RESUMO
This study analyzes the temporal-spatial correlation between angio-and corticogenesis in the developing chick optic tectum (OT) by means of NADPH-diaphorase and immunolabeling methods. Qualitative and quantitative parameters were used to analyze a new vessels formation and growth of preexisting ones as a function of time and space...
Assuntos
Embrião de Galinha , Movimento Celular/fisiologia , Neovascularização Patológica/patologia , Proliferação de Células , Sistema Nervoso Central/embriologia , Veias Cerebrais/patologia , Vias Visuais/anatomia & histologia , AnisotropiaRESUMO
Matrix metalloproteinases (MMPs) play an important role in tissue remodeling that accompanies the rapid growth, differentiation, and structural changes of the placenta and several fetal organs. In the present study, we investigated whether the diabetic maternal environment may alter the regulatory homeostasis exerted by nitric oxide (NO) on MMPs activity in the feto-placental unit from rats at midgestation. We found that NADPH-diaphorase activity, which reflects the distribution and activity of NO synthases (NOS), was increased in both placenta and fetuses from diabetic rats when compared with controls. In addition, while a NO donor enhanced MMP2 and MMP9 activities, a NOS inhibitor reduced these activities in the maternal side of the placenta from control rats. This regulatory effect of NO was only observed on MMP9 in the diabetic group. On the other hand, the NO donor did not modify MMP2 and MMP9 activities, while the NOS inhibitor reduced MMP9 activity in the fetal side of both control and diabetic placentas. In the fetuses, MMP2 was enhanced by the NO donor and reduced by the NO inhibitor in both fetuses from control and diabetic rats. Overall, this study demonstrates that NO is able to modulate the activation of MMPs in the feto-placental unit, and provides supportive evidence that increased NOS activity leads to NO overproduction in the feto-placental unit from diabetic rats, an alteration closely related to the observed MMPs dysregulation that may have profound implications in the formation and function of the placenta and the fetal organs.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Feto/enzimologia , Metaloproteinases da Matriz/metabolismo , Óxido Nítrico/fisiologia , Placenta/enzimologia , Animais , Western Blotting/métodos , Eletroforese em Gel de Poliacrilamida , Feminino , Feto/efeitos dos fármacos , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/análise , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz/análise , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo III/análise , Nitroprussiato/farmacologia , Placenta/efeitos dos fármacos , Gravidez , Ratos , Ratos WistarRESUMO
Continuous illumination (CI) of the retina induces an oxidative stress followed by the degeneration of photoreceptors. This phenomenon may be partially related to the excessive production of nitric oxide (NO). In order to confirm this hypothesis, the aims of this work are to determine NO levels during the illumination of the retina by electron paramagnetic resonance (EPR), and if an increase of NO is found, to characterize the NOS isoform responsible of the increment by using Western blot. Sprague-Dawley rats were continuously illuminated with white light (12,000 lux) for 2, 24, 48 h, 5 and 7 days while control rats were maintained at light/dark cycles of 12/12 h. Using EPR, an increase of NO signal was observed in the light exposed retinas peaking at 24 h of CI. Western blot analysis showed the expression of iNOS in the illuminated retinas with a peak after 24 h of CI, but did not show significant differences of nNOS among illuminated and control retinas. In summary, there is an increase of NO during CI. Further studies will reveal whether this mechanism is responsible for light induced photoreceptor degeneration.
Assuntos
Óxido Nítrico/metabolismo , Retina/fisiologia , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Luz , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Ratos , Valores de Referência , Retina/efeitos da radiaçãoRESUMO
Matrix metalloproteinases (MMPs) are involved in placental remodelling throughout pregnancy. Diabetes mellitus induces alterations in tissue production of NO, a regulator of MMPs activity. The present work evaluates placental and fetal MMPs and NO levels during midpregnancy in neonatal streptozotocin-induced diabetic rats. MMP-2 and MMP-9 immunolabelling was increased both in the labyrinth zone (p<0.001) and in the giant trophoblast cells of the junctional zone (p<0.001) from diabetic placenta, when compared with controls. Also MMP-2 (p<0.01) and MMP-9 (p<0.005) activities were increased in both maternal and fetal sides of diabetic placenta when related to controls. In both sides of the diabetic placenta, nitrate/nitrite concentrations (which indicate NO production) were higher than in controls (p<0.05). An intense immunostaining for nitrotyrosine, indicating peroxynitrite-induced damage, was found in both labyrinth (p<0.001) and junctional zones (p<0.001) of diabetic placenta. Enhanced MMP-2 activity (p<0.05) and NO production were also higher in the fetuses from diabetic rats when compared to controls (p<0.005). These findings demonstrate alterations in MMPs and NO in the feto-placental unit of diabetic rats, anomalies that are likely to be involved in the developmental alterations induced by maternal diabetes.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Placenta/metabolismo , Animais , Diabetes Mellitus Experimental/enzimologia , Feminino , Idade Gestacional , Imuno-Histoquímica , Nitratos/metabolismo , Nitritos/metabolismo , Placenta/química , Placenta/enzimologia , Gravidez , Ratos , Ratos Wistar , Tirosina/análogos & derivados , Tirosina/análise , Tirosina/metabolismoRESUMO
Distribution of GAP-43 was studied in the retinas of rats after continuous illumination followed by different darkness periods. GAP-43 immunoreactivity was maximum in regenerating outer photoreceptor segments of rats kept in total darkness for 10 days, while in the inner plexiform layer, immunoreactivity was maximum immediately after illumination. Changes in GAP-43 expression could participate in retinal repair/regeneration after light-induced damage.
Assuntos
Proteína GAP-43/metabolismo , Luz , Retina/metabolismo , Retina/efeitos da radiação , Animais , Escuridão , Imuno-Histoquímica , Regeneração Nervosa/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Ratos , Ratos Wistar , Fatores de Tempo , Distribuição TecidualRESUMO
Serotonin (5HT) containing cell bodies are localized in mesencephalic and rhombencephalic raphe nuclei. It has been proposed that 5HT could be involved in neuronal development and plasticity. In the central nervous system, nitric oxide (NO) has been postulated as a neurotransmitter and neuromodulator, and has been implicated in neurotoxicity as well as in neuroprotection. Using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) technique, NO synthesizing neurons were described in raphe nuclei. By immunohistochemistry, nitric oxide synthase (NOS) was found colocalized with 5HT in some dorsal raphe nucleus (DRN) neurons. In a model of inhibition of 5HT synthesis produced by daily administration of parachlorophenilalanine during 14 days, we have studied the relationship between 5HT and NO systems after 5HT depletion by histochemical and immunocytochemical methods. After the treatment, we observed an important reduction of 5HT immunostaining in the DRN and enhanced NOS activity demonstrated by NADPH-d technique, especially in the dorsomedial and ventromedial subgroups. In spite of the increased NOS activity, we could not observe significant changes in the NOS-immunoreactivity in the DRN after 5HT depletion. These results could indicate that 5HT depletion is concomitant with changes in NOS activity without affecting NOS expression in the DRN.
Assuntos
Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Núcleos da Rafe/metabolismo , Serotonina/metabolismo , Animais , Fenclonina/farmacologia , Masculino , NADPH Desidrogenase , Neurônios/química , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/efeitos dos fármacos , Núcleos da Rafe/química , Núcleos da Rafe/efeitos dos fármacos , Ratos , Ratos Wistar , Serotonina/análise , Antagonistas da Serotonina/farmacologiaRESUMO
The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.
Assuntos
Animais , Movimento Celular , Óxido Nítrico Sintase , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Trypanosoma cruzi , Compartimento Celular , Membrana Celular , FlagelosRESUMO
The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.(AU)
Assuntos
Animais , RESEARCH SUPPORT, NON-U.S. GOVT , Movimento Celular/fisiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Transdução de Sinais/fisiologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/ultraestrutura , Compartimento Celular/fisiologia , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Flagelos/enzimologia , Flagelos/ultraestruturaRESUMO
Nitric oxide (NO) is a gas involved in neurotransmission in the central nervous system (CNS) and in vertebrate retinas. This paper describes five types of nitrergic neurons in developing and adult chick retina using the nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) reaction. Three of them, nitrergic types 1, 2 and 3, were observed in the inner nuclear layer, while nitrergic type 4 was observed in the ganglion cell layer; nitrergic type 5 were the retinal photoreceptors. Cell processes formed four nitrergic networks, which could be observed in the inner plexiform layer (IPL), at sublayers 1, 3a, 3b and 4. Another nitrergic network was observed in the outer plexiform layer (OPL). From hatching, the dendritic branches were completely developed in the IPL and in the OPL, forming the mentioned networks. Current evidence suggests that NO is coexpressed with other neurotransmitters in neurons of the CNS. Double-staining procedures, using NADPHd and 5HT immunohistochemistry in chicken retina, in a sequential or in an alternative manner, did not reveal the coexistence of these two neurotransmitters in the same neurons, but their networks matched in sublayers 1 and 4 of the IPL.
Assuntos
Neurônios Aferentes/enzimologia , Óxido Nítrico/análise , Células Fotorreceptoras de Vertebrados/enzimologia , Retina/embriologia , Animais , Tamanho Celular , Embrião de Galinha , Galinhas , NADPH Desidrogenase/análise , Neurônios Aferentes/química , Neurônios Aferentes/citologia , Células Fotorreceptoras de Vertebrados/química , Células Fotorreceptoras de Vertebrados/citologia , Retina/citologia , Retina/enzimologia , Serotonina/análiseRESUMO
The localization and subcellular distribution of Trypanosoma cruzi nitric oxide synthase was investigated in epimastigote cells by immunocytochemistry at electron and light microscope level, using a polyclonal antibody to neuronal nitric oxide synthase, and also, at light microscope level, by the nicotinamide adenine dinucleotide phosphate-diaphorase histochemical reaction. The immunoreactivity was ultrastructurally localized by electron microscopy in the inner surface of cell membranes and in free cytosolic clusters in the body, flagellum and apical extreme. Light microscopy showed that immunoprecipitates, specific for the Trypanosoma cruzi nitric oxide synthase, co-localized with the formazan precipitates generated by the diaphorase reaction in the same areas identified by electron microscopy. These results, taken together with previous finding from our laboratory could help to explain the involvement of the nitric oxide transduction pathway in T. cruzi epimastigote motility.