RESUMEN
Vitamin E and polyphenols could exhibit a therapeutic role in the treatment of oxidative stress-induced neurodegenerative diseases. Therefore, their ability to cross the blood-brain barrier (BBB) represents an important issue to be explored by different diet combinations. In this study, we have evaluated the ability of α-tocopherol to support epigallocatechin-3-gallate (EGCG), quercetin and rutin to cross the BBB, following oral administration. Eighteen rats were fed a standard diet (C), a diet supplemented with α-tocopherol (A), with a mixture of EGCG, quercetin and rutin (P); or with a mixture of α-tocopherol and the three flavonoids (AP). Flavonoids and their conjugated derivatives were assayed in brain and plasma by HPLC-MS, whereas α-tocopherol was detected by RP-HPLC. The oxidative damage, due to the potential pro-oxidant activity of flavonoids, was evaluated by the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in hippocampal Cornus Ammonis, one of the most vulnerable sites in the brain. Our results indicate that α-tocopherol is able to promote quercetin transport across the BBB. The mixture of rutin and quercetin seems to favour the accumulation of quercetin and/or its conjugated derivatives in the brain. In contrast, α-tocopherol does not affect EGCG transport across the BBB. The densitometric analysis of 8-OHdG immunoreactivity does not reveal any difference of oxidative damage among the experimental groups. Our results suggest that α-tocopherol may promote quercetin transport across the BBB, leading to a significant increase of α-tocopherol and quercetin concentration in the brain.
Asunto(s)
Barrera Hematoencefálica/fisiología , Catequina/análogos & derivados , Quercetina/administración & dosificación , Rutina/administración & dosificación , alfa-Tocoferol/administración & dosificación , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Antioxidantes/administración & dosificación , Antioxidantes/farmacocinética , Catequina/administración & dosificación , Catequina/farmacocinética , Cromatografía Líquida de Alta Presión , Desoxiguanosina/análogos & derivados , Desoxiguanosina/química , Hipocampo/fisiología , Masculino , Espectrometría de Masas , Estrés Oxidativo/efectos de los fármacos , Quercetina/farmacocinética , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Rutina/farmacocinética , alfa-Tocoferol/farmacocinéticaRESUMEN
It is known that the MyoD family members (MyoD, Myf5, myogenin, and MRF4) play a pivotal role in the complex mechanism of skeletal muscle cell differentiation. However, fragmentary information on transcription factor-specific regulation is available and data on their post-transcriptional and post-translational behavior are still missing. In this work, we combined mRNA and protein expression analysis with their subcellular localization. Each myogenic regulator factor (MRF) revealed a specific mRNA trend and a protein quantitative analysis not overlapping, suggesting the presence of post-transcriptional mechanisms. In addition, each MRF showed a specific behavior in situ, characterized by a differentiation stage-dependent localization suggestive of a post-translational regulation also. Consistently with their transcriptional activity, immunogold electron microscopy data revealed MRFs distribution in interchromatin domains. Our results showed a MyoD and Myf5 contrasting expression profile in proliferating myoblasts, as well as myogenin and MRF4 opposite distribution in the terminally differentiated myotubes. Interestingly, MRFs expression and subcellular localization analysis during C2C12 cell differentiation stages showed two main MRFs regulation mechanisms: (i) the protein half-life regulation to modulate the differentiation stage-dependent transcriptional activity and (ii) the cytoplasmic retention, as a translocation process, to inhibit the transcriptional activity. Therefore, our results exhibit that MRFs nucleo-cytoplasmic trafficking is involved in muscle differentiation and suggest that, besides the MRFs expression level, also MRFs subcellular localization, related to their functional activity, plays a key role as a regulatory step in transcriptional control mechanisms.
Asunto(s)
Diferenciación Celular , Músculo Esquelético/citología , Mioblastos/metabolismo , Factores Reguladores Miogénicos/análisis , Factores Reguladores Miogénicos/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Ratones , Músculo Esquelético/metabolismoRESUMEN
Hippocampus dentate gyrus (DG) is characterized by neuronal plasticity processes in adulthood, and polysialylation of NCAM promotes neuronal plasticity. In previous investigations we found that alpha-tocopherol increased the PSA-NCAM-positive granule cell number in adult rat DG, suggesting that alpha-tocopherol may enhance neuronal plasticity. To verify this hypothesis, in the present study, structural remodeling in adult rat DG was investigated under alpha-tocopherol supplementation conditions. PSA-NCAM expression was evaluated by Western blotting, evaluation of PSA-NCAM-positive granule cell density, and morphometric analysis of PSA-NCAM-positive processes. In addition, the optical density of synaptophysin immunoreactivity and the synaptic profile density, examined by electron microscopy, were evaluated. Moreover, considering that PSA-NCAM expression has been found to be related to PKCdelta activity and alpha-tocopherol has been shown to inhibit PKC activity in vitro, Western blotting and immunohistochemistry followed by densitometry were used to analyze PKC. Our results demonstrated that an increase in PSA-NCAM expression and optical density of DG molecular layer synaptophysin immunoreactivity occurred in alpha-tocopherol-treated rats. Electron microscopy analysis showed that the increase in synaptophysin expression was related to an increase in synaptic profile density. In addition, Western blotting revealed a decrease in phospho-PKC Pan and phospho-PKCdelta, demonstrating that alpha-tocopherol is also able to inhibit PKC activity in vivo. Likewise, immunoreactivity for the active form of PKCdelta was lower in alpha-tocopherol-treated rats than in controls, while no changes were found in PKCdelta expression. These results demonstrate that alpha-tocopherol is an exogenous factor affecting neuronal plasticity in adult rat DG, possibly through PKCdelta inhibition.
Asunto(s)
Giro Dentado/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Proteína Quinasa C-delta/antagonistas & inhibidores , alfa-Tocoferol/farmacología , Animales , Antioxidantes/farmacología , Dendritas/metabolismo , Dendritas/ultraestructura , Giro Dentado/enzimología , Giro Dentado/ultraestructura , Inhibidores Enzimáticos/farmacología , Inmunohistoquímica , Masculino , Microscopía Electrónica de Transmisión , Molécula L1 de Adhesión de Célula Nerviosa/efectos de los fármacos , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Plasticidad Neuronal/fisiología , Terminales Presinápticos/enzimología , Terminales Presinápticos/ultraestructura , Proteína Quinasa C-delta/metabolismo , Ratas , Ratas Sprague-Dawley , Ácidos Siálicos/metabolismo , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/enzimología , Membranas Sinápticas/ultraestructura , Sinaptofisina/efectos de los fármacos , Sinaptofisina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The resistance to oxidative stress is a multifactorial reaction involving the clustering of transcriptionally regulated genes. Because glucose-6-phosphate dehydrogenase (G6PD), the principal enzyme responsible for reducing power, is highly expressed in the olfactory bulb (OB), it is of interest to verify whether other enzymes utilizing NADPH are also highly expressed. The level and localization of G6PD- and NADPH-consuming enzymes, such as NADPH-cytochrome P450 oxidoreductase (P450R), glutathione reductase (GR), and NADPH-diaphorase (NADPH-d), were analyzed in the rat olfactory bulb (OB) by quantitative histochemistry and immunohistochemistry. The highest concentration of G6PD, P450R, and GR was observed in the olfactory nerve layer (ONL), suggesting a correlation in the expression of these enzymes at the gene level. Correlation in staining intensity between G6PD and NADPH-d activities occurred only in part of the ONL, some glomeruli, and scattered periglomerular cells. This peculiar distribution of NADPH-d could reflect a spatial patterning of the nose to bulb projections. Taken together, these results indicate that G6PD expression in the ONL could be related to the importance of generating a substantial supply of NADPH to sustain the detoxifying systems represented by GR and P450R reactions and, only in discrete zones, by NADPH-d activity.
Asunto(s)
Glucosafosfato Deshidrogenasa/metabolismo , NADP/metabolismo , Neuronas/enzimología , Bulbo Olfatorio/enzimología , Estrés Oxidativo/fisiología , Animales , Metabolismo Energético/fisiología , Glutatión Reductasa/metabolismo , Inmunohistoquímica , Masculino , NADPH Deshidrogenasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Bulbo Olfatorio/citología , Ratas , Ratas Sprague-DawleyRESUMEN
In cerebellum of the adult rat, glucose-6-phosphate dehydrogenase (G6PD) activity is particularly localized in Purkinje cells, showing lower activity in the molecular and granule cell layers. G6PD is the first and rate-limiting step of the hexose monophosphate shunt (HMS), which has the physiological role of providing NADPH for reductive biosynthesis and detoxifying reactions. In this study, we searched for a possible correlation between G6PD and other NADPH-consuming enzymes, such as NADPH-cytochrome P450 reductase (P450R), glutathione reductase (GR) and NADPH-diaphorase (NADPH-d). This study was performed by means of immunohistochemistry and enzyme histochemistry followed by quantitative densitometric and confocal laser scanning microscopic analyses. Our results demonstrated that G6PD, P450R and GR have a similar distribution pattern characterized by the highest concentration of these enzymes in the somata of Purkinje cells, and by lower concentrations in the molecular and the granule cell layers. Moreover, in Purkinje cells, G6PD colocalized with both P450R and GR. NADPH-d activity showed a different distribution pattern when compared to the other enzymes, revealing the highest activity in the molecular layer and the lowest in Purkinje cells. Our results suggest a coordinated regulative mechanism of G6PD, P450R and GR based on the request of NADPH or on specific transcription factors.
Asunto(s)
Corteza Cerebelosa/citología , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Reductasa/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , NADP/metabolismo , Células de Purkinje/enzimología , Análisis de Varianza , Animales , Corteza Cerebelosa/enzimología , Densitometría/métodos , Proteína Ácida Fibrilar de la Glía/metabolismo , Histocitoquímica/métodos , Inmunohistoquímica/métodos , Masculino , Microscopía Confocal/métodos , Células de Purkinje/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
This review describes the variation of glucose-6-phosphate dehydrogenase (G6PD) activity in the main neurons of the molecular and granular layers as well as in the deep nuclei of the cerebellum as observed so far by optical and electron microscopy studies. Light microscopy and semiquantitative microphotometry of histochemical staining showed that the highest G6PD activity was expressed by Purkinje cells and neurons of the deep cerebellar nuclei; the elements of the molecular layer showed a diffuse G6PD staining, while the granular layer displayed only scattered G6PD activity. Electron microscopy analysis showed that the basket and stellate cells, as well as the Golgi cells, have a remarkable G6PD activity, while in the granule cells the enzyme was barely detectable. The results show that cerebellar G6PD activity changes with different neuron types as a function of its role in sustaining NADPH dependent pathways in these cells.
Asunto(s)
Cerebelo/metabolismo , Glucosafosfato Deshidrogenasa/biosíntesis , NADP/metabolismo , Neuronas/metabolismo , Animales , Cerebelo/citología , Cerebelo/enzimología , Humanos , Microscopía/métodos , Neuronas/enzimologíaRESUMEN
In previous work, we found that adult hippocampal neurogenesis in rat is affected by vitamin E deficiency. Because vitamin E deficiency is a complex condition involving numerous biological systems, it is possible that its effect on postnatal new neuron production could be mediated by unknown changes in different factors that in turn play a role in this process. To clarify if vitamin E plays a direct role in regulating hippocampal neurogenesis, we studied the neurogenesis in adult control rats and in adult rats under supplementation with alpha-tocopherol, the most important compound of vitamin E. The alpha-tocopherol level in control and supplemented rats was monitored. Qualitative and quantitative analysis of cell proliferation and death was carried out and expression of immature neuron markers PSA-NCAM, TUC 4, and DCX was investigated in hippocampus dentate gyrus. alpha-Tocopherol levels increased significantly in both plasma and brain after supplementation. Cell proliferation was inhibited in alpha-tocopherol-supplemented rats, the number of dying cells was reduced, and the number of cells expressing the immature neuron markers was increased. The results obtained confirm and extend the idea that vitamin E is an exogenous factor playing a direct role in regulation of different steps of adult hippocampal neurogenesis. Some hypotheses about the possible mechanisms underlying the complex action of alpha-tocopherol, related to its antioxidant and molecule-specific non-antioxidant properties, are proposed and discussed.
Asunto(s)
Antioxidantes/farmacología , Proteínas de Drosophila , Hipocampo/efectos de los fármacos , Proteínas Asociadas a Microtúbulos , Neuronas/efectos de los fármacos , Ubiquitina-Proteína Ligasas , alfa-Tocoferol/farmacología , Animales , Química Encefálica , Bromodesoxiuridina/metabolismo , Bromodesoxiuridina/farmacocinética , Proteínas de Caenorhabditis elegans/metabolismo , Calbindinas , Recuento de Células , Muerte Celular , División Celular , Cromatografía Líquida de Alta Presión/métodos , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Técnica del Anticuerpo Fluorescente , Hipocampo/fisiología , Etiquetado Corte-Fin in Situ/métodos , Ligasas/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/citología , Neuronas/fisiología , Neuropéptidos/metabolismo , Fármacos Sensibilizantes a Radiaciones/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacocinética , Ratas , Ratas Sprague-Dawley , Proteína G de Unión al Calcio S100/metabolismo , Ácidos Siálicos/metabolismo , Factores de Tiempo , alfa-Tocoferol/sangreRESUMEN
We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.
Asunto(s)
Apoptosis/efectos de los fármacos , Giro Dentado/efectos de los fármacos , Giro Dentado/patología , Fármacos Neuroprotectores/farmacología , Deficiencia de Vitamina E/patología , Vitamina E/farmacología , Envejecimiento/metabolismo , Envejecimiento/patología , Animales , Apoptosis/fisiología , Bromodesoxiuridina , Recuento de Células , División Celular/efectos de los fármacos , División Celular/fisiología , Giro Dentado/fisiopatología , Etiquetado Corte-Fin in Situ , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/sangre , Fármacos Neuroprotectores/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-Dawley , Vitamina E/sangre , Vitamina E/metabolismo , Deficiencia de Vitamina E/tratamiento farmacológico , Deficiencia de Vitamina E/fisiopatologíaRESUMEN
Thiocolchicine is a colchicine-derivative used in the therapy of some diseases and extensively studied in the field of oncological research as antimitotic agent. Here we studied the activity of thiocolchicine on the cytoskeleton of the peroneus nerve, performing a histological and ultrastructural analysis. We observed a decrease in mean myelinated fiber area in thiocolchicine-treated rats in comparison to controls; this was due to a decrease in mean axoplasm area, while myelin thickness was constant. In the ultrastructural analysis a decrease in microtubule density and an increase in neurofilaments were found; moreover, the myelinated fibers seemed to be more affected in comparison to the unmyelinated axons. These findings are in agreement with the capability of binding to microtubule skeleton shared by all the colchicinoids.
Asunto(s)
Axones/ultraestructura , Colchicina/análogos & derivados , Colchicina/farmacología , Citoesqueleto/ultraestructura , Nervio Peroneo/efectos de los fármacos , Animales , Inyecciones Intraperitoneales , Masculino , Microtúbulos/ultraestructura , Vaina de Mielina/ultraestructura , Nervio Peroneo/ultraestructura , Ratas , Ratas Sprague-DawleyRESUMEN
The adult hippocampal neurogenesis is affected by vitamin E deficiency. In the present investigation we examined if neural precursor proliferation, newborn cell survival or both are altered by vitamin E deficiency. 5-Bromo-2'-deoxyuridine (BrdU) was employed as a marker of proliferating cells. BrdU-labelled cells were revealed 1 and 30 days after BrdU administration in order to evaluate proliferation and newborn cell survival, respectively. Cell proliferation decreased in controls from juvenile to adult age, and the decrease was lesser in vitamin E deficiency. Thus we found a higher number of proliferating cells in vitamin E-deficient rats than in age-matched controls at 5 months of age. Comparing the number of BrdU-positive cells between 1 and 30 days after the last BrdU injection revealed a remarkable decrease in all groups; this is the greatest in vitamin E-deficient rats and the lowest in control rats. Consistently cell death in the dentate gyrus, assessed by TUNEL technique, was found to decrease from 1 to 5 months of age, but at 5 months it was significantly higher in vitamin E-deficient rats than in age-matched controls. These data show that vitamin E deficiency enhances neural precursor proliferation and cell death during adult neurogenesis.
Asunto(s)
Giro Dentado/crecimiento & desarrollo , Giro Dentado/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Deficiencia de Vitamina E/metabolismo , Animales , Animales Recién Nacidos , Bromodesoxiuridina , Recuento de Células , Muerte Celular/fisiología , División Celular/fisiología , Supervivencia Celular/fisiología , Giro Dentado/citología , Técnica del Anticuerpo Fluorescente , Alimentos Formulados , Etiquetado Corte-Fin in Situ , Masculino , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Deficiencia de Vitamina E/patología , Deficiencia de Vitamina E/fisiopatología , alfa-Tocoferol/sangreRESUMEN
In the present study we show that a reduction in the number of neural precursor cells enhances survival of new granule cells in the dentate gyrus allowing the recovery of the proper granule cell layer structure. To diminish the number of newborn cells methylazoxymethanol (MAM), a toxic agent for proliferating cells, was injected during neonatal life. Proliferation of precursor cells and survival of newborn cells were assessed by BrdU administration to 1-month-old rats when granule cell layer still shows a reduction in granule cell number in treated animals. Treatment with MAM reduced cell proliferation by 30% and enhanced cell progeny survival: so that the final number of newborn cells exceeded control ones by 38%. Consistently, dentate granule cell death, assessed by the TUNEL method, was significantly decreased in the MAM rats. The enhanced survival of newborn granule cells and the consistent reduced cell death suggest a link between neurogenesis and regulation of granule cell number. A comparison with previous findings shows that the recovery in the long-term of granule cell layer may be due to the re-establishing of the progenitor pool size and/or to the rescue of cell progeny.
Asunto(s)
Giro Dentado/citología , Acetato de Metilazoximetanol/análogos & derivados , Neuronas/fisiología , Células Madre/fisiología , Animales , Muerte Celular , División Celular , Supervivencia Celular , Masculino , Acetato de Metilazoximetanol/farmacología , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Células Madre/efectos de los fármacosRESUMEN
In the dentate gyrus of the mammalian hippocampus, neurogenesis carries on throughout postnatal life. The aim of this work was to identify an exogenous control factor of adult neurogenesis. Neurogenesis in the adult dentate gyrus was previously found to be enhanced in vitamin E-deficient rats. The effects of alpha- or beta-tocopherol supplementation on neurogenesis in the adult dentate gyrus were investigated by 5-bromo-2'-deoxyuridine labeling. Tocopherol was found to increase the survival of newborn cells and the total number of granule cells in the adult rat dentate gyrus. Newborn cells were phenotypically characterized by expression of the immature neuron marker TOAD-64 (turned on after division-64). Therefore tocopherol in high doses possibly increases the number of granule cells in the dentate gyrus by saving newborn cells from death.
Asunto(s)
División Celular/efectos de los fármacos , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Neuronas/citología , alfa-Tocoferol/administración & dosificación , beta-Tocoferol/administración & dosificación , Animales , Química Encefálica , Recuento de Células , Técnica del Anticuerpo Fluorescente Indirecta , Masculino , Microscopía Fluorescente , Ratas , Ratas Sprague-Dawley , Vitamina E/análisis , Vitamina E/sangreRESUMEN
In order to investigate the role of postnatal neurogenesis in granule cell number control in the rat dentate gyrus, we administered Methylazoxymethanol (MAM), a drug able to prevent cells from dividing, on P3, P5, P7, P9, when the most granule cells are produced. The effect of MAM on the number of proliferating precursors and of granule cells was examined at P16 and P90. We used 5-bromo-2'-deoxyuridine administration to label proliferating cells and immunohistochemistry to characterize the cell phenotype using neuron markers TUC 4, PSA-NCAM, Calbindin D28K and glial marker GFAP. At 16 days of age in MAM-treated rats we observed a significant decrease of BrdU-positive cells. Consistently, a decrease in density and number of granule cells was found compared to the controls. At 90 days the dentate gyrus of treated rats showed a complete recovery: no differences in the density, total number of neurons, the BrdU- and TUC 4-positive cells were revealed with respect to the controls. No deficits were evident in performance on the water maze in MAM-treated rats. These data suggest that the dentate gyrus is able to re-establish the proliferative zone and to rebuild the granule cell layer following neonatal MAM administration.