Purpose
Retinopathy of prematurity (ROP) causes neonatal vision loss that accounts globally for 50,000 annual cases. The eNOS enzyme plays a prominent role in regulation of the retinal vasculature and in modulating vascular tone in response to changes in oxygen tension. This study aimed to establish a correlation between the extent of retinal vascularity, VEGF and eNOS expression, and post-translationally modified Serine-1177 phosphorylated eNOS (P-eNOS), in an oxygen-induced rat model of ROP.
Methods
Sprague-Dawley pups were placed in an oxygen chamber (cycling every 24 hours at 50% and 10% FiO2) for post-natal (P) days P1-P14. On P15 pups were exposed to normaxia (21% oxygen). Littermate non-oxygen treated naïve pups were used as controls. Rats were euthanized on P15, P18 & P20. One retina of each rat was flat-mounted and ADPase stained for evaluation of the vasculature. Contralateral retinas were homogenized and evaluated using real time RT PCR for VEGF and eNOS mRNA levels or using Western Blots (WB) for eNOS and P-eNOS. A subset of the samples were fixed and cross-sectioned for eNOS or P-eNOS immmunohistochemistry.
Results
RT PCR data demonstrates a relative increase of VEGF mRNA at P15, 18 and P20. The eNOS mRNA levels, in conjunction with immunohistochemistry (IHC) for eNOS immunoreactivity in retinal vessels demonstrated an appreciable progressive reduction at P15, 18 and 20 relative to controls. No apparent reduction of P-eNOS was observed. WB evaluations for eNOS and P-eNOS protein expressions are pending.
Conclusions
During the normoxic phase of the ROP model, progressive increases in VEGF mRNA expression are consistent and temporally correlate with the extent of neovascularization. However, despite eNOS maintaining its phosphorylation state both eNOS mRNA and protein expressions (IHC) decline during the normoxia period. This reduction in eNOS expression could lead to exacerbation of tissue hypoxia resulting in an augmentation of the neovascular response in this ROP model.
Keywords: 700 retinal neovascularization •
617 nitric oxide •
748 vascular endothelial growth factor