RESUMO
PURPOSE: To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²âº signaling in ONHAs lacking functional endothelin B (ETB) receptors. METHODS: ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10â»7 or 10â»9 M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²âº](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. RESULTS: ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10â»7 M and by 33% at 10â»9 M) and 72 hours (by 117% at 10â»7 M and by 100% at 10â»9 M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10â»7 M and by 48% at 10â»9 M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. CONCLUSIONS: ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²âº](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²âº](i) elevation.
Assuntos
Astrócitos/patologia , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endotelina-1/farmacologia , Disco Óptico/patologia , Receptor de Endotelina B/deficiência , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Separação Celular , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Técnica Indireta de Fluorescência para Anticorpo , Fura-2/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Microscopia Confocal , Disco Óptico/metabolismo , Ratos , Ratos Endogâmicos WKY , Ratos Transgênicos , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismoRESUMO
AIM: To characterise the influence of endothelin-1 (ET-1), a vasoactive peptide, and its receptors (endothelin B (ETB) and endothelin A (ETA)) on rat optic nerve head astrocyte (ONHA) proliferation. METHODS: ONHAs were isolated from adult Brown Norway rats. ONHA specificity was determined with immunohistochemistry for: glial fibrillary acidic protein (GFAP); A2B5, a marker of type II astrocytes located outside the ONH; and myelin basic protein (MBP). ONHA proliferation was quantified following treatment with ET-1 (1x10(-6), 1x10(-7), 1x10(-9) or 1x10(-11) M) or vehicle for 24, 48 or 72 h. ETB and ETA antagonists were used to assess the role of each receptor in ONHA proliferation. RESULTS: ONHA specificity was confirmed with positive labelling for GFAP, and negative labelling for A2B5 and MBP. ONHAs also expressed ETB and ETA. Cell percentages increased significantly beginning 48 h after ET-1 exposure with 1x10(-7) (20%) and 1x10(-9) M (15%). After 72 h, ONHA percentages increased significantly at all ET-1 concentrations (25%, 21%, 29%, 28% increases relative to vehicle, for 1x10(-6), 1x10(-7), 1x10(-9) and 1x10(-11) M, respectively). No significant proliferation occurred in the presence of either antagonist. CONCLUSION: ONHAs proliferated following 48 h or more of exposure to ET-1. The proliferation required both ETB and ETA receptors.
Assuntos
Astrócitos/citologia , Endotelina-1/fisiologia , Disco Óptico/citologia , Receptor de Endotelina A/fisiologia , Receptor de Endotelina B/fisiologia , Animais , Proliferação de Células , Antagonistas do Receptor de Endotelina A , Antagonistas do Receptor de Endotelina B , Masculino , Oligopeptídeos/farmacologia , Piperidinas/farmacologia , RatosRESUMO
PURPOSE: During development, all retinal cells express polysialylated neural cell adhesion molecule (PSA-NCAM). PSA is localized only on glia in the adult retina, but as Müller glial processes ensheathe most retinal neurons, PSA remains in the extracellular environment of adult neurons. The authors sought to investigate the influence of endogenous PSA on the survival of neonatal as well as adult normal and injured retinal ganglion cells (RGCs). METHODS: Endogenous retinal PSA was selectively degraded by application of endoneuraminidase. PSA presence and removal were confirmed by immunohistochemistry and levels were assessed by Western Blot analysis. Neonatal RGC survival after PSA removal was assessed in vitro in RGCs immunopanned from rat pups. Adult RGC survival was assessed in vivo in mice by investigating RGC densities after removal of PSA in normal retinas and after optic nerve transection. RESULTS: Virtually all neonatal RGCs express PSA-NCAM and survive well in vitro; however, removal of PSA resulted in a 42% loss of these cells 3 days after the treatment. Similarly, removal of PSA in the adult retina in vivo induced a loss of 25% of RGCs at 14 days, and significantly reduced RGC densities after optic nerve transection by an additional 27% (relative to injured retinas with a vehicle injection) at 7 days. CONCLUSIONS: Together, these findings demonstrate that endogenous PSA supports the survival of neonatal as well as injured and normal adult RGCs and provide the first functional evidence of a role for PSA in the adult retina.
Assuntos
Molécula L1 de Adesão de Célula Nervosa/fisiologia , Células Ganglionares da Retina/citologia , Ácidos Siálicos/fisiologia , Animais , Western Blotting , Contagem de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase , Traumatismos do Nervo Óptico , Ratos , Ratos Long-EvansRESUMO
Despite the widespread use of caffeine, the neuronal mechanisms underlying its stimulatory effects are not completely understood. By using c-Fos immunohistochemistry as a marker of neuronal activation, we recently showed that stimulant doses of caffeine activate arousal-promoting hypothalamic orexin (hypocretin) neurons. In the present study, we investigated whether other key neurons of the arousal system are also activated by caffeine, via dual immunostaining for c-Fos and transmitter markers. Rats were administered three doses of caffeine or saline vehicle during the light phase. Caffeine at 10 and 30 mg/kg, i.p., increased motor activities, including locomotion, compared with after saline or a higher dose, 75 mg/kg. The three doses of caffeine induced distinct dose-related patterns of c-Fos immunoreactivity in several arousal-promoting areas, including orexin neurons and adjacent neurons containing neither orexin nor melanin-concentrating hormone; tuberomammillary histaminergic neurons; locus coeruleus noradrenergic neurons; noncholinergic basal forebrain neurons that do not contain parvalbumin; and nondopaminergic neurons in the ventral tegmental area. At any dose used, caffeine induced little or no c-Fos expression in cholinergic neurons of the basal forebrain and mesopontine tegmentum; dopaminergic neurons of the ventral tegmental area, central gray, and substantia nigra pars compacta; and serotonergic neurons in the dorsal raphe nucleus. Saline controls exhibited only few c-Fos-positive cells in most of the cell groups examined. These results indicate that motor-stimulatory doses of caffeine induce a remarkably restricted pattern of c-Fos expression in the arousal-promoting system and suggest that this specific neuronal activation may be involved in the behavioral arousal by caffeine.
Assuntos
Nível de Alerta/efeitos dos fármacos , Encéfalo , Cafeína/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Nível de Alerta/fisiologia , Comportamento Animal , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Contagem de Células/métodos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica/métodos , Masculino , Atividade Motora/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Ratos Wistar , Tirosina 3-Mono-Oxigenase/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/metabolismoRESUMO
The purpose of the present experiment was to characterize changes in TrkB signaling in the rat visual system resulting from exposure to enriched environment. Female Sprague-Dawley rats were placed in enriched or impoverished conditions for 1, 7 or 28 days. Levels of BDNF protein and its predominant receptor TrkB were examined in the retina, superior colliculus and visual cortex. In the retina, 1 day of enrichment increased full-length TrkB and after 28 days increased BDNF. In the superior colliculus, enrichment for 7 days reduced full-length TrkB and after 28 days increased BDNF and full-length TrkB. One day of enrichment significantly increased BDNF, reduced full-length TrkB and increased truncated TrkB in the visual cortex. Consequently, we further investigated whether exposure to enriched environment and the subsequent changes in BDNF and TrkB translates into a neuroprotective effect on retinal ganglion cells (RGCs) following transection of the optic nerve. Although exogenous intraocular application of BDNF provides neuroprotection to RGCs after axotomy, the endogenous increase in BDNF in the retina after 28 days of enrichment had no effect on RGC survival. While enriched housing conditions offer a model of non-invasive rehabilitation treatment for injury and modulates changes in BDNF and TrkB levels, these molecular changes did not translate into a neuroprotective effect on RGCs following transection of the optic nerve.