Abstract
Purpose: :
We recently developed an Experimental Autoimmune Glaucoma model, where we could show that systemic immunization with specific ocular antigens causes significant retinal ganglion cell (RGC) loss. But the mechanism in how antibodies cause cell loss is so far unknown. Therefore, we investigated possible binding sights of antibodies in the eye.
Methods: :
Rats were systemically immunized with bovine retinal ganglion cell-layer homogenate (RGH; n=10) or optic nerve homogenate (ONH; n=10). Antigens were suspended in an equal volume of Freund’s adjuvant plus pertussis toxin as an additional adjuvans. An additional group received NaCl and served as control group (n=10). Blood was collected from all animals after 6 or 10 weeks right before they were euthanized. The serum was used for indirect immunohistochemical techniques on retinae. Retinal cross-sections of healthy animals were incubated with sera from study animals as first antibody and an anti-rat IgG secondary antibody. Sections were scored ranging from 0 (no antibody binding) to 4 (severe binding) and data analyzed by ANOVA and post-hoc. Eyes from immunized animals were fixed in formaldehyde at the end of the study and flatmounts were stained with creysyl to detect RGC nuclei. RGCs were counted and data analyzed by ANOVA and post-hoc test. Some study eyes from each group were used for cross-sections to detect GFAP expression.
Results: :
Retinal flatmount analysis revealed significant RGC loss in animals immunized with RGH (P=0.024) or ONH (P=0.003). Animals immunized with RGH (P=0.002; mean score 1.2) or ONH (P=0.0001; mean score 2.3) developed anti-retinal antibodies, their binding was significantly stronger compared to control sera. ONH sera bound especially in epiretinal sections, while no staining could be detected in other layers of the retina. RGH sera on the other hand bound especially between the retinal ganglion cell and amacrin cell layer. No binding was detected for sera from control animals (mean 0). Animals immunized with RGH or ONH also showed more severe gliosis similar to the one observed in human glaucoma.
Conclusions: :
These results suggest that antibodies directly bind to retinal structures after immunization with ONH or RGH, which probably causes RGC loss. Therefore, adaptive immunity seems to play a role in RGC loss in this model of Experimental Autoimmune Glaucoma.
Keywords: retinal degenerations: cell biology • optic nerve • ganglion cells