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BACKGROUND: To develop and validate a nomogram model based on Gd-EOB-DTPA enhanced MRI for differentiation between hepatocellular carcinoma (HCC) and focal nodular hyperplasia (FNH) showing iso- or hyperintensity in the hepatobiliary phase (HBP). METHODS: A total of 75 patients with 49 HCCs and 26 FNHs randomly divided into a training cohort (n = 52: 34 HCC; 18 FNH) and an internal validation cohort (n = 23: 15 HCC; 8 FNH). A total of 37 patients (n = 37: 25 HCC; 12 FNH) acted as an external test cohort. The clinical and imaging characteristics between HCC and FNH groups in the training cohort were compared. The statistically significant parameters were included into the FAE software, and a multivariate logistic regression classifier was used to identify independent predictors and establish a nomogram model. Receiver operating characteristic (ROC) curves were used to evaluate the prediction ability of the model, while the calibration and decision curves were used for model validation. Subanalysis was used to compare qualitative and quantitative characteristics of patients with chronic hepatitis and cirrhosis between the HCC and FNH groups. RESULTS: In the training cohort, gender, age, enhancement rate in the arterial phase (AP), focal defects in uptake were significant predictors for HCC showing iso- or hyperintensity in the HBP. In the training cohort, area under the curve (AUC), sensitivity and specificity of the nomogram model were 0.989(95%CI: 0.967-1.000), 97.1% and 94.4%. In the internal validation cohort, the above three indicators were 0.917(95%CI: 0.782-1.000), 93.3% and 87.5%. In the external test cohort, the above three indicators were 0.960(95%CI: 0.905-1.000), 84.0% and 100.0%. The results of subanalysis showed that age was the independent predictor in the patients with chronic hepatitis and cirrhosis between HCC and FNH groups. CONCLUSIONS: Gd-EOB-DTPA enhanced MRI nomogram model may be useful for discriminating HCC and FNH showing iso- or hyperintensity in the HBP before surgery.
Assuntos
Carcinoma Hepatocelular , Meios de Contraste , Hiperplasia Nodular Focal do Fígado , Gadolínio DTPA , Neoplasias Hepáticas , Imageamento por Ressonância Magnética , Nomogramas , Humanos , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Feminino , Masculino , Hiperplasia Nodular Focal do Fígado/diagnóstico por imagem , Pessoa de Meia-Idade , Imageamento por Ressonância Magnética/métodos , Diagnóstico Diferencial , Adulto , Idoso , Estudos Retrospectivos , Curva ROCRESUMO
Two rearranged norditerpenoids with novel tricyclic carbon skeletons, strophiofimbrin A (1) and strophiofimbrin B (2), were isolated from Strophioblachia fimbricalyx. Their structures were established by 1D/2D NMR spectroscopy, HRESIMS, quantum chemistry calculations, and X-ray diffraction analyses. 1 and 2 represented the first examples of diterpenoids with unprecedented 5/6/7-fused ring systems. In the proposed biosynthetic pathway, they were suspected to derive from cleistanthane norditerpenoids via ring opening, expansion, cyclization, and rearrangement based on the existence of phenanthrenone and cleistanthane diterpenoids from Strophioblachia and Trigonostemon, two closely related genera of the Euphorbiaceae family. Furthermore, compounds 1 and 2 exhibited significant proliferation inhibition and obvious neuroprotective effects.
Assuntos
Diterpenos , Euphorbiaceae , Estrutura Molecular , Carbono/química , Diterpenos/farmacologia , Diterpenos/química , Espectroscopia de Ressonância Magnética , Euphorbiaceae/químicaRESUMO
An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.
Assuntos
Modelos Animais de Doenças , Degeneração Macular/genética , Estresse Oxidativo , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Superóxido Dismutase/genética , Animais , Bestrofinas , Eletrorretinografia , Proteínas do Olho/genética , Feminino , Humanos , Imuno-Histoquímica , Canais Iônicos/genética , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Oftalmoscópios , Oftalmoscopia/métodos , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/fisiopatologia , Superóxido Dismutase/deficiência , Superóxido Dismutase/metabolismo , Tomografia de Coerência ÓpticaRESUMO
Chronic oxidative stress contributes to age related diseases including age related macular degeneration (AMD). Earlier work showed that the 5-hydroxy-tryptamine 1a (5HT1a) receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) protects retinal pigment epithelium (RPE) cells from hydrogen peroxide treatment and mouse retinas from oxidative insults including light injury. In our current experiments, RPE derived cells subjected to mitochondrial oxidative stress were protected from cell death by the up-regulation of anti-oxidant enzymes and of the metal ion chaperone metallothionein. Differentiated RPE cells were resistant to oxidative stress, and the expression of genes for protective proteins was highly increased by oxidative stress plus drug treatment. In mice treated with 8-OH-DPAT, the same genes (MT1, HO1, NqO1, Cat, Sod1) were induced in the neural retina, but the drug did not affect the expression of Sod2, the gene for manganese superoxide dismutase. We used a mouse strain deleted for Sod2 in the RPE to accelerate age-related oxidative stress in the retina and to test the impact of 8-OH-DPAT on the photoreceptor and RPE degeneration developed in these mice. Treatment of mice with daily injections of the drug led to increased electroretinogram (ERG) amplitudes in dark-adapted mice and to a slight improvement in visual acuity. Most strikingly, in mice treated with a high dose of the drug (5 mg/kg) the structure of the RPE and Bruch's membrane and the normal architecture of photoreceptor outer segments were preserved. These results suggest that systemic treatment with this class of drugs may be useful in preventing geographic atrophy, the advanced form of dry AMD, which is characterized by RPE degeneration.
Assuntos
8-Hidroxi-2-(di-n-propilamino)tetralina/uso terapêutico , Mitocôndrias/metabolismo , Estresse Oxidativo , Retina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/uso terapêutico , Animais , Linhagem Celular , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Metalotioneína/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor 5-HT1A de Serotonina/metabolismo , Retina/metabolismo , Retina/fisiopatologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Superóxido Dismutase/genética , Tomografia de Coerência Óptica , Acuidade Visual/efeitos dos fármacosRESUMO
A group of phenanthrene derivatives with different deformed types, including four previously undescribed derivatives (1-4), an undescribed natural product (5) and five known compounds (6-10), were isolated from the leaves and stems of Strophioblachia fimbricalyx by molecular networking based on UPLC-MS/MS method. Their structures were established by 1D/2D NMR spectroscopy, HRESIMS, quantum chemistry calculation, and single crystal X-ray diffraction. In biogenic pathways, series of deformed phenanthrenes were all suspected to be derived from 6/6/6 tricyclic phenanthrenes with a gem-dimethyl unit in one ring as characteristic components of Strophioblachia. Fimbricalyxone (1) and trigoxyphin M (6) with a 6/6/5 tricyclic carbon skeleton were reported for the first time from the genus and fimbricalyxanhydride C (2) is the first example of anhydride type bearing a rare 8,9-oxycycle. All the isolates were evaluated for their cytotoxic activity against three tumor cell lines, and compounds 8 and 10 exhibited significant activity with IC50 values of 4.65-9.02 µM, and the structure-activity relationship of the deformed phenanthrenes was discussed. In addition, the X-ray structure of 8 and 10 and the antineoplastic activity of 10 are reported herein for the first time. Trigohowilol G (10) inhibiting the proliferation of A549 cells might be related to cell cycle distribution and the induction of S phase arrest, and it induced cell apoptosis through Bad/Bax/Cleaved PARP1 pathway.
Assuntos
Antineoplásicos Fitogênicos , Antineoplásicos , Fenantrenos , Estrutura Molecular , Antineoplásicos Fitogênicos/química , Fenantrenos/química , Cromatografia Líquida , Espectrometria de Massas em Tandem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , ApoptoseRESUMO
A kinematics analysis of a new hybrid mechanical leg suitable for bipedal robots was carried out and the gait of the robot walking on flat ground was planned. Firstly, the kinematics of the hybrid mechanical leg were analyzed and the applicable relevant models were established. Secondly, based on the preliminary motion requirements, the inverted pendulum model was used to divide the robot walking into three stages for gait planning: mid-step, start and stop. In the three stages of robot walking, the forward and lateral robot centroid motion trajectories and the swinging leg joint trajectories were calculated. Finally, dynamic simulation software was used to simulate the virtual prototype of the robot, achieving its stable walking on flat ground in the virtual environment, and verifying the feasibility of the mechanism design and gait planning. This study provides a reference for the gait planning of hybrid mechanical legged bipedal robots and lays the foundation for further research on the robots involved in this thesis.
RESUMO
In recent years, significant progress has been made in employing reinforcement learning for controlling legged robots. However, a major challenge arises with quadruped robots due to their continuous states and vast action space, making optimal control using simple reinforcement learning controllers particularly challenging. This paper introduces a hierarchical reinforcement learning framework based on the Deep Deterministic Policy Gradient (DDPG) algorithm to achieve optimal motion control for quadruped robots. The framework consists of a high-level planner responsible for generating ideal motion parameters, a low-level controller using model predictive control (MPC), and a trajectory generator. The agents within the high-level planner are trained to provide the ideal motion parameters for the low-level controller. The low-level controller uses MPC and PD controllers to generate the foot-end force and calculates the joint motor torque through inverse kinematics. The simulation results show that the motion performance of the trained hierarchical framework is superior to that obtained using only the DDPG method.
RESUMO
N-glycosylation has been revealed to be tightly associated with cancer metastasis. As a key transferase that catalyzes the formation of ß1,4 N-acetylglucosamine (ß1,4GlcNAc) branches on the mannose core of N-glycans, N-acetylglucosaminyltransferase IVa (GnT-IVa) has been reported to be involved in hepatocellular carcinoma (HCC) metastasis by forming N-glycans; however, the underlying mechanisms are largely unknown. In the current study, we found that GnT-IVa was upregulated in HCC tissues and positively correlated with worse outcomes in HCC patients. We found that GnT-IVa could promote tumor growth in mice; notably, this effect was attenuated after mutating the enzymatic site (D445A) of GnT-IVa, suggesting that GnT-IVa regulated HCC progression by forming ß1,4GlcNAc branches. To mechanistically investigate the role of GnT-IVa in HCC, we conducted GSEA and GO functional analysis as well as in vitro experiments. The results showed that GnT-IVa could enhance HCC cell migration, invasion and adhesion ability and increase ß1,4GlcNAc branch glycans on integrin ß1 (ITGB1), a tumor-associated glycoprotein that is closely involved in cell motility by interacting with vimentin. Interruption of ß1,4GlcNAc branch glycan modification on ITGB1 could suppress the interaction of ITGB1 with vimentin and inhibit cell motility. These results revealed that GnT-IVa could promote HCC cell motility by affecting the biological functions of ITGB1 through N-glycosylation. In summary, our results revealed that GnT-IVa is highly expressed in HCC and can form ß1,4GlcNAc branches on ITGB1, which are essential for interactions with vimentin to promote HCC cell motility. These findings not only proposed a novel mechanism for GnT-IVa in HCC progression but also revealed the significance of N-glycosylation on ITGB1 during the process, which may provide a novel target for future HCC therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , N-Acetilglucosaminiltransferases , Animais , Camundongos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Glicosilação , Integrina beta1/genética , Integrina beta1/metabolismo , Neoplasias Hepáticas/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Vimentina/genética , Vimentina/metabolismo , HumanosRESUMO
Gene therapy for dominantly inherited genetic disease is more difficult than gene-based therapy for recessive disorders, which can be treated with gene supplementation. Treatment of dominant disease may require gene supplementation partnered with suppression of the expression of the mutant gene either at the DNA level, by gene repair, or at the RNA level by RNA interference or transcriptional repression. In this review, we examine some of the gene delivery approaches used to treat animal models of autosomal dominant retinitis pigmentosa, focusing on those models associated with mutations in the gene for rhodopsin. We conclude that combinatorial approaches have the greatest promise for success.
Assuntos
Terapia Genética , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Rodopsina/genética , Adenoviridae/genética , Animais , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Transferência de Genes , Genes Dominantes , Vetores Genéticos , Humanos , Camundongos , Mutação , Interferência de RNA , Retinose Pigmentar/patologia , Transcrição GênicaRESUMO
Oxidative stress in the retinal pigment epithelium (RPE) is hypothesized to be a major contributor to the development of age-related macular degeneration (AMD). Mitochondrial manganese superoxide dismutase (MnSOD) is a critical antioxidant protein that scavenges the highly reactive superoxide radical. We speculated that specific reduction of MnSOD in the RPE will increase the level of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to geographic atrophy. To test this hypothesis, an Sod2-specific hammerhead ribozyme (Rz), delivered by AAV2/1 and driven by the human VMD2 promoter was injected subretinally into C57BL/6J mice. Dark-adapted full field electroretinogram (ERG) detected a decrease in the response to light. We investigated the age-dependent phenotypic and morphological changes of the outer retina using digital fundus imaging and SD-OCT measurement of ONL thickness. Fundus microscopy revealed pigmentary abnormalities in the retina and these corresponded to sub-retinal and sub-RPE deposits seen in SD-OCT B-scans. Light and electron microscopy documented the localization of apical deposits and thickening of the RPE. In RPE flat-mounts we observed abnormally displaced nuclei and regions of apparent fibrosis in the central retina of the oldest mice. This region was surrounded by enlarged and irregular RPE cells that have been observed in eyes donated by AMD patients and in other mouse models of AMD.
Assuntos
Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica/fisiologia , Atrofia Geográfica/patologia , Mitocôndrias/enzimologia , Estresse Oxidativo , Epitélio Pigmentado da Retina/ultraestrutura , Superóxido Dismutase/genética , Animais , Dependovirus/genética , Eletrorretinografia , Angiofluoresceinografia , Inativação Gênica/fisiologia , Vetores Genéticos , Atrofia Geográfica/enzimologia , Atrofia Geográfica/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Catalítico/genética , Epitélio Pigmentado da Retina/enzimologia , Tomografia de Coerência ÓpticaRESUMO
Genomic selection in plants and animals has become a standard tool for breeding because of the advantages of high accuracy and short generation intervals. Implementation of this technology is hindered by the high cost of genotyping and other factors. The aim of this study was to determine an optional marker density panel and reference population size for using genomic selection of goats, with speculation on the number of QTLs that affect the important economic traits of goats. In addition, the effect of buck population size in the reference population on the accuracy of genomic estimated breeding value (GEBV) was discussed. Based on the previous genetic evaluation results of Inner Mongolia White Cashmere Goats, live body weight (LBW, h 2 = 0.11) and fiber diameter (FD, h 2 = 0.34) were chosen to perform genomic selection in this study. Reasonable genome parameters and generation transmission processes were set, and phenotypic and genotype data of the two traits were simulated. Then, different sizes of the reference population and validation population were selected from progeny. The GEBVs were obtained by six methods, including GBLUP (Genomic Best Linear Unbiased Prediction), ssGBLUP (Single Step Genomic Best Linear Unbiased Prediction), BayesA, BayesB, Bayesian ridge regression, and Bayesian LASSO. The correlation coefficient between the predicted and realized phenotypes from simulation was calculated and used as a measure of the accuracy of GEBV in each trait. The results showed that the medium marker density Panel (45 K) could be used for genomic selection in goats, which can ensure the accuracy of the GEBV. The reference population size of 1,500 can achieve greater genetic progress in genomic selection for fiber diameter and live body weight in goats by comparing with the population size below this level. The accuracy of the GEBV for live body weight and fiber diameter was better when the number of QTLs was 100 and 50, respectively. Additionally, the accuracy of GEBV was discovered to be good when the buck population size was up to 200. Meanwhile, the accuracy of the GEBV for medium heritability traits (FDs) was found to be higher than the accuracy of the GEBV for low heritability traits (LBWs). These findings will provide theoretical guidance for genomic selection in goats by using real data.
RESUMO
Coriolus versicolor is an edible medicinal mushroom in China. Two polysaccharides, named as CVPn and CVPa were separated from the dried fruiting bodies of Coriolus versicolor by water extraction and ethanol precipitation. Their chemical structures were well elucidated with overall consideration of monosaccharide composition, methylation analysis and 1D/2D-NMR spectra data. The bioactivities on RAW 264.7 macrophages cells were evaluated, and further structure-bioactivity relationships were concluded. With molecular weight of 29.7 kDa for CVPn and 50.8 kDa for CVPa, the two isolated polysaccharides were both composed of (l â 4)-ß-/(1 â 3)-ß-d-glucopyranosyl group as backbone with branches attached at O-6 site. Comparing to CVPn, CVPa with relative high molecular weight and less branches showed significant induction of NO production, obvious augmentation of iNOS and TNF-α mRNA expression level, and phagocytosis on RAW 264.7 cells. These results clarified that CVP polysaccharides with less branches and high molecular weight possessed enhanced immunomodulatory ability, and this finding could be a reference for the utilization of Coriolus versicolor.
Assuntos
Polissacarídeos Fúngicos/química , Fatores Imunológicos/química , Polyporaceae/química , Animais , Carpóforos/química , Polissacarídeos Fúngicos/farmacologia , Fatores Imunológicos/farmacologia , Camundongos , Fagocitose/efeitos dos fármacos , Células RAW 264.7RESUMO
Exposure to pesticides has been speculated to contribute to the development of sporadic Parkinson's disease (PD) characterized by a progressive degeneration of the nigrostriatal dopaminergic pathway. Activation of brain microglia that produce various neurotoxic factors including cytokines and reactive oxygen species (ROS) has been increasingly associated with dopaminergic neurodegeneration induced by various toxicants. Dieldrin, a highly persistent organochlorinated pesticide found enriched in the substantia nigra of some postmortem PD brains, has been shown to be toxic to dopamine neurons. In this study, we set out to determine the effect of dieldrin on the production of ROS and the underlying mechanism of action in murine microglia. Treatment of microglial cells with 0.1 nM to 1 microM dieldrin for 24 h resulted in a concentration-dependent generation of ROS. The dieldrin-induced microglial ROS generation was time-dependent in that significant ROS production was observed in cells 12-24 h, but not 6 h after dieldrin treatment. Furthermore, the dieldrin-induced microglial ROS generation was significantly reduced by inhibitors of NADPH oxidase, gene transcription and protein synthesis. In addition to immortalized microglial cells, dieldrin induced a concentration-dependent ROS generation in primary microglia, but not in primary astroglia. These results demonstrate that nanomolar concentrations of dieldrin can stimulate microglia to produce ROS that may contribute to the degeneration of dopamine neurons known to be vulnerable to oxidative damage. These findings provide important information on the potential role of microglia in dieldrin-induced neurodegeneration in relevance to the development of idiopathic PD.
Assuntos
Dieldrin/toxicidade , Microglia/metabolismo , Praguicidas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Dieldrin/administração & dosagem , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Poluentes Ambientais/administração & dosagem , Poluentes Ambientais/toxicidade , Hidrocarbonetos Clorados/administração & dosagem , Hidrocarbonetos Clorados/toxicidade , Camundongos , Microglia/efeitos dos fármacos , NADPH Oxidases/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Neurônios/metabolismo , Ratos , Ratos Endogâmicos F344 , Fatores de TempoAssuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Retinose Pigmentar/genética , Retinose Pigmentar/terapia , Rodopsina/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Genes Dominantes/genética , Camundongos , Periferinas , Células Fotorreceptoras de Vertebrados/fisiologiaRESUMO
Mutations in the gene for rhodopsin, RHO, cause autosomal dominant retinitis pigmentosa, a disease characterized by death of rod photoreceptor cells. At the end stage, when most rods are gone, cones die too, taking central vision with them. One goal of gene therapy, therefore, is to preserve central vision by promoting rod survival in the vicinity of the macula. Dominance in RHO mutations is associated with two phenomena: interference with the function of normal rhodopsin and intrinsic toxicity of the mutant protein. In the case of interference, increased production of the wild-type protein may be therapeutic, but in the case of toxicity, suppression of the mutant protein may also be needed. RHO augmentation has made use of advances in gene delivery to the retina using adeno-associated virus (AAV). Several strategies have been developed for suppression of rhodopsin expression, but because of the heterogeneity of RHO mutations they are not specific for the mutant allele: They suppress both mutant and wild-type RHO. Experiments in autosomal dominant retinitis pigmentosa (adRP) mouse models suggest that both RHO augmentation and supplementation plus suppression preserve the survival of rod cells.
Assuntos
Proteínas de Fase Aguda/genética , DNA/genética , Mutação , Retinose Pigmentar/genética , Rodopsina/genética , Proteínas de Fase Aguda/metabolismo , Animais , Genes Dominantes , Vetores Genéticos , Humanos , Fenótipo , Retinose Pigmentar/metabolismo , Rodopsina/metabolismoRESUMO
PURPOSE: Oxidative stress in the RPE is widely accepted as a contributing factor to AMD. We have previously shown that ribozyme-mediated reduction in the antioxidant enzyme manganese superoxide dismutase (MnSOD) leads to some of the features of geographic atrophy in mice. To develop a mouse model independent of viral injection, we used a conditional knockout of the Sod2 gene in the RPE to elevate mitochondrial oxidative stress in that cell layer. METHODS: Experimental mice in which exon 3 of Sod2 was flanked by loxP sites were also transgenic for PVMD2-rtTA and tetO-PhCMV cre, so that cre recombinase was expressed only in the RPE. Pups of this genotype (Sod2(flox/flox)VMD2cre) were induced to express cre recombinase by feeding doxycycline-laced chow to nursing dams. Controls included mice of this genotype not treated with doxycycline and doxycycline-treated Sod2(flox/flox) mice lacking the cre transgene. Expression of cre in the RPE was verified by immunohistochemistry, and deletion of Sod2 exon 3 in the RPE was confirmed by PCR. Mice were followed up over a period of 9 months by spectral-domain optical coherence tomography (SD-OCT), digital fundus imaging, and full-field ERG. Following euthanasia, retinas were examined by light and electron microscopy or by immunohistochemistry. Contour length of rod outer segments and thickness of the RPE layer were measured by unbiased stereology. RESULTS: Following doxycycline induction of cre, Sod2(flox/flox) cre mice demonstrated increased signs of oxidative stress in the RPE and accumulation of autofluorescent material by age 2 months. They showed a gradual decline in the ERG response and thinning of the outer nuclear layer (by SD-OCT), which were statistically significant by 6 months. In addition, OCT and electron microscopy revealed increased porosity of the choroid. At the same interval, hypopigmented foci appeared in fundus micrographs, and vascular abnormalities were detected by fluorescein angiography. By 9 months, the RPE layer in Sod2(flox/flox) cre mice was thicker than in nontransgenic littermates, and the rod outer segments were significantly longer over most of the retina, although localized atrophy of photoreceptors was also obvious in some eyes. CONCLUSIONS: Conditional tissue-specific reduction in MnSOD induced oxidative stress in mouse RPE, leading to RPE dysfunction, damage to the choroid, and death of photoreceptor cells. The RPE oxidative stress did not cause drusen-like deposits, but the model recapitulated certain key aspects of the pathology of dry AMD and may be useful in testing therapies.
Assuntos
Modelos Animais de Doenças , Atrofia Geográfica/etiologia , Mitocôndrias/metabolismo , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Doxiciclina/toxicidade , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Feminino , Angiofluoresceinografia , Deleção de Genes , Expressão Gênica/efeitos dos fármacos , Atrofia Geográfica/metabolismo , Atrofia Geográfica/patologia , Integrases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Epitélio Pigmentado da Retina/ultraestrutura , Segmento Externo da Célula Bastonete/ultraestrutura , Superóxido Dismutase/genética , Tomografia de Coerência ÓpticaRESUMO
Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.
Assuntos
Autofagia , Degeneração Macular/patologia , Estresse Oxidativo , Epitélio Pigmentado da Retina/citologia , Adenina/análogos & derivados , Adenina/química , Animais , Apolipoproteína E4/genética , Sobrevivência Celular , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/química , Lipofuscina/química , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismo , Retina/metabolismoRESUMO
Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.
Assuntos
Dependovirus/genética , RNA/genética , Retina/patologia , Retina/fisiopatologia , Rodopsina/genética , Animais , Terapia Genética , Vetores Genéticos , Camundongos , Camundongos Transgênicos , RNA/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/terapiaRESUMO
Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.
Assuntos
8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Lipofuscina/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptores de Serotonina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Agonistas do Receptor de Serotonina/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Antioxidantes/metabolismo , Linhagem Celular , Citoproteção/efeitos dos fármacos , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Epitélio Pigmentado da Retina/citologia , Superóxidos/metabolismo , Visão Ocular/efeitos dos fármacos , Visão Ocular/fisiologiaRESUMO
Autosomal dominant retinitis pigmentosa (ADRP) is frequently caused by mutations in RHO, the gene for rod photoreceptor opsin. Earlier, a study on mice carrying mutated rhodopsin transgenes on either RHO + / + or RHO + /- backgrounds suggested that the amount of wild-type rhodopsin affected survival of photoreceptors. Therefore, we treated P23H RHO transgenic mice with adeno-associated virus serotype 5 (AAV5) expressing a cDNA clone of the rhodopsin gene (RHO301) that expressed normal opsin from the mouse opsin promoter. Analysis of the electroretinogram (ERG) demonstrated that increased expression of RHO301 slowed the rate of retinal degeneration in P23H mice: at 6 months, a-wave amplitudes were increased by 100% and b-wave amplitudes by 79%. In contrast, nontransgenic mice injected with AAV5 RHO301 demonstrated a decrease in the ERG, confirming the damaging effect of rhodopsin overproduction in normal photoreceptors. In P23H mice, the increase in the ERG amplitudes was correlated with improvement of retinal structure: the thickness of the outer nuclear layer in RHO301-treated eyes was increased by 80% compared with control eyes. These findings suggest that the wild-type RHO gene can be delivered to rescue retinal degeneration in mice carrying a RHO mutation and that increased production of normal rhodopsin can suppress the effect of the mutated protein. These findings make it possible to treat ADRP caused by different mutations of RHO with the expression of wild-type RHO.