RESUMO
Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood-brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10 k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)-positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO-1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight-junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB.
Assuntos
Envelhecimento/fisiologia , Barreira Hematoencefálica/fisiologia , Permeabilidade Capilar/fisiologia , Órgãos Circunventriculares/irrigação sanguínea , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Órgãos Circunventriculares/anatomia & histologia , Órgãos Circunventriculares/ultraestrutura , Claudina-5/metabolismo , Difusão , Fluoresceína-5-Isotiocianato/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ocludina/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
OBJECTIVE: There are conflicting reports regarding the effects of cytochrome P450 (P450, CYP) genotypes on the plasma concentrations of risperidone and its pharmacologically active metabolite, 9-hydroxyrisperidone (paliperidone), in clinical patients. The aim of this study was to investigate individual differences in the metabolic clearance of risperidone in vitro and in vivo. METHODS: In vitro liver microsomal risperidone 9-hydroxylation activities and in vivo plasma concentrations of risperidone and paliperidone were investigated in 15 male and 12 female Japanese subjects (mean age 52 years, range: 24-75 years) genotyped for CYP2D6 and CYP3A5. RESULTS: CYP2D6 intermediate and poor metabolizers showed significantly lower liver microsomal risperidone 9-hydroxylation activities than extensive metabolizers did at 5 µM of risperidone; this difference was not evident at 50 µM of risperidone. The recombinant CYP3A5 Vmax/Km value for risperidone 9-hydroxylation was 30% that of CYP3A4, and liver microsomes from CYP3A5 expressers had similar risperidone 9-hydroxylation activities to those of CYP3A5 poor expressers. The plasma concentration/dose ratios for risperidone and paliperidone in 27 Japanese patients were not significantly influenced by the CYP2D6 or CYP3A5 genotypes. CONCLUSIONS: Individual differences in metabolic clearance of risperidone under the present conditions were not significantly influenced by the genotypes of CYP2D6 or CYP3A5.
Assuntos
Antipsicóticos/farmacocinética , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP3A/genética , Risperidona/farmacocinética , Fumar/genética , Fumar/metabolismo , Adulto , Idoso , Antipsicóticos/uso terapêutico , Povo Asiático/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Feminino , Técnicas de Genotipagem , Humanos , Japão , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Pessoa de Meia-Idade , Proteínas Recombinantes/metabolismo , Risperidona/uso terapêutico , Adulto JovemRESUMO
In our previous study, the CS-56 antibody, which recognizes a chondroitin sulfate moiety, labeled a subset of adult brain astrocytes, yielding a patchy extracellular matrix pattern. To explore the molecular nature of CS-56-labeled glycoproteins, we purified glycoproteins of the adult mouse cerebral cortex using a combination of anion-exchange, charge-transfer, and size-exclusion chromatographies. One of the purified proteins was identified as tenascin-R (TNR) by mass spectrometric analysis. When we compared TNR mRNA expression patterns with the distribution patterns of CS-56-positive cells, TNR mRNA was detected in CS-56-positive astrocytes. To examine the functions of TNR in astrocytes, we first confirmed that cultured astrocytes also expressed TNR protein. TNR knockdown by siRNA expression significantly reduced glutamate uptake in cultured astrocytes. Furthermore, expression of mRNA and protein of excitatory amino acid transporter 1 (GLAST), which is a major component of astrocytic glutamate transporters, was reduced by TNR knockdown. Our results suggest that TNR is expressed in a subset of astrocytes and contributes to glutamate homeostasis by regulating astrocytic GLAST expression.
Assuntos
Astrócitos/metabolismo , Córtex Cerebral/citologia , Ácido Glutâmico/metabolismo , Homeostase/fisiologia , Tenascina/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Astrócitos/ultraestrutura , Células Cultivadas , Córtex Cerebral/metabolismo , Sulfatos de Condroitina/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Glicoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Tenascina/genéticaRESUMO
The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.
Assuntos
Envelhecimento/metabolismo , Órgãos Circunventriculares/irrigação sanguínea , Neovascularização Fisiológica , Sensação , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Órgãos Circunventriculares/citologia , Órgãos Circunventriculares/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Sensação/efeitos dos fármacosRESUMO
Old astrocyte specifically induced substance (OASIS), a basic leucine zipper transcription factor of the cAMP response element binding/Activating transcription factor family, is induced in reactive astrocytes in vivo and has important roles in quality control of protein synthesis at the endoplasmic reticulum. Reactive astrocytes produce a non-permissive environment for regenerating axons by up-regulating chondroitin sulfate proteoglycans (CSPGs). In this study, we focus on the potential role of OASIS in CSPG production in the adult mouse cerebral cortex. CS-C immunoreactivity, which represents chondroitin sulfate moieties, was significantly attenuated in the stab-injured cortices of OASIS knockout mice compared to those of wild-type mice. We next examined expression of the CSPG-synthesizing enzymes and core proteins of CSPGs in the stab-injured cortices of OASIS knockout and wild-type mice. The levels of chondroitin 6-O-sulfotransferase 1 (C6ST1, one of the major enzymes involved in sulfation of CSPGs) mRNA and protein increased after cortical stab injury of wild-type, but not of OASIS knockout, mice. A C-terminal deletion mutant OASIS over-expressed in rat C6 glioma cells increased C6ST1 transcription by interacting with the first intron region. Neurite outgrowth of cultured hippocampal neurons was inhibited on culture dishes coated with membrane fractions of epidermal growth factor-treated astrocytes derived from wild type but not from OASIS knockout mice. These results suggest that OASIS regulates the transcription of C6ST1 and thereby promotes CSPG sulfation in astrocytes. Through these mechanisms, OASIS may modulate axonal regeneration in the injured cerebral cortex. OASIS, an ER stress-responsive CREB/ATF family member, is up-regulated in the reactive astrocytes of the injured brain. We found that the up-regulated OASIS is involved in the transcriptional regulation of C6ST1 gene, which promotes chondroitin sulfate proteoglycan (CSPG) sulfation. We conclude that OASIS functions as an anti-regenerative transcription factor by establishing a non-permissive microenvironment to regenerating axons.
Assuntos
Lesões Encefálicas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Sulfotransferases/biossíntese , Animais , Astrócitos/metabolismo , Western Blotting , Lesões Encefálicas/genética , Córtex Cerebral/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfotransferases/genética , Transcrição Gênica , Carboidrato SulfotransferasesRESUMO
Accumulating evidence indicates that the medial prefrontal cortex (mPFC) is a site of myelin and oligodendrocyte abnormalities that contribute to psychotic symptoms of schizophrenia. The development of therapeutic approaches to enhance remyelination, a regenerative process in which new myelin sheaths are formed on demyelinated axons, may be an attractive remedial strategy. Geissoschizine methyl ether (GM) in the Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan (Yi-gan san), is one of the active components responsible for the psychotropic effects of yokukansan, though little is known about the mechanisms underlying the effects of either that medicine or GM itself. In the present study, we employed a cuprizone (CPZ)-induced demyelination model and examined the cellular changes in response to GM administration during the remyelination phase in the mPFC of adult mice. Using the mitotic marker 5-bromo-2'-deoxyuridine (BrdU), we demonstrated that CPZ treatment significantly increased the number of BrdU-positive NG2 cells, as well as microglia and mature oligodendrocytes in the mPFC. Newly formed oligodendrocytes were increased by GM administration after CPZ exposure. In addition, GM attenuated a decrease in myelin basic protein immunoreactivity caused by CPZ administration. Taken together, our findings suggest that GM administration ameliorated the myelin deficit by mature oligodendrocyte formation and remyelination in the mPFC of CPZ-fed mice. The present findings provide experimental evidence supporting the role for GM and its possible use as a remedy for schizophrenia symptoms by promoting the differentiation of progenitor cells to and myelination by oligodendrocytes.
Assuntos
Cuprizona/farmacologia , Indóis/farmacologia , Córtex Pré-Frontal/metabolismo , Animais , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Alcaloides Indólicos , Camundongos , Proteína Básica da Mielina/fisiologia , Córtex Pré-Frontal/efeitos dos fármacos , Regeneração/efeitos dos fármacosRESUMO
The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.
Assuntos
Antígenos/metabolismo , Área Postrema/metabolismo , Permeabilidade Capilar , Hipotálamo/metabolismo , Pericitos/metabolismo , Proteoglicanas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Órgão Subfornical/metabolismo , Animais , Antígenos/genética , Área Postrema/irrigação sanguínea , Área Postrema/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Hipotálamo/irrigação sanguínea , Hipotálamo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Osmorregulação , Pericitos/citologia , Proteoglicanas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Cloreto de Sódio/farmacologia , Órgão Subfornical/irrigação sanguínea , Órgão Subfornical/citologiaRESUMO
The circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) sense a variety of blood-borne molecules because they lack typical blood-brain barrier. Though a few signaling pathways are known, it is not known how endogenous ligands for transient receptor potential vanilloid receptor 1 ion channel (TRPV1) are sensed in the CVOs. In this study, we aimed to examine whether or not astrocytic TRPV1 senses directly blood-borne molecules in the OVLT, SFO, and AP of adult mice. The reverse transcription-polymerase chain reaction and Western analysis revealed the expression of TRPV1 in the CVOs. Confocal microscopic immunohistochemistry further showed that TRPV1 was localized prominently at thick cellular processes of astrocytes rather than fine cellular processes and cell bodies. TRPV1-expressing cellular processes of astrocytes surrounded the vasculature to constitute dense networks. The expression of TRPV1 was also found at neuronal dendrites but not somata in the CVOs. The intravenous administration of a TRPV1 agonist resiniferatoxin (RTX) prominently induced Fos expression at astrocytes in the OVLT, SFO, and AP and neurons in adjacent related nuclei of the median preoptic nuclei (MnPO) and nucleus of the solitary tract (Sol) of wild-type but not TRPV1-knockout mice. The intracerebroventricular infusion of RTX induced Fos expression at both astrocytes and neurons in the CVOs, MnPO, and Sol. Thus, this study demonstrates that blood-borne molecules are sensed directly by astrocytic TRPV1 of the CVOs in adult mammalians.
Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Encéfalo/irrigação sanguínea , Hipotálamo/irrigação sanguínea , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Órgão Subfornical/irrigação sanguínea , Órgão Subfornical/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Growth-associated protein 43 (GAP-43), a novel axonal phosphoprotein, is originally identified as a growth-cone-specific protein of developing neurons in vitro. The expression of GAP-43 is also shown to be up-regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP-43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP-43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP-43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double-labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP-43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double-labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP-43 was observed mostly at weak synapsin- and synaptotagmin-positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP-43 and either synapsin or synaptotagmin expression. These data indicate that GAP-43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis.
Assuntos
Proteína GAP-43/genética , Hipocampo/metabolismo , Morfogênese/genética , Neurônios/metabolismo , Sinapses/genética , Animais , Embrião de Mamíferos , Proteína GAP-43/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Imuno-Histoquímica , Neurônios/citologia , Cultura Primária de Células , Ratos , Ratos Wistar , Sinapses/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMO
Blood-derived molecules are able to access to the median eminence (ME) and arcuate hypothalamic nucleus (Arc) due to the lack of the blood-brain barrier. In the present study, we examined the accessibility of low-molecular-mass (LMM) molecules into parenchyma in the ME and Arc of adult mice by administration of Dextran 3000 (Dex3k), Dex10k, Evans blue (EB) and fluorescein isothiocyanate (FITC). In the external zone of the ME, the fluorescence of Dex3k, EB and FITC tracers generated an intensity gradient from fenestrated capillary, but that of Dex10k was detected only between the inner and outer basement membrane of pericapillary space. The fluorescence of FITC in the external zone of the ME was closely associated with axonal terminals and surrounded by cellular processes of tanycytes-like cells and astrocytes. In the ependymal/internal zone of the ME and Arc, the fluorescence of all LMM tracers was seen at tanycytes-like cells and neurons. The fluorescence of EB and FITC in these regions was not detected when brains were fixed during or before the administration of tracers. The inhomogeneity of accessibility for fluorescent tracers depended on routes for tracer administration. Thus, the present study indicates that the accessibility of LMM blood-derived molecules to parenchyma depends on fenestration of the capillary in the external zone of the ME and active transport of ependymal cells in the ependymal/internal zone of the ME and Arc.
Assuntos
Núcleo Arqueado do Hipotálamo/metabolismo , Dextranos/farmacocinética , Azul Evans/farmacocinética , Corantes Fluorescentes/farmacocinética , Eminência Mediana/metabolismo , Animais , Dextranos/química , Azul Evans/química , Fluorescência , Corantes Fluorescentes/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Peso Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The blood-brain barrier (BBB) prevents free access of circulating molecules to the brain and maintains a specialized brain environment to protect the brain from blood-derived bioactive and toxic molecules; however, the circumventricular organs (CVOs) have fenestrated vasculature. The fenestrated vasculature in the sensory CVOs, including the organum vasculosum of lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows neurons and astrocytes to sense a variety of plasma molecules and convey their information into other brain regions and the vasculature in the secretory CVOs, including median eminence (ME) and neurohypophysis (NH), permits neuronal terminals to secrete many peptides into the blood stream. The present study showed that vascular permeability of low-molecular-mass tracers such as fluorescein isothiocyanate (FITC) and Evans Blue was higher in the secretory CVOs and kidney as compared with that in the sensory CVOs. On the other hand, vascular permeability of high-molecular-mass tracers such as FITC-labeled bovine serum albumin and Dextran 70,000 was lower in the CVOs as compared with that in the kidney. Prominent vascular permeability of low- and high-molecular-mass tracers was also observed in the arcuate nucleus. These data demonstrate that vascular permeability for low-molecular-mass molecules is higher in the secretory CVOs as compared with that in the sensory CVOs, possibly for large secretion of peptides to the blood stream. Moreover, vascular permeability for high-molecular-mass tracers in the CVOs is smaller than that of the kidney, indicating that the CVOs are not totally without a BBB.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Permeabilidade Capilar , Azul Evans/farmacocinética , Fluoresceína-5-Isotiocianato/farmacocinética , Sistemas Neurossecretores/irrigação sanguínea , Animais , Encéfalo/metabolismo , Bovinos , Dextranos/química , Dextranos/farmacocinética , Fluoresceína-5-Isotiocianato/química , Rim/irrigação sanguínea , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Sistemas Neurossecretores/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/farmacocinéticaRESUMO
IgLON family is a subgroup of the immunoglobulin superfamily cell adhesion molecules and composed of limbic system-associated protein (LAMP), opioid binding cell adhesion molecule (OBCAM), neurotrimin (Ntm) and Kilon. In the present study, we investigated the overexpression of LAMP, OBCAM, Ntm and Kilon on the proliferation and cell size of type-1 astrocytes in vitro. Quantitative analysis using bromodeoxyuridine immunocytochemistry revealed that the expression of OBCAM had greater inhibitory effect on astrocytic proliferation as compared with LAMP, Ntm and Kilon ones. OBCAM overexpression increased the cell size of astrocytes as compared with the control. The treatment of FGF-2 had greater proliferative effect on OBCAM-transfected astrocytes as compared with the control. These results suggest that OBCAM is more potent regulator for controlling the proliferation and cell size of astrocytes as compared with other IgLON proteins possibly through FGF-2 receptor-mediated pathway.
Assuntos
Astrócitos/citologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Tamanho Celular , Córtex Cerebral/citologia , Animais , Astrócitos/metabolismo , Proliferação de Células , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
Although the tissue plasminogen activator/plasminogen system contributes to numerous brain functions, such as learning, memory, and anxiety behavior, little attention has as yet been given to the localization of plasminogen in the brain. We have investigated the localization of plasminogen in the adult mouse brain by using immunohistochemistry. In the hippocampus, plasminogen immunoreactivity was seen in the pyramidal cell layer as numerous punctate structures in neuronal somata. An electron-microscopic study further demonstrated that the plasminogen-immunoreactive punctate structures represented secretory vesicles and/or vesicle clusters. In the cerebral cortex, plasminogen immunoreactivity was evident in the somata of the layer II/III and V neurons. A quantitative analysis revealed that parvalbumin (PV)-positive neurons had more plasminogen-immunoreactive puncta compared with those of PV-negative neurons in the hippocampus and cerebral cortex. Plasminogen immunoreactivity was present throughout the hypothalamus, being particularly prominent in the neuronal somata of the organum vasculosum laminae terminalis, ventromedial preoptic nucleus, supraoptic nucleus, subfornical organ, medial part of the paraventricular nucleus (PVN), posterior part of the PVN, and arcuate hypothalamic nucleus. Thus, plasminogen is highly expressed in specific populations of hippocampal, cortical, and hypothalamic neurons, and plasminogen-containing vesicles are mainly observed at neuronal somata.
Assuntos
Córtex Cerebral/química , Hipocampo/química , Hipotálamo/química , Plasminogênio/análise , Animais , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/química , Núcleo Hipotalâmico Paraventricular/química , Plasminogênio/metabolismoRESUMO
Pleiotrophin (PTN) is a secreted heparin-binding cytokine that signals diverse functions, including lineage-specific differentiation of glial progenitor cells, axonal outgrowth and angiogenesis. Neurotoxicity mediated by glutamate receptor is thought to play a role in various neurodegenerative disorders. In the present study, we examined the effect of PTN on the neuronal viability of hippocampal neurons in vitro by using the immunostaining of MAP2 and permeability of propidium iodide. PTN significantly prevented glutamate-induced neurotoxicity when hippocampal neurons were treated with PTN after the glutamate stimulation. PTN significantly promoted glutamate-induced neurotoxicity, when cells were incubated with PTN before and after the glutamate stimulation. Thus, the effect of PTN on the neuronal viability of hippocampal neurons largely depends on the timing of the treatment of PTN. The treatment of PTN promoted dendrite-specific expression of MAP2, indicating that PTN stabilizes microtubule system at dendrites. The data suggest that PTN may be relevant to be concerned with glutamate-induced neurotoxicity of hippocampal neurons.
Assuntos
Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Ácido Glutâmico/toxicidade , Hipocampo/citologia , Neurônios/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Systemic osmotic homeostasis is regulated mainly by neuroendocrine system of arginine-vasopressin (AVP) in mammalians. In the present study, we demonstrated that the immunoreactivity of tissue plasminogen activator (tPA) was observed specifically at neurosecretory granules of AVP-positive magnocellular terminals and that of plasminogen was seen at astrocytes in the neurohypophysis (NH). Both tPA and plasminogen knockout (KO) mice revealed higher plasma osmolarity upon water deprivation, a chronic osmotic stimulation, as compared with their wild-type (WT) animals, indicating abnormal osmotic control in these KO mice. tPA KO mice but not plasminogen ones revealed lower ability in secreting AVP into the blood circulation upon an acute osmotic stimulation. Both tPA and plasminogen KO animals showed lower ability in secreting AVP into the blood circulation upon a chronic osmotic stimulation. The recombinant tPA was able to promote the release of AVP from isolated NH. Chronic osmotic stimulation decreased the laminin expression level of neurohypophysial microvessel in WT mice but not in plasminogen KO ones. We suggest that AVP secretion is critically regulated by tPA-dependent facilitation of AVP release from terminals and plasminogen-dependent increase of AVP permeability across microvessels possibly via laminin degradation.
Assuntos
Arginina Vasopressina/metabolismo , Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Arginina Vasopressina/sangue , Astrócitos/metabolismo , Permeabilidade Capilar/fisiologia , Colágeno Tipo IV/metabolismo , Fibrinolisina/metabolismo , Homeostase/fisiologia , Laminina/metabolismo , Camundongos , Microvasos/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Concentração Osmolar , Neuro-Hipófise/irrigação sanguínea , Neuro-Hipófise/metabolismo , Neuro-Hipófise/ultraestrutura , Plasminogênio/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Tempo , Ativador de Plasminogênio Tecidual/genética , Privação de Água/fisiologiaRESUMO
Toll-like receptor 2 (TLR2) recognizes a wide range of microbial molecules and plays critical roles in the initiation of innate immune responses. In the present study, we aimed to investigate whether the depletion of microglia and macrophages with clodronate liposomes (Clod-Lips) attenuates the activation of mouse brain circuits for TLR2-mediated inflammation and hypothermia. The peripheral administration of the TLR2 agonist zymosan induced nuclear factor-κB activation in microglia and macrophages and Fos expression in astrocytes/tanycytes and neurons in the circumventricular organs (CVOs). The depletion of microglia and macrophages with Clod-Lips markedly decreased zymosan-induced Fos expression in astrocytes/tanycytes and neurons in the CVOs. The treatment with Clod-Lips significantly attenuated zymosan-induced hypothermia. These results indicate that microglia and macrophages in the CVOs participate in the initiation and transmission of inflammatory responses after the peripheral administration of zymosan.
Assuntos
Ácido Clodrônico/administração & dosagem , Hipotermia/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Proteínas Proto-Oncogênicas c-fos/biossíntese , Zimosan/toxicidade , Fatores Etários , Animais , Conservadores da Densidade Óssea/administração & dosagem , Portadores de Fármacos/administração & dosagem , Expressão Gênica , Hipotermia/induzido quimicamente , Hipotermia/prevenção & controle , Lipossomos , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/metabolismoRESUMO
The purpose of this research is to clarify the nursing diagnostic processes of nursing students. The study subjects were 20 nursing students who were in bedside learning and took charge of patients suffering from depression. We analyzed their training records and extracted the information on which their nursing diagnoses were made. The information was classified into the categories based on the nursing diagnosis of North American Nursing Diagnosis Association (NANDA). As a result, the students were more likely to make their nursing diagnoses based mainly on subjective complaints of the patients, without logical reasoning. The results indicated an insufficient knowledge of the disease and a shortage of opportunity for the students to collect objective information of the patients. In conclusion, our study results suggest that bedside learning students should have the chance to reconfirm their knowledge and the nursing diagnosis procedures; that arrangements have to be made for students and ward staff to confer with each other; and that students should be advised on how to make effective use of the meetings to share their experiences and increase their knowledge. The implications of these findings for nursing students'education are discussed.
Assuntos
Depressão/enfermagem , Educação em Enfermagem , Diagnóstico de Enfermagem , Aprendizagem Baseada em Problemas , Estudantes de Enfermagem , Adulto , Bacharelado em Enfermagem , Humanos , Conhecimento , Avaliação em Enfermagem , Processo de EnfermagemRESUMO
Choroidal neovascularization is one of the major pathological changes in age-related macular degeneration, which causes devastating blindness in the elderly population. The molecular mechanism of choroidal neovascularization has been under extensive investigation, but is still an open question. We focused on sonic hedgehog signaling, which is implicated in angiogenesis in various organs. Laser-induced injuries to the mouse retina were made to cause choroidal neovascularization. We examined gene expression of sonic hedgehog, its receptors (patched1, smoothened, cell adhesion molecule down-regulated by oncogenes (Cdon) and biregional Cdon-binding protein (Boc)) and downstream transcription factors (Gli1-3) using real-time RT-PCR. At seven days after injury, mRNAs for Patched1 and Gli1 were upregulated in response to injury, but displayed no upregulation in control retinas. Immunohistochemistry revealed that Patched1 and Gli1 proteins were localized to CD31-positive endothelial cells that cluster between the wounded retina and the pigment epithelium layer. Treatment with the hedgehog signaling inhibitor cyclopamine did not significantly decrease the size of the neovascularization areas, but the hedgehog agonist purmorphamine made the areas significantly larger than those in untreated retina. These results suggest that the hedgehog-signaling cascade may be a therapeutic target for age-related macular degeneration.
RESUMO
The sensory circumventricular organs (CVOs) comprise the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), and area postrema (AP) and lack the blood-brain barrier. The expression of Toll-like receptor 4 (TLR4) was seen at astrocytes throughout the sensory CVOs and at microglia in the AP and solitary nucleus around the central canal. The peripheral and central administration of lipopolysaccharide induced a similar pattern of nuclear translocation of STAT3. A microglia inhibitor minocycline largely suppressed lipopolysaccharide-induced astrocytic nuclear translocation of STAT3 in the OVLT and AP, but its effect was less in the SFO.
Assuntos
Encéfalo/anatomia & histologia , Órgãos Circunventriculares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fator de Transcrição STAT3/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/metabolismo , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Proteína Glial Fibrilar Ácida/metabolismo , Laminina/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Minociclina/farmacologia , Fatores de TempoRESUMO
Expression of the LIM homeodomain transcription factor Lhx8 is restricted to and up-regulated in the mesenchyme of the upper face prominence before lip fusion. Msx1/2 acts in early development to control cell proliferation and differentiation. Deficiency of these genes is associated with nonsyndromic cleft lip with/without cleft palate. Since retinoid is a potential patterning influence on the developing face, we have examined whether retinoic acid (RA) signaling regulated Lhx8, Msx1 and Msx2 transcription through fibroblast growth factor (FGF) signals in the maxillary prominence. Application of exogenous RA caused severe defects of the maxilla. Citral also induced a specific loss of derivatives from the maxillary prominences by blocking RA synthesis. Real-time RT-PCR and semi-quantitative RT-PCR analysis of the maxillary mesenchyme revealed that the expressions of Lhx8, Msx1 and Msx2 were significantly down-regulated by RA as well as by citral. The downregulated Lhx8 was rescued by combined treatment with FGF-8b, which indicated a downstream of RA signaling. FGF-8b induced up-regulated Lhx8 expression whereas SU5402, a pan-FGF family antagonist, down-regulated and caused defective maxillary morphogenesis and cleft lip. Our data suggest that Lhx8 is regulated by RA signaling through FGF signals and the level window of RA and FGF-8b could control the upper jaw morphogenesis.