RESUMEN
Normal pressure hydrocephalus (NPH) compromises the morphology of the corpus callosum (CC). This study aims to determine whether 60- or 120-day NPH disrupts the cytoarchitecture and functioning of white matter (WM) and oligodendrocyte precursor cells (OPCs) and establish whether these changes are reversible after hydrocephalus treatment. NPH was induced in CD1 adult mice by inserting an obstructive lamina in the atrium of the aqueduct of Sylvius. Five groups were assembled: sham-operated controls (60 and 120 days), NPH groups (60 and 120 days), and the hydrocephalus-treated group (obstruction removal after 60-d hydrocephalus). We analyzed the cellular integrity of the CC by immunohistochemistry, TUNEL analysis, Western blot assays, and transmission electron microscopy (TEM). We found a reduction in the width of the CC at 60 and 120 days of NPH. TEM analysis demonstrated myelin abnormalities, degenerative changes in the WM, and an increase in the number of hyperdense (dark) axons that were associated with significant astrogliosis, and microglial reactivity. Hydrocephalus also caused a decrease in the expression of myelin-related proteins (MOG and CNPase) and reduced proliferation and population of OPCs, resulting in fewer mature oligodendrocytes. Hydrocephalus resolution only recovers the OPC proliferation and MOG protein density, but the rest of the WM abnormalities persisted. Interestingly, all these cellular and molecular anomalies occur in the absence of behavioral changes. The results suggest that NPH severely disrupts the myelin integrity and affects the OPC turnover in the CC. Remarkably, most of these deleterious events persist after hydrocephalus treatment, which suggests that a late treatment conveys irreversible changes in the WM of CC.
Asunto(s)
Hidrocéfalo Normotenso , Células Precursoras de Oligodendrocitos , Ratones , Animales , Cuerpo Calloso , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , Vaina de Mielina , Oligodendroglía , Proteínas de la Mielina , Proliferación CelularRESUMEN
CNP::EGFP transgenic mice, genetically engineered to express the enhanced green fluorescent protein (EGFP) under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase) promoter in oligodendroglial and Schwann cells, constitute a very important and widely used tool for the study of oligodendrocyte (OLG) development and function in young mice. Our results showed that CNP::EGFP mice were significantly more susceptible to CPZ-induced demyelination, as evaluated by MBP immunostaining, oligodendroglial progenitor cell (OPC) recruitment and astroglial, microglial and nestin response. This enhanced vulnerability was a consequence of their hypomyelination. CNP::EGFP control mice also displayed a significant decrease in corpus callosum (CC) thickness and MBP immunoreactivity. Morphometric analysis further showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio carried out in the optic nerve (ON) and CC of CNP::EGFP, as compared to WT mice. Moreover, our results showed a decrease in the number of axons of small caliber, concomitantly with an increase in the number of axons of bigger size with more and enlarged mitochondria, which suggests a high energy demand. These findings and those displaying that MBP+ cells and NF200 staining in the CNP::EGFP cortex were more sparsely distributed provide evidence of axonal loss, which was supported by a decreased number of NeuN+ cells in the CA3 fields of the hippocampus. Transgenic mice also showed an increase in microglial and astroglial activation, accompanied by enhanced lipid peroxidation and recruitment of morphologically altered OPC. Finally, CNPase protein levels proved to be lower than MBP in the CC, which might indicate an altered pattern in myelin proteins with a CNPase deficiency. Behavioral analysis of adult CNP::EGFP transgenic mice supported our results, as it revealed a decrease in locomotion, exploratory activity and motor impairment, as compared to their WT littermates. Our data highlight the relevance of confronting results obtained in adult CNP::EGFP mice with those observed in WT mice. According to our findings, CNP::EGFP hypomyelination might be triggered by the cellular stress induced by the high level of EGFP expression in mature OLG. Adult CNP::EGFP mice could be considered a useful tool to evaluate future therapies for demyelinating diseases such as multiple sclerosis (MS), since these animals present chronic demyelination with axonal degeneration, a characteristic of such pathologies.
Asunto(s)
Cuprizona/toxicidad , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Inhibidores de la Monoaminooxidasa/toxicidad , Proteínas de la Mielina/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , Aldehídos/metabolismo , Animales , Encéfalo/patología , Encéfalo/ultraestructura , Antígeno CD11b/metabolismo , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Modelos Animales de Enfermedad , Ectodisplasinas/metabolismo , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Interleucina-1beta/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Mitocondrias/metabolismo , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Proteínas del Tejido Nervioso/metabolismo , Oligodendroglía/efectos de los fármacos , Oligodendroglía/patología , Desempeño Psicomotor/efectos de los fármacos , Desempeño Psicomotor/fisiología , Especies Reactivas de Oxígeno/metabolismo , Células Madre/efectos de los fármacos , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Cuprizone (bis-cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well-known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone-treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration.
Asunto(s)
Encéfalo/patología , Enfermedades Desmielinizantes/patología , Oligodendroglía/patología , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/metabolismo , Recuento de Células , Proliferación Celular , Cuprizona , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Gliosis/metabolismo , Gliosis/patología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Proteína Básica de Mielina/metabolismo , Neuronas/metabolismo , Neuronas/patología , Oligodendroglía/metabolismo , Regiones Promotoras GenéticasRESUMEN
Apotransferrin (aTf), intracranially administered into newborn rats, produces increased myelination with marked increases in the levels of myelin basic protein (MBP), phospholipids and galactolipids, and mRNAs of MBP and 2', 3' cyclic nucleotide 3'-phosphohydrolase (CNPase). Cytoskeletal proteins such as tubulin, actin, and microtubule-associated proteins are also increased after aTf injection. In contrast, almost no changes are observed in myelin proteolipid protein (PLP) or in its mRNA or cholesterol. In the present study, we used brain-tissue slices and cell cultures highly enriched for oligodendroglia to investigate signaling pathways involved in the action of aTf, and to find out whether cytoskeletal integrity and dynamics were essential for its action upon the neural expression of certain genes. Treatment of brain-tissue slices with aTf produced a marked increase in the expression of MBP, CNPase, and tubulin mRNAs. Colchicine, cytochalasin, and taxol severely reduced the effect of aTf. Addition to cultures of an antibody against transferrin receptor (TfR), protein kinase inhibitors, or a cyclic AMP (cAMP) analogue showed that a functionally intact TfR was necessary, and that tyrosine kinase, protein kinase C and A, as well as calcium-calmodulin-dependent kinase (Ca-CaMK) activities appeared to mediate aTf actions upon the expression of the above mentioned genes. Changes in the levels of phosphoinositides and cAMP induced by aTf in oligodendroglial cell (OLGc) cultures correlated with these results and coincide with an activation of the cyclic response element binding protein (CREB) and of mitogen activated protein kinases. The increased expression of certain myelin genes produced by aTf appear to be mediated by interaction of this glycoprotein with its receptor, by the cytoskeleton of the OLGc, and by a complex activation of protein kinases which lead to CREB phosphorylation.
Asunto(s)
Apoproteínas/metabolismo , Sistema Nervioso Central/crecimiento & desarrollo , Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Sistemas de Mensajero Secundario/genética , Transferrina/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/efectos de los fármacos , 2',3'-Nucleótido Cíclico Fosfodiesterasas/genética , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citoesqueleto/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína Básica de Mielina/efectos de los fármacos , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/genética , Oligodendroglía/citología , Oligodendroglía/efectos de los fármacos , Técnicas de Cultivo de Órganos , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Transferrina/efectos de los fármacos , Receptores de Transferrina/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Tubulina (Proteína)/efectos de los fármacos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
Thyroid hormones are critical for maturation of the central nervous system. In a previous study, we showed a change in the pattern of mature myelinated nerve fibers by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in developing hypothyroid animals, which suggests a possible role for thyroid hormones in myelin compaction. The classical myelin markers myelin basic protein (MBP) and proteolipidic protein (PLP) are expressed later in oligodendroglial development, when myelin sheath formation is in progress. A myelin constituent designated myelin-associated/oligodendrocytic basic protein (MOBP) has been identified and related to myelin compaction. We assessed the developmental sequence of appearance of CNPase, MBP, MOPB, and PLP proteins in cerebellum (Cb) and corpus callosum (cc) in an experimental hypothyroidism model. The appearance of both MOBP isoforms occurred at postnatal day (P)25 and P30 in cc and Cb, respectively, followed by an increase with age in the control group. However, all the MOBP isoforms were weakly detectable in both regions at P30 from the hypothyroid (H) group, and the higher molecular weight isoform remains decreased in cc, even at P90. The developmental pattern of expression of CNPase, MBP, and PLP proteins was also delayed in the H group. CNPase and MBP expression was recovered in cc and Cb, whereas PLP remained below control levels at P90 in cc. Our data show that the experimental hypothyroidism affects the developmental pattern of the oligodendrocytic/myelin markers. Furthermore, thyroid hormone may modulate specific genes, as demonstrated by permanent down-regulation of MOBP and PLP expression in adulthood.