Abstract
Purpose::
In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress in the mitochondria before leukocyte infiltration. The generation of nitric oxide reaction products in the early phase of EAU damages both lipids and protein in mitochondria. The purpose of this study is to determine whether oxidative mitochondrial DNA (mtDNA) damage occurs early in EAU before leukocyte infiltration.
Methods::
A group of 6 Lewis rats were immunized with S-antigen mixed with complete Freund’s adjuvant (CFA) to induce EAU. Animals injected with CFA along and nonimmunized animals served as control. Three animals were euthanized on day 4 and 7 after immunization. The retinas were isolated and processed to obtain the DNA. Damage to mtDNA was assessed using quantitative polymerase chain reaction. DNA damage was estimated in mitochondria by amplifying 13.5 kb fragments of mtDNA. DNA lesion frequencies were calculated assuming a random distribution of lesions and using the Poisson equation [f(x)=e-x/x!, where = the average lesion frequency for the nondamaged template. The amplification of damaged samples (AD) was normalized to the amplification of nondamaged control (A0). The average lesion per DNA strand was determined as: =-ln AD / A0. Statistical analysis was performed with a Student’s t test with p values <0.05 considered significant. A positive control for oxidative DNA damage involving exposure of rat retina to Sin1 for six hours in vitro was also performed.
Results::
The relative DNA amplification ratios were the following- control: 1±0, CFA: 1±0.16, Sin1: 0.73±0.33 and S-Antigen Day 7: 0.70±0.09. The DNA lesions per 10 kb were calculated for control: 0, CFA: 0, Sin1: 0.313 and S-Antigen Day 7: 0.360. The increase in DNA lesions was significant at day 7 in animals immunized with S-antigen (p<0.03).
Conclusions::
There is increased oxidative mtDNA damage at day 7 in EAU. This further supports that oxidative stress selectively occurs in the mitochondria in the early phase of EAU before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal damage in EAU.
Keywords: uveitis-clinical/animal model • oxidation/oxidative or free radical damage • mitochondria