RESUMO
INTRODUCTION: Non-invasive diagnosis of pulmonary hypertension (PH) relies on estimation of pulmonary arterial pressure (PAP) via Doppler echocardiographic measurement of tricuspid regurgitation pressure gradient (TRPG). The pulmonary vein-to-right pulmonary artery ratio (PV/PA) recently has been described for the detection of pulmonary venous congestion. Whether this variable could be used to detect the presence of precapillary PH is unknown. The objective of the present study was to investigate the diagnostic value of PV/PA for prediction of TRPG, as a surrogate of PAP, in dogs with PH of precapillary origin. ANIMALS: Sixty-seven client-owned dogs were included in the study. METHODS: This was a retrospective study. Dogs with a measurable TRPG were included and classified into group 1 (TRPG < 30 mmHg), group 2 (TRPG 30-49 mmHg), group 3 (TRPG 50-80 mmHg), or group 4 (TRPG > 80 mmHg). The PV/PA, acceleration time-to-ejection time ratio of pulmonary artery flow, main pulmonary artery diameter-to-aortic diameter ratio, and right pulmonary artery distensibility index were measured retrospectively from cineloops in each dog. RESULTS: The PV/PA measured by both two-dimensional (2D) and time-motion mode(MM) echocardiography decreased proportionally with PH severity. Using regression analysis, PV/PA (2D) was identified as the strongest predictor for TRPG (R2 = 0.70, p < 0.0001) among other variables studied, with a good diagnostic accuracy (area under the curve = 0.94) for moderate PH (TRPG > 50 mmHg) using a cutoff value of < 0.70 (sensitivity = 96%, specificity = 82%). CONCLUSIONS: Results of the present study suggest that PV/PA can be useful as an additional, non-invasive, and indirect variable to identify precapillary PH in dogs.
Assuntos
Doenças do Cão/diagnóstico , Hipertensão Pulmonar/diagnóstico , Artéria Pulmonar/diagnóstico por imagem , Animais , Doenças do Cão/diagnóstico por imagem , Cães , Ecocardiografia Doppler/veterinária , Feminino , Hipertensão Pulmonar/diagnóstico por imagem , Masculino , Sensibilidade e EspecificidadeRESUMO
INTRODUCTION: Headache is usually considered a key symptom of intracranial hypertension (ICHT). However, there are no published experimental data to support the concept that increased intracranial pressure (ICP) is painful in humans. MATERIALS AND METHODS: This prospective study was performed in 16 patients with suspected normal-pressure hydrocephalus, necessitating a lumbar infusion test with measurement of cerebrospinal fluid (CSF) hydrodynamics. During the test, ICP was increased from baseline to a plateau. Headache was scored on a visual analog scale (VAS) (0 = no pain, 10 = very severe pain) at baseline ICP and when ICP plateaued. RESULTS: At baseline, mean ICP was 11 ± 3.6 mmHg and VAS was 0. At plateau, mean ICP was 28 ± 9.5 mmHg and VAS was 0. There was a significant increase in ICP (p <0.001), but no increase in headache intensity (VAS). An acute (20-min) moderate increase in ICP was not accompanied by a headache. DISCUSSION: We demonstrate that an acute, isolated increase in CSF pressure does not produce a headache. To occur, a headache needs activation of the pain-sensitive structures (dura and venous sinuses) or central activation of the cerebral nociceptive structures. This peripheral or central activation does not occur with an isolated increase in CSF pressure.
Assuntos
Cefaleia/etiologia , Hipertensão Intracraniana/complicações , Idoso , Feminino , Humanos , Hidrocefalia de Pressão Normal/diagnóstico , Infusões Parenterais/métodos , Hipertensão Intracraniana/etiologia , Masculino , Medição da Dor , Estudos Prospectivos , Punção Espinal/métodosRESUMO
Guillain-Barré syndrome (GBS) is an acute-onset inflammatory polyradiculoneuropathy usually triggered by an infectious disease. In some cases, GBS can occur without any preceding infectious episode, like after vaccination, epidural anaesthesia or surgery. A 73 years old woman had head and spine trauma. Body-TDM showed bilateral temporal and right frontal haematomas and fracture of the first lumbar vertebrae. Sextant and kyphoplasty were performed. She presented 14 days after surgery tetraparesis, swallowing difficulties and bilateral facial palsy. Electromyography was consistent with demyelinating neuropathy. Cerebrospinal fluid examination found albumino-cytological dissociation. Viral and bacterial serology and antiganglioside antibodies were negative. She was treated with intravenous immunoglobulins. Four months after discharge she had fully recovered except left peripheral facial palsy. GBS can rarely be triggered by head trauma or spine surgery. Physician must keep in mind this diagnosis whenever their patients present acute-onset neurological worsening in such context.
Assuntos
Síndrome de Guillain-Barré/etiologia , Traumatismos Cranianos Fechados/complicações , Cifoplastia , Vértebras Lombares/cirurgia , Complicações Pós-Operatórias/etiologia , Fraturas da Coluna Vertebral/cirurgia , Acidentes por Quedas , Idoso , Transtornos de Deglutição/etiologia , Paralisia Facial/etiologia , Feminino , Síndrome de Guillain-Barré/terapia , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Traumatismo Múltiplo , Condução Nervosa , Paralisia/etiologia , Complicações Pós-Operatórias/terapia , Tempo de Reação , Fraturas da Coluna Vertebral/etiologiaRESUMO
The dorsal vagal complex (DVC) is the brainstem integrative center that mediates the satiety reflex and relays autonomic neural responses to stress. The DVC displays adult neurogenesis, intrinsic neural stem cells and a high brain-derived neurotrophic factor (BDNF) content, effectors of plasticity that are modulated by stress in the hippocampus. In this study we asked whether neurogenesis and BDNF expression in the DVC are altered by stress, in parallel with food intake reduction. To this end, neurogenesis was assessed in adult rats in vivo by repetitive 5-bromo-2'-deoxyuridine (BrdU) administration without (controls) or with daily sessions of immobilization stress (1 h/day), and were allowed to survive for 2 weeks after the end of BrdU treatment. Neurogenic proliferation in the brainstem was detected by immunohistochemistry and confocal microscopy mainly in the area postrema and the nucleus tractus solitarius; newly formed neurons amounted to about 35% of all BrdU-labeled cells in the DVC of control rats. Chronic immobilization stress induced a significant decrease in neurogenic proliferation in the DVC which reached 50% in the area postrema. The number of newly-formed neurons was also decreased by chronic immobilization stress in the DVC, and this effect was again maximal in the area postrema; the proportion of BrdU-labeled cells that were neurons was unchanged. In vitro neurosphere assay was then performed on microdissected DVC tissue from another cohort of chronically stressed and control rats. Chronic immobilization stress induced a significant decrease of the total neurosphere number per rat DVC in both primary and secondary cultures, indicating that intrinsic neural stem cell frequency was decreased by chronic stress in DVC tissue. Tissue BDNF concentration in the DVC, as assessed by enzyme-linked immunosorbent assay, was not significantly altered when compared with controls after 3, 6, 9 or 13 days of chronic immobilization stress. These results further characterize neurogenesis in the DVC and suggest its involvement in the long-term regulation of food intake.
Assuntos
Área Postrema/fisiopatologia , Neurogênese/fisiologia , Restrição Física/métodos , Núcleo Solitário/fisiopatologia , Estresse Psicológico/patologia , Animais , Peso Corporal/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Ingestão de Alimentos/fisiologia , Ensaio de Imunoadsorção Enzimática/métodos , Masculino , Análise Multivariada , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Wistar , Estresse Psicológico/fisiopatologia , Fatores de TempoRESUMO
The dorsal vagal complex, located in the brainstem, is the major integrative center of the autonomic nervous system. By combining in vivo bromodeoxyuridine incorporation and phenotypic immunolabeling, we have previously reported that neurogenesis occurs in the adult rat dorsal vagal complex [Bauer S, Hay M, Amilhon B, Jean A, Moyse E (2005) In vivo neurogenesis in the dorsal vagal complex of the adult rat brainstem. Neuroscience 130:75-90.]. In the present study we asked whether adult dorsal vagal complex contains proliferative and/or neural stem cells. Using Ki-67 immunolabeling and cyclin D1 Western blot, we showed intrinsic cell proliferation in the dorsal vagal complex and its stimulation by vagotomy. Detailed time-course analysis revealed that vagotomy-induced proliferation in the dorsal vagal complex peaked three days after lesion. In order to directly assess the presence of intrinsic stem cells, primary cell cultures from adult rat dorsal vagal complex were performed in the presence of epidermal growth factor and basic fibroblast growth factor (neurosphere assay). A discrete subpopulation of dorsal vagal complex cells proliferated as neurospheres, self-renewed when passaged, and differentiated into neurons, astrocytes and oligodendrocytes. Proliferation and neuron-differentiating potentials of dorsal vagal complex neurospheres were both lower than those of subventricular zone neurospheres from the same rats. The relationship between in vitro neurosphere-forming cells of dorsal vagal complex and in vivo dorsal vagal complex neurogenesis is discussed and remains to be directly addressed. The present data demonstrate the occurrence of neural stem cells in the dorsal vagal complex of adult rat brain.
Assuntos
Neurônios/fisiologia , Células-Tronco/fisiologia , Nervo Vago/fisiologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Ciclina D1/biossíntese , Ciclina D1/genética , Fator de Crescimento Epidérmico/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/metabolismo , Masculino , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Wistar , Regulação para Cima/efeitos dos fármacos , Vagotomia , Nervo Vago/citologiaRESUMO
The dorsal vagal complex (DVC) encompasses the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMX) and the area postrema (AP), that altogether provide the major integrative center for the mammalian autonomic nervous system. The adult rat DVC has been reported to contain afferent-dependent concentration of the plasticity-promoting polysialylated form of neural cell adhesion molecule [J Neurosci 21 (2001) 4721; Eur J Neurosci 14 (2001) 1194]. This prompted us to assess the occurrence of neurogenesis in the DVC of adult rats. Cumulative in vivo labeling of cell proliferation with i.p. bromodeoxyuridine (BrdU) injections was combined with phenotypic markers and confocal microscopy on serial brainstem sections throughout the DVC extent. In basal condition, sparse BrdU+ nuclei were selectively detected in the DVC according to a discrete and reproducible pattern. Some of them were found to colocalize with the neuronal markers doublecortin, HuC/D, or neuronal-specific antigen (NeuN), demonstrating that neurogenesis does occur within the DVC of adult rat. In the NTS, 10% of the BrdU+ nuclei were also NeuN+. A comparable proportion of astrogliogenesis was found in the DVC. Nestin immunohistochemistry yielded a highly specific labeling pattern at the border between AP and NTS. These data may relate to the neural stem cells that have been reported in the floor of the IVth ventricle [J Neurosci 16 (1996) 7599]. In order to assess a possible modulation of neurogenesis by afferent input in vivo, unilateral vagotomy was performed prior to cumulative BrdU treatment. Such DVC deafferentation triggered a large increase of BrdU incorporation in the ipsilateral DVC, which was associated with microglial proliferation in the DMX and with increased genesis of neurons and astrocytes in the NTS. These findings establish DVC as a novel model of adult neurogenesis that is reactive to deafferentation.
Assuntos
Proliferação de Células , Bulbo/citologia , Neurônios/citologia , Animais , Bromodesoxiuridina/metabolismo , Antígeno CD11b/metabolismo , Contagem de Células/métodos , Proteínas do Domínio Duplacortina , Proteína Duplacortina , Lateralidade Funcional/fisiologia , Imuno-Histoquímica/métodos , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Bulbo/metabolismo , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Ratos , Ratos Sprague-Dawley , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo , Distribuição Tecidual , Vagotomia/métodosRESUMO
A unique feature of the olfactory system is its efficiency to produce new neurons in the adult. Thus, destruction of the olfactory receptor neurons (ORNs) using chemical (intranasal perfusion with ZnSO4) or surgical (axotomy or bulbectomy) methods, leads to an enhanced rate of proliferation of their progenitors and to complete ORNs regeneration. The aim of our study was to identify new factors implied in this regenerative process. Using an electrophoretic method, we observed the accumulation of a 42 kDa protein after axotomy in the olfactory mucosa, but not in the olfactory bulb. Its expression started after a few days following injury and increased massively during the phase of ORN regeneration. The purification and the sequence characterization revealed that this protein was Ym1/2, recently identified in activated macrophages present in various tissues during inflammation. Western blotting analysis of Ym1/2 confirmed the accumulation of this protein in the regenerating olfactory mucosa consecutively to olfactory axotomy or bulbectomy but also after ZnSO4 irrigation of the nasal cavity. In the olfactory mucosa of control mice, Ym1/2 was hardly detectable in young animals and became more and more abundant with increasing age. In injured and aged mice, Ym1/2 mainly accumulates in the cytoplasm of supporting cells as well as in other cells located throughout the olfactory epithelium. Our results suggest that Ym1/2 is involved in olfactory epithelium remodeling following several kinds of lesions of the adult olfactory mucosa and support the view of a critical role of inflammatory cues in neurodegeneration and aging.
Assuntos
Envelhecimento/metabolismo , Lectinas/metabolismo , Regeneração Nervosa/fisiologia , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Axotomia/métodos , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting/métodos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Bulbo Olfatório/lesões , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Sulfato de Zinco/toxicidadeRESUMO
A dysfunction of noradrenergic mechanisms originating in the locus coeruleus has been hypothesised to be the critical factor underlying the evolution of central neurodegenerative diseases [Colpaert FC (1994) Noradrenergic mechanism Parkinson's disease: a theory. In: Noradrenergic mechanisms in Parkinson's disease (Briley M, Marien M, eds) pp 225-254. Boca Raton, FL, USA: CRC Press Inc.]. alpha(2)-Adrenoceptor antagonists, presumably in part by facilitating central noradrenergic transmission, afford neuroprotection in vivo in models of cerebral ischaemia, excitotoxicity and devascularization-induced neurodegeneration. The present study utilised the rat olfactory bulb as a model system for examining the effects of the selective alpha(2)-adrenoceptor antagonist dexefaroxan upon determinants of neurogenesis (proliferation, survival and death) in the adult brain in vivo. Cell proliferation (5-bromo-2'-deoxyuridine labelling) and cell death associated with DNA fragmentation (terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling assay) were quantified following a 7-day treatment with either vehicle or dexefaroxan (0.63 mg/kg i.p., three times daily), followed by a 3-day washout period. The number of terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling-positive nuclei in the olfactory bulb was lower in dexefaroxan-treated rats, this difference being greatest and significant in the subependymal layer (-52%). In contrast, 5-bromo-2'-deoxyuridine-immunoreactive nuclei were more numerous (+68%) in the bulbs of dexefaroxan-treated rats whilst no differences were detected in the proliferating region of the subventricular zone. Terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling combination with glial fibrillary acidic protein or neuronal-specific antigen immunohistochemistry revealed that terminal dideoxynucleotidyl transferase-catalysed 2'-deoxyuridine-5'-triphosphate nick end-labelling-positive nuclei were associated primarily with a neuronal cell phenotype. These findings suggest that dexefaroxan increases neuron survival in the olfactory bulb of the adult rat in vivo, putatively as a result of reducing the apoptotic fate of telencephalic stem cell progenies.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2 , Antagonistas Adrenérgicos alfa/farmacologia , Benzopiranos/farmacologia , Imidazóis/farmacologia , Neurônios/efeitos dos fármacos , Bulbo Olfatório/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Masculino , Neurônios/citologia , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Adrenérgicos alfa 2/fisiologiaRESUMO
The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout adulthood by local, restricted neuronal progenitor cells. The molecular signals that control this neurogenesis in vivo are unknown. Using olfactory bulb ablation (OBX) in adult mice to trigger synchronous mitotic stimulation of neuronal progenitors in the OE, we show the in vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA expression peaks at 8 hr after OBX, and in situ RT-PCR and immunocytochemistry indicate that LIF is upregulated, in part, in the injured neurons themselves. This rise in LIF is necessary for injury-induced neurogenesis, as OBX in the LIF knock-out mouse fails to stimulate cell proliferation in the OE. Moreover, delivery of exogenous LIF to the intact adult OE using an adenoviral vector stimulates BrdU labeling in the apical OE. Taken together, these results suggest that injured OSNs release LIF as a stimulus to initiate their own replacement.
Assuntos
Inibidores do Crescimento/deficiência , Inibidores do Crescimento/metabolismo , Interleucina-6 , Linfocinas/deficiência , Linfocinas/metabolismo , Neurônios/metabolismo , Mucosa Olfatória/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Bromodesoxiuridina , Morte Celular , Divisão Celular , Citocinas/biossíntese , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Inibidores do Crescimento/genética , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/biossíntese , Fator Inibidor de Leucemia , Linfocinas/genética , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Procedimentos Neurocirúrgicos , Bulbo Olfatório/fisiologia , Bulbo Olfatório/cirurgia , Mucosa Olfatória/citologia , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/lesões , RNA Mensageiro/biossínteseRESUMO
The olfactory epithelium of adult mouse, where primary sensory neurons are massively committed to apoptosis by removal of their synaptic target, was used as a model to determine in vivo mechanisms for neuronal cell death induction. A macro-array assay revealed that the death of olfactory neurons is accompanied with over-expression of the serine protease inhibitor Spi2. This over-expression is associated with decreased serine protease activity in the olfactory mucosa. Moreover, in vitro or in vivo inhibition of serine proteases induced apoptotic death of olfactory neuronal cells. Interestingly, Spi2 over-expression is not occurring in olfactory neurons but in cells of the lamina propria, suggesting that Spi2 may act extracellularly as a cell death inducer. In that sense, we present evidence that in vitro Spi2 overexpression generates a secreted signal for olfactory neuron death. Hence, taken together these results document a possible novel mechanism for apoptosis induction that might occur in response to neurodegenerative insults.
Assuntos
Apoptose/genética , Proteínas Nucleares/metabolismo , Bulbo Olfatório/lesões , Neurônios Receptores Olfatórios/metabolismo , Serina Endopeptidases/metabolismo , Serpinas , Animais , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Proteínas Nucleares/genética , Bulbo Olfatório/cirurgia , Condutos Olfatórios/citologia , Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/citologia , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The distribution of delta sleep-inducing peptide immunoreactive cell bodies, fibers, and terminal-like structures was investigated in the normal human hypothalamus during the first postnatal year, using immunohistofluorescence and peroxidase anti-peroxidase techniques. Immunolabeled perikarya were relatively few and were mostly scattered through the anterior (preoptic) and mediobasal regions (infundibular nucleus) of the hypothalamus. DSIP-immunoreactive fibers and terminal-like fibers were observed throughout the entire rostrocaudal extent of the hypothalamus. They exhibit high densities in the preoptic region, the organum vasculosum of lamina terminalis, infundibular nucleus and median eminence. Moderate to low densities of DSIP-immunoreactive fibers were observed in the other hypothalamic structures, located in the anterior and mediobasal regions of hypothalamus, such as periventricular, paraventricular, suprachiasmatic, ventromedial, dorsomedial and parafornical nuclei. In the present study, the analysis of the immunohistochemical pattern of DSIP-immunoreactive neuronal elements in the human infant hypothalamus during the first postnatal year provided evidence of the presence of several differences. We have found qualitative age-related changes in the density of DSIP immunoreactivity in several hypothalamic structures such as the anterior region and the median eminence.
Assuntos
Peptídeo Indutor do Sono Delta/análise , Hipotálamo/química , Feminino , Técnica Indireta de Fluorescência para Anticorpo/métodos , Humanos , Técnicas Imunoenzimáticas/métodos , Lactente , Recém-Nascido , Masculino , Neurônios/químicaRESUMO
The distribution of DSIP-IR cell bodies and fibers was investigated in the normal human hypothalamus during the first postnatal year using the indirect immunofluorescence technique. The analysis of the immunohistochemical patterns obtained in the seven cases analyzed showed regional differences in the localization of cell bodies and fibers. Immunoreactive perikarya were relatively few, and were mostly scattered throughout the anterior and the mediobasal hypothalamus. DSIP-IR fibers and terminal-like structures were observed throughout the rostro-caudal extent of the hypothalamic region. In the present study, we noticed qualitative changes in the density of DSIP immunoreactivity in several hypothalamic structures such as the preoptic area and the median eminence with respect to age. These postnatal differences observed for DSIP could be related to neuronal maturation processes occurring at this period in the central nervous system as well as other physiological processes controlling the evolution of DSIP concentrations. These data are compatible with the proposed role of the neuropeptide in the regulation of many postnatal physiological functions.
Assuntos
Peptídeo Indutor do Sono Delta/metabolismo , Hipotálamo/crescimento & desenvolvimento , Neurônios/fisiologia , Envelhecimento , Causas de Morte , Peptídeo Indutor do Sono Delta/análise , Feminino , Humanos , Hipotálamo/patologia , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Eminência Mediana/crescimento & desenvolvimento , Eminência Mediana/patologia , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Neurônios/patologia , Área Pré-Óptica/crescimento & desenvolvimento , Área Pré-Óptica/patologiaRESUMO
Using in vitro quantitative autoradiography and [3H]flunitrazepam we examined the rostrocaudal distribution of benzodiazepine binding sites in the human neonate/infant hypothalamus. The autoradiographic analysis shows the presence of a heterogeneous distribution throughout the rostrocaudal extent of this brain structure. High [3H]flunitrazepam binding corresponds primarily to the diagonal band of Broca and the preoptic region. The labelling in the preoptic region showed a rostrocaudal increase, contrasting in that with the other hypothalamic structures. Intermediate densities were present in the septohypothalamic, suprachiasmatic, periventricular and paraventricular nuclei as well as in the mammillary complex. Low binding was observed in the other hypothalamic structures. The benzodiazepine binding sites analyzed belong mostly to type II receptors. In an attempt to unravel possible differences related to age, we compared the autoradiographic distribution in three postnatal age ranges. The topographical distribution of these binding sites was almost identical in each period analyzed. We found, however, that benzodiazepine binding is generally low in the neonatal period and a tendency in increasing densities is observed during development. Taken together, these results provide evidence for a large distribution of benzodiazepine binding sites in neonate/infant hypothalamus, suggesting their implication in the development of this brain structure and the maintenance of its various functions.
Assuntos
Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Fatores Etários , Núcleo Hipotalâmico Anterior/citologia , Núcleo Hipotalâmico Anterior/crescimento & desenvolvimento , Núcleo Hipotalâmico Anterior/metabolismo , Ansiolíticos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Feminino , Flunitrazepam/farmacocinética , Humanos , Hipotálamo/citologia , Hipotálamo Médio/citologia , Hipotálamo Médio/crescimento & desenvolvimento , Hipotálamo Médio/metabolismo , Hipotálamo Posterior/citologia , Hipotálamo Posterior/crescimento & desenvolvimento , Hipotálamo Posterior/metabolismo , Lactente , Recém-Nascido , Masculino , Neurônios/citologia , Ensaio Radioligante , Trítio/farmacocinética , Ácido gama-Aminobutírico/metabolismoRESUMO
Normal apoptosis occurs continuously in the olfactory neuroepithelium of adult vertebrates, making it a useful model for studying neuronal apoptosis. Here we demonstrate that overexpression of the anti-apoptotic Bag-1 gene in olfactory neuronal cells confers a strong resistance to apoptosis. Conversely decreased levels of Bag-1 were found to precede a massive wave of olfactory neuronal apoptosis triggered by synaptic target ablation. We show that the decrease is brought about by ubiquitination and subsequent degradation of the Bag-1 protein. The ring finger protein Siah-2 is a likely candidate for the ubiquitination reaction since Siah-2 mRNA accumulated in lesioned olfactory neuroepithelium and overexpression of Siah-2 stimulated Bag-1 ubiquitination and degradation in transient expression assays. These results together identify destabilization of Bag-1 as a necessary step in olfactory neuronal apoptosis.
Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Neurônios/citologia , Mucosa Olfatória/citologia , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Chlorocebus aethiops , Proteínas de Ligação a DNA , Dopamina/farmacologia , Regulação para Baixo , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fatores de Transcrição , Ubiquitina-Proteína Ligases , Ubiquitinas/genética , Regulação para CimaRESUMO
The netrins, a family of laminin-related secreted proteins, are critical in controlling axon elongation and pathfinding. The DCC (for deleted in colorectal cancer) protein was proposed as a receptor for netrin-1 in the light of many observations including the inhibition of netrin-1-mediated axon outgrowth and attraction in the presence of an anti-DCC antiserum, the similitude of nervous system defects in DCC and netrin-1 knockout mice and the results of receptor swapping experiments. Previous studies have failed to show a direct interaction of DCC with netrin-1 (ref. 10), suggesting the possibility of an additional receptor or co-receptor. Here we show that DCC interacts with the membrane-associated adenosine A2b receptor, a G-protein-coupled receptor that induces cAMP accumulation on binding adenosine. We show that A2b is actually a netrin-1 receptor and induces cAMP accumulation on binding netrin-1. Finally, we show that netrin-1-dependent outgrowth of dorsal spinal cord axons directly involves A2b. Together our results indicate that the growth-promoting function of netrin-1 may require a receptor complex containing DCC and A2b.
Assuntos
Axônios/fisiologia , AMP Cíclico/biossíntese , Fatores de Crescimento Neural/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Purinérgicos P1/fisiologia , Proteínas Supressoras de Tumor , Animais , Encéfalo/fisiologia , Moléculas de Adesão Celular/metabolismo , Divisão Celular , Linhagem Celular , Galinhas , Técnicas de Cultura , Receptor DCC , Cones de Crescimento , Humanos , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/genética , Receptores de Netrina , Netrina-1 , Ratos , Receptor A2B de Adenosina , Receptores Purinérgicos P1/genética , Medula Espinal/citologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: To evaluate viral and cytokine signaling correlates of the persistent inflammation associated with chronic otitis media with effusion (OME). DESIGN: Prospective study. METHOD: Reverse transcriptase-polymerase chain reaction targeting RNA viruses frequently associated with OME (respiratory syncytial virus and parainfluenza virus type 3, the proinflammatory cytokines interleukin 8 and interleukin 1beta, and RANTES [regulated upon activation, normal T cell expressed and secreted]) was performed on mucosal biopsy samples and on samples of the liquid and cellular compartments of inflammatory exudates obtained from 26 children (49 ears) with infected middle ears. Ribonucleic acid extracted from rapidly frozen samples was reverse transcribed by Moloney murine leukemia virus reverse transcriptase and amplified for 35 cycles using previously validated primers. Amplicons were evaluated by molecular size after agarose gel electrophoresis with ethidium bromide. RESULTS: Most children had evidence of the presence of an RNA virus in at least one specimen. Respiratory syncytial virus was present in 40% and parainfluenza virus type 3 in 8% of effusions. Interleukin 8 messenger RNA was present in 21% of inflammatory exudates but never in cells from the mucosal biopsy samples. CONCLUSIONS: Our data support a viral contribution to the cause of OME and suggest that the inflammatory cytokines observed derive more from cells in the inflammatory exudate than from those in the middle ear mucosa.
Assuntos
Orelha Média/química , Exsudatos e Transudatos/química , Otite Média com Derrame/virologia , Vírus da Parainfluenza 3 Humana/isolamento & purificação , RNA Viral/análise , Vírus Sinciciais Respiratórios/isolamento & purificação , Biópsia , Quimiocina CCL5/análise , Criança , Pré-Escolar , Doença Crônica , Orelha Média/virologia , Exsudatos e Transudatos/virologia , Feminino , Humanos , Interleucina-1/análise , Interleucina-8/análise , Masculino , Mucosa/química , Mucosa/virologia , Vírus da Parainfluenza 3 Humana/genética , Reação em Cadeia da Polimerase , Estudos Prospectivos , RNA Mensageiro/análise , Vírus Sinciciais Respiratórios/genéticaRESUMO
The molecular mechanisms underlying cell cycle control in neuronal progenitors have been investigated with adult mouse olfactory epithelium as a model system. Odor receptive neurons of mammalian olfactory epithelium are short-lived and renewed in the adult by mitotic division of intrinsic neuronal progenitors. Ablation of the synaptic target, olfactory bulb, induces sequentially extensive apoptosis of sensory neurons and then stimulation of progenitor proliferation, peaking at 36 h and 4 days, respectively, postlesion. Known molecular effectors of G1 phase entry have been assessed on protein extracts of olfactory organs sampled at various postbulbectomy times in adult mice. The decay of betaIII-tubulin and olfactory marker protein levels and the rise of proliferating cell nuclear antigen (PCNA) levels, starting 1 and 3 days, respectively, postlesion, provided the kinetic frame of neuronal dynamics. Cyclin D1, cyclin E, and cyclin-dependent kinase cdk2 levels, low in olfactory organ of intact mice, increased 3 days after bulbectomy in parallel with PCNA levels; cdk4 content was initially high and unaffected by lesioning. Western blots of the known cdk inhibitors revealed proliferation-related decreases of p18, p21, and p27 from high expression in intact organs. Immunoprecipitation of cdk2 and cdk4 fractions of protein extracts at 4 days postlesion (mitotic reaction peak) versus control, followed by cyclin D1 immunoblotting, and vice versa, revealed that levels of both cyclin D1/cdk2 and cyclin D1/cdk4 complexes, as well as their kinase activities, were dramatically increased after lesion. In vivo proliferation of olfactory neuronal lineage cells thus involves functional binding of cyclin D1 with cdk2 and cdk4, with differential activation mechanisms for cdk2 and cdk4. In addition, the RT-PCR-detected cyclin D1 mRNA level remained unaffected after bulbectomy, which indicated that the cyclin D1 rise should involve posttranscriptional mechanisms in this in vivo neuronal system. These observations are discussed, along with their relevance to cell cycle control and to olfactory neuron dynamics.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Bulbo Olfatório/citologia , Neurônios Receptores Olfatórios/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Células-Tronco/citologia , Fatores Etários , Animais , Western Blotting , Divisão Celular/fisiologia , Ciclina D1/análise , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/análise , Denervação , Fase G1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/fisiologia , Bulbo Olfatório/cirurgia , Neurônios Receptores Olfatórios/enzimologia , Testes de Precipitina , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Serina-Treonina Quinases/análise , Fase S/fisiologia , Células-Tronco/enzimologiaRESUMO
Measles virus (MV) infection causes acute childhood disease, associated in certain cases with infection of the central nervous system (CNS) and development of neurological disease. To develop a murine model of MV-induced pathology, we generated several lines of transgenic mice ubiquitously expressing as the MV receptor a human CD46 molecule with either a Cyt1 or Cyt2 cytoplasmic tail. All transgenic lines expressed CD46 protein in the brain. Newborn transgenic mice, in contrast to nontransgenic controls, were highly sensitive to intracerebral infection by the MV Edmonston strain. Signs of clinical illness (lack of mobility, tremors, and weight loss) appeared within 5 to 7 days after infection, followed by seizures, paralysis, and death of the infected animals. Virus replication was detected in neurons from infected mice, and virus was reproducibly isolated from transgenic brain tissue. MV-induced apoptosis observed in different brain regions preceded the death of infected animals. Similar results were obtained with mice expressing either a Cyt1 or Cyt2 cytoplasmic tail, demonstrating the ability of different isoforms of CD46 to function as MV receptors in vivo. In addition, maternally transferred immunity delayed death of offspring given a lethal dose of MV. These results document a novel CD46 transgenic murine model where MV neuronal infection is associated with the production of infectious virus, similarly to progressive infectious measles encephalitis seen in immunocompromised patients, and provide a new means to study pathogenesis of MV infection in the CNS.
Assuntos
Encéfalo/patologia , Encefalite Viral/patologia , Vírus do Sarampo/fisiologia , Sarampo/patologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose , Encéfalo/metabolismo , Encéfalo/virologia , Modelos Animais de Doenças , Encefalite Viral/virologia , Feminino , Humanos , Imunidade Materno-Adquirida , Sarampo/imunologia , Sarampo/virologia , Vírus do Sarampo/isolamento & purificação , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Gravidez , Receptores Virais/genética , Receptores Virais/metabolismo , Transgenes , Replicação ViralRESUMO
Dopamine binding sites of D1 and D2/D3 subtypes had been detected in the rat peripheral olfactory system and postulated to account for dopamine-dependent enhancement of olfactory memory and retro-inhibition of olfactory input within the olfactory bulb, respectively. We further assessed, in the present study, the mechanisms of these dopamine actions by using adenylyl cyclase activity assay and [35S]GTP radioautography in rat olfactory bulb and mucosa. The D1 agonist SKF 38393 increased adenylyl cyclase activity on membranes of the olfactory bulb, but not on those of the olfactory mucosa. Stimulation of adenylyl cyclase by SKF 38393 in the olfactory bulb was dose dependent, with a half-maximal effect (EC50) at 0.16 microM SKF 38393, reaching 40% over basal adenylyl cyclase activity, and was blocked by the D1 antagonist SCH 23390. The D2 agonists bromocriptine and quinpirole inhibited both basal and forskolin-stimulated adenylyl cyclase activities in the olfactory bulb and mucosa. These adenylyl cyclase inhibitions were dose dependent, with EC50 values of 0.1-0.3 microM for bromocriptine and 1-3 microM for quinpirole, equal to 25% of basal enzyme activity at concentrations of 1-10 microM, and were blocked by the D2 antagonist eticlopride. The D2 antagonist was devoid of any effect on basal and forskolin-stimulated adenylyl cyclase activities in the olfactory bulb and mucosa. Odorant-induced stimulation of adenylyl cyclase was blocked by D2 agonist in olfactory mucosa membranes, which suggests dopaminergic regulation of odor detection in the olfactory mucosa. By using microdissected fractions of the olfactory mucosa, D2 agonist-induced inhibition of adenylyl cyclase was shown to occur only in lamina propria, thus co-localizing with D2 binding sites. [35S]GTP radioautography on tissue sections revealed D2 agonist-induced G-protein activation in olfactory nerve and glomerular layers of the olfactory bulb, and in the chorion of the olfactory mucosa. Taken together, these data demonstrate functional coupling of the dopamine receptors with adenylyl cyclase in both the olfactory bulb and mucosa, and document novel aspects of dopamine's physiological involvement in olfaction and of D2-mediated signal transduction.
Assuntos
Adenilil Ciclases/fisiologia , Dopamina/fisiologia , Bulbo Olfatório/fisiologia , Mucosa Olfatória/fisiologia , Condutos Olfatórios/fisiologia , Receptores de Dopamina D1/fisiologia , Receptores de Dopamina D2/fisiologia , Transdução de Sinais/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Autorradiografia/métodos , Benzazepinas/farmacologia , Bromocriptina/farmacologia , Carbacol/farmacologia , Colforsina/farmacologia , Corpo Estriado/enzimologia , Corpo Estriado/fisiologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/fisiologia , Guanosina Trifosfato/fisiologia , Masculino , Agonistas Muscarínicos/farmacologia , Nitrilas/farmacologia , Odorantes , Bulbo Olfatório/enzimologia , Mucosa Olfatória/enzimologia , Condutos Olfatórios/enzimologia , Quimpirol/farmacologia , Ratos , Ratos Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/antagonistas & inibidores , Salicilamidas/farmacologiaRESUMO
Using in vitro labelling and autoradiographic techniques, we have analyzed the fine and the detailed distribution of benzodiazepine binding sites in the post-mortem human hypothalamus. Binding sites were labelled in mounted tissue sections from adult brains, using the selective high affinity ligand [3H]-Flunitrazepam. A heterogeneous distribution of benzodiazepine binding sites was found throughout the rostrocaudal extent of human hypothalamus. The autoradiographic labelling was shown in the three hypothalamic parts, i.e., anterior, mediobasal and posterior levels. At the anterior level, the highest densities were present in the diagonal band of Broca, the preoptic area (medial and lateral parts) and the septohypothalamic nucleus. At the mediobasal hypothalamic level, the highest densities were mainly localized in the ventromedial nucleus, whereas the other structures were moderately labelled with [3H]-Flunitrazepam. The mammillary complex as well as the posterior hypothalamic area represented the most heavily labelled structures in the posterior hypothalamus. The results obtained in this study, indicate the presence of a large and heterogeneous distribution of benzodiazepine binding sites in human adult hypothalamus. This could support their implication in the control of distinct neural functions (like neuroendocrine role).