Abstract
Purpose:
Extensive remodeling of extracellular matrix (ECM) and changes in the expression of integrins are typical to glaucoma and associated with RGC death and degeneration. We recently showed that laminin-integrin-FAK-Akt pathway is disrupted in retinal ischemia. The purpose of this study is to investigate the role of FAK in RGC in glaucoma.
Methods:
Experimental glaucoma was induced in the left eye of 30 Sprague Dawley rats by blocking the ocular outflow and producing increased intraocular pressure (IOP) using a diode laser. IOP was measured periodically in both eyes after laser photocoagulation (LP). A group of rats (n=8) was euthanized 9 weeks post LP. Their optic nerves and retinas were removed and processed for histology. Semi-automated counting of the axons was performed in optic nerve cross sections stained with toluidine blue. Corresponding flat mounted retinas were processed for immunofluorescence with a TUJ1 (βIII tubulin) antibody, and TUJ1 labeled RGC were counted with an inverted fluorescence microscope. Another group of rats (n=8) was euthanized 4 weeks post-LP and fluorogold (FG) labeled RGC were counted in flat mounted retinas. Retinal flat-mounts (n=4) from control and experimental glaucoma eyes 2 weeks post-LP, and human normal (n=2) and glaucomatous (n=4) retinas and optic nerves were processed for immunohistochemsitry with β1-integrin, FAK, phospho-FAK antibodies
Results:
Mean IOP was increased in the treated eye from 8.66±1.03 to 18.34±5.86 to a peak value of 25.73± 9.73 at 1 day, 16.44 ± 6.98 at 1, and 20.75 ± 7.27 mm Hg at 2 weeks post LP (*p<0.05). Axonal loss at 9 weeks post-LP, was 64.89% ± 11.05 (*p<0.05), in good correlation with RGC survival, 52.39% ± 4.74 (*p<0.01),Loss of FG labeled RGCs was aparent at 2 weeks, with significantly reduced FG+ RGCs in the glaucomatous eye at 4 weeks post LP (12.66% ± 9.21; *p<0.05), suggesting that many surviving RGCs are dysfunctional and have impaired retrograde active transport. Toluidine blue staining in optic nerve cross-sections showed swollen axons, collapsed myelin sheets, and axon bundles disarray 9 weeks post-LP in treated eyes. Immunohistochemistry showed decreased expression of β1-integrin and significantly reduced phospho/activated FAK in both human and experimental glaucoma.
Conclusions:
Our results suggest that β1 integrin signaling is disrupted with significantly reduced FAK activation in RGC in glaucoma.