Triamcinolone in small-gauge vitrectomy for epiretinal membrane peeling.

OBJECTIVE: We compared visual and macular morphological outcomes after epiretinal membrane (ERM) peeling, with and without IVTA treatment. DESIGN: Interventional, retrospective, consecutive case-control study. PARTICIPANTS: Forty-one eyes of 41 participants (17 men, 24 women) were included. Twenty-one were treated by standard vitrectomy and peeling (controls) and 20 patients received intravitreal triamcinolone after vitrectomy and peeling. METHODS: Pre-and postoperative letter score and central foveal thickness (CFT) through the foveal centre were compared between both groups. Best-corrected visual acuity (BCVA) was measured using Snellen charts and converted to logMAR for statistical analyses. RESULTS: CFT and BCVA had improved by the 6-month follow-up from baseline. In the control group, the mean logMAR BCVA improved from 0.57 (SD: 0.22) to 0.21 (0.17) (p < 0.01), and the mean CFT reduced from 462.5 (98.6) µm to 329.8 (82.7) µm (p < 0.01). The mean logMAR BCVA of the IVTA group improved from 0.73 (0.17) to 0.36 (0.31) (p < 0.01), and the mean CFT reduced from 561.45 (131.0) µm to 339.25 (72.6) µm (p < 0.01). Visual improvement and CFT did not differ significantly at follow up (p = 0.583; p= 0.85). Significant reduction of CFT is seen in the IVTA group (p = 0.048). CONCLUSIONS: Visual acuity and macular morphology improved after ERM peeling, with or without IVTA. Although conjunctive IVTA did not significantly influence visual outcome at 6 months, a significant decrease in CFT was observed after IVTA administration.

Changes in the size of the foveal avascular zone after vitrectomy with internal limiting membrane peeling for a macular hole.

PURPOSE: To determine the size of the foveal avascular zone (FAZ) before and after vitrectomy for a macular hole (MH). STUDY DESIGN: Retrospective case series study. METHODS: Twenty-five eyes of 25 patients with a unilateral MH that had undergone vitrectomy with internal limiting membrane peeling were studied. The unaffected 17 fellow eyes were studied in the same way. En face images of the parafoveal region were obtained by optical coherence tomography angiography, and the images were used to measure the FAZ before and 1 month after the vitrectomy. The relationships between the different FAZ sizes and the ocular parameters were determined by Pearson product moment correlation analysis. RESULTS: Compared with the preoperative superficial FAZ (sFAZ), the postoperative sFAZ was significantly reduced (P < 0.001). The postoperative sFAZ was significantly smaller than that of the fellow eye (P < 0.001). The size of the postoperative sFAZ was significantly correlated with that of the preoperative sFAZ, the postoperative foveal thickness (FT), and the sFAZ of the fellow eyes (r = 0.520, P = 0.008; r = -0.515, P = 0.012; and r = 0.702, P = 0.002, respectively). The size of the postoperative deep FAZ (dFAZ) was significantly correlated with the postoperative FT and the dFAZ of the fellow eyes (r = -0.441, P = 0.035; and r = 0.499, P = 0.049, respectively). However, no significant correlation was found between the size of the postoperative FAZ and the size of the preoperative MH. CONCLUSIONS: MH closure leads to a significant decrease in the size of the FAZ symmetrical to the size of the fellow eye. The size of the postoperative FAZ is influenced by the postoperative FT independently of the size of the MH.

MULTICOLOR IMAGING OF INNER RETINAL ALTERATIONS AFTER INTERNAL LIMITING MEMBRANE PEELING.

PURPOSE: To characterize the appearance of inner retinal alterations after internal limiting membrane (ILM) peeling using multicolor confocal scanning laser ophthalmoscopy (cSLO). METHODS: Retrospective review of two eyes that underwent pars plana vitrectomy with internal limiting membrane peeling and postoperative multicolor cSLO with spectral-domain optical coherence tomography. Infrared, green, blue, standard multicolor, and blue-green enhanced multicolor reflectance images were evaluated alongside spectral-domain optical coherence tomography for inner retinal alterations. RESULTS: Two eyes of 2 patients, aged 70 and 65 years, were identified. Preoperative diagnoses were epiretinal membrane with lamellar macular hole for Case 1 and full-thickness macular hole for Case 2. Time from surgery to initial multicolor cSLO imaging was 9 years in Case 1 and 3 weeks in Case 2. Inner retinal alterations were best visualized on blue reflectance, moderately visualized on green and blue-green enhanced multicolor, and less evident on infrared and standard multicolor. In Case 2, serial multicolor cSLO imaging demonstrated the emergence of inner retinal alterations between 3 weeks and 5 weeks postoperatively. CONCLUSION: Multicolor cSLO is a novel imaging modality capable of detecting inner retinal alterations in patients with a history of internal limiting membrane peeling, and may be clinically useful for monitoring anatomical changes associated with internal limiting membrane peeling.

A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization.

PURPOSE: Therapeutic efficacy is routinely assessed by measurement of lesion size using flatmounted choroids and confocal microscopy in the laser-induced choroidal neovascularization (L-CNV) rodent model. We investigated whether optical coherence tomography (OCT) quantification, using an ellipsoid volume measurement, was comparable to standard ex vivo evaluation methods for this model and whether this approach could be used to monitor treatment-related lesion changes. METHODS: Bruch's membrane was ruptured by argon laser in the dilated eyes of C57BL/6J mice, followed by intravitreal injections of anti-VEGF164 or vehicle, or no injection. In vivo OCT images were acquired using Micron III or InVivoVue systems at 7, 10, and/or 14 days post-laser and neovascular lesion volume was calculated as an ellipsoid. Subsequently, lesion volume was compared to that calculated from confocal Z-stack images of agglutinin-stained choroidal flatmounts. RESULTS: Ellipsoid volume measurement of orthogonal 2-dimensional OCT images obtained from different imaging systems correlated with ex vivo lesion volumes for L-CNV (Spearman's ρ=0.82, 0.75, and 0.82 at days 7, 10, and 14, respectively). Ellipsoid volume calculation allowed temporal monitoring and evaluation of CNV lesions in response to antivascular endothelial growth factor treatment. CONCLUSIONS: Ellipsoid volume measurements allow rapid, quantitative use of OCT for the assessment of CNV lesions in vivo. This novel method can be used with different OCT imaging systems with sensitivity to distinguish between treatment conditions. It may serve as a useful adjunct to the standard ex vivo confocal quantification, to assess therapeutic efficacy in preclinical models of CNV, and in models of other ocular diseases.

Assessment of laser induction of Bruch's membrane disruption in monkey by spectral-domain optical coherence tomography.

PURPOSE: Laser-induced choroidal neovascularisation is a widely used model for age-related macular degeneration. The success rates of induction have been relatively low in large animals such as monkeys. Our study aimed to investigate the laser-induced damages to the Bruch's membrane of monkeys using the spectral-domain optical coherence tomography (OCT). METHODS: Laser photocoagulation was performed in the posterior and peripheral fundus of a rhesus monkey using a 532 nm laser. The lesions were examined by fundus photography and spectral-domain OCT immediately after the procedure. Fluorescein angiography was performed after 3 and 4 weeks in the animal to assess the development of choroidal neovascularisation. RESULTS: A total of 44 lesions were produced in both eyes of the animal. Subretinal bubbles with or without haemorrhage were observed at 41 spots during the procedure. Spectral-domain OCT showed that laser damages varied considerably among lesions and the disruption of the Bruch's membrane could be visualised at 23 spots on the OCT images. Leakage of fluorescein was only observed after 3 and 4 weeks within the macular area at lesions where Bruch's membrane disruptions had been detected by OCT. CONCLUSIONS: The presence of subretinal bubbles with haemorrhage is not an accurate indicator for successful disruption of the Bruch's membrane. Instead, spectral-domain OCT provides a better alternative to assess the retinal damages to the Bruch's membrane during laser induction of choroidal neovascularisation in monkeys.

Effect of chromogranin A-derived vasostatin-1 on laser-induced choroidal neovascularization in the mouse.

PURPOSE: To verify the effect of vasostatin-1 (VS-1), an anti-angiogenic fragment of chromogranin A, in the prevention of choroidal neovascularization (CNV) in an established mouse model of laser-induced ocular neovascularization. METHODS: Bruch's membrane, the innermost layer of the choroid, was broken by laser photocoagulation in C57/Bl6 mice, to induce CNV. Mice were then treated daily for 14 days by intraperitoneal injection of VS-1 or vehicle (6 mice/group). CNV and vascular leakage were measured at three time-points (day 0, 7 and 14) in vivo by spectral domain optical coherence tomography (OCT) and fluorescein angiography (FA). Ex vivo analysis of CNV was also performed at day 14 by confocal microscopy analysis of dextran-perfused choroidal flat-mounts. RESULTS: In vivo analyses showed that VS-1 significantly reduced CNV at day 14 (p = 0.03) and vascular leakage at day 7 (p = 0.01) and 14 (p = 0.04). Ex vivo confocal microscopy analysis of CNV performed on dextran-perfused choroidal flat-mounts at day 14 confirmed the protective activity of VS-1 (p = 0.01). A significant correlation between the results of in vivo and ex vivo analyses of CNV was also observed (p = 0.001, R(2) = 0.81). CONCLUSION: The results indicate that VS-1 can prevent CNV and vascular leakage in a mouse model of ocular neovascularization, suggesting that this polypeptide might have therapeutic activity in human ocular diseases that are complicated by neovascularization or excessive vascular permeability.

Evaluation of the ability of a photocoagulator to rupture the retinal vein and Bruch's membrane for potential vein bypass in retinal vein occlusion.

BACKGROUND AND OBJECTIVE: To evaluate the laser power required to rupture both the retinal vein and Bruch's membrane in a porcine model using a new laser system for treatment of retinal vein occlusion. MATERIALS AND METHODS: The retina was treated in areas with and without an overlying vein. Sections through the laser sites were stained for light and transmission electron microscopy. The percentage of the laser sites with rupture of the retinal vein and Bruch's membrane, the degree of tissue damage, and the diameter of the rupture in Bruch's membrane were determined. RESULTS: The rate of vein rupture was 75% at 3 W, and rupture of Bruch's membrane was achieved at all powers tested. The mean diameter of the rupture in Bruch's membrane increased with laser power. CONCLUSION: The Integre Duo laser system (Ellex Medical Lasers, Adelaide, Australia) is capable of rupturing the retinal vein and Bruch's membrane at lower powers than prior laser systems.

Laser clearance of drusen deposit in patients with autosomal dominant drusen (p.Arg345Trp in EFEMP1).

PURPOSE: To assess whether laser treatment to the retinal pigment epithelium anterior to drusen in eyes of patients with EFEMP1-related maculopathy affects visual acuity, deposit volume, and retinal sensitivity. DESIGN: Prospective, interventional case series. METHODS: In 11 patients with autosomal dominant drusen and confirmed disease-causing EFEMP1 mutation, the worse-seeing eye was treated with Argon green laser (10 to 15 laser spots; 200-µm spot size, 0.1-second duration, 80 to 120 mW). Patients were examined before treatment as well as 1, 3, 6, and 12 months after the procedure. Clinical assessment included visual acuity, fundus-controlled perimetry, spectral-domain optical coherence tomography, and autofluorescence imaging. Custom-made software allowed for coregistration of fundus-controlled perimetry and spectral-domain optical coherence tomography data sets. The main outcome measures were change in visual acuity, retinal sensitivity, and drusen volume. RESULTS: The untreated eyes lost an average of 0.8 letters, whereas the treated eyes gained an average of 4.9 letters. For fundus-controlled perimetry, locus-by-locus differences in sensitivity were calculated between pretreatment and posttreatment assessments; subsequently, the overall difference in the treated and untreated eye was compared. Five patients showed significant improvement in retinal sensitivity, 5 patients showed no change, and 1 patient showed significant deterioration. An increase in mean drusen thickness was observed in the untreated eyes, but not in the treated eyes (P = .0322). The thickness of the drusen correlated with retinal sensitivity (ρ = -0.49; P < .0001). Safety was demonstrated and no adverse events were observed. CONCLUSIONS: Low-energy laser treatment is safe and may be effective in the treatment of autosomal dominant drusen. Further evaluation with long-term assessment is required to confirm the benefits.

Histology and immunochemistry evaluation of autologous translocation of retinal pigment epithelium-choroid graft in porcine eyes.

PURPOSE: To evaluate structure and cellular functionality of retinal pigment epithelium (RPE)-choroid grafts after autologous translocation in porcine eyes. METHODS: Retinal pigment epithelium-choroid grafts were obtained from the nasal midperiphery donor site and translocated to the central area in 12 pigs (12 eyes). Grafts were placed under the central retina through a retinotomy. Ophthalmoscopic and pathological evaluations were performed immediately (n = 1) and at 15 (n = 3) and 30 (n = 3) days after surgery. Untranslocated nasal RPE-choroid grafts were obtained at time of surgery and used as controls. Specimens were evaluated by standard histology and by immunochemical studies of RPE65, CRALBP and GFAP. RESULTS: Five animals were lost to follow-up owing to surgery or anaesthesia complications. Ophthalmoscopic examination revealed that the grafts remained in place at all time-points studied. Fifteen and thirty days postsurgery, some areas of the transplanted RPE maintained a monolayered structure. Retinal pigment epithelium cells were firmly attached to Bruch's membrane and predominantly preserved polarity and pigment distribution. However, RPE65, CRALBP and GFAP patterns of expression and distribution were diminished and modified during follow-up. Ophthalmoscopic retinal detachment and proliferative vitreoretinopathy (PVR), confirmed by microscopic evaluation, complicated all cases at 30 days of follow-up. CONCLUSION: Autologous RPE-choroid grafts survived up to 30 days in porcine eyes. Histological and immunochemical evaluation revealed preserved transplanted RPE cells morphology accompanied by alterations in the immunoreactivity expression of functional proteins, and development of significant PVR. The data presented in this manuscript provide insights into the fate, viability and cellular functionality of the transplanted RPE-choroid graft, serving as foundation for further knowledge and improvement of this technique.

Optimization of laser-induced choroidal neovascularization in African green monkeys.

We developed and validated a new nonhuman primate model of laser-induced choroidal neovascularization (CNV) that addresses study design limitations prevalent in laser-induced CNV-based efficacy studies. Laser-induced Bruch's membrane disruption triggers CNV and has been widely utilized in animals to model neovascular ("wet") age-related macular degeneration (AMD). Despite widespread use of the approach, detailed assessment of experimental parameters and their influence on pathophysiological endpoints critical for disease modeling has been extremely limited and largely based on anecdotal observations. We evaluated laser power parameters and endpoint measures to optimize methods for CNV formation and quantification to facilitate drug efficacy screening in African green monkeys. Six laser spots of 350, 550, 750, 950 or 1500 mW laser power were positioned bilaterally 1.5 disc diameters from the fovea, within the macula. Fluorescein angiograms were collected 3-5 weeks later and scored by trained masked investigators using graded (I-IV) and densitometric methods. Histopathology assessments were also performed, including determination of CNV area. Test system sensitivity to angiogenesis inhibition was subsequently assessed by evaluating the effect of intravitreal bevacizumab (Avastin) pretreatment (one day prior to laser photocoagulation) on incidence of CNV. Grade III and grade IV lesions were considered clinically relevant, demonstrating early hyperfluorescence and late leakage within or beyond the lesion borders. By 4 weeks post-laser all treatment groups demonstrated evidence of grade III lesions with greatest incidence observed in lesions induced by 750 and 950 mW laser power (72.9% and 69.4% respectively). Grade IV lesions were confined to eyes receiving 550 mW laser power or higher, with highest incidence of grade IV lesions observed in eyes receiving 950 (19.4%) and 1500 mW (31%) laser spots, incidence peaking 4 weeks post-laser photocoagulation. Densitometric analyses of angiograms corroborated visual scoring. Bevacizumab completely abolished grade IV lesion development and significantly lowered lesion fluorescein signal intensity (P < 0.0001) and CNV area (P = 0.038) compared to vehicle-treated controls. Our studies demonstrate that laser power of 950-1500 mW and angiography analysis 4 weeks post-laser are optimal parameters to evaluate treatment effects on CNV induction following laser photocoagulation. Bevacizumab significantly attenuated CNV development, as determined by fluorescein angiography and histopathology assessments in this model, supporting the application of African green monkeys in preclinical modeling of CNV. Laser parameters and time points for therapeutic dosing and angiography endpoints are critical factors to the laser-induced CNV model and must be validated for robust assessment of efficacy. The newly optimized nonhuman primate model described will facilitate preclinical efficacy assessments of novel therapeutics for CNV.

In vivo evaluation of laser-induced choroidal neovascularization using spectral-domain optical coherence tomography.

PURPOSE: To describe the in vivo evolution of laser-induced choroidal neovascularization (CNV) in mice using spectral domain optical coherence tomography (SD-OCT). METHODS: Laser photocoagulation was applied to the mouse fundus using a 532-nm diode laser (100, 150, and 200 mW; 100-µm diameter, 0.1-second duration). SD-OCT examination was performed immediately after laser application and at days 3, 5, 7, 14, 21, and 28 after laser. Fluorescein angiography (FA) was performed at day 5, 7, 14, and 28. Acquired SD-OCT images were analyzed to describe morphologic features, measure CNV size and retinal thickness, and assess the frequency of lesions resulting in fluid accumulation. Finally, SD-OCT images were compared to fluorescein angiograms and histologic sections with immunostaining at similar time points. RESULTS: SD-OCT allowed visualization of the initial laser damage and the subsequent stages of the injury response. CNV formation reached its maximum size at day 5. By day 7, significant size reduction was observed (P < 0.001), continuing through days 14 and 28. Exudation signs, such as fluid accumulation and increase in retinal thickness, followed the same time course, with a peak at day 5 and a decrease by day 7. Delivery of higher laser energy levels to the RPE/choroid complex resulted in a significant percentage of lesions demonstrating excessive chorioretinal damage without CNV formation. CONCLUSIONS: SD-OCT is a fast and reliable tool for the in vivo evaluation of laser-induced CNV, allowing quantification of lesion size and exudation parameters. Moreover, it provides morphologic information that correlates with histologic findings.

Differential role of tumor necrosis factor (TNF)-alpha receptors in the development of choroidal neovascularization.

PURPOSE: Tumor necrosis factor alpha (TNF)-alpha contributes to inflammation-associated angiogenesis. This study investigates the role of TNF-alpha receptors 1a and 1b in the development of choroidal neovascularization (CNV). METHODS: CNV was induced in Tnfrsf1a(-/-) and Tnfrsf1b(-/-) mice with C57Bl6/J background and their wild-type (WT) (C57Bl/6J) controls by laser damage to the Bruch's membrane. TNF-alpha expression in RPE/choroid was determined by Western blot analysis. Pathologic angiogenesis was estimated qualitatively and quantitatively by fluorescein angiography and histology on choroidal flat mounts and paraffin cross-sections. Inflammatory cell invasion was investigated by clodronic acid depletion of circulating macrophages and immunochemistry, and the apoptotic activity was investigated by TUNEL assay and by caspase-3 and caspase-8 expression. Receptor 1b-specific Bmx/Etk kinase was detected by immunochemistry. RESULTS: TNF-alpha levels were elevated after laser treatment. Severe CNV lesions and increased macrophage invasion were observed in Tnfrsf1a(-/-) compared with WT and Tnfrsf1b(-/-) mice. Increased immunoreactivity for Bmx/Etk kinase corresponded to the severity of CNV formation. Reduced pathologic angiogenesis and macrophage invasion in Tnfrsf1b(-/-) mice (vs. WT and Tnfrsf1a(-/-)) was accompanied by enhanced endothelial cell apoptosis and by caspase-3 and caspase-8 activation. CONCLUSIONS: Receptor 1b promotes the recruitment of inflammatory cells to the site of injury and exacerbated pathologic angiogenesis probably by way of the Bmx/Etk-kinase-dependent pathway in the absence of receptor 1a. On the other hand, receptor 1a-dependent apoptosis in the absence of receptor 1b leads to reduced inflammatory response and CNV lesions after laser treatment. This demonstrates the potential for specific targeting of TNF-alpha receptors for future therapies of inflammation-associated choroidal neovascularization.

Evaluation of CXCR4 inhibition in the prevention and intervention model of laser-induced choroidal neovascularization.

PURPOSE. Endothelial precursor cells (EPCs) derived from hematopoietic stem cells (HSCs) have been shown to contribute to choroidal neovascularization by signaling through the SDF-1/CXCR4 axis. In a prevention and treatment/intervention modality of the laser choroidal neovascularization (CNV) model, the efficacy of CXCR4 inhibition on reducing choroidal leakage and angiogenesis was evaluated. METHODS. CNV in rats was generated by focal rupture of Bruch's membrane with an 810-nm diode laser. In the prevention mode, a CXCR4 antagonist (AMD3100) was delivered via an osmotic pump 1 day after laser induction. In the intervention mode, AMD3100 delivery commenced 14 days after laser induction. Inhibition of CXCR4 was determined through leukocyte and SDF-1 actin polymerization blood biomarker assays. Leakage was assessed by fluorescein angiography, and CNV lesion size was quantified after isolectin B4 endothelial cell staining. SU14813, an anti-VEGFR, PDGFR-beta, KIT, and FLT3 inhibitor, was also assessed in an intervention study protocol. RESULTS. Inhibition of CXCR4 was demonstrated by an increase in the number of blood leukocytes, and diminished SDF-1 induced actin polymerization in whole blood. CNV leakage and neovascularization were inhibited when the dose regimen was initiated 1 day after laser-induced CNV induction. AMD3100 did not show efficacy when administered 14 days after lasering. Treatment with SU14813 significantly decreased CNV leakage and lesion size in an intervention modality. CONCLUSIONS. Inhibition of CXCR4 may be useful in preventing neovascularization but does not appear to have an effect on already established angiogenesis. A multiple receptor tyrosine kinase (RTK) inhibitor approach shows promise for the treatment of wet age-related macular degeneration.

Natural history of choroidal neovascularization after surgical induction in an animal model.

PURPOSE: To study an expanded time course of surgically induced choroidal neovascularization (CNV) in a porcine model applying fluorescence angiography and immunohistology. METHODS: Twenty-two porcine eyes underwent vitrectomy, a retinal bleb was raised and the detached retina perforated using endodiathermy. Bruch's membrane was perforated with a retinal perforator at a site where the overlying neuroretina was normal. Eyes were enucleated in a time interval between 30 min and 42 days after the perforation, and the pigs were subsequently killed. Immediately prior to enucleation, fundus photographs and fluorescein angiograms were obtained. Sections of paraffin-embedded eyes were immunohistochemically stained. RESULTS: On fluorescein angiography, membranes aged 14 days or less exhibited leakage in 10/11 cases while the remaining showed persistent staining. The propensity to leak diminished with time and only 1/3 of the oldest membranes leaked. In eyes enucleated immediately after surgery, neuroretinas overlying the induced lesions were intact without apparent atrophy of cells. At day 3, macrophages and myofibroblasts formed membrane-like structures in the subretinal space. At day 7, the outer surface of the membrane was covered by retinal pigment epithelium (RPE) cells and the neuroretinas had suffered damage in the form of outer segment loss. In the time period 14-42 days, the CNV membrane became completely enveloped by RPE cells. The degree of membrane vascularization increased with time and was at its maximum after 42 days. Intact outer segments were identified over the oldest membranes. CONCLUSION: The formation of surgical CNV membranes followed the normal reparatory pathway and the degree of vascularization of CNV membranes continued to increase during the 42 days. However, propensity to leak diminished with time. We believe that this was because of the fact that RPE cells completely enveloped older membrane and thus prevented leakage from the newly formed vessels. Photoreceptor outer segments, which had atrophied after 7 days, were able to regenerate over CNV membranes and could be identified again after 42 days.

Staining and peeling of the internal limiting membrane using a fluorescent dye (Rhodamine 6 G).

AIM: To assess whether low concentrations of a fluorescent dye such as Rhodamine 6G would help the unaided human eye visualise the vitreous and the internal limiting membrane (ILM) under standard halogen illumination. MATERIAL/METHODS: The UV/Vis absorption (E) and fluorescence (I) spectra of Rhodamine 6G in water were measured and compared with Indocyanine Green (ICG). Surgery was performed in two rhesus monkeys and consisted of standard pars plana vitrectomy with halogen light source used for illumination. Rhodamine 6G was diluted in balanced salt solution (BSS). A few drops of the dye in a concentration of 0.1% (307 mOsm) were applied over the posterior pole in the air-filled globe and washed out by irrigation after 1 min. Immediately after surgery, the globes were enucleated, fixated and prepared for histological evaluation. RESULTS: In contrast to ICG, both the maximum of the absorption and emission of Rhodamin 6G are very much within the spectral sensitivity of the human eye. The Rhodamine 6G-BSS itself appears red in colour. Using a dye concentration of 0.1%, there was no visible red-staining of the ILM as such. As the dye was irrigated out with BSS, a marked green fluorescence of the fluid within the vitreous cavity was noted. With halogen illumination through a standard 20-gauge light pipe, the dye provided a sufficient green fluorescence to identify and safely remove the ILM and to clearly differentiate areas of peeled from non-peeled ILM. During light microscopy, eyes revealed a peeled ILM demarcation with no signs of acute retinal toxicity. CONCLUSION: The findings indicate that a fluorescent dye can be used for ILM peeling. Assuming that the fluorophore provides a high enough fluorescence quantum yield after adsorption to the ILM, much lower dye concentrations could be used compared with absorbent dyes, thereby minimising toxic effects.

Autologous transplantation of RPE with partial-thickness choroid after mechanical debridement of Bruch membrane in the rabbit.

PURPOSE: An improved translocation technique for autologous retinal pigment epithelium (RPE) transplantation is presented. The graft consists of a sheet of a partial-thickness choroid with RPE attached. METHODS: Twenty-seven pigmented rabbits were used in this study. After mechanical debridement of Bruch membrane, partial-thickness RPE-choroid sheets were transplanted onto the subretinal space in 25 rabbits. The animals were examined by fundus photographs and fluorescein angiographs and were killed postoperatively at 1, 2, 4, 12, and 24 weeks. Eyecups containing the grafts were examined by light microscopy and immunohistochemistry. In addition, two partial-thickness RPE-choroid sheets were analyzed by transmission electron microscopy (TEM). RESULTS: TEM revealed that the partial-thickness RPE-choroid graft consisted of retinal pigment epithelial cells, Bruch membrane, choriocapillaris, and ruptured middle vessels. The thickness of the graft was approximately 50 to 60 microm. Fluorescein angiography revealed neither fluorescein leakage nor staining in the graft at early or late phase. Light microscopy revealed that in 17 experiments in which the graft survived, the neural retina remained intact; however, in eight experiments with unsuccessful grafts, the neural retina degenerated. The surviving graft showed revascularization and monolayered retinal pigment epithelial cells. Furthermore, in sections in which the neural retina over the graft remained intact, all retinal pigment epithelial cells in the graft and rhodopsin in photoreceptor outer segments were positively labeled with anticellular retinaldehyde-binding protein antibodies and anti-opsin antibodies, respectively. CONCLUSIONS: A partial-thickness RPE-choroid graft showed improved integration with the host choroid and photoreceptors. This technique has the potential to be a treatment for age-related macular degeneration.

Determination of human lens capsule permeability and its feasibility as a replacement for Bruch's membrane.

We have investigated human anterior lens capsule as a potential replacement for Bruch's membrane as a treatment for age-related macular degeneration. Any substrate to replace Bruch's membrane should possess certain characteristics to maintain proper function of the overlying retina. One of the important properties of Bruch's membrane is allowing the flow of nutrients and waste between the retinal pigment epithelium and the choriocapillaris. Here, we measured the permeability of the lens capsule by studying the diffusion of various molecular weight FITC-dextran molecules. Expressions for extraction of diffusion coefficients from concentration vs. time data from a blind-well chamber apparatus were derived for both a single and double membrane experiments. The diffusion coefficients in the lens capsule were found to be in the range of 10(-6) to 10(-10)cm2/s. We demonstrated a power law relationship, with the diffusion coefficient possessing a -0.6 order dependence on molecular weight. The molecular weight exclusion limit was determined to be 150+/-40 kDa. We have compared this value with reported values of Bruch's membrane molecular weight exclusion limit and find that the lens capsule has the potential to act as a substitute Bruch's membrane.

Suppression and regression of choroidal neovascularization by polyamine analogues.

PURPOSE: Polyamine analogues inhibit tumor growth in vitro and in vivo, and oligoamines with a chain length of 10, 12, or 14 are particularly potent. This study was conducted to investigate the effect of the decamines CGC-11144 and CGC-11150 in a mouse model of choroidal neovascularization (CNV). METHODS: Mice with laser-induced rupture of Bruch's membrane were given intraperitoneal, intravitreous, or periocular injection of CGC-11144, CGC-11150, or vehicle, and after 14 days, they were perfused with fluorescein-labeled dextran, and the area of CNV was measured on choroidal flatmounts by image analysis. In some groups of mice, treatments were started 7 days after rupture of Bruch's membrane to determine the effect of the agent on established CNV. Electroretinograms (ERGs) were performed to assess the effects on retinal function, and histopathology was used to evaluate retinal structure. RESULTS: Intraperitoneal injection of 10 or 20 mg/kg CGC-11144 or CGC-11150 resulted in small but significant reductions in the area of CNV. Intravitreous injection of 20 microg CGC-11144 or CGC-11150 on days 0 and 7 after rupture of Bruch's membrane resulted in a approximately 40% reduction in the area of CNV, with a similar reduction after periocular injections of 0.2 mg CGC-11144 three times a week for 2 weeks. Both intravitreous and periocular delivery of CGC-11144 also caused significant regression of established CNV. Within 2 days of periocular injection of CGC-11144, there was apoptosis in CNV lesions, but not in normal blood vessels or other retinal cells. Periocular injections of d,l-alpha-difluoromethyl-ornithine (DFMO), which decreases polyamine levels by a different mechanism, also inhibited CNV. There was no decline in ERG amplitudes or abnormal retinal morphology after daily injections of 0.2 mg CGC-11144 for 2 weeks, but a single intravitreous injection compromised retinal structure and function. CONCLUSIONS: Periocular delivery of the polyamine analogues may be a useful approach for the treatment of CNV.