As previously shown, immunization with ocular antigens, like heat shock protein 27 (HSP 27), leads to retinal ganglion cell loss in an autoimmune glaucoma model. Aim of this study was to assess how immunization with S100 alone and in combination with HSP 27 affects retinal ganglion and macroglia cells. Rats were immunized with S100 or S100 plus HSP 27 (COMB). Neuronal cell density was evaluated on Nissl-stained flatmounts. Immunized groups showed a significant neuronal cell loss (S100, p = 0.005; COMB, p = 0.0005). A significant loss of retinal ganglion cells was also observed in both immunized groups on Brn-3a stained retinal cross-sections (S100, p = 0.003; COMB, p = 0.001). An increase in GFAP(+) area was noted in both groups (S100, p = 0.01; COMB, p = 0.001). In contrary, vimentin staining was not altered (S100/COMB, p > 0.05). In summary, immunization with solely S100 leads to retinal ganglion cell damage and reactive gliosis. While the combination of S100 plus HSP 27 also caused retinal ganglion cell loss and a glia response, the combination of the two antigens did not cause additional damage or more severe cell loss. We assume that both antigens might interact, possibly having inhibitory effects on each other and thus preventing additional damage to the retina.