RESUMO
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.
Assuntos
Astrócitos/metabolismo , Proteína Glial Fibrilar Ácida/biossíntese , Vimentina/fisiologia , Animais , Astrócitos/citologia , Astrócitos/ultraestrutura , Sequência de Bases , Lesões Encefálicas/metabolismo , Células Cultivadas , Primers do DNA , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Transfecção , Vimentina/deficiência , Vimentina/genéticaRESUMO
For several decades, the reactive gliosis that occurs after an injury to the CNS has been considered one of the major impediments to axonal regeneration. Nevertheless, recent studies have suggested that in certain conditions, reactive astrocytes may provide a permissive substratum to support axonal regrowth. The important criteria, allowing for the distinction between permissive and non-permissive gliosis, are the ultrastructural 3D organization of the scar and more importantly the recognition molecules expressed by reactive astrocytes. Reactive astrocytes express surface molecules and produce various neurotrophic factors and cytokines. The latter in turn might modulate the production of recognition molecules by reactive astrocytes, allowing them to support post-lesional axonal regrowth. Although numerous recent articles have focused on cytokines and cell adhesion molecules, scant attention has been paid to reactive astrocytes. Reactive astrocytes should be considered a key element, like neurons, of a dynamic environment, thus forming with neurons a functional unit involved in homeostasis, plasticity and neurotransmission. Attempts are in progress to identify molecular markers for reactive astrocytes.
Assuntos
Astrócitos/fisiologia , Encéfalo/citologia , Animais , Astrócitos/metabolismo , Axônios/fisiologia , HumanosRESUMO
In the past year, the roles and mechanisms of molecules involved in cell survival (glial-derived neurotrophic growth factor), growth cone guidance (netrins and semaphorins), axonal outgrowth and sorting (neural cadherin, polysialylated neural cell adhesion molecules, and L1), and neuronal connectivity (cell adhesion molecules, dystroglycan, and agrin) have been described during development and, to a limited extent, in the mature CNS. Evidence is now emerging that some developmental events, such as the expression of polysialylated neural cell adhesion molecule and L1, are recapitulated during adult CNS regeneration. These results suggest new avenues to address more accurately the challenges of axonal regrowth in the adult mammalian CNS.
Assuntos
Axônios/fisiologia , Sistema Nervoso Central/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Animais , Sobrevivência Celular/fisiologiaRESUMO
The combination of gene transfer techniques and cell transplantation is a promising approach to deliver therapeutic molecules into the CNS. To optimize gene transfer systems, several neural and nonneural cell types are currently under investigation. Among these cells, astrocytes are particularly well suited because of their CNS origin, their efficient secretory mechanisms, and their role as neuronal support. Most importantly, the use of human adult astrocytes as cellular vehicles for ex vivo gene transfer may open the way to autologous transplantation, thus obviating immunological rejection and the side effects of immunosuppressors. In the present study, we report the ability of these cells to be expanded and genetically modified in vitro. Astrocytes derived from human adult cerebral cortex were grown and maintained in vitro as pure primary cultures for at least 10 months. In addition, cells were efficiently transduced by an adenoviral vector encoding human tyrosine hydroxylase (hTH) under the negative control of the tetracycline-based regulatory system (tet-off). The infected cells synthesized large amounts of active hTH and released L-dopa. In addition, doxycycline, a potent analog of tetracycline, efficiently regulated transgene expression. This work is a first step toward the development of therapeutic strategies based on the use of genetically engineered human adult astrocytes for autologous transplantation in human neurodegenerative diseases and CNS trauma.
Assuntos
Astrócitos/transplante , Transplante de Células , Córtex Cerebral/citologia , Terapia Genética , Adenoviridae/genética , Adulto , Idoso , Astrócitos/enzimologia , Divisão Celular , Células Cultivadas , Congelamento , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Tetraciclinas/farmacologia , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
A previous report has shown that small diameter serotoninergic (5-HT) axons innervating the forebrain are selectively eliminated by treatment with an amphetamine derivative, (+/-)p-chloroamphetamine (PCA; Mamounas et al., [1991] J. Comp. Neurol. 314:558-586). It is well known that the spinal cord is the target of numerous monoaminergic fibers of different types. We have previously shown that the dorsal and ventral horns and the intermediolateral cell column are innervated by numerous serotonin-, noradrenaline- and dopamine-containing axons, including both thin axons with small varicosities and beaded axons with large varicosities. In all these regions, the large majority of fine indolaminergic fibers do not establish synaptic contacts, contrasting with the beaded axons which mostly exhibit synapses. The present work was conducted to study the effect of PCA on the monoaminergic innervation of the adult rat spinal cord. Animals received two subcutaneous doses of PCA 24 hours apart and were perfused 3 weeks later. Immunocytochemistry was performed to detect 5-HT, noradrenergic and dopaminergic fibers by using light and electron microscopy. Light microscopy revealed that PCA treatment caused a marked and selective elimination of the fine 5-HT-immunoreactive fibers, mainly found in the dorsal horn, but spared all other larger axons. This selective effect on the dorsal horn innervating thin 5-HT fibers was confirmed with the electron microscope by calculating the synaptic incidence(s) of monoaminergic innervation. These results suggest that fine and beaded 5-HT axons correspond to two anatomically, biochemically and pharmacologically different types of fibers, which could arise from two subpopulations of brainstem neurons. In addition, this drug could be used to provide an experimental animal, devoid of 5-HT nonsynaptic fibers, thereby facilitating a study on the role of dorsal horn nonsynaptic system in pain modulation.
Assuntos
Dopamina/metabolismo , Norepinefrina/metabolismo , Serotonina/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , p-Cloroanfetamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-DawleyRESUMO
The objective of the present study was to evaluate the expression of polysialic acid (PSA) and the cell adhesion molecule L1 during axonal regeneration and sprouting after injury to the adult rat brain. All animals received a complete lesion of the fimbria-fornix (FF). Grafts of nerve growth factor (NGF)- or beta-galactosidase (betaGal)-producing fibroblasts were placed in the FF lesion cavity and induced septohippocampal cholinergic regeneration or sympathetic tyrosine hydroxylase (TH)-positive sprouting, respectively. Cholinergic regeneration was evaluated from 2 to 8 weeks following grafting of NGF-producing fibroblasts in the FF lesion cavity. In the graft area, choline acetyltransferase (ChAT)-positive fibers expressed L1 and PSA. Once cholinergic axons reached the hippocampal formation (HF), they no longer expressed L1 or PSA. Eight weeks after a lesion of the FF and transplantation of betaGal-producing fibroblasts, TH-positive fibers sprouted in the denervated HF and expressed L1 but not PSA. At the zone of reactive gliosis, PSA but not L1 expression was increased following a lesion of the FF and transplantation of NGF- or betaGal-producing fibroblasts. In animals that received a graft of NGF-producing fibroblasts in the FF lesion cavity, numerous ChAT-positive axons were observed along these areas rich in PSA and reactive astrocytes. Taken together, these results suggest that the expression of PSA and L1 is upregulated on regenerating cholinergic axons during axonal elongation and downregulated upon target innervation. In contrast, TH-positive fibers that sprout in the denervated HF express and maintain their expression of L1. Finally, the expression of PSA in the area of reactive gliosis may contribute to a permissive environment for axonal regrowth.
Assuntos
Transplante de Tecido Encefálico , Hipocampo/metabolismo , Hipocampo/transplante , Glicoproteínas de Membrana/biossíntese , Moléculas de Adesão de Célula Nervosa/biossíntese , Ácidos Siálicos/biossíntese , Fatores Etários , Animais , Moléculas de Adesão Celular/análise , Colina O-Acetiltransferase/análise , Fibras Colinérgicas/química , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/enzimologia , Colágeno/farmacologia , Feminino , Fibroblastos/química , Fibroblastos/transplante , Fibroblastos/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Genes Virais , Proteína Glial Fibrilar Ácida/análise , Hipocampo/química , Complexo Antígeno L1 Leucocitário , Glicoproteínas de Membrana/análise , Microscopia Confocal , Microscopia Eletrônica , Degeneração Neural/metabolismo , Regeneração Nervosa/fisiologia , Moléculas de Adesão de Célula Nervosa/análise , Neurônios/química , Neurônios/transplante , Neurônios/ultraestrutura , Ratos , Ratos Endogâmicos F344 , Núcleos Septais/química , Núcleos Septais/citologia , Núcleos Septais/metabolismo , Ácidos Siálicos/análise , Tirosina 3-Mono-Oxigenase/análiseRESUMO
We investigated the synaptic incidence of the contacts established by serotonergic and noradrenergic descending fibers in the dorsal horn of the rat spinal cord. Serial electron microscopic sections were performed. Synapses were scarce. The majority of serotonergic and noradrenergic varicosities (more than 60%) are characterized by nonsynaptic contacts. Numerous glial profiles, and particularly astrocytic profiles, were observed in apposition with serotonergic and noradrenergic varicosities. The proportion of astroglia was higher around serotonergic and noradrenergic varicosities devoid of synaptic specialization. The length of the contact between immunoreactive nonsynaptic varicosities and astrocytes was twice as long as that between synaptic varicosities and astrocytes. Thus, the modulation of sensitive messages by serotonin and noradrenaline through pauci-synaptic varicosities in the dorsal horn of the spinal cord could be an example of the concept of "volume transmission" [Fuxe and Agnati (1991) Volume Transmission in the Brain: Novel Mechanisms for Neural Transmission, Advances in Neuroscience, Vol. 1, pp. 1-9.] in the central nervous system. Analysis of the microenvironment of serotonergic and noradrenergic varicosities led us to make the hypothesis that glial cells, particularly astrocytes, could play some role in volume transmission.
Assuntos
Neuroglia/fisiologia , Neurônios/fisiologia , Norepinefrina/fisiologia , Serotonina/fisiologia , Medula Espinal/citologia , Sinapses/fisiologia , Animais , Imuno-Histoquímica , Masculino , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Norepinefrina/imunologia , Ratos , Ratos Sprague-Dawley , Serotonina/imunologia , Medula Espinal/fisiologia , Medula Espinal/ultraestrutura , Sinapses/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor disturbances caused by an alteration of the dopaminergic nigrostriatal system. Current symptomatic treatments for PD include dopaminergic drug administration, deep brain stimulation, ablative surgery and fetal cell transplantation. Though these approaches have significant beneficial effects, they are hampered by limiting side-effects, but more importantly they do not change the disease progression. Alternative restorative and neuroprotective strategies have therefore to be considered. Neuroprotective effects of neurotrophic factors, anti-apoptotic and antioxidant molecules are currently being investigated for this purpose. Among neurotrophic molecules, the potential of the glial cell line-derived neurotrophic factor (GDNF) to protect the nigral dopaminergic neurons and/or rescue striatal dopamine levels has been extensively documented. For GDNF to become a clinical reality, appropriate delivery techniques will have to be developed. This chapter focuses on the potential of encapsulated cells and viral vectors to locally release neurotrophic factors in experimental models of PD.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Fatores de Crescimento Neural , Proteínas do Tecido Nervoso/genética , Fármacos Neuroprotetores , Doença de Parkinson/terapia , Animais , Fator Neurotrófico Derivado de Linhagem de Célula Glial , HumanosRESUMO
The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within non-dopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltransferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the noradrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.
Assuntos
Fibras Adrenérgicas/química , Dopamina/análise , Bulbo/química , Mesencéfalo/química , Neurônios/química , Norepinefrina/análise , Animais , Axônios/química , Dopamina/fisiologia , Técnica Indireta de Fluorescência para Anticorpo , Imunoglobulina G/imunologia , Masculino , Bulbo/citologia , Mesencéfalo/citologia , Microscopia Confocal , Feniletanolamina N-Metiltransferase/análise , Ratos , Ratos Sprague-Dawley , Medula Espinal/químicaRESUMO
The mapping of the spinal dopaminergic innervation has been performed in the adult rat using an anti-dopamine antiserum. Immunoreactive fibers were detected with the light microscope in the dorsal horn (mainly in laminae III-IV), in the intermediolateral cell column (IML), in the peri-ependymal region and in the ventral horn. The ultrastructural analysis of dopaminergic innervation showed mainly axodendritic contacts and fewer axosomatic ones. In the ventral horn and the IML, the pattern of dopaminergic innervation exhibited a majority of classical synapses. In the dorsal horn, dopaminergic innervation was partly non-synaptic (at cervical level), whereas numerous axodendritic synapses were observed at thoraco-lumbar level. Previous studies described the non-synaptic organization of serotonergic and noradrenergic projections in the dorsal horn. It is thus hypothesized that the monoaminergic systems, involved in pain modulation within the dorsal horn, act partly through volume transmission. In contrast, these systems would modulate the motor and autonomic functions through classical synapses.
Assuntos
Dopamina/análise , Medula Espinal/química , Sinapses/química , Animais , Soros Imunes/imunologia , Imuno-Histoquímica , Masculino , Microscopia , Microscopia Eletrônica , Ratos , Ratos Sprague-DawleyRESUMO
To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.
Assuntos
Glicoconjugados/metabolismo , Lectinas/metabolismo , Análise em Microsséries , Células CACO-2 , Glicoconjugados/química , Humanos , Lectinas/química , Estrutura Molecular , Polissacarídeos/química , Polissacarídeos/metabolismoRESUMO
Over the last decade, the combination of molecular biology and cell transplantation techniques has given rise to a powerful method for gene therapy. The implantation of genetically modified cultured cells has been extensively used in the central nervous system (CNS) in various experimental models of neurologic disorders. More recently, viral and chemical methods have been developed to further efforts to shuttle transgenes into the relatively inaccessible brain. Adenoviral and liposomal synthetic vectors carry transgenes into neural tissue in situ and are beginning to show promise as new methods for CNS therapy.
Assuntos
Doenças do Sistema Nervoso Central/terapia , Terapia Genética , Animais , Transplante de Células , Técnicas de Transferência de Genes , Vetores Genéticos , HumanosRESUMO
In the present study, we performed the immunodetection of serotonergic (5-HT) receptor subtypes in the spinal cord by using an anti-idiotypic antiserum (TH8) at light and electron microscopic levels. This antibody has been shown to recognize 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes (Tamir et al.: J Neurochem 57:930-942, 1991). The TH8 immunoreactivity was observed in the dorsal and ventral horns of the gray matter. Light microscopy revealed that small cell bodies located in laminae I-III of the dorsal horn were intensely immunolabeled. A more homogenous and discrete staining was also observed throughout the entire dorsal horn. In the ventral horn, motoneurons were also immunoreactive (IR). Peroxidase deposits were observed as numerous patches covering the motoneuronal surface. Numerous interneurons were moderately and homogeneously immunostained. With the electron microscope, most of the labeled structures were identified as neurons (dendrites and perikarya) in both the dorsal and ventral horns. In the dorsal horn, immunoreactivity was present in dendrites and neuronal perikarya. A large majority of the immunoreactivity found in dendrites was not associated with synaptic differentiations. Indeed, the dendrites, in which peroxidase deposit was seen, were not locally involved in synapses. Very scarce synaptic varicosities were observed in close apposition with IR dendrites. In the ventral horn, TH8 immunoreactivity was present in dendrites, with an accumulation of peroxidase deposit on the active zone of synapses, facing presynaptic membranes. Both the postsynaptic membrane and the submembrane area were IR. In addition, a few astroglial fine processes were immunostained; most of them were observed in the dorsal horn. Scarce IR astroglial profiles were observed in the ventral horn. These observations show that such an antiserum constitutes a useful tool for the ultrastructural analysis of 5-HT receptor distribution. Finally, correlation between the immunocytochemical localization of 5-HT receptor subtypes and the modes of 5-HT transmission in the spinal cord (wiring and volume transmissions) is discussed in the present report.
Assuntos
Receptores de Serotonina/metabolismo , Medula Espinal/metabolismo , Animais , Especificidade de Anticorpos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Neurônios Motores/imunologia , Neurônios Motores/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Serotonina/imunologia , Medula Espinal/citologiaRESUMO
At the transition between central nervous system (CNS) and peripheral nervous system (PNS), the CNS compartment forms cone-shaped incursions into the peripheral part of the dorsal root. The ultrastructural study of the CNS-PNS transitional zone shows that this region is particularly rich in astrocyte processes. In an attempt to investigate the possible role of the CNS-PNS interface astrocytes in myelin formation, a photonic microscopy immunocytochemical study has been done with anti-GFAP and anti-MBP sera. The CNS glial expansion shows an important GFAP immunoreactivity with intimate association between astrocyte processes and myelinated axons. This may indicate that the transitional myelin originates from astrocytes. The same region is also MBP-positive. Two explanations are considered: some astrocytes form transitional myelin sheathes and express MBP epitopes, or oligodendrocytes, with cell bodies distant from the CNS-PNS interface, send myelinating cytoplasmic expansions which are not shown by the techniques we used.
Assuntos
Astrócitos/fisiologia , Sistema Nervoso Central/fisiologia , Neuroglia/fisiologia , Nervos Periféricos/fisiologia , Animais , Astrócitos/ultraestrutura , Sistema Nervoso Central/ultraestrutura , Epitopos/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Neuritos/metabolismo , Neuritos/ultraestrutura , Neuroglia/ultraestrutura , Nervos Periféricos/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
Astrocytes are a good candidate cell type for brain transplantation: They are endogenous to the CNS, they have efficient secretory machinery, and they play a major role in neuronal support. We assessed the potential of genetically modified primary adult human astrocytes as vehicles for the delivery of secreted molecules in the mammalian CNS. We report that such cells can be efficiently transduced by a recombinant adenoviral vector carrying the human beta-glucuronidase cDNA (Ad/CMV*beta-glu) and that the transduced astrocytes produce large amounts of the enzyme. Released beta-glucuronidase could be captured, in vitro, by primary neurons and astrocytes and by a neuroblastoma cell line and beta-glucuronidase-deficient fibroblasts. Following grafting into the mouse striatum, adult human astrocytes survived and expressed the transgene for at least 8 weeks. Moreover, the dosage of beta-glucuronidase activity within the grafted brains revealed high enzymatic levels at a long distance from the graft. These experiments document the grafting of engineered primary adult human astrocytes, allowing the release of a secreted therapeutic factor throughout the brain.
Assuntos
Adenoviridae/genética , Astrócitos/enzimologia , Transplante de Células , Corpo Estriado/cirurgia , Glucuronidase/genética , Adulto , Animais , Astrócitos/transplante , Divisão Celular , Células Cultivadas , Corpo Estriado/enzimologia , Expressão Gênica , Terapia Genética/métodos , Vetores Genéticos , Glucuronidase/metabolismo , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Transdução Genética , TransgenesRESUMO
A specific monoclonal antiserum (Mab 6.17) inducing a strong immunostaining of the neuromuscular junction has been used to detect the possible occurrence of the corresponding antigen throughout the intact or lesioned central nervous system of adult rats. In intact animals, 6.17-immunolabeling was essentially detected in astrocyte-like structures located in white matter fasciculi of the brain, such as the optic tract, corpus callosum, fornix, and in the white matter of the spinal cord. The astroglial nature of such 6.17-immunolabeled profiles was verified by performing double or triple immunofluorescent labeling with Mab 6.17 and with specific antisera against astrocytic markers, such as S100 protein, glial fibrillary acidic protein and vimentin. In the white matter, all the structures reactive to Mab 6.17 were also reactive to antibodies against S100 protein, glial fibrillary acidic protein and vimentin. On the other hand, astrocytes of the grey matter that were immunoreactive to S100 and glial fibrillary acidic protein but negative to vimentin, were devoid of 6.17-immunoreactivity. After lesions including stab wound through the diencephalon or transection of the spinal cord, a marked increase of 6.17-immunostaining was noted in the regions surrounding the lesions. In these regions, 6.17-immunolabeling was associated with S100-, GFAP- and vimentin-positive astrocytes constituting the glial scar. The ultrastructural localization of 6.17-immunoreactivity indicated that, similar to glial fibrillary acidic protein and vimentin, the recognized antigen was mainly associated with gliofilaments. These observations indicate that, in the central nervous system of adult rats, Mab 6.17 recognizes a molecule associated with gliofilaments, which is essentially associated to reactive astrocytes expressing high levels of vimentin.
Assuntos
Astrócitos/química , Biomarcadores/análise , Proteínas/análise , Animais , Anticorpos Monoclonais , Encéfalo/citologia , Química Encefálica , Técnica Direta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/análise , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Proteínas/imunologia , Ratos , Ratos Sprague-Dawley , Proteínas S100/análise , Medula Espinal/química , Medula Espinal/citologia , Vimentina/análiseRESUMO
Parkinson's disease (PD) is characterized by the progressive loss of substantia nigra dopaminergic neurons and the presence of cytoplasmic inclusions named Lewy bodies. Two missense mutations of the alpha-synuclein (alpha-syn; A30P and A53T) have been described in several families with an autosomal dominant form of PD. alpha-Syn also constitutes one of the main components of Lewy bodies in sporadic cases of PD. To develop an animal model of PD, lentiviral vectors expressing different human or rat forms of alpha-syn were injected into the substantia nigra of rats. In contrast to transgenic mice models, a selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum was observed in animals expressing either wild-type or mutant forms of human alpha-syn. This neuronal degeneration correlates with the appearance of abundant alpha-syn-positive inclusions and extensive neuritic pathology detected with both alpha-syn and silver staining. Lentiviral-mediated expression of wild-type or mutated forms of human alpha-syn recapitulates the essential neuropathological features of PD. Rat alpha-syn similarly leads to protein aggregation but without cell loss, suggesting that inclusions are not the primary cause of cell degeneration in PD. Viral-mediated genetic models may contribute to elucidate the mechanism of alpha-syn-induced cell death and allow the screening of candidate therapeutic molecules.
Assuntos
Dopamina , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/patologia , Doença de Parkinson/patologia , Animais , Animais Geneticamente Modificados , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos , HIV-1 , Humanos , Lentivirus , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Ratos , Substância Negra/metabolismo , Sinucleínas , Células Tumorais Cultivadas , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-SinucleínaRESUMO
The persistence of high levels of B-50 (GAP-43) in fibers innervating various regions of the adult central nervous system is generally thought to characterize neuronal systems capable of undergoing morphological plasticity. In a recent series of in situ hybridization studies, it has been shown that most catecholaminergic and serotonergic neurons of the adult rat brain express high levels of B-50 mRNA. The present study addresses the question whether high expression of B-50 mRNA in the catecholaminergic and serotonergic perikarya corresponds with detectable high levels of the B-50 protein in the efferent axonal fibers that innervate various regions of the adult rat brain and spinal cord. For this purpose, vibratome sections were doubly immunostained for B-50 and for tyrosine hydroxylase or serotonin and were analyzed by laser scanning confocal microscope. Colocalizations were investigated either (1) in regions of intact rat brain and spinal cord in which particular concentrations of B-50 immunoreactive fibers appeared codistributed with catecholaminergic or serotonergic fibers or (2) in intrahypothalamic portions of the medial forebrain bundle in which a surgical lesion was made. In the intact brain, frequent colocalizations of B-50 and tyrosine hydroxylase were detected in fibers innervating both the mediobasal hypothalamus and the neurointermediate hypophysial lobe. In all the other regions examined, the analysis of thin optical sections demonstrated that immunoreactivity to B-50 was only rarely associated with axonal profiles immunoreactive to tyrosine hydroxylase or to serotonin. By contrast, in the lesioned medial forebrain bundle B-50 immunoreactivity was found to be associated with numerous catecholaminergic and serotonergic axonal sprouts that regenerate around the surgical lesion. These data indicate that the majority of intact catecholaminergic and serotonergic axons innervating the adult rat brain and spinal cord contains low levels of B-50. However, following axotomy, B-50 is immunocytochemically detectable in the regenerating sprouts produced by both types of axonal fibers. This suggests that under basal conditions the relatively high content of B-50 mRNA in monoaminergic perikarya does not lead to appreciable accumulation of B-50 within corresponding axonal fibers and terminals, whereas conditions of morphological reorganization induce increased production of B-50 that accumulates within monoaminergic axonal sprouts.
Assuntos
Axônios/metabolismo , Encéfalo/metabolismo , Catecolaminas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serotonina/metabolismo , Medula Espinal/metabolismo , Animais , Encéfalo/ultraestrutura , Denervação , Imunofluorescência , Proteína GAP-43 , Imuno-Histoquímica/métodos , Masculino , Microscopia Confocal , Ratos , Ratos Sprague-Dawley , Medula Espinal/ultraestrutura , Coloração e Rotulagem , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The mapping of noradrenergic innervation was performed in transverse and longitudinal sections of the adult rat spinal cord using noradrenaline immunocytochemistry. Noradrenergic fibres and terminals were distributed in the dorsal horn (mainly in the superficial part), in the vicinity of the different groups of motoneurons, and concentrated in the intermediolateral cell column and around the central canal. The ultrastructural study showed principally axodendritic synapses in the ventral horn and in the intermediolateral cell column. Fewer axosomatic synapses were detected. In the dorsal horn, noradrenaline-innervation was predominantly non-synaptic. It is hypothesized that the noradrenergic modulation of nociception is not mediated through classical synapses. The concept of 'volume transmission' can explain such an influence. Conversely, noradrenaline may be involved in the control of locomotion and automatic functions through conventional synapses.
Assuntos
Técnicas Imunoenzimáticas , Norepinefrina/análise , Medula Espinal/ultraestrutura , Animais , Axônios/ultraestrutura , Dendritos/ultraestrutura , Masculino , Microscopia Eletrônica , Neurônios Motores/ultraestrutura , Terminações Nervosas/ultraestrutura , Fibras Nervosas/ultraestrutura , Norepinefrina/imunologia , Norepinefrina/fisiologia , Ratos , Ratos Endogâmicos , Sinapses/ultraestruturaRESUMO
Gene delivery to the central nervous system is central to the development of gene therapy for neurological diseases. We developed a baculovirus-derived vector, the Bac-CMV-GFP vector, containing a reporter gene encoding for the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. Two neuroblastomal cell lines and three human primary neural cultures could be efficiently transduced. In all cases, addition of butyrate, an inhibitor of histone deacetylase, increased the level of expression in terms of the number of GFP-expressing cells and the intensity of fluorescence. The level of expression in a human telencephalic culture was over 50% of transduced cells with a multiplicity of infection of 25. GFP expression was demonstrated to be genuine expression and not pseudotransduction of the reporter protein. Most interestingly, Bac-CMV-GFP could transduce neural cells in vivo when directly injected into the brain of rodents and was not inactivated by the complement system. Thus, baculovirus is a promising tool for gene transfer into the central nervous system both for studies of the function of foreign genes and the development of gene therapy strategies.