RESUMO
The treatment of posterior eye segment diseases through intravitreal injection requires repeated injections of an active molecule, which may be associated with serious side effects and poor patient compliance. One brilliant strategy to overcome these issues is the use of drug-loaded microparticles for sustained release, aiming at reducing the frequency of injections. Therefore, the aim of this work was to assess the safety features of poly(lactic-co-glycolic acid) (PLGA)-based, hyaluronic acid-decorated microparticles loaded with palmitoylethanolamide (PEA), citicoline (CIT), or glial-cell-derived neurotrophic factor (GDNF). Microparticles were prepared by double emulsion-solvent evaporation and fully characterized for their technological features. Microparticles possessed a satisfactory safety profile in vitro on human retinal pigment epithelial (ARPE-19) cells. Interestingly, the administration of free GDNF led to a loss of cell viability, while GDNF sustained release displayed a positive effect in that regard. In vivo results confirmed the safety profile of both empty and loaded microparticles. Overall, the outcomes suggest that the produced microparticles are promising for improving the local administration of neuroprotective molecules. Further studies will be devoted to assess the therapeutic ability of microparticles.
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Central serous chorioretinopathy (CSCR) is a retinal disease affecting the retinal pigment epithelium (RPE) and the choroid. This is a recognized side-effect of glucocorticoids (GCs), administered through nasal, articular, oral and dermal routes. However, CSCR does not occur after intraocular GCs administration, suggesting that a hypothalamic-pituitary-adrenal axis (HPA) brake could play a role in the mechanistic link between CSCR and GS. The aim of this study was to explore this hypothesis. To induce HPA brake, Lewis rats received a systemic injection of dexamethasone daily for five days. Control rats received saline injections. Baseline levels of corticosterone were measured by Elisa at baseline and at 5 days in the serum and the ocular media and dexamethasone levels were measured at 5 days in the serum and ocular media. The expression of genes encoding glucocorticoid receptor (GR), mineralocorticoid receptors (MR), and the 11 beta hydroxysteroid dehydrogenase (HSD) enzymes 1 and 2 were quantified in the neural retina and in RPE/ choroid. The expression of MR target genes was quantified in the retina (Scnn1A (encoding ENac-α, Kir4.1 and Aqp4) and in the RPE/choroid (Shroom 2, Ngal, Mmp9 and Omg, Ptx3, Plaur and Fosl-1). Only 10% of the corticosterone serum concentration was measured in the ocular media. Corticosterone levels in the serum and in the ocular media dropped after 5 days of dexamethasone systemic treatment, reflecting HPA axis brake. Whilst both GR and MR were downregulated in the retina without MR/GR imbalance, in the RPE/choroid, both MR/GR and 11ß-hsd2/11ß-hsd1 ratio increased, indicating MR pathway activation. MR-target genes were upregulated in the RPE/ choroid but not in the retina. The psychological stress induced by the repeated injection of saline also induced HPA axis brake with a trend towards MR pathway activation in RPE/ choroid. HPA axis brake causes an imbalance of corticoid receptors expression in the RPE/choroid towards overactivation of MR pathway, which could favor the occurrence of CSCR.
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Glucocorticoides/metabolismo , Mineralocorticoides/metabolismo , Retina/metabolismo , Animais , Coriorretinopatia Serosa Central/tratamento farmacológico , Coriorretinopatia Serosa Central/fisiopatologia , Corioide/efeitos dos fármacos , Corioide/metabolismo , Corticosterona/sangue , Dexametasona/metabolismo , Dexametasona/farmacologia , Olho/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Fenômenos Fisiológicos Oculares/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Glucocorticoides/metabolismo , Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in the Crb1 gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-ß and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia.
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Células Ependimogliais/patologia , Proteínas do Olho/genética , Mutação/genética , Degeneração Retiniana , Telangiectasia/complicações , Telangiectasia/genética , Fatores Etários , Animais , Animais Recém-Nascidos , Células Cultivadas , Modelos Animais de Doenças , Eletrorretinografia , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Proteínas do Olho/metabolismo , Angiofluoresceinografia , Proteína Glial Fibrilar Ácida/metabolismo , Neurônios/patologia , Neurônios/ultraestrutura , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos , Ratos Mutantes , Degeneração Retiniana/etiologia , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Vasos Retinianos/patologia , Vasos Retinianos/ultraestrutura , Transdução de Sinais/fisiologia , Vias Visuais/patologia , Vias Visuais/ultraestruturaRESUMO
Mast cells are important in the initiation of ocular inflammation, but the consequences of mast cell degranulation on ocular pathology remain uncharacterized. We induced mast cell degranulation by local subconjunctival injection of compound 48/80. Initial degranulation of mast cells was observed in the choroid 15 minutes after the injection and increased up to 3 hours after injection. Clinical signs of anterior segment inflammation paralleled mast cell degranulation. With the use of optical coherence tomography, dilation of choroidal vessels and serous retinal detachments (SRDs) were observed and confirmed by histology. Subconjunctival injection of disodium cromoglycate significantly reduced the rate of SRDs, demonstrating the involvement of mast cell degranulation in posterior segment disorders. The infiltration of polymorphonuclear and macrophage cells was associated with increased ocular media concentrations of tumor necrosis factor-α, CXCL1, IL-6, IL-5, chemokine ligand 2, and IL-1ß. Analysis of the amounts of vascular endothelial growth factor and IL-18 showed an opposite evolution of vascular endothelial growth factor compared with IL-18 concentrations, suggesting that they regulate each other's production. These findings suggest that the local degranulation of ocular mast cells provoked acute ocular inflammation, dilation, increased vascular permeability of choroidal vessels, and SRDs. The involvement of mast cells in retinal diseases should be further investigated. The pharmacologic inhibition of mast cell degranulation may be a potential target for intervention.
Assuntos
Degranulação Celular/efeitos dos fármacos , Corioide/patologia , Mastócitos/patologia , Retina/patologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Quimiocinas/metabolismo , Corioide/efeitos dos fármacos , Corioide/metabolismo , Citocinas/metabolismo , Feminino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Ratos , Ratos Endogâmicos Lew , Retina/efeitos dos fármacos , Retina/metabolismo , Tomografia de Coerência Óptica , p-Metoxi-N-metilfenetilamina/farmacologiaRESUMO
PURPOSE: Intravitreal recombinant tissue plasminogen activator (rtPA) is used off-label for the surgical management of submacular hemorrhage, a severe complication of neovascular age-related macular degeneration. rtPA is approved for coronary and cerebral thrombolysis. However, in ischemic stroke rtPA is known to increase excitotoxic neural cell death by interacting with the N-methyl-D-aspartate (NMDA) receptor. We therefore investigated the retinal toxicity of rtPA in healthy rats and in a model of NMDA-induced retinal excitotoxicity. METHODS: First, rtPA at three different doses (2.16 µg/5 µl, 0.54 µg/5 µl, and 0.27 µg/5 µl) or vehicle (NaCl 0.9%) was injected intravitreally in healthy rat eyes. Electroretinograms (ERGs) were performed at 24 h or 7 days. Annexin V-fluorescein isothiocyanate (FITC)-labeled apoptotic retinal ganglion cells (RGCs) were counted on flatmounted retinas at 24 h or 7 days. Next, NMDA + vehicle or NMDA + rtPA (0.27 µg/5 µl) was injected intravitreally to generate excitotoxic conditions. Apoptotic annexin V-FITC-labeled RGCs and surviving Brn3a-labeled RGCs were quantified on flatmounted retinas and radial sections, 18 h after treatment. RESULTS: In healthy rat eyes, the number of apoptotic RGCs was statistically significantly increased 24 h after the administration of rtPA at the highest dose (2.16 µg/5 µl; p = 0.0250) but not at the lower doses of 0.54 and 0.27 µg/5 µl (p = 0.36 and p = 0.20), compared to vehicle. At day 7, there was no difference in the apoptotic RGC count between the rtPA- and vehicle-injected eyes (p = 0.70, p = 0.52, p = 0.11). ERG amplitudes and implicit times were not modified at 24 h or 7 days after injection of any tested rtPA doses, compared to the baseline. Intravitreal administration of NMDA induced RGC death, but under these excitotoxic conditions, coadministration of rtPA did not increase the number of dead RGCs (p = 0.70). Similarly, the number of surviving RGCs on the flatmounted retinas and retinal sections did not differ between the eyes injected with NMDA + vehicle and NMDA + rtPA (p = 0.59 and p = 0.67). CONCLUSIONS: At low clinical equivalent doses corresponding to 25 µg/0.1 ml in humans, intravitreal rtPA is not toxic for healthy rat retinas and does not enhance NMDA-induced excitotoxicity. Vitreal equivalent doses ≥200 µg/0.1 ml should be avoided in patients, due to potential RGC toxicity.
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Neurotoxinas/toxicidade , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tecidual/efeitos adversos , Ativador de Plasminogênio Tecidual/farmacologia , Animais , Apoptose/efeitos dos fármacos , Eletrorretinografia , Injeções Intravítreas , Masculino , Ratos Long-Evans , Proteínas Recombinantes/administração & dosagem , Retina , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Ativador de Plasminogênio Tecidual/administração & dosagemRESUMO
PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Microglia/patologia , Retina/patologia , Uveíte/tratamento farmacológico , Uveíte/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anticorpos Neutralizantes/farmacologia , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Testes de Neutralização , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Photoreceptors and retinal pigment epithelial cells (RPE) targeting remains challenging in ocular gene therapy. Viral gene transfer, the only method having reached clinical evaluation, still raises safety concerns when administered via subretinal injections. We have developed a novel transfection method in the adult rat, called suprachoroidal electrotransfer (ET), combining the administration of nonviral plasmid DNA into the suprachoroidal space with the application of an electrical field. Optimization of injection, electrical parameters and external electrodes geometry using a reporter plasmid, resulted in a large area of transfected tissues. Not only choroidal cells but also RPE, and potentially photoreceptors, were efficiently transduced for at least a month when using a cytomegalovirus (CMV) promoter. No ocular complications were recorded by angiographic, electroretinographic, and histological analyses, demonstrating that under selected conditions the procedure is devoid of side effects on the retina or the vasculature integrity. Moreover, a significant inhibition of laser induced-choroidal neovascularization (CNV) was achieved 15 days after transfection of a soluble vascular endothelial growth factor receptor-1 (sFlt-1)-encoding plasmid. This is the first nonviral gene transfer technique that is efficient for RPE targeting without inducing retinal detachment. This novel minimally invasive nonviral gene therapy method may open new prospects for human retinal therapies.
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Corioide/metabolismo , Técnicas de Transferência de Genes , Retina/metabolismo , Transfecção/métodos , Animais , Feminino , Masculino , RatosRESUMO
The pre-symptomatic stage of Alzheimer's disease (AD) is associated with increased amyloid-ß (Aß) precursor protein (APP) processing and Aß accumulation in the retina and hippocampus. Because neuronal dysfunctions are among the earliest AD-related alterations, we asked whether they are already detectable in the retina during the pre-symptomatic stage in a APPswePS1dE9 (APP/PS1) mouse model. The age chosen for the study (3-4 months) corresponds to the pre-symptomatic stage because no retinal Aß was detected, in spite of the presence of ßCTF (the first cleavage product of APP). We observed an increase in ERG amplitudes in APP/PS1 mice in comparison to the controls, which indicated an increased retinal neuron activity. These functional changes coincided with an increased expression of retinal TNFα and its receptors type-1 (TNFR1). Consistently, the IkB expression increased in APP/PS1 mice with a greater proportion of the phosphorylated protein (P-IkB) over total IkB, pointing to the putative involvement of the NFkB pathway. Because TNFα plays a crucial role in the control of neuronal excitability, it is likely that, as in the hippocampus, TNFα signaling via the TNFR1/NFkB pathway may be also involved in early, AD-associated, retinal neuron hyperexcitability. These results further demonstrate the interest of the retina for early disease detection with a potential to assess future therapeutic strategies.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Retina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids have shown neuroprotective properties in neurodegenerative diseases, but differential effects of the two bile acids have been poorly explored. The aim of this study was to evaluate the neuroprotective effects of UDCA versus TUDCA in a neuroretinal degeneration model and to compare transcriptionally regulated pathways. The WERI-Rb-1 human cone-like cell line and retinal explants were exposed to albumin and TUDCA or UDCA. Viability, cell death, and microglial activation were quantified. Transcriptionally regulated pathways were analyzed after RNA sequencing using the edgeR bioconductor package. Pre-treatment of cone-like cells with UDCA or TUDCA significantly protected cells from albumin toxicity. On retinal explants, either bile acid reduced apoptosis, necroptosis, and microglia activation at 6 h. TUDCA induced the regulation of 463 genes, whilst 31 genes were regulated by UDCA. Only nineteen common genes were regulated by both bile acids, mainly involved in iron control, cell death, oxidative stress, and cell metabolism. As compared to UDCA, TUDCA up-regulated genes involved in endoplasmic reticulum stress pathways and down-regulated genes involved in axonal and neuronal development. Either bile acid protected against albumin-induced cell loss. However, TUDCA regulated substantially more neuroprotective genes than UDCA.
RESUMO
PURPOSE: To compare the effect of a rat anti-VEGF antibody, administered either by topical or subconjunctival (SC) routes, on a rat model of corneal transplant rejection. METHODS: Twenty-four rats underwent corneal transplantation and were randomized into four treatment groups (n=6 in each group). G1 and G2 received six SC injections (0.02 ml 10 µg/ml) of denatured (G1) or active (G2) anti-VEGF from Day 0 to Day 21 every third day. G3 and G4 were instilled three times a day with denatured (G3) or active (G4) anti-VEGF drops (10 µg/ml) from Day 0 to Day 21. Corneal mean clinical scores (MCSs) of edema (E), transparency (T), and neovessels (nv) were recorded at Days 3, 9, 15, and 21. Quantification of neovessels was performed after lectin staining of vessels on flat mounted corneas. RESULTS: Twenty-one days after surgery, MCSs differed significantly between G1 and G2, but not between G3 and G4, and the rejection rate was significantly reduced in rats receiving active antibodies regardless of the route of administration (G2=50%, G4=66.65% versus G1 and G3=100%; p<0.05). The mean surfaces of neovessels were significantly reduced in groups treated with active anti-VEGF (G2, G4). However, anti-VEGF therapy did not completely suppress corneal neovessels. CONCLUSIONS: Specific rat anti-VEGF antibodies significantly reduced neovascularization and subsequent corneal graft rejection. The SC administration of the anti-VEGF antibody was more effective than topical instillation.
Assuntos
Córnea/imunologia , Transplante de Córnea/métodos , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Neovascularização da Córnea/prevenção & controle , Edema/patologia , Feminino , Rejeição de Enxerto , Sobrevivência de Enxerto , Masculino , Neovascularização Patológica/prevenção & controle , Distribuição Aleatória , Ratos , Ratos Endogâmicos Lew , Fator A de Crescimento do Endotélio Vascular/químicaRESUMO
The aim of this study was to evaluate the potential anti-angiogenic effect of MTRN (meteorin) in the laser-induced CNV rat model and explore its mechanisms of action. MTRN, thrompospondin-1, glial cell markers (GFAP, vimentin), and phalloidin were immuno-stained in non-human primate flat-mounted retinas and human retina cross sections. The effect of MTRN at different doses and time points was evaluated on laser-induced CNV at 14 days using in vivo fluorescein angiography and ex vivo quantification of CNV. A pan transcriptomic analysis of the retina and the RPE/choroid complex was used to explore MTRN effects mechanisms. In human retina, MTRN is enriched in the macula, expressed in and secreted by glial cells, and located in photoreceptor cells, including in nuclear bodies. Intravitreal MTRN administered preventively reduced CNV angiographic scores and CNV size in a dose-dependent manner. The highest dose, administered at day 7, also reduced CNV. MTRN, which is regulated by mineralocorticoid receptor modulators in the rat retina, regulates pathways associated with angiogenesis, oxidative stress, and neuroprotection. MTRN is a potential novel therapeutic candidate protein for wet AMD.
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Diabetic retinopathy remains a major cause of vision loss worldwide. Mineralocorticoid receptor (MR) pathway activation contributes to diabetic nephropathy, but its role in retinopathy is unknown. In this study, we show that MR is overexpressed in the retina of type 2 diabetic Goto-Kakizaki (GK) rats and humans and that cortisol is the MR ligand in human eyes. Lipocalin 2 and galectin 3, two biomarkers of diabetes complications regulated by MR, are increased in GK and human retina. The sustained intraocular delivery of spironolactone, a steroidal mineralocorticoid antagonist, decreased the early and late pathogenic features of retinopathy in GK rats, such as retinal inflammation, vascular leakage, and retinal edema, through the upregulation of genes encoding proteins known to intervene in vascular permeability such as Hey1, Vldlr, Pten, Slc7a1, Tjp1, Dlg1, and Sesn2 but did not decrease VEGF. Spironolactone also normalized the distribution of ion and water channels in macroglial cells. These results indicate that MR is activated in GK and human diabetic retina and that local MR antagonism could be a novel therapeutic option for diabetic retinopathy.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/etiologia , Receptores de Mineralocorticoides/metabolismo , Retina/patologia , Neurônios Retinianos/patologia , Espironolactona/farmacologia , Animais , Preparações de Ação Retardada , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrocortisona/metabolismo , Masculino , Antagonistas de Receptores de Mineralocorticoides/administração & dosagem , Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ratos , Ratos Endogâmicos , Receptores de Mineralocorticoides/genética , Neurônios Retinianos/efeitos dos fármacos , Espironolactona/administração & dosagem , Espironolactona/química , Regulação para Cima , Corpo VítreoRESUMO
Diabetic retinopathy (DR) remains a major cause of vision loss, due to macular edema, retinal ischemia and death of retinal neurons. We previously demonstrated that acute administration of glibenclamide into the vitreous, or given orally at a non-hypoglycemic dose, protected the structure and the function of the retina in three animal models that each mimic aspects of diabetic retinopathy in humans. In this pilot study, we investigated whether one year of chronic oral glibenclamide, in a non-hypoglycemic regimen (Amglidia®, 0.4 mg/kg, Ammtek/Nordic Pharma, 5 d/week), could alleviate the retinopathy that develops in the Goto-Kakizaki (GK) rat. In vivo, retinal function was assessed by electroretinography (ERG), retinal thickness by optical coherence tomography (OCT) and retinal perfusion by fluorescein and indocyanin green angiographies. The integrity of the retinal pigment epithelium (RPE) that constitutes the outer retinal barrier was evaluated by quantitative analysis of the RPE morphology on flat-mounted fundus ex vivo. Oral glibenclamide did not significantly reduce the Hb1Ac levels but still improved retinal function, as witnessed by the reduction in scotopic implicit times, limited diabetes-induced neuroretinal thickening and the extension of ischemic areas, and it improved the capillary coverage. These results indicate that low doses of oral glibenclamide could still be beneficial for the prevention of type 2 diabetic retinopathy. Whether the retinas ofpatients treated specifically with glibenclamideare less at risk of developing diabetic complications remains to be demonstrated.
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Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial.
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Apoptose/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , Colagogos e Coleréticos/farmacologia , Retina/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Degeneração Retiniana/terapia , Descolamento Retiniano/terapia , Ácido Ursodesoxicólico/farmacologia , Administração Oral , Albuminas/metabolismo , Animais , Disponibilidade Biológica , Linhagem Celular , Colagogos e Coleréticos/metabolismo , Criocirurgia , Feminino , Humanos , Técnicas In Vitro , Terapia a Laser , Masculino , Pessoa de Meia-Idade , Necrose , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Retina/patologia , Retina/cirurgia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Descolamento Retiniano/metabolismo , Descolamento Retiniano/patologia , Líquido Sub-Retiniano/química , Ácido Ursodesoxicólico/metabolismo , VitrectomiaRESUMO
Sulfonylureas, widely used as hypoglycemic agents in adults with type 2 diabetes, have neuroprotective effects in preclinical models of central nervous system injury, and in children with neuropsychomotor impairments linked to neonatal diabetes secondary to ATP-sensitive potassium channel mutations. In the human and rodent retina, we show that the glibenclamide-activated channel sulfonylurea receptor 1 (SUR1) is expressed in the retina and enriched in the macula; we also show that it colocalizes with the potassium channel Kir6.2, and with the cation channel transporter TRPM4. Glibenclamide (glyburide), administered at doses that did not decrease the glycemia, or injected directly into the eye, protected the structure and the function of the retina in various models of retinal injury that recapitulate the pathogenic neurodegenerative events in the diabetic retina. The downregulation of SUR1 using a siRNA suppressed the neuroprotective effects of glibenclamide on excitotoxic stress-induced cell death. The glibenclamide effects include the transcriptional regulation of antioxidant and neuroprotective genes. Ocular glibenclamide could be repurposed for diabetic retinopathy.
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Glibureto/farmacologia , Fármacos Neuroprotetores/farmacologia , Doenças Retinianas/tratamento farmacológico , Neurônios Retinianos/efeitos dos fármacos , Administração Oral , Animais , Chlorocebus aethiops , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Feminino , Glibureto/administração & dosagem , Humanos , Hiperglicemia/metabolismo , Hipoglicemiantes/farmacologia , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/administração & dosagem , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos Endogâmicos Lew , Ratos Wistar , Doenças Retinianas/etiologia , Doenças Retinianas/patologia , Neurônios Retinianos/patologia , Receptores de Sulfonilureias/metabolismo , Canais de Cátion TRPM/metabolismoRESUMO
BACKGROUND: We have developed a nonviral gene therapy method based on the electrotransfer of plasmid in the ciliary muscle. These easily accessible smooth muscle cells could be turned into a biofactory for any therapeutic proteins to be secreted in a sustained manner in the ocular media. METHODS: Electrical conditions, design of electrodes, plasmid formulation, method and number of injections were optimized in vivo in the rat by localizing ß-galactosidase expression and quantifying reporter (luciferase) and therapeutic (anti-tumor necrosis factor) proteins secretion in the ocular media. Anatomical measurements were performed via human magnetic resonance imaging to design a human eye-sized prototype that was tested in the rabbit. RESULTS: In the rat, transscleral injection of 30 µg of plasmid diluted in half saline (77 mM NaCl) followed by application of eight square-wave electrical pulses (15 V, 10 ms, 5.3 Hz) using two platinum/iridium electrodes, an internal wire and an external sheet, delivered plasmid efficiently to the ciliary muscle fibers. Gene transfer resulted in a long-lasting (at least 5 months) and plasmid dose-/injection number- dependent secretion of different molecular weight proteins mainly in the vitreous, without any systemic exposure. Because ciliary muscle anatomical measurements remained constant among ages in adult humans, an integrated device comprising needle-electrodes was designed and manufactured. Its usefulness was validated in the rabbit. CONCLUSIONS: Plasmid electrotransfer to the ciliary muscle with a suitable medical device represents a promising local and sustained protein delivery system for treating posterior segment diseases, avoiding repeated intraocular injections.
Assuntos
Corpo Ciliar/metabolismo , Eletroporação/métodos , Técnicas de Transferência de Genes , Terapia Genética , Músculo Liso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Humor Aquoso/metabolismo , Criança , Pré-Escolar , Olho/anatomia & histologia , Olho/metabolismo , Feminino , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Animais , Plasmídeos , Coelhos , Ratos , Ratos Endogâmicos Lew , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.
Assuntos
Corioide/patologia , Fator H do Complemento/metabolismo , Macrófagos/imunologia , Degeneração Macular/metabolismo , Alelos , Animais , Corioide/irrigação sanguínea , Neovascularização de Coroide , Ativação do Complemento , Complemento C3/metabolismo , Fator H do Complemento/genética , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos , Risco , Trombospondina 1/metabolismoRESUMO
In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.
Assuntos
Oftalmopatias Hereditárias/metabolismo , Ferro/metabolismo , Ferro/toxicidade , Neuroproteção , Descolamento Retiniano/metabolismo , Transferrina/metabolismo , Idoso , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Oftalmopatias Hereditárias/cirurgia , Feminino , Humanos , Ferro/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Necrose , Células Fotorreceptoras de Vertebrados/metabolismo , Ratos , Ratos Long-Evans , Ratos Wistar , Retina/metabolismo , Descolamento Retiniano/cirurgia , Epitélio Pigmentado da Retina/metabolismo , Líquido Sub-Retiniano/metabolismo , Transferrina/genéticaRESUMO
Age Related Macular Degeneration (AMD) is the first cause of social blindness in people aged over 65 leading to atrophy of retinal pigment epithelial cells (RPE), photoreceptors and choroids, eventually associated with choroidal neovascularization. Accumulation of undigested cellular debris within RPE cells or under the RPE (Drusen), oxidative stress and inflammatory mediators contribute to the RPE cell death. The major risk to develop AMD is the Y402H polymorphism of complement factor H (CFH). CFH interacting with oxidized phospholipids on the RPE membrane modulates the functions of these cells, but the exact role of CFH in RPE cell death and survival remain poorly understood. The aim of this study was to analyze the potential protective mechanism of CFH on RPE cells submitted to oxidative stress. Upon exposure to oxidized lipids 4-HNE (4-hydroxy-2-nonenal) derived from photoreceptors, both the human RPE cell line ARPE-19 and RPE cells derived from human induced pluripotent stem cells were protected from death only in the presence of the full length human recombinant CFH in the culture medium. This protective effect was independent from the membrane attack complex (MAC) formation. CFH maintained RPE cells tight junctions' structure and regulated the caspase dependent apoptosis process. These results demonstrated the CFH anti-oxidative stress functions independently of its capacity to inhibit MAC formation.
Assuntos
Fator H do Complemento/farmacologia , Complexo de Ataque à Membrana do Sistema Complemento/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Aldeídos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Eletrônica de Transmissão , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
Choroidal neovascularization (CNV) is a major cause of visual impairment in patients suffering from wet age-related macular degeneration (AMD), particularly when refractory to intraocular anti-VEGF injections. Here we report that treatment with the oral mineralocorticoid receptor (MR) antagonist spironolactone reduces signs of CNV in patients refractory to anti-VEGF treatment. In animal models of wet AMD, pharmacological inhibition of the MR pathway or endothelial-specific deletion of MR inhibits CNV through VEGF-independent mechanisms, in part through upregulation of the extracellular matrix protein decorin. Intravitreal injections of spironolactone-loaded microspheres and systemic delivery lead to similar reductions in CNV. Together, our work suggests MR inhibition as a novel therapeutic option for wet AMD patients unresponsive to anti-VEGF drugs.