Baseline characteristics and progression of neovascular age-related macular degeneration in patients receiving over 60 intravitreal injections of anti-vascular endothelial growth factor.

Objective: Age-related macular degeneration (AMD) is the leading cause of vision loss in older populations of industrialized countries. Antibody-based therapy inhibiting the vascular endothelial growth factor (VEGF) has been very successful in the treatment of the neovascular form of AMD. This retrospective clinical study investigates the baseline characteristics and progression of neovascular age-related macular degeneration (nAMD) in patients who received over 60 anti-VEGF intravitreal injections. Methods: Retrospective analysis of 6812 eyes of 5678 patients undergoing anti-VEGF treatment at our clinic between November 2006 and December 2017 yielded 12 eyes of 12 patients who had received more than 60 intravitreal injections into one eye. We re-evaluated the baseline characteristics of visual acuity, intraocular pressure, optical coherence tomography, fluorescein angiography, as well as autofluorescence and analyzed the documented disease progress as monitored in our daily clinical practice. Data on the fellow eye were also analyzed. Results: Each of our 12 patients had the injected anti-VEGF agent (bevacizumab, ranibizumab, or aflibercept) changed at least once during treatment. After initial improvement, visual acuity decreased in most patients over time. The 2 patients with the best visual acuity at the beginning also showed the best visual acuity at the end of the study. No significant change was observed in the intraocular pressure. Conclusions: After the initial improvement, visual acuity decreased over time. Good visual acuity at the beginning of the study increased the chances of maintaining the same level throughout the treatment. Intravitreal treatment did not affect intraocular pressure. Abbreviations: AMD = age-related macular degeneration, nAMD = neovascular age-related macular degeneration, VEGF = vascular endothelial growth factor, OCT = optical coherence tomography, VA = visual acuity, PDT = photodynamic therapy.

ERG Alteration Due to the rd8 Mutation of the Crb1 Gene in Cln3 +/+ rd8-/rd8- Mice.

Mattapallil et al. described that vendor lines for C57BL/6 N mice may carry the rd8 mutation that leads to an ocular phenotype, which could be mistaken for an induced retinal degeneration. This mouse strain is widely used in ophthalmic research as a background for modeling retinal degeneration. In the process of studying Cln3Δex7/8 knock-in mice on a C57BL/6 N background, we became aware of this issue. The aim of this study thus was to use electroretinography to investigate the age-dependent functional loss in Cln3+/+ rd8-/rd8- mice and compare it to C57BL/6 J mice.The scotopic and photopic amplitudes of the a-wave and b-wave decrease significantly in mutant mice with increasing age, and the implicit time is prolonged. Especially the oscillatory potentials arising from inner retinal interaction seem to be notably affected by the rd8 mutation. Surprisingly, the amplitudes in young C57BL/6 J mice were lower than those measured in C57BL/6 N at any time point.Our results indicate that the rd8 mutation present in C57BL/6 N mice affects the function of the inner and outer retina. This is surprising given that the major retinal morphological alterations due to the rd8 mutation are found in the outer retina.We conclude that the rd8 mutation does affect the retinal function in Cln3+/+ rd8-/rd8- mice in a variable manner. Epigenetic factors and modifying genes lead to a phenotype shift in these mice. Interpreting the results of previous studies in mutant mice on C57BL/6 N background is challenging as comparing results obtained in independent studies or on other mouse backgrounds may be misleading. Using littermates as controls remains the only valid option.

Spectral Domain Optical Coherence Tomography Allows the Unification of Clinical Decision Making for the Evaluation of Choroidal Neovascularization Activity.

PURPOSE: This prospective observational clinical study investigated the benefits of spectral domain optical coherence tomography for specialists and residents in the management of neovascular age-related macular degeneration (AMD). PROCEDURES: The study involved 49 eyes of 44 patients. Patients were advised to present for reevaluation 4 weeks after the administration of the loading dose of vascular endothelial growth factor (VEGF)-inhibitors (3 intravitreal injections every 4 weeks after diagnosis). They were examined by residents (3-4 years' experience in ophthalmology) and specialists (> 5 years' experience). Each examiner evaluated the clinical situation and the spectral domain optical coherence tomography (SD-OCT) scan. After each evaluation, the examiners independently stated if further anti-VEGF treatment was recommended. The "true outcome" was defined as the specialist decision based on clinical evaluation and SD-OCT. RESULTS: Specialists and residents did not significantly differ in their accuracy in deciding on the correct treatment (p = 0.705 and p = 1), with or without the aid of SD-OCT. Both groups benefited from using SD-OCT to support their recommendations (p = 0.001 and p = 0.0002) and achieved a similar level of accuracy (p = 1 for difference). CONCLUSIONS: Residents benefited more than specialists by using SD-OCT to substantiate their recommendation on how to manage exudative AMD after the administration of the loading dose.

Further Characterization of the Predominant Inner Retinal Degeneration of Aging Cln3 Δex7/8 Knock-In Mice.

Neuronal ceroid lipofuscinosis (NCL) is the most common group of neurogenetic storage diseases typically beginning in childhood. The juvenile form (JNCL), also known as Batten disease, is the most common form. Vision-related problems are often an early sign, appearing prior to motor and mental deficits. We have previously investigated disease progression with age in the Cln3 Δex7/8 KI mouse model for JNCL and showed a decline of visual acuity and a predominant decline of the inner retinal function in mice, similar to human disease. The aim of this study was to further characterize this degeneration by means of flicker ERGs. For the scotopic flicker ERG, we found a significantly lower magnitude for Cln3 Δex7/8 KI mice already at 6 months of age for low stimulus frequencies, while the difference declines with increasing frequency. Under photopic conditions there was no magnitude difference at 6 months, but a cumulative magnitude reduction with further aging. For both conditions the phases were similar for both groups. There was a similar magnitude reduction for the responses of both the slow and fast rod pathway in the 15 Hz experiments, and there were no differences in response phase. Low-frequency flicker responses seem to be sensitive to very early disease manifestations, and while the degeneration is associated with a reduction of predominating inner retinal responses in the scotopic flash ERG, this predominance seems not to be related to a selective involvement of the slow and fast rod pathways.

Suppression of SNARE-dependent exocytosis in retinal glial cells and its effect on ischemia-induced neurodegeneration.

Nervous tissue is characterized by a tight structural association between glial cells and neurons. It is well known that glial cells support neuronal functions, but their role under pathologic conditions is less well understood. Here, we addressed this question in vivo using an experimental model of retinal ischemia and transgenic mice for glia-specific inhibition of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis. Transgene expression reduced glutamate, but not ATP release from single Müller cells, impaired glial volume regulation under normal conditions and reduced neuronal dysfunction and death in the inner retina during the early stages of ischemia. Our study reveals that the SNARE-dependent exocytosis in glial cells contributes to neurotoxicity during ischemia in vivo and suggests glial exocytosis as a target for therapeutic approaches.

Mutated olfactomedin 1 in the interphotoreceptor matrix of the mouse retina causes functional deficits and vulnerability to light damage.

Olfactomedin 1 (OLFM1) is a secreted glycoprotein and member of the olfactomedin protein family, which is preferentially expressed in various areas throughout the central nervous system. To learn about the functional properties of OLFM1 in the eye, we investigated its localization in the mouse and pig eye. In addition, we analyzed the ocular phenotype of Olfm1 mutant mice in which 52 amino acids were deleted in the central part (M2 region) of OLFM1. OLFM1 was detected in cornea, sclera, retina, and optic nerve of both wild-type and Olfm1 mutant littermates. By immunohistochemistry and double labeling with the lectin peanut agglutinin, OLFM1 was found in the interphotoreceptor matrix (IPM) of mouse and pig retina where it was directly localized to the inner segments of photoreceptors. Western blotting confirmed the presence of the OLFM1 isoforms pancortin 1 (BMY) and pancortin 2 (BMZ) in the IPM. The retinal phenotype of Olfm1 mutant mice did not obviously differ from that of wild-type littermates. In addition, outer nuclear layer (ONL) and total retinal thickness were not different, and the same was true for the area of the optic nerve in cross sections. Functional changes were observed though by electroretinography, which showed significantly lower a- and b-wave amplitudes in Olfm1 mutant mice when compared to age-matched wild-type mice. When light damage experiments were performed as an experimental paradigm of photoreceptor apoptosis, significantly more TUNEL-positive cells were observed in Olfm1 mutant mice 30 h after light exposure. One week after light exposure, the ONL was significantly thinner in Olfm1 mutant mice than in wild-type littermates indicating increased photoreceptor loss. No differences were observed when rhodopsin turnover or ERK1/2 signaling was investigated. We conclude that OLFM1 is a newly identified IPM molecule that serves an important role for photoreceptor homeostasis, which is significantly compromised in the eyes of Olfm1 mutant mice.

Acid sphingomyelinase (aSMase) deficiency leads to abnormal microglia behavior and disturbed retinal function.

Mutations in the acid sphingomyelinase (aSMase) coding gene sphingomyelin phosphodiesterase 1 (SMPD1) cause Niemann-Pick disease (NPD) type A and B. Sphingomyelin storage in cells of the mononuclear phagocyte system cause hepatosplenomegaly and severe neurodegeneration in the brain of NPD patients. However, the effects of aSMase deficiency on retinal structure and microglial behavior have not been addressed in detail yet. Here, we demonstrate that retinas of aSMase(-/-) mice did not display overt neuronal degeneration but showed significantly reduced scotopic and photopic responses in electroretinography. In vivo fundus imaging of aSMase(-/-) mice showed many hyperreflective spots and staining for the retinal microglia marker Iba1 revealed massive proliferation of retinal microglia that had significantly enlarged somata. Nile red staining detected prominent phospholipid inclusions in microglia and lipid analysis showed significantly increased sphingomyelin levels in retinas of aSMase(-/-) mice. In conclusion, the aSMase-deficient mouse is the first example in which microglial lipid inclusions are directly related to a loss of retinal function.

Deletion of ocular transforming growth factor ß signaling mimics essential characteristics of diabetic retinopathy.

Diabetic retinopathy, a major cause of blindness, is characterized by a distinct phenotype. The molecular causes of the phenotype are not sufficiently clear. Here, we report that deletion of transforming growth factor ß signaling in the retinal microenvironment of newborn mice induces changes that largely mimic the phenotype of nonproliferative and proliferative diabetic retinopathy in humans. Lack of transforming growth factor ß signaling leads to the formation of abundant microaneurysms, leaky capillaries, and retinal hemorrhages. Retinal capillaries are not covered by differentiated pericytes, but by a coat of vascular smooth muscle-like cells and a thickened basal lamina. Reactive microglia is found in close association with retinal capillaries. In older animals, loss of endothelial cells and the formation of ghost vessels are observed, findings that correlate with the induction of angiogenic molecules and the accumulation of retinal hypoxia-inducible factor 1α, indicating hypoxia. Consequently, retinal and vitreal neovascularization occurs, a scenario that leads to retinal detachment, vitreal hemorrhages, neuronal apoptosis, and impairment of sensory function. We conclude that transforming growth factor ß signaling is required for the differentiation of retinal pericytes during vascular development of the retina. Lack of differentiated pericytes initiates a scenario of structural and functional changes in the retina that mimics those of diabetic retinopathy strongly indicating a common mechanism.

Antibody therapies and their challenges in the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of vision loss in the western world. This multifactorial disease results from the combined contributions of age, environment and genetic predisposition. Antibody-based treatment of late-stage neovascular AMD with inhibitors of vascular endothelial growth factor has had great success, which is now the goal for currently untreatable AMD manifestations. The existence of an immune-privileged environment in the eye supports the feasibility of localized antibody therapy. Many different antibodies against various targets are being developed for the treatment of AMD, which reflects the etiological complexity of the disease. This review provides an overview of 19 potential therapeutic antibodies targeting angiogenesis, the complement system, inflammation or amyloid beta deposition in the eye. It summarizes the immunoglobulin structure, the specific target and study outcomes for each approach. The latter include beneficial results or adverse effects in AMD models and patients. Finally, this article discusses the challenges in the development of antibody-based drugs to treat degenerative processes in the posterior eye. In spite of these difficulties, to date, the following four antibodies have overcome the technical and preclinical hurdles and are being tested in active clinical studies: Lampalizumab, Sonepcizumab, GSK933776 and LFG316. We conclude that, while there are some antibody-based drugs that have made it into clinical practice, a successful transfer from bench to beside is still pending for many promising approaches.

Myocilin modulates programmed cell death during retinal development.

Mutations in the myocilin gene (MYOC) are causative for 10% of cases with juvenile open-angle glaucoma and 3-4% of those with primary open-angle glaucoma. Myocilin is a secreted protein with relatively ill-defined matricellular properties. Despite its high expression in the eye, myocilin-deficient mice have originally been reported to have no obvious ocular phenotype. Here we revisited the ocular phenotype of myocilin-deficient mice and detected a higher number of neurons in their inner (INL) and outer (ONL) nuclear layers, as well as a higher number of retinal ganglion cells (RGC) and their axons. The increase in retinal neurons appears to be caused by a decrease in programmed developmental cell death, as apoptosis of retinal neurons between postnatal days 4 and 10 was found to be attenuated when compared to that of wildtype littermates. In contrast, when Myoc(-/-) mice were crossed with ßB1-crystallin-MYOC mice with ectopic overexpression of myocilin in the eye, no differences in developmental apoptosis, RGC number and INL thickness were observed when compared to wildtype littermates. The amounts of the anti-apoptotic Bcl-2-like protein 1 (BCL2L1, Bcl-xL) and its mRNA were increased in retinae of Myoc(-/-) mice, while lower amounts of BCL2L1 and its mRNA were detected in mixed Myoc(-/-)/ßB1-crystallin-MYOC mice. The structural differences between Myoc(-/-) mice and wildtype littermates did not result in functional differences as measured by electroretinography. Noteworthy though mixed Myoc(-/-)/ßB1-crystallin-MYOC mice with ocular overexpression of myocilin had significant cone function deficits. Myocilin appears to modulate apoptotic death of retinal neurons likely by interacting with the intrinsic apoptotic pathway.

Retinal function in aging homozygous Cln3 (Δex7/8) knock-in mice.

Neuronal ceroid lipofuscinoses (NCL) are characterized by lysosomal accumulation of autofluorescent material and lead to degeneration of the central nervous system. Patients affected by the juvenile form of NCL (JNCL), the most common form of the disease, develop visual failure prior to mental and motor deficits. It is currently unclear if the corresponding mouse model, Cln3 (Δex7/8) knock-in, develops the same retinal phenotype and electroretinogram (ERG) measurements as affected patients. The aim of our study was to investigate the visual disease progression in the Cln3 (Δex7/8) mice using scotopic and photopic ERGs as well as optokinetic tracking (OKT) at different ages. The results were then compared with age-matched controls.The amplitudes of the a-wave and b-wave (scotopic ERG) decrease significantly in Cln3 (Δex7/8) mice starting at the age of 12 months. A reduction in the b/a-amplitude ratio indicates a degeneration preferentially of the inner retina. An amplitude reduction observed in the Cln3 (+/+) control mice may be attributed to an additional Crb1 (rd8) mutation. Using optokinetic tracking (OKT) the Cln3 (Δex7/8) mice show a progressive decline in visual acuity after 12 months of age.

Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6 (nclf) mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6 (nclf) retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6 (nclf) retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6 (nclf) mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6 (nclf) retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6 (nclf) retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.

Constitutive overexpression of Norrin activates Wnt/ß-catenin and endothelin-2 signaling to protect photoreceptors from light damage.

Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/ß-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65-Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/ß-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/ß-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/ß-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury.

Evaluation of intraocular pressure elevation in a modified laser-induced glaucoma rat model.

The main drawbacks of currently described pressure induced glaucoma animal models are, that intraocular pressure (IOP) either rises slowly, leading to a heterogeneous onset of glaucoma in the treated animals or that IOP normalizes before significant damage occurs, necessitating re-treatment. Furthermore, a variable magnitude of IOP increase often results when particles are introduced into the anterior chamber. In order to develop a simple and reproducible rat glaucoma model with sustained IOP elevation after a single treatment we induced occlusion of the chamber angle by anterior chamber paracentesis and subsequent laser coagulation of the limbal area with 35, 40 or 45 laser burns. Right eyes served as controls. IOP was measured three times weekly using TonoLab rebound tonometry in awake animals. After four weeks, retinal tissue was harvested and processed for whole mount preparation. The number of prelabeled, fluorogold-positive retinal ganglion cells (RGCs) was analyzed under a fluorescence microscope. The eyes were further analyzed histologically. Results are expressed as means and standard deviation. Amplitude and duration of the IOP elevation increased with the number of laser burns. Two weeks after 35, 40 or 45 translimbal laser burns the IOP difference between treated and control eye was 7.5 ± 5, 14 ± 8 or 19 ± 9 mmHg, respectively; the RGC density/mm(2) 28 days after treatment was 1488 ± 238 for control eyes (n = 31) and 1514 ± 287 (n = 10), 955 ± 378 (n = 10) or 447 ± 350 (n = 11) for the respective laser groups. Mean IOP of all control eyes over the observation period was 12.4 ± 0.8 mmHg. The chamber angle showed pigment accumulation in the trabecular meshwork of all laser groups and confluent peripheral anterior synechia after 40 and 45 laser burns. Histologic examination of the retina revealed increasing glia activation in a pressure dependant manner. In this study, >91% of laser treated rats developed secondary glaucoma with sustained IOP elevation for at least 2 weeks. The amount of IOP elevation and RGC loss correspond with the number of laser burns applied. This relatively high success rate after a single procedure may constitutes an advantage over established glaucoma models, as this decreases the risk of complications (e.g. corneal decompensation, intraocular bleeding or inflammation) and, thus, improves the outcome.