Measurement of Reactive Oxygen Species Byproduct in Leber’s Hereditary Optic Neuropathy Affected and Carrier Patients

Abstract

Purpose:: It has been postulated that in Leber’s Hereditary Optic Neuropathy (LHON), a defect in mtDNA results in the build up of reactive oxygen species and byproducts, causing optic nerve degeneration via apoptosis. Some carriers may be able to better compensate, accounting for variable penetrance. Nitrotyrosine is a byproduct of reactive oxygen species and hence may be a marker for this pathophysiology.

Methods:: Tissues were previously fixed in 10% neutral buffered formalin, processed, and embedded in paraffin. Longitudinal sections of 4 control optic nerve heads, 4 Leber’s affected optic nerve heads, and 2 carrier optic nerve heads were used. The sections were baked overnight in a 60°C oven, deparaffinized, treated with antigen retrieval citra, then with glycine for aldehyde binding, and then with 1% BSA and 0.1% triton X-100 in PBS for blocking. The sections were labeled using either primary antibody consisting of monoclonal mouse anti nitrotyrosine in 1% BSA and 0.1% triton X-100 in PBS, or this buffer alone without primary antibody. Secondary rabbit anti mouse antibody conjugated to rhodamine was then bound. The sections were mounted with Vectashield plus DAPI and then photographed using a confocal microscope. Rhodamine labeling of nitrotyrosine was quantified using a densitometer. The mean values for control, carrier, and affected optic nerves were compared.

Results:: We found increased nitrotyrosine staining in LHON carrier and affected optic nerves compared to controls. The carrier patient’s optic nerves appear to have more nitrotyrosine staining than the affected patients’ optic nerves.

Conclusions:: We found increased nitrotyrosine staining in LHON optic nerves, indicating that reactive oxygen species are involved in the process of this disease. In so far as the LHON carrier showing more nitrotyrosine staining than the LHON affected, this did not support our hypothesis regarding the carriers’ ability to effect compensatory strategy. However, given our limited sample size, it also did not definitively rule out this possibility. Our carrier patient was 86 years of age at the time of death compared to an average age of 56 for our affected patients. Further studies will need to be performed.

Keywords: pathology: human • visual impairment: neuro-ophthalmological disease • oxidation/oxidative or free radical damage