RESUMO
Vitreous alterations occur from early stages and continue through the normal aging, with gradual lamellae formation and the appearance of liquefied spaces, which eventually leads to complications, such as retinal tear, retinal detachment, and intravitreal hemorrhage. The aim of the present study was to investigate the expression of let-7 miRNA family in the vitreous and retina in newborn (1-3- day-old), young adult (2-month-old), and aging (12-month-old) rats, as well as their role as regulators of vitreous components. MicroRNAs are small, non-coding RNAs that post-transcriptionally regulate gene expression. Our results showed detection of all investigated let-7 isoforms (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f and let-7i) in the retina and vitreous. Although most let-7 members were significantly upregulated in the vitreous during development, only let-7b, let-7c, and let-7e followed this same expression pattern in the retina. Let-7b and -7c increased in aging vitreous as well, and were expressed in vitro by Müller glial cells and their extracellular vesicles. Moreover, let-7 targeted hyaluronan synthase 2 (Has2) mRNA, a synthesizing enzyme of hyaluronan. These observations indicate that let-7 function is important during retina and vitreous development, and that isoforms of let-7 increased with aging, potentially modulating hyaluronan content.
Assuntos
Envelhecimento/fisiologia , Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Retina/metabolismo , Corpo Vítreo/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Células Ependimogliais/metabolismo , Humanos , Hialuronan Sintases/genética , Masculino , Microscopia Eletrônica de Transmissão , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Ratos , Ratos Wistar , Retina/crescimento & desenvolvimento , Corpo Vítreo/crescimento & desenvolvimentoRESUMO
PURPOSE: To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes. METHODS: PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1-8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka-Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized. RESULTS: After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants. CONCLUSIONS: Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.
Assuntos
Imunossupressores/farmacocinética , Imunossupressores/toxicidade , Epitélio Pigmentado da Retina/efeitos dos fármacos , Sirolimo/farmacocinética , Sirolimo/toxicidade , Corpo Vítreo/metabolismo , Implantes Absorvíveis , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular , Sistemas de Liberação de Medicamentos , Implantes de Medicamento , Eletrorretinografia , Células-Tronco Embrionárias/efeitos dos fármacos , Humanos , Injeções Intravítreas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Retina/efeitos dos fármacosRESUMO
Etoposide-loaded poly(lactic-co-glycolic acid) implants were developed for intravitreal application. Implants were prepared by a solvent-casting method and characterized in terms of content uniformity, morphology, drug-polymer interaction, stability, and sterility. In vitro drug release was investigated and the implant degradation was monitored by the percent of mass loss. Implants were inserted into the vitreous cavity of rabbits' eye and the in vivo etoposide release profile was determined. Clinical examination and the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method were performed to evaluate the implant tolerance. The original chemical structure of the etoposide was preserved after incorporation in the polymeric matrix, which the drug was dispersed uniformly. In vitro, implants promoted sustained release of the drug and approximately 57% of the etoposide was released in 50 days. In vivo, devices released approximately 63% of the loaded drug in 42 days. Ophthalmic examination and HET-CAM assay revealed no evidence of toxic effects of implants. These results tend to show that etoposide-loaded implants could be potentially useful as an intraocular etoposide delivery system in the future.
Assuntos
Implantes de Medicamento/metabolismo , Etoposídeo/metabolismo , Ácido Láctico/metabolismo , Ácido Poliglicólico/metabolismo , Corpo Vítreo/metabolismo , Animais , Galinhas , Implantes de Medicamento/administração & dosagem , Implantes de Medicamento/química , Etoposídeo/administração & dosagem , Etoposídeo/química , Injeções Intravítreas , Ácido Láctico/administração & dosagem , Ácido Láctico/química , Masculino , Ácido Poliglicólico/administração & dosagem , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Corpo Vítreo/efeitos dos fármacosRESUMO
BACKGROUND: Drug ocular toxicity is a field that requires attention. Clindamycin has been injected intravitreally to treat ocular toxoplasmosis, the most common cause of eye posterior segment infection worldwide. However, little is known about the toxicity of clindamycin to ocular tissues. We have previously showed non intraocular toxicity in rabbit eyes of poly(lactic-co-glycolic acid) (PLGA) implants containing clindamycin hydrochloride (CLH) using only clinical macroscotopic observation. In this study, we investigated the in vivo biocompatibility of CLH-PLGA implants at microscotopic, cellular and molecular levels. METHODS: Morphology of ARPE-19 and MIO-M1 human retinal cell lines was examined after 72 h exposure to CLH-PLGA implant. Drug delivery system was also implanted in the vitreous of rat eyes, retinal morphology was evaluated in vivo and ex vivo. Morphology of photoreceptors and inflammation was assessed using immunofluorescence and real-time PCR. RESULTS: After 72 h incubation with CLH-PLGA implant, ARPE-19 and MIO-M1 cells preserved the actin filament network and cell morphology. Rat retinas displayed normal lamination structure at 30 days after CLH-PLGA implantation. There was no apoptotic cell and no loss in neuron cells. Cones and rods maintained their normal structure. Microglia/macrophages remained inactive. CLH-PLGA implantation did not induce gene expression of cytokines (IL-1ß, TNF-α, IL-6), VEGF, and iNOS at day 30. CONCLUSION: These results demonstrated the safety of the implant and highlight this device as a therapeutic alternative for the treatment of ocular toxoplasmosis.
Assuntos
Clindamicina/administração & dosagem , Clindamicina/química , Ácido Láctico/química , Ácido Poliglicólico/química , Retina/efeitos dos fármacos , Animais , Materiais Biocompatíveis/química , Linhagem Celular , Sistemas de Liberação de Medicamentos/métodos , Células Ependimogliais , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Injeções Intravítreas/métodos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Próteses e Implantes , Ratos , Ratos Endogâmicos Lew , Retina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Corpo Vítreo/efeitos dos fármacos , Corpo Vítreo/metabolismoRESUMO
Chronic inflammation develops in the retinal microvasculature under sustained hyperglycemia and is implicated in the pathogenesis of diabetic retinopathy. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 have been reported to promote pro-inflammatory cytokines, which are involved in the pathogenesis of proliferative diabetic retinopathy (PDR). It is therefore possible that the TWEAK/Fn14 pathway can play a regulatory role in PDR. In the present study, we examined the expression of TWEAK and Fn14 in vitreous fluid from PDR patients. To confirm the correlation between the TWEAK expression and clinical pathological characteristics of PDR, we investigated the regulatory role of the TWEAK/Fn14 pathway in cell proliferation and collagen synthesis in retinal ARPE-19 cells. The results demonstrated that vitreous fluid from patients with PDR had higher levels of TWEAK and Fn14 than that from T2DM patients without PDR, thus suggesting an important regulatory role of TWEAK/Fn14 signaling in the pathogenesis of PDR. Furthermore, overexpression of TWEAK in ARPE-19 cells also promoted proliferation of and collagen synthesis in these retinal cells. It is possible that TWEAK/Fn14 upregulation in PDR may contribute to PDR progression by promoting the proliferation or fibrosis of retinal cells.
Assuntos
Colágeno/metabolismo , Retinopatia Diabética/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Retina/metabolismo , Fatores de Necrose Tumoral/metabolismo , Apoptose/genética , Apoptose/fisiologia , Linhagem Celular , Proliferação de Células/fisiologia , Citocina TWEAK , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Receptores do Fator de Necrose Tumoral/genética , Retina/patologia , Receptor de TWEAK , Fatores de Necrose Tumoral/genética , Corpo Vítreo/metabolismoRESUMO
The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-D-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.
Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Neuropeptídeos/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Doenças Retinianas/tratamento farmacológico , Venenos de Aranha/uso terapêutico , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Eletrorretinografia , Ácido Glutâmico/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , N-Metilaspartato , Neuropeptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/metabolismo , Doenças Retinianas/fisiopatologia , Venenos de Aranha/farmacologia , Corpo Vítreo/metabolismoRESUMO
PURPOSE: To determine the in vivo release profile and retinal safety of cyclosporine A (CsA) delivered from a biodegradable poly-lactide-co-glycolide (PLGA) device in the vitreous cavity of rabbits' eyes. METHODS: A total of 60 animals (60 eyes) divided into two groups were used. For the in vivo release study, 32 eyes received PLGA implants containing 350 µg of CsA, and 16 eyes received the implants without drug (control). Four animals of CsA group and two of the control group were killed weekly until 8 weeks; the vitreous was removed, and CsA concentration was evaluated. Ophthalmological examination was performed in the animals prior to implant placement and weekly during the study period. Electroretinography (ERG) was performed in other six animals for each group, treated and control, at the beginning and at the end of the study (8 weeks) when they were killed and had their eyes processed for histology. RESULTS: No sign of inflammation was noticed on slit lamp examinations and the IOP maintained stable during the study period in CsA and control groups. CsA concentration in the vitreous (ng/ml) was 257.07 ± 117.23, 271.15 ± 98.96, 296.66 ± 86.25, 256.27 ± 99.22, 304.50 ± 88.18, 326.35 ± 105.24, 491.25 ± 119.90 and 589.93 ± 132.55 after 1, 2, 3, 4, 5, 6, 7 and 8 weeks of implantation, respectively. At the end of the study, 21.67 % of mass loss was found. The retina did not show any histological alteration in either group, but a significant reduction in dark-adapted b-wave amplitude was observed in the CsA group, with no changes in a-wave amplitude. CONCLUSIONS: These data show that the PLGA system is safe, but the selective reduction in ERG b-wave amplitude indicates that the PLGA with 350 µg CsA causes retinal function impairment, specifically on the rod postreceptor pathway, 8 weeks after implantation. These ERG changes were not associated with any histological damage as seen at the light microscopy level.
Assuntos
Ciclosporina/farmacocinética , Ciclosporina/toxicidade , Portadores de Fármacos , Imunossupressores/farmacocinética , Imunossupressores/toxicidade , Retina/efeitos dos fármacos , Corpo Vítreo/metabolismo , Implantes Absorvíveis , Animais , Cromatografia Líquida de Alta Pressão , Implantes de Medicamento , Eletrorretinografia , Ácido Láctico , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Retina/fisiopatologiaRESUMO
This work describes the evaluation of metals and (metallo)proteins in vitreous humor samples and their correlations with some biological aspects in different post-mortem intervals (1-7 days), taking into account both decomposing and non-decomposing bodies. After qualitative evaluation of the samples involving 26 elements, representative metal ions (Fe, Mg and Mo) are determined by inductively coupled plasma mass spectrometry after using mini-vial decomposition system for sample preparation. A significant trend for Fe is found with post-mortem time for decomposing bodies because of a significant increase of iron concentration when comparing samples from bodies presenting 3 and 7 days post-mortem interval. An important clue to elucidate the role of metals is the coupling of liquid chromatography with inductively coupled plasma mass spectrometry for identification of metals linked to proteins, as well as mass spectrometry for the identification of those proteins involved in the post-mortem interval.
Assuntos
Metaloproteínas/análise , Metais/análise , Corpo Vítreo/química , Sequência de Aminoácidos , Autopsia/métodos , Cromatografia Líquida/métodos , Humanos , Espectrometria de Massas/métodos , Metais/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas/análise , Proteínas/metabolismo , Corpo Vítreo/metabolismoRESUMO
PURPOSE: To review the ocular pharmacology and antitumor activity of topotecan for the treatment of retinoblastoma by an evaluation of different routes of administration. METHODS: Systematic review of studies available at PubMed using the keywords retinoblastoma, topotecan, and camptothecins, including preclinical data such as cell lines and animal models, as well as clinical studies in patients with retinoblastoma. RESULTS: Forty-two available studies were reviewed. Evidence of antitumor activity against retinoblastoma as a single agent is based on data on cell lines and a limited number of affected patients with intraocular and extraocular disease when given in a protracted schedule. Evidence of additive or synergistic activity in combination with other agents such as carboplatin, melphalan, and vincristine was reported in preclinical and clinical models. In animal models, pharmacokinetic evaluation of topotecan administered by the periocular route shows that most of the drug reaches the vitreous through the systemic circulation. Topotecan administered by intravitreal injection shows high and sustained vitreal concentrations with limited systemic exposure and lack of retinal toxicity at a dose of up to 5 µg. Topotecan administered intraophthalmic artery shows higher passage to the vitreous compared with periocular administration in a swine model. CONCLUSION: Topotecan alone or in combination is active against retinoblastoma. It shows a favorable passage to the vitreous when given intravenously and intraarterially, and ocular toxicity is minimal by all routes of administration. However, its clinical role, optimal dose, and route of administration for the treatment of retinoblastoma are to be determined.