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Age-related macular degeneration (AMD) is a leading cause of blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids of human RPE cells constructed a well-differentiated monolayer of RPE with a Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between the RPE and Bruch's membrane with expression of many drusen-associated proteins, such as amyloid β and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases, such as AMD., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Proliferative vitreoretinopathy (PVR) is a challenging pathological condition, often causing failure of retinal detachment surgery. The purpose of this study was to evaluate the feasibility of a delivery system of bioactive proteins using anionic and cationic gelatin microspheres and to establish a new PVR model in rabbits by intraocular sustained delivery of basic fibroblast growth factor (bFGF) and interferon-beta (IFNβ). Anionic and cationic gelatin microspheres were prepared and immersed in bFGF and IFNβ solution, respectively, to yield a polyion complex between gelatin matrix and a bioactive protein. The bFGF-impregnated microspheres were injected into the subretinal space in rabbit eyes. At week 2, the IFNβ-impregnated microspheres also were injected into the same space. Control eyes received gelatin microspheres without bFGF or IFNß, or both. The eyes then were observed for 8 weeks by ophthalmoscopy, fundus photography, and fluorescein angiography. The eyes also were evaluated histologically. In the group with both bFGF and IFNβ, the number of eyes with more severe PVR increased over time. Histologic examination showed retinal folds. In contrast, no proliferative changes were seen in any control groups. Subretinal implantation of bFGF and IFNβ-impregnated gelatin microspheres induced reproducible PVR in rabbit eyes. This study guaranteed delivery of bioactive proteins with gelatin microspheres., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: To investigate the association between retinal neovascularization and the CC chemokine receptor-3 (CCR3) in a mouse model of oxygen-induced retinopathy (OIR)., Methods: An OIR model in C57BL/6J mice was used as a retinal neovascularization model. An enzyme-linked immunosorbent assay was performed to evaluate the chronological change in vascular endothelial growth factor A (VEGF-A) and eotaxin expressions. CCR3 and VEGF subtype expression in the retina was examined using real-time RT-PCR, and CCR3, eotaxin, VEGF-A, and CD31 expression was examined immunohistochemically. A CCR3 neutralizing antibody (Ab) was injected into the vitreous humor on both postnatal days 12 (P12) and 14 (P14). Retinal neovascularizations were quantified by measurement of the percentages of neovascular area., Results: The mean eotaxin and VEGF-A protein level was significantly downregulated at P10 and P12 and was significantly upregulated at P14 and P17 (p < 0.05). CCR3 mRNA expression was significantly upregulated at P12 (p < 0.05). VEGF164 mRNA expression was significantly upregulated at P14 (p < 0.05). The areas of vaso-obliteration and neovascularization were significantly suppressed in anti-CCR3 Ab-treated eyes (p < 0.05). Anti-CCR3 Ab treatment suppressed VEGF and eotaxin but not monocyte chemoattractant protein-1. And VEGF 164 mRNA but not VEGF120 mRNA was suppressed by anti-CCR3 Ab treatment., Conclusions: The present data suggest that anti-CCR3 treatment can suppress retinal neovascularization. Anti-CCR3 treatment may have potential as a new therapy for retinopathies with retinal neovascularization such as diabetic retinopathy and retinopathy of prematurity., (© 2017 S. Karger AG, Basel.)

Purpose: We investigate the antiangiogenic efficacy of tissue plasminogen activator (tPA) on experimental laser-induced choroidal neovascularization (CNV) in mice., Methods: After CNV was induced by laser photocoagulation in 92 C57BL/6J wild-type mice, tPA (4 or 40 international units [IU]/μl) or PBS was injected intravitreally immediately after laser injury. Fluorescein angiography was performed on day 7 to grade CNV leakage. The CNV volume was measured by confocal microscopy in eyes enucleated 7 days after laser injury. Immunohistochemical studies were performed 3 days after laser injury to evaluate fibrin/fibrinogen and CD31 expression. The possible adverse effects of tPA were assessed by electroretinography (ERG) and histology on day 7., Results: Intravitreal administration of tPA significantly suppressed CNV leakage and CNV volume in a dose-dependent manner (P < 0.01). Intravitreal injection of tPA suppressed fibrin/fibrinogen and CD31 expression in laser-induced lesions. Histologic examination and ERG showed no evidence of retinal toxicity in eyes injected with tPA., Conclusions: Intravitreal injection of tPA suppressed fibrin/fibrinogen expression and laser-induced CNV. The current results suggested that tPA may be a potential therapeutic adjuvant for treating CNV.

This report describes a 19-year-old patient with the rare association of multiple evanescent white dot syndrome (MEWDS) and choroidal neovascularization (CNV). Despite the initial diagnosis of MEWDS, her vision significantly decreased. Fluorescein angiography showed dye leakage and pooling at the macula, whereas optical coherence tomography (OCT) showed cystoid macular edema and serous neuroepithelial elevation. The dye leakage and pooling made it impossible to diagnose CNV. However, OCT angiography (OCTA) revealed a vascular structure in the outer retina, thereby leading to the CNV diagnosis. Visual acuity improved after an anti-vascular endothelial growth factor injection. OCTA proved to be useful in the diagnosis of CNV. [Ophthalmic Surg Lasers Imaging Retina. 2016;47:580-584.]., (Copyright 2016, SLACK Incorporated.)

Purpose: To observe changes in fundus autofluorescence 2 years after implantation of blue light-filtering (yellow-tinted) and ultraviolet light-filtering (colorless) intraocular lenses (IOLs)., Setting: Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan, and the Department of Ophthalmology, University of Bern, Bern, Switzerland., Design: Prospective comparative observational study., Methods: Patients were enrolled who had cataract surgery with implantation of a yellow-tinted or colorless IOL and for whom images were obtained on which the fundus autofluorescence was measurable using the Heidelberg Retina Angiogram 2 postoperatively. The fundus autofluorescence in the images was classified into 8 abnormal patterns based on the classification of the International Fundus Autofluorescence Classification Group, The presence of normal fundus autofluorescence, geographic atrophy, and wet age-related macular degeneration (AMD) also was recorded. The fundus findings at baseline and 2 years postoperatively were compared., Results: Fifty-two eyes with a yellow-tinted IOL and 79 eyes with a colorless IOL were included. Abnormal fundus autofluorescence did not develop or increase in the yellow-tinted IOL group; however, progressive abnormal fundus autofluorescence developed or increased in 12 eyes (15.2%) in the colorless IOL group (P = .0016). New drusen, geographic atrophy, and choroidal neovascularization were observed mainly in the colorless IOL group. The incidence of AMD was statistically significantly higher in the colorless IOL group (P = .042)., Conclusions: Two years after cataract surgery, significant differences were seen in the progression of abnormal fundus autofluorescence between the 2 groups. The incidence of AMD was lower in eyes with a yellow-tinted IOL., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.)

Aims: The purpose of this study was to evaluate the effect of pulse duration on the expression of inflammatory cytokines in the murine retina after laser photocoagulation treatment with a PASCAL(®) pattern scan laser photocoagulator and conventional laser treatment., Methods: Retinal scatter laser photocoagulation was performed on C57BL/6J mice using a short pulse (10 ms) with a PASCAL laser or conventional settings (100 ms) with a multicolor laser. Eyes were enucleated before treatment (control) and 1 day, 3 days and 7 days after treatment. The levels of inflammatory cytokines (i.e., VEGF, MCP-1, RANTES and IL-6) in the retina/choroid were quantified by an ELISA. The expression patterns of VEGF and macrophages (i.e., F4/80) in the retina/choroid were evaluated by immunohistochemistry., Results: The levels of RANTES, IL-6 and MCP-1 after PASCAL and conventional laser treatments were significantly elevated compared with controls (p < 0.05). Conventional laser treatment, but not PASCAL treatment, resulted in the up-regulation of VEGF. RANTES and IL-6 levels on day 1 and MCP-1 levels on day 3 in the sensory retina were also significantly up-regulated with conventional laser treatment compared with PASCAL treatment (p < 0.05). Immunohistochemical analysis showed that PASCAL treatment was associated with lower VEGF and F4/80 expression levels compared with conventional laser treatment., Conclusions: Our data suggested that the short pulse duration induced fewer inflammatory cytokines in the sensory retina compared with the conventional pulse duration. Short pulse laser photocoagulation with the PASCAL may prevent macular edema after panretinal photocoagulation.

Purpose: To study the correlation between the visual fields (VF) and wide-field fundus autofluorescence (FAF) in patients with retinitis pigmentosa (RP)., Design: Retrospective, observational, consecutive case series., Methods: Twenty-four eyes of 12 patients diagnosed with RP were enrolled. The VFs measured by Goldmann perimetry and wide-field FAF images were compared for each eye. The relationship between the areas of hypoautofluorescence on the wide-field FAF images and scotoma on Goldmann perimetry were evaluated. The VF and FAF images in the central 60 degrees were trimmed and superimposed to calculate the percentage agreement between the hypoautofluorescence and the scotomas and between the isoautofluorescence and hyperautofluorescence and the remaining VFs., Results: The areas of hypoautofluorescence on the FAF images were correlated significantly (R = 0.86, P < .001) with the areas of the VF defects on Goldmann perimetry. The mean percentage agreement between the hypoautofluorescence and the scotomas was 91.0% ± 7.7% and that of the isoautofluorescence and hyperautofluorescence with the remaining VFs was 84.5% ± 7.4%. The areas of geographic hypoautofluorescence with or without hyperautofluorescent bands reflected the VF defects, while nummular or mottled hypoautofluorescence without VF defects was seen in 7 eyes., Conclusions: These results suggested that wide-field FAF imaging is useful to evaluate the remaining retinal function in patients with RP. Abnormal fundus autofluorescence precedes loss of retinal function and is helpful for monitoring disease progression., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: We investigated the vascular system in the far peripheral retina in eyes with pathologic myopia by ultra-widefield fluorescein angiography (FA)., Methods: We analyzed retrospectively 230 with pathologic myopia (myopic refractive error >8 diopters [D] or axial length >26.5 mm) and 42 emmetropic (refractive error < ± 2 D) controls who were examined with ultra-widefield FA by the Optos P200 system. Far peripheral retina was defined as the area anterior to the ampullae of the vortex veins., Results: Retinal capillary telangiectasia was observed in the far periphery of 34 of 42 (81.0%) emmetropic eyes and in 90 of 115 (78.3%) highly myopic eyes. Retinal capillary microaneurysms were observed in 13 of 42 (31.0%) emmetropic eyes and in 60 of 115 (52.2%) eyes with pathologic myopia. The differences in the incidences of these two lesions were not significant. Areas of nonperfusion in the far periphery were found in two of 42 (4.8%) emmetropic eyes and in 95 of 115 (82.6%) eyes with pathologic myopia. In these myopic eyes, the arterioles and venules had an abrupt ending, and in advanced cases, the perfused area was limited to just beyond the staphyloma border. None of the eyes developed retinal neovascularization. Statistical analyses showed that the highly myopic patients with avascular areas in the far periphery were significantly older, and had significantly longer axial length., Conclusions: Areas of nonperfusion in the far periphery are common in eyes with pathologic myopia. Retinal vasculature in the far periphery is significantly altered in eyes with pathologic myopia, and this may be due to a mechanical stretching.

Purpose: Macular edema is one of the serious side effects associated with panretinal photocoagulation (PRP). The inhibitory effect of triamcinolone acetonide (TA) on leukocyte-endothelial cell interactions in vivo after PRP was evaluated., Methods: Argon laser photocoagulation was performed in one half of the retinas in male Brown Norway rats. Experimental rats were injected with 2 mg TA (50-microL volume) in the posterior sub-Tenon space, and the vehicle-treated rats were injected with the same amount of saline (50 microL) immediately after PRP. Untreated rats were used as the control. Leukocyte dynamics in retinal microcirculation and retinal vessel diameters were evaluated 1 day after laser photocoagulation with the use of acridine orange digital fluorography. Retinal thickness was evaluated with optical coherence tomography., Results: The number of rolling leukocytes and accumulating leukocytes in the retina decreased by 66% in the TA-treated rats (P < 0.01) and by 24% (P < 0.05), respectively, compared with the number in the vehicle-treated rats. Retinal thickness in the vehicle-treated rats was significantly thicker than that in control rats 1 day after laser photocoagulation (P < 0.01). Retinal thickness in the TA-treated rats was significantly suppressed compared with that in the vehicle-treated rats (P < 0.05)., Conclusions: Sub-Tenon administration of TA significantly suppressed leukocyte dynamics in rat retinal microcirculation and decreased retinal edema after laser photocoagulation. The results suggest that the suppression of leukocyte-endothelial cell interactions in retinal microcirculation may be one mechanism responsible for the therapeutic effect of sub-Tenon TA on postlaser retinal edema.

Purpose: This study was conducted to determine changes in the expression of vascular endothelial growth factor (VEGF) in murine retina after retinal scatter laser photocoagulation., Methods: Photocoagulation (PHC) was performed on wild-type C57BL/6J mice using a diode laser, and the eyes were enucleated 1, 2, 3, 4, 7, and 14 days after laser treatment. VEGF and monocyte chemoattractant protein (MCP)-1 levels in the sensory retina and retinal pigmented epithelial (RPE) cells in both tissues were measured by ELISA. The VEGF mRNA was measured by real-time RT-PCR. Leukocyte infiltration into the RPE-choroid was determined by flow cytometry. VEGF comparisons between mice subjected to PHC and those treated with monocyte recruitment inhibitor (anti-MCP-1) were performed and statistically analyzed. The expression of VEGF and MCP-1 in the retina was determined by immunohistochemistry., Results: VEGF protein levels significantly increased 1 day after PHC in both the RPE-choroid and the sensory retina. VEGF concentrations were maximum at day 3 after photocoagulation and stayed elevated until day 7. The number of choroid-infiltrating macrophages was markedly increased in mice with laser treatment compared with those without laser treatment. VEGF expression decreased after treatment with neutralized antibody to monocyte recruitment. We demonstrate that MCP-1 expression in the retina increased markedly after scatter laser photocoagulation by immunohistochemistry and ELISA., Conclusions: Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes.

Purpose: The biogenesis of drusen, a hallmark of age-related macular degeneration (AMD), is still unclear. Lipofuscin, which extensively accumulates with age in RPE cells, is hardly soluble, derived in part from oxidation byproducts of the photoreceptor outer segments. The purpose of the current study is to develop a new AMD model in rabbits using glycoxidized particles as imitation lipofuscin, and determine whether accumulation of lipofuscin as insoluble material may play a role in drusen biogenesis and other pathogenesis of AMD., Methods: To mimic the accumulation of insoluble lipofuscin, glycoxidized microspheres (glycox-MS) were made through a glycoxidation process with albumin and glycolaldehyde, alpha-hydroxy aldehyde. As a control, microspheres made with glutaraldehyde (cMS) and soluble glycoxidized (glycox-) albumin were prepared. Each material was implanted into the subretinal space in rabbits. The implanted area was assessed by funduscopy, fluorescein angiography, histology, and transmission electron microscopy (TEM)., Results: Compared with control microspheres, glycox-MS stagnated for a prolonged period in the cytoplasm of RPE cells. Eyes implanted with glycox-MS produced drusen-like deposits at a significantly higher frequency, when compared with the controls. Glycox-MS were observed at the margin of or beneath the drusen-like deposits in all cases. In some eyes with glycox-MS, late-onset sub-RPE choroidal neovascularization was observed, while control groups did not have these findings., Conclusions: These results suggest that the accumulation of indigestible granules such as lipofuscin in RPE or subsequent depositions toward Bruch's membrane may play a role in drusen biogenesis as a trigger of inflammation or via other mechanisms. This model of AMD may be useful to elucidate drusen biogenesis and pathogenesis of AMD.

Purpose: To investigate the intraocular distribution of betamethasone (BM) after implantation of transscleral drug delivery devices at three different sites in the eye: anterior episclera, posterior episclera, and anterior intrasclera., Methods: Nonbiodegradable implants designed to release BM for at least 1 month were placed at three different sites in albino rabbit eyes. The concentrations of BM in the aqueous humor, vitreous, and retina-choroid were determined by high-performance liquid chromatography at 4 weeks after placement. The distribution of BM in the retina-choroid was also evaluated., Results: In the in vitro and in vivo release study, the release of BM from implants in vivo correlated comparatively with those in vitro in all studied devices. The concentrations of BM in ocular tissues were measured after the implantations. The concentrations of BM after anterior-intrascleral placement of the implants were highest in the vitreous and retina-choroid among the three sites. The concentrations of BM in the three different sections of retina-choroid, which were around the placement site, opposite the placement site and posterior pole, were also determined. The concentration of BM in the retina-choroid around the placement site was higher than that in other parts of the retina-choroid after anterior-episcleral and anterior-intrascleral placement of implants. The implants at the anterior-intrascleral site yielded the highest concentrations of BM in the anterior part and posterior pole of the retina-choroid among the three placement sites. Only the implant at the posterior-episcleral site showed a higher concentration of BM at the posterior pole of the retina-choroid than at the other anterior part., Conclusions: Transscleral drug delivery devices may be feasible systems to deliver BM to intraocular tissues. The placement site of the device should be selected according to the location of the targeted lesions.

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte-mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Purpose: To describe the long-term retention of indocyanine green (ICG) in the fundus after ICG-assisted internal limiting membrane peeling., Design: Case report. Two patients underwent vitrectomy including ICG-assisted internal limiting membrane peeling. The fundus was examined with a 780-nm infrared illumination of a scanning laser ophthalmoscope after surgery., Results: No ICG staining of the fundus was visible ophthalmoscopically. Examination with a scanning laser ophthalmoscope, however, detected fluorescence from residual ICG until 6 months after surgery in case 1 and 9 months in case 2. No complication related to the residual ICG was observed., Conclusions: The results suggested that ICG remains in the fundus for a long period after surgery. Clearance of the dye from the diabetic retina may be prolonged.

Purpose: Macular edema is one of the most serious adverse effects after retinal scatter laser photocoagulation. It has been suggested that the changes in the distribution of retinal blood flow or the inflammatory reaction after photocoagulation may be involved in the pathogenesis of macular edema, but little information is available about its exact mechanism. This study was designed to evaluate quantitatively leukocyte-endothelial cell interactions and vascular permeability in the nonphotocoagulated portions of the retina after partial scatter laser photocoagulation., Methods: Argon laser photocoagulation was performed in one half of the retina in male pigmented rats (n = 90). In the other half of the retina, leukocyte dynamics after photocoagulation were evaluated in vivo with acridine orange digital fluorography. Retinal vessel permeability was quantified by using Evans blue dye., Results: Scatter laser photocoagulation caused significant inflammatory leukocyte-endothelial interactions not only in the photocoagulated but also in the untreated half of the retina. In the nonphotocoagulated half of the retina, the number of leukocytes rolling along the major retinal veins increased after photocoagulation and peaked at 12 hours (14.3 +/- 4.5 cells/min per vessel). Leukocyte accumulation in the untreated half of the retina increased after photocoagulation, with a peak of 47.5 +/- 13.0 cells/mm(2) 24 hours after photocoagulation. Retinal vascular permeability in the untreated half of the retina gradually increased after photocoagulation., Conclusions: Scatter laser photocoagulation increased leukocyte rolling and subsequent accumulation in both the photocoagulated and the untreated portions of the retina. The accumulated leukocytes may be involved in the augmented vascular permeability in the untreated retina, resulting in retinal edema after photocoagulation.

Glia maturation factor plays important roles in the development and growth of glia and neurons. We investigated the expression and localization of Glia maturation factor-beta (GMFB) and Glia maturation factor-gamma (GMFG) in the rat retina. By northern blot analysis, both GMFB and GMFG mRNAs were detected in retina as early as embryonic day (E) 18 and persisted until adult. The expression of GMFB mRNA was always much greater than that of GMFG mRNA. In situ hybridization showed that the GMFB mRNA signal was positive in the retina from E14 till adult. Immunostaining revealed that GMFB protein was present in the inner layer of retina at E14 and P1, and in Müller cells in adult. GMFG immunoreactivity was observed only in the inner limiting membrane from E14 to P1 rat retina, and was not detected in the adult retina. These results show that GMFs are synthesized and localized mainly in Müller cells in the rat retina, and suggest that they may contribute to the development and growth of glia and neurons.

Purpose: To evaluate the retinal thickness analyzer, a new image analyzer involving laser-slit biomicroscopy, for quantitative assessment of retinal thickness in patients with macular edema secondary to retinal vein occlusion., Methods: Fifty-eight patients (central retinal vein occlusion, 22 eyes; branch retinal vein occlusion, 36 eyes) with a clinical diagnosis of macular edema associated with retinal vein occlusion were evaluated. Retinal thickness analysis was performed with the retinal thickness analyzer. A green helium-neon laser slit directed into the eye was scanned to generate optical cross-sections of the retina. Images were analyzed by an automated software algorithm, and a detailed map of the retinal thickness was obtained., Results: Laser-slit images obtained with the retinal thickness analyzer in patients with macular edema associated with retinal vein occlusion disclosed intraretinal changes such as retinal edema, cystoid spaces, and hemorrhages. Linear regression analysis showed a significant relationship between the macular thickness in the central fovea and corrected visual acuity reported on a logMAR scale (P = .002, adjusted R2 = 0.40 in central retinal vein occlusion; P = .002, adjusted R2 = 0.34 in branch retinal vein occlusion)., Conclusion: We confirmed the capability of the retinal thickness analyzer to provide rapid and precise evaluation of macular thickening in patients with retinal vein occlusion.

Purpose: To perform acridine orange digital fluorography on rats after scatter photocoagulation to investigate alterations of retinal microcirculation at the capillary level, the authors used leukocyte dynamics as a parameter., Methods: Twenty-five pigmented rats (Long-Evans) were studied. Argon laser photocoagulation, extending 6 disc diameters from the optic disc, was delivered to one half of the retina, and the other half was untreated. The total number of burns was 200 +/- 10. Leukocyte hemodynamics in retinal microcirculation were evaluated 4, 7, 14, and 28 days after photocoagulation by acridine orange digital fluorography. The fundus image was obtained by using an argon laser in a scanning laser ophthalmoscope and was recorded on magnetic tapes at a video rate. The images were analyzed by a personal computer-based image analysis system., Results: Leukocyte velocities in the retinal capillaries were significantly decreased immediately after photocoagulation. In the laser-treated area, mean capillary leukocyte velocities were 0.73, 0.92, 1, and 1.3 mm/second on days 4, 7, 14, and 28, respectively (velocity in normal control animals 1.4 mm/second). In addition, leukocyte hemodynamics were compromised in the untreated retina: Mean capillary leukocyte velocities were 0.88, 1.1, 1.2, and 1.3 mm/second on days 4, 7, 14, and 28, respectively. Twenty-eight days after photocoagulation, the velocities recovered to normal values in the treated and the untreated areas of the retina., Conclusions: Retinal capillary hemodynamics were impaired after scatter photocoagulation, and the hemodynamics in the untreated retina were also affected. Photocoagulation to the retina may influence capillary hemodynamics by diffusible chemical substances and by direct tissue injury.

Purpose: To develop a highly sensitive method for in vivo quantitation of intravitreal peroxides by vitreous fluorophotometry with 2',7'-dichlorofluorescin (DCFH), a hydrogen peroxide (H2O2)-sensitive fluorescent dye, and to measure peroxides in the vitreous humor after panretinal laser photocoagulation (PRP)., Methods: In the presence of H2O2 and lipid hydroperoxides, nonfluorescent DCFH was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF; excitation, 495 nm; emission, 520 nm), which is detectable by vitreous fluorophotometry. Reactions of DCFH, including hematin with various concentrations of H2O2, were investigated in vivo. Fluorophotometry with DCFH was performed 1, 7, 14, and 28 days and 2 months after argon laser PRP. Untreated eyes served as the controls., Results: Exogenously applied H2O2 oxidized DCFH to DCF in a dose-dependent manner, ranging from 6 x 10(-8) mol/l to 6 x 10(-5) mol/l in concentration in vivo. Intravitreal DCF concentration was 83.7 +/- 6.8 nmol/l in control eyes. A significant increase of DCF was detected 1 day after PRP (330.7 +/- 123.8 nmol/l, P < 0.002). The increase peaked on day 7 (352.4 +/- 239.5 nmol/l, P < 0.002) and remained elevated at 2 months after PRP (161.8 +/- 51.4 nmol/l, P < 0.01)., Conclusions: This method allowed a highly sensitive quantitation of intravitreal peroxides in vivo. The authors' findings indicated that PRP induces increased production of peroxides in rabbit vitreous for 2 months. The data suggested that persistently high levels of peroxides in the vitreous humor affect the development of vitreous liquefaction after PRP.

Purpose: This study sought to measure foveal retinal thickness in patients with diabetic retinopathy and to investigate the relationship between foveal thickness and visual acuity, biomicroscopic findings, and angiographic features., Methods: A commercial scanning retinal thickness analyzer was used to measure retinal thickness. A laser slit was projected onto the retina and scanned in 400 milliseconds across the central area of the fundus. The image where the laser slit intersects with the retina was digitally recorded and analyzed. Retinal thickness was measured in 35 patients (35 eyes; patient age, 57 +/- 13 years) with diabetic retinopathy. Patients also were examined by fluorescein angiography and slit-lamp biomicroscopy to detect foveal thickening., Results: Linear regression analysis indicated a significant correlation between foveal thickness and visual acuity (adjusted R2 = 0.72, P < 0.001). Foveal thickness was abnormal in 6 (100%) of 6 eyes in which foveal thickening was detected with slit-lamp biomicroscopy. Foveal thickness also was abnormal in 9 (31%) of 29 eyes that appeared normal by biomicroscopic examination. Foveal thickness was 136 +/- 65 microns in 7 eyes without leakage, 175 +/- 35 microns in 13 eyes with questionable leakage, and 291 +/- 120 microns in 7 eyes with definite leakage (P = 0.0075)., Conclusions: Retinal thickness analysis is shown to be more sensitive than slit-lamp biomicroscopy for detecting small changes in retinal thickness. Retinal thickness analysis may prove to be a useful, noninvasive modality for the development or regression of macular edema.

Purpose: To establish a new fluorophotometric method to quantitate oxidative stress in the retina in vivo with a hydrogen peroxide (H2O2)-sensitive fluorescent dye., Methods: For in vitro fluorophotometric study, nonfluorescent 2',7'-dichlorofluorescein (DCFH) was incubated with H2O2 (10 pM to 100 nM), and the production of fluorescent 2',7'-dichlorofluorescein (DCF) was measured with fluorophotometric analysis. The inhibitory effect of catalase was also examined. For in vivo fluorophotometric study, rabbit eyes received vitrectomy and were perfused with 5 microM 2',7'-dichlorofluorescein diacetate (DCF-DA) or 2',7'-dichlorofluorescein diacetate (DCFH-DA). For oxidative stress, 300 microM H2O2 was infused after perfusion of DCFH-DA. Fluorophotometric measurements of the chorioretinal peak were performed. The eyes were enucleated for fluorescent microscopic examination to determine the localization of DCF fluorescence., Results: H2O2 converted DCFH to DCF in a dose-dependent manner, which was inhibited by catalase dose dependently. In vivo fluorophotometric study showed DCF-DA and DCFH-DA caused production of 2006 +/- 274 picomole/ml (mean +/- SD, n = 5) and 8.35 +/- 1.11 picomole/ml (n = 5), respectively, in the chorioretinal peak. DCFH-DA with stimulation by H2O2 induced 30.7 +/- 13.1 (n = 4) picomole/ml DCF. Fluorescent microscopy showed DCF production in the retina was significant in the eye treated with DCF-DA and minimal in the eye treated with DCFH-DA. Moderate DCF production in the nerve fiber layer was observed in the eye treated with DCFH-DA and H2O2., Conclusions: This new fluorophotometric method with DCFH-DA may be useful in quantitatively evaluating oxidative stress in the retina in vivo.

Phototoxic retinopathy due to light from the operating microscope has been reported during cataract surgery, as well as during vitreous surgery. A neutral density filter for the coaxial light or coverage of the cornea with various materials has been advocated to prevent retinal complications. However, it is difficult to actually monitor the fundus during surgery with usual methods. The author used an endoscope to demonstrate how intensively the light from the operating microscope is focused on the retinal surface during vitreous surgery.

Purpose: To establish a new model of subretinal neovascularization (SRN) in the rabbit by implanting basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres beneath the retina., Methods: Basic fibroblast growth factor-impregnated gelatin microspheres were prepared by forming a polyion complex between gelatin and bFGF. The microspheres, containing 2.5 micrograms of bFGF, were injected into the subretinal space of rabbit eyes (n = 29). Control eyes (n = 10) received bFGF-free gelatin microspheres. Eyes were followed up for 3 days to 8 weeks by ophthalmoscopy, photography, fluorescein angiography, light microscopy, and transmission electron microscopy., Results: Twenty of 24 experimental eyes (83%) showed fluorescein leakage from SRN 2 weeks after implantation of the bFGF-impregnated microspheres. This leakage continued for 2 to 6 more weeks. In striking contrast, control eyes showed no fluorescein leakage. Histologic examination revealed SRN in all the experimental eyes but in none of the control eyes., Conclusions: Subretinal implantation of bFGF-impregnated gelatin microspheres induces reproducible SRN in the rabbit.

Laser photocoagulation has been used successfully for the treatment of clinically significant macular oedema to reduce the risk of loss of vision in diabetic patients. A quantitative method for measuring retinal thickness was applied to 20 patients with diabetic macular oedema before and 4 months after focal laser treatment to assess the reduction in retinal thickening and its relation to visual acuity. The degree of thickening at each location, defined by thickness index, was determined relative to the corresponding average value in normal subjects. Comparison of quantitative retinal thickness measurements before and after treatment demonstrated that treatment at thickness indices of approximately 1.6 (60% thickening) has nearly 50% probability for reversal of thickening to within the normal range (< or = 1.3), whereas at thickness indices greater than 2.8 (180% thickening) there is less than 2.5% probability that reversal will occur. The level of foveal thickening before treatment strongly correlated with the degree of thickening after treatment. Most of the eyes with an improvement in visual acuity had a foveal thickness within the normal range at 4 months' follow up. These findings suggest that quantitative retinal thickness measurement provides an objective assessment of the degree of macular oedema and can be useful for monitoring the efficacy of focal laser treatment in reducing the thickening and relating the latter to visual outcome.

Purpose: An incidence of and risk factors for retinal nerve fiber layer defect were investigated in patients with type II diabetes mellitus and compared with that of age-matched control subjects., Methods: The authors photographed the retinal nerve fiber layer of the right eye in each of 137 patients with diabetes and 144 healthy control subjects. The level of diabetic retinopathy ranged from levels 1 (no microaneurysm) to 4 (eyes with localized intra-retinal microvascular abnormalities or venous beading). Risk factors for the nerve fiber layer defect analyzed were age of patients, visual acuity, axial length, optic disc size, glycosylated hemoglobin, systolic blood pressure, and level of diabetic retinopathy., Results: Defects of the retinal nerve fiber layer were found in 6/30 (20%) eyes with level 1 retinopathy, 8/14 (57%) eyes with level 2 retinopathy, 24/47 (51%) eyes with level 3 retinopathy, and 36/46 (78%) eyes with level 4 retinopathy. These defect incidences were significantly higher than that of the control group, which had 5/144 (3.5%) defects (P < or = 0.001). Risk factors for this nerve defect were level of diabetic retinopathy (P = 0.002), high systolic blood pressure (P = 0.0232), and patient's age (P = 0.0478). Not correlated with the incidence of the retinal nerve fiber layer defect were visual acuity, disc size, axial length, and glycosylated hemoglobin level at the time of examination., Conclusion: These findings suggest that the retinal nerve fiber layer defect is common in patients with early diabetic retinopathy. Risk factors for this defect were higher level of diabetic retinopathy, systemic hypertension, and advanced age.

Background: The use of slit-lamp biomicroscopy provides valuable information for the diagnosis and management of vitreoretinal disorders. However, intense backscatter from the fundus often precludes the visualization of fine structures in the vitreoretinal interface., Methods: Laser biomicroscopy is a new method designed to improve the visualization of fine vitreoretinal structures at the macula. This method was applied to eyes suspected of traction maculopathies. With this method, the contrast and the lateral separation between the vitreal and retinal images are optimized while preserving information on the location of the slit on the fundus., Results: The results indicated that fine structures in the vitreoretinal interface, which were difficult to observe with conventional slit-lamp biomicroscopy, could be clearly visualized., Conclusion: Laser biomicroscopy may be a useful tool in the diagnosis and management of diseases with vitreoretinal abnormalities.

Diabetic macular edema is a major cause of vision loss and is evaluated with qualitative or semiquantitative techniques. A new quantitative method for assessment of macular edema using retinal thickness analysis was applied to 19 patients with diabetic macular edema. Foveal thickening was frequently coupled with poor visual acuity. Slit-lamp biomicroscopy and stereophotography detected 80% and 78% of local areas of thickening, respectively, but failed to detect locations with average thicknesses of 1.5 and 1.6 times normal, respectively. Fluorescein leakage on angiography was generally associated with retinal thickening, but locations with similar degrees of leakage had widely varying retinal thickening. Fluorescein leakage in the posterior vitreous correlated poorly with the degree of foveal thickening. These results indicate that quantitative measurement of retinal thickness may become useful in the management of diabetic patients with macular edema.

A new method was developed to deliver locally a bolus dose of a drug to the retinal vasculature. The targeted delivery system was based on encapsulating the drug in heat-sensitive liposomes, which are injected intravenously and lysed in the retinal vessels by a heat pulse generated by a laser. To test if substances delivered in the vessels could also penetrate into the surrounding tissue, 6-carboxyfluorescein was encapsulated in liposomes and used as a marker for drug penetration. Moderate argon laser pulses were applied to the retinal vessels of Dutch pigmented rabbits to induce breakdown of the blood-retinal barrier (BRB). A suspension of liposomes at a dose of 2 ml/kg body weight, corresponding to a carboxyfluorescein dose of 12 mg/kg, was injected into the ear vein. The dye was released from the liposomes proximal to the damaged portion of the vessel. Fundus fluorescein angiograms were recorded with a video camera and digitized for subsequent image analysis. The penetration of carboxyfluorescein into the retinal tissue was evaluated by comparing the fluorescence intensity of the area around the damaged vessel with that of an adjacent control area. The dye penetration increased with the numbers of laser applications (P less than 0.001). The leakage was localized distally to the released site and was restricted to areas with a disrupted BRB. The mass of carboxyfluorescein that penetrated gradually spread with time. Both veins and arteries could be used for the targeted delivery. These results indicated that this delivery system, which is fully controllable by laser through the pupil, can deliver drugs inside the vasculature and into the retinal tissue wherever the BRB is disrupted.