Balaiya Sankarathi, Malyapa Robert, Hsi Wen, Murthy Ravi K, Chalam Kakarla V
Department of Ophthalmology, University of Florida, Jacksonville, FL 32209, USA.
Cutan Ocul Toxicol. 2012 Mar;31(1):14-9. doi: 10.3109/15569527.2011.594697. Epub 2011 Aug 24.
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.
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.
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).
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).
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.
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.
质子束疗法具有精确给药且对健康组织损伤有限的优势，目前正在渗出性年龄相关性黄斑变性（AMD）的治疗中进行试验。然而，所测试的剂量是经验性的，并非基于临床前研究。
在本研究中，我们使用克隆形成试验评估不同剂量的质子束辐射对脉络膜内皮细胞（CEC）和视网膜神经节细胞（RGC）的影响，以确定差异敏感性。
每种细胞类型具有不同的复制效率（接种效率（PE））。通过在培养瓶中分别接种250个细胞（无任何处理）并在13天后计数集落数量，来确定培养瓶中生长的CEC（RF/6A）和RGC（RGC-5）的PE。通过将半汇合的RF/6A和RGC-5细胞暴露于0（对照）、2、4、8和12钴灰当量（CGE）剂量的质子束来确定辐射诱导的敏感性。在辐射13天后，用结晶紫染色分析细胞修复和复制形成集落的能力，并计算存活率。使用方差分析（Graphpad Instat.3）分析存活率的显著性。
CEC和RGC的PE分别为12.96±0.29%和40.7±1.48%。在暴露于2、4、8和12 CGE质子辐射时，CEC在2、4、8和12 CGE质子辐射下的存活率分别为66.0±8.6%、44.3±6.5%、7.6±0.3%和1.14±0.06%，p<0.01）。RGC在2、4、8和12 CGE剂量下的存活率分别为71.1±22.4%（p=0.05）、40.2±7.9%、8.89±2.6%和0.78±0.31%（p<0.001）。
CEC显示出存活率呈剂量依赖性下降，在所有辐射剂量下其值均具有显著性。相比之下，RGC具有相对的放射抗性，在质子束辐射后能够在较低剂量下复制，而在较高剂量下敏感。
由于CEC在新生血管形成过程中增殖，这种克隆形成试验是评估CEC对辐射敏感性的有用试验。本研究确定，在所有剂量下，CEC对质子束辐射比RGC更敏感。这可能为在AMD治疗中给予质子束辐射提供一个治疗窗口。
