Role of HIF1α and HIF2α in Cre Recombinase-Induced Retinal Pigment Epithelium Pathology and Its Secondary Effect on Choroidal Neovascularization.

CreTrp1 mice are widely used for conditional retinal pigment epithelium (RPE) gene function studies. Like other Cre/LoxP models, phenotypes in CreTrp1 mice can be affected by Cre-mediated cellular toxicity, leading to RPE dysfunction, altered morphology and atrophy, activation of innate immunity, and consequent impairment of photoreceptor function. These effects are common among the age-related alterations of RPE that feature in early/intermediate forms of age-related macular degeneration. This article characterizes Cre-mediated pathology in the CreTrp1 line to elucidate the impact of RPE degeneration on both developmental and pathologic choroidal neovascularization. Nonredundant roles of the two major components of the hypoxia-inducible factor (HIF) family of transcription regulators, HIF1α and HIF2α, were identified. Genetic ablation of Hif1a protected against Cre-induced degeneration of RPE and choroid, whereas ablation of Hif2a exacerbated this degeneration. Furthermore, HIF1α deficiency protected CreTrp1 mice against laser-induced choroidal neovascularization, whereas HIF2α deficiency exacerbated the phenotype. Cre-mediated degeneration of the RPE in CreTrp1 mice offers an opportunity to investigate the impact of hypoxia signaling in the context of RPE degeneration. These findings indicate that HIF1α promotes Cre recombinase-mediated RPE degeneration and laser-induced choroidal neovascularization, whereas HIF2α is protective.

Retinal detachment after post-operative endophthalmitis: clinical characteristics, outcomes and risk factors.

PURPOSE: To describe clinical characteristics, risk factors, and outcomes of rhegmatogenous retinal detachment (RRD) following treatment of postoperative endophthalmitis (PE). METHODS: Analysis of cross-referenced data from two service reviews of patients with RRD and bacterial PE treated between 01/01/2013 and 01/07/2020. The main outcome measure was final best-corrected visual acuity (BCVA). Secondary measures include proportion of patients with BCVA of ≤ 0.3 logMAR and ≥ 1.0 logMAR, rate of phthsis, and rate of eye removal. RESULTS: Ninety-four cases of PE were analysed finding 21 cases of RRD (22%). Seven (35%) experienced recurrent RRD. Seven eyes (35%) were left with permanent silicone oil fill. All RRD cases had vitrectomy. After PE with RRD the median BCVA was 1.1 logMAR, compared with 0.4 logMAR for PE without RRD (p < 0.04). Fifty-seven percent (12/21) of RRD eyes attained BCVA of ≥ 1.0 logMAR vs. 29% (21/73) of PE without RRD (p = 0.01). Nineteen percent (4/21) of eyes with RRD attained BCVA of ≤ 0.3 logMAR, whereas those without RRD did so in 43% (31/73) of cases (p = 0.02). Five eyes with RRD (24%) and 2 eyes without RRD (3%) developed phthisis (p < 0.01). Three non-RRD cases required removal of the eye (4%, p = 0.46). Higher bacterial virulence was associated with worse final BCVA (2.1 logMAR vs. 0.3 logMAR; p < 0.01). RRD rate did not differ by bacterial virulence (OR 1.9; CI95: 0.6-6.9; p = 0.24). CONCLUSIONS: RRD following PE leads to worse clinical outcomes. Eyes which developed RRD were more likely to have undergone vitrectomy. Final BCVA was worse in cases with more virulent micro-organisms.

Pathological Angiogenesis Requires Syndecan-4 for Efficient VEGFA-Induced VE-Cadherin Internalization.

[Figure: see text].

Late neuroprogenitors contribute to normal retinal vascular development in a Hif2a-dependent manner.

In the adult central nervous system, endothelial and neuronal cells engage in tight cross-talk as key components of the so-called neurovascular unit. Impairment of this important relationship adversely affects tissue homeostasis, as observed in neurodegenerative conditions including Alzheimer's and Parkinson's disease. In development, the influence of neuroprogenitor cells on angiogenesis is poorly understood. Here, we show in mouse that these cells interact intimately with the growing retinal vascular network, and we identify a novel regulatory mechanism of vasculature development mediated by hypoxia-inducible factor 2a (Hif2a). By Cre-lox gene excision, we show that Hif2a in retinal neuroprogenitor cells upregulates the expression of the pro-angiogenic mediators vascular endothelial growth factor and erythropoietin, whereas it locally downregulates the angiogenesis inhibitor endostatin. Importantly, absence of Hif2a in retinal neuroprogenitor cells causes a marked reduction of proliferating endothelial cells at the angiogenic front. This results in delayed retinal vascular development, fewer major retinal vessels and reduced density of the peripheral deep retinal vascular plexus. Our findings demonstrate that retinal neuroprogenitor cells are a crucial component of the developing neurovascular unit.

Comparison of Perfluorodecalin and Silicone Oil as Initial Tamponade for Giant Retinal Tear-Associated Retinal Detachment.

INTRODUCTION: To compare visual outcomes and complication rates of giant retinal tear-associated retinal detachment (GRT-RD) cases treated with short-term perfluorodecalin (PFD) tamponade versus silicone oil (SiO). METHODS: Database analysis of patients with GRT-RD operated on in the period from 1 January 2014 to 31 December 2019. RESULTS: Forty-five patients were operated for GRT-RD using PFD or SiO during this period. Two children, 7 patients receiving gas tamponade, and 2 lost to follow-up were excluded. Eighteen eyes (40%) received PFD and 27 (60%) received SiO. There were 15/18 (83%) macula-sparing cases in the PFD group and 18/27 (67%) in the SiO group (p = 0.13). The mean duration of oil tamponade was 91 days for SiO and 7.6 days for PFD (p < 0.0001). The mean length of follow-up was 274.5 days for PFD and 668.9 days for SiO. The mean BCVA was 6/18 (63.4 ± 26.0 ETDRS letters) for SiO and 6/12 (72.9 ± 12.7 ETDRS letters) for PFD (p = 0.42). Analysing macula-sparing pseudophakic eyes, the BCVA was 6/12 (67.4 ± 25.9 letters, n = 18) for SiO eyes and 6/9 (76.8 ± 9.9 letters, n = 11) for PFD eyes (p = 0.54). The recurrence rate was 22% (6/27) for SiO and 6% (1/18) for PFD (p = 0.12). The rate of cystoid macular oedema (CMO) was 22% for SiO and 22% for PFD. Epiretinal membrane (ERM) was found in 26% of SiO cases and 22% of PFD cases. Loss of vision after oil removal was not observed. Seven eyes (26%) receiving SiO and none receiving PFD developed chronic ocular hypertension (OHT) (p = 0.02). CONCLUSIONS: Short-term tamponade with PFD for GRT-RD appears similar to tamponade with SiO in terms of the visual outcomes and complication rates.

Stabilization of myeloid-derived HIFs promotes vascular regeneration in retinal ischemia.

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.

A Study of the Natural History of Vitreomacular Traction Syndrome by OCT.

PURPOSE: To examine the natural history of vitreomacular traction syndrome (VMTS) in the absence of other ocular comorbidities. DESIGN: Retrospective clinical case series. PARTICIPANTS: A total of 183 eyes of 159 patients diagnosed with VMTS with no other ocular comorbidity. METHODS: Patients with VMTS were identified from an OCT database at Moorfields Eye Hospital, London. Sequential OCT scans and patient notes were reviewed over a minimum period of 6 months. Data collected included patient demographics, best-corrected visual acuity, and OCT features of vitreomacular adhesion. Contingency tests and binary logistic modeling were used to identify baseline predictors of stability and progression. MAIN OUTCOME MEASURES: The rates of spontaneous resolution (defined by release of traction), progression to full-thickness macular hole, and surgical intervention were analyzed. RESULTS: Presenting visual acuity was 0.3±0.3 logMAR units. The mean length of follow-up was 17.4±12.1 months. During this period, VMTS persisted in 60% and resolved in 20% (occurring on average at 15 months). Of the remainder, 12% developed a macular hole and 8% elected to proceed with surgery for symptoms. Focal adhesion <1500 µm was present in 87%. A premacular membrane with macular pucker (PMM) was present in 20%. With persistent VMTS, vision and central foveal thickness remained unchanged. The relative risk of resolution increased in those cases with better presenting visual acuities, lesser foveal thicknesses, and no associated PMMs; vision significantly improved in those cases with resolution. CONCLUSIONS: VMTS persists in the majority of patients but despite this, visual acuities did not deteriorate significantly over the study period unless patients developed a full-thickness macular hole or required surgical intervention for symptoms. Resolution spontaneously occurred in 20%, with an improvement in vision.

Myeloid-Derived Vascular Endothelial Growth Factor and Hypoxia-Inducible Factor Are Dispensable for Ocular Neovascularization--Brief Report.

OBJECTIVE: Ocular neovascularization (ONV) is a pathological feature of sight-threatening human diseases, such as diabetic retinopathy and age-related macular degeneration. Macrophage depletion in mouse models of ONV reduces the formation of pathological blood vessels, and myeloid cells are widely considered an important source of the vascular endothelial growth factor A (VEGF). However, the importance of VEGF or its upstream regulators hypoxia-inducible factor-1α (HIF1α) and hypoxia-inducible factor-2α (HIF2α) as myeloid-derived regulators of ONV remains to be determined. APPROACH AND RESULTS: We used 2 mouse models of ONV, choroidal neovascularization and oxygen-induced retinopathy, to show that Vegfa is highly expressed by several cell types, but not myeloid cells during ONV. Moreover, myeloid-specific VEGF ablation did not reduce total ocular VEGF during choroidal neovascularization or oxygen-induced retinopathy. In agreement, the conditional inactivation of Vegfa, Hif1a, or Epas1 in recruited and resident myeloid cells that accumulated at sites of neovascularization did not significantly reduce choroidal neovascularization or oxygen-induced retinopathy. CONCLUSIONS: The finding that myeloid cells are not a significant local source of VEGF in these rodent models of ONV suggests that myeloid function in neovascular eye disease differs from skin wound healing and other neovascular pathologies.

Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models.

Myeloid cells make a pivotal contribution to tissue homeostasis during inflammation. Both tissue-specific resident populations and infiltrating myeloid cells can cause tissue injury through aberrant activation and/or dysregulated activity. Reliable identification and quantification of myeloid cells within diseased tissues is important to understand pathological inflammatory processes. Flow cytometry is a valuable technique for leukocyte analysis, but a standardized flow cytometric method for myeloid cell populations in the eye is lacking. Here, we validate a reproducible flow cytometry gating approach to characterize myeloid cells in several commonly used models of ocular inflammation. We profile and quantify myeloid subsets across these models, and highlight the value of this strategy in identifying phenotypic differences using Ccr2-deficient mice. This method will aid standardization in the field and facilitate future investigations into the roles of myeloid cells during ocular inflammation.

PRIMARY SCLERAL BUCKLING FOR PEDIATRIC RHEGMATOGENOUS RETINAL DETACHMENT.

PURPOSE: To evaluate the anatomical outcomes of primary scleral buckling (SB) procedures for pediatric rhegmatogenous retinal detachments. METHODS: Retrospective consecutive case series. One hundred and four eyes of 99 consecutive nonselected pediatric patients undergoing primary SB were identified. Baseline factors recorded were demographics, presenting clinical examination findings, previous ocular surgery, predisposing factors. Intraoperative factors recorded were the type of buckle, number and distribution of retinal breaks, number of retinal quadrants detached, macular status (involved vs. uninvolved), the use of subretinal fluid drainage, and surgical complications. Anatomical reattachment rate at last follow-up. Subgroup analysis was carried out to identify any predisposing factors for failure of primary surgery, effect of age on outcome, intraoperative pathology, effect of posterior versus anterior SB, and redetachment and secondary-procedure complications specific to SB. RESULTS: The initial surgery was segmental SB alone in 87 eyes (83.6%). Retinal reattachment was achieved with 1 operation in 73% (76 of 104 eyes). Of the 28 cases that redetached, 14 eyes underwent a repeat SB procedure (success rate of this second operation: 85.7% [12 of 14 eyes]), 13 eyes underwent vitrectomy (success rate of this second operation: 38.4% [5 of 13 eyes]), and 1 case was not reoperated. Overall, the final success rate was 94% (98 of 104 eyes). Factors associated with a statistically significant increased risk of failure included more than one break; three or more quadrants of detachment; horseshoe tears; no breaks seen on preoperative examination; Stickler syndrome. CONCLUSION: In selected cases, primary SB is an effective treatment for pediatric, rhegmatogenous retinal detachment.

RHEGMATOGENOUS RETINAL DETACHMENT AFTER INJECTION OF TISSUE PLASMINOGEN ACTIVATOR AND GAS FOR SUBMACULAR HEMORRHAGE SECONDARY TO AGE-RELATED MACULAR DEGENERATION.

PURPOSE: The purpose of this study was to describe the rate, clinical characteristics, and outcomes of rhegmatogenous retinal detachment (RRD) after injection of tissue plasminogen activator (TPA) and gas for submacular hemorrhage displacement. METHODS: Retrospective analysis of consecutive cases developing RRD after TPA injection and gas for submacular hemorrhage displacement. The rate of RRD was calculated, and a description of RRD clinical characteristics was performed. Anatomic and visual outcomes after RRD repair were analyzed. RESULTS: Ninety eyes of 90 patients were analyzed. Tissue plasminogen activator was given intravitreally in 53 eyes (59%) and subretinally in 37 eyes (41%). RRD occurred in 6 of 90 eyes (7%). Of these, one had intravitreal TPA and five had vitrectomy with subretinal TPA ( P = 0.04). The mean age was 75 (64-93) years. The median time of RRD occurrence was 42 (1-134) days. All cases had macular involvement. Two cases had PVR at presentation. Vitrectomy was performed in all cases and silicone oil used in five, all of which resulted in permanent silicone oil retention. One case (17%) achieved primary single surgery success. The median final visual acuity was 1.8 logMAR (20/1,260 Snellen). CONCLUSION: The RRD rate after submacular hemorrhage displacement was 7% in our case series. Rhegmatogenous retinal detachment occurred more commonly after vitrectomy with subretinal TPA injection. The visual and anatomic outcomes were poor, with a high rate of retained silicone oil and recurrent RRD.

Using spectral-domain optical coherence tomography imaging to identify the presence of retinal silicone oil emulsification after silicone oil tamponade.

PURPOSE: To describe small hyperreflective areas using spectral-domain optical coherence tomography (SD-OCT) imaging in eyes that have had silicone oil tamponade. METHODS: Retrospective case series of 11 eyes of 11 patients. The authors retrospectively identified patients who underwent vitrectomy and silicone oil tamponade secondary to a rhegmatogenous retinal detachment (nine patients), panuveitis with retinal necrosis (one patient), or recurrent full-thickness macular hole surgery (one patient) who had manifestations of silicone oil emulsion on SD-OCT imaging. Patients were monitored during the postoperative period by clinical examination and using SD-OCT. A model eye in which emulsified silicone oil had been injected in the anterior chamber was used to obtain anterior segment SD-OCT images for comparison. RESULTS: The mean age of our patients was 50 years (range, 39-76 years). In eight eyes, the SD-OCT examination was carried out after silicone oil removal, and in three eyes, the SD-OCT examination was carried out with the oil in situ. Of the nine eyes treated for rhegmatogenous retinal detachment, five had a relieving retinectomy for advanced anterior proliferative vitreoretinopathy or for traumatic retinal incarceration (one eye). The eye treated for full-thickness macular hole had a vitrectomy, internal limiting membrane peel, and silicone oil injection for recurrent macular hole. Ten eyes showed hyperreflective, spherical, tiny droplets using SD-OCT imaging. These were thought to represent silicone oil droplets intraretinally or underneath epiretinal membranes, and one eye showed hyperreflective areas subretinally (retina detached). One additional patient was found to have tiny intravitreal silicone oil droplets after silicone oil removal. Similarly, the silicone oil appeared as multiple hyperreflective spherical droplets as detected by SD-OCT. Anterior segment studies of silicone oil emulsification in the experimental model revealed a similar appearance to that seen with in vivo SD-OCT imaging. CONCLUSION: The authors have found small hyperreflective areas intraretinally, subretinally, and underneath epiretinal membranes on SD-OCT in eyes that have had silicone oil tamponade for a variety of indications. The authors have seen a similar appearance when silicone oil emulsification is examined in vivo. The authors conclude that the hyperreflective areas are likely (but not certain) to be very small bubbles of emulsified silicone. Further studies are required to determine the incidence, clinicopathologic, and functional significance of probable silicone oil emulsification and deposition within the retinal layers.

Pars Plana Vitrectomy for Vitreomacular Traction Syndrome: Analysing the Preoperative Prognostic Factors.

PURPOSE: To identify the prognostic factors affecting the surgical outcomes in patients with vitreomacular traction syndrome undergoing pars plana vitrectomy. METHODS: This was a retrospective clinical study of 67 eyes of 67 patients with vitreomacular traction syndrome who underwent pars plana vitrectomy. Demographic, clinical, and optical coherence tomography (OCT) characteristics were collected and analyzed. Univariate and multivariate linear regression analysis were used to examine the effect of parameters on change in best-corrected visual acuity (BCVA). RESULTS: At a mean follow-up period of 15.9±12 months (mean±SD), the BCVA improved from 0.7±0.3 LogMAR (mean±SD) to 0.5±0.3. Seven patients developed full-thickness macular hole intraoperatively and tamponade (air, 20% SF6 or 12% C3F8) was used in 41 patients. Retinal breaks were identified intraoperatively in four patients. Regression analysis demonstrated that the preoperative BCVA was the only parameter affecting the postoperative visual outcome. CONCLUSION: In the present study, the preoperative BCVA plays a predictive role in the surgical outcome of patients with VMT undergoing pars plana vitrectomy. No other preoperative OCT characteristics demonstrated prognostic potential. Further prospective studies are needed in order to examine the role of several factors that could potentially facilitate preoperative patient counselling.

Conjunctival sweeping with a squint hook to reduce chemosis.

We describe a simple technique for reducing surgical chemosis. The conjunctival sweeping technique uses a squint hook and tissue scissors and can be used effectively by surgeons at all levels of experience.

Hypoxia inducible factors are dispensable for myeloid cell migration into the inflamed mouse eye.

Hypoxia inducible factors (HIFs) are ubiquitously expressed transcription factors important for cell homeostasis during dynamic oxygen levels. Myeloid specific HIFs are crucial for aspects of myeloid cell function, including their ability to migrate into inflamed tissues during autoimmune disease. This contrasts with the concept that accumulation of myeloid cells at ischemic and hypoxic sites results from a lack of chemotactic responsiveness. Here we seek to address the role of HIFs in myeloid trafficking during inflammation in a mouse model of human uveitis. We show using mice with myeloid-specific Cre-deletion of HIFs that myeloid HIFs are dispensable for leukocyte migration into the inflamed eye. Myeloid-specific deletion of Hif1a, Epas1, or both together, had no impact on the number of myeloid cells migrating into the eye. Additionally, stabilization of HIF pathways via deletion of Vhl in myeloid cells had no impact on myeloid trafficking into the inflamed eye. Finally, we chemically induce hypoxemia via hemolytic anemia resulting in HIF stabilization within circulating leukocytes to demonstrate the dispensable role of HIFs in myeloid cell migration into the inflamed eye. These data suggest, contrary to previous reports, that HIF pathways in myeloid cells during inflammation and hypoxia are dispensable for myeloid cell tissue trafficking.

VEGF165-induced vascular permeability requires NRP1 for ABL-mediated SRC family kinase activation.

The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.

Multimodal analysis of ocular inflammation using the endotoxin-induced uveitis mouse model.

Endotoxin-induced uveitis (EIU) in rodents is a model of acute Toll-like receptor 4 (TLR4)-mediated organ inflammation, and has been used to model human anterior uveitis, examine leukocyte trafficking and test novel anti-inflammatory therapeutics. Wider adoption has been limited by the requirement for manual, non-specific, cell-count scoring of histological sections from each eye as a measure of disease severity. Here, we describe a comprehensive and efficient technique that uses ocular dissection and multimodal tissue analysis. This allows matched disease scoring by multicolour flow cytometric analysis of the inflammatory infiltrate, protein analysis on ocular supernatants and qPCR on remnant tissues of the same eye. Dynamic changes in cell populations could be identified and mapped to chemokine and cytokine changes over the course of the model. To validate the technique, dose-responsive suppression of leukocyte infiltration by recombinant interleukin-10 was demonstrated, as well as selective suppression of the monocyte (CD11b+Ly6C+) infiltrate, in mice deficient for eitherCcl2orCcr2 Optical coherence tomography (OCT) was used for the first time in this model to allowin vivoimaging of infiltrating vitreous cells, and correlated with CD11b+Ly6G+ counts to provide another unique measure of cell populations in the ocular tissue. Multimodal tissue analysis of EIU is proposed as a new standard to improve and broaden the application of this model.

Cd59a deficiency in mice leads to preferential innate immune activation in the retinal pigment epithelium-choroid with age.

Dysregulation of the complement system has been implicated in the pathogenesis of age-related macular degeneration. To investigate consequences of altered complement regulation in the eye with age, we examined Cd59a complement regulator deficient (Cd59a(-/-)) mice between 4 and 15 months. In vivo imaging revealed an increased age-related accumulation of autofluorescent spots in Cd59a(-/-) mice, a feature that reflects accumulation of subretinal macrophages and/or microglia. Despite this activation of myeloid cells in the eye, Cd59a(-/-) mice showed normal retinal histology and function as well as normal choroidal microvasculature. With age, they revealed increased expression of activators of the alternative complement pathway (C3, Cfb, Cfd), in particular in the retinal pigment epithelium (RPE)-choroid but less in the retina. This molecular response was not altered by moderately-enhanced light exposure. Cd59a deficiency therefore leads to a preferential age-related dysregulation of the complement system in the RPE-choroid, that alone or in combination with light as a trigger, is not sufficient to cause choroidal vascular changes or retinal degeneration and dysfunction. This data emphasizes the particular vulnerability of the RPE-choroidal complex to dysregulation of the alternative complement pathway during aging.