Abstract
Purpose: :
To investigate the role of complement in the protection of retinal ganglion cells in chronic ocular hypertension model of glaucoma.
Methods: :
Intraocular pressure (IOP) was elevated in the left eye of Lewis rats by laser photocoagulation (two treatments, 7 days apart) of episcleral and limbal veins. The right eye did not receive laser treatment and served as control. Lewis rats were injected with cobra venom factor (CVF, >30 units/rat, i.p.) at day 7, 14, 21, 28, 35 and 49 post-laser, to deplete the complement system and these animals were sacrificed on day 49 post-laser. Control animals received similar treatment with PBS. The retina was harvested from laser-treated eyes as well as untreated control eyes and processed for flow cytometry, paraffin embedding, RNA extraction and protein extraction. Paraffin sections were used for immunostaing for glial fibrillary acidic protein (GFAP), Brn3a, complement component C3, complement membrane attack complex (MAC), TUNEL and caspase-3.
Results: :
Immunofluorescent staining demonstrated that CVF treatment resulted in decreased deposition of C3 split products and MAC in the retina of the animals with increased IOP compared to the animals with treated with PBS. Drastic decrease in C3 split products, and MAC in the retina of CVF treated Lewis rats were further demonstrated by Western blot analysis. Depletion of complement by CVF resulted in decrease in GFAP staining in the retina of animals with elevated IOP. Furthermore CVF treatment resulted in reduction of TUNEL and active caspase-3 staining in ganglionic cell layer. Flow cytometric analysis showed that the number of apoptotic cells significantly decreased in retina of CVF-treated rats with increased IOP compared to control rats. Complement depletion also resulted in the protection of RGCs as indicated by increased number of Brn3a+ cells (marker for RGC) in CVF treated rats compared to PBS treated rats with increased IOP.
Conclusions: :
Our results provide evidence that complement activation plays a critical role in the development of glaucoma in Lewis rats and the loss of retinal ganglion cells in these animals can be prevented by complement depletion.
Keywords: inflammation • apoptosis/cell death • ganglion cells