Abstract
Purpose: :
The purpose of this study was to determine in human primary open angle glaucoma whether oxidative injury occurs in pre-laminar optic nerve blood vessels and glial cells.
Methods: :
Following IRB approval, sections from post-mortem primary open angle glaucoma eyes (n=5) with mean age of 77 ± 9 yrs (±SD) were compared to age-matched normal control eyes (n=5) with mean age 73 ± 10 yrs (Eye Bank of Canada). Immunostaining with nitrotyrosine, a footprint for peroxynitrite-mediated injury, was performed and sections were double-labelled with markers for vascular endothelial cells, perivascular smooth muscle cells, and astrocytes with CD34, smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP), respectively. Immunostaining was captured in a masked fashion using confocal microscopy, in defined regions of interest for blood vessels and glial tissue. Intensity measurements of supra-threshold area in pixels as percent of the total number of pixels were measured using Image J (NIH) and compared using two-tailed Mann-Whitney nonparametric tests between glaucoma and control groups. Co-localization coefficients with cell specific markers were determined and compared with random coefficients of correlation.
Results: :
Increased nitrotyrosine immunoreactivity was observed in pre-laminar optic nerve head blood vessels of primary open angle glaucoma eyes compared to controls and this difference was statistically significant (1.35 ± 1.11 % [± SD] vs. 0.01 ± 0.01 %, P = 0.008). NT immunoreactivity was also increased in the glial tissue surrounding the pre-laminar optic nerve head in the glaucoma group and compared to controls; the difference was statistically significant (18.37 ± 12.80 % vs. 0.09 ± 0.05 %, P = 0.006). Co-localization studies demonstrated nitrotyrosine staining in vascular endothelial and smooth muscle cells, in addition to astrocytes. Correlation coefficients for CD34, SMA, and GFAP were 0.37, 0.52, and 0.64 respectively.
Conclusions: :
Oxidative injury is present in pre-laminar optic nerve head in human primary open angle glaucoma. The presence of peroxynitrite-mediated oxidative injury in vascular endothelial cells and smooth muscle cells, in addition to astrocytes, implicates these tissue elements in the pathobiology of glaucoma disease.
Keywords: astrocytes: optic nerve head • oxidation/oxidative or free radical damage • blood supply