Mei Cheng

Effects of anti-VEGF treatment on the recovery of the developing retina following oxygen-induced retinopathy.

PURPOSE Inhibition of VEGF is widely used in patients to control neovascularization and decrease vascular permeability. To date, the effect of VEGF inhibition has not been evaluated in the developing retina such as that seen in premature infants. The goal of this study was to address the effect of anti-VEGF treatment on retinal development of a mouse model of retinopathy. METHODS C57BL/6J mice were evaluated using a model of oxygen-induced retinopathy. Test animals were treated at postnatal day (P) 14 with intravitreal injections of the VEGF inhibitor aflibercept (2.5 or 10 μg) in one eye. Control animals were treated with injection of PBS in one eye. The noninjected fellow eyes were used as internal controls. Areas of avascular retina and neovascular tufts in injected (treated) eyes and noninjected fellow eyes were determined at P17, and the difference related to these characteristics was obtained among them. To evaluate the effect of VEGF inhibition on neurogenesis, focal ERG was performed at P21 and P42. Histologic evaluation of the retinal structure was also evaluated at P42. RESULTS Aflibercept treatment reduced the amount of neovascular tufts but significantly increased the area of avascular retina (low dose and high dose) at P17. The delayed vascular growth corresponded to decreased ERG amplitudes (at P21 and P42) and structural changes in the retinal layers that persisted (at P42), despite vascular recovery. CONCLUSIONS Inhibition of VEGF in developing eyes has the short-term effect of delayed vascular growth and the long-term effects of decreased function with persistent changes in the neuroretinal structures.

Posterior Vitreous Detachment With Microplasmin Alters the Retinal Penetration of Intravitreal Bevacizumab (Avastin) in Rabbit Eyes

Purpose: Intravitreal bevacizumab (BV) (Avastin, Genentech Inc., South San Francisco, CA) is frequently used for the treatment of age-related macular degeneration. Previous studies have demonstrated full-thickness retinal penetration. Intravitreal recombinant microplasmin (MP) has been shown to successfully induce a posterior vitreous detachment (PVD) and vitreous liquefaction in animals. It has been suggested that a PVD may alter the retinal penetration of molecules in the vitreous cavity. The aim of this study was to compare BV retinal penetration in rabbit eyes with and without an MP-induced PVD. Methods: Twelve adult rabbits were injected with 0.1 mL (0.4 mg) of MP into the vitreous cavity of 1 eye. One week later, the rabbits were injected with 0.05 mL (1.25 mg) of BV into both eyes. Both eyes of 3 rabbits were harvested at 6 hours, 12 hours, 24 hours, and 72 hours after the BV injection. Frozen retinal cross sections were prepared, and BV retinal penetration was evaluated with immunohistochemistry using a fluorescence-labeled antibody against BV. Two eyes from one rabbit were not injected with either agent and used as controls to compare the background autofluorescence. Peripapillary retinal sections were recorded with a digital camera, and intraretinal BV fluorescence-labeled antibody was measured by qualitative photographic interpretation. Two additional rabbits received an intravitreal injection of 0.1 mL of MP in 1 eye. One week later, both eyes from each rabbit were enucleated, and frozen retinal sections were prepared and analyzed with light microscopy to evaluate histologic damage. Results: Full-thickness BV retinal penetration was observed throughout the retina in both eyes of each rabbit. All the MP-injected eyes exhibited increased antibody labeling in retinas evaluated at 6 hours, 12 hours, and 24 hours after BV injection when compared with the contralateral non-MP-injected eyes. By 3 days after BV injection, all eyes demonstrated decreased antibody labeling compared with earlier periods. At 3 days, 1 rabbit showed increased antibody labeling in the retina of the non-MP-injected eye compared with the contralateral MP-injected eye, and 2 rabbits exhibited similar antibody labeling in both eyes. When compared with control eyes, light microscopy demonstrated normal retinal histologic findings in eyes injected only with MP. Conclusion: Increased BV retinal penetration is observed initially in eyes with an MP-induced PVD, and the mechanism is likely multifactorial. By 3 days, retinal penetration is similar in eyes with and without a PVD. Although it is difficult to directly extrapolate to humans, our study suggests that a PVD may alter the retinal penetration of BV.

Norrin treatment improves ganglion cell survival in an oxygen-induced retinopathy model of retinal ischemia

ABSTRACT Treatment of a mouse model of oxygen‐induced retinopathy (OIR) with recombinant human Norrin (Norrie Disease Protein, gene: NDP) accelerates regrowth of the microvasculature into central ischemic regions of the neural retina, which are generated after treatment with 75% oxygen. While this reduces the average duration and severity of ischemia overall, we do not know if this accelerated recovery of the microvasculature results in any significant survival of retinal ganglion cells (RGCs). The purpose of this study was to investigate ganglion cell survival with and without the intravitreal injection of Norrin in the murine model of oxygen induced retinopathy (OIR), using two strains of mice: C57BL/6J and Thy1‐YFP mice. Intravitreal injections of Norrin or vehicle were done after five days of exposure to 75% oxygen from ages P7 to P12. The C57BL/J mice were followed by Spectral‐Domain Optical Coherence Tomography (SD‐OCT), and the average nerve fiber layer (NFL) and inner‐plexiform layer (IPL) thicknesses were measured at twenty‐four locations per retina at P42. Additionally, some C57BL/J retinas were flat mounted and immunostained for the RGC marker, Brn3a, to compare the population density of surviving retinal ganglion cells. Using homozygous Thy1‐YFP mice, single intrinsically fluorescent RGCs were imaged in live animals with a Micron‐III imaging system at ages P21, 28 and P42. The relative percentage of YFP‐fluorescent RGCs with dendritic arbors were compared. At age P42, the NFL was thicker in Norrin‐injected OIR eyes, 14.4 &mgr;m, compared to Vehicle‐injected OIR eyes, 13.3 &mgr;m (p = 0.01). In the superior retina, the average thickness of the IPL was greater in Norrin‐injected OIR eyes, 37.7 &mgr;m, compared to Vehicle‐injected OIR eyes, 34.6 &mgr;m (p = 0.04). Retinas from Norrin injected OIR mice had significantly more surviving RGCs (p = 0.03) than vehicle‐injected mice. Based upon NFL thickness and counts of RGCs, we conclude that Norrin treatment, early in the ischemic phase, increased the relative population density of surviving RGCs in the central retinas of OIR mice. HighlightsNorrin treatment accelerates recovery of the mouse OIR model from ischemic insult.SD‐OCT can compare NFL/GCL (nerve fiber layer/ganglion cell layer) thickness in vivo.Norrin treatment counters thinning of the NFL/GCL in the mouse OIR model.Norrin treatment increases the surviving population density of RGCs in OIR retinas.

Decreased Expression of DREAM Promotes the Degeneration of Retinal Neurons

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells.

Re: Infectious endophthalmitis after intravitreal injection of antiangiogenic agents.

To the Editor: We read with great interest the article by Nagpal et al. In the article, the authors have prospectively compared the effects of 20-millisecond Pattern Scanning Laser (Pascal) with 200-millisecond single-spot, 532-nm, solid-state green laser (GLX) in nonproliferative and proliferative diabetic retinopathy. They claimed that Pascal panretinal photocoagulation (PRP) showed lesser collateral damage and was less painful for the patient compared with GLX. We would like to address several points in this study that may provide a more open interpretation of their findings to the journal readership. Currently, the standard laser pulse durations for PRP range from 20 milliseconds to 100 milliseconds. The use of 200-millisecond pulse laser appears rather excessive and not in keeping with current laser practice. Inevitably, the laser–tissue interactions and pain responses using 200-millisecond pulse laser will be considerably greater when compared with 20-millisecond PRP. ‘‘The Pascal and GLX systems showed average fluences of 191 and 40.33 J/cm, respectively.’’ These data are clearly erroneous because shorter pulse duration will deliver a lower fluence compared with higher pulse durations. These data may be a typographic error as the abstract results conflict with the data presented in the article’s results section and Table 1. The authors report significant differences in fluence between Pascal and GLX; however, these data would appear inconsistent to readers and require clarification by the authors. A Mainster 165 PRP lens (magnification, 31.96) was used in the study, but there is no mention of adjusted laser spot size and fluence calculations by the authors. The current Pascal system has an integrated laser lens correction mode that will automatically adjust the fluence according to the laser lens used by the operator. This lens magnification factor requires clarification by the authors in order that these laser parameter data may clearly be interpreted. The fluence data presented by the authors would suggest that a 100-mm spot size was used for PRP, and the final 200-mm spot would be as a result of laser spot magnification. Otherwise, the reported fluence values should be corrected by 31.96 magnification, and this will produce lower fluence levels. GLX fluence range would be 33.1 J/cm to 66.3 J/cm (average, approximately 48 J/cm), and Pascal fluence range would be 6.6 J/cm to 16.6 J/cm (average, approximately 11 J/cm). The corrected fluence for 20-millisecond Pascal PRP would significantly be less than the authors’ report, and further clarification is required by Nagpal et al. To achieve threshold Grade 3 burns, the Pascal system required an average power of 630 mW (range 400–1000 mW), whereas the GLX needed 288 mW. The power used at 20-millisecond appears excessively high for threshold 20-millisecond PRP. The Pascal photocoagulator system was introduced to Manchester in 2006. We undertook a large retrospective evaluation of Pascal laser at Manchester Royal Eye Hospital and have recently published our experience using 20-millisecond to 30-millisecond Pascal PRP in proliferative diabetic retinopathy. On average, laser powers between 350 mW and 400 mW were required for 20-millisecond to 30-millisecond PRP, and levels of PRP power greater than 800 mW have not been encountered in our practice. There may be a higher risk of choroidal rupture and subretinal neovascularization using very high power at short pulse duration. The Manchester Pascal Study is a randomized clinical trial that evaluated the clinical effects of Pascal 20-millisecond versus 100-millisecond PRP (Archives of Ophthalmology 2010, in press). The laser powers used in our study were significantly lower than the Nagpal study, and we were able to demonstrate effective PRP responses using these lower laser power parameters. Furthermore, we performed 100-millisecond laser using much lower power settings than the Nagpal study, and so we would welcome the authors’ views on laser burn titration for PRP in their study. The authors report the changes in laser burn size over time. The laser burns in both groups increased up to 3 months, with doubling of the 200-millisecond burns and 1.5 times increase in average sizes of 20-millisecond burns. The expansion of 20-millisecond burns in this study is in contrast to the observations of laser–tissue interactions in animal studies. In the last few years, short-pulse laser burns have been found to progressively reduce in size, and this has been reported in vivo and in histopathologic work. We recently completed a randomized controlled trial of laser–tissue interactions, comparing 20-millisecond and 100-millisecond PRP burns. We observed a reduction in burn size using 20-millisecond laser lesions in the short-term. We presume that the enlargement of 20-millisecond burns in the study by Nagpal is related to the higher powers Supported in part by the Manchester Academic Health Sciences Centre (MAHSC) and NIHR Manchester Biomedical Research Centre. P.E.S. has received financial support from Optimedica Corporation.

Ocular coherence tomography image data of the retinal laminar structure in a mouse model of oxygen-induced retinopathy

The data presented in this article are related to the research paper entitled “Norrin treatment improves ganglion cell survival in an oxygen-induced model of retinal ischemia” (Dailey et al., 2017) [1] This article describes treatment with the human Norrin protein, an atypical Wnt-protein, to improve the survival of retinal ganglion cells in a murine model of Oxygen-Induced Retinopathy (OIR). That study utilized Optical coherence tomography (OCT) to visualize retinal layers at high resolution in vivo, and to quantify changes to nerve fiber layer thickness. Organization of the laminar structure of other retinal layers in this model in vivo, were not known because of uncertainties regarding potential artifacts during the processing of tissue for traditional histology. The OCT image data provided here shows researchers the retinal laminar structural features that exist in vivo in this popular mouse OIR model. Traditional H&E stained retinal tissue sections are also provided here for comparison.