RESUMO
Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100ß and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.
Assuntos
Astrócitos/metabolismo , Astrócitos/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nanotecnologia/métodos , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Masculino , Microscopia/métodos , Ratos , Ratos Sprague-DawleyRESUMO
We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.
Assuntos
Citoesqueleto/ultraestrutura , Proteína Glial Fibrilar Ácida/ultraestrutura , Filamentos Intermediários/ultraestrutura , Animais , Cátions Bivalentes , Cristalografia , Técnicas In Vitro , Camundongos , Microscopia Eletrônica , Fragmentos de Peptídeos , Conformação Proteica , Proteínas Recombinantes de Fusão , Proteínas RecombinantesRESUMO
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
Decreased beta-amyloid clearance in Alzheimer's disease and increased blood-brain barrier permeability in aged subjects have been reported in several articles. However, morphological and functional characterization of blood-brain barrier and its membrane transporter activity have not been described in physiological aging yet. The aim of our study was to explore the structural changes in the brain microvessels and possible functional alterations of P-glycoprotein at the blood-brain barrier with aging. Our approach included MR imaging for anatomical orientation in middle aged rats, electronmicroscopy and immunohistochemistry to analyse the alterations at cellular level, dual or triple-probe microdialysis and SPECT to test P-glycoprotein functionality in young and middle aged rats. Our results indicate that the thickness of basal lamina increases, the number of tight junctions decreases and the size of astrocyte endfeet extends with advanced age. On the basis of microdialysis and SPECT results the P-gp function is reduced in old rats. With our multiparametric approach a complex regulation can be suggested which includes elements leading to increased permeability of blood-brain barrier by enhanced paracellular and transcellular transport, and factors working against it. To verify the role of P-gp pumps in brain aging further studies are warranted.
Assuntos
Envelhecimento/fisiologia , Barreira Hematoencefálica/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/ultraestrutura , Fatores Etários , Animais , Área Sob a Curva , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/ultraestrutura , Cromatografia Líquida , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Imageamento por Ressonância Magnética , Masculino , Microdiálise , Microvasos/metabolismo , Microvasos/ultraestrutura , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Using the smallest subunit (NF-L) of a neurofilament and a glial fibrillary acidic protein, the subunit arrangement in intermediate filaments was studied by low-angle rotary shadowing. NF-L formed a pair of 70 to 80 nm rods in a low ionic strength solution at pH 6.8. Two 70 to 80 nm rods appeared to associate in an antiparallel manner with an overlap of about 55 nm, almost the same length as the alpha-helix-rich central rod domain of intermediate filament proteins. The overlap extended for three-beaded segments, present at 22 nm intervals along the pairs of rods. The observations that (1) 70 to 80 nm rods were a predominant structure in a low ionic strength solution at pH 8.5, (2) the molecular weights of the rod and the pair were measured by sedimentation equilibrium as 190,000 and 37,000 respectively, and (3) the rods formed from the trypsin-digested NF-L had a length of about 47 nm, indicated that the 70 to 80 nm rod is the four-chain complex and the pair of rods is the eight-chain complex. Similar structures were observed with glial fibrillary acidic protein, indicating that these oligomeric structures are common to other intermediate filament proteins. NF-L assembled into short intermediate-sized filaments upon dialysis against a low-salt solution containing 1 to 2 mM-MgCl2 at 4 degrees C. The majority of these short filaments possessed four or five-beaded segments, suggesting that the pair of rods were arranged in a half-staggered fashion in neurofilaments. On the basis of these observations, we propose the following model for the intermediate filament subunit arrangement. (1) The four-chain complex is the 70 to 80 nm rod, in which two coiled-coil molecules align in parallel and in register. (2) Two four-chain complexes form the eight-chain complex by associating in an antiparallel fashion with the overlap of the entire central rod domain. (3) The eight-chain complex is the building block of the intermediate filament. The eight-chain complexes are arranged in a half-staggered fashion within the intermediate filament.
Assuntos
Citoesqueleto/ultraestrutura , Proteínas de Filamentos Intermediários/ultraestrutura , Filamentos Intermediários/ultraestrutura , Proteínas de Neurofilamentos , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Proteína Glial Fibrilar Ácida/ultraestrutura , Proteínas de Filamentos Intermediários/isolamento & purificação , Substâncias Macromoleculares , Microscopia Eletrônica , Peso Molecular , Concentração Osmolar , Medula Espinal/ultraestruturaRESUMO
Astrocytosis is a hallmark of damage that frequently occurs during aging in human brain. Astrocytes proliferate in elderly subjects, becoming hypertrophic and highly immunoreactive for glial fibrillary acidic protein (GFAP). These cells are one type that actively responds in the repair and reorganization of damage to the neural parenchyma and are a source of several peptides and growth factors. One of these biomolecules is apolipoprotein D (apo D), a member of the lipocalin family implicated in the transport of small hydrophobic molecules. Although the role of apo D is unknown, increments in brain apo D expression have been observed in association with aging and with some types of neuropathology. We have found an overexpression of apo D mRNA in reactive astrocytes by in situ hybridization in combination with immunohistochemistry for apo D in normal aged human brains. The number of double-labeled cells varied according to the cerebral area and the gliosis grade. The possible significance of this increased synthesis of apo D in reactive astrocytes is discussed in relation to the role of apo D in aging and in glial function.
Assuntos
Apolipoproteínas/biossíntese , Astrócitos/metabolismo , Córtex Cerebral/citologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas D , Astrócitos/citologia , Astrócitos/ultraestrutura , Córtex Cerebral/metabolismo , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Imuno-Histoquímica , Hibridização In Situ , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/biossínteseRESUMO
We identified the phosphorylation sites of glial fibrillary acidic protein (GFAP) for cdc2 kinase and Ca(2+)-calmodulin (CaM)-dependent protein kinase II. GFAP was phosphorylated to approximately 0.2 mol of phosphate/mol of GFAP by cdc2 kinase, and this phosphorylation did not induce disassembly of the filament structure. On the other hand, GFAP was phosphorylated to approximately 1.9 mol of phosphate/mol of GFAP by Ca(2+)-CaM-dependent protein kinase II, and this phosphorylation did induce disassembly of the filament. Sequential analysis of the purified phosphopeptides revealed that only Ser8 on GFAP was phosphorylated by cdc2 kinase, whereas Ser13, Ser17, Ser34, and Ser389 on GFAP were phosphorylated by Ca(2+)-CaM-dependent protein kinase II.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Filamentos Intermediários/metabolismo , Mitose/fisiologia , Dados de Sequência Molecular , Fosforilação , RatosRESUMO
Temporal cortical sections from postmortem brains of individuals without any dementing condition and with different degrees of severity of Alzheimer's disease (AD) evaluated by the Clinical Dementia Rating scale (CDR 0-CDR 3) were analyzed using immunohistochemical procedures. To demonstrate the amyloid-beta-peptide (Abeta) deposition and the neurofibrillary pathology, two monoclonal antibodies were used, a human CERAD Abeta (10D5) antibody raised against the N-terminal region of the Abeta-peptide, and an antibody raised against paired helical filaments (PHF-1). The neuron cell bodies and the glial cells were also recognized by two polyclonal antibodies raised, respectively, against the protein gene peptide (PGP 9.5) and glial fibrillary acidic protein (GFAP). Directly related to severity of AD, progressive deposits of Abeta-peptide were found within cortical pyramidal-like neurons and forming senile plaques. Ultrastructurally, Abeta-peptide deposits were related to neuronal intracytoplasmic organelles, such as the ER, the mitochondria, the Nissl bodies and lipofuscin. We have also found that the intracellular deposition of the Abeta peptide is a neuropathological finding prior to the appearance of PHF-immunoreactive structures. We suggest that the intracellular Abeta deposition in cortical pyramidal neurons is a first neurodegenerative event in AD development and that it is involved in cell dysfunction, neuronal death, and plaque formation.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais/metabolismo , Índice de Gravidade de Doença , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/ultraestrutura , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/ultraestrutura , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/ultraestrutura , Biomarcadores , Proteína Glial Fibrilar Ácida/imunologia , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Neurônios/patologia , Neurônios/ultraestrutura , Lobo Temporal/metabolismo , Lobo Temporal/patologia , Lobo Temporal/ultraestrutura , Ubiquitina Tiolesterase/imunologia , Ubiquitina Tiolesterase/metabolismoRESUMO
During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.
Assuntos
Sistema Nervoso Central/patologia , Citoesqueleto/patologia , Neuroglia/patologia , Enjoo devido ao Movimento em Voo Espacial/fisiopatologia , Ausência de Peso/efeitos adversos , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Apoptose/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Núcleo Celular/ultraestrutura , Tamanho Celular/fisiologia , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Imunofluorescência , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Filamentos Intermediários/metabolismo , Filamentos Intermediários/patologia , Filamentos Intermediários/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica de Varredura , Microtúbulos/metabolismo , Microtúbulos/patologia , Microtúbulos/ultraestrutura , Mitose/fisiologia , Neuroglia/metabolismo , Neuroglia/ultraestrutura , Ratos , Rotação/efeitos adversos , Enjoo devido ao Movimento em Voo Espacial/patologia , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestrutura , Células Tumorais Cultivadas , Vimentina/metabolismo , Vimentina/ultraestrutura , Simulação de Ausência de PesoRESUMO
The consequences of the neurotoxic insult induced by 3,4-methylenedioxymethamphetamine (MDMA, an amphetamine derivative with specific action on the serotonergic system) were compared with those of methamphetamine (a derivative with specific action on dopaminergic system) in rats. Both drugs induced a very similar loss of body weight, especially evident 24 h after treatment. Their hyperthermic profile was also very similar and was dependent on ambient temperature, corroborating the thermo-dysregulatory effect of both substances. Methamphetamine (four injections of 10 mg kg(-1) s.c. at 2-h intervals) induced the loss of dopaminergic (35%) but not of serotonergic, terminals in the rat striatum and, simultaneously, a significant increase in striatal peripheral-type benzodiazepine receptor density, pointing to a glial reaction. Evidence for this drug-induced astrogliosis was the increased heat shock protein 27 (HSP27) expression in striatum, cortex and hippocampus. MDMA (20 mg kg(-1) s.c. b.i.d. for 4 days) induced a similar dopaminergic lesion in the striatum 3 days post-treatment, which reversed 4 days later. An important neurotoxic effect on serotonergic terminals was also observed in the cortex, striatum and hippocampus 3 days post-treatment, which partially reversed 4 days later in the striatum and hippocampus. No microglial activation was noticeable at either 3 or 7 days after MDMA treatment. This lack of effect on microglial cells was assessed by [(3)H]PK 11195 binding and OX-6 immunostaining, which were unchanged in the striatum and cortex after MDMA treatment. A non-significant tendency to increase was noted in the hippocampus 3 days after MDMA treatment. Furthermore, in MDMA-treated rats, neither HSP27 expression nor an increase in HSP27 immunoreactivity were detected. This result, together with the lack of increase in glial fibrilliary acidic protein (GFAP) immunoreactivity, indicate no astroglial activation at either 3 or 7 days post-treatment. Without microglial activation, an inflammatory process would not accompany the lesion induced by MDMA. The differences in glial activation between methamphetamine and MDMA observed in the present study could have implications for the prognosis of the injury induced by these drugs.
Assuntos
Doenças do Sistema Nervoso Central/induzido quimicamente , Glicoproteínas de Membrana , Metanfetamina/toxicidade , N-Metil-3,4-Metilenodioxianfetamina/toxicidade , Proteínas do Tecido Nervoso , Neuroglia/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Esquema de Medicação , Febre/induzido quimicamente , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/ultraestrutura , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/imunologia , Masculino , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Metanfetamina/administração & dosagem , Metanfetamina/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/administração & dosagem , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Neuroglia/patologia , Neuroglia/ultraestrutura , Ratos , Ratos Sprague-Dawley , Serotonina/metabolismo , Fatores de TempoRESUMO
A monoclonal antibody was developed, using cultured subepithelial fibroblasts of rat duodenal villi as the antigenic material, by in vitro immunization. Hybridomas were selected on cryosections of rat brain and small intestine using indirect immunofluorescence techniques. The monoclonal antibody, termed 8E1, was very useful to label GFAP-positive astrocytes in the adult rat brain, subepithelial fibroblasts of intestinal villi and Thuneberg's fibroblast-like interstitial cells associated with the myenteric plexus layer. These cells are known to be stellate in shape, forming a cellular network with extended cell processes that communicate via gap junctions. Immature astrocytes such as radial fibers were not immunolabeled with mAb-8E1. Immunoelectron microscopy revealed clustered gold labeling of 8E1 antigen on the intermediate filaments of cultured astrocytes and cultured subepithelial fibroblasts. This staining pattern was different from the continuous and linear gold labeling of GFAP and vimentin. MAb-8E1 did not immunoblot purified human brain GFAP nor bovine lens vimentin, and so was not neutralized by preabsorption with these molecules. Immunoblot analysis after SDS-PAGE showed that the antigen recognized by mAb-8E1 was a Mr 50K protein in the rat cerebra, and a Mr 56K protein in the cultured subepithelial fibroblasts. This study showed that astrocytes and some types of stellate cells share distinct antigenic properties in the components of their intermediate filaments.
Assuntos
Anticorpos Monoclonais/análise , Astrócitos/imunologia , Duodeno/imunologia , Fibroblastos/imunologia , Plexo Mientérico/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Astrócitos/citologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Células Cultivadas , Duodeno/citologia , Eletroforese em Gel de Poliacrilamida , Epitopos/imunologia , Fibroblastos/citologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Proteínas de Filamentos Intermediários/imunologia , Microscopia Confocal , Microscopia Imunoeletrônica , Microvilosidades/imunologia , Peso Molecular , Plexo Mientérico/citologia , Ratos , Vimentina/metabolismo , Vimentina/ultraestruturaRESUMO
OBJECTIVE: To determine whether a heterogeneous collection of salivary gland tumors shared common antigenic characteristics and growth patterns in tissue culture. DESIGN: Cell cultures were derived from benign and malignant salivary gland neoplasms, cultured conservatively, and serially analyzed for epithelial, myoepithelial, and neuroectodermal antigens. SUBJECTS: Nineteen samples reflecting the spectrum of salivary tumor pathologic characteristics were established in tissue culture. Most were derived from benign pleomorphic adenomas, and several were from carcinomas, including carcinoma ex pleomorphic adenoma, and mucoepidermoid and adenoid cystic carcinoma. RESULTS: All cultures were epithelial as determined by morphologic and antigenic examination, using antibodies for cytokeratin. The phenotype of cells derived from benign tumors, especially the pleomorphic adenomas, resembled those in the original neoplasm. Those from carcinomas were similar, with less differentiated characteristics. Manipulation of growth conditions altered the phenotypes shown in culture. Some cultures contained cells expressing vascular smooth-muscle actin and glial fibrillary acidic protein or nestin. CONCLUSIONS: This model cell system containing proliferative cells from several tumor types is consistent with a stem-cell theory of salivary gland tumor origin. Our data were not consistent with the bicellular or multicellular theory. We hypothesize a neuroectodermal origin for this group of apparently heterogeneous tumors. These cultured cells will be valuable for in-depth investigation of the loss of proliferation controls in benign and malignant tumors of the salivary gland.
Assuntos
Antígenos de Neoplasias/análise , Tumores Neuroectodérmicos/imunologia , Neoplasias das Glândulas Salivares/imunologia , Glândulas Salivares/imunologia , Anticorpos Monoclonais , Divisão Celular , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Imuno-Histoquímica , Neoplasias das Glândulas Salivares/patologia , Neoplasias das Glândulas Salivares/ultraestrutura , Glândulas Salivares/patologia , Glândulas Salivares/ultraestrutura , Células Tumorais CultivadasRESUMO
Report concerns the uncommon cerebellar neoplasm of choroid plexus in 57-year-old woman, which was devoid of some anaplastic features causing doubts about degree of tumor differentiation. Immuno- and histochemical study presented positive CK, VIM, GFAP, S-100 immunostaining and positive PAS-dimedone reaction conforming histological diagnosis.
Assuntos
Carcinoma/patologia , Neoplasias do Plexo Corióideo/patologia , Plexo Corióideo/patologia , Carcinoma/diagnóstico , Neoplasias do Plexo Corióideo/diagnóstico , Feminino , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Pessoa de Meia-IdadeRESUMO
We established a cell line (TS) from adenoma sebaceum of a patient with tuberous sclerosis. Through our previous studies, the abnormal cell division and dysfunction of TS cells were indicated. Glial fibrillary acidic protein (GFAP) and 55 kd protein had been to be major cytoskeletal proteins in these cells. This time we have examined the structure and distribution of cytoskeletons in TS cells with immunoelectron microscopy. TS cells were found to coexpress GFAP and vimentin-like substance; both structures seem to be closely related. The coexistence of a few kinds of proteins integrated in the cytoskeletons might lead to the abnormal behavior of the nucleus during the process of cell division.
Assuntos
Proteína Glial Fibrilar Ácida/metabolismo , Filamentos Intermediários/metabolismo , Esclerose Tuberosa/patologia , Adenoma/metabolismo , Adenoma/ultraestrutura , Divisão Celular , Linhagem Celular , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Filamentos Intermediários/ultraestrutura , Microscopia Eletrônica , Neoplasias das Glândulas Sebáceas/metabolismo , Neoplasias das Glândulas Sebáceas/ultraestruturaRESUMO
INTRODUCTION: A central neurocytoma (CN) is a rare tumor, of neuronal origin, well-differentiated and found intraventricularly. It mainly affects young adults. Firm diagnosis is made on immunohistochemical (IHQ) and ultrastructural studies, since on optic microscopy it is similar in appearance to an oligodendroglioma or to an ependymoma. PATIENTS AND METHODS: We studied 4 cases, three after surgical resection and one on autopsy. The average age was 29, ranging from 3 to 63. Both sexes were equally affected. In all cases IHQ techniques were used (GFAP, neurofilament, synaptophysin and specific neuronal enolase) and they were studied by electron microscopy. RESULTS: IHQ was negative for GFAP and neurofilament, but intensely positive for synaptophysin and specific neuronal enolase. On ultrastructural study there were few neurofilaments, microtubules and dense central granules typical of neural differentiation. CONCLUSIONS: The findings in our cases lead to diagnosis of NC and confirm that this tumor is a distinct clinicopathological entity.
Assuntos
Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/ultraestrutura , Neurocitoma/diagnóstico , Neurocitoma/ultraestrutura , Adolescente , Adulto , Neoplasias Encefálicas/cirurgia , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Masculino , Pessoa de Meia-Idade , Neurocitoma/cirurgia , Fosfopiruvato Hidratase/ultraestrutura , Sinaptofisina/ultraestrutura , Tomografia Computadorizada por Raios XRESUMO
Though intrinsically of much higher frequency than open-field blast overpressures, high-intensity focused ultrasound (HIFU) pulse trains can be frequency modulated to produce a radiation pressure having a similar form. In this study, 1.5-MHz HIFU pulse trains of 1-ms duration were applied to intact skulls of mice in vivo and resulted in blood-brain barrier disruption and immune responses (astrocyte reactivity and microglial activation). Analyses of variance indicated that 24 h after HIFU exposure, staining density for glial fibrillary acidic protein was elevated in the parietal and temporal regions of the cerebral cortex, corpus callosum and hippocampus, and staining density for the microglial marker, ionized calcium binding adaptor molecule, was elevated 2 and 24 h after exposure in the corpus callosum and hippocampus (all statistical test results, p < 0.05). HIFU shows promise for the study of some bio-effect aspects of blast-related, non-impact mild traumatic brain injuries in animals.
Assuntos
Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/metabolismo , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Animais , Barreira Hematoencefálica/fisiopatologia , Lesões Encefálicas/fisiopatologia , Modelos Animais de Doenças , Azul Evans , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Histocitoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The pharyngeal tonsil has recently been identified as a new participant in airborne contamination by the ovine scrapie agent. In the context of scrapie pathogenesis, we conducted a three-dimensional reconstruction of the innervation pattern in the lymphoid compartments of this tonsil. This model confirmed that very few nerve fibres penetrated the lymphoid follicles and suggested that the nerve fibre distribution in the interfollicular and subepithelial areas is more suitable with neuro-invasion through direct contact between these nerve fibres and prion-transporting cells prior to or after prion amplification in the germinal centre of the pharyngeal tonsil lymphoid follicles.
Assuntos
Tonsila Faríngea/inervação , Tomografia com Microscopia Eletrônica/métodos , Tonsila Faríngea/patologia , Tonsila Faríngea/ultraestrutura , Animais , Proteína Glial Fibrilar Ácida/ultraestrutura , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Proteínas PrPSc/patogenicidade , Scrapie/patologia , Ovinos , Carneiro DomésticoRESUMO
Sex differences in the structure and organization of the corpus callosum (CC) can be attributed to genetic, hormonal or environmental effects, or a combination of these factors. To address the role of gonadal hormones on axon myelination, functional axon conduction and immunohistochemistry analysis of the CC in intact, gonadectomized and hormone-replaced gonadectomized animals were used. These groups were subjected to cuprizone diet-induced demyelination followed by remyelination. The myelinated component of callosal compound action potential was significantly decreased in ovariectomized and castrated animals under normal myelinating condition. Compared to gonadally intact cohorts, both gonadectomized groups displayed more severe demyelination and inhibited remyelination. Castration in males was more deleterious than ovariectomy in females. Callosal conduction in estradiol-supplemented ovariectomized females was significantly increased during normal myelination, less attenuated during demyelination, and increased beyond placebo-treated ovariectomized or intact female levels during remyelination. In castrated males, the non-aromatizing steroid dihydrotestosterone was less efficient than testosterone and estradiol in restoring normal myelination/axon conduction and remyelination to levels of intact males. Furthermore, in both sexes, estradiol supplementation in gonadectomized groups increased the number of oligodendrocytes. These studies suggest an essential role of estradiol to promote efficient CC myelination and axon conduction in both sexes.
Assuntos
Corpo Caloso/patologia , Doenças Desmielinizantes/sangue , Doenças Desmielinizantes/patologia , Hormônios Esteroides Gonadais/sangue , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Animais Recém-Nascidos , Castração , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/ultraestrutura , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Estradiol/farmacologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/ultraestrutura , Proteínas de Fluorescência Verde/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Proteína Proteolipídica de Mielina/genética , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Caracteres SexuaisRESUMO
Primary open angle glaucoma (POAG), the most common form of glaucoma, is characterized by irreversible loss of axons from the optic nerve. The site of damage to the axons is at the level of the lamina cribrosa in the optic nerve head. It has been hypothesized that structural and biochemical abnormalities in the extracellular matrix (ECM) of the lamina cribrosa underlie the progressive compressive and remodelling of this connective tissue in glaucoma. In this review, we present evidence of specific changes in collagen and elastic fibers, major ECM components in the lamina cribrosa of glaucomatous eyes.
Assuntos
Matriz Extracelular/ultraestrutura , Glaucoma de Ângulo Aberto/patologia , Disco Óptico/ultraestrutura , Colágeno/ultraestrutura , Elastina/ultraestrutura , Imunofluorescência , Proteína Glial Fibrilar Ácida/ultraestrutura , Humanos , Microscopia Imunoeletrônica , Nervo Óptico/ultraestruturaRESUMO
Glial fibrillary acidic protein (GFAP) is soluble in low ionic strength solutions but shows a strong tendency toward assembly with increasing ionic strength as revealed by electron microscopy and turbidity measurements. Increasing K+, Na+, and Li+ concentrations cause an increase followed by a decrease in GFAP turbidity with a maximum at 200 mM, but their effects are much weaker than effects of divalent cations at the same ionic strength. Ca2+, Mg2+, Mn2+, and Ba2+ promote assembly at millimolar concentrations, and 10 microM Cu2+ causes rapid aggregation. The critical concentration for GFAP assembly was 0.08 +/- 0.04 mg/mL in 2 mM Tris-HCl, 60 mM KCl, and 1 mM CaCl2, pH 6.8. The Mr 38,000 rod domain of GFAP obtained by limited chymotryptic digestion is more soluble in 100 mM imidazole hydrochloride buffer, pH 6.8, than the intact molecule, and removal of the end pieces greatly reduces the ability of GFAP to form filaments. BNPS-skatole (2-[(2-nitrophenyl)sulfenyl]-3-methyl-3-bromoindolenine) treatment releases a Mr 30,000 N-terminus and a Mr 20,000 C-terminus. The Mr 30,000 polypeptide shows a higher affinity than the Mr 20,000 fragment for intact GFAP. Arginine and lysine at low concentrations slightly accelerate GFAP assembly, but above 100 mM both amino acids inhibit assembly. ATP, GTP, CTP, and UTP do not show significant effects on GFAP assembly. Dephosphorylation by alkaline phosphatase slightly reduces the assembly ability of GFAP, but phosphatase-treated GFAP still is assembly competent.