Purpose: To determine whether intravitreal unconjugated tissue plasminogen activator (tPA) (alteplase) can penetrate the intact neural retina and reach the subretinal space in an experimental model. Methods: This study was performed in 24 Sprague-Dawley rats aged 12 weeks. Under general anesthesia, the right eye was injected with either 0.75 μg of 3 μL tPA (14 rats; study group) or saline (10 rats, control group) into the vitreous. Animals were euthanized at 3, 24, and 48 h. The eyes were enucleated, and cryosections were prepared for immunofluorescence staining. Goat anti-tPA antibody was used to detect tPA. Results: In the study group, staining for tPA was detected in the deep retinal layers in all eyes. The staining was deeper and more intense at 3 and 24 h than at 48 h. There was no tPA staining in the retina of eyes injected with saline. Conclusions: This experimental study shows that unconjugated tPA administered into the vitreous is capable of penetrating the deep retinal layers and the subretinal space. These findings suggest that further clinical research is warranted on the benefits of intravitreal tPA in the treatment of submacular hemorrhage.

Purpose:To evaluate whether NETosis is involved in cytokine-induced ocular inflammation and to track neutrophil extracellular traps (NET) complexes in patients with proliferative diabetic retinopathy (PDR).Methods:For the animal model, the eyes of C57BL/6J mice were intravitreally injected with interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), or saline. Histology and immunofluorescence staining for CD11b, neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone 3 (H3Cit), and net-like structure were performed. Vitreous samples were collected from patients with PDR; the PDR1 group had no need for repeated surgical intervention, and the PDR2 group had repeated vitreous bleeding or other complication and controls. Levels of MPO, H3Cit-MPO, and NE-MPO complex were measured with enzyme-linked immunosorbent assay (ELISA).Results:Massive influx of CD11+ inflammatory cells, involving the anterior and posterior chambers, was observed in the murine eyes 24 h after the IL-8 or TNF-α injections. Cells excreted to their surroundings an extracellular net-like structure positive for NE, MPO, and H3Cit. H3Cit staining was abolished with the DNase I treatment, indicating the presence of extracellular DNA in the net-like structures. The vitreous samples of the patients with PDR2 contained statistically significantly higher levels of MPO (173±230) compared to those of the patients with PDR1 (12.0±33.0, p<0.05) or the controls (0.00, p<0.01). The levels of H3Cit-MPO and NE-MPO complexes were also statistically significantly higher in the patients with PDR2 (776.0±1274, 573.0±911.0, respectively) compared to those in the patients with PDR1 (0, p<0.05) and the controls (0, p<0.05).Conclusions:This study showed the existence of NETosis in cytokine-induced ocular inflammation in a mouse model and human samples. Furthermore, the extent of NET complex formation was higher in a subset of patients who exhibited more complicated PDR.

Program Number: 3321 Poster Board Number: D0124 Presentation Time: 8:30 AM–10:15 AM AAV2 Mediated Expression of Intraceptor Flt23k Shows No Endoplasmic Reticulum Stress Response Austin Bohner, Xiaohui Zhang, Hironori Uehara, Bonnie Archer, Balamurali Ambati. John Moran Eye Center, University of Utah School of Medicine, Salt Lake City, UT. Purpose: Current therapy of neovascular age-related macular degeneration is a multiple intravitreal injection of an anti-VEGF-A drug. Previously we demonstrated that single subretinal injections of a AAV2 mediated expression of Flt23k was able to inhibit vascular endothelial growth factor (VEGF) in mice and effectively inhibit laser-induced murine choroidal neovascularization (CNV) for up to six months. Because our novel treatment method acts by maintaining VEGF in the endoplasmic reticulum (ER) and thus blocks extracellular release of VEGF, further study has now been conducted to determine the effect of sequestered VEGF on ER stress. Methods: An overexpression of protein in the ER has been shown to activate signalling pathways that lead to an increase in the production of molecular chaperones GRP78/BiP and result in increased splicing of XBP-1 mRNA. We examined whether Flt23k overexpression upregulated GRP78/BiP and XBP-1 mRNA splicing as markers for ER stress in Hela cells via western blot and RT-PCR compared with soluble Flt-1 overexpression. To investigate the effects on ER stress of AAV2.Flt23k in a murine model, mice were given a single subretinal injection of 1 μl (5x108 vector genomes) of AAV2.Flt23k. Control groups received a single injection of an equivalent amount of AAV2.AcGFP, PBS or no injection. Six months post subretinal injection all groups had RPE/ choroid tissues harvested, sectioned, and analyzed. ER stress and apoptosis was assessed in RPE cells using Anti-GRP78/BiP staining and TUNEL staining method. Results: AAV2.Flt23k overexpression in Hela cells did not induce an upregulated response of ER stress markers at mRNA or protein level compared to positive control cells. Mice treated with a subretinal injection AAV2.Flt23k also did not exhibit increased GRP78 BiP expression or apoptosis in RPE of treated mice compared to positive control mice. Conclusions: The absence of ER stress increases confidence in the safety of AAV2.Flt23k as a novel treatment approach to neovascular AMD. Potentially, AAV2.Flt23k may induce efficient degradation of VEGF-A. Future study will focus on the cellular methods of the degradation of sequestered VEGF which prevent the cell from experiencing ER stress. Commercial Relationships: Austin Bohner, None; Xiaohui Zhang, None; Hironori Uehara, None; Bonnie Archer, None; Balamurali Ambati, None Support: RPB Unrestricted Award and NEI 5R01EY017182

The aim of this experimental study was to compare the efficacy of topical aflibercept and topical bevacizumab in preventing corneal neovascularization. A chemical burn was created in the right central cornea of male Sprague-Dawley rats, followed immediately by instillation of one drop (25 mg/ml, 20 μl volume) of aflibercept (15 eyes), bevacizumab (14 eyes), or saline (15 eyes). Treatment was repeated twice daily for 7 days. Corneal neovascularization was determined using corneal photographs (ImageJ) on days 1, 4, 7, 10, and histological and immunofluorescence studies, on day 10. Stromal immunoreactivity was evaluated 2 days after injury in 6 rats treated singly with bevacizumab or aflibercept. Corneal neovascularization was observed clinically on day 4 in all groups. In the aflibercept group, the area of neovascularization increased from 7.38 ± 2.23% on day 4 to 21.73 ± 14.59% on day 7 and 31.0 ± 23.61% on day 10. Corresponding values in the bevacizumab group were 6.04% ± 1.81%, 51.27 ± 15.50%, and 54.4 ± 11.33%, and in the control group, 8.99 ± 1.93%, 42.6 ± 19.59%, and 55.15 ± 11.54%. The area of neovascularization was significantly smaller on days 7 and 10 in the aflibercept group than in the control and bevacizumab groups (P < 0.001, all analyses), with no significant differences between the latter two groups (day 7, P = 0.868; day 10, P = 0.213). Clinical findings were compatible with the histological data and supported by immunofluorescence and corneal flat-mount staining. Both drugs demonstrated variable penetration into the corneal stroma. Topical aflibercept effectively inhibits corneal neovascularization in a rat model of chemical burn. These findings may have important therapeutic implications for humans.

Antivascular endothelial growth factor (Anti-VEGF) agents have been widely used for a variety of ocular disorders. The etiology of sustained ocular hypertension following intravitreal administration of anti-VEGF agents is yet to be unraveled. Our study investigates and characterizes the presence of intravitreally injected bevacizumab in the aqueous outflow channels of a rat model. Choroidal neovascularization (CNV) was induced by diode laser photocoagulation to the right eye of twelve Brown Norway rats. Bevacizumab (25 mg/ml) was injected intravitreally after 3 days. Immediately after bevacizumab injection, and 3, 6, 24 and 48 h later, animals were euthanized for immunofluorescence staining. Donkey anti-human IgG labeled with Alexa Fluor(®) 488 was used for bevacizumab immunoreactivity detection. Anti-CD31 antibody was used as a marker for Schlemm's canal endothelial cells. Untreated eyes were used as negative controls. The intensity of the immunostaining was analyzed qualitatively. Bevacizumab immunoreactivity was found in the aqueous outflow channels including the trabecular meshwork and Schlemm's canal immediately after injection, and declined incrementally within the following hours. Forty-eight hours after the injection, no bevacizumab staining was detected in the aqueous outflow channel structures. Our manuscript demonstrates the presence of bevacizumab in the trabecular meshwork and Schlemm's canal structures after intravitreal injection in a CNV induced rat model. Bevacizumab molecules passed through the aqueous outflow channels within 48 h after intravitreal bevacizumab injection.