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PURPOSE: To investigate the potential antiproliferative effect of cyclin-dependent kinase inhibitor 1B (CDKN1B) overexpression in a rabbit model of glaucoma filtration surgery (GFS). METHODS: The recombinant adenovector expressing exogenous CDKN1B was delivered to Tenon's capsule by subconjunctival injection during unilateral filtration surgery. The time course of CDKN1B expression was monitored by immunohistochemistry and Western blot analysis. Evaluation of proliferating activity was performed by proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizing region (AgNOR) staining, and fibroblast-specific protein 1 (FSP-1). Cyclin-dependent kinase 2 (Cdk2) and Cdk4 expression were detected with immunohistochemical analysis. RESULTS: The overexpression of CDKN1B in Tenon's capsule was monitored throughout the experimental period. Immunoreactivity to CDKN1B was mainly observed in the nucleus of fibroblasts. The increased expression of CDKN1B in sclera was detected up to 21 days after viral infection, whereas the level of CDKN1B protein in corneal stroma was not significantly increased. The overexpression of CDKN1B induced a significant decrease in AgNOR number/nucleus and area/nucleus, PCNA staining, FSP-1 positive cells, and the decreased expressions of Cdk2 and Cdk4, as evidenced by nuclear and cytoplasmic immunoreactivity to Cdk2 and Cdk4 antibodies in positive fibroblasts. CONCLUSIONS: The persistent overexpression of CDKN1B mediated by the recombinant adenovector expressing exogenous CDKN1B in Tenon's fibroblasts after GFS may lead to the inhibition of fibroblast proliferation and the downregulation of Cdk2 and Cdk4 activity, thereby reducing the severity of scar formation and the surgical outcome.
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Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Fibroblastos/metabolismo , Cirugía Filtrante , Regulación de la Expresión Génica , Glaucoma/metabolismo , Animales , Western Blotting , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/patología , Glaucoma/patología , Glaucoma/cirugía , Inmunohistoquímica , ConejosRESUMEN
AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-ß1 (TGF-ß1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 in vitro were induced by TGF-ß1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-ß1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-ß1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-ß1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-ß1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750µmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-ß1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.
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PURPOSE: To investigate postoperative inhibition of cell proliferation in animals treated with exogenous p27Kip1 during an experimental glaucoma filtration surgery (GFS) by histological and ultrastructural examinations. METHODS: The primer was designed according to p27Kip1 gene sequence of GenBank. The p27Kip1-expressing adenovirus-mediated Ad-p27 was constructed according to standard techniques. Gene therapy was performed by subconjunctival delivery of Ad-p27, mitomycin C (MMC) and PBS served as controls. Histological and ultastructural changes at surgical sites were observed for 28 days postoperatively. RESULTS: Histologically, evident cellular proliferation induced by Ad-p27 was observed on day 7, with appreciable filtering cavity. However, thin conjunctival layers were noted, and the number of fibroblasts decreased in the Tenon's capsule on day 14. On day 28, the filtering cavity partially disappeared. The histological features of the animals with MMC delivery were similar to those treated by Ad-p27. In contrast, in the PBS treatment group, thick conjunctival layers were observed, and the number of goblet cells and fibroblasts, increased markedly. The filtering cavity disappeared at postoperative day 21. Twenty eight day postoperatively, ultrastructural findings showed that most conjunctival epithelial cells in the Ad-p27 treatment group were under static state, and that organelles were inactive. The number of goblet cells, and the secretion of mucin, were decreased. The amount of fibroblasts also decreased with partial apoptotic cells. The ultrastructural features presented by rabbits with the MMC delivery resembled those treated with Ad-p27. In the PBS treatment group, the number of conjunctival epithelial cells increased; the amount of goblet cells increased as the secretion of mucin was strengthened, and a substantial amount of fibroblasts were observed with active and productive organelles. CONCLUSION: Ad-p27 inhibits the proliferation of Tenon's capsule fibroblasts at surgical sites, and suppresses the formation of scars, thereby promoting surgical efficacy.
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Proliferación Celular/efectos de los fármacos , Conjuntiva/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/uso terapéutico , Cirugía Filtrante , Terapia Genética/métodos , Glaucoma/cirugía , Cápsula de Tenon/patología , Adenoviridae/genética , Animales , Cicatriz/prevención & control , Conjuntiva/ultraestructura , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Fibroblastos/citología , Mitomicina/uso terapéutico , ConejosRESUMEN
OBJECTIVE: To investigate the effects of hydrogen peroxide (H2O2) on the barrier function and expression of tight junction protein in human retinal pigment epithelium (RPE) cells. METHODS: Experimental study. The human RPE cell line (D407) were cultured and treated with (H2O2 treated group) or without H2O2 (normal control group). The effect of H2O2 on cell viability of RPE cells was determined by MTT test. After treated with low concentration of H2O2 for 24 h to 72 h, transepithelial electrical resistance (TER) of confluent RPE cells was measured by epithelial voltmeter. The permeability of RPE cells to sodium fluorescein was measured. The expressions of the occludin and claudin-1 to -4 were determined by real-time polymerase chain reaction and Western blot analysis.t-text and one-way ANOVA were used to assess statistical significance between H2O2 treated and normal control groups. RESULTS: H2O2 at 0.2 mmol/L showed no decrease of cell viability of D407 cells, and this concentration was selected for the present study. The TER of D407 cells gradually increased, peaking at day 8 and then remained stable for 1 week. As compared to the control group, a reduction in the TER was first evident after 3 hours of treatment. Continuous culturing of cells for longer periods further reduced the TER, with a maximum effect after 24 hours of treatment and was maintained to 72 hours (24 h: 11.86 ± 1.19 vs. 24.13 ± 1.26, t = 12.260, P = 0.000; 72 h: 11.56 ± 1.47 vs. 24.33 ± 1.52, t = 10.460, P = 0.000). At any time point after adding sodium fluorescein, the permeability values of cells after treated with H2O2 for 24 hours were significantly higher than those of cells without H2O2 treatment (20 min: 25% ± 3% vs. 12% ± 4%, t = -4.50, P = 0.011; 40 min: 36% ± 4% vs. 16% ± 5%, t = -5.41, P = 0.006; 60 min: 51% ± 5% vs. 29% ± 6%, t = -4.88, P = 0.008). The expression of mRNA and protein in claudin-1, -3, and -4 were all downregulated in D407 cells treated with H2O2, whereas the expression of claudin-2 was upregulated (claudin-1 mRNA: 0.98 ± 0.18 vs. 0.28 ± 0.12, t = 5.60, P = 0.005, claudin-1 protein, 48 ± 10 vs. 100 ± 12, t = 5.77, P = 0.004; claudin-3 mRNA: 0.37 ± 0.12 vs.1.03 ± 0.15, t = 5.95, P = 0.004; claudin-3 protein: 63 ± 13 vs. 100 ± 15, t = 3.23, P = 0.032; claudin-4 mRNA: 0.38 ± 0.11 vs.0.99 ± 0.17, t = 5.22, P = 0.002, claudin-4 protein, 57 ± 12 vs. 100 ± 13, t = 4.21, P = 0.014). However, the expression of these occluding did not differ between cells treated with and without H2O2 (mRNA:1.30 ± 0.21 vs. 1.02 ± 0.16, t = -1.84, P = 0.140; protein: 109 ± 15 vs. 100 ± 14, t = -0.76, P = 0.490). CONCLUSION: Oxidative stress causes increase in the paracellular permeability of RPE cells in vitro, which may depends on the changes in expression of certain transmembrane proteins associated with the tight junction.
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Estrés Oxidativo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiopatología , Línea Celular , Permeabilidad de la Membrana Celular , Células Cultivadas , Claudina-1/metabolismo , Claudina-2/metabolismo , Claudina-3/metabolismo , Claudina-4/metabolismo , Humanos , Peróxido de Hidrógeno , Ocludina/metabolismo , ARN Mensajero/genética , Epitelio Pigmentado de la Retina/citología , Uniones Estrechas/metabolismoRESUMEN
PURPOSE: Our aim was to explore the effects and mechanism of 17-alpha-estradiol (17α-E2) on oxygen-induced retinopathy (OIR) in a murine model. METHODS: Newborn mice exposed to hyperoxia underwent subcutaneous injections of different doses of 17α-E2 from postnatal days (PND) 7 to 17. The retinal flat mounts were scored for avascular/total retinal area on PND 17. Vascular endothelial growth factor (VEGF), malondialdehyde (MDA) concentrations, and intensity, activity, and quality of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the retina were determined on PND 9, 13 (14), and 17. RESULTS: The avascular area, which is found in retinas of hyperoxia-exposed pups but not in retinas of normoxia-exposed ones, was significantly smaller in retinas of 17α-E2-treated pups. MDA and VEGF concentrations and intensity, activity, and quality of NADPH oxidase were stable in retinas of normoxia pups on PND 9, 13 (14), and 17, whereas in retinas of hyperoxia-exposed and 17α-E2-treated pups, they fluctuated markedly. VEGF concentrations were lower in retinas of hyperoxia-exposed pups than in those of normoxia ones on PND 9. Elevated VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 9 and in hyperoxia-exposed pups on PND 14 and 17. Low VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 14 and 17. MDA concentrations and NADPH oxidase concentration and activity, which were higher in retinas of hyperoxia-exposed pups, were lower in retinas of 17α-E2-treated pups on PND 9, 13, and 17. The most effective outcome in retinas of 1.0 µg 17α-E2-treated pups was markedly reversed by ICI182780. CONCLUSIONS: We found that 17α-E2 mitigates oxidative stress reactions and ameliorates OIR severity by decreasing NADPH oxidase expression and activity via the receptor and other pathways.
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Modelos Animales de Enfermedad , Estradiol/farmacología , Estrógenos/farmacología , Retinopatía de la Prematuridad/prevención & control , Animales , Animales Recién Nacidos , Permeabilidad Capilar , Dextranos , Relación Dosis-Respuesta a Droga , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Fluoresceínas , Fulvestrant , Humanos , Técnicas para Inmunoenzimas , Recién Nacido , Peroxidación de Lípido , Malondialdehído/metabolismo , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxígeno/toxicidad , Retina/metabolismo , Vasos Retinianos/patología , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: A noninferiority trial was conducted to evaluate the efficacy of a single evening dose of fixed-combination latanoprost 50 µg/mL and timolol 0.5 mg/mL (Xalacom®; LTFC), in Chinese patients with primary open-angle glaucoma (POAG) or ocular hypertension (OH) who were insufficiently controlled on ß-blocker monotherapy or ß-blocker-based dual therapy. METHODS: This 8-week, randomized, open-label, parallel-group, noninferiority study compared once-daily evening dosing of LTFC with the unfixed combination of latanoprost, one drop in the evening, and timolol, one drop in the morning (LTuFC). The primary efficacy endpoint was the mean change from baseline to week 8 in diurnal intraocular pressure (IOP; mean of 8 AM, 10 AM, 2 PM, 4 PM IOPs). LTFC was considered noninferior to LTuFC if the upper limit of the 95% confidence interval (CI) of the difference was < 1.5 mmHg (analysis of covariance). RESULTS: Baseline characteristics were similar for LTFC (N = 125; POAG, 70%; mean IOP, 25.8 mmHg) and LTuFC (N = 125; POAG, 69%; mean IOP, 26.0 mmHg). Mean diurnal IOP changes from baseline to week 8 were -8.6 mmHg with LTFC and -8.9 mmHg with LTuFC (between-treatment difference: 0.3 mmHg; 95%-CI, -0.3 to 1.0). Both treatments were well tolerated. CONCLUSIONS: A single evening dose of LTFC was at least as effective as the unfixed combination of latanoprost in the PM and timolol in the AM in reducing IOP in Chinese subjects with POAG or OH whose IOP was insufficiently reduced with ß-blocker monotherapy or ß-blocker-based dual therapy. LTFC is an effective and well tolerated once-daily treatment for POAG and OH.
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Glaucoma de Ángulo Abierto/tratamiento farmacológico , Hipertensión Ocular/tratamiento farmacológico , Prostaglandinas F Sintéticas/administración & dosificación , Timolol/administración & dosificación , Adolescente , Antagonistas Adrenérgicos beta/administración & dosificación , Adulto , Anciano , Antihipertensivos/administración & dosificación , China/epidemiología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Femenino , Estudios de Seguimiento , Glaucoma de Ángulo Abierto/etnología , Glaucoma de Ángulo Abierto/fisiopatología , Humanos , Incidencia , Presión Intraocular/efectos de los fármacos , Latanoprost , Masculino , Persona de Mediana Edad , Hipertensión Ocular/etnología , Hipertensión Ocular/fisiopatología , Soluciones Oftálmicas , Prevalencia , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
OBJECTIVE: Thymoquinone (TQ) is an active and potent compound in the oil of Nigella sativa, which has anti-inflammatory properties. The aim of this study was to evaluate the effects of TQ on ovalbumin (OVA)-induced allergic conjunctivitis (AC) in Balb/c. METHODS: The mice were divided into seven experimental groups; PBS Con, OVA Con, Conj, 0.05%TQ, 0.1%TQ, 0.5%TQ and Dex group. The mice were immunized and exposed to OVA, and eyes were treated with TQ and dexamethasone. Ocular symptoms were observed after last exposure to OVA. Eosinophils count in blood and Ophthalmic lavage fluid (OLF), recruitment of inflammatory cells in conjunctiva, serum IgE and OVA-specific IgE were evaluated by Giemsa and HE staining, and Enzyme-Linked Immunosorbent Assay (ELISA) respectively. High Performance Liquid Chromatography (HPLC) was used to determine the histamine level in OLF. The mRNA expression and protein level of cytokines were examined by real time RT-PCR and ELISA respectively. RESULTS: Ocular symptoms of AC and other characteristics of allergic inflammation including IgE and OVA-specific immunoglobulin E (IgE) level, recruitment of eosinophils, histamine level, mRNA expressions and protein level of cytokines were remarkably increased in OVA-exposed mice compared with the control groups. Administration of TQ suppressed the ocular symptoms, inflammatory cell infiltration in conjunctiva, blood and OLF, increased level of serum IgE and OVA-specific IgE, and OLF histamine level in OVA-exposed mice. Furthermore, TQ abrogated the mRNA expression and serum level of interleukin including 1L-4, IL-5, IL-13 and transforming growth factor beta (TGF-ß) in mice immunized and exposed to OVA. CONCLUSION: Administration of TQ significantly reduced the ocular symptoms in AC by attenuating the recruitment of eosinophils, level of IgE, histamine and cytokines. Our findings suggest that TQ might be a useful intimation for the treatment and future research for AC.
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Benzoquinonas/farmacología , Conjuntivitis Alérgica/prevención & control , Animales , Benzoquinonas/administración & dosificación , Cromatografía Líquida de Alta Presión , Conjuntivitis Alérgica/inducido químicamente , Conjuntivitis Alérgica/inmunología , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Eosinófilos/inmunología , Histamina/metabolismo , Inmunoglobulina E/inmunología , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Ovalbúmina , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND AND OBJECTIVE: To study the drainage pathway using a tracer after a modified filtering surgery in rabbit eyes. MATERIALS AND METHODS: The authors performed trabeculectomy combined with deep sclerectomy on one eye (TS group) and trabeculectomy on the other eye (T group) of 20 rabbits. Cationic ferritin was injected intracamerally just before killing the rabbits at postsurgical week 2, 4, 8, 12, or 24. The opposite side of the surgical site was used as the control. Histologic study was conducted under light microscopy. RESULTS: In both groups, significantly more intrascleral and episcleral drainage vessels were observed than in the paired controls (P < .001). Intrascleral drainage canals were more often manifested in the TS group than in the T group (P < .05), especially after week 12. CONCLUSION: Intrascleral and episcleral outflow were the main drainage routes after filtering surgeries in this model. Compared with trabeculectomy, the modified surgery was superior for forming an intrascleral "bleb" and seemed to be better maintained at a later stage.
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Humor Acuoso/metabolismo , Ferritinas , Esclerostomía , Trabeculectomía , Animales , Cámara Anterior/efectos de los fármacos , ConejosRESUMEN
AIM: To study the role of connective tissue growth factor (CTGF) antibody in inhibiting bleb scarring after glaucoma filtration surgery (GFS) in rabbit model. METHODS: GFS was performed on both eyes in five rabbits. One eye of each rabbit was chosen randomly as antibody group and received subconjunctival injection of 0.1mL CTGF antibody (50mg/L) immediately after GFS applied and on the 5 th day after GFS. The other eye of each rabbit as control group was received subconjunctival injection of 0.1mL PBS at the same time as antibody group. On postoperative days 1, 3, 5, 7, 10, and 14, the appearance of filtrating blebs was observed under slit lamp, the area and the intraocular pressure (IOP) were measured with micrometer and applanation tonometer, respectively. RESULTS: On postoperative days 1, 3, 5, 7, 10, and 14, areas of filtrating blebs in antibody group were all larger comparing with the control group (P<0.05) and IOPs of antibody group were lower than the control group (P<0.05). CONCLUSION: Subconjunctival injection of CTGF antibody can maintain larger bleb area and lower IOP after GFS in rabbit.
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NAP (NAPVSIPQ) is a short peptide derived from activity-dependent neuroprotective protein (ADNP) sequence, whose potent and direct neuroprotective capabilities have been widely accepted. However, due to the high risk and inconvenience of intraocular injections, NAP is difficult to be clinically administered as therapeutic agent in treating retinal diseases. Currently, stable transfection of this octapeptide into cells has not been reported, partly because of its small size and lacking of 5' signal sequence. Here, we have developed a novel NT4-NAP fusion gene by attaching the 5' nonfunctional preproregion of neurotrophin 4 (NT4) to NAP cDNA. Recombinant adeno-associated virus was established to introduce NT4-NAP construct into cultured rat retinal Müller cells (RMC), resulting in sustained high level NAP production from stable transfection. Functional analyses of RMC cells transfected with NAP revealed the remarkably reduced cytotoxicity and apoptosis of the cells under hypoxia. Furthermore, coculturing of transfected RMC-NAP cells with primary rat retinal neural cells offer marked protection to the latter against hypoxia induced cellular damages. Together our data indicate that stable transfection of NAP into retinal Müller cells with constant NAP production is possible. NAP produced from cellular transfection maintained its biological neuroprotective activities. This targeted gene expression may provide an effective treatment for retinal diseases in the near future.
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Hipoxia de la Célula , Terapia Genética , Oligopéptidos/genética , Retina/metabolismo , Enfermedades de la Retina/terapia , Neuronas Retinianas/fisiología , Animales , Proliferación Celular , Células Cultivadas , Dependovirus/genética , Fusión Génica , Factores de Crecimiento Nervioso/genética , Fármacos Neuroprotectores , Ratas , Ratas Sprague-Dawley , Retina/citología , TransfecciónRESUMEN
This project was aimed to develop a simple, convenient and reliable computer image processing software for the measurement of lens dimensions, including the radius of curvature of anterior lens surface (RCALS), the radius of curvature of posterior lens surface (RCPLS) and the lens volume (LV). On the basis of lens images captured by B ultrasound, our computer software was designed to calculate the three parameters of lens in accordance to geometry principle. This software comprises Program I and Program II, and they both possess different calculation methods. Then they were used in a group of normal volunteers who were recruited via randomization and the outcomes were compared. The results showed that the two programs were developed successfully, and the outcomes of RCALS, RCPLS and LV calculated by the two programs were similar. The standard deviation of Program II is smaller than that of Program I. The computer software for calculating RCALS, RCPLS and LV was characterized by scientific design and easy-to-use. In respect of calculating repetition, Program II was better than Program I, and it possesses prosperous prospect in terms of clinical research and application.
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Procesamiento de Imagen Asistido por Computador/métodos , Cristalino/anatomía & histología , Cristalino/diagnóstico por imagen , Refracción Ocular , Programas Informáticos , Adulto , Anciano , Femenino , Humanos , Masculino , Cómputos Matemáticos , Persona de Mediana Edad , Óptica y Fotónica , UltrasonografíaRESUMEN
OBJECTIVE: To investigate the effect of recombinant human erythropoietin (rhEPO) on the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure and explore the mechanism underlying the protective effect of rhEPO on the retina against ischemia-reperfusion injury. METHODS: rhEPO was injected subcutaneously in the ear of a rabbit model of acute high intraocular pressure induced by physiological saline perfusion into the anterior chamber. Bcl-2 protein expression in the retina of the rabbits was observed by immunohistochemical staining on days 1, 3, 7, and 14 after retinal ischemia-reperfusion and compared with that in normal rabbits and untreated rabbit models. RESULTS: bcl-2-positive cells were observed in the retina of normal rabbits with a mean positive cell number of 10.5-/+1.2 in each high-power visual field. Compared with that in the normal control group, the number of the positive cells decreased significantly in both the model group and EPO group (P<0.05, P<0.01), but the latter group showed a significantly greater number than the former (P<0.05 at day 7 and P<0.01 at day 14). CONCLUSION: Systemic administration of rhEPO can up-regulate the expression of bcl-2 protein in the retina of rabbits with acute high intraocular pressure, which is probably one of the mechanisms for the protective effect of rhEPO on the retina against ischemia-reperfusion injury.
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Eritropoyetina/uso terapéutico , Hipertensión Ocular/tratamiento farmacológico , Hipertensión Ocular/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Retina/metabolismo , Animales , Eritropoyetina/farmacología , Femenino , Humanos , Masculino , Conejos , Distribución Aleatoria , Proteínas RecombinantesRESUMEN
OBJECTIVE: Retinopathy of prematurity is becoming obvious with the improvement of neonatal ambulance. However there is still not a good treatment. The present study is to observe the effect of 17 beta-estradiol (E2) on oxygen-induced retinopathy (OIR), and explore the relationship between the changes of avascular area and malondialdehyde (MDA) in retina. METHODS: Newborn oxygen-exposed mice underwent subcutaneous injections of different dose of E2 (0.1 µg, 1.0 µg, 10.0 µg ), tamoxifen or phosphate buffered saline (PBS; controls)everyday from post-natal day (p)7 to p17. At p17, retinal flat mounts were scored for the percentage of avascular/total retinal area, and pathological changes during revascularization. The MDA concentration in the retina was determined also. In the most efficacious E2 group (10.0 µg), 100.0 µg tamoxifen was also administered, and the percentage of capillary-free/total retinal area determined, and the retinal malondialdehyde concentration assayed. RESULTS: The mean percentage of capillary-free area over total retinal area was 0(PBS, in room air), 34.197±1.301(PBS, in hyperoxia), 23.685±0.407 (0.1 µg E2), 14.648±0.355 (1.0 µg E2), 4.693±0.450 (10.0 µg E2) and 32.240±0.654 (10.0 µg E2 +100.0 µg tamoxifen). The difference was significant (F = 2778.759, P < 0.01), and the difference between any two groups were also significant (all P value were less than 0.01). The predilection of tufts and clusters during revascularization was mainly aggregated in zones 2 and 3, but the difference of retinal neovascular clusters and tufts in fourth zone among different groups were significant [clusters (F = 44.719, P < 0.01) vs tufts (F = 39.997, P < 0.01)]. The mean MDA concentration were 0.711±0.037(PBS, in room air), 2.084±0.066 (PBS, in hyperoxia), 1.829±0.091(0.1 µg E2), 1.152±0.067(1.0 µg E2), 0.796±0.027(10.0 µg E2), 1.988±0.049(10.0 µg E2 +100.0 µg tamoxifen) (F = 628.103, P < 0.01). The difference between any two groups were also significant (all P value were less than 0.05). The close relation between the percentage of avascular/total retinal area and MDA concentration was also verified (r = 0.981, P < 0.01). CONCLUSION: Oxidative stress responses play a pivotal role in OIR, by means of receptor pathway. E2 can alleviate oxidative stress reaction, and thus ameliorate the severity of oxygen induced retinopathy.
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AIM: To investigate the effect of lentivirus-mediated integrin-linked kinase (ILK) RNA interference (RNAi) on human retinal Müller cells transdifferentiation into contractile myofibroblasts. METHODS: A lentiviral vector expressing ILK-specific shRNA was constructed and introduced into cultured retinal Müller cells. Silencing of the ILK gene was identified by real time RT-PCR and Western blot. The Müller cell phenotype change was confirmed by immunodetection of alpha-smooth muscle actin (alpha-SMA) stress fiber formation. The generation of tractional force was assessed using a tissue culture assay with cells incubated in three-dimensional collagen gels; cell migration was determined by the Boyden chamber method, using 10% FBS as a chemotactic factor. RESULTS: Significant decreases in ILK mRNA and protein expression were detected in Müller cells carrying lentiviral ILK-shRNA vector. Cells treated with anti-ILK siRNA showed less alpha-SMA stress fiber formation under hypoxic conditions or cell subcultivation. Lentiviral ILK-shRNA vector transfection also significantly reduced cell migration and cell-mediated gel contraction. CONCLUSION: Lentivirus-mediated ILK RNAi decreased cell migration and contractile force generation by inhibiting alpha-SMA stress fiber formation in human retinal Müller cells. This tool might be useful to treat ocular fibroproliferative diseases associated with transdifferentiated Müller cells.
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Diferenciación Celular , Fibroblastos/fisiología , Neuroglía/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Retina/citología , Fibras de Estrés/metabolismo , Actinas/metabolismo , Movimiento Celular , Células Cultivadas , Regulación hacia Abajo , Fibroblastos/patología , Vectores Genéticos , Humanos , Lentivirus , Músculo Liso/metabolismo , Neuroglía/citología , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARNRESUMEN
OBJECTIVE: To observe the changes in the expression of brain derived neurotrophic factor (BDNF) gene in the retina of rabbits with acute high intraocular pressure (IOP) after injection of recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-hBDNF), and investigate the neuroprotective mechanism of rAAV-hBDNF. METHODS: The unilateral eyes of 24 white rabbits were randomly chosen as the model group with high IOP induced by saline perfusion into the anterior chamber, and the contralateral eyes served as the control group without treatment. In another 24 white rabbits, 10 microl rAAV-BDNF was injected into the vitreous body of one of the eyes 3 days before induction of high IOP. On days 1, 3, 7, and 14 after perfusion, the bilateral eyes of 6 rabbits were excised for immunohistochemistry for the expression of endogenous BDNF gene in the retina. RESULTS: The number of BDNF-positive cells in the retina decreased after induction of high IOP, and injection of rAAV-hBDNF resulted in a significant increase in BDNF-positive cells as compared with the positive cell number in the high IOP model and control groups (P<0.05, P<0.01). CONCLUSION: rAAV-mediated BDNF gene transfection can increase endogenous BDNF expression in the retina of rabbits with acute high IOP. Intravitreous injection is an effective pathway for rAAV-hBDNF gene transfection into the retina.
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Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Dependovirus/genética , Hipertensión Ocular/metabolismo , Retina/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Factor Neurotrófico Derivado del Encéfalo/genética , Dependovirus/metabolismo , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Conejos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , TransfecciónRESUMEN
OBJECTIVE: To investigate the neuroprotective effect of human brain-derived neurotrophic factor gene transfection into rabbit retina against acute high intraocular pressure (HIOP). METHODS: Acute HIPO was induced in one eye of 24 white rabbits via saline perfusion into the anterior chamber (model group), and the contralateral eye without treatment served as the control group. In another 24 rabbits, 10 microl recombinant adeno-associated virus (rAAV) vector containing human BDNF gene (rAAV-BDNF) was injected into the vitreous body of one of the eyes 3 days before the operation for HIPO (BDNF group). At 1, 3, 7, and 14 days after HIOP model establishment, 6 eyes in each group were excised to observe the number of retinal ganglion cells (RGCs) and the thickness of the inner retina layer. For the eyes dissected on day 14, electroretinogram b (ERG-b) wave was detected 30 min before (baseline) and on days 1, 3, 7 and 14 after HIOP. Another 5 rabbits were used for ultrastructural observation of the RGCs using transmission electron microscopy, including 1 without treatment, 2 with unilateral HIOP and 2 with rAAV-BDNF transfection before HIOP. RESULTS: The amplitude of ERG-b wave showed no significant difference between the 3 groups before HIOP (P>0.05). In HIOP model group and BDNF group, the amplitude decreased to the lowest at 1 day after HIOP and failed to recover the baseline level at 14 days (P<0.01); at the end of the observation, the amplitude was significantly higher in BDNF group than in the model group (P<0.01). Decreased number of RGCs and thickness of inner retina layer occurred in the model group, but these changes were milder in BDNF group (P<0.05, P<0.01). Electron microscopy revealed ultrastructural changes in the RGCs following acute HIOP, and transfection with rAAV-BDNF ameliorated these changes. CONCLUSION: rAAV-BDNF transfection protects the retinal structure and improves the amplitude of ERG-b wave after acute high IOP suggesting its neuroprotective effects.
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Factor Neurotrófico Derivado del Encéfalo/genética , Dependovirus/genética , Hipertensión Ocular/terapia , Enfermedades de la Retina/prevención & control , Transfección , Animales , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Dependovirus/metabolismo , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Hipertensión Ocular/complicaciones , Conejos , Retina/patología , Enfermedades de la Retina/etiologíaRESUMEN
PURPOSE: The purpose of this study is to assess the change of uveoscleral outflow induced by moxonidine and to investigate whether the increase of uveoscleral outflow induced by moxonidine is mediated by alpha1, alpha2, or I1 receptors. METHODS: 0.05% moxonidine was topically and unilaterally administered in rabbit eyes with or without pretreatment of prazosin, yohimbine, efaroxan, or AGN 192403, as indicated. We injected fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) into the anterior chamber and observed the fluorescence intensity of the uveoscleral outflow. Finally, the volume of uveoscleral outflow was calculated based on the fluorescence intensities captured. RESULTS: A bilateral increase of fluorescence intensity was observed along the uveoscleral outflow pathway following moxonidine administration, especially in the ciliary body and supraciliochoroidal space. Pretreatment with prazosin further enhanced the bilateral increase of fluorescence intensity at between 2 and 4 hours after moxonidine administration. The response of moxonidine was antagonized by either yohimbine, an alpha2 receptor antagonist, or efaroxan, an I1/alpha2 receptor antagonist. The antagonizing effect of yohimbine was more potent than that of efaroxan. The moxonidne-induced response was not antagonized by AGN 192403, an I1 receptor antagonist. The bilateral volumes of aqueous humor within the uveoscleral pathway increased significantly induced by moxonidine (p < 0.01 versus control). The increased bilateral volumes of uveoscleral outflow were 0.381 +/- 0.073 and 0.376 +/- 0.095 mu l/min, respectively. CONCLUSION: These results suggest that topical, unilateral administration of moxonidine causes a bilateral increase of aqueous humor via the uveoscleral outflow pathway. The moxonidine-induced increase of uveoscleral outflow is mediated by alpha2 adrenergic receptors, not by I1 imidazoline receptors.
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Humor Acuoso/metabolismo , Imidazoles/farmacología , Receptores de Imidazolina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Esclerótica/metabolismo , Úvea/metabolismo , Administración Tópica , Antagonistas Adrenérgicos alfa/farmacología , Animales , Humor Acuoso/efectos de los fármacos , Benzofuranos/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Sinergismo Farmacológico , Fluoresceína-5-Isotiocianato/análogos & derivados , Heptanos/farmacología , Imidazoles/administración & dosificación , Receptores de Imidazolina/antagonistas & inhibidores , Prazosina/farmacología , Conejos , Esclerótica/efectos de los fármacos , Albúmina Sérica Bovina , Úvea/efectos de los fármacos , Yohimbina/farmacologíaRESUMEN
AIM: The aim of the study was to evaluate the outcome of adenovirus-mediated p27(KIP1) (Ad-p27) expression on wound healing after filtration surgery and to investigate the inhibition of cell proliferation induced by Ad-p27. METHODS: We constructed the adenovirus recombinant vector Ad-p27 and administered it to a rabbit model of glaucoma filtration surgery by subconjunctival injection; phosphate-buffered saline (PBS) and mitomycin C (MMC) were used as controls. Intraocular pressure (IOP), bleb scores, and anterior chamber depths were observed during a 28-d period. Histological examinations, fluorescence observations and Western blot analyses were evaluated. RESULTS: Ad-p27 enhanced the surgical outcome and inhibited cell proliferation when compared with PBS. Bleb scores in the Ad-p27-treated eyes were higher than those in the PBS-treated eyes on d 7 (P<0.01), 14 (P<0.01) and 21 (P<0.05). On d 28, IOP remained significantly decreased in the Ad-p27 group compared with the PBS group (P<0.05). However, no differences in bleb scores or IOPs were observed between the Ad-p27 and MMC groups. Histological analysis showed that total cell numbers were markedly reduced, and less scar tissue was observed at the surgical site in eyes treated with Ad-p27. The number of fibroblasts was decreased in Tenon's capsule in Ad-p27-treated eyes; however, a marked and diffuse signal from the green fluorescent protein (GFP) was observed in fibroblasts. Western blot analysis revealed a high level of p27(KIP1) expression in conjunctival epithelium (P<0.01), relatively high expression in superficial scleral stroma (P<0.01), and low expression in corneal epithelium in the Ad-p27 group. CONCLUSIONS: Ad-p27 administration significantly improves the outcome of filtration surgery and inhibits postoperative proliferation in rabbit eyes. These findings suggest that p27(KIP1) is a potential adjunctive agent for inhibition of wound healing after filtration surgery.
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Adenoviridae/genética , Cirugía Filtrante/efectos adversos , Terapia Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Cicatrización de Heridas , Animales , Western Blotting , Línea Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Presión Intraocular , Microscopía Electrónica de Transmisión , Mitomicina/farmacología , ConejosRESUMEN
OBJECTIVE: To observe the effect of recombinant human erythropoietin (rhEPO) on the expression of hypoxia inducible factor-1alpha (HIF-1alpha) in the retina of rabbits with acute high intraocular pressure and investigate the mechanism of rhEPO in protecting the retina from ischemia-reperfusion injury. METHODS: Acute high intraocular pressure was induced in the rabbits by perfusion of normal saline into the anterior chamber, and rhEPO was injected subcutaneously. The changes in HIF-1alpha protein expression in the retina was observed by immunohistochemistry on days 1, 3, 7, and 14 after retinal ischemia- reperfusion. RESULTS: HIF-1alpha expression was not observed in the retina of the normal control rats, but intense HIF-1alpha expression was found in the model group (P<0.01). In rabbits with rhEPO injection and those in the model group, the patterns of HIF-1alpha expression alterations were similar, but the HIF-1alpha-positive cells in the retina were significantly fewer in rhEPO group (P<0.05). CONCLUSION: rhEPO can down-regulate HIF-1alpha expression in the retina of rabbits with acute high intraocular pressure, which may be one of the mechanisms that rhEPO protects the retina from ischemia-reperfusion injury.
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Eritropoyetina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipertensión Ocular/metabolismo , Daño por Reperfusión/prevención & control , Retina/metabolismo , Animales , Regulación hacia Abajo , Humanos , Fármacos Neuroprotectores/farmacología , Conejos , Proteínas Recombinantes , Vasos Retinianos/metabolismoRESUMEN
AIM: To determine the release characteristics of a 5-fluorouracil-loaded poly (lactic acid) disc (5-FU-PLA-DS) and the effect of sustained drug delivery on the success of glaucoma filtration surgery in rabbit eyes. METHODS: A method of microspheres accumulated by excessive carriers was used in the preparation of the 5-FU-PLA-DS. The disc was characterized for drug loading, entrapment efficiency, in vitro release, and external morphology. It was then implanted subconjunctivally into rabbit eyes with trabeculectomy. Intraocular pressure, ocular inflammatory reaction, filtration bleb appearance, and persistence were evaluated up to postoperative d 90. A quantitative analysis of 5-fluorouracil (5-FU) was performed in the aqueous humor. Ultrasound biomicroscopy was used to assess the appearance of the filtering fistula. RESULTS: The 5-FU-PLA-DS was produced with the drug-loading of 3.07+/-0.08 mg (mean+/-SD). 5-FU was released for 91 d with suppressive concentrations. The decrease in intraocular pressure from baseline was significantly more marked in the 5-FU-PLA-DSimplanted eyes during postoperative d 3-90, and the persistence of bleb and filtration fistula was longer than the control eyes (P<0.05). Corneal toxicity and hyperemia triggered by 5-FU was lower in the 5-FU-PLA-DS-implanted eyes than those exposed to 5-FU intraoperatively. The 5-FU concentration in the aqueous humor was insufficient for corneal endothelial damage. No evidence of toxic reaction was found in the conjunctival biopsy. CONCLUSION: 5-FU-PLADS displaying sustained intraocular release of 5-FU, reduced intraocular pressure, and prolonged bleb persistence, while significantly reducing 5-FU toxicity.