Real-time in vivo detection of retinal reactive oxygen species

Abstract

Purpose: We characterized and optimized the use of a chemiluminescent probe L-012 as a non-invasive in vivomethod to assessoptic nerve crush(ONC)-induced accumulation of reactive oxygen species (ROS) in the mouseretina.

Methods: We performed ONC unilaterally in adult C57BL/6J mice to induce retinal damage. L-012-dependent luminescencein the eye was acquired in a small animalin vivo imaging system. Optimal detection time and dose of L-012 were empirically determined.Histology of the retina was performed by H&E staining of cross-sections.Protein carbonyl content in retina was measured byenzyme-linked immunosorbent assay.

Results: Ocular L-012 chemiluminescence was observed at Day 1 and Day 3 in the ONC-injured eye, but not in the contralateral uninjured control orsham surgery eye. The signal was dependent on the dose of L-012; 75mg/kg of L-012 providedhighest signalcompared with that of 25mg/kg or 7.5mg/kg.The peak chemiluminescence occurredbetween15min and20 min after intraperitoneal L-012injection (Peak photon radiance of ONC eye = 120 ± 61 x 10³ p/s/cm²/sr;control eye = 8 ± 1 x 10³ p/s/cm²/sr; mean ± SD, n = 5; p < 0.05).The signal correlated with oxidative changes in the retina: retinal protein carbonyl content was 1.36±0.16nmol/mg proteinat Day 1 after ONC, which was significantly (p < 0.05) higher than that ofthe control retina(0.51±0.05nmol/mg). Thechemiluminescence signal of Day 3 was significantly(p < 0.05) lower than that of Day 1.Intraperitoneal treatment with tempol (100mg/kg), a ROS scavenger, or apocynin (50mg/kg), a NADPH oxidase inhibitor significantly(p < 0.05) reduced ocular L-012 chemiluminescence. Histological evaluation of H&E stained retina cross-sections showed no evidence of retinal toxicity by L-012.

Conclusions: Currently available detection methods of ROS require post-mortem, ex vivo tissue or cell samples. Our data show thatL-012is a useful non-invasive in vivo tool to assess the accumulation of ROS in the retina. Thisreal-time in vivo imaging technology can significantly aid our understanding of the involvement of oxidative damage in various retinal injuries.