Abstract
Purpose::
In experimental autoimmune uveitis (EAU) the retinal damage is alleged to be caused by generation of nitric oxide reaction products, which can oxidize lipids. The lipid peroxidation product, 4-hydroxynonenal (HNE) can disturb the protein function following adducts with proteins. We hypothesized that mitochondrial oxidative stress occurring in the early phase of EAU before inflammatory cell infiltration leads to selective mitochondrial membrane lipid peroxidation
Methods::
A group of 12 Lewis rats were immunized with S-antigen mixed with complete Freund’s adjuvant (CFA) to induce uveitis. Animals injected with CFA alone and nonimmunized animals served as control. Six animals from each group were euthanized on day 7 after immunization. The retinas were isolated and processed to obtain mitochondrial and cytosol components and assayed by gel electrophoresis and by Western blotting probed with HNE antibody. Positive band excised from the coomassie gel were analyzed by LC-MS/MS after in gel trypsin digestion. Three rats from each group were euthanized on day 7 and day 14 after immunization and Enucleated eyes were submitted for histologic analysis using hematoxylin eosin stain.
Results::
Compared to the control group, immunoblots of mitochondria of EAU rats demonstrated a 1.5 fold increase in formation of 4-HNE adducts in the 50kDa band. Mitochondria of EAU rats also showed HNE adducts in the 20±2kDa bands; such products were absent in the control mitochondria. LC-MS/MS analyses of the 20±2 kDa bands revealed the presence of Mn superoxide dismutase, ATP synthase O subunit, and crystallins. Presence of alphaA-crystallin was confirmed by Immunoblot. Histologically none of the animals showed inflammatory cell infiltration on day 7; but on day 14 there was inflammatory cell infiltration in the retina and Uvea.
Conclusions::
Presence of 4-HNE adducts involving mitochondrial related proteins suggests that selective mitochondrial lipid peroxidation takes place in the early phase of EAU before leukocyte infiltration; such oxidative damage in the mitochondria may be an initial event leading to retinal damage seen in EAU.
Keywords: oxidation/oxidative or free radical damage • retina • mitochondria