PURPOSE:Retinal injuries that affect the photoreceptors and/or the retinal pigment epithelium (RPE) may result in the leakage of retinal proteins into the systemic circulation. This study was designed to determine whether an immune response is elicited after an acute retinal injury resulting in circulating anti-retinal antibodies in the serum.METHODS:Fifty laser burns of different grades (minimally visible lesion [MVL], grade II [GII], or grade III [GIII] lesions) were created in the retinas of Dutch Belted rabbits. The degree of laser burns was confirmed by fundus imaging and histology. Serum samples were collected from the animals 3 months after the retinal injury. Candidate autoantigens were identified by two-dimensional (2-D) Western blots of rabbit retinal lysate probed with sera from either control or laser-treated animals. Candidate autoantigens were further characterized by immunostaining to confirm their retinal localization.RESULTS:Seven and 11 protein spots were selected from the MVL and GII laser-treated samples, respectively, for autoantigen identification. No protein spots were detected in the GIII laser-treated samples. Four candidate autoantigens were common to both MVL and GII lesions: dihydropyrimidinase-related protein 2, fructose-bisphosphate aldolase C, chaperonin-containing T-complex polypeptide 1 subunit zeta, and pyruvate kinase isozyme.CONCLUSIONS:Laser-induced retinal injuries resulted in circulating anti-retinal antibodies that were detectable 3 months after the injury. The response appeared to vary with the severity of the laser retinal damage. The identification of the candidate antigens in this study suggest that this approach may permit future development of new diagnostic methods for retinal injuries.

Purpose To identify candidate protein biomarkers in sera indicative of acute retinal injury. Methods We used laser photocoagulation as a model of acute retinal injury in Rhesus macaques. In a paired-control study design, we collected serum from each animal (n=6) at 4 h, 1 day, and 3 days following a mock procedure and then again following retinal laser treatment that produced mild lesions. Samples were fractionated by isoelectric focusing, digested with trypsin, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Spectral counting was used to determine relative protein abundances and identify proteins with statistically significant differences between control and treated sera. Results Mild retinal injury was confirmed by fundus photography and histological examination. The average number of total proteins detected by LC-MS/MS was 908±82 among samples from all three time points. Following statistical analysis and employing stringent filtering criteria, a total of 19 proteins were identified as being significantly more abundant in sera following laser-induced retinal injury, relative to control sera. Many of the proteins detected were unique to one time point. However, four proteins (phosphoglycerate kinase 1, keratin 18, Lewis alpha-3-fucosyltransferase, and ephrin receptor A2) showed differences that were significant at both 4 h and 1 day after laser treatment, followed by a decrease to baseline levels by day 3. Conclusions A serum biomarker response to mild retinal laser injury was demonstrated in a primate model. Among the proteins detected with highest significant differences, most are upregulated within 24 h, and their appearance in the serum is transient. It is conceivable that a panel of these proteins could provide a means for detecting the acute-phase response to retinal injury. Further investigation of these candidate biomarkers and their correlation to retinal damage is warranted.

PURPOSE:Transforming growth factor-β (TGF-β) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-β receptor type I) could suppress TGF-β activity and thereby promote filtering bleb survival after GFS in a rabbit model.METHODS:An ALK-5 inhibitor, SB-505124, was used. A docking study was performed to investigate the interaction between the inhibitor and the receptor. Immunofluorescence for connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA) was performed in cultured rabbit subconjunctival fibroblasts. Immunoblotting for phosphorylated Smad2 (pSmad2), CTGF, and α-SMA was also performed. In an in vivo rabbit GFS model, SB-505124 was delivered in a lactose tablet during surgery. Eyes were examined by slit-lamp and intraocular pressure (IOP) was measured until the time of bleb failure or up to 28 days after surgery. Tissue sections on day 5 after surgery were histologically evaluated after staining with hematoxylin and eosin. The sections were also immunostained for CTGF and α-SMA. In addition, cell outgrowth from dissected subconjunctival tissues placed in a cell culture flask with media was investigated.RESULTS:The docking study indicated hydrogen bond interactions between SB-505124 and amino acids His-283 and Ser-280 of ALK-5. Suppression of pSmad2, CTGF, and α-SMA by SB-505124 was observed in cultured fibroblasts. Filtering blebs in the GFS with SB-505124 group were maintained for more than 10 days, and the period of bleb survival was significantly longer than that in controls. IOP levels after surgery seemed to be related to bleb survival. Histologically, subconjunctival cell infiltration and scarring at the surgical site in the GFS with SB-505124 and mitomycin C (MMC) groups were much subsided compared to controls. Suppression of CTGF and α-SMA by SB-505124 was also observed by immunofluorescence. Cell outgrowth from explants dissected from eyes to which SB-505124 was applied during GFS was robust while outgrowth was poor from those treated with MMC.CONCLUSIONS:The ALK-5 inhibitor SB-505124 was efficacious both in vitro and in vivo in suppressing the TGF-β action. The inhibitor may provide a novel therapy for preventing ocular inflammation and scarring.