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Background: Pathological angiogenesis is the hallmark of many vision-threatening diseases. Anti-VEGF is a primary treatment with substantial beneficial effects. However, such agents require frequent intravitreal injections. Our previous work established a method for effectively modifying exosomes (EXOs) for loading therapeutic peptides. Here, we used this system to load the anti-angiogenic peptide KV11, aiming to establish an EXO-based therapy strategy to suppress neovascularization in the retina. Methods: Using an anchoring peptide, CP05, we linked KV11 to endothelial cell (EC) derived EXOs, yielding EXO KV11 . We tested the delivery efficiency of EXO KV11 via two commonly used ocular injection methods: retro-orbital injection and intravitreal injection. Deploying an oxygen-induced retinopathy (OIR) model and a VEGF injection model, we tested the effects of EXO KV11 on neovascular formation, EC proliferation, and vascular permeability. In vitro experiments were used to test the mechanism and to analyze the effects of EXO KV11 on EC proliferation, migration, and sprouting. Results : By using the EXO loading system, KV11 was more efficiently delivered to the blood vessels of the mouse retina via retro-orbital injection. In both OIR model and VEGF injection model, EXO KV11 was more effective than KV11 alone in inhibiting neovascularization and vessel leakage. The therapeutic effect of retro-orbital injection of EXO KV11 was comparable to the intravitreal injection of VEGF-trap. Mechanistically, KV11 alone inhibited VEGF-downstream signaling, while EXO KV11 showed a stronger effect. Conclusions: We used EXOs as a carrier for intraocular delivery of KV11. We showed that KV11 itself has an anti-angiogenic effect through retro-orbital injection, but that this effect was greatly enhanced when delivered with EXOs. Thus, this system has the potential to treat proliferative retinopathy via retro-orbital injection which is a less invasive manner compared with intravitreal injection., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

To evaluate the effects of intravitreal ranibizumab injection (IVR) on metamorphopsia in patients with branch retinal vein occlusion (BRVO), and to assess the relationship between metamorphopsia and inner retinal microstructure and other factors. Thirty-three treatment-naïve eyes of 33 patients with macular edema caused by BRVO with at least 12 months of follow-up were included. The degree of metamorphopsia was quantified using the M-CHARTS. Retinal microstructure was assessed with spectral-domain optical coherence tomography. Disorganization of the retinal inner layers (DRIL) at the first month after resolution of the macular edema (early DRIL) and at 12 months after treatment (after DRIL) was studied. Central retinal thickness (CRT), and status of the external limiting membrane as well as ellipsoid zone were also evaluated. IVR treatment significantly improved best-corrected visual acuity (BCVA) and CRT, but the mean metamorphopsia score did not improve even after 12 months. Post-treatment metamorphopsia scores showed a significant correlation with pre-treatment metamorphopsia scores (P < 0.005), the extent of early DRIL (P < 0.05) and after DRIL (P < 0.05), and the number of injections (P < 0.05). Multivariate analysis revealed that the post-treatment mean metamorphopsia score was significantly correlated with the pre-treatment mean metamorphopsia score (P < 0.05). IVR treatment significantly improved BCVA and CRT, but not metamorphopsia. Post-treatment metamorphopsia scores were significantly associated with pre-treatment metamorphopsia scores, the extent of DRIL, and the number of injections. Prognostic factor of metamorphopsia was the degree of pre-treatment metamorphopsia.

In current clinical settings, frequent intravitreal (IVT) injections of anti-vascular endothelial growth factors are used due to their short in-vivo half-life and rapid clearance from the back of the eye. The IVT injections are associated with pain, risk of infection, retinal detachment, and financial burden. Biologics molecules can undergo physical, chemical, and enzymatic degradation during formulation development and in the biological environment. Moreover, the complex ocular structures also act as a rate-limiting barrier for these biologics. Thus, delivering stable and clinically relevant biologics concentration to the back of the eye is still a challenge. Compare to other drug delivery platforms, injectable in-situ gelling depot systems (IISGDs) have emerged as an effective system for biologics delivery. In this review, we have discussed various biologics used in ocular therapeutics and their associated challenges. Different routes of delivery and associated tissue barriers are also discussed. Different types of IISGDs developed to date for biologics delivery to the back of the eye were also covered. To conclude, various critical parameters related to the formulation development process and injectable depot systems that need careful consideration and further investigations were highlighted.

Proliferative vitreoretinopathy (PVR) is a significant threat for vision recovery from retinal detachment or ocular trauma. Currently, no approved pharmacological intervention to prevent PVR. Daunorubicin (DNR) and dexamethasone (DEX) were sequentially loaded into oxidized porous silicon (pSiO 2 ) particles by covalent conjugation. The DNR + DEX-loaded particles, and control particles loaded with DNR only and DEX only were incubated with RPE-populated collagen for daily gel surface quantitation. Toxicity was monitored by ophthalmic examinations and histological evaluation 21 days after injection. At 3rd week following intravitreal injection, a localized retinal detachment (RD) was created by subretinal injection of Healon in all pretreated eyes in addition to 3 non-interventional control eyes. 10 µg of bromodeoxyuridine (BrdU) was injected into the vitreous 4 h before sacrifice on day 3 after RD induction. Retinal sections were stained for glial fibrillary green protein (GFAP) and BrdU to identify activated glial cells and retinal cell proliferation. The studies demonstrated that all three pSiO 2 particle types were well tolerated in vivo. DNR alone and DNR + DEX combination formulations demonstrated equally strong suppression on gel contraction (least square mean area of the gel: control = 1.71 vs. 30DNR = 1.85 or 30/40Dual = 1.83, p  < .05). Eyes pretreated with pSiO 2 -DNR + DEX exhibited the least GFAP activation (least square mean intensity mm -2 : Dual = 4.03, DNR = 7.76, Dex = 16.23, control = 29.11, p  < .05) and BrdU expression (Mean number of BrdU positive cells per mm of retina: Dual = 2.77, DNR = 4.58, Dex = 4.01, control = 6.16, p  < .05). The synergistic effect of a sustained release pSiO 2 -DNR/DEX showed promise for the prevention of PVR development while reducing the necessary therapeutic concentration of each drug.

Purpose: To estimate the diffusion coefficients of an IgG antibody (150 kDa) and its antigen-binding fragment (Fab; 50 kDa) in the neural retina (D ret ) and the combined retinal pigment epithelium-choroid (D RPE-cho ) with a 3-dimensional (3D) ocular pharmacokinetic (PK) model of the rabbit eye., Methods: Vitreous, retina, and aqueous humor concentrations of IgG and Fab after intravitreal injection in rabbits were taken from Gadkar et al. (2015). A least-squares method was used to estimate D ret and D RPE-cho with the 3D finite element model where mass transport was defined with diffusion and convection. Different intraocular pressures (IOP), initial distribution volumes (V init ), and neural retina/vitreous partition coefficients (K ret/vit ) were tested. Sensitivity analysis was performed for the final model., Results: With the final IgG model (IOP 10.1 Torr, V init 400 μl, K ret/vit 0.5), the estimated D ret and D RPE-cho were 36.8 × 10 -9 cm 2 s -1 and 4.11 × 10 -9 cm 2 s -1 , respectively, and 76% of the dose was eliminated via the anterior chamber. Modeling of Fab revealed that a physiological model parameter "aqueous humor formation rate" sets constraints that need to be considered in the parameter estimation., Conclusions: This study extends the use of 3D ocular PK models for parameter estimation using simultaneously macromolecule concentrations in three ocular tissues.

Accumulated evidence indicates that platelet-derived growth factor (PDGF) contributes to various types of tissue regeneration. However, the effects and mechanisms of PDGF signaling for retina regeneration have not been sufficiently investigated. To clarify this, we investigated the role of PDGF signaling in retina regeneration process after needle puncture in zebrafish. Time-course analysis showed a spike peak of pdgf-a at 6 h after injury and a broad peak of pdgf-b during 6-96 h after injury. Inhibition of PDGF signaling with AG1295 suppressed BrdU-positive proliferative cell numbers at 4 days after injury. At the same time, retina regeneration-associated transcription factors, ascl1a and pax6b, were down-regulated by AG1295 treatment. Intravitreal injection of human recombinant PDGF-AA or -BB into intact zebrafish induced the cell proliferation. PDGF-BB injection induced the Müller glia-derived neurogenic cluster; PDGF-AA increased the 4C4-positive microglia. These findings indicate that PDGF signaling contributes to retina regeneration in zebrafish and causes different types of cell proliferation, depending on each subtype of PDGF. (160 words)., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

S100B is a glial protein, which belongs to calcium-binding protein family. Alterations of S100B level were noted in various neurodegenerative diseases. In a new glaucoma-like animal model S100B was injected intravitreally, which led to neuronal degeneration in retina and optic nerve. The pathological mechanisms are still unknown. Therefore, S100B protein was intravitreally injected in rats. At days 14 and 21, retina, optic nerve, serum, and aqueous humor were investigated. S100B injection led to an increase of retinal NF-κB at day 14. Furthermore, higher IL-1β levels in retina, serum, and aqueous humor were measured. A co-localization of microglia and IL-1β was noted, which correlated with an increased microglia response in retina and optic nerve at day 14. At the same point in time, more apoptotic RGCs and a decline in RGC numbers were observed. At 21 days, this damage was still present, but no signal pathway activations were detectable anymore. Interestingly, macroglia were not affected at any point in time. We conclude that S100B activated the NF-κB signal pathway, which then regulated IL-1β production and release from microglia. A positive feedback loop of IL-1β likely stimulates microglia in a pro-inflammatory manner. These microglia probably induce apoptotic damage in retina and optic nerve. Meanwhile, the injected S100B protein was naturally degraded, which explains the resting state of the pro-inflammatory signal pathways with constant damage later on. The inhibition of S100B release or microglia response could potentially decrease the damage in degenerative diseases, like glaucoma.

Objective: This study was conducted to evaluate the effects of anti-vascular endothelial growth factor (VEGF) antibody (bevacizumab) on vascular leakage and fibrosis in a monkey choroidal neovascularization (CNV) model. The relationship between fibrotic tissue and subretinal hyper-reflective material (SHRM), in optical coherence tomography (OCT) images, was also investigated., Methods: Experimental CNV was induced in male cynomolgus monkeys by laser photocoagulation. Intravitreal injection of bevacizumab at 0.5 mg/eye/dosing was initiated 2 weeks before or after laser irradiation and thereafter, conducted intermittently at 2- or 3-week intervals. Fluorescein fundus angiography (FA) and OCT imaging were conducted weekly from 2 to 7 weeks after laser irradiation. CNV leakage was evaluated by an established grading method using FA images. To assess the fibrosis and scarring, Masson's trichrome specimens of each CNV lesion were prepared, and morphometric analysis was conducted using an image analysis software., Results: The effects of bevacizumab on vascular leakage were shown using an established evaluation method. Morphometric analysis of Masson's trichrome-stained (MT) specimens revealed that collagen fiber synthesis was suppressed by bevacizumab pre-treatment (-29.2%) or post-treatment (-19.2%). SHRM was detected in OCT images in a monkey CNV model, and a significant correlation between the SHRM area in the OCT images and the collagen fiber area in the MT specimens was noted., Conclusion: In the established cynomolgus monkey CNV model, bevacizumab prevented blood leakage but could not completely suppress fibrosis. SHRM in the OCT images reflected retinal fibrous tissue in a laser-induced CNV monkey model. This model might be useful for elucidating the pathology and development therapy for neovascularization or fibrosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Considerable research over the last few years has revealed dysregulation of growth factors in various retinal diseases, such as glaucoma, diabetic retinopathy and photoreceptor degenerations. The use of messengerRNA (mRNA) to transiently overexpress a specific factor could compensate for this imbalance. However, a critical challenge of this approach lies in the ability to efficiently deliver mRNA molecules to the retinal target cells. In this study we found that intravitreal (IVT) injection is an attractive approach to deliver mRNA to the retina, providing two critical barriers can be overcome: the vitreous and the inner limiting membrane (ILM). We demonstrated that the vitreous is indeed a major hurdle in the delivery of the cationic mRNA-complexes to retinal cells, both in terms of vitreal mobility and cellular uptake. To improve their intravitreal mobility and avoid unwanted extracellular interactions, we evaluated the use of hyaluronic acid (HA) as an electrostatic coating strategy. This HA-coating provided the complexes with a negative surface charge, markedly enhancing their mobility in the vitreous humor, without reducing their cellular internalization and transfection efficiency. However, although this coating strategy allows the mRNA-complexes to successfully overcome the vitreal barrier, the majority of the particles accumulated at the ILM. This study therefore underscores the crucial barrier function of the ILM toward non-viral retinal gene delivery and the need to smartly design mRNA-carriers able to surmount the vitreous as well as the ILM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Intravitreal injection is one of the major administration routes for the treatment of posterior ocular diseases. Intravitreal therapeutics usually suffer from unsatisfactory efficacy owing to fast clearance from the vitreous humour and insufficient distribution into the retina. Engineered nanoparticles have been applied for specific tissue targeting over the past decades. In this review, we summarize the most recent research utilizing intravitreal nanoparticles to deliver therapeutics to the retina. Herein, the achievement made in preclinical research and challenges remaining in the field are highlighted. Parameters including size, charge, stability and choice of modified ligand on intraocular distribution and transport are also systematically discussed based on a proposed pharmacokinetic model. We provide insights for rational design principles for intravitreal nanoparticles for targeted retinal delivery., (Copyright © 2019. Published by Elsevier Ltd.)

Objective: N-methyl-D-aspartate (NMDA) excitotoxicity has been proposed to mediate apoptosis of retinal ganglion cells (RGCs) in glaucoma. Taurine (TAU) has been shown to have neuroprotective properties, thus we examined anti-apoptotic effect of TAU against retinal damage after NMDA exposure., Methodology: Sprague-Dawley rats were divided into 5 groups of 33 each. Group 1 was administered intravitreally with PBS and group 2 was similarly injected with NMDA (160 nmol). Groups 3, 4 and 5 were injected with TAU (320 nmol) 24 hours before (pre-treatment), in combination (co-treatment) and 24 hours after (post-treatment) NMDA exposure respectively. Seven days after injection, rats were sacrificed; eyes were enucleated, fixed and processed for morphometric analysis, TUNEL and caspase-3 staining. Optic nerve morphology assessment was done using toluidine blue staining. The estimation of BDNF, pro/anti-apoptotic factors (Bax/Bcl-2) and caspase-3 activity in retina was done using ELISA technique., Results: Severe degenerative changes were observed in retinae after intravitreal NMDA exposure. The retinal morphology in the TAU pre-treated group appeared more similar to the control retinae and demonstrated a higher number of nuclei than the NMDA group both per 100 μm length (by 1.5-fold, p < 0.001) and per 100 μm 2 area (by 1.41-fold, p < 0.05) of the GCL. After NMDA exposure, visible axonal swelling was observed in optic nerve sections. In comparison with the changes observed in the NMDA treated group, the TAU treated group showed fewer prominent changes; axonal swelling was less frequent and less marked. Additionally, no marked glial cell changes were observed in the TAU-pretreated group. All TAU treated groups, particularly the pre-treated group, showed a significant decrease in the NMDA-induced optic nerve damage, with a 50% reduction (p < 0.001) in the mean grading compared to NMDA group. For the same, there was 25% decrease in co- and post-treatment groups, as compared with the NMDA group. Pre-treatment with TAU abolished apoptotic response to NMDA as indicated by decrease in the number of TUNEL- and caspase-3-positive cells. TAU pre-treatment also increased the Bcl-2 level (by 2.80-fold, p < 0.001) and decreased the level of Bax (by 34%, p < 0.01), and activity of caspase-3 (by 36%, p < 0.001) compared to NMDA group., In Conclusion: our study revealed that pre-treatment with TAU prevents NMDA-induced retinal cell apoptosis more effectively than co- and post-treatment with TAU., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: To evaluate safety and efficacy of the vascular endothelial growth factor binding protein abicipar pegol (abicipar) versus ranibizumab for neovascular age-related macular degeneration. Methods: Phase 2, multicenter, randomized, double-masked comparison (REACH study, stage 3). Patients ( n  = 64) received intravitreal injections of abicipar 1 mg or 2 mg at baseline, week 4, and week 8 (3 injections) or ranibizumab 0.5 mg at baseline and monthly (5 injections). Results: In the abicipar 1 mg ( n  = 25), abicipar 2 mg ( n  = 23), and ranibizumab ( n  = 16) arms, respectively, least-squares mean best-corrected visual acuity (BCVA) change from baseline was +6.2, +8.3, and +5.6 letters at week 16 (primary endpoint) and +8.2, +10.0, and +5.3 letters at week 20. Least-squares mean central retinal thickness (CRT) reduction from baseline was 134, 113, and 131 μm at week 16 and 116, 103, and 138 μm at week 20. Intraocular inflammation adverse events (AEs), reported in 5/48 (10.4%) abicipar-treated patients, resolved without sustained vision loss or other sequelae. Conclusions: Abicipar demonstrated durability of effect: BCVA and CRT improvements were similar between abicipar and ranibizumab at weeks 16 and 20 (8 and 12 weeks after the last abicipar injection and 4 weeks after the last ranibizumab injection). No serious AEs were reported.

Background: Drug ocular toxicity is a field that requires attention. Clindamycin has been injected intravitreally to treat ocular toxoplasmosis, the most common cause of eye posterior segment infection worldwide. However, little is known about the toxicity of clindamycin to ocular tissues. We have previously showed non intraocular toxicity in rabbit eyes of poly(lactic-co-glycolic acid) (PLGA) implants containing clindamycin hydrochloride (CLH) using only clinical macroscotopic observation. In this study, we investigated the in vivo biocompatibility of CLH-PLGA implants at microscotopic, cellular and molecular levels., Methods: Morphology of ARPE-19 and MIO-M1 human retinal cell lines was examined after 72 h exposure to CLH-PLGA implant. Drug delivery system was also implanted in the vitreous of rat eyes, retinal morphology was evaluated in vivo and ex vivo. Morphology of photoreceptors and inflammation was assessed using immunofluorescence and real-time PCR., Results: After 72 h incubation with CLH-PLGA implant, ARPE-19 and MIO-M1 cells preserved the actin filament network and cell morphology. Rat retinas displayed normal lamination structure at 30 days after CLH-PLGA implantation. There was no apoptotic cell and no loss in neuron cells. Cones and rods maintained their normal structure. Microglia/macrophages remained inactive. CLH-PLGA implantation did not induce gene expression of cytokines (IL-1β, TNF-α, IL-6), VEGF, and iNOS at day 30., Conclusion: These results demonstrated the safety of the implant and highlight this device as a therapeutic alternative for the treatment of ocular toxoplasmosis.

Purpose: To study the suitability of injectable microspheres based on poly(ester amide) (PEA) or poly lactic-co-glycolic acid (PLGA) as potential vehicles for intravitreal drug delivery in rat eyes. Dexamethasone-loaded PEA microspheres (PEA + DEX) were also evaluated., Methods: Forty male Sprague Dawley rats were divided into four groups that received different intravitreally injected microspheres: PEA group (n = 12); PLGA group (n = 12); PEA + DEX group (n = 8); and control group (no injection, n = 8). Electroretinography (ERG), fundus autofluorescence (FAF), and spectral domain optical coherence tomography (sdOCT) were performed at baseline, weeks 1 and 2, and months 1, 2, and 3 after intravitreal injection. Eyes were histologically examined using light microscopy and transmission electron microscopy at the end of the in vivo study., Results: There were no statistically significant changes in ERG among the groups. Abnormal FAF pattern and abnormal deposits in OCT were observed after injection but almost completely disappeared between week 2 and month 3 in all injected groups. GFAP staining showed that Müller glia cell activation was most pronounced in PLGA-injected eyes. Increased cell death was not observed by TUNEL staining at month 1. In electron microscopy at month 3, the remnants of microparticles were found in the retinal cells of all injected groups, and loss of plasma membrane was seen in the PLGA group., Conclusions: Although morphological changes such as mild glial activation and material remnants were observed histologically 1 month and 3 months after injection in all injected groups, minor cell damage was noted only in the PLGA group at 3 months after injection. No evidence of functional abnormality relative to untreated eyes could be detected by ERG 3 months after injection in all groups. Changes observed in in vivo imaging such as OCT and FAF disappeared after 3 months in almost all cases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Recent studies have shown that Connexin43 mimetic peptide (Cx43 MP) can prevent secondary damage following several retinal ischemic and inflammatory disorders by blocking the pathological opening of gap junction hemichannels. However, the poor stability of peptides and the presence of various intraocular barriers limit efficient retinal delivery in the clinical setting. The present study aimed to prolong the bioactivity of Cx43 MP and achieve targeted delivery to the retina by loading the peptide into hyaluronic acid (HA) coated human serum albumin nanoparticles (HSA NPs). Two different loading methods, adsorption and incorporation, were used with the peptide released slowly over a period of up to four months. Compared to uncoated particles, HA coated HSA NPs exhibited enhanced in vitro cellular uptake and ex vivo retinal penetration via HA-CD44 receptor mediated interactions. Furthermore, cell viability and Cx43 MP functionality assays showed that NPs protected Cx43 MP from degradation, sustained its release, and thus prolonged its bioactivity without reducing cell viability at concentrations used for Cx43 hemichannel blocking. Therefore, HA coated HSA NPs could have great potential for sustained and targeted delivery of Cx43 MP to treat various retinal inflammatory conditions.

There is increasing use of the vitreous cavity as a reservoir for drug delivery. We study the intraocular migration and distribution of triamcinolone acetonide (TA) after injection into silicone oil tamponade agent during and after vitrectomy surgery ex vivo (pig eye) and in vitro (glass bottle). For ex vivo assessment, intraocular migration of TA was imaged using real-time FLASH MRI scans and high-resolution T2W imaging and the in vitro model was monitored continuously with a video camera. Results of the ex vivo experiment showed that the TA droplet sank to the interface of silicone oil and aqueous almost immediately after injection and remained inside the silicone oil bubble for as long as 16 minutes. The in vitro results showed that, after the shrinkage of the droplet, TA gradually precipitated leaving only a lump of whitish crystalline residue inside the droplet for about 100 minutes. TA then quickly broke the interface and dispersed into the underlying aqueous within 15 seconds, which may result in a momentary increase of local TA concentration in the aqueous portion and potentially toxic to the retina. Our study suggests that silicone oil may not be a good candidate as a drug reservoir for drugs like TA.

Retinal ocular toxicity is among the leading causes of drug development attrition in the pharmaceutical industry. Electroretinography (ERG) is a non-invasive functional assay used to assess neuro-retinal physiological integrity by measuring the electrical responses. To directly assess the utility of ERG, a series of studies was conducted following intravitreal and/or iv administration of pan-cyclin-dependent kinase inhibitors: AG-012,986 and AG-024,322 in rats. Both compounds have previously shown to induce retinal toxicity. Retinal injury was evaluated by ERG, histopathology and TUNEL staining. Intravitreal injection of AG-012,986 at ≥ 10 μg/eye resulted in decreases (60%) in ERG b-wave and microscopic changes of mild to moderate retinal degeneration, and at 30 μg/eye led to additional ophthalmic findings. Intravenous administration of AG-012,986 daily at ≥ 5 mg/kg resulted in dose-related decreases (25 to 40%) in b-wave and sporadic to intense positive TUNEL staining. Intravitreal injection of AG-024,322 at 30 μg/eye also resulted in decreases (50 to 60%) in b-wave, mild to marked retinal degeneration and mild vitreous debris. These experiments demonstrate that ERG can be used as a sensitive and reliable functional tool to evaluate retinal toxicity induced by test compounds in rats complementing other classical ocular safety measurements., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques., Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR) was used to evaluate expression of apoptosis-inducing caspases (8 and 3)., Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo) following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10%) or necrotic (<1%) activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group., Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

Purpose: To describe retinal changes during Spectral Domain Optical Coherence Tomography (SD-OCT) guided bevacizumab treatment for neovascular age- related macular degeneration (AMD)., Settings and Design: Single center observational study., Materials and Methods: We confirmed wet AMD in 47 eyes of 45 patients by fluorescein angiography and SD-OCT. After bevacizumab injection, we examined the patients at 4-week intervals. During each follow-up control, we performed SD-OCT and a complete ophthalmic examination. Criteria for reinjection were visual acuity loss of more than five ETDRS letters, and/or increase of central retinal thickness, sub-retinal fluid, intra-retinal fluid, pigment epithelium detachment. If reinjection criteria were not met, we advised the patient to return in 4 weeks' time for the next scheduled follow-up. We used 3-dimensional SD-OCT to measure photoreceptor defects and sub-retinal fibrosis. The main efficacy endpoints were the SD-OCT measurements of the size of photoreceptor defects, the size of external membrane defects and the central retinal thickness., Results: Over the 12 months study period, the percentage of scans in 3-D imaging mode showing visible defects of the junction between inner and outer segments of photoreceptors increased from 38.96 to 53.8%. The percentage of scans in 3-D imaging mode with visible sub-retinal fibrosis increased from 33 to 52% and mean central retinal thickness decreased from 333 μm (96-900 μm) to 272 μm (P = 0.011)., Conclusion: In long-term anti- Vascular endothelial growth factor (VEGF) treatment for neovascular AMD, photoreceptor defects and fibrosis progress despite a decrease in central retinal thickness and improvements in visual acuity. We would encourage further discussion as to whether this is the natural course of the disease or a result of the treatment.

Purpose: To evaluate the efficacy of intravitreal bevacizumab (IVB) injection combined with anterior retinal cryotherapy (ARC) in the treatment of proliferate diabetic retinopathy (PDR) with vitreous hemorrhage (VH)., Methods: A retrospective case controlled study was performed on 67 cases (67 patients) with PDR and persistent VH obscuring the fundus detail for ≧3 weeks. The follow-up period was ≧6 months. Cases with extensive vitreo-retinal adhesion by ultrasonography were excluded. In the study group, transconjunctival ARC (a total of 12 spots) followed by IVB 1.25 mg was performed in the same setting; in the control group, only IVB was given. Panretinal photocoagulation was performed when possible. Repeated IVB was performed in both groups if VH persisted for >4 weeks following the initial treatment. Vitrectomy was performed if VH showed no improvement for 12 to 16 weeks. The vitreous clear-up time (VCUT) in the posterior pole and the rate of vitrectomy were compared between the 2 groups., Results: In the study group (n=35), second treatment was required in 7 out of 35 patients (20.0%). Vitrectomy was performed in 5 (14.3%) patients. In the control group (n=32), 8 out of 32 eyes (25.0%) underwent second injection. Four eyes (12.5%) had vitrectomy within the follow-up time. VCUT in the study and the control group was 6.9±8.7 weeks and 13.0±9.3 weeks respectively (P=0.003)., Conclusion: Combined treatment of ARC and IVB is associated with more rapid clearing of VH in eyes with PDR compared with IVB alone.

Purpose: To study the visual outcomes and change in central macular thickness (CMT) in patients with neovascular age-related macular degeneration (AMD) who were previously treated with ranibizumab (Lucentis) and/or bevacizumab (Avastin) and were subsequently switched to aflibercept (VEGF Trap-Eye; Eylea)., Methods: Retrospective study of patients who received intravitreal aflibercept from December 2011 to December 2012 and had previous anti-vascular endothelial growth factor treatment for AMD. The main outcome measures were best-corrected visual acuity (BCVA) and CMT as measured by optical coherence tomography., Results: The study population included 30 patients aged 80.4±1.45 (mean±SEM) who received 6.27±0.37 (range 4-11) aflibercept injections. Eighteen patients had previously received only bevacizumab (12.4±2.18 injections), 2 had received only ranibizumab (19±6 injections), and 10 had received both ranibizumab and bevacizumab (mean 19.3 injections). BCVA logMAR at the initial visit (aflibercept initiation) was 0.506±0.054 (mean VA 20/64), and then, follow ups at 1-month 0.504±0.055 (20/64) P=0.903, 3-months 0.458±0.061 (20/57) P=0.112, 6-months 0.413±0.071 (20/52) P=0.036, and 12-months 0.521±0.076 (20/66) P=0.836. CMT at the initial visit was 261±10.9, and then, at 1-month 238±12.4 P=0.021, 3-months 245±10.6 P=0.102, 6-months 245±10.4 P=0.099, and 12-months 237±10.2 P=0.012. Results were similar in a subset of patients (n=15) with central macular edema or submacular fluid at aflibercept initiation. While on aflibercept, 2 patients developed intraocular pressure increases that required treatment., Conclusions: These findings demonstrate a significant decrease in CMT but no statistically significant improvement in BCVA through the 12-month follow up in patients previously treated who were switched to aflibercept for AMD. Patients may develop ocular hypertension after multiple aflibercept injections.

To investigate a simple and effective intraocular xenotransplant technique of rhesus monkey neural progenitor cells to rats, mechanical injury was induced in the rat's right retina. And the GFP-labeled rhesus monkey neural progenitor cells suspension was slowly injected into the vitreous space of the right injured and left control eye. Confocal image suggested that the xenografted cells survived in both the injured and control eye, meanwhile the cells integrated in the injured right retina. The results demonstrated that intravitreal xenotransplant could be adopted as a simple and reliable method.

Purpose: To assess serum level changes of antiretinal antibodies (ARA) in patients with exudative age-related macular degeneration (AMD), treated with transpupillary thermotherapy (TTT) alone and with TTT combined with injection of triamcinolone acetonide (sTTA) under posterior Tenon's capsule and to compare the efficacy of TTT and TTT+sTTA. The purpose of the study was also to estimate if serum ARA may act as the biomarker of AMD., Material and Methods: This prospective study comprised 46 patients (46 eyes) with exudative AMD. Patients were assigned into: group I (n = 24) received TTT alone and group II (n = 20) received TTT with sTTA. Follow-up was at 3, 6, 9 and 12 months, when best-corrected visual acuity (BCVA), Amsler grid-test, intraocular pressure (IOP), fluorescein angiography (FA) and central retinal thickness (CRT) by optical coherence tomography (OCT) were assessed. In all patients serum ARA was determined using indirect immunofluorescence (IIF) method on normal monkey retina as antigens substrate and FITC--labelled goat's anti-human IgA, G, M serum as the secondary antibody., Results: Baseline serum ARA titres in group I ranged from 1: 40 to 1: 5120 and in group II--1: 40 to 1: 1280 (p = 0.1). In control group serum ARA was present in 46.4% of sera in titres from 1: 10 to 1: 40. These differences were statistcally significant (p < 0.001). Nine fluorescence patterns of ARA were detected by IIF method in both groups of AMD patients, while control sera showed only three types of reaction. Statistically significant correlation was found between CNV size, CRT and serum ARA titres in both groups of patients (p < 0.01). In a follow-up period decreasing serum ARA titres were noted, specially for subjects treated with combined therapy, however it was not statistically significant at 3, 6 and 9 months while achieved significance (p < 0.01) at month 12. BCVA improvement or stabilization was observed in 64% of eyes in group I and in 75%--in group II (p = 0.1). No leakage on FA was found in 66.7% and 70% of cases, in group I and II, respectively (p = 0.01). CRT reduction was observed in 50% (group I) and in 70% (group II) of eyes (p = 0.01) at month 12., Conclusions: Our preliminary observations indicate that serum ARA changes may reflect the activity of CNV in a course of AMD and may act as the biomarker of treatment efficacy. The use of sTTA in conjunction with TTT for CNV in AMD showed a tendency towards lower serum ARA titres and better functional results after treatment as compared with eyes treated with TTT alone.

Purpose: To determine whether the co-injection of extracellular matrix degrading enzymes improves retinal transduction following intravitreal delivery of adeno-associated virus-2 (AAV2)., Methods: AAV2 containing cDNA encoding enhanced green fluorescent protein (GFP), under the control of a chicken β-actin promoter, was delivered by intravitreal injection to adult mice in conjunction with enzymes including collagenase, hyaluronan lyase, heparinase III, or chondroitin ABC lyase. Two weeks later, retinal flatmounts were examined for GFP expression using confocal microscopy., Results: Without the addition of enzymes, transduction was limited to occasional cells in the retinal ganglion cell layer. The addition of heparinase III or chondroitin ABC lyase greatly enhanced transduction of the retinal ganglion cell layer and increased the depth of transduction into the outer retina. Hyaluronan lyase had a limited effect and collagenase was ineffective. Electroretinograms survived with higher concentrations of heparinase III and chondroitin ABC lyase than were required for optimal retinal transduction., Conclusions: AAV2-mediated retinal transduction is improved by co-injection of heparinase III or chondroitin ABC lyase. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to both the inner and outer retina., (© 2011 Molecular Vision)