Nitric Oxide and Retinal Damage by Uveitis: Studies in a Novel Model System, Platelet-activating Factor-induced Uveitis in Chick Retina

Abstract

Abstract: : Purpose: Uveitis is an ocular inflammatory disorder that can damage pigment epithelium (RPE) and photoreceptors, causing blindness. The mechanism of damage is unknown, but it could involve excessive nitric oxide (NO) produced by inflammatory cells. We have induced uveitis in chick eyes with platelet-activating factor (PAF, a bioactive phospholipid) to create a model for testing this hypothesis. Methods: We injected PAF, 2 fmol-20 pmol in 20 µL vehicle (0.5% bovine serum albumin [BSA] in phosphate-buffered saline [PBS]), or vehicle alone intravitreally in the left eye of 7-day chicks, and vehicle alone in the right (control) eye. A nitric oxide synthase (NOS)-inhibitor, N₅-(1-Iminoethyl)-L-ornithine (L-NIO) or N₆-(1-Iminoethyl)-L-lysine (L-NIL), 300 nmol in 20µL vehicle, was co-injected with PAF and re-injected in vehicle 1 day later. Inflammation and retinal integrity were assessed 3 days post initial injection by ophthalmoscopy, inspection of the opened eye, paraformaldehyde fixation and cryosectioning, and staining with 0.1% toluidine blue or indirect immunofluorescence for retinal cell markers. Results: Vehicle caused no inflammation or damage, but PAF caused both in a dose-dependent manner (ED₅₀=20 fmol). After injecting 20 fmol PAF the vitreous was cloudy, the vitreous and outer retina were infiltrated with leukocytes, the RPE was discontinuous, melanotic cells were present in the outer nuclear layer, and rods and cones were severely damaged. L-NIO and L-NIL eliminated most of these effects. Retinas treated with L-NIO or L-NIL alone were indistinguishable from vehicle controls. L-NIO at this dose is known to inhibit NOS-activity in intact chick retina and RPE by 61-90% (Wellard et al., 1995; Gudgeon et al., 2002). Conclusion: Chicks are tractable experimental animals, with large, diurnally-adapted eyes that can be injected with drugs and examined easily. The uveitis caused by PAF in chicks closely resembles experimental autoimmune uveitis in mammals and should be advantageous for studying how uveitis damages the RPE and outer retina. Our data implicate nitric oxide as a mediator of photoreceptor and RPE damage in uveitis.

Keywords: 612 uveitis-clinical/animal model • 491 nitric oxide • 554 retina