Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3-/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3-/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Increased susceptibility to fundus camera-delivered light-induced retinal degeneration in mice deficient in oxidative stress response proteins.

Oxidative stress is an important contributor to the pathogenesis of many retinal diseases including age-related macular degeneration and retinal dystrophies. Light-induced retinal degeneration (LIRD) can serve as a model in which to study the response of the retina to stress. Of note, many genetic mutant mice are in a C57BL/6 J background and are thus resistant to the usual LIRD models. We recently developed a new model of fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) which is effective in strains of mice expressing the light-resistant variant of RPE65 (450Met), including C57BL/6 J. In this work we investigated whether FCD-LIRD would be useful as a model in which to test the effect of genetic mutations on the response of the retina to stress. Furthermore, we tested whether oxidative stress plays an important role in the setting of this new FCD-LIRD model. FCD-LIRD was applied to C57BL/6 J mice and to mice simultaneously deficient in three proteins that are important in the response of the retina to oxidative stress (SOD1, DJ-1 and Parkin). Using fundus photography, we found that retinal damage was dramatically increased in the SOD1/DJ-1/Parkin deficient mice compared to C57BL/6 J. Outer retinal OCT volume and RPE cell morphology analysis in ZO-1-stained flat mounts added support to these findings. Gene expression analysis confirmed a strong oxidative stress response after FCD-LIRD, which was differentially altered in the SOD1/DJ1/Parkin deficient mice. We conclude that FCD-LIRD is useful to study the effect of genetic mutations on the response of the retina to light stress in light-resistant strains of mice. Furthermore, oxidative stress seems to be an important component of FCD-LIRD. Finally, we have established protocols to quantify the effect of FCD-LIRD on the retina and RPE which will be useful for future studies. Further dissection of the mechanisms by which the retina responds to light-induced oxidative stress may result in new strategies to modulate this response, which could lead to a reduction in retinal and RPE damage.

Differences in the distribution, phenotype and gene expression of subretinal microglia/macrophages in C57BL/6N (Crb1 rd8/rd8) versus C57BL6/J (Crb1 wt/wt) mice.

BACKGROUND: Microglia/macrophages (MG/MΦ) are found in the subretinal space in both mice and humans. Our goal was to study the spatial and temporal distribution, the phenotype, and gene expression of subretinal MG/MΦ in mice with normal retinas and compare them to mice with known retinal pathology. METHODS: We studied C57BL/6 mice with (C57BL/6N), or without (C57BL/6J) the rd8 mutation in the Crb1 gene (which, in the presence of yet unidentified permissive/modifying genes, leads to a retinal degeneration), and documented their fundus appearance and the change with aging. Immunostaining of retinal pigment epithelium (RPE) flat mounts was done for 1) Ionized calcium binding adaptor (Iba)-1, 2) FcγIII/II Receptor (CD16/CD32, abbreviated as CD16), and 3) Macrophage mannose receptor (MMR). Reverse-transcription quantitative PCR (RT-qPCR) was done for genes involved in oxidative stress, complement activation and inflammation. RESULTS: The number of yellow fundus spots correlated highly with subretinal Iba-1+ cells. The total number of subretinal MG/MΦ increased with age in the rd8 mutant mice, but not in the wild-type (WT) mice. There was a centripetal shift in the distribution of the subretinal MG/MΦ with age. Old rd8 mutant mice had a greater number of CD16+ MG/MΦ. CD16+ cells had morphological signs of activation, and this was most prominent in old rd8 mutant mice (P < 1 × 10(-8) versus old WT mice). Subretinal MG/MΦ in rd8 mutant mice also expressed iNOS and MHC-II, and had ultrastructural signs of activation. Finally, rd8 mutant mouse RPE/ MG/MΦ RNA isolates showed an upregulation of Ccl2, CFB, C3, NF-kß, CD200R and TNF-alpha. The retinas of rd8 mutant mice showed upregulation of HO-1, C1q, C4, and Nrf-2. CONCLUSIONS: When compared to C57BL/6J mice, C57BL/6N mice demonstrate increased accumulation of subretinal MG/MΦ, displaying phenotypical, morphological, and gene-expression characteristics consistent with a pro-inflammatory shift. These changes become more prominent with aging and are likely due to the combination of the rd8 mutation and yet unidentified permissive/modulatory genes in the C57BL/6N mice. In contrast, aging leads to a scavenging phenotype in the C57BL/6J subretinal microglia/macrophages.

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Nonfunctional variant 3 factor H binding proteins as meningococcal vaccine candidates.

Neisseria meningitidis is a human-specific pathogen and leading cause of meningitis and septicemia. Factor H binding protein (fHbp), a virulence factor which protects N. meningitidis from innate immunity by binding the human complement regulator factor H (fH) with high affinity, is also a key antigen in vaccines being developed to prevent meningococcal disease. fHbp can be divided into three variant groups (V1, V2, and V3) that elicit limited immunological cross-reactivity. The interaction of fH with fHbp could impair the immunogenicity of this antigen by hindering access to the antigenic epitopes in fHbp, providing the rationale for the development of nonfunctional fHbps as vaccine candidates. Here, we characterized the two nonfunctional V3 fHbps, fHbp(T286A) and fHbp(E313A), which each contains a single amino acid substitution that leads to a marked reduction in affinity for fH without affecting the folding of the proteins. The immunogenicity of the nonfunctional fHbps was assessed in transgenic mice expressing a single chimeric fH containing domains of human fH involved in binding to fHbp. No differences in anti-V3 fHbp antibody titers were elicited by the wild-type V3 fHbp, V3 fHbp(T286A), and V3 fHbp(E313A), demonstrating that the nonfunctional fHbps retain their immunogenicity. Furthermore, the nonfunctional V3 fHbps elicit serum bactericidal activity that is equivalent to or higher than that observed with the wild-type protein. Our findings provide the basis for the rational design of next-generation vaccines containing nonfunctional V3 fHbps.

Design and evaluation of meningococcal vaccines through structure-based modification of host and pathogen molecules.

Neisseria meningitis remains a leading cause of sepsis and meningitis, and vaccines are required to prevent infections by this important human pathogen. Factor H binding protein (fHbp) is a key antigen that elicits protective immunity against the meningococcus and recruits the host complement regulator, fH. As the high affinity interaction between fHbp and fH could impair immune responses, we sought to identify non-functional fHbps that could act as effective immunogens. This was achieved by alanine substitution of fHbps from all three variant groups (V1, V2 and V3 fHbp) of the protein; while some residues affected fH binding in each variant group, the distribution of key amino underlying the interaction with fH differed between the V1, V2 and V3 proteins. The atomic structure of V3 fHbp in complex with fH and of the C-terminal barrel of V2 fHbp provide explanations to the differences in the precise nature of their interactions with fH, and the instability of the V2 protein. To develop transgenic models to assess the efficacy of non-functional fHbps, we determined the structural basis of the low level of interaction between fHbp and murine fH; in addition to changes in amino acids in the fHbp binding site, murine fH has a distinct conformation compared with the human protein that would sterically inhibit binding to fHbp. Non-functional V1 fHbps were further characterised by binding and structural studies, and shown in non-transgenic and transgenic mice (expressing chimeric fH that binds fHbp and precisely regulates complement system) to retain their immunogenicity. Our findings provide a catalogue of non-functional fHbps from all variant groups that can be included in new generation meningococcal vaccines, and establish proof-in-principle for clinical studies to compare their efficacy with wild-type fHbps.

Regulation of angiogenesis and choroidal neovascularization by members of microRNA-23~27~24 clusters.

MicroRNAs (miRNAs) modulate complex physiological and pathological processes by repressing expression of multiple components of cellular regulatory networks. Here we demonstrate that miRNAs encoded by the miR-23∼27∼24 gene clusters are enriched in endothelial cells and highly vascularized tissues. Inhibition of miR-23 and miR-27 function by locked nucleic acid-modified anti-miRNAs represses angiogenesis in vitro and postnatal retinal vascular development in vivo. Moreover, miR-23 and miR-27 are required for pathological angiogenesis in a laser-induced choroidal neovascularization mouse model. MiR-23 and miR-27 enhance angiogenesis by promoting angiogenic signaling through targeting Sprouty2 and Sema6A proteins, which exert antiangiogenic activity. Manipulating miR-23/27 levels may have important therapeutic implications in neovascular age-related macular degeneration and other vascular disorders.

Impact of COVID-19-related lifestyle changes on diabetic macular edema.

AIM: To assess diabetic macular edema (DME) progression during the early phases of the COVID-19 pandemic, when severe societal restrictions raised the concern of possible deterioration of health in patients with systemic conditions, particularly those requiring frequent office visits. METHODS: This is a multicenter retrospective chart review of 370 patients (724 eyes) with an established diagnosis of DME seen on 3 separate visits between January 2019 and July 2021. Period 1 was January 2019 to February 2020 (considered pre-COVID-19), period 2 was March 2020 to December 2020 (considered the height of the pandemic; highest level of pandemic-related clinical and societal regulations) and period 3 was January 2021 to July 2021 (re-adjustment to the new "pandemic norms"). Main outcome measures included visual acuity, body mass index (BMI), blood pressure (BP), hemoglobin A1c (HbA1c), macular thickness, patient adherence to scheduled ophthalmology visits, and DME treatment(s) received at each visit. To facilitate measurement of macular thickness, each macula was divided into 9 Early Treatment Diabetic Retinopathy Study (ETDRS)-defined macular sectors as measured by OCT imaging. RESULTS: There was no change of BMI, systolic BP, and diastolic BP between any of the time periods. HbA1c showed a very small increase from period 1 (7.6%) to period 2 (7.8%, P=0.015) and decreased back to 7.6% at period 3 (P=0.12). Macular thickness decreased for 100% of macular regions. The central macular thickness decreased across all 3 periods from 329.5 to 316.6 µm (P=0.0045). After analysis of multiple variables including HbA1c, BMI, adherence to scheduled appointments, different clinic centers, and treatment interventions, there was no easily identifiable subgroup of patients that experienced the increase in DME. CONCLUSION: DME doesn't worsen during the COVID-19 pandemic, instead sustaining a very small but statistically significant improvement. While identifying a mechanism behind our findings is beyond the scope of this study, potential explanations may include a delay in retinal changes beyond our study period, an unexpected increase in treatment frequency despite pandemic restrictions, and an unanticipated pandemic-related improvement in some lifestyle factors that may have had a positive impact on DME.

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration.

Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin-proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.

Single Cell RNA Sequencing Analysis of Mouse Retina Identifies a Subpopulation of Muller Glia Involved in Retinal Recovery From Injury in the FCD-LIRD Model.

Purpose: Although retinal light injury models have been useful in understanding aspects of retinal degeneration and retinal oxidative stress, information on retinal recovery from oxidative/photoinflammatory retinal injury is scarce. The fundus camera-delivered light-induced retinal degeneration model is a simple and reproducible retinal light injury model developed to recapitulate not only the retinal degeneration aspect, but also the retinal recovery from injury. In this study, we used the fundus camera-delivered light-induced retinal degeneration model to perform cell type-specific analyses of the acute and subacute retinal responses to light injury. Methods: C57BL/6J eyes were collected before or after light injury (4 hours, 48 hours, and day 5). Retina samples were processed into single-cell suspensions. Droplet-based encapsulation of single cells was performed to generate libraries for sequencing. Results: Gene expression analysis generated 23 clusters encompassing all known major retinal cell populations. Using unbiased analyses, we identified genes and pathways that were significantly altered in each cell type after light injury, including some cellular processes suggestive of activation of pathways for retinal recovery (e.g., synaptogenesis signaling, ephrin receptor signaling, and Reelin signaling in neurons). More importantly, our data show that a subpopulation of Muller glia cells may play an important role in the cellular recovery process. Conclusions: This work identifies acute and subacute cell type-specific responses to retinal photo-oxidative injury. A subpopulation of Muller glia seems to initiate the cellular recovery process. A better understanding of these responses may be helpful in identifying therapeutic approaches to minimize retinal damage and maximize recovery after exposure to injury.

Comparison of Surgical Outcomes for Uncomplicated Primary Retinal Detachment Repair.

Purpose: To compare the outcomes of primary uncomplicated rhegmatogenous retinal detachment (RRD) repair using pars plana vitrectomy (PPV), scleral buckling (SB), or combined scleral buckling with vitrectomy (SB/PPV). Patients and Methods: Single-institution, retrospective, observational study of 179 patients with primary RRD managed at a large academic hospital system. We excluded patients with less than 6 months of follow-up, previous vitrectomy or buckle, giant retinal tears, aphakia, recurrent forms of RRD, or extensive proliferative vitreoretinopathy (Grade C or worse) documented on exam or requiring membrane peel. Outcome measures included primary anatomical success at 6 months, functional success defined as BCVA ≥ 20/200, and best corrected visual acuity (BCVA) using logMAR scoring. Subgroup analysis was performed in the following patient groups: phakic, pseudophakic, inferior detachments, and prior pneumatic retinopexy. Results: Primary anatomical success was achieved in 145 of 179 eyes (81.0%), with SB/PPV showing a significantly greater success rate (p = 0.046) when compared to SB and PPV. Functional success was achieved in 137 of the 145 anatomically successful eyes (94.5%), with values ranging between 92% and 97% amongst the interventions (p = 0.552). No difference was found in final BCVA (p = 0.367). Patients with inferior detachment had an odds ratio of 2.15 for primary anatomic failure. Prior pneumatic retinopexy did not significantly affect any of the primary outcomes. Conclusion: SB/PPV yielded a significantly better primary anatomical success rate when compared to SB and PPV. Functional success and final BCVA was similar amongst the interventions. Inferior detachments were associated with worse primary anatomic outcomes. Prior pneumatic retinopexy did not significantly affect surgical outcomes.

Forward genetic screening using fundus spot scale identifies an essential role for Lipe in murine retinal homeostasis.

Microglia play a role in the pathogenesis of many retinal diseases. Fundus spots in mice often correlate with the accumulation of activated subretinal microglia. Here we use a semiquantitative fundus spot scoring scale in combination with an unbiased, state-of-the-science forward genetics pipeline to identify causative associations between chemically induced mutations and fundus spot phenotypes. Among several associations, we focus on a missense mutation in Lipe linked to an increase in yellow fundus spots in C57BL/6J mice. Lipe-/- mice generated using CRISPR-Cas9 technology are found to develop accumulation of subretinal microglia, a retinal degeneration with decreased visual function, and an abnormal retinal lipid profile. We establish an indispensable role of Lipe in retinal/RPE lipid homeostasis and retinal health. Further studies using this new model will be aimed at determining how lipid dysregulation results in the activation of subretinal microglia and whether these microglia also play a role in the subsequent retinal degeneration.

Novel method for the rapid isolation of RPE cells specifically for RNA extraction and analysis.

RPE cells are involved in the pathogenesis of many retinal diseases. Accurate analysis of RPE gene expression profiles in different scenarios will increase our understanding of disease mechanisms. Our objective in this study was to develop an improved method for the isolation of RPE cells, specifically for RNA analysis. Mouse RPE cells were isolated using different techniques, including mechanical dissociation techniques and a new technique we refer to here as "Simultaneous RPE cell Isolation and RNA Stabilization" (SRIRS method). RNA was extracted from the RPE cells. An RNA bioanalyzer was used to determine the quantity and quality of RNA. qPCR was used to determine contamination with non-RPE-derived RNA. Several parameters with a potential impact on the isolation protocol were studied and optimized. A marked improvement in the quantity and quality of RPE-derived RNA was obtained with the SRIRS technique. We could get the RPE in direct contact with the RNA protecting agent within 1 min of enucleation, and the RPE isolated within 11 min of enucleation. There was no significant contamination with vascular, choroidal or scleral-derived RNA. We have developed a fast, easy and reliable method for the isolation of RPE cells that leads to a high yield of RPE-derived RNA while preserving its quality. We believe this technique will be useful for future studies looking at gene expression profiles of RPE cells and their role in the pathophysiology of retinal diseases.

Parafoveal acute middle maculopathy (PAMM) in sickle cell disease after discontinuation of hydroxyurea.

Purpose: Paracentral acute middle maculopathy (PAMM) is a rare ophthalmologic emergency involving the intermediate and deep retinal capillary plexus that supply the retina's middle layers. This case report describes an episode of PAMM in a patient with sickle cell disease (SCD) to demonstrate the importance of early diagnosis, review potential pathophysiologic mechanisms, and finally discuss appropriate management in this patient population. Observations: A 33-year-old black female with SCD, who had recently discontinued disease-modifying therapy with hydroxyurea, presented with a central scotoma of the left eye. Examination showed superficial opacification and whitening of the temporal perifoveal macula. After an initial diagnosis of central retinal artery occlusion she was admitted for a stroke workup. MRI was negative for stroke, and the patient was discharged after undergoing a red blood cell exchange (RBCX). Follow-up exam and optical coherence tomography (OCT) findings were more consistent with PAMM. Conclusions and Importance: To our knowledge, this is the first report of PAMM after discontinuation of hydroxyurea in preparation for pregnancy. It highlights the importance of a multidisciplinary approach when treating peripartum patients with SCD and the need for further research regarding vaso-occlusive prophylactic agents and their effects in pregnancy to minimize morbidity during family planning.

Utility of the DHFR-based destabilizing domain across mouse models of retinal degeneration and aging.

The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4 -/- mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.

Unexpected Etiology in a Case of Bilateral Maculopathy.

A 74-year-old woman with a history of rheumatoid arthritis using hydroxychloroquine presented with gradually progressive decreased vision in both eyes and was found to have a bilateral maculopathy. Initial genetic testing was negative, and after discussing the low likelihood of her severe findings being secondary to her relatively low hydroxychloroquine exposure, the possibility of an autoimmune retinopathy was entertained. Updated data on the genetic testing reclassified one of her mutations in HGSNAT as pathogenic. This case highlights the value of genetic testing and the need to keep a high index of suspicion even after initial negative results, given the fact that our knowledge of mutations leading to retinal degeneration is constantly evolving.

Delivery of Antisense Oligonucleotides to the Cornea.

Antisense oligonucleotides (ASOs) are synthetic nucleic acids that recognize complementary RNA sequences inside cells and modulate gene expression. In this study, we explore the feasibility of ASO delivery to the cornea. We used quantitative polymerase chain reaction to test the efficacy of a benchmark ASO targeting a noncoding nuclear RNA, Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), in a human corneal endothelial cell line, ex vivo human corneas, and in vivo in mice. In vivo delivery was via intravitreal or intracameral injections as well as topical administration. The anti-MALAT1 ASO significantly reduced expression of MALAT1 in a corneal endothelial cell line. We achieved a dose-dependent reduction of target gene expression in endothelial tissue from ex vivo human donor corneas. In vivo mouse experiments confirmed MALAT1 reduction in whole corneal tissue via intravitreal and intracameral routes, 82% and 71% knockdown, respectively (P < 0.001). Effects persisted up to at least 21 days, 32% (P < 0.05) and 43% (P < 0.05) knockdown, respectively. We developed protocols for the isolation and analysis of mouse corneal endothelium and observed reduction in MALAT1 expression upon both intravitreal and intracameral administrations, 64% (P < 0.05) and 63% (P < 0.05) knockdown, respectively. These data open the possibility of using ASOs to treat corneal disease.

Mice With a Combined Deficiency of Superoxide Dismutase 1 (Sod1), DJ-1 (Park7), and Parkin (Prkn) Develop Spontaneous Retinal Degeneration With Aging.

Purpose: Chronic oxidative stress is an important mechanism of disease in aging disorders. We do not have a good model to recapitulate AMD and other retinal disorders in which chronic oxidative stress plays an important role. We hypothesized that mice with a combined deficiency in superoxide dismutase 1 (Sod1), DJ-1 (Park-7), and Parkin (Prkn) (triple knock out, TKO) would have an increased level of chronic oxidative stress in the retina, with anatomic and functional consequences just with aging. Methods: Eyes of TKO and B6J control mice were (1) monitored with optical coherence tomography (OCT) and electroretinography (ERG) over time, and (2) collected for oxidative marker protein analysis by ELISA or immunohistochemistry and for transmission electron microscopy studies. Results: TKO mice developed qualitative disruptions in outer retinal layers in OCT by 3 months, increased accumulation of fundus spots and subretinal microglia by 6 months of age, significant retinal thinning by 9 months, and decreased ERG signal by 12 months. Furthermore, we found increased accumulation of the oxidative marker malondialdehyde (MDA) in the retina and increased basal laminal deposits (BLD) and mitochondria number and size in the retinal pigment epithelium of aging TKO mice. Conclusions: TKO mice can serve as a platform to study retinal diseases that involve chronic oxidative stress, including macular degeneration, retinal detachment, and ischemic retinopathies. In order to model each of these diseases, additional disease-specific catalysts or triggers could be superimposed onto the TKO mice. Such studies could provide better insight into disease mechanisms and perhaps lead to new therapeutic approaches.

Vitreoretinal lymphoma, secondary to non-CNS systemic lymphoma, masquerading as an infectious retinitis.

PURPOSE: To report an atypical case of vitreoretinal lymphoma, secondary to non-central nervous system (non-CNS) systemic lymphoma, masquerading as an infectious retinitis. OBSERVATIONS: A 76-year-old female with a history of cecal diffuse large B-cell lymphoma with two prior occurrences of posterior segment ocular involvement presented with a complaint of blurry vision in the right eye. Exam findings were significant for large areas of retinal whitening and retinal hemorrhages in the absence of choroidal lesions or significant vitritis. The clinical suspicion of an infectious retinitis, was supported by a presumptive immunosuppressive state secondary to her recent treatment (within 1 month) with both intravitreal and systemic rituximab plus high-dose methotrexate. Aggressive treatment with intravitreal and systemic antivirals and antibiotics was initiated. However, polymerase chain reaction (PCR) testing of aqueous fluid was negative for cytomegalovirus (CMV), herpes simplex virus, herpes zoster virus and toxoplasma, and her condition continued to worsen, so suspicion was raised for a masquerading recurrent malignancy. She was treated empirically with serial intravitreal injections of methotrexate and showed dramatic clinical improvement. A subsequent relapse occurred that responded rapidly to intravitreal methotrexate in the absence of antiviral/antibiotics. CONCLUSION: It is important for clinicians to be aware of atypical presentations of vitreoretinal lymphoma. This case emphasizes the fact that secondary ocular lymphoma after systemic lymphoma can have a vitreoretinal presentation rather than the more common choroidal involvement. Furthermore, it shows that recurrences of this disease in the same patient can have very different manifestations, including an appearance indistinguishable from a viral retinitis.

A Mouse Model of Retinal Recovery From Photo-Oxidative/Photo-Inflammatory Injury: Nrf2, SOD1, DJ-1, and Parkin Are Not Essential to Recovery.

Purpose: To determine if there is structural and functional recovery of the retina from light induced retinal degeneration, and to evaluate the role of the oxidative stress response elements Nrf2, SOD1, DJ-1, and Parkin in such a recovery process. Methods: Eyes from C57BL/6J (B6J) mice and from oxidative stress response-deficient strains of mice were treated with intense light using the fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) model. Fundus photographs, optical coherence tomography (OCT) images, and electroretinography (ERG) responses were obtained before the injury, during the "maximal injury phase" (days 4-7) and during the "recovery phase" (days 14-16) post light exposure and were evaluated for retinal damage and assessed for evidence of recovery from the injury. Results: We demonstrate that mice treated with a sub-lethal FCD-LIRD protocol show an initial acute retina injury phase peaking between days 4 to 7 followed by a recovery phase in which the outer retinal thickness/volume and retinal function partially recover. These observations are reproduced in B6J mice and in mice lacking oxidative stress response enzymes (SOD1, DJ-1, and Parkin) or the oxidative stress response master regulator Nrf2. Conclusions: Our data indicate that retinal recovery from injury can proceed via pathways that are independent from the common oxidative stress response elements Nrf2, SOD1, DJ-1, and Parkin. Furthermore, the model of retinal recovery from injury that we describe here mimics changes seen in a variety of clinical entities and may provide an excellent platform for dissecting general pathways of retinal recovery from sub-lethal injury.