Tyrosinase Function Determines Retinal Vascular Regeneration and Retinal Vascular Endothelial Progenitor Cell Recruitment in the Oxygen-Induced Retinopathy Model

Abstract

Purpose: : We have previously reported a quantitative trait locus (QTL) on chromosome 7 with a LOD score of 8.9 that influences the extent of central retinal avascularity in the oxygen-induced retinopathy (OIR) model. The LOD score was determined in an F2 intercross between BALB/cByJ and C57BL/6ByJ where retinal avascular area was measured 96 hours after mice were returned to normoxia. Tyrosinase lies within the QTL. Tyrosinase is functional in C57BL/6ByJ mice and non-functional in BALB/cByJ mice. We sought to determine whether tyrosinase may be one of the quantitative trait genes (QTGs) underlying the QTL, and whether control of recruitment of endothelial progenitor cells into the retina may be part of the mechanism by which tyrosinase influences avascular area.

Methods: : B6(Cg)-Tyr^c-2J/J ("B6(Cg)") is a commercially available strain of mice derived from the C57BL/6J strain with a spontaneous loss of function mutation in tyrosinase that has been bred to homozygosity. Pups from both C57BL/6 and B6(Cg) strains were exposed 75% inspired oxygen for 120 hours, beginning on post-natal day 7 (P7). Mice were anesthetized, perfused with fluorescein-labeled dextran, and enucleated either immediately upon return to normoxia on P12 or 96 hours later on P16. Retinas were dissected and flatmounted, and the avascular area was measured using ImageJ software.Additional retinas were harvested 24 hours after return to normoxia and stained for AC133+ progenitor cells, which were counted by an observer blind to strain.

Results: : Tyrosinase-deficient B6(Cg) had smaller avascular areas at both P12 and P16 (p = 0.02 and p = 2.3 x 10^-9, respectively). B6(Cg) were also found to have more EPCs in perivascular areas of the retina compared to C57BL/6J mice on P13 (87.9 cells/mm² vs. 49.0 cells/mm², p < 0.01).

Conclusions: : Examination of avascular area at both P12 and P16 demonstrated that tyrosinase-deficient B6(Cg)-Tyr^c-2J/J mice revascularize more rapidly after return to normoxia than do C57BL/6J mice. This increased rate of revascularization is associated with greater retinal recruitment of EPCs. These data confirm the role of tyrosinase as a QTG in OIR revascularization and suggest that tyrosinase activity modifies angiogenic repair mechanisms

Keywords: retinopathy of prematurity • genetics • regeneration