Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD)1-15. However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina16-21. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD)

Advances in Age-related Macular Degeneration Understanding and Therapy

While the development of anti-vascular endothelial growth factor (anti-VEGF) as a therapy for neovascular age-related macular degeneration (AMD) was a great success, the pathologic processes underlying dry AMD that eventually leads to photoreceptor dysfunction, death, and vision loss remain elusive to date, with a lack of effective therapies and increasing prevalence of the disease. There is an overwhelming need to improve the classification system of AMD, to increase our understanding of cell death mechanisms involved in both neovascular and non-neovascular AMD, and to develop better biomarkers and clinical endpoints to eventually be able to identify better therapeutic targets—especially early in the disease process. There is no doubt that it is a matter of time before progress will be made and better therapies will be developed for non-neovascular AMD

Receptor interacting protein kinases mediate retinal detachment-induced photoreceptor necrosis and compensate for inhibition of apoptosis

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders

ThicknessTool: automated ImageJ retinal layer thickness and profile in digital images

To develop an automated retina layer thickness measurement tool for the ImageJ platform, to quantitate nuclear layers following the retina contour. We developed the ThicknessTool (TT), an automated thickness measurement plugin for the ImageJ platform. To calibrate TT, we created a calibration dataset of mock binary skeletonized mask images with increasing thickness masks and different rotations. Following, we created a training dataset and performed an agreement analysis of thickness measurements between TT and two masked manual observers. Finally, we tested the performance of TT measurements in a validation dataset of retinal detachment images. In the calibration dataset, there were no differences in layer thickness between measured and known thickness masks, with an overall coefficient of variation of 0.00%. Training dataset measurements of immunofluorescence retina nuclear layers disclosed no significant differences between TT and any observer's average outer nuclear layer (ONL) (p = 0.998), inner nuclear layer (INL) (p = 0.807), and ONL/INL ratio (p = 0.944) measurements. Agreement analysis showed that bias between TT vs. observers' mean was lower than between any observers' mean against each other in the ONL (0.77 ± 0.34 µm vs 3.25 ± 0.33 µm) and INL (1.59 ± 0.28 µm vs 2.82 ± 0.36 µm). Validation dataset showed that TT can detect significant and true ONL thinning (p = 0.006), more sensitive than manual measurement capabilities (p = 0.069). ThicknessTool can measure retina nuclear layers thickness in a fast, accurate, and precise manner with multi-platform capabilities. In addition, the TT can be customized to user preferences and is freely available to download

EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

EGF-Like-Domain-7 Is Required for VEGF-Induced Akt/ERK Activation and Vascular Tube Formation in an Ex Vivo Angiogenesis Assay

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization

CFH and ARMS2 genetic risk determines progression to neovascular age-related macular degeneration after antioxidant and zinc supplementation

We evaluated the influence of an antioxidant and zinc nutritional supplement [the Age-Related Eye Disease Study (AREDS) formulation] on delaying or preventing progression to neovascular AMD (NV) in persons with age-related macular degeneration (AMD). AREDS subjects (n = 802) with category 3 or 4 AMD at baseline who had been treated with placebo or the AREDS formulation were evaluated for differences in the risk of progression to NV as a function of complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) genotype groups. We used published genetic grouping: a two-SNP haplotype risk-calling algorithm to assess CFH, and either the single SNP rs10490924 or 372_815del443ins54 to mark ARMS2 risk. Progression risk was determined using the Cox proportional hazard model. Genetics–treatment interaction on NV risk was assessed using a multiiterative bootstrap validation analysis. We identified strong interaction of genetics with AREDS formulation treatment on the development of NV. Individuals with high CFH and no ARMS2 risk alleles and taking the AREDS formulation had increased progression to NV compared with placebo. Those with low CFH risk and high ARMS2 risk had decreased progression risk. Analysis of CFH and ARMS2 genotype groups from a validation dataset reinforces this conclusion. Bootstrapping analysis confirms the presence of a genetics–treatment interaction and suggests that individual treatment response to the AREDS formulation is largely determined by genetics. The AREDS formulation modifies the risk of progression to NV based on individual genetics. Its use should be based on patient-specific genotype