RESUMEN
Purpose: Choroidal and retinal neovascularization plays an essential role in various ocular diseases. In this study, we examined the role of nestin in this process. Nestin is an intermediate filament protein known to play several roles, including as a marker of neural progenitor and proliferating endothelial cells. Methods: We used Brown Norway rats, in which choroidal and retinal neovascularization was induced using intraocular laser impacts. The role of nestin was examined using angiography, western blot from the second to the 14th day after laser impacts, and intraocular injection of nestin siRNA. The localization of the protein was specified by co-immunoreactivity with glial fibrillary protein (GFAP), glutamine synthetase (GS), and von Willebrand factor (vWF). Results: In the control retina, nestin was found principally in glial structures in the ganglion cell layer, as confirmed by nestin/GFAP immunolabeling. Two days after the laser impacts, the nestin expression extended to numerous radial processes at the site of the impacts. With Bruch's membrane ruptured, these processes penetrated into the choroid. Nestin immunolabeling remained high from the third to the seventh day but appeared reduced on the 14th day. The nature of these processes was not clearly defined, but co-immunolabeling with GFAP suggested that they were principally in activated Müller cells from the third day after the laser impacts. However, the co-immunoreactivity of nestin and GS, a marker of mature functional Müller cells, could be observable only from the seventh day. Nestin was also observed in some vascular cells, as demonstrated by the co-immunoreactivity of the protein with vWF in the choroid and retina. As observed on angiography, the numbers of choroidal and retinal blood vessels were significantly increased (principally on the seventh day) after the laser impacts. An intraocular injection of nestin siRNAs led to a significant decrease in the number of blood vessels. Conclusions: Our results confirmed the presence of nestin in glial (e.g., astrocytes), reactive Müller, and endothelial cells. They demonstrated their critical involvement in a rat model of retinal and choroidal neovascularization experimentally induced using ocular laser impacts.
Asunto(s)
Neovascularización Coroidal , Neovascularización Retiniana , Ratas , Animales , Nestina/genética , Nestina/metabolismo , Neovascularización Retiniana/metabolismo , Factor de von Willebrand/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Células Endoteliales/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Coroides/metabolismo , Retina/metabolismo , Neovascularización Coroidal/metabolismo , Rayos LáserRESUMEN
PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels. CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedades Hereditarias del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Anciano , Animales , Western Blotting , Preescolar , Modelos Animales de Enfermedad , Enfermedades Hereditarias del Ojo/metabolismo , Femenino , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratas , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/crecimiento & desarrolloRESUMEN
Nestin is found in radial glia and neuronal/glial progenitor cells during retinal development, and is re-expressed after acute damage in the retina of adult mammals. We have investigated nestin expression in the retina of the Royal College of Surgeons (RCS) rat model of human inherited blindness, Retinitis pigmentosa (RP). During the first postnatal week, nestin immunoreactivity was located in elongated processes resembling radial glia in both control and dystrophic animals. During the second postnatal week, the density of nestin immunoreactive radial processes decreased progressively starting in the outer retina. At postnatal day 20 (PNd20), Nestin immunoreactive radial processes were no longer visible, with immunoreactivity restricted to structures resembling Müller end-feet and/or astrocytes located in the ganglion cell layer (GCL) in both control and dystrophic rats. These morphological results were confirmed by Western blotting and qPCR analysis. The level of nestin remained low in control animals at different time points up to 1 year, but we observed a re-expression of this protein from PNd30 in the dystrophic animals. The morphology of cells re-expressing nestin resembled that of radial glia and/or Muller cells, but co-localization of nestin and glutamine synthetase (GS: a marker of mature Müller cells) was only partial. Interestingly, whereas Western blot analysis confirmed the increase in protein levels from PNd30 onwards, mRNA levels remained low in dystrophic rats. Additional studies demonstrated that the discrepancy between protein and mRNA contents could be due to a dysfunction in proteasome activity as often observed in neurodegenerative pathologies. In conclusion, because of its localization in astrocytes and in radial processes resembling radial glia in the pathologic adult retina, nestin may be involved in mechanisms such as cell migration, generation of new neurons or glial cells and/or in retinal (re)modeling in dystrophic adult animals. The lack of concomitant up-regulation of mRNAs in adult dystrophic animals suggests that the pathology could lead to transcriptional and/or metabolic changes involving the stabilization of the half-life and/or dysregulation of degradation processes of nestin protein.
Asunto(s)
Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Retinitis Pigmentosa/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Astrocitos/patología , Biomarcadores , Western Blotting , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Glutamato-Amoníaco Ligasa/metabolismo , Nestina , Neuroglía/metabolismo , Neuroglía/patología , ARN Mensajero/metabolismo , Ratas , Ratas Mutantes , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/patología , Retinitis Pigmentosa/patologíaRESUMEN
In lower vertebrates, like fishes and amphibians, retina is able to self-regenerate whereas Mammalian retina has lost this property. Nevertheless, recently, it has turned out that retinal glial Müller cells were playing a role in neuronal regeneration of the adult rodent retina, in case of acute damages, by dedifferentiating and redifferentiating in glial and neural cells. The purpose of this study was to analyse the ability of mammalian Müller cells for forming neurospheres. First of all, rats Müller cells were isolated in a primary culture. Second, these cells were resuspended in two different culture media: the cells cultures in the Neurobasal-A medium kept a typical Müller cells morphology even after 15 days of EGF treatment, and the cells plated in the DMEM-F12 medium formed neurospheres from the third day in culture. The neurosphere cells expressed nestin, cellular retinaldehyde binding protein (CRALBP) and glial fibrillary acidic protein (GFAP). These results showed our capacity to isolate and propagate Müller cells-derived progenitor cells. Moreover, it allows us to control the number of progenitor cells and, in the future, to study their differentiation capacity.
Asunto(s)
Neuroglía/metabolismo , Retina/citología , Células Madre/metabolismo , Animales , Animales Recién Nacidos , Proteínas Portadoras/metabolismo , Células Cultivadas , Medios de Cultivo/farmacología , Factor de Crecimiento Epidérmico/farmacología , Proteína Ácida Fibrilar de la Glía/metabolismo , Neuroglía/efectos de los fármacos , Ratas , Ratas Long-EvansRESUMEN
In this study, we used a neuronal cell line generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus-2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Time-dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by retinoic acid (RA) were concomitant. Indeed, RA altered the cell proliferation rate, but it also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein-immunoreactive neurons. To characterize the nature of the cells we used immunocytochemistry, optical imaging, scanning electron microscopy and atomic force microscopy.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Mucosa Olfatoria/efectos de los fármacos , Tretinoina/farmacología , Adenilil Ciclasas/análisis , Animales , Apoptosis/efectos de los fármacos , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/análisis , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/análisis , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Proteínas del Tejido Nervioso/análisis , Nestina , Neuronas/química , Neuronas/ultraestructura , Mucosa Olfatoria/citología , Mucosa Olfatoria/ultraestructura , Ratas , Factores de Tiempo , Tubulina (Proteína)/análisisRESUMEN
Noda epileptic rats (NER) exhibit frequent spontaneous tonic-clonic convulsions which represent a valuable model of human epilepsy. If implication of brain neurotransmitters was largely reported, little is known about retina. However, it has been reported that human epilepsy syndrome varies not only with the location of seizure foci but also according to rhythmic patterns, for which retina has a major role in the transmission of external light-dark cycle information. The purpose of this work was to evaluate dopamine (DA), DA metabolites, serotonin (5-HT), and amino acid [glutamate, aspartate, glycine, gamma aminobutyric acid (GABA), and taurine] level variations in retina from NER, at two different nycthemeral periods (11 a.m. and 11 p.m.) and at different ages (2, 6, and 12 months). In NER, retinal dopaminergic function was decreased as soon as 2 months, whereas GABA levels were increased, even if no differences among the different ages could be distinguished. These variations were associated to a slight increase in 5-HT. Other amino acids tested were not affected by epilepsy, whereas taurine decreased with aging in NER as well as in control rats. Retinal 5-HT occurs principally as a precursor of melatonin (MEL). A triangular interaction may be hypothesized: MEL could decrease DA synthesis or release by enhancing GABA activity. Taken together, these results suggest that the retinal physiology is affected by the epileptic status and that information transmitted from retina to the brain should be affected by epilepsy in NER.
Asunto(s)
Aminoácidos/metabolismo , Dopamina/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Retina/metabolismo , Serotonina/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Monoaminas Biogénicas/metabolismo , Ritmo Circadiano , Dopamina/análogos & derivados , Femenino , Ácido Homovanílico/metabolismo , Masculino , Ratas , Ratas Mutantes , Ratas WistarRESUMEN
We have previously identified in some mouse strains (e.g. BALB/c, DBA/2) a murine Intracisternal A-particle (IAP) transposable element specifically expressed in the liver. This IAP sequence is inserted within a gene, mCCR4/m. nocturnin, the sequence of which is related to the circadian Xenopus nocturnin gene. Here we show, using real-time quantitative RT-PCR, that both the IAP sequence and the m. nocturnin gene display strong circadian expression in the liver, with peak abundance after dusk. Circadian oscillations of m. nocturnin RNA are maintained in mice without the IAP insertion (e.g. CBA/J, 129/sv), are free-running under constant light and dark conditions, and persist upon food and water privation, demonstrating that m. nocturnin is a circadian gene. In situ hybridization analyses (in 129/sv mice) further show circadian expression of m. nocturnin also in the retina, precisely at the level of the photoreceptors, a result consistent with the previously described circadian expression of the Xenopus gene. These results strengthen the strong conservation of the nocturnin gene with the identification of a functional mouse ortholog of the Xenopus gene, and demonstrate the reciprocal influence of nearby genes on the expression of transposable elements via "position effects".
Asunto(s)
Genes de Partícula A Intracisternal , Hígado/metabolismo , Células Fotorreceptoras/metabolismo , Proteínas/genética , Animales , Ritmo Circadiano , Elementos Transponibles de ADN , Ingestión de Líquidos , Ingestión de Alimentos , Expresión Génica , Hibridación in Situ , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos CBA , Proteínas Nucleares , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TranscripciónRESUMEN
Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Mucosa Olfatoria/enzimología , Mucosa Olfatoria/patología , Oxidorreductasas/biosíntesis , Sulfato de Zinc/farmacología , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Mucosa Olfatoria/efectos de los fármacos , Oxidorreductasas/genética , Sulfato de Zinc/químicaRESUMEN
PURPOSE: To observe the morphology and physiology of the retina in rats 11 weeks after a constant (24-hour) but moderate (500-lux) illumination for 1 week. METHODS: Levels of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, and taurine were measured by high-pressure liquid chromatography (HPLC) in the retina and vitreous humor of albino (Wistar) and pigmented (Long-Evans) rats. Semithin sections were used to determine retinal morphology. The TUNEL method was used to detect cells degenerating by apoptosis. Because the GABAergic system has been shown to be particularly sensitive to the loss of photoreceptors, an additional immunohistochemical study using anti-GABA, anti-glutamate decarboxylase (GAD)(67) and anti-GAD(65) antibodies was performed. RESULTS: No apparent morphologic changes were found in the retina of pigmented rats after constant illumination, whereas in albino rats disappearance of photoreceptors (except in the extreme retinal periphery) and cell bodies was observed. A significant number of TUNEL-positive nuclei also occurred in the remaining nuclear and ganglion cell layers. However, no change in the distribution of GABA, GAD(67), and GAD(65) immunoreactivities was found in either strain under constant illumination compared with control animals. Constant illumination affected the retinal levels of aspartate, glutamate, glutamine, glycine in both strains, whereas GABA contents did not change and taurine was decreased only in albino rats. A significant increase of vitreal glutamate levels was also found in both strains and of taurine levels only in albino rats. CONCLUSIONS: Phototoxicity can provoke durable retinal alterations beyond the period of lighting, suggesting progressive and probably continuous modifications of retinal physiology, even in pigmented animals in which the retina seems morphologically normal.
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Luz/efectos adversos , Traumatismos Experimentales por Radiación/etiología , Retina/efectos de la radiación , Degeneración Retiniana/etiología , Animales , Apoptosis , Ácido Aspártico/metabolismo , Cromatografía Líquida de Alta Presión , Femenino , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Etiquetado Corte-Fin in Situ , Estudios Longitudinales , Masculino , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Ratas , Ratas Long-Evans , Ratas Wistar , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Taurina/metabolismo , Cuerpo Vítreo/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Migration of stem/progenitor cells is a crucial event for homing toward tissue where cells need to be renewed. The neurotransmitter gamma-aminobutyric acid (GABA) has been shown to have a crucial role in migration of neuronal stem/progenitor cells. Since human umbilical cord blood (HUCB) contains stem/progenitor cells able to generate either neuronal or hematopoietic cells, we evaluated the effect of GABA on this type of cells. While whole fraction of mononuclear cells expressed GABA(A) and GABA(B) receptor subunits (GABA-R), only GABA(B)R subunits were found to be expressed on immature CD133+ cells. Functional experiments revealed that both cell fractions of HUCB were attracted by a gradient of GABA concentration and furthermore were blocked by specific antagonists of GABA(A)R and GABA(B)R bicuculline and saclofen, respectively. Moreover, through GABA(B)R activation the migrating fraction was highly enriched by both hematopoietic progenitors and cells able to generate neuron- like cells in culture. Therefore, GABA is a potent chemoattractant of HUCB stem/progenitor cells specifically through GABA(B)R activation.
Asunto(s)
Quimiotaxis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre/citología , Ácido gamma-Aminobutírico/farmacología , Antígeno AC133 , Antígenos CD/metabolismo , Baclofeno/análogos & derivados , Baclofeno/farmacología , Bicuculina/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Sangre Fetal/citología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antagonistas del GABA/farmacología , Antagonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-B , Expresión Génica , Glicoproteínas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Neuronas/citología , Neuronas/metabolismo , Péptidos/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismoRESUMEN
Previous studies described that neurons could be generated in vitro from human umbilical cord blood cells. However, there are few data concerning their origin. Notably, cells generating neurons are not well characterized. The present study deals with the origin of cord blood cells generating neurons and mechanisms allowing the neuronal differentiation. We studied neuronal markers of both total fractions of cord blood and stem/progenitor cord blood cells before and after selections and cultures. We also compared neuronal commitment of cord blood cells to that observed for the neuronal cell line SK-N-BE(2). Before cultures, neuronal markers are found within the total fraction of cord blood cells. In CD133+ stem/progenitor cell fraction only immature neuronal markers are detected. However, CD133+ cells are unable to give rise to neurons in cultures, whereas this is achieved when total fraction of cord blood cells is used. In fact, mature functional neurons can be generated from CD133+ cells only in cell-to-cell close contact with either CD133- fraction or a neurogenic epithelium. Furthermore, since CD133+ fraction is heterogenous, we used several selections to precisely identify the phenotype of cord blood-derived neuronal stem/progenitor cells. Results reveal that only CD34- cells from CD133+ fraction possess neuronal potential. These data show the phenotype of cord blood neuronal stem/progenitor cells and the crucial role of direct cell-to-cell contact to achieve their commitment. Identifying the neuron supporting factors may be beneficial to the use of cord blood neuronal stem/progenitor cells for regenerative medicine.
Asunto(s)
Antígenos CD34/metabolismo , Antígenos CD/metabolismo , Comunicación Celular , Sangre Fetal/citología , Glicoproteínas/metabolismo , Neuronas/citología , Péptidos/metabolismo , Células Madre/citología , Antígeno AC133 , Diferenciación Celular , Línea Celular , Humanos , Fracciones Subcelulares/metabolismoRESUMEN
Chameleons (Order, Reptilia: Family, Lacertilia) are unique among vertebrates in being able to make independent eye movements. The organisation of their retina, however, closely ressembles that of other diurnal lizards; based on morphological studies, it is typically described as containing only cone photoreceptors. We show here that a subpopulation of the photoreceptors are immunolabelled by an antibody directed against rhodopsin, suggesting the presence of rods. We conclude that in the nonmammalian retina, rods and cones cannot be exclusively distinguished on purely morphological grounds.
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Lagartos/metabolismo , Células Fotorreceptoras Retinianas Conos/química , Rodopsina/análisis , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Colorantes Fluorescentes , Inmunohistoquímica/métodos , Indoles , Microscopía Fluorescente , Aglutinina de Mani , Células Fotorreceptoras Retinianas Bastones/química , Rodopsina/inmunologíaRESUMEN
Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.