RESUMO
Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.
Assuntos
Envelhecimento/metabolismo , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Hiperprolactinemia/genética , Hiperprolactinemia/terapia , Adenoviridae/genética , Animais , Antígenos Nucleares/metabolismo , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Contagem de Células , Células Cultivadas , Doença Crônica/terapia , Feminino , Genes Reporter/genética , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Hiperprolactinemia/metabolismo , Lactotrofos/metabolismo , Microinjeções/métodos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Prolactina/análise , Prolactina/sangue , Prolactina/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/genética , Resultado do Tratamento , Túber Cinéreo/metabolismo , Túber Cinéreo/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismo , beta-Galactosidase/genéticaRESUMO
PURPOSE: In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. METHODS: Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. RESULTS: Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [(3)H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. CONCLUSIONS: These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.
Assuntos
Ácidos Docosa-Hexaenoicos/farmacologia , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/farmacologia , Células Fotorreceptoras de Vertebrados/citologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina G , Ciclina G1 , Ciclinas/metabolismo , DNA/biossíntese , Combinação de Medicamentos , Sinergismo Farmacológico , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Mitocôndrias/fisiologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Ratos Wistar , Opsinas de Bastonetes/metabolismoRESUMO
OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.
Assuntos
Técnicas de Transferência de Genes , Adeno-Hipófise/fisiologia , Hormônios Hipofisários/metabolismo , Animais , Células Cultivadas , Feminino , Expressão Gênica , Vetores Genéticos , Hormônio do Crescimento/metabolismo , Herpesvirus Humano 1/genética , Hiperplasia , Mutação , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Adeno-Hipófise/patologia , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Prolactina/sangue , Prolactina/metabolismo , Prolactinoma/genética , Prolactinoma/metabolismo , Prolactinoma/patologia , Ratos , Ratos Sprague-Dawley , Valores de Referência , Transgenes/fisiologiaRESUMO
Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells.
Assuntos
Fatores de Crescimento Neural/farmacologia , Neuroblastoma/patologia , Neurônios/citologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Diferenciação Celular , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Camundongos , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Neurotrofina 3/farmacologia , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
Previous studies have shown that extracts from the target optic tectum stimulate neurite outgrowth from retinal explants. The present study indicates that the choroid coat is an even richer source of retinotrophic activity. We thus studied the effects of recombinant rat ciliary neurotrophic factor (CNTF) on primary cultures of dissociated chick ciliary ganglion neurons and retinal explants for a comparison with choroid coat extract from the E18 chick. For our assays, E9 ciliary neurons were incubated in collagen gels and retinal explants were cultured on collagen gels with the addition of the trophic factors and maintained for two or four days. Survival of ciliary neurons per area as well as maximal neurite length in retinal cultures were determined. Growth responses occurred in a dose-dependent manner both to CNTF and choroid extract. Immunofluorescence examination of cells and developing processes showed 200 kdal neurofilament positivity demonstrating that the cells studied were neurons with neurites. It is concluded that a trophic activity of the choroid as well as the recombinant CNTF stimulate retinal neuron survival and neurite extension. The results suggest that CNTF may have developmental functions in the establishment of the visual pathways.
Assuntos
Corioide/fisiologia , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neuritos/efeitos dos fármacos , Retina/crescimento & desenvolvimento , Extratos de Tecidos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Fator Neurotrófico Ciliar , Imunofluorescência , Imuno-Histoquímica , Neuritos/ultraestrutura , Ratos , Proteínas Recombinantes/farmacologia , Retina/efeitos dos fármacos , Retina/ultraestrutura , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/transplante , Estimulação QuímicaAssuntos
Integrinas/análise , Retina/química , Animais , Imunofluorescência , Imuno-Histoquímica , Integrinas/química , Ratos , Ratos EndogâmicosRESUMO
Labeled retinal ganglion cells from neonatal rats extended neurites in dissociated cell culture as a cell type-specific response to the influence of a superior collicular extract. The molecule responsible for this neuritogenic effect is soluble and non-dialysable (> 12 kDa). Nerve growth factor had a neuritogenic effect both on ganglion cells and other types of retinal cells.