RESUMO
PURPOSE: Brilliant Blue G is used as a surgical adjuvant for retinal surgery. Although BBG double or multiple staining was reported, the effectiveness and safety of repeated staining is still elusive. To further examine the effectiveness and safety, we examined BBG in clinical cases in vivo, primary cell culture in vitro, and surgically resected specimen ex vivo. METHODS: A retrospective interventional case series with in vitro and ex vivo studies were performed. Vitrectomy was performed in 28 cases of epiretinal membrane with BBG single to multiple staining. The surgically resected membranes were stained by BBG with or without cellular fixation. Primary cell cultures were examined with BBG and live/death cell markers, such as Calcein AM and TUNEL. RESULTS: Single staining provided satisfactory staining in seven cases. Double or multiple staining substantially visualized internal limiting membrane (21 cases), especially the edges of remaining internal limiting membrane (11 cases). Adverse retinal staining was not noted and the final visual acuity showed no difference with multiple staining. The live cells barely stained with BBG, while some dead cells were stained. CONCLUSION: Brilliant Blue G multiple staining substantially enhanced the visualization of internal limiting membrane. The absence of abnormal staining supports the safety of repeated BBG staining.
Assuntos
Membrana Basal/patologia , Membrana Epirretiniana/cirurgia , Indicadores e Reagentes , Corantes de Rosanilina , Animais , Membrana Basal/metabolismo , Sobrevivência Celular , Células Cultivadas , Membrana Epirretiniana/diagnóstico , Membrana Epirretiniana/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Permeabilidade , Receptores Purinérgicos P2X7/fisiologia , Retina/citologia , Retina/metabolismo , Estudos Retrospectivos , Coloração e Rotulagem , Tomografia de Coerência Óptica , VitrectomiaRESUMO
Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.
Assuntos
Trifosfato de Adenosina/farmacologia , Degeneração Macular/metabolismo , Células Fotorreceptoras/efeitos dos fármacos , Receptores Purinérgicos P2X7/metabolismo , Hemorragia Retiniana/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Corioide/irrigação sanguínea , Corioide/metabolismo , Corioide/patologia , Neovascularização de Coroide , Técnicas de Cocultura , Fragmentação do DNA/efeitos dos fármacos , Humanos , Degeneração Macular/patologia , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Cultura Primária de Células , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X7/genética , Hemorragia Retiniana/patologia , Epitélio Pigmentado da Retina , Corantes de Rosanilina/farmacologia , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/metabolismo , Corpo Vítreo/patologiaRESUMO
A phase 1 clinical trial evaluating the safety of gene therapy for patients with wet age-related macular degeneration (AMD) or retinoblastoma has been completed without problems. The efficacy of gene therapy for Leber's congenital amaurosis (LCA) was reported by three groups. Gene therapy may thus hold promise as a therapeutic method for the treatment of intractable ocular diseases. However, it will first be important to precisely evaluate the efficiency and safety of alternative gene transfer vectors in a preclinical study using large animals. In the present study, we evaluated the acute local (ophthalmic) and systemic toxicity of our simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vectors carrying human pigment epithelium-derived factor (SIV-hPEDF) for transferring genes into nonhuman primate retinas. Transient inflammation and elevation of intraocular pressure were observed in some animals, but these effects were not dose dependent. Electroretinograms (ERGs), including multifocal ERGs, revealed no remarkable change in retinal function. Histopathologically, SIV-hPEDF administration resulted in a certain degree of inflammatory reaction and no apparent structural destruction in retinal tissue. Regarding systemic toxicity, none of the animals died, and none showed any serious side effects during the experimental course. No vector leakage was detected in serum or urine samples. We thus propose that SIVagm-mediated stable gene transfer might be useful and safe for ocular gene transfer in a clinical setting.