RESUMO
Prenatal stress (PS) represents a critical variable affecting lifetime health trajectories, metabolic and vascular functions. Beneficial experiences may attenuate the effects of PS and its programming of health outcomes in later life. Here we investigated in a rat model (1) if PS modulates recovery following cortical ischemia in adulthood; (2) if a second hit by adult stress (AS) exaggerates stress responses and ischemic damage; and (3) if tactile stimulation (TS) attenuates the cumulative effects of PS and AS. Prenatally stressed and non-stressed adult male rats underwent focal ischemic motor cortex lesion and were tested in skilled reaching and skilled walking tasks. Two groups of rats experienced recurrent restraint stress in adulthood and one of these groups also underwent daily TS therapy. Animals that experienced both PS and AS displayed the most severe motor disabilities after lesion. By contrast, TS promoted recovery from ischemic lesion and reduced hypothalamic-pituitary-adrenal axis activity. The data also showed that cumulative effects of adverse and beneficial lifespan experiences interact with disease outcomes and brain plasticity through the modulation of gene expression. Microarray analysis of the lesion motor cortex revealed that cumulative PS and AS interact with genes related to growth factors and transcription factors, which were not affected by PS or lesion alone. TS in PS+AS animals reverted these changes, suggesting a critical role for these factors in activity-dependent motor cortical reorganization after ischemic lesion. These findings suggest that beneficial experience later in life can moderate adverse consequences of early programming to improve cerebrovascular health.
Assuntos
Encéfalo/metabolismo , Recuperação de Função Fisiológica/genética , Sensação , Estresse Psicológico/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/fisiopatologia , Transcriptoma/genética , Envelhecimento/genética , Envelhecimento/fisiologia , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Sistema Hipotálamo-Hipofisário/fisiopatologia , Masculino , Atividade Motora , Córtex Motor/fisiopatologia , Movimento , Análise de Sequência com Séries de Oligonucleotídeos , Estimulação Física , Sistema Hipófise-Suprarrenal/fisiopatologia , Ratos Long-Evans , Reprodutibilidade dos Testes , Estresse Psicológico/fisiopatologia , Tato , CaminhadaRESUMO
Tuberculosis (TB) is a serious public health problem. Development of experimental models and vaccines are essential to elucidate physiopathological mechanisms and to control the disease. Vascular endothelial growth factor (VEGF) is a potent activator of vascular permeability and angiogenesis. VEGF seems to participate in breakdown of the blood brain-barrier (BBB) in tuberculous meningitis (TBM), contributing to worsening of disease. Therefore, the objective here was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB) by describing the VEGF participation in the CNS disease, and suggesting a vaccination plan in mice. Plasmid encoding DNA protein antigen DNA-hsp65 has been described as a protector against TB infection and was used here to test its effectiveness in the prevention of VEGF production and TB disease. Vaccinated mice and its controls were injected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in cerebellum. Four weeks after BCG injection, mice were perfused and brains were paraffin-embedded for VEGF expression analysis. We observed VEGF immunohistochemical expression in TBM and granulomas in non-vaccinated mice. The DNA-hsp65 treatment blocked the expression of VEGF in mice TBM. Therefore, our murine model indicated the VEGF participation in the physiopathology of CNS-TB and the potential prevention of the DNA-hsp65 in the disease progression.
Assuntos
Proteínas de Bactérias/imunologia , Chaperonina 60/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose do Sistema Nervoso Central/metabolismo , Vacinas de DNA/imunologia , Fator A de Crescimento do Endotélio Vascular/biossíntese , Animais , Proteínas de Bactérias/genética , Doenças Cerebelares/metabolismo , Doenças Cerebelares/prevenção & controle , Cerebelo/metabolismo , Chaperonina 60/genética , Modelos Animais de Doenças , Esquemas de Imunização , Masculino , Camundongos , Mycobacterium bovis , Tuberculoma Intracraniano/metabolismo , Tuberculoma Intracraniano/prevenção & controle , Tuberculose do Sistema Nervoso Central/prevenção & controle , Tuberculose Meníngea/metabolismo , Tuberculose Meníngea/prevenção & controleRESUMO
Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB, characterized morphologically by brain granulomas and tuberculous meningitis (TBM). Experimental strategies for the study of the host-pathogen interaction through the analysis of granulomas and its intrinsic molecular mechanisms could provide new insights into the neuropathology of TB. To verify whether cerebellar mycobacterial infection induces the main features of the disease in human CNS and better understand the physiological mechanisms underlying the disease, we injected bacillus Calmette-Guerin (BCG) into the mouse cerebellum. BCG-induced CNS-TB is characterized by the formation of granulomas and TBM, a build up of bacterial loads in these lesions, and microglial recruitment into the lesion sites. In addition, there is an enhanced expression of signaling molecules such as nuclear factor-κB (NF-κB) and there is a presence of inducible nitric oxide synthase (iNOS) in the lesions and surrounding areas. This murine model of cerebellar CNS-TB was characterized by cellular and biochemical immune responses typically found in the human disease. This model could expand our knowledge about granulomas in TB infection of the cerebellum, and help characterize the physiological mechanisms involved with the progression of this serious illness that is responsible for killing millions people every year.
Assuntos
Compreensão , Modelos Animais de Doenças , Granuloma/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Mycobacterium bovis/patogenicidade , Tuberculose do Sistema Nervoso Central/microbiologia , Animais , Granuloma/etiologia , Granuloma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/microbiologia , Microglia/patologia , Tuberculose do Sistema Nervoso Central/complicações , Tuberculose do Sistema Nervoso Central/patologiaRESUMO
Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900 W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.
Assuntos
Córtex Cerebelar/enzimologia , Óxido Nítrico Sintase/metabolismo , Isoformas de Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Arteríolas/enzimologia , Linhagem Celular , Pré-Escolar , Feminino , Feto , Granuloma/enzimologia , Granuloma/patologia , Humanos , Lactente , Recém-Nascido , Pulmão/citologia , Masculino , Pessoa de Meia-Idade , Mudanças Depois da Morte , Ratos , Fatores de TempoRESUMO
Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50mM TrisHCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10mMsodium citrate buffer,pH6.0, for 15 min at 900 W.Inflammatory cells in ahumanlung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli.Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.