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Gauthier AC, Wiggs JL. Childhood glaucoma genes and phenotypes: Focus on FOXC1 mutations causing anterior segment dysgenesis and hearing loss. Exp Eye Res 2020;190:107893.Abstract
Childhood glaucoma is an important cause of blindness world-wide. Eleven genes are currently known to cause inherited forms of glaucoma with onset before age 20. While all the early-onset glaucoma genes cause severe disease, considerable phenotypic variability is observed among mutations carriers. In particular, FOXC1 genetic variants are associated with a broad range of phenotypes including multiple forms of glaucoma and also systemic abnormalities, especially hearing loss. FOXC1 is a member of the forkhead family of transcription factors and is involved in neural crest development necessary for formation of anterior eye structures and also pharyngeal arches that form the middle ear bones. In this study we review the clinical phenotypes reported for known FOXC1 mutations and show that mutations in patients with reported ocular anterior segment abnormalities and hearing loss primarily disrupt the critically important forkhead domain. These results suggest that optimal care for patients affected with anterior segment dysgenesis should include screening for FOXC1 mutations and also testing for hearing loss.
Miyajima T, Melangath G, Zhu S, Deshpande N, Vasanth S, Mondal B, Kumar V, Chen Y, Price MO, Price FW, Rogan EG, Zahid M, Jurkunas UV. Loss of NQO1 generates genotoxic estrogen-DNA adducts in Fuchs Endothelial Corneal Dystrophy. Free Radic Biol Med 2020;147:69-79.Abstract
Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of corneal endothelial cells (HCEnCs). FECD has a greater incidence and advanced phenotype in women, suggesting a possible role of hormones in the sex-driven differences seen in the disease pathogenesis. In this study, catechol estrogen (4-OHE), the byproduct of estrogen metabolism, induced genotoxic estrogen-DNA adducts formation, macromolecular DNA damage, and apoptotic cell death in HCEnCs; these findings were potentiated by menadione (MN)-mediated reactive oxygen species (ROS). Expression of NQO1, a key enzyme that neutralizes reactive estrogen metabolites, was downregulated in FECD, indicating HCEnC susceptibility to reactive estrogen metabolism in FECD. NQO1 deficiency in vitro exacerbated the estrogen-DNA adduct formation and loss of cell viability, which was rescued by the supplementation of N-acetylcysteine, a ROS scavenger. Notably, overexpression of NQO1 in HCEnCs treated with MN and 4-OHE quenched the ROS formation, thereby reducing the DNA damage and endothelial cell loss. This study signifies a pivotal role for NQO1 in mitigating the macromolecular oxidative DNA damage arising from the interplay between intracellular ROS and impaired endogenous estrogen metabolism in post-mitotic ocular tissue cells. A dysfunctional Nrf2-NQO1 axis in FECD renders HCEnCs susceptible to catechol estrogens and estrogen-DNA adducts formation. This novel study highlights the potential role of NQO1-mediated estrogen metabolite genotoxicity in explaining the higher incidence of FECD in females.
Liu C, Miyajima T, Melangath G, Miyai T, Vasanth S, Deshpande N, Kumar V, Ong Tone S, Gupta R, Zhu S, Vojnovic D, Chen Y, Rogan EG, Mondal B, Zahid M, Jurkunas UV. Ultraviolet A light induces DNA damage and estrogen-DNA adducts in Fuchs endothelial corneal dystrophy causing females to be more affected. Proc Natl Acad Sci U S A 2020;117(1):573-583.Abstract
Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of -acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.
Veronese C, Pellegrini M, Maiolo C, Morara M, Armstrong GW, Ciardella AP. Multimodal ophthalmic imaging of staphylococcus aureus bacteremia associated with chorioretinitis, endocarditis, and multifocal brain abscesses. Am J Ophthalmol Case Rep 2020;17:100577.Abstract
Purpose: bacteriemia (SAB) as critical condition for the life and occasionally involves the eyes. The aim of this report is to describe the ocular involvement with multimodal imaging. Observations: A patient admitted for evaluation of acute onset of confusion, disorientation, and generalized malaise and found to have methicillin-resistant staphylococcus aureus (MRSA)-associated endocarditis and multifocal brain abscesses was evaluated by the ophthalmology service. The patient's visual acuity was 20/20 OU without relative afferent pupillary defect and normal intraocular pressures. Bedside anterior segment examination was normal. Posterior segment examination revealed intraretinal hemorrhages and Roth spots in the posterior pole of the right eye, and two deep well-defined focal white chorioretinal infiltrates and a hemorrhagic pigment epithelium detachment in the temporal quadrant of the left eye. Multimodal imaging was utilized to document these findings and ensure adequate antibiotic therapy. Conclusion: SAB has the potential for poor visual outcomes as well as significant morbidity and mortality. Multimodal imaging of SAB-related chorioretinitis allows for accurate diagnosis as well as assessment of response to antimicrobial therapy.
Freitag SK, Tanking T. A Nomenclature to Describe the Sequence of Visual Field Defects in Progressive Thyroid Eye Disease-Compressive Optic Neuropathy (An American Ophthalmological Society Thesis). Am J Ophthalmol 2020;213:293-305.Abstract
PURPOSE: To create a novel nomenclature to characterize the longitudinal sequence of visual field (VF) defects in patients with progression of thyroid eye disease-compressive optic neuropathy (TED-CON). METHODS: A retrospective review of records from 1 institution identified patients with progressive Humphrey VF defects secondary to TED-CON. The VF defects were analyzed by 2 independent reviewers and classified into 1 of 10 categories, divided into 3 stages that reflect the observed progression pattern, plus a miscellaneous category (stage X). Stage 1 VF defects are the earliest detectable and involve the inferior visual field with 3 levels of severity. Stage 2 VF defects include 2 distinguishable levels of severity and occur as the inferior defects advance above the horizontal midline to involve the superior VF. Stage 3 involves progression of stage 2 VF defects to complete loss of inferior and superior hemifields. RESULTS: Of 234 VFs in 37 eyes of 23 subjects, inferior defects were most common, including stage 1a (small inferior paracentral defect) in 22 of 234 VFs (9.4%), stage 1b (large inferior paracentral defect) in 112 of 234 VFs (47.9%), and stage 1c (inferior altitudinal defect) in 11 of 234 VFs (4.7%). Stage 2a (inferior altitudinal with superior advancement above the horizontal meridian) occurred in 41 of 234 VFs (17.5%), stage 2b (inferior altitudinal with superior arcuate) occurred in 6 of 234 VFs (2.6%), and stage 3 (total loss) occurred in 5 of 234 VFs (2.1%). The longitudinal sequence of VF defects from the 37 eyes of 23 patients was analyzed. Thirty-one of 37 eyes (83.8%) demonstrated a predictable progression pattern from least to more severe: stage 1a, stage 1b, stage 1c, stage 2a, stage 2b, and stage 3. A reverse order of VF defect progression was noted in 15 eyes with improving TED-CON. A minority of progression patterns (16.2%) originated from stage X (central/paracentral, enlarged blind spot, and scatter). CONCLUSIONS: Humphrey VF defects resulting from TED-CON are most often inferior, often have a predictable pattern of progression, and can be categorized into a novel descriptive nomenclature system. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.
Jackson CJ, Myklebust Ernø IT, Ringstad H, Tønseth KA, Dartt DA, Utheim TP. Simple limbal epithelial transplantation: Current status and future perspectives. Stem Cells Transl Med 2020;9(3):316-327.Abstract
Damage to limbal stem cells as a result of injury or disease can lead to limbal stem cell deficiency (LSCD). This disease is characterized by decreased vision that is often painful and may progress to blindness. Clinical features include inflammation, neovascularization, and persistent cornea epithelial defects. Successful strategies for treatment involve transplantation of grafts harvested from the limbus of the alternate healthy eye, called conjunctival-limbal autograft (CLAU) and transplantation of limbal cell sheets cultured from limbal biopsies, termed cultured limbal epithelial transplantation (CLET). In 2012, Sangwan and colleagues presented simple limbal epithelial transplantation (SLET), a novel transplantation technique that combines the benefits of CLAU and CLET and avoids the challenges associated with both. In SLET a small biopsy from the limbus of the healthy eye is divided and distributed over human amniotic membrane, which is placed on the affected cornea. Outgrowth occurs from each small explant and a complete corneal epithelium is typically formed within 2 weeks. Advantages of SLET include reduced risk of iatrogenic LSCD occurring in the healthy cornea at harvest; direct transfer circumventing the need for cell culture; and the opportunity to perform biopsy harvest and transplantation in one operation. Success so far using SLET is comparable with CLAU and CLET. Of note, 336 of 404 (83%) operations using SLET resulted in restoration of the corneal epithelium, whereas visual acuity improved in 258 of the 373 (69%) reported cases. This review summarizes the results of 31 studies published on SLET since 2012. Progress, advantages, challenges, and suggestions for future studies are presented.
McKay TB, Hutcheon AEK, Zieske JD. Biology of corneal fibrosis: soluble mediators, integrins, and extracellular vesicles. Eye (Lond) 2020;34(2):271-278.Abstract
Corneal fibrosis develops in response to injury, infection, postsurgical complications, or underlying systemic disease that disrupts the homeostasis of the tissue leading to irregular extracellular matrix deposition within the stroma. The mechanisms that regulate corneal scarring are focused heavily on the canonical transforming growth factor-β pathway and relevant activators, and their role in promoting myofibroblast differentiation. In this paper, we discuss the biochemical pathways involved in corneal fibrosis in the context of different injury models-epithelial debridement, superficial keratectomy, and penetrating incision. We elaborate on the interplay of the major pro-fibrotic factors involved in corneal scar development (e.g., transforming growth factor-β1, thrombospondin-1, and ανβ6), and explore a novel role for extracellular vesicles secreted by the wounded epithelium and the importance of the basement membrane.
Shoshany TN, Michalak S, Staffa SJ, Chinn RN, Bishop K, Hunter DG. Effect of Primary Occlusion Therapy in Asymmetric, Bilateral Amblyopia. Am J Ophthalmol 2020;211:87-93.Abstract
PURPOSE: Many bilateral amblyopia patients have asymmetric visual acuity (VA). There is no standard treatment for these patients, and outcomes have not been well described. Our goal is to compare VA outcomes in this group based on timing of occlusion therapy. DESIGN: Retrospective interventional comparative case series. METHODS: Setting: Institutional practice. PatientPopulation: Patients diagnosed with amblyopia at Boston Children's Hospital between 2010 and 2014. InclusionCriteria: VA ≥ 0.3 logMAR bilaterally by objective optotype-based measures, interocular difference (IOD) ≥ 0.18 logMAR, age 2-12 years. ExclusionCriteria: Loss to follow-up, managed surgically, deprivation amblyopia. Patients had either primary or secondary occlusion (primary = initiated when VA ≥ 0.3 logMAR bilaterally; secondary = initiated to correct residual IOD once VA improved to ≤0.18 logMAR in the stronger eye). ObservationProcedure: Patient demographics, VA, IOD, and stereopsis were compared between groups. OutcomeMeasures: VA improvement at 12-18 months and at last visits. RESULTS: Of 2,200 patients reviewed, 167 (7.6%) had asymmetric, bilateral amblyopia; 98 met inclusion and exclusion criteria. Patients were equally divided between primary (n = 50) and secondary (n = 48) occlusion groups. There were no differences in demographics, baseline VA, or IOD between groups (P ≥ .22), although the primary occlusion group had a higher proportion of strabismic amblyopia (P = .007). VA in both eyes, IOD, and stereopsis improved similarly between groups, even after stratifying by amblyopia subtype (P ≥ .48). The secondary occlusion group was more likely to achieve 20/30 bilaterally and IOD ≤ 1 line at 12-18 months (P ≤ .4), although this equalized by the last visit. CONCLUSION: In patients with asymmetric, bilateral amblyopia, VA improved by 4 lines in the weaker eye and 2 lines in the stronger eye, while IOD improved by 2 lines, irrespective of occlusion status. Primary occlusion thus provided no further benefit over spectacle correction alone.
Cohen LM, Habib LA, Yoon MK. Post-traumatic enophthalmos secondary to orbital fat atrophy: a volumetric analysis. Orbit 2020;39(5):319-324.Abstract
PURPOSE: To investigate via volumetric analysis whether orbital fat atrophy occurs in late post-traumatic enophthalmos. METHODS: An IRB-approved retrospective cohort study identified patients with diagnoses of both orbital fracture and enophthalmos with a CT orbits >3 months after injury. Exclusion criteria were surgical repair, other orbital disease or surgery, adjacent sinus disease, and an abnormal contralateral orbit. Images were analyzed using OsiriX imaging software (v.9.0.2, Pixmeo, Switzerland). Total orbital volume and orbital fat volume for the fractured and normal contralateral orbits were measured via three-dimensional volume rendering assisted region-of-interest computation. Enophthalmos was measured radiographically. Paired samples -tests were used to compare orbital fat and total orbital volumes between the fractured and normal contralateral orbits. RESULTS: Thirteen patients met the inclusion criteria. The numbers of patients with each fracture pattern were floor (4), medial wall (4), floor/medial wall (3), zygomaticomaxillary complex (floor+lateral wall) (1), zygomaticomaxillary complex+medial (inferior/medial/lateral walls) (1). Mean time from injury to CT scan was 21.8 ± 16.3 months. Comparing the fractured and normal contralateral orbits, there was a statistically significant decrease in orbital fat volume (mean difference 0.9 ml (14.2%), = .0002) and increase in total orbital volume (mean difference 2.0 ml (7.0%), = .0001). One ml orbital volume change was responsible for 0.83 mm enophthalmos. CONCLUSIONS: In addition to an increase in total orbital volume, orbital fat loss occurs with late post-traumatic enophthalmos due to unrepaired fractures. This suggests correction of bony change alone may be insufficient in some cases, and the use of custom implants may compensate for fat atrophy.
Amamoto R, Zuccaro E, Curry NC, Khurana S, Chen H-H, Cepko CL, Arlotta P. FIN-Seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system. Nucleic Acids Res 2020;48(1):e4.Abstract
Thousands of frozen, archived tissue samples from the human central nervous system (CNS) are currently available in brain banks. As recent developments in RNA sequencing technologies are beginning to elucidate the cellular diversity present within the human CNS, it is becoming clear that an understanding of this diversity would greatly benefit from deeper transcriptional analyses. Single cell and single nucleus RNA profiling provide one avenue to decipher this heterogeneity. An alternative, complementary approach is to profile isolated, pre-defined cell types and use methods that can be applied to many archived human tissue samples that have been stored long-term. Here, we developed FIN-Seq (Frozen Immunolabeled Nuclei Sequencing), a method that accomplishes these goals. FIN-Seq uses immunohistochemical isolation of nuclei of specific cell types from frozen human tissue, followed by bulk RNA-Sequencing. We applied this method to frozen postmortem samples of human cerebral cortex and retina and were able to identify transcripts, including low abundance transcripts, in specific cell types.
Greiner JV, Glonek T, Korb DR, Lindsay ME, Oliver PJ, Olson MCD. Corneal Cryopreservation Using Glycerylphosphorylcholine-Enriched Medium. Cornea 2020;39(3):370-375.Abstract
PURPOSE: To determine the effects of prolonged cryopreservation at subzero-degree temperatures on corneal transparency and histology after treatment with preservation medium containing the phosphodiester glycerylphosphorylcholine (GPC). METHODS: Rabbit corneas (n = 30) were immersed for 3 hours in K-Sol preservation medium containing 30 mM GPC. Three groups with 6 corneas each were refrigerated at -8°C for 2 weeks and liquid nitrogen temperature for 2 and 6 weeks, respectively. Two groups with 6 corneas each immersed in K-Sol preservation medium only were refrigerated at -8°C for 2 weeks and liquid nitrogen temperature for 6 weeks, respectively. Postthawing corneal transparency was measured on a grading scale after which corneas were prepared for and analyzed by light and transmission electron microscopy. RESULTS: All 3 groups of corneas preserved with GPC maintained a greater degree of corneal transparency compared with corneas preserved without GPC. The number of corneas retaining epithelial and endothelial layers increased in all groups where corneas were preserved in medium containing GPC, in contrast to corneas preserved in medium without GPC. Cytoplasmic vacuolization or nuclear damage was greater in corneas preserved without GPC. Similar findings were found in corneas stored at -8°C and liquid nitrogen temperatures. CONCLUSIONS: This study demonstrates a cryoprotective effect of corneas preserved in K-Sol containing the phosphodiester GPC at subzero-degree temperatures. In corneas immersed in preservation medium containing GPC, a higher degree of transparency is maintained and a lesser degree of histopathologic changes is observed with storage at both -8°C and in liquid nitrogen.
Silva RNE, Taniguchi EV, Cruzat A, Paschalis EI, Pasquale LR, Colby KA, Dohlman CH, Chodosh J, Shen LQ. Angle Anatomy and Glaucoma in Patients With Boston Keratoprosthesis. Cornea 2020;39(6):713-719.Abstract
PURPOSE: To quantitatively analyze the angle anatomy in eyes with a Boston type 1 keratoprosthesis (KPro) using anterior segment optical coherence tomography (AS-OCT) and to assess the diagnostic ability of AS-OCT in KPro-associated glaucoma. METHODS: AS-OCT (RTVue) images from KPro eyes with and without glaucoma were reviewed. The angle opening distance at 500 μm from the scleral spur (AOD500), trabecular-iris angle at 500 μm from the scleral spur (TIA500), and trabecular-iris surface area at 500 μm from the scleral spur (TISA500) were measured by 2 observers masked to the diagnosis. The measurements for each visible quadrant were compared between KPro eyes with and without glaucoma. RESULTS: Twenty-two eyes with glaucoma and 17 eyes without glaucoma from 39 patients with KPro were included. Of the 4 quadrants imaged, the temporal angle was the most visible (79.5%) and angle measurements of the temporal quadrant were the only ones that differentiated the 2 groups: the mean AOD500, TIA500, and TISA500 were significantly lower in KPro eyes with glaucoma than without glaucoma (388.2 ± 234.4 μm vs. 624.5 ± 310.5 μm, P = 0.02; 26.1 ± 14.0 degrees vs. 39.1 ± 17.1 degrees, P = 0.03; and 0.15 ± 0.09 mm vs. 0.23 ± 0.12 mm, P = 0.03; respectively). The highest area under the receiver operating characteristic curve for detecting glaucoma was 0.75 for temporal TIA500 (95% confidence interval 0.57-0.94, P = 0.02) with 50% specificity at 80% of sensitivity and a cutoff value of 37 degrees. CONCLUSIONS: The temporal angle was the most visible on AS-OCT in eyes with a KPro. Significant narrowing of the temporal angle detected on AS-OCT was associated with glaucoma in these eyes.
Dudek AM, Zabaleta N, Zinn E, Pillay S, Zengel J, Porter C, Franceschini JS, Estelien R, Carette JE, Zhou GL, Vandenberghe LH. GPR108 Is a Highly Conserved AAV Entry Factor. Mol Ther 2020;28(2):367-381.Abstract
Adeno-associated virus (AAV) is a highly promising gene transfer vector, yet major cellular requirements for AAV entry are poorly understood. Using a genome-wide CRISPR screen for entry of evolutionarily divergent serotype AAVrh32.33, we identified GPR108, a member of the G protein-coupled receptor superfamily, as an AAV entry factor. Of greater than 20 divergent AAVs across all AAV clades tested in human cell lines, only AAV5 transduction was unaffected in the GPR108 knockout (KO). GPR108 dependency was further shown in murine and primary cells in vitro. These findings are further validated in vivo, as the Gpr108 KO mouse demonstrates 10- to 100-fold reduced expression for AAV8 and rh32.33 but not AAV5. Mechanistically, both GPR108 N- and C-terminal domains are required for transduction, and on the capsid, a VP1 unique domain that is not conserved on AAV5 can be transferred to confer GPR108 independence onto AAV2 chimeras. In vitro binding and fractionation studies indicate reduced nuclear import and cytosolic accumulation in the absence of GPR108. We thus have identified the second of two AAV entry factors that is conserved between mice and humans relevant both in vitro and in vivo, further providing a mechanistic understanding to the tropism of AAV gene therapy vectors.

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