RESUMO
Y-box-binding protein (YB-1) is a member of the cold-shock protein family and participates in a wide variety of DNA/RNA-dependent cellular processes including DNA repair, transcription, mRNA splicing, packaging, and translation. At the cellular level, YB-1 is involved in cell proliferation and differentiation, stress responses, and malignant cell transformation. A general role for YB-1 during inflammation has also been well described; however, there are minimal data concerning YB-1 expression in microglia, which are the immune cells of the brain. Therefore, we studied the expression of YB-1 in a clinically relevant global ischemia model for neurological injury following cardiac arrest. This model is characterized by massive neurodegeneration of the hippocampal CA1 region and the subsequent long-lasting activation of microglia. In addition, we studied YB-1 expression in BV-2 cells, which are an accepted microglia culture model. BV-2 cells were stressed by oxygen/glucose deprivation (OGD), OGD-relevant mediators, lipopolysaccharide (LPS), and phagocytosis-inducing cell debris and nanoparticles. Using quantitative polymerase chain reaction (PCR), we show constitutive expression of YB-1 transcripts in unstressed BV-2 cells. The functional upregulation of the YB-1 protein was demonstrated in microglia in vivo and in BV-2 cells in vitro. All stressors except for LPS were potent enhancers of the level of YB-1 protein, which appears to be regulated primarily by proteasomal degradation and, to a lesser extent, by the activation (phosphorylation) of the translation initiation factor eIF4E. The proteasome of BV-2 cells is impaired by OGD, which results in decreased protein degradation and therefore increased levels of YB-1 protein. LPS induces proteasome activity, which enables the level of YB-1 protein to remain at control levels despite enhanced protein ubiquitination. The proteasome inhibitor MG-132 was able to increase YB-1 protein levels in control and LPS-treated cultures. YB-1 upregulation was not accompanied by its translocation from the cytoplasm to the nucleus. YB-1 induction appeared to be related to microglial proliferation because it was partially co-regulated with Ki67. In addition, YB-1 protein levels correlated with microglia phagocytic activity because its upregulation could also be induced by inert NPs.
Assuntos
Regulação da Expressão Gênica/fisiologia , Parada Cardíaca/patologia , Microglia/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Asfixia/complicações , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Transformada , Modelos Animais de Doenças , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Glucose/deficiência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Parada Cardíaca/etiologia , Antígeno Ki-67/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Oxigênio/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Proteína 1 de Ligação a Y-Box/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons which leads to progressive paralysis and death by respiratory failure. Although the cause of sporadic ALS is still unknown, oxidative stress is suggested to play a major role in the pathogenesis of this disease and of the rare familial form, which often exhibits mutations of the superoxide dismutase 1 (SOD1) gene. Since enhanced iron levels are discussed to participate in oxidative stress and neuronal death, we analyzed the expression levels of Fe-related mRNAs in a cell culture ALS model with the G93A mutation of SOD1. We observed an increased total iron content in G93A-SOD1 SH-SY5Y neuroblastoma cells compared to wild-type (WT)-SOD1 cells. mRNA expression for transferrin receptor 1 (TfR1) and divalent metal transporter 1 was increased in G93A-SOD1 cells, which was in accordance with higher iron uptake. Experiments with the iron chelator deferoxamine revealed a normal reaction of WT and mutant cells to cytoplasmic iron depletion, i.e. TfR1 upregulation, suggesting a basically conserved function of the iron-responsive element/iron regulatory protein (IRE/IRP) pathway, designed to adapt gene expression to iron levels. Expression levels of mitoferrin 1 and 2, frataxin, and iron-sulfur cluster scaffold protein were also significantly increased in G93A-SOD1 cells, suggesting higher mitochondrial iron import and utilization in biosynthetic pathways within the mitochondria. Moreover, expression of these transcripts was further enhanced, if G93A-SOD1 cells were differentiated by retinoic acid (RA). Since RA treatment increased cytoplasmic reactive oxygen species (ROS) levels in these cells, an IRE/IRP independent, ROS-mediated mechanism may account for dysregulation of iron-related genes.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Mitocondriais/metabolismo , Receptores da Transferrina/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Transporte de Cátions/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fluoresceínas/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Neuroblastoma/patologia , Neuroblastoma/ultraestrutura , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , RNA Mensageiro , Espécies Reativas de Oxigênio , Receptores da Transferrina/genética , Superóxido Dismutase/genética , Transfecção , Tretinoína/farmacologiaRESUMO
Recent meta-analyses showed consistently elevated levels of S100B in serum and cerebrospinal fluid of schizophrenic patients. This finding has been attributed to glial pathology because S100B is produced by astrocytes and oligodendrocytes. However, S100B may be likewise associated with schizophrenia-related disturbances in glial cell as well as adipocyte energy supply and glucose metabolism. The influence of antipsychotic drugs on S100B levels remains unclear, and some studies have suggested that treatment with these drugs may actually contribute to the elevated S100B levels observed in schizophrenic patients. In this study, we explored the effects of the typical antipsychotic haloperidol and the atypical prototype drug clozapine on the release of S100B by astrocytic C6 cells and oligodendrocytic OLN-93 cells. Because of the association between schizophrenia and disturbances in energy metabolism, we assessed the effects of these drugs under basal condition (BC) compared to serum and glucose deprivation (SGD). We found that treatment of C6 and OLN-93 cells with haloperidol and clozapine reduced the release of S100B from C6 and OLN-93 cells under BC and SGD in vitro at a tissue concentration corresponding to the assumed therapeutic dose range of these drugs. These data suggest that elevated levels of S100B in bodily fluids of schizophrenic patients are normalized rather than increased by the effects of antipsychotic drugs on glial cells.
Assuntos
Antipsicóticos/farmacologia , Clozapina/farmacologia , Haloperidol/farmacologia , Fatores de Crescimento Neural/biossíntese , Neuroglia/efeitos dos fármacos , Proteínas S100/biossíntese , Animais , Linhagem Celular Tumoral , Meios de Cultura , Meios de Cultura Livres de Soro , Glucose/metabolismo , Imuno-Histoquímica , Neuroglia/metabolismo , Ratos , Receptores de Neurotransmissores/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100RESUMO
At low concentration H(2)O(2) is an important signal molecule in proliferation of tumour cells. We report about a study investigating the effect of an ethanolic extract from Gynostemma pentaphyllum on proliferation of C6 glioma tumour cells and cellular H(2)O(2) concentration. The proliferation of these cells was maximal at about 1 muM extracellular H(2)O(2). HPLC-finger prints of the extract revealed a set of saponines as essential components. In C6 glioma cells the extract caused increase in super oxide dismutase (SOD) activity, in the amount of SOD protein, and in cellular H(2)O(2) concentration. It inhibited cell proliferation and induced activation of caspase 3 as indication of apoptosis. No effect of the extract was observed on the proliferation of astrocytes of a primary cell culture. From these findings we suggest that the ethanolic extract from Gynostemma pentaphyllum may selectively shift the H(2)O(2) concentration to toxic levels exclusively in tumour cells due to increased SOD activity. It may have a high potency in cancer therapy and cancer prophylaxis.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Glioma/tratamento farmacológico , Gynostemma/química , Peróxido de Hidrogênio/metabolismo , Extratos Vegetais/uso terapêutico , Animais , Antineoplásicos Fitogênicos/farmacologia , Astrócitos/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Glioma/metabolismo , Humanos , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ratos , Ratos Wistar , Saponinas/análise , Saponinas/farmacologia , Saponinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase/metabolismoAssuntos
Síndrome do Hamartoma Múltiplo/complicações , Síndrome do Hamartoma Múltiplo/genética , PTEN Fosfo-Hidrolase/genética , Transtornos Psicóticos/genética , Adulto , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Feminino , Ganglioneuroma/complicações , Ganglioneuroma/patologia , Ganglioneuroma/cirurgia , Mutação em Linhagem Germinativa , Síndrome do Hamartoma Múltiplo/psicologia , Humanos , Imageamento por Ressonância MagnéticaRESUMO
S100B (member of a family of proteins that are 100% soluble in ammonium sulfate at neutral pH) has been widely used as astrocyte marker in animal models and in human brain diseases. Recent studies revealed S100B-immunopositivity in oligodendrocytes and O2A oligodendroglial progenitor cells. It is unknown, however, if oligodendrocytes produce S100B themselves, or if the S100B-immunolabeling is caused by binding or absorption of the protein. To address this question, S100B expression and protein release were analyzed in a highly pure oligodendrocytic OLN-93 cell line (from rat), in the astrocytic C6 cell line (from rat) and primary astrocytes. S100B was gene expressed in all cultures, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. OLN-93 cells and glial fibrillary acidic protein (GFAP)-negative astrocytes expressed the multiligand receptor for advanced glycation end products (RAGE). S100B protein levels were determined in supernatants and cell homogenates by immunoluminometry under normal conditions and after serum and glucose deprivation (SGD). SGD led to a several-fold increased release of S100B (after 6 and 24 h), which was particularly pronounced in primary astrocytes. Increased S100B in cell homogenates was most notable in OLN-93 cells under SGD, indicating activated S100B synthesis. These cells also showed the highest percentage of dead cells, as determined by propidium iodide-positivity, after SGD. Incubation with 0.5, 2 and 5 microg/l exogenous S100B was not toxic to OLN-93 cells. In conclusion, OLN-93 cells produce more S100B under SGD than astrocytes and are more susceptible to cell death upon SGD, which provokes leakage of S100B. Our data indicate active S100B secretion from astrocytes under SGD since highly elevated levels of S100B were detected in the supernatant despite a low percentage of dead cells. The experimental results provide further evidence for a production/release of S100B in/from oligodendrocytes, e.g. in metabolic stress conditions like cerebral ischemia. Studies on S100B in bodily fluids should be carefully interpreted in order to avoid misleading hypotheses concerning the specific involvement of astrocytes, due to the various cellular sources of S100B.
Assuntos
Glucose/deficiência , Fatores de Crescimento Neural/biossíntese , Oligodendroglia/metabolismo , Proteínas S100/biossíntese , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Linhagem Celular Tumoral , Células Cultivadas , Corantes , Meios de Cultura Livres de Soro , Fluoresceínas , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Fatores de Crescimento Neural/metabolismo , Oligodendroglia/ultraestrutura , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismoRESUMO
Treatment with the phencyclidine derivative ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist and a well known anesthetic, has recently been introduced to mimic schizophrenia in animals. Using rats repeatedly treated with sub-anesthetic doses we demonstrate in the hippocampal formation the cellular distribution patterns of proteins being relevant to the pathogenesis of schizophrenia. Compared with controls an increase in the density of reduced nicotinamide adenine dinucleotide phosphate diaphorase-, neuronal nitric oxide synthase- and cFOS-positive hippocampal interneurons was found, whereas the density of parvalbumin expressing cells was decreased. Our experiments show that repeated injections of sub-anesthetic doses of ketamine induce significant changes in the nitrergic and GABAergic system which, in part, resemble those described in postmortem brains of human schizophrenics indicating that sub-chronic treatment with sub-anesthetic doses of ketamine might be a useful animal model to study schizophrenia.
Assuntos
Genes fos/fisiologia , Hipocampo/efeitos dos fármacos , Ketamina/farmacologia , Óxido Nítrico Sintase/biossíntese , Parvalbuminas/biossíntese , Esquizofrenia/fisiopatologia , Animais , Antagonistas de Aminoácidos Excitatórios , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Masculino , NADPH Desidrogenase/efeitos dos fármacos , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo I , Parvalbuminas/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Nonneural derived nerve conduits fail to support regeneration over larger gaps due to lacking viable Schwann cells. Thus, tissue engineering of nerves is focusing on implantation of viable Schwann cells into suitable scaffolds. We established grafts made from acellular muscles and veins, respectively, seeded with cultured Schwann cells. As timing of revascularization is crucial to determine Schwann cell survival and depending axonal regeneration we studied establishment of vascular architecture in a rat sciatic nerve model (2-cm gap) after 3, 5, 7, and 10 days postoperatively, using albumin bound Evans blue. Additionally, macrophage recruitment was immunohistochemically assessed. Engineered grafts showed a delayed revascularization, starting between day 5 and 7 in comparison to normal autografts, that revascularized by day 3. Macrophage recruitment in autologous nerve grafts was evident by day 3. The engineered groups revealed no macrophage invasion until day 7. As Schwann cells survive up to 7 days in autologous grafts without blood supply, depending purely on diffusion, establishment of vascular structure between day 5 and 7 is rapid enough to support Schwann cell survival in engineered grafts. As these grafts are lacking Wallerian degeneration delayed macrophage invasion may not impair degeneration-dependent regeneration, but presence of macrophage derived or induced growth factors may be decreased.
Assuntos
Macrófagos/fisiologia , Neovascularização Fisiológica , Regeneração Nervosa , Nervos Periféricos/transplante , Células de Schwann/transplante , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/administração & dosagem , Movimento Celular , Células Cultivadas , Feminino , Microscopia de Fluorescência , Músculo Esquelético/transplante , Nervos Periféricos/irrigação sanguínea , Nervos Periféricos/fisiologia , Ratos , Ratos Wistar , Nervo Isquiático/irrigação sanguínea , Nervo Isquiático/transplante , Fatores de Tempo , Veias/transplanteRESUMO
The relevance of NO in neuroendocrine signalling has been investigated by analysis of cellular expression of pro-opiomelanocortin (POMC) and the POMC-derived peptides beta-endorphin, alpha-melanocyte stimulating hormone and adrenocorticotropin. Expression patterns were studied in the pituitary gland of 150-day old wild-type and neuronal-NOS (nNOS) knock-out mice by using immunohistochemistry, in situ hybridization and Northern blot analysis. Remaining NO-generating capacities in the knock-out mice were demonstrated by immunohistochemical localization of inducible, endothelial and neuronal NOS isoforms. Quantitative analysis revealed that cellular expression of POMC mRNA was drastically reduced in the pituitary of knock-out mice in comparison to controls. In situ hybridization studies demonstrated that this reduction was most pronounced in the intermediate lobe, while the anterior lobe was much less affected. Immunostaining for the proteolytic fragments of POMC was significantly reduced in the intermediate lobe cells of knock-out mice. A moderate reduction of immunostaining for these peptides was also observed in adenopituitary cells of nNOS knock-out mice. Our data demonstrate that the lack of nNOS substantially affects cellular levels of pituitary opioid peptides, which may have consequences for the response of these animals to stress and pain.
Assuntos
Regulação da Expressão Gênica/fisiologia , Óxido Nítrico Sintase/metabolismo , Hipófise/metabolismo , Pró-Opiomelanocortina/genética , Hormônio Adrenocorticotrópico/genética , Animais , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , RNA Mensageiro/genética , Transcrição Gênica , alfa-MSH/genética , beta-Endorfina/genéticaRESUMO
Previous studies showed that proanthocyanidins provide potent protection against oxidative stress. Here we investigate the effects of grape seed proanthocyanidin extract (GSPE) as a novel natural antioxidant on the generation and fate of nitric oxide (NO) in rat primary glial cell cultures. GSPE treatment (50 mg/L) increased NO production (measured by NO(2-) assay) by stimulation of the inducible isoform of NOS. However, GSPE failed to affect the LPS/IFN-gamma-induced NO production or iNOS expression. Similar responses were found in the murine macrophage cell line RAW264.7. GSPE did not show any effect on dihydrodichlorofluorescein fluorescence (ROS marker with high sensitivity toward peroxynitrite) either in control or in LPS/IFN-gamma-induced glial cultures even in the presence of a superoxide generator (PMA). GSPE treatment alone had no effect on the basal glutathione (GSH) status in glial cultures. Whereas the microglial GSH level declined sharply after LPS/IFN-gamma treatment, the endogenous GSH pool was protected when such cultures were treated additionally with GSPE, although NO levels did not change. Glial cultures pretreated with GSPE showed higher tolerance toward application of hydrogen peroxide (H(2)O(2)) and tert-butylhydroperoxide. Furthermore, GSPE-pretreated glial cultures showed improved viability after H(2)O(2)-induced oxidative stress demonstrated by reduction in lactate dehydrogenase release or propidium iodide staining. We showed that, in addition to its antioxidative property, GSPE enhances low-level production of intracellular NO in primary rat astroglial cultures. Furthermore, GSPE pretreatment protects the microglial GSH pool during high output NO production and results in an elevation of the H(2)O(2) tolerance in astroglial cells.
Assuntos
Antocianinas/farmacologia , Neuroglia/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Proantocianidinas , Rosales , Sementes , Animais , Antioxidantes/farmacologia , Células Cultivadas , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Peróxido de Hidrogênio/toxicidade , Imuno-Histoquímica , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Microscopia de Fluorescência , Neuroglia/enzimologia , Neuroglia/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/imunologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Nitrosação/efeitos dos fármacos , Oxidantes/farmacologia , Oxidantes/toxicidade , Ratos , Ratos Wistar , terc-Butil Hidroperóxido/farmacologiaRESUMO
Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.
Assuntos
Diferenciação Celular/fisiologia , Músculo Esquelético/inervação , Músculo Liso Vascular/inervação , Regeneração Nervosa/fisiologia , Nervos Periféricos/transplante , Células de Schwann/citologia , Animais , Células Cultivadas , Masculino , Microcirurgia , Ratos , Ratos WistarRESUMO
Schwann cells are used in combination with biological matrices as tissue engineered nerve grafts in animal models offering a new therapeutic approach for treatment of lesions of the peripheral nervous system. A high yield of human Schwann cells from adult donors is only achieved by pharmacological stimulation, which should, however, be avoided in clinical therapy. Here, we establish cultures of activated human Schwann cells which were isolated from peripheral nerve neuroma which developed after a median nerve lesion. To allow nerve reconstruction neuroma have to be resected. Such neuroma tissue is virtually predegenerated and shows activation of Schwann cells, implying good adherence and high mitotic activity. This allows, irrespective of donor age, growing within a short time period and without any pharmacological treatment.
Assuntos
Envelhecimento/fisiologia , Transplante de Células/fisiologia , Neuroma/patologia , Células de Schwann/transplante , Doadores de Tecidos , Adulto , Idoso , Western Blotting , Divisão Celular/fisiologia , Células Cultivadas , Criança , Eletroforese em Gel de Poliacrilamida , Humanos , Nervo Mediano/citologia , Proteínas do Tecido Nervoso/metabolismo , Neuroma/metabolismo , Células de Schwann/metabolismoRESUMO
Cultivation of human cells is well established. The cultivation of human Schwann cells may offer a new therapeutic approach for treatment of degenerative and traumatic lesions of the peripheral nervous system. Currently, Schwann cells in combination with other biological matrices are used as tissue-engineered biological nerve grafts in animal models. Cultivation of human Schwann cells, however, is more difficult than cultivation of rodent cells. A high cell yield is only achieved by pharmacological stimulation, which should not be used in clinical therapy. Thus, we aimed to establish an easy method of cultivating human Schwann cells from peripheral nerve neuromas that have developed after a complete nerve lesion. As these neuromas have to be resected in any case to allow proper nerve reconstruction, their removal does not lead to additional neurological defects. Schwann cells were cultivated from the neuromas of eleven patients, aged 5 to 75 years. All patients suffered from a complete median nerve lesion at the level of the distal forearm, which could not be treated primarily. Two weeks after trauma, the patients underwent secondary reconstruction by autologous sural nerve grafts. During this operation, the neuromas were resected. Control cultures were established from remaining parts of the sural nerve. Cell yield was determined on the first, third and seventh day in vitro. Schwann cells were stained for S100. Viability was assessed with fluoresceine-fluorescence. The cell count was assessed with regard to the donor age. The growth rate of Schwann cells was found to be donor-age dependent. The highest cell yield was obtained from adult neuromas. By the third day in vitro, they showed a 1.5 fold increased cell count compared with juvenile nerves and neuromas. By the seventh day in vitro, Schwann cells from adult neuromas were increased 2.5 times compared with cells from juvenile nerves and 7 times compared to cells from adult sural nerves. Cultivation of Schwann cells taken from sural nerves of patients older than 65 years was not possible. The utilization of neuromas as a source for human Schwann cells allows an age-independent cultivation within a short time period without any pharmacological treatment. These neuromas are virtually predegenerated and show an activation of Schwann cells implying good adherence and high mitotic activity in culture. Normal nerve tissue as a source for Schwann cells for tissue-engineered nerves is only sufficient in young patients due to its greater proliferative potential. The age-dependent proliferation underlines the need for alternative sources for Schwann cells.
Assuntos
Traumatismos dos Nervos Periféricos , Doenças do Sistema Nervoso Periférico/cirurgia , Células de Schwann/transplante , Adolescente , Adulto , Fatores Etários , Idoso , Divisão Celular/fisiologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Nervo Mediano/lesões , Nervo Mediano/patologia , Nervo Mediano/cirurgia , Pessoa de Meia-Idade , Neuroma/patologia , Nervos Periféricos/patologia , Nervos Periféricos/cirurgia , Doenças do Sistema Nervoso Periférico/patologia , Células de Schwann/patologia , Nervo Sural/patologia , Nervo Sural/transplanteRESUMO
Primary glial cultures are able to express the inducible isoform of nitric oxide synthase (i-NOS) upon stimulation by bacterial lipopolysaccharides (LPS) and gamma-interferon (gamma-IfN). Immunocytochemical studies revealed, that under our experimental conditions i-NOS is expressed exclusively by the microglial cells and not in the astrocytes. Nitric oxide (NO) formation represents an oxidative load for the microglial cells, as observed by the oxidation rate of the ROS- and peroxynitrite indicator dichloro-dihydrofluorescein (DCF-H) in these cells. However, cell viability was not affected by the nitric oxide formation, indicating some form of protection against the higher oxidative load. Upregulation of Mn-SOD in the mitochondria in the course of the induction of i-NOS and, compared to the astrocytes, higher GSH levels in the microglial cells probably explain the resistance of the cultures against nitrosative stress. Increased SOD-activities in the mitochondria could lower the superoxide concentration in this organelle and may prevent an oxidative and/or nitrosative damage via a decreased peroxynitrite formation. The higher GSH-levels in the microglial cells of unstimulated cultures represents a buffer which, under the conditions of i-NOS catalyzed NO-formation, prevents a decline of the microglial GSH-levels below that of the astrocytes.
Assuntos
Neuroglia/enzimologia , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo , Animais , Animais Recém-Nascidos , Células Cultivadas , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Interferon gama/farmacologia , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Nitritos/metabolismo , Nitrosação , Oxirredução , Ratos , Ratos Wistar , Superóxido Dismutase/metabolismoRESUMO
Because glutathione (GSH) levels in glia play an important role in cellular defense against oxidative and nitrosative stress, the present study was designed to study GSH levels in the primary glial cell cultures. Here we used fluorescence microscopy and spectroscopy with monochlorobimane for measurement of intracellular glutathione content. Monochlorobimane showed high specificity for GSH with very little binding to protein sulphydryls as ascertained from the low fluorescence intensity of the protein fraction of the cells as well as from the low fluorescence of the GSH-depleted cells. The formation of the monochlorobimane-glutathione conjugate was observed to be enzymatically catalyzed as seen from its higher rate of formation in the presence of cell homogenate. A monochlorobimane concentration of 60 microM was used for conjugation of cellular GSH; at higher mBCl concentrations there was no appreciable increase in fluorescence. Therefore, cultures were treated with 60 microM mBCl for an incubation time of 20 min (beyond this time, export of the bimane-glutathione adduct was significantly large) and examined by fluorescence microscopy. This adduct could be fixed with a mixture of paraformaldehyde and glutaraldehyde, and excellent fixation was observed with 4% paraformaldehyde and 0.2% glutaraldehyde. Analysis of the fluorescence images revealed differences in fluorescence intensity between astro- and microglial cells, which were identified by glial fibrilliary acidic protein and OX42 staining, respectively. Microglial cells isolated from primary glial cultures were found to have higher GSH content than astrocytes. Biochemical determination of GSH levels in microglia isolated from primary glial cultures corroborated this fact. From our findings it seems that owing to the greater intracellular concentration of reactive oxygen and nitrogen species to which microglia are subjected, especially under conditions of inflammation, this cell type is fortified with higher GSH levels as a means to combat oxidative and nitrosative stress.
Assuntos
Glutationa/análise , Neuroglia/química , Pirazóis/análise , Animais , Animais Recém-Nascidos , Biomarcadores , Células Cultivadas , Fixadores/farmacologia , Corantes Fluorescentes , Formaldeído/farmacologia , Glutaral/farmacologia , Microglia/química , Microscopia Confocal , Microscopia de Fluorescência , Polímeros/farmacologia , Ratos , Ratos Wistar , Fixação de TecidosRESUMO
Nerve allografts are highly antigenic and require the continuous use of immunosuppressive drugs. Neurotoxic complications from immunosuppressant therapy with FK 506 have been noted in the central and peripheral nervous system although an increased rate of axonal regeneration has also been noted. Regeneration of peripheral nerve grafts was assessed in a rat model clinically and morphometrically after treatment for 2 and 6 weeks with two different doses of FK 506. Good regeneration was noted in all groups at 6 weeks. A significantly higher axon count was observed in both the FK 506 groups after 2 weeks regeneration compared with controls. This beneficial effect was not evident after 6 weeks of regeneration. Whether this is related to a pruning mechanism or to a down-regulation of regenerative processes in the nerve due to possible neurotoxic effects of FK 506 remains unknown.
Assuntos
Imunossupressores/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Nervo Isquiático/transplante , Tacrolimo/farmacologia , Animais , Masculino , Ratos , Ratos Wistar , Nervo Isquiático/fisiologia , Transplante HomólogoRESUMO
Inhibitors of dihydroorotate dehydrogenase (DHO-DH), such as brequinar or leflunomide, have been intensively tested for their antitumour and immunomodulating effects. Polyporic acid (PA) from the mushroom Hapalopilus rutilans (H. r.) also is a DHO-DH inhibitor (50% inhibitory concn., IC50, 10(-4)-10(-3) M). As three people had been poisoned following ingestion of H. r. we wanted to investigate the effects of PA in rats and in cell cultures. Rats given PA via probang (100-800 mg/ kg) within 24 h developed strongly reduced locomotor activity, depressed visual placing response and impaired wire manoeuvre. Laboratory investigation of blood revealed hepatorenal failure, metabolic acidosis as well as hypokalaemia and hypocalcaemia. All symptoms closely paralleled the effects seen in the poisoned people. Proliferation of cultured cells (including rat brain neurons and glia, fibroblasts, tumour cells) was depressed at 10(-4)-10(-3) M PA. We conclude that the intoxication of people poisoned with H. r. is due to the high content of the DHO-DH inhibitor PA.