Caspase Pathway In Model Of Autoimmune Glaucoma

Abstract

Purpose: : An apoptotic death of retinal ganglion cells (RGC) has been shown in glaucoma patients, caspase-3 being a key player. Caspase-8 and caspase-9 are markers of different previous branches in this pathway. Our former studies using a model of experimental autoimmune glaucoma have shown that immunization with ocular antigens causes RGC loss. To learn more about the pathophysiology of this cell death we investigated the steps leading to apoptosis.

Methods: : Rats were immunized with a retinal ganglion cell-layer homogenate (RGA; n=5) in incomplete Freund’s adjuvant and pertussis toxin. We generated 2 control groups, one was injected with NaCl plus Freund’s adjuvant and pertussis toxin (CO; n=5) and the second one were native rats (NA; n=5). After 7 weeks the animals were euthanized and retina cross-sections were generated. We used anti-caspase-3; -8 and -9 antibodies to stain different stages of apoptosis. The rate of apoptotic RGC was scored ranging from 0 (no stain) to 3 (severe stain) on entire sections and group comparison was performed applying Tukey HSD test. Antibody-deposits were analyzed using an anti-IgG-antibody and also visualized via transmission electron microscopy.

Results: : Immunofluorescence proof of caspases showed positive stained scattered nuclei, likely RGCs. In caspase-3 stained sections a significant higher amount of apoptotic cells in the RGA group (score: 1.40) compared to the NA group (score: 0.50; p=0.03) and the CO group (score: 0.30; p=0.01). Caspase-8 staining showed a similar result: RGA: 2.00, NA: 0.90 (p=0.02) and CO: 0.60 (p=0.005). In contrast the staining intensity for caspase-9 did not differ between groups (RGA: 0.70; NA: 0.40 (p=0.5); CO: 0.20 (p=0.1)). IgG immunostaining showed visible differences between groups concerning IgG-deposits in the retinal ganglion cell layer. These deposits as well as apoptotic cells were confirmed by electron microscopy.

Conclusions: : These results suggest that IgG antibodies are involved in the observed apoptosis of RGC in this model. Further more we can suspect that caspase-3 is activated predominantly via caspase-8. Perhaps the TNFα-receptor and TNFα itself are playing an important role in apoptosis in our model.

Keywords: autoimmune disease • ganglion cells • immunohistochemistry