Abstract

DO.05.12

Complex protein profiles in aqueous humor of Experimental Autoimmune Glaucoma animal model (EAG)

Gramlich O. W., Joachim S. C., Reichmann J., Pfeiffer N., Grus F. H.
Experimentelle Ophthalmologie, Universitäts-Augenklinik, Johannes Gutenberg-Universität, Mainz

Objective: An autoimmune involvement in the pathogenesis of glaucoma has been recently discussed. In this project aqueous humor of animals with Experimental Autoimmune Glaucoma (EAG) was analyzed with proteomic methods. Previous studies showed, that in the EAG model a loss of retinal ganglion cells (RGCs) is caused by immunization with ocular antigens.
Methods: Lewis rats were immunized with myelin basic protein (MBP) or optic nerve homogenate (ONH) in combination with pertussis toxin and Freund’s adjuvant. In study I rats were euthanized 5 weeks after immunization with MBP (n=10) or ONH (n=10). A control group (n=10) received only pertussis and Freund’s. In study II (MBP, ONH, and control group; n=11) animals received an additional 50% dose of immunization after 4 and 8 weeks and were euthanized after 10 weeks. Aqueous humor was collected in all animals at the end of the studies. In study II eyes were enucleated, fixed, and retinal flatmounts were stained with Kresylblue to detect and count RGCs. All aqueous humor samples (5 µl) were fractionated using C8 beads (Bruker) and analyzed with a SELDI-TOF-MS (Biorad). Data were analyzed by multivariate statistical techniques.
Results: Study I revealed a significant difference between protein profiles of samples from animals immunized with ONH and control animals (P=0.05). No significant difference was observed between profiles of MBP immunized animals and controls (P=0.11). In study II a significant RGC loss was observed in rats immunized with MBP (P=0.0001) and ONH (P=0.003). Protein patterns in animals immunized with MBP deviated significantly from controls (P=0.02), whereas profiles of the ONH group showed no significant difference (P=0.27). Three significant biomarkers (7120, 12297, and 19436 Da) were detected in the studies.
Conclusions: A significant RGC loss could be observed after immunization with MBP or optic nerve homogenate as well as changes in the aqueous humor protein pattern. The study shows that both duration and the number of immunizations seem to influence the aqueous humor protein profiles depending on the antigen in the EAG animal model.
Support: Boehringer Ingelheim Foundation, MAIFOR

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