RESUMO
Ischemic stroke is the leading cause of human disability and mortality in the world. Neuroinflammation is the main pathological event following ischemia which contributes to secondary brain tissue damage and is driven by infiltration of circulating immune cells such as macrophages. Because of neuroprotective properties against ischemic brain damage, estrogens have the potential to become of therapeutic interest. However, the exact mechanisms of neuroprotection and signaling pathways is not completely understood. In the current study, 12-week-old male Wistar rats underwent an experimental ischemia by occluding the middle cerebral artery transiently (tMCAO) for 1 h. Male rats subjected to tMCAO were randomly assigned to receive 17ß-estradiol or vehicle treatment. The animals were sacrificed 72 h post tMCAO, transcardially perfused and the brains were proceeded either for TTC staining and gene analysis or for flow cytometry (CD45, CD11b, CD11c, CD40). We found that 17ß-estradiol substitution significantly reduced the cortical infarct which was paralleled by an improved Garcia test scoring. Flow cytometry revealed that CD45+ cells as well as CD45+CD11b+CD11c+ cells were massively increased in tMCAO animals and numbers were nearly restored to sham levels after 17ß-estradiol treatment. Gene expression analysis showed a reperfusion time-dependent upregulation of the markers CD45, CD11b and the activation marker CD40. The reduction in gene expression after 72 h of reperfusion and simultaneous 17ß-estradiol substitution did not reach statistical significance. These data indicate that 17ß-estradiol alleviated the cerebral ischemia-reperfusion injury and selectively suppressed the activation of the neuroinflammatory cascade via reduction of the number of activated microglia or infiltrated monocyte-derived macrophages in brain.
Assuntos
Estradiol/farmacologia , Infarto da Artéria Cerebral Média/imunologia , Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Acidente Vascular Cerebral/imunologia , Animais , Citocinas/imunologia , Estradiol/uso terapêutico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Masculino , Fármacos Neuroprotetores/uso terapêutico , Ratos Wistar , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219-2230.
Assuntos
Astrócitos/metabolismo , Doenças Desmielinizantes/etiologia , Regulação da Expressão Gênica/fisiologia , Esclerose Múltipla/complicações , Esclerose Múltipla/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Encéfalo/patologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Cuprizona/toxicidade , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Esclerose Múltipla/induzido quimicamente , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismoRESUMO
BACKGROUND AND PURPOSE: Modulation of the sphingosine 1-phosphate receptor is an approved treatment for relapsing multiple sclerosis because of its anti-inflammatory effect of retaining lymphocytes within the lymph nodes. Here, we evaluated the potential of an agonist at this receptor, FTY720 (fingolimod), to activate the promyelinating pathways within the brain to encourage remyelination and neuroprotection. EXPERIMENTAL APPROACH: In this study, we used the cuprizone model in male C57BL/6 mice and tested the promyelinating and neuroprotective effects of FTY720 after acute and chronic toxin-induced experimental demyelination. We used histological, immunohistochemical and gene expression methods. KEY RESULTS: The midline of the corpus callosum was severely demyelinated after acute and chronic cuprizone-induced demyelination. Robust endogenous remyelination was evident after acute, but impaired after chronic, demyelination. FTY720 treatment modestly accelerated myelin recovery after acute but not chronic cuprizone exposure. Markers of gliosis (astrocyte and microglia activation) were not affected by FTY720 treatment. Remarkably, the accumulation of amyloid precursor protein-positive spheroids in axons was less distinct in FTY720-treated animals, indicating that this compound alleviated ongoing axonal damage. CONCLUSIONS AND IMPLICATIONS: We show that even during endogenous remyelination, axonal degeneration continued at a low level, accumulating over time. This continuous neurodegenerative process was ameliorated by FTY720 treatment. FTY720 preserved CNS integrity by direct interaction with brain resident cells, the actions of which are still to be defined.
Assuntos
Doenças Desmielinizantes/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Propilenoglicóis/uso terapêutico , Receptores de Lisoesfingolipídeo/agonistas , Esfingosina/análogos & derivados , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/patologia , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Cloridrato de Fingolimode , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Fosfatidato Fosfatase/genética , Monoéster Fosfórico Hidrolases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Propilenoglicóis/farmacologia , Esfingosina/farmacologia , Esfingosina/uso terapêuticoRESUMO
The cuprizone model is a suitable animal model of de- and remyelination secondary to toxin-induced oligodendrogliopathy. From a pharmaceutical point of view, the cuprizone model is a valuable tool to study the potency of compounds which interfere with toxin-induced oligodendrocyte cell death or boost/inhibit remyelinating pathways and processes. The aim of this study was to analyze the vulnerability of neighboring white mater tracts (i.e., the fornix and cingulum) next to the midline of the corpus callosum which is the region of interest of most studies using this model. Male mice were fed cuprizone for various time periods. Different white matter areas were analyzed for myelin (anti-PLP), microglia (anti-IBA1), and astrocyte (anti-GFAP) responses by means of immunohistochemistry. Furthermore, Luxol fast blue-periodic acid Schiff stains were performed to validate loss of myelin-reactive fibers in the different regions. Cuprizone induced profound demyelination of the midline of the corpus callosum and medial parts of the cingulum that was paralleled by a significant astrocyte and microglia response. In contrast, lateral parts of the corpus callosum and the cingulum, as well as the fornix region which is just beneath the midline of the corpus callosum appeared to be resistant to cuprizone exposure. Furthermore, resistant areas displayed reduced astrogliosis and microgliosis. This study clearly demonstrates that neighboring white matter tracts display distinct vulnerability to toxin-induced demyelination. This important finding has direct relevance for evaluation strategies in this frequently used animal model for multiple sclerosis.
Assuntos
Quelantes/toxicidade , Corpo Caloso/patologia , Cuprizona/toxicidade , Fibras Nervosas Mielinizadas/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/patologia , Fórnice/química , Fórnice/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Proteínas da Mielina/análise , Proteínas da Mielina/efeitos dos fármacos , Fibras Nervosas Mielinizadas/efeitos dos fármacosRESUMO
Brain inflammation plays a central role in multiple sclerosis (MS). Besides lymphocytes, the astroglia and microglia mainly contribute to the cellular composition of the inflammatory infiltrate in MS lesions. Several studies were able to demonstrate that cortical lesions are characterized by lower levels of inflammatory cells among activated microglia/macrophages. The underlying mechanisms for this difference, however, remain to be clarified. In the current study, we compared the kinetics and extent of microglia and astrocyte activation during early and late cuprizone-induced demyelination in the white matter tract corpus callosum and the telencephalic gray matter. Cellular parameters were related to the expression profiles of the chemokines Ccl2 and Ccl3. We are clearly able to demonstrate that both regions are characterized by early oligodendrocyte stress/apoptosis with concomitant microglia activation and delayed astrocytosis. The extent of microgliosis/astrocytosis appeared to be greater in the subcortical white matter tract corpus callosum compared to the gray matter cortex region. The same holds true for the expression of the key chemokines Ccl2 and Ccl3. The current study defines a model to study early microglia activation and to investigate differences in the neuroinflammatory response of white vs. gray matter.
Assuntos
Córtex Cerebral/imunologia , Quimiocina CCL2/genética , Quimiocina CCL3/genética , Corpo Caloso/imunologia , Cuprizona/toxicidade , Doenças Desmielinizantes/imunologia , Encefalite/imunologia , Animais , Córtex Cerebral/patologia , Quimiocina CCL2/imunologia , Quimiocina CCL3/imunologia , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/genética , Regulação da Expressão Gênica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/imunologia , Microglia/patologia , Inibidores da Monoaminoxidase/toxicidade , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Oligodendroglia/imunologia , Oligodendroglia/patologiaRESUMO
Sex steroids have been demonstrated as powerful compounds to protect neurones and neural tissue from neurotoxic challenges and during neurodegeneration. A multitude of cellular actions have been attributed to female gonadal steroid hormones, including the regulation of pro-survival and anti-apoptotic factors, bioenergetic demands and radical elimination, growth factor allocation and counteracting against excitotoxicity. In recent years, immune-modulatory and anti-inflammatory characteristics of oestrogen and progesterone have also come under scrutiny. To date, each of these physiological responses has been considered to be partially and selectively integrated in the mediation of steroid-mediated cell protection and tested in suitable animal models and in vitro systems. To what extent these individual effects contribute to the overall neural protection remains sketchy. One idea is that a battery of cellular mechanisms operates at the same time. On the other hand, interactions and the control of the brain-intrinsic and peripheral immune system may play an additional and perhaps pioneering function in this scenario, notwithstanding the importance of secondary adjuvant mechanisms. In the present review, we highlight neuroprotective effects of oestrogen and progesterone in two different disease models of the brain, namely acute ischaemic and demyelination damage, which represent the most common acute and degenerative neurological disorders in humans. Besides other inflammatory parameters, we discuss the idea that chemokine expression and signalling appear to be early hallmarks in both diseases and are positively affected by sex steroids. In addition, the complex interplay with local brain-resident immune-competent cells appears to be controlled by the steroid environment.
Assuntos
Isquemia Encefálica/metabolismo , Doenças Desmielinizantes/metabolismo , Encefalite/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Animais , Quimiocinas/metabolismoRESUMO
Over a century ago, hyperplasia and hypertrophy of astrocytes was noted as a histopathological hallmark of multiple sclerosis and was hypothesized to play an important role in the development and course of this disease. However until today, the factual contribution of astrocytes to multiple sclerosis is elusive. Astrocytes may play an active role during degeneration and demyelination by controlling local inflammation in the CNS, provoking damage of oligodendrocytes and axons, and glial scarring but might also be beneficial by creating a permissive environment for remyelination and oligodendrocyte precursor migration, proliferation, and differentiation. Recent findings from our lab suggest that brain lipid binding protein (FABP7) is implicated in the course of multiple sclerosis and the regulation of astrocyte function. The relevance of our findings and data from other groups are highlighted and discussed in this paper in the context of myelin repair.
Assuntos
Astrócitos/metabolismo , Proteínas de Transporte/metabolismo , Doenças Desmielinizantes/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Animais , Astrócitos/patologia , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Proteína 7 de Ligação a Ácidos Graxos , Humanos , Esclerose Múltipla/patologia , Bainha de Mielina/metabolismoRESUMO
Several lines of evidence indicate that remyelination represents one of the most effective mechanisms to achieve axonal protection. For reasons that are not yet understood, this process is often incomplete or fails in multiple sclerosis (MS). Activated astrocytes appear to be able to boost or inhibit endogenous repair processes. A better understanding of remyelination in MS and possible reasons for its failure is needed. Using the well-established toxic demyelination cuprizone model, we created lesions with either robust or impaired endogenous remyelination capacity. Lesions were analyzed for mRNA expression levels by Affymetrix GeneChip® arrays. One finding was the predominance of immune and stress response factors in the group of genes which were classified as remyelination-supporting factors. We further demonstrate that lesions with impaired remyelination capacity show weak expression of the radial-glia cell marker brain lipid binding protein (BLBP, also called B-FABP or FABP7). The expression of BLBP in activated astrocytes correlates with the presence of oligodendrocyte progenitor cells. BLBP-expressing astrocytes are also detected in experimental autoimmune encephalomyelitis during the remission phase. Furthermore, highest numbers of BLBP-expressing astrocytes were evident in lesions of early MS, whereas significantly less are present at the rim of (chronic)-active lesions from patients with long disease duration. Transfection experiments show that BLBP regulates growth factor expression in U87 astrocytoma cells. In conclusion, we provide evidence that expression of BLBP in activated astrocytes negatively correlates with disease duration and in parallel with remyelination failure.
Assuntos
Astrócitos/metabolismo , Proteínas de Transporte/biossíntese , Doenças Desmielinizantes/metabolismo , Proteínas de Ligação a Ácido Graxo/biossíntese , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Adulto , Idoso , Animais , Western Blotting , Contagem de Células , Linhagem Celular Tumoral , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Encefalomielite Autoimune Experimental/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Feminino , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Osteopontina/biossíntese , Fator de Crescimento Derivado de Plaquetas/biossíntese , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
OBJECTIVE AND DESIGN: It has been demonstrated that changes in the normal-appearing white matter (NAWM) in multiple sclerosis precede the appearance of classical lesions. The understanding of NAWM biology in an established disease model might help to clarify why some of them progress to active demyelinating lesions. MATERIAL OR SUBJECTS: C57BL6 male mice (19-21 g) were used in this study. TREATMENT: Demyelination was induced by feeding mice a diet containing 0.2% cuprizone for up to 5 weeks. METHODS: Routine stainings (luxol fast blue, and hematoxylin and eosin) and immunohistochemistry were performed to assess myelin status and the inflammatory infiltrate. RESULTS: We demonstrated that, in the toxic demyelination cuprizone model, the corpus callosum is severely demyelinated after a 5-week cuprizone challenge (acute demyelination) whereas the fimbria of the hippocampus appear normal in routine myelin stainings. Microgliosis but not astrogliosis is evident after acute demyelination in the fimbria. Interestingly, both regions, the fimbria and the corpus callosum, demonstrated early oligodendrocyte apoptosis as well as intense microglia accumulation and activation. However, only the corpus callosum progresses to actively demyelination lesions whereas the fimbria does not. CONCLUSIONS: The applied model appears suitable for elucidating pathways which promote progression of affected tissue to an active lesion.
Assuntos
Cuprizona/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Esclerose Múltipla/induzido quimicamente , Esclerose Múltipla/patologia , Fármacos Neuroprotetores/farmacologia , Animais , Quelantes/farmacologia , Quelantes/uso terapêutico , Corpo Caloso/citologia , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Cuprizona/uso terapêutico , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Hipocampo/anatomia & histologia , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Fibras Nervosas Mielinizadas/patologia , Fármacos Neuroprotetores/uso terapêutico , Adulto JovemRESUMO
Corticosteroids (CS) are effective in the treatment of many brain disorders, such as multiple sclerosis (MS) or traumatic brain injury. This has been scrutinised in different experimental animal models. However, neither the mechanisms, nor the site of CS action are fully understood. Short-term high-dose CS treatment improves MS symptoms and severity of clinical disability during an acute inflammatory exacerbation of disease. In the present study, we analysed the influence of CS on the expression of cellular and molecular markers of spontaneous endogenous remyelination in the toxic non-immune cuprizone animal model at early (9 days) and intermediate (21 days) remyelination, as well as steroidal effects in primary astrocytes and oligodendrocyte progenitor cultures. Dexamethasone (Dex) and methylprednisolone (MP) induced a higher expression of the differentiation markers myelin basic protein and proteolipid protein (PLP) in cultured oligodendrocyte progenitor cells (OPC). CS exposure of primary cultured astrocytes resulted in a greater expression of those genes involved in OPC proliferation [fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF)-αα] and a reduced expression of the pro-maturation factor insulin-like growth factor 1. Pro-maturating effects of CS were completely blocked by FGF2 and PDGF-αα co-application in OPC cultures. MP treatment in vivo resulted in a reduced recovery of PLP-staining intensity, whereas the re-population of the demyelinated corpus callosum with adenomatous polyposis coli-expressing oligodendrocytes was not affected. The numbers of brain intrinsic inflammatory cells, microglia and astrocytes during remyelination were similar in placebo and MP-treated animals. Our findings suggest that treatment with CS might have, in addition to the well-known benefical effects on inflammatory processes, a negative influence on remyelination.