the semi-confluent RF/6A and RGC-5 cells to proton beam at the doses of 0 (control), 2, 4, 8 and 12 cobalt gray equivalent (CGE). The ability of the cells to repair and replicate to form colonies were analyzed 13 days after radiation with crystal violet stain and the survival ratio was calculated. The significance of survival was analyzed using ANOVA (Graphpad Instat.3).
Results: The PE of CEC and RGC was 12.96 ± 0.29% and 40.7 ± 1.48%, respectively. A survival ratio of CEC at 2, 4, 8 and 12 CGE proton radiation was 66.0 ± 8.6%, 44.3 ± 6.5%, 7.6 ± 0.3% and 1.14 ± 0.06% on exposure to 2, 4, 8 and 12 CGE proton radiation, respectively, p < 0.01). Survival ratio of RGC was 71.1 ± 22.4% (p = 0.05), 40.2 ± 7.9%, 8.89 ± 2.6% and 0.78 ± 0.31% at 2, 4, 8 and 12 CGE dosages (p < 0.001).
Discussion: CEC showed dose-dependent decrease in survival rate with values attaining significance at all radiation dosages. In contrast, RGC was comparatively radio resistant and were able to replicate at lower doses and sensitive at higher doses after proton beam radiation.
Conclusion: Since CECs proliferate during neovascularization, this clonogenic assay is a useful assay to assess the sensitivity of CEC to radiation. This study identified that CEC were more sensitive to proton beam radiation than RGC at all doses. This may provide a therapeutic window for administration of proton beam radiation in the management of AMD.
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NOTICE OF CORRECTION
The Early Online version of this article published online ahead of print on 24 August 2011 contained an error on page 1.
The author list and affilations Sankarathi Balaiya1, Malyapa Robert2, Hsi Wen1, Ravi K Murthy1, and Kakarla V Chalam1
1University of Florida, Jacksonville, FL, USA and 2Radiation oncology, University of Florida, Jacksonville, FL, USA.
Should have read
Sankarathi Balaiya1, Robert Malyapa2, Wen Hsi2, Ravi K Murthy1, and Kakarla V Chalam1
1Department of Ophthalmology, University of Florida, Jacksonville, FL, USA and 2University of Florida Proton Therapy Institute, Jacksonville, FL, USA.
This has been corrected for the current version.