Comparison of effectiveness and safety between conbercept and ranibizumab for treatment of neovascular age-related macular degeneration. A retrospective case-controlled non-inferiority multiple center study.

PurposeTo compare the efficacy and safety of conbercept and ranibizumab when administered according to a treat-and-extend (TREX) protocol for the treatment of neovascular age-related macular degeneration (AMD) in China.Patients and methodsBetween May 2014 and May 2015, 180 patients were treated in a 1 : 1 ratio using conbercept or ranibizumab from four hospitals. Patients received either conbercept 0.5 mg or ranibizumab 0.5 mg intravitreal injections. Follow-up time was 1 year and treated based on a TREX approach. Main outcomes and measures include best-corrected visual acuity (BCVA), using Early Treatment Diabetic Retinopathy Study (ETDRS); number of injections; central retinal thickness (CRT); and leakage of choroidal neovascularization before and after the treatment was analyzed by fluorescein fundus angiography and indocyanine green angiography.ResultsThe 1-year visit was completed by 168 (93.3%) of patients. Mean BCVA was equivalent between two cohorts, and were improved by 12.7±7.770 and 12.3±7.269 letters in the conbercept and ranibizumab cohorts, respectively (P=0.624). There was no significant difference in measured CRT, with a mean decrease of 191.5 µm for conbercept and 187.8 µm for ranibizumab (P=0.773). There was a statistically significant difference (P=0.001) between the drugs regarding the number of treatments: 7.4 for conbercept and 8.7 for ranibizumab. The difference in the distribution of injection intervals was statistically significant between two groups (P=0.011). During the study, there were no cases of endophthalmitis or intraocular inflammation.ConclusionBoth drugs had equivalent effects in visual and anatomic gains at 1 year when administered. In the conbercept group, longer treatment intervals were achieved with more patients.

Persistent expression of cyclin D1 disrupts normal photoreceptor differentiation and retina development.

The differentiation of neuronal cells in the developing mammalian retina is closely coupled to cell cycle arrest and proceeds in a highly organized manner. Cyclin D1, which regulates cell proliferation in many cells, also drives the proliferation of photoreceptor progenitors. In the mouse retina, cyclin D1 protein normally decreases as photoreceptors mature. To study the importance of the down-regulation of cyclin D1 during photoreceptor development, we generated a transgenic mouse in which cyclin D1 was persistently expressed in developing photoreceptor cells. We observed numerous abnormalities in both photoreceptors and other nonphotoreceptor cells in the retina of these transgenic mice. In particular, we observed delayed opsin expression in developing photoreceptors and alterations in their number and morphology in the mature retina. These alterations were accompanied by disorganization of the inner nuclear and plexiform layers. The expression of cyclin D1 caused excess photoreceptor cell proliferation and apoptosis. Loss of the p53 tumor suppressor gene decreased cyclin D1-induced apoptosis and led to microscopic hyperplasia in the retina. These findings are distinct from other mouse models in which the retinoblastoma gene pathway is disrupted and suggest that the IRBP-cyclin D1 mouse model may recapitulate an early step in the development of retinoblastoma.

Investigating the mechanisms of retinal degenerations with antisense oligonucleotides.

Utilizing antisense oligonucleotides coupled with an intact Xenopus eye rudiment model, we have effectively demonstrated that we are able to downregulate the expression of a photoreceptor-specific protein, rds/peripherin, and generate a loss-of-function model upon which to further study the function of the rds/peripherin gene. The ultrastructure and protein expression patterns very closely resemble those previously documented in both the rds mouse and in human autosomal dominant retinitis pigmentosa due to peripherin/RDS mutations. An identical strategy can be applied to any gene correlated with a degenerative retinal phenotype. As the entire array of genes is revealed through the various genome projects, including human and mouse, it is becoming increasingly critical to evaluate and determine the function of the corresponding gene products. Discovering which gene is responsible for a particular clinical phenotype is only the first of many steps in the development of a treatment or cure for that particular disease. Using our in vitro model, in which the retina is readily accessible to the antisense oligonucleotide yet the normal three-dimensional ultrastructure of the retina is maintained, we can evaluate the function of virtually any gene as the sequence becomes available. A thorough understanding of the function of individual genes will provide insights on the role of gene product in retinal health and pathophysiology. This experimental approach will also allow for specific therapeutic interventions to be evaluated so that targeted treatments can be designed for individuals with specific genetic mutations.

Pigment epithelium-derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epithelium.

In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments.

Reliability assessment of a rod photoreceptor outer segment grading system.

The purpose of this study was to assess the reliability of a rod photoreceptor outer segment (PR-OS) grading system based on the analysis of 1 microm thick retinal sections obtained from Xenopus laevis whole-eye organ cultures. Digitally captured images, representative of the entire spectrum of rod PR-OS organization levels, were selected and coded numerically. A total of 102 individual rod PR-OS profiles were graded according to a six-step classification scheme based on the percentage of rod PR-OS membrane organization. Unweighted (exact agreement) and weighted kappa (kappa) coefficients (for use with ordered categorical rating scales) were calculated. Differences between kappa coefficients were tested for by chi-square analysis. To investigate the intra- and inter-rater variability and the possible presence of an interaction of the measurements with time, a repeated-measures analysis of variance was performed. The overall unweighted and weighted intra-rater kappa coefficients were 0.78 and 0.92, respectively. The overall unweighted and weighted inter-rater kappa coefficients were 0.73 and 0.90, respectively. There was no significant difference between raters or between first and second reading, nor was interaction between raters and time of rating documented. Individual kappa coefficients were equivalent both between raters and between sessions. Intra- and inter-rater agreement was within one step in 100% of cases. The estimated values of the kappa coefficients are consistent with a good to excellent degree of reliability and reproducibility of this rod PR-OS grading system. This system will be useful in the assessment of rod PR-OS morphology in studies of photoreceptor physiology and pathology.

Lactose supports Muller cell protein expression patterns in the absence of the retinal pigment epithelium.

PURPOSE: We have previously shown that lactose, but not mannose, promotes the assembly of nascent photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of the present study was to determine if, in addition to the improved outer segment assembly observed in the presence of lactose, biosynthetic changes in Muller cells could also be detected. METHODS: The RPE was removed from intact isolated Xenopus embryonic eyes that were allowed to complete differentiation in Niu-Twitty medium, Niu-Twitty with mannose, or Niu-Twitty with lactose. Control retinas matured in vitro with an adherent RPE. Retinal morphology was evaluated for organized folding of outer segment membranes and cell loss. In addition, the expression of three Muller cell proteins, glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), and glutamine synthetase, was examined. RESULTS: In control retinas, GFAP is undetectable, CRALBP heavily immunolabels Muller cells, and radial patterns of glutamine synthetase immunoreactivity are present. In the absence of the RPE, Muller cells upregulate GFAP expression, CRALBP labeling is present at a slightly reduced level, and glutamine synthetase immunolabeling is negligible. Neither mannose nor lactose modify significantly the expression of CRALBP. Similarly, both compounds completely prevent the upregulation of GFAP. However, normal glutamine synthetase expression was observed only in the presence of lactose, but not in the presence of mannose. Statistical analyses of slot blot-based protein quantification confirmed our immunochemical results. CONCLUSIONS: In RPE-deprived retinas supplemented with lactose, Muller cells were morphologically normal. The proper photoreceptor outer segment morphogenesis observed under these conditions was uniquely associated with normal levels of glutamine synthetase expression. The exact significance of this finding with respect to photoreceptor outer segment morphogenesis is unknown. We suggest that glutamine synthetase may be a marker of Muller cell metabolic or structural integrity that may reflect the enhanced ability of these cells, in the presence of lactose, to support photoreceptor outer segment morphogenesis.

Pigment epithelium-derived factor supports normal development of photoreceptor neurons and opsin expression after retinal pigment epithelium removal.

Dysfunction of the retinal pigment epithelium (RPE), its loss, or separation from the underlying neural retina results in severe photoreceptor degeneration. Pigment epithelium-derived factor (PEDF) is a glycoprotein with reported neuroprotective and differentiation properties that is secreted in abundance by RPE cells. The "pooling" of PEDF within the interphotoreceptor matrix places this molecule in a prime physical location to affect the underlying neural retina. The purpose of this study was to analyze the morphogenetic activity of PEDF in a model of photoreceptor dysmorphogenesis induced by removal of the RPE. Eyes were dissected from embryonic Xenopus laevis, and the RPE was removed before culturing in medium containing PEDF, PEDF plus anti-PEDF antibodies, or medium alone. Control retinas were maintained with an adherent RPE. Light and electron microscopic analysis was used to examine retinal ultrastructure. Opsin was localized immunocytochemically and quantified as an index of outer segment membranous material and photoreceptor protein expression. Removal of the RPE resulted in an aberrant assembly of photoreceptor outer segments, loss of fine subcellular ultrastructure in photoreceptors, and a reduction in opsin protein levels when compared with control retinas. The addition of PEDF prevented the dysmorphic photoreceptor changes induced by RPE removal. In particular, photoreceptor ultrastructure, outer segment membrane assembly, and steady-state levels of opsin were equivalent to control conditions. Anti-PEDF antibodies completely blocked the morphogenetic activity of PEDF. These results indicate that PEDF is able to mimic the supportive role of the RPE on photoreceptors during the final stages of retinal morphogenesis.

Targeted disruption of Müller cell metabolism induces photoreceptor dysmorphogenesis.

Within the retina, the Müller cells and photoreceptors are in close physical proximity and are metabolically coupled. It is unknown, however, whether Müller cells affect photoreceptor differentiation and outer segment membrane assembly. The objective of this study was to determine whether targeted disruption of Müller cell metabolism would induce photoreceptor dysmorphogenesis. Intact isolated Xenopus laevis embryonic eyes were cultured in medium with or without Müller cell-specific inhibitors (i.e., alpha-aminoadipic acid and fluorocitrate). To assess Müller cell injury, the gross retinal morphology was examined along with immunocytochemical assessment of Müller cell-specific protein expression patterns. The steady-state levels of opsin were quantified to determine whether the Müller cell inhibitors negatively affected photoreceptor protein synthesis. Müller and photoreceptor cell ultrastructure was scrutinized and the organization of the outer segment membranes was graded. In control retinas, there was no swelling of Müller cell cytoplasm. Glial fibrillary acidic protein (GFAP) was undetectable, whereas glutamine synthetase was abundant. The steady-state level of opsin was high and photoreceptors elaborated properly folded outer segments. Exposure to both Müller cell-specific inhibitors induced swelling of Müller cell endfeet, cytoplasmic paling and alterations of Müller cell-specific protein expression patterns. The steady-state level of opsin in retinas exposed to alpha-aminoadipic acid was unchanged compared with control eyes, whereas, in eyes exposed to fluorocitrate, opsin levels were slightly reduced. The most significant finding was that targeted disruption of Müller cell metabolism adversely affected photoreceptor outer segment membrane assembly, causing dysmorphogenesis of nascent outer segments. These results suggest that the termination signal(s) necessary for proper outer segment folding were disrupted by targeted inhibition of Müller cells and support the hypothesis that Müller cells interact with photoreceptors through mechanisms that may regulate, at least in part, the assembly of photoreceptor outer segment membranes.

Closer look at lactose-mediated support of retinal morphogenesis.

We have previously shown in intact isolated eye rudiments from Xenopus laevis that lactose, but not mannose, permits the formation of organized photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine, using electron microscopic analysis, the key ultrastructural differences between healthy retinas, lactose-protected retinas, and retinas that developed aberrantly to reveal which subcellular structures were exclusively present in healthy retinas. Filamentous actin was also localized in retinas to determine its distribution under the various conditions. In healthy retinas, calycal processes extending approximately three-fourths of the length of the outer segment surrounded highly organized photoreceptor outer segments. Adherens junctions were localized between adjacent photoreceptors and Müller cells at the outer limiting membrane. In addition, Müller cells possessed apical processes that extended for a short distance beyond the adherens junctions. These fine cytoarchitectural details were missing in retinas that completed differentiation in the absence of the RPE; both calycal and apical processes were no longer present and adherens junctions were sparsely intermittent. Müller cells appeared atrophic. Similarly, mannose promoted none of the fine cytoarchitectural details of the retina. Lactose, however, supported the formation of the proper subcellular cytoarchitecture of both photoreceptor and Müller cells. These results suggest that these subcellular structures may be fundamental for the proper assembly and stability of organized outer segments and are necessary to allow for normal cytogenesis of the outer retina.

Lactose promotes organized photoreceptor outer segment assembly and preserves expression of photoreceptor proteins in retinal degeneration.

PURPOSE: We have previously shown that lactose promotes the proper assembly of photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of this study was to determine if the difference between organized and disorganized membranes was a variation in the amounts of two structural proteins, opsin and rds/peripherin. METHODS: Eye rudiments were dissected from Xenopus laevis embryos and the RPE was removed prior to culturing in the following media: Niu-Twitty medium; Niu-Twitty with mannose; Niu-Twitty with lactose. Controls included retinas that matured in vitro with an adherent RPE. Photoreceptor ultrastructure was evaluated with emphasis on outer segment membrane organization. The relative amounts of opsin and rds/ peripherin, two outer segment-specific proteins, were determined, as were their immunolabeling patterns. RESULTS: In control retinas, outer segments were composed of stacked, flattened membranous saccules. Opsin labeling of rod outer segments was very dense, indicative of normally organized disc membranes, and rds/peripherin labeling was heavy at the outer segment disc periphery and incisures. In the absence of the RPE, a whorl-like profile of outer segments is present in what would be the sub-retinal space. Opsin immunolabeling was patchy and disorganized. Immunolabeling of rds/peripherin was present, but in a disorderly array. Mannose showed no protective effect. In contrast, lactose promoted the formation of organized outer segments and allowed for near normal expression of both photoreceptor markers. In retinas with disorganized outer segments, the expression of opsin is downregulated while the expression of rds/peripherin is maintained or upregulated. CONCLUSIONS: Lactose protects against the retinal degeneration induced by RPE removal by preserving the outer segment structure and the photoreceptor immunolabeling patterns. It also maintains constant the relative amounts of opsin and rds/peripherin. It is possible that in degenerating retinas, photoreceptors upregulate rds/peripherin expression in attempt to provide additional support for the proper folding of nascent membranes, however this is insufficient to permit organization of the photoreceptor outer segments. Our results suggest that rescue-effect of lactose is mediated by a non-rds/peripherin related mechanism.

Comparison of conjunctival and corneal surface areas in rabbit and human.

This comparative study shows the surface area ratio of conjunctiva to cornea to be two times larger in humans than in rabbits. This large heretofore unrecognized interspecies difference may affect the applicability of drug pharmacokinetic data obtained using rabbit models and should be taken into consideration in topical drug development and future comparative drug penetration studies between rabbit and man.