Abstract
Purpose: :
Glaucoma is characterized by apoptotic death of retinal ganglion cells (RGCs). Caspase-3, a key player involved in the caspase-dependant apoptotic pathway, is an established apoptosis marker. Our studies indicate that immunization with ocular antigens leads to RGC loss in a model of Experimental Autoimmune Glaucoma. To gain further knowledge about the mechanism of RGC loss time-course of cell death and possible antibody appearance were evaluated.
Methods: :
We immunized Lewis rats with optic nerve homogenate (ONH; n=15) in incomplete Freund’s adjuvant and pertussis toxin. The control group was injected with NaCl (CO; n=15). RGC density was quantified in retinal flatmounts after cresyl stain and compared 8, 14, and 22 days after immunization via t-test. We stained retina cross-sections from the 3 time-points using anti-caspase-3 antibody to investigate apoptosis rate. Apoptotic RGCs were scored ranging from 0 (no stain) to 3 (severe stain) on entire sections and group comparison was performed applying t-tests. Antibody deposits were detected in cross-sections via anti-rat IgM stain.
Results: :
Immunofluorescence detection of caspase-3 revealed scattered nuclei with positive staining in the RGC layer of immunized animals. We detected no difference in apoptosis score 8 days after immunization (p=0.22) and no significant RGC loss. At 14 days ONH animals had a mean apoptosis score of 2±0.7, compared to a mean score of 0.4±0.5 for controls (p=0.004), and distinct IgM deposits were at this time observed epiretinally. But groups showed no difference in RGC density. At 21 days we detected a significantly higher number of apoptotic RGCs in ONH animals (mean scores: ONH=2.1±0.1 and CO=0.4±0.6; p=0.001) and concurrent a lower RGC density (p=0.001). Immunized animals had a mean RGC density of 2456 cells/mm2 compared to 2900 cells/mm2 in the CO group.
Conclusions: :
This data suggest that immunization with ocular antigens leads to apoptotic RGC death. Antibody deposits are detectable when apoptosis occurs. Therefore, we conclude that antibodies are engaged in eliciting RGC apoptosis in this model. The slow apoptotic dissolution of RGCs we observed in animals with autoimmune glaucoma is comparable to slow progressive RGC loss in glaucoma patients, thus making this a useful model to develop neuroprotective therapies in the future.
Keywords: apoptosis/cell death • pathology: experimental • inner retina dysfunction: biochemistry and cell biology