RESUMO
In mice with experimental autoimmune encephalomyelitis (EAE) pretreatment with progesterone improves clinical signs and decreases the loss of myelin basic protein (MBP) and proteolipid protein (PLP) measured by immunohistochemistry and in situ hybridization. Presently, we analyzed if progesterone effects in the spinal cord of EAE mice involved the decreased transcription of local inflammatory mediators and the increased transcription of myelin proteins and myelin transcription factors. C57Bl/6 female mice were divided into controls, EAE and EAE receiving progesterone (100mg implant) 7 days before EAE induction. Tissues were collected on day 17 post-immunization. Real time PCR technology demonstrated that progesterone blocked the EAE-induced increase of the proinflammatory mediators tumor necrosis factor alpha (TNFα) and its receptor TNFR1, the microglial marker CD11b and toll-like receptor 4 (TLR4) mRNAs, and increased mRNA expression of PLP and MBP, the myelin transcription factors NKx2.2 and Olig1 and enhanced CC1+oligodendrocyte density respect of untreated EAE mice. Immunocytochemistry demonstrated decreased Iba1+microglial cells. Confocal microscopy demonstrated that TNFα colocalized with glial-fibrillary acidic protein+astrocytes and OX-42+microglial cells. Therefore, progesterone treatment improved the clinical signs of EAE, decreased inflammatory glial reactivity and increased myelination. Data suggest that progesterone neuroprotection involves the modulation of transcriptional events in the spinal cord of EAE mice.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Mediadores da Inflamação/metabolismo , Bainha de Mielina/efeitos dos fármacos , Progesterona/farmacologia , Medula Espinal/metabolismo , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Regulação para Baixo/efeitos dos fármacos , Encefalomielite Autoimune Experimental/patologia , Feminino , Proteína Homeobox Nkx-2.2 , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Microglia/metabolismo , Microscopia Confocal , Proteínas da Mielina/biossíntese , Bainha de Mielina/patologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Medula Espinal/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
The hypoxic-ischemic encephalopathy caused by peripartum asphyxia is a serious disease in newborn infants, and effective therapies need to be developed to reduce injury-related disorders. We evaluated the effects of NEP1-40 and fasudil on Nogo-A expression in neonatal hypoxic-ischemic brain damage (HIBD) rats. Seven-day-old Wistar rats were randomly divided into control, HIBD, NEP1-40, and fasudil groups. NEP1-40 and fasudil groups were injected intraperitoneally with these compounds. Rat brains at 6, 24, 72 h, and 7 days after HIBD were collected to determine histopathological damage and the expression levels of Nogo-A. Histopathological damage was reduced in NEP1-40 and fasudil groups compared with the untreated HIBD group. The expression of Nogo-A in the HIBD group was significantly higher than that in control, NEP1-40 and fasudil groups at the same times. Compared with the fasudil group, the expression levels of Nogo-A were significantly reduced in the NEP1-40 group. We conclude that NPE1-40 and fasudil have potential for neuroprotective effects in the neonatal rat HIBD model, mediated by inhibiting Nogo-A/ Rho pathways.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Encéfalo/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/prevenção & controle , Proteínas da Mielina/biossíntese , Proteínas da Mielina/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Encéfalo/patologia , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Feminino , Expressão Gênica/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Imuno-Histoquímica , Hibridização In Situ , Injeções Intraperitoneais , Ligadura/métodos , Masculino , Proteínas da Mielina/administração & dosagem , Proteínas da Mielina/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Proteínas Nogo , Fragmentos de Peptídeos/administração & dosagem , Ratos , Ratos WistarRESUMO
OBJECTIVES: The prognosis of breast cancer patients depends on primary tumor resection and axillary lymph nodes examination. The purpose of this study was to analyze by molecular biology techniques the presence of mammaglobin A and B messenger RNA in breast sentinel lymph node (SLN) by reverse-transcription polymerase chain reaction (RT-PCR). METHODS: Sentinel lymph nodes from 50 patients with a diagnosis of breast cancer were prospectively studied between June 2004 and August 2006. Lymph nodes were all examined every 2 mm by intraoperative cytology. Hematoxylin-eosin (HE), immunohistochemistry (IHC) with cytokeratin (clone AE1-AE3, DAKO, dilution 1:100), and molecular biology techniques were used in all cases. RESULTS: Deferred study with routine techniques showed subcapsular metastasis in 3/50 cases. Out of 50 cases, 5 were detected with IHC, and 2 of them were negative for HE. Multiplex RT-PCR allowed the detection of 18/50 positive SLN, which included the 5 above-mentioned cases. The other SLN studied (32/50) showed no metastases with the methods herein implemented. CONCLUSIONS: The epidemiologic impact of incomplete SLN study has been observed, as the HE technique fails to identify all SLN with micrometastases. In our opinion, SLN should be studied with IHC and molecular biology techniques. The multiplex RT-PCR technique for A and B mammaglobin proves to be specific and sensitive. This study will serve to formulate hypotheses. Further research, including a larger population and a longer-term follow-up period, will be required to confirm these hypotheses. Should our findings be confirmed in the future, molecular biology determinations could modify patients' staging and treatment.
Assuntos
Neoplasias da Mama/diagnóstico , Carcinoma/diagnóstico , Perfilação da Expressão Gênica , Linfonodos/patologia , Proteínas da Mielina/biossíntese , Proteínas de Neoplasias/biossíntese , Proteolipídeos/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Uteroglobina/biossíntese , Sequência de Bases , Feminino , Histocitoquímica , Humanos , Imuno-Histoquímica , Mamoglobina A , Dados de Sequência Molecular , SecretoglobinasRESUMO
We have previously shown that a single intracranial injection of apotransferrin (aTf) in neonatal rats produces an accelerated mylinogenesis and increases the expression of certain myelin proteins such as myelin basic protein (MBP). In the present work, we studied the effects of aTf upon oligodendrocyte progenitor cell (Opc) cultures. In the presence of aTf, cells developed a multipolar morphology and showed an increased expression of O(4), MBP, O(1) and myelin-associated glycoprotein compared to controls. Migration studies using the agarose drop assay showed that aTf strongly inhibited OPc migration. This effect was not observed when an antibody against the transferrin receptor was added. The expression of two cell adhesion molecules, neural cell adhesion molecule (NCAM), N-cadherin and of polysialylated NCAM (PSA-NCAM) was evaluated by immunocytochemistry and by Western blot. Although NCAM expression did not change, there was a significant increase in N-cadherin expression and a decrease in PSA-NCAM in the aTf-treated cells. Time lapse studies of the expression of PSA-NCAM as an indicator of migration and of MBP as a marker of differentiation showed that in the cultures treated with aTf there is first a decrease in the percentage of cells expressing the former molecule which is followed by an increase in the percentage of cells expressing MBP. These results suggest that aTf added in vitro to cultured OPcs inhibits first their migration and then enhances their differentiation.
Assuntos
Apoproteínas/farmacologia , Oligodendroglia/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Transferrina/farmacologia , Animais , Caderinas/biossíntese , Caderinas/genética , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Moléculas de Adesão de Célula Nervosa/biossíntese , Moléculas de Adesão de Célula Nervosa/genética , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The targeting of polypeptides to restricted cytoplasmic domains by means of mRNA sorting is a widespread phenomena utilized by many cell types. In the central nervous system, in situ hybridization analysis has shown previously that the mRNAs encoding several myelin-specific proteins are specifically located within the myelinating processes of oligodendrocytes. Here, by means of biochemical and subcellular fractionation methods, we show that a myelin fraction is selectively enriched in those mRNAs. The four major myelin basic protein (MBP) mRNAs that arise by alternative splicing of exons II and VI of the MBP gene are concentrated in this subcellular fraction. Furthermore, an interaction of MBP and MOBP 81A mRNAs with the cytoskeleton was observed. This interaction might serve to mediate the anchoring of these messengers after translocation to the subcellular site of translation.
Assuntos
Citoesqueleto/metabolismo , Proteínas da Mielina/biossíntese , RNA Mensageiro/biossíntese , Frações Subcelulares/metabolismo , Processamento Alternativo , Animais , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Citoesqueleto/química , Éxons/fisiologia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Proteínas da Mielina/análise , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Pressão Osmótica , Plasmídeos , Sondas RNA , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/químicaRESUMO
Expression of myelin P0 protein by myelinating Schwann cells in vivo is dependent on axonal influences. This report describes P0 gene expression during development of rat sciatic nerve and spinal nerve roots using Northern blotting, in situ hybridization and immunohistochemistry. We demonstrate that: (1) the appearance of P0 mRNA and P0 protein in Schwann cells during nerve development in the rat begins prenatally, at day 18 post-fertilization (E18); (2) P0 mRNA and P0 protein have essentially identical developmental profiles, and are expressed in Schwann cells that are many days prior to myelin formation; (3) initial P0 gene expression is greatest in Schwann cells at the periphery of nerve bundles and in Schwann cells in contact with motor axons; (4) the decline in P0 expression with nerve maturation is accompanied by a sharp decline in P0 message levels in most Schwann cells, but a small subpopulation of these cells continue to synthesize very high levels of P0 mRNA. This study provides data on myelin P0 protein gene expression and distribution during PNS development and adds further insights into the axonal influences controlling Schwann cell behaviour during myelination of the rat PNS.
Assuntos
Envelhecimento/fisiologia , Desenvolvimento Embrionário e Fetal , Expressão Gênica , Proteínas da Mielina/biossíntese , Neurônios/metabolismo , RNA Mensageiro/biossíntese , Nervo Isquiático/metabolismo , Raízes Nervosas Espinhais/metabolismo , Animais , Northern Blotting , Moléculas de Adesão Celular Neuronais/biossíntese , Idade Gestacional , Hibridização In Situ , Proteína P0 da Mielina , Proteínas da Mielina/análise , Neurônios/citologia , Neurônios/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/embriologia , Nervo Isquiático/crescimento & desenvolvimento , Raízes Nervosas Espinhais/embriologia , Raízes Nervosas Espinhais/crescimento & desenvolvimentoRESUMO
Thyroid hormones have a significant influence on the development and maturation of the central nervous system. Among their actions, T3 and T4 have effects on the differentiation of various cell types in the rat brain and cerebellum as well as on the process of myelination. Recently, several investigators have shown effects of thyroid hormones on myelin protein gene expression. Thyroid hormones seem to have a regulatory role with regard to life span. Hyperthyroid animals appear to have a shorter life and, at advanced age, show a myelin deficit. This may be due to the damage produced by the oxidative stress generated by an excess of thyroid hormones.
Assuntos
Encéfalo/fisiologia , Tiroxina/fisiologia , Tri-Iodotironina/fisiologia , Envelhecimento/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Cerebelo/fisiologia , Expressão Gênica , Homeostase , Hipertireoidismo/fisiopatologia , Hipotireoidismo/fisiopatologia , Proteínas da Mielina/biossíntese , Bainha de Mielina/fisiologia , Estresse Oxidativo , Ratos , Tiroxina/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
1. The myelin protein profiles in the CNS and PNS of three species of amphibians were analyzed by biochemical and immunohistochemical methods. 2. The CNS myelin of the African clawed frog (Xenopus) and the Mexican salamander (axolotl) contained, in addition to proteolipid protein, a unique protein zero (P0)-like protein, whereas the adult bullfrog did not. 3. A strong expression of the P0-like protein in the bullfrog CNS myelin was found transiently at ontogenetically early phases including at the time of metamorphosis. 4. The CNS P0-like protein and the PNS P0 protein showed a difference in reactivity with lectins and anti-L2/HNK-1 antibodies, suggesting that the two proteins differ in some aspects of their carbohydrate structures.