RESUMO
PURPOSE: The purpose of this article was to evaluate how human trabecular meshwork (TM) is influenced by the chronic presence of trabecular bypass implants. METHODS: Human TM samples were obtained intraoperatively from 3 patients who had previously undergone implantation of a trabecular micro-bypass stent. Trabecular strips were obtained with a goniotomy blade from areas directly adjacent to the stent after stent removal. Tissue samples were preserved, processed, cut, and stained according to standardized laboratory protocol. Harvested samples were compared with human cadaveric TM from an eye without ocular disease as well as TM obtained from a glaucomatous eye without prior stent placement. RESULTS: In all samples, a significant increase in the amount of fibrous material compared with cellular material was noted when compared with controls. In a single strip, a basement membrane-like structure was noted, which correlated with a semiopaque membrane noted intraoperatively overlying the stent and adjacent TM. Further, TM cells were absent from areas adjacent to the stent implantation site with related collapse of collagen beams. CONCLUSIONS: These findings indicate that inflammatory and fibrotic changes are present surrounding the device with clear differences noted when compared with both healthy and glaucomatous controls. These changes suggest a possible etiology for device failure over time. Further studies are necessary to tease out differences in TM tissue reaction to various implant materials as well as to make comparisons to procedures that excise TM.
Assuntos
Glaucoma/cirurgia , Implantação de Prótese , Stents , Malha Trabecular/diagnóstico por imagem , Malha Trabecular/patologia , Malha Trabecular/cirurgia , Trabeculectomia , Fibrose/diagnóstico , Fibrose/patologia , Glaucoma/diagnóstico , Glaucoma/patologia , Glaucoma/fisiopatologia , Técnicas Histológicas , Humanos , Pressão Intraocular , Microscopia , Período Pós-Operatório , Implantação de Prótese/reabilitação , Trabeculectomia/instrumentação , Trabeculectomia/métodos , Trabeculectomia/reabilitaçãoRESUMO
PURPOSE: To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to the vesicating agent, nitrogen mustard (NM), a bifunctional alkylating analog of the chemical warfare agent sulfur mustard. METHODS: Toxic effects and associated mechanisms were examined in maximally affected corneal tissue using corneal cultures and human corneal epithelial (HCE) cells exposed to NM. RESULTS: Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation, and levels of vascular endothelial growth factor, cyclooxygenase 2, and matrix metalloproteinase-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53, and H2A.X ser139 levels. NM exposure also induced caspase-3 and poly ADP ribose polymerase cleavage, suggesting their involvement in NM-induced apoptotic death in the rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in cyclooxygenase 2, matrix metalloproteinase-9, and vascular endothelial growth factor levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation, and neovascularization. NM exposure also induced activation of activator protein 1 transcription factor proteins and upstream signaling pathways including mitogen-activated protein kinases and Akt protein kinase, suggesting that these could be key factors involved in NM-induced corneal injury. CONCLUSIONS: Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant-induced ocular injuries, which could be helpful in the development of targeted therapies.
Assuntos
Substâncias para a Guerra Química/toxicidade , Córnea/efeitos dos fármacos , Neovascularização da Córnea/induzido quimicamente , Substância Própria/patologia , Dano ao DNA , Epitélio Corneano/patologia , Mecloretamina/toxicidade , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Técnicas de Cultura de Órgãos , Coelhos , Ruptura , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities. METHODS: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei. RESULTS: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules. CONCLUSIONS: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.
Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Retina/anatomia & histologia , Retina/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Desenho de Equipamento , Humanos , Metabolismo dos Lipídeos , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Gambás , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Epitélio Pigmentado da Retina/anatomia & histologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
PURPOSE: To examine the protective effects of antioxidants in cultured trabecular meshwork (TM) cells exposed to oxidative stress. METHODS: Primary porcine TM cells were pretreated with 50 µM resveratrol, 0.2 mM urate, 1 mM ascorbate, 1 mM reduced glutathione (rGSH), or 1 mM ρ-coumarate followed by exposure to hydrogen peroxide (0.5-4 mM). Cell metabolism was determined by mitochondrial enzyme activity and cell viability by uptake of the vital dye calcein, a fluorescent calcium binding dye. Reactive oxygen species (ROS), which may reflex oxidative damage, were determined by 2'7'-dichlorofluorescein diacetate. RESULTS: Trabecular meshwork cell metabolism was reduced to 72 ± 5% of control levels with 1 mM hydrogen peroxide (H2O2) treatment. TM cells that co-incubated with ascorbate (85% ± 5%), ρ-coumarate (98 ± 11%) or rGSH (103 ± 17%) had significantly increased metabolism compared to 1 mM H2O2 treatment. Resveratrol significantly increased TM cell metabolism at both 2 mM (102 ± 14% live) and 4 mM H2O2 (27 ± 12% live), with H2O2-treated cultures containing mostly metabolically inactive cells (3% at 2 mM; 2% at 4 mM). Similar results were obtained in cell viability assays. Ascorbate and resveratrol, but not ρ-coumarate or rGSH, decreased ROS levels in TM cells exposed to a sublethal dose of H2O2 (0.5 mM). Urate had no protective effect against H2O2 damage in any of the assays. CONCLUSIONS: Increased oxidative damage was demonstrated in the TM of patients with primary open angle glaucoma. The antioxidants (resveratrol, ascorbate, ρ-coumarate) and the antioxidant enzyme cofactor (rGSH) protected TM cells from H2O2-induced damage. TRANSLATIONAL RELEVANCE: Future experiments are needed to determine whether addition of antioxidants may maintain TM cell viability in vivo. Antioxidants could be applied either topically or coupled with extended-release vehicles for intraocular injection to reduce free radical formation leading to enhanced therapeutic outcomes. Ultimately, studies using animal models could determine whether application of antioxidants can ameliorate progression in diseases such as glaucoma and macular degeneration.