RESUMEN
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.
Asunto(s)
Proteínas de Transporte de Catión/genética , Degeneración Retiniana/genética , Segmento Externo de las Células Fotorreceptoras Retinianas/patología , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrorretinografía , Etilnitrosourea/toxicidad , Femenino , Humanos , Masculino , Ratones , Microscopía Electrónica , Mutagénesis , Mutación/efectos de los fármacos , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/patología , Segmento Externo de las Células Fotorreceptoras Retinianas/ultraestructura , Epitelio Pigmentado de la Retina/diagnóstico por imagen , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/ultraestructura , Tomografía de Coherencia ÓpticaRESUMEN
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.
Asunto(s)
Estrés Oxidativo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Animales , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Angiografía con Fluoresceína , Fondo de Ojo , Luz/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Tomografía de Coherencia Óptica , Proteína de la Zonula Occludens-1/genética , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica/genética , Macrófagos/metabolismo , Microglía/metabolismo , Mutación/genética , Proteínas del Tejido Nervioso/genética , Retina/patología , Degeneración Retiniana/patología , Factores de Edad , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Recuento de Células , Modelos Animales de Enfermedad , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/ultraestructura , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Microglía/ultraestructura , Proteínas del Tejido Nervioso/metabolismo , Vías Nerviosas/metabolismo , Vías Nerviosas/patología , Fenotipo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Streptococcus pyogenes/inmunología , Streptococcus pyogenes/patogenicidad , Animales , Sitios de Unión , Proteína de Unión al Complemento C4b/metabolismo , Factor H de Complemento/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fagocitosis , Unión Proteica , Estructura Terciaria de Proteína , Especificidad de la Especie , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/metabolismoRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Infecciones Meningocócicas/inmunología , Vacunas Meningococicas/inmunología , Neisseria meningitidis/inmunología , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Epítopos/genética , Epítopos/inmunología , Vacunas Meningococicas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neisseria meningitidis/genética , Unión Proteica/genética , Unión Proteica/inmunologíaRESUMEN
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.
RESUMEN
Introduction: We aimed to describe a clinical presentation of central serous retinopathy that poses a diagnostic and management dilemma. Case Presentation: A 30-year-old male patient presented with bilateral vision loss and multifocal serous retinal detachments involving the posterior pole of both eyes. Optical coherence tomography revealed prominent bilateral bacillary layer detachments. The patient complained of recent headaches and tinnitus. However, the clinical exam did not reveal overt inflammation and the patient admitted to being under significant stress. The clinical presentation raised concerns for both central serous retinopathy (CSR) and Vogt-Koyanagi-Harada (VKH). Additional findings, including white fundus spots and focal areas of retinal vascular leakage, were seen in our patient. We highlight these because, while they have been described in CSR, they are not commonly discussed and could add to the diagnostic dilemma. After a conservative approach that avoided steroids, our patient showed marked improvement over the following month, supporting a diagnosis of CSR. Conclusion: CSR can mimic VKH disease. A high level of suspicion is needed to avoid instituting steroid therapy that could induce a severe iatrogenic exacerbation of the disease.
RESUMEN
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.
Asunto(s)
Microglía , Degeneración Retiniana , Animales , Humanos , Ratones , Microglía/metabolismo , Retina/patología , Degeneración Retiniana/patología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
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.
Asunto(s)
Degeneración Retiniana , Animales , Ratones , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Ratones Endogámicos C57BL , Retina/metabolismo , Neuroglía/metabolismo , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Degeneración Retiniana , Animales , Ratones , Modelos Animales de Enfermedad , Pruebas Genéticas , Lípidos , Ratones Endogámicos C57BL , Degeneración Retiniana/genética , Degeneración Retiniana/patologíaRESUMEN
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.
Asunto(s)
Separación Celular/métodos , ARN/análisis , ARN/aislamiento & purificación , Epitelio Pigmentado de la Retina/citología , Animales , Biomarcadores/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Estabilidad del ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/química , Epitelio Pigmentado de la Retina/metabolismo , Transcriptoma , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Enfermedades de la Córnea/terapia , Distrofia Endotelial de Fuchs/terapia , Oligonucleótidos Antisentido/farmacología , ARN Largo no Codificante/genética , Animales , Córnea/efectos de los fármacos , Córnea/patología , Enfermedades de la Córnea/genética , Enfermedades de la Córnea/patología , Modelos Animales de Enfermedad , Endotelio Corneal/efectos de los fármacos , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patología , Humanos , Ratones , ARN Largo no Codificante/antagonistas & inhibidoresRESUMEN
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.
RESUMEN
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.
Asunto(s)
Modelos Animales de Enfermedad , Factor 2 Relacionado con NF-E2/metabolismo , Traumatismos Experimentales por Radiación/fisiopatología , Recuperación de la Función/fisiología , Degeneración Retiniana/fisiopatología , Superóxido Dismutasa-1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Electrorretinografía , Femenino , Luz/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 2 Relacionado con NF-E2/deficiencia , Estrés Oxidativo , Fotograbar , Traumatismos Experimentales por Radiación/metabolismo , Retina/diagnóstico por imagen , Retina/fisiopatología , Retina/efectos de la radiación , Degeneración Retiniana/metabolismo , Tomografía de Coherencia ÓpticaRESUMEN
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.
Asunto(s)
Envejecimiento/fisiología , Proteína Desglicasa DJ-1/deficiencia , Degeneración Retiniana/metabolismo , Superóxido Dismutasa-1/deficiencia , Ubiquitina-Proteína Ligasas/deficiencia , Animales , Biomarcadores/metabolismo , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Estrés Oxidativo/fisiología , Proteína Desglicasa DJ-1/genética , Retina/metabolismo , Retina/fisiopatología , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Superóxido Dismutasa-1/genética , Tomografía de Coherencia Óptica , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
PURPOSE: To report a case of cytomegalovirus (CMV) retinitis after placement of a fluocinolone acetonide (Retisert) implant. DESIGN: Interventional case report. METHODS: Retrospective chart review. RESULTS: A 65-year-old man with a history of Adamantiades-Behcet disease and bilateral recurrent uveitis that was unresponsive to systemic corticosteroid-sparing immunosuppressive therapy developed clinical evidence of CMV retinitis after receiving his second intravitreal Retisert implant in the left eye, while on no systemic immunosuppression. He did not develop CMV retinitis in the right eye despite multiple intraocular and periocular steroid injections. The patient responded well to intravitreal foscarnet followed by placement of an intravitreal ganciclovir implant. CONCLUSIONS: Ophthalmologists should be aware of the potential risk for development of CMV retinitis after local ocular immunosuppressive therapy.
Asunto(s)
Retinitis por Citomegalovirus/etiología , Fluocinolona Acetonida/efectos adversos , Glucocorticoides/efectos adversos , Anciano , Antivirales/uso terapéutico , Síndrome de Behçet/complicaciones , Retinitis por Citomegalovirus/diagnóstico , Retinitis por Citomegalovirus/tratamiento farmacológico , Implantes de Medicamentos , Fluocinolona Acetonida/administración & dosificación , Foscarnet/uso terapéutico , Ganciclovir/uso terapéutico , Glucocorticoides/administración & dosificación , Humanos , Huésped Inmunocomprometido , Edema Macular/tratamiento farmacológico , Masculino , Estudios Retrospectivos , Uveítis/complicacionesRESUMEN
PURPOSE: Oxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD. METHODS: The retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed. RESULTS: Both protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition. CONCLUSIONS: We characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.
Asunto(s)
Apoptosis , Regulación de la Expresión Génica , Estrés Oxidativo , Células Fotorreceptoras de Vertebrados/patología , ARN/genética , Degeneración Retiniana/genética , cis-trans-Isomerasas/genética , Animales , Modelos Animales de Enfermedad , Angiografía con Fluoresceína , Fondo de Ojo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Luz/efectos adversos , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Microglía/patología , Células Fotorreceptoras de Vertebrados/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Tomografía de Coherencia Óptica , cis-trans-Isomerasas/biosíntesisRESUMEN
PURPOSE: Variants of complement factor H (Cfh) affecting short consensus repeats (SCRs) 6 to 8 increase the risk of age-related macular degeneration. Our aim was to explore the effect of expressing a Cfh variant on the in vivo susceptibility of the retina and RPE to oxidative stress and inflammation, using chimeric Cfh transgenic mice (chCfhTg). METHODS: The chCfhTg and age-matched C57BL/6J (B6) mice were subjected to oxidative stress by either normal aging, or by exposure to a combination of oral hydroquinone (0.8% HQ) and increased light. Eyes were collected for immunohistochemistry of RPE-choroid flat mounts and of retinal sections, ELISA, electron microscopy, and RPE/microglia gene expression analysis. RESULTS: Aging mice to 2 years led to an increased accumulation of basal laminar deposits, subretinal microglia/macrophages (MG/MΦ) staining for CD16 and for malondialdehyde (MDA), and MDA-modified proteins in the retina in chCfhTg compared to B6 mice. The chCfhTg mice maintained on HQ diet and increased light showed greater deposition of basal laminar deposits, more accumulation of fundus spots suggestive of MG/MΦ, and increased deposition of C3d in the sub-RPE space, compared to controls. In addition, chCfhTg mice demonstrated upregulation of NLRP3, IP-10, CD68, and TREM-2 in the RNA isolates from RPE/MG/MΦ. CONCLUSIONS: Expression of a Cfh transgene introducing a variant in SCRs 6 to 8 was sufficient to lead to increased retinal/RPE susceptibility to oxidative stress, a proinflammatory MG/MΦ phenotype, and a proinflammatory RPE/MG/MΦ gene expression profile in a transgenic mouse model. Our data suggest that altered interactions of Cfh with MDA-modified proteins may be relevant in explaining the effects of the Cfh variant.