RESUMO
In recent years, the simultaneous monitoring of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in vivo is advancing due to innovations in miniaturized and fast chromatographic techniques. However, the determination of the most hydrophilic compound, noradrenaline, in microdialysis samples by (ultra-)high performance liquid chromatography ((U)HPLC) with electrochemical detection (ECD) is impeded by a broad solvent front, caused by the addition of antioxidative agents. Hence, an elaborate reassessment of currently used antioxidative mixtures is necessary for further analytical improvements. The proposed mixture, containing 100mM acetic acid, 0.27mM Na2EDTA and 12.5µM ascorbic acid (pH 3.2), is less complex than previously described mixtures and shows minimal ECD interference. It stabilizes the three monoamines in standard solutions and in microdialysis samples, considering both autosampler stability at 4°C for 48h and long term stability at -20°C for a duration of six months. An in vivo microdialysis experiment demonstrates the possibility to monitor changes in extracellular levels of the three monoamines simultaneously in the rat hippocampus with UHPLC-ECD using the optimized antioxidative mixture.
Assuntos
Antioxidantes/química , Dopamina/química , Neurotransmissores/química , Norepinefrina/química , Serotonina/química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Estabilidade de Medicamentos , Hipocampo/química , Masculino , Microdiálise , Ratos , Ratos WistarRESUMO
Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 µg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets.
RESUMO
AIM: An ultrasensitive nano UHPLC-ESI-MS/MS method is developed to simultaneously monitor three low-concentration neuromedin-like peptides in microdialysates. RESULTS: Peptide preconcentration and sample desalting is performed online on a trap column. A shallow gradient slope at 300 nl/min on the analytical column maintained at 35°C, followed by two saw-tooth column wash cycles, results in the highest sensitivity and the lowest carryover. The validated method allows the accurate and precise quantification of 0.5 pM neurotensin and neuromedin N (2.5 amol on column), and of 3.0 pM neuromedin B (15.0 amol on column) in in vivo microdialysates without the use of internal standards. CONCLUSION: The assay is an important tool for elucidating the role of these neuromedin-like peptides in the pathophysiology of neurological disorders.
Assuntos
Cromatografia Líquida/métodos , Microdiálise/métodos , Neurotensina/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process.
Assuntos
Dependovirus/genética , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/genética , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Comportamento Animal , Progressão da Doença , Dopamina/metabolismo , Estudos Longitudinais , Microdiálise , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/psicologia , Tomografia por Emissão de Pósitrons , Agregados Proteicos , Substância Negra/patologia , Fatores de Tempo , alfa-Sinucleína/administração & dosagem , alfa-Sinucleína/metabolismoRESUMO
There is considerable preclinical and clinical evidence indicating that abnormal changes in glutamatergic signaling underlie the development of mood disorders. Astrocytic glutamate dysfunction, in particular, has been recently linked with the pathogenesis and treatment of mood disorders, including anxiety and depression. System xc- is a glial cystine/glutamate antiporter that is responsible for nonvesicular glutamate release in various regions of the brain. Although system xc- is involved in glutamate signal transduction, its possible role in mediating anxiety or depressive-like behaviors is currently unknown. In the present study, we phenotyped adult and aged system xc- deficient mice in a battery of tests for anxiety and depressive-like behavior (open field, light/dark test, elevated plus maze, novelty suppressed feeding, forced swim test, tail suspension test). Concomitantly, we evaluated the sensorimotor function of system xc- deficient mice, using motor and sensorimotor based tests (rotarod, adhesive removal test, nest building test). Finally, due to the presence and potential functional relevance of system xc- in the eye, we investigated the visual acuity of system xc- deficient mice (optomotor test). Our results indicate that loss of system xc- does not affect motor or sensorimotor function, in either adult or aged mice, in any of the paradigms investigated. Similarly, loss of system xc- does not affect basic visual acuity, in either adult or aged mice. On the other hand, in the open field and light/dark tests, and forced swim and tail suspension tests respectively, we could observe significant anxiolytic and antidepressive-like effects in system xc- deficient mice that in certain cases (light/dark, forced swim) were age-dependent. These findings indicate that, under physiological conditions, nonvesicular glutamate release via system xc- mediates aspects of higher brain function related to anxiety and depression, but does not influence sensorimotor function or spatial vision. As such, modulation of system xc- might constitute the basis of innovative interventions in mood disorders.
Assuntos
Sistema y+ de Transporte de Aminoácidos/deficiência , Ansiedade/genética , Ansiedade/fisiopatologia , Depressão/genética , Depressão/fisiopatologia , Percepção Espacial/fisiologia , Adaptação Ocular/genética , Envelhecimento , Sistema y+ de Transporte de Aminoácidos/genética , Análise de Variância , Animais , Comportamento Exploratório/fisiologia , Comportamento Alimentar , Genótipo , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora , Desempenho Psicomotor , Natação/psicologia , Fatores de Tempo , Acuidade Visual/genéticaRESUMO
Obtaining maximal sensitivity of nano UHPLC-MS/MS methods is primordial to quantify picomolar concentrations of neuropeptides in microdialysis samples. Since aspecific adsorption of peptides to Eppendorf tubes, pipette tips and UHPLC vials is detrimental for method sensitivity, a strategy is presented to reduce adsorption of these peptides during standard preparation. Within this respect, all procedural steps from dissolution of the lyophilized powder until the injection of the sample onto the system are investigated. Two peptides of the neuromedin family, i.e. neuromedin B and neuromedin N, and a neuromedin N-related neuropeptide, neurotensin, are evaluated. The first part of this study outlines a number of parameters which are known to affect peptide solubility. The main focus of the second part involves the optimization of the sample composition in the UHPLC vial by using design of experiments. Contradictory findings are observed concerning the influence of acetonitrile, salts and matrix components. They are found important for injection of the peptides into the system, but crucially need to be excluded from the dilution solvent. Furthermore, the type of surface material, temperature and the pipetting protocol considerably affect the adsorption phenomenon. Statistical analysis on the results of the central composite design reveals that the highest peptide responses are obtained with the injection solvent consisting of 13.1% V/V ACN and 4.4% V/V FA. This aspect of the optimization strategy can be identified as the main contributor to the gain in method sensitivity. Since the reduction of peptide adsorption and the optimization of the injection solvent resulted in a clear and quantifiable signal of the three peptides, optimization of both issues should be considered in the early stage of method development, in particular when the analysis of low-concentration peptide solutions is envisaged.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Neurotensina/análise , Fragmentos de Peptídeos/análise , Espectrometria de Massas em Tandem/métodos , Adsorção , Fenômenos Químicos , Solventes/química , Propriedades de SuperfícieRESUMO
In the drug-discovery setting, the development of new peptide and protein-based biopharmaceuticals attracts increased attention from the pharmaceutical industry and consequently demands the development of high-throughput LC-MS methods. Regulatory guidelines require bioanalytical methods to be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity and stability, but also in terms of carryover. Carryover results from the aspecific adsorption of analyte(s) to parts of the analytical system and thus introduces bias in both identification and quantification assays. Moreover, nonspecific binding occurs at the surface of materials used during sample preparation, such as pipette tips, sample tubes and LC-vials. Hence, linearity, sensitivity and repeatability of the analyses are negatively affected. Due to the great diversity in physicochemical properties of biomolecules, there is no general approach available to minimize adsorption phenomena. Therefore, we aim to present different strategies which can be generically applied to reduce nonspecific binding of peptides and proteins. In the first part of this review, a systematic approach is proposed to guide the reader through the different solvents which can be used to dissolve the analyte of interest. Indeed, proper solubilization is one of the most important factors for a successful analysis. In addition, alternative approaches are described to improve analyte recovery from the sample vial. The second part focuses on strategies to efficiently reduce adsorption at components of the autosampler, column and mass spectrometer. Thereby carryover is reduced while maintaining a sufficiently wide dynamic range of the assay.
Assuntos
Peptídeos/química , Proteínas/química , Adsorção , Animais , Cromatografia Líquida/métodos , Descoberta de Drogas , Humanos , Espectrometria de Massas/métodos , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Long term treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) is associated with several motor complications. Clinical improvement of this treatment is therefore needed. Lesions or high frequency stimulation of the hyperactive subthalamic nucleus (STN) in Parkinson's disease (PD), alleviate the motor symptoms and reduce dyskinesia, either directly and/or by allowing the reduction of the L-DOPA dose. N-methyl-D-aspartate (NMDA) receptor antagonists might have similar actions. However it remains elusive how the neurochemistry changes in the STN after a separate or combined administration of L-DOPA and a NMDA receptor antagonist. By means of in vivo microdialysis, the effect of L-DOPA and/or MK 801, on the extracellular dopamine (DA) and glutamate (GLU) levels was investigated for the first time in the STN of sham and 6-hydroxydopamine-lesioned rats. The L-DOPA-induced DA increase in the STN was significantly higher in DA-depleted rats compared to shams. MK 801 did not influence the L-DOPA-induced DA release in shams. However, MK 801 enhanced the L-DOPA-induced DA release in hemi-parkinson rats. Interestingly, the extracellular STN GLU levels remained unchanged after nigral degeneration. Furthermore, administration of MK 801 alone or combined with L-DOPA did not alter the STN GLU levels in both sham and DA-depleted rats. The present study does not support the hypothesis that DA-ergic degeneration influences the STN GLU levels neither that MK 801 alters the GLU levels in lesioned and non-lesioned rats. However, NMDA receptor antagonists could be used as a beneficial adjuvant treatment for PD by enhancing the therapeutic efficacy of l-DOPA at least in part in the STN.
Assuntos
Antiparkinsonianos/farmacologia , Dopamina/metabolismo , Levodopa/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Núcleo Subtalâmico/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/metabolismo , Lateralidade Funcional , Ácido Glutâmico/metabolismo , Masculino , Microdiálise , Oxidopamina , Transtornos Parkinsonianos/fisiopatologia , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Substância Negra/efeitos dos fármacos , Substância Negra/fisiopatologia , Núcleo Subtalâmico/fisiopatologiaRESUMO
Cortistatin (CST)-14, a neuropeptide that is structurally and functionally related to somatostatin-14 (SRIF) binds all five somatostatin receptor subtypes (sst1-sst5). Using in vivo microdialysis and telemetry-based electroencephalographic recordings, we provide the first experimental evidence for anticonvulsive effects of CST-14 in a pilocarpine-induced seizure model in rats and mice and for the involvement of sst2 and sst3 receptors in these anticonvulsant actions of CST-14. Both receptor subtypes are required for the anticonvulsant effects of CST-14 given that co-perfusion of a selective sst2 antagonist (cyanamid15486) or a selective sst3 antagonist (SST3-ODN-8) reversed anticonvulsant effect of CST-14, and this, independently of each other. Next, as the ghrelin receptor has been proposed as a target for the biological effects of CST-14, we used ghrelin receptor knockout mice and their wild type littermates to study the involvement of this receptor in the anticonvulsive actions of CST-14. Our results show a significant decrease in seizure duration in both genotypes when CST-14 treated mice were compared with corresponding control animals receiving only pilocarpine. In addition, this CST-14-induced decrease was comparable in both genotypes. We here thus provide the first evidence that ghrelin receptors are not involved in mediating anticonvulsant actions of CST-14 in vivo.
Assuntos
Anticonvulsivantes/uso terapêutico , Neuropeptídeos/uso terapêutico , Peptídeos Cíclicos/uso terapêutico , Receptores de Grelina/fisiologia , Receptores de Somatostatina/fisiologia , Convulsões/tratamento farmacológico , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pilocarpina/toxicidade , Ratos , Ratos Wistar , Receptores de Grelina/antagonistas & inibidores , Receptores de Grelina/deficiência , Receptores de Somatostatina/antagonistas & inibidores , Convulsões/induzido quimicamente , Convulsões/metabolismoRESUMO
The use of ion-pair ultra-high performance liquid chromatography (UHPLC) coupled with electrochemical detection (ECD) is of great interest for the fast and sensitive determination of the monoamine neurotransmitters dopamine, noradrenaline and serotonin in microdialysis samples. However, when applying high flow rates in ion-pair UHPLC, other peaks than the initial compound peaks appear on the chromatogram. This peak-splitting phenomenon is caused by disturbed ion-pair retention mechanisms. The influence of several chromatographic parameters is investigated. Peak-splitting is delayed to higher flow rates when increasing the concentration of ion-pair reagent or buffering agent in the mobile phase, when decreasing the percentage of organic modifier in the mobile phase, when applying a stationary phase with a smaller amount of packing material or when increasing the separation temperature. One or a combination of these conditions can be applied to analyze the monoamine neurotransmitters using ion-pair UHPLC-ECD at high flow rates.
Assuntos
Monoaminas Biogênicas/análise , Cromatografia Líquida de Alta Pressão/métodos , Íons , Microdiálise , Padrões de Referência , SolventesRESUMO
Mild hypothermia is a promising neuroprotective therapy in stroke management. However, little is known about its effects on the global protein expression patterns in brain regions affected by ischemic stroke. We investigated protein expression changes associated with the neuroprotective effects of hypothermia via a functional proteomics approach through the analysis of the core (striatum) and the penumbra (cortex) after an ischemic insult in rats induced by endothelin-1 (Et-1). Functional outcome, infarct volume and related global protein expression changes were assessed 24h after the insult using two-dimensional difference gel electrophoresis. Mild hypothermia, induced 20 min after endothelin-1 infusion, improved the neurological outcome, reflected by a 36% reduction in infarct volume and a significantly better neurological deficit score. Hypothermia was typically associated with opposite protein expression changes inthe cortex to those induced by stroke under normothermic conditions, but not in the striatum. The main cellular processes rescued by hypothermia and potentially involved in the protection of the cortex are cellular assembly and organization, followed by cell signaling, thereby confirming that hypothermia is neuroprotective through multiple molecular and cellular pathways.
Assuntos
Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Endotelina-1/administração & dosagem , Hipotermia Induzida , Proteínas do Tecido Nervoso/metabolismo , Proteômica , Animais , Western Blotting , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/terapia , Masculino , Ratos , Ratos Wistar , Eletroforese em Gel Diferencial BidimensionalRESUMO
L-Theanine, an ethylamide derivate of glutamate found in abundance in green tea, has been shown to exert beneficial actions in animal models for several neurological disorders. We here investigated for the first time the effect of L-theanine intake on seizure susceptibility using acute pilocarpine and pentylenetetrazol (PTZ) mouse models for studying, respectively, limbic seizures or primarily generalized seizures. Moreover, we studied the effect of l-theanine intake on extracellular hippocampal and cortical glutamate and gamma-aminobutyric acid (GABA) levels, using in vivo microdialysis. Feeding mice with a 4% L-theanine solution significantly decreased their susceptibility to pilocarpine-induced seizures whereas susceptibility to PTZ-induced seizures was increased. The latter effect was linked to decreased extracellular GABA concentrations in frontal cortex.
Assuntos
Glutamatos/farmacologia , Convulsões/tratamento farmacológico , Ácido gama-Aminobutírico/metabolismo , Animais , Modelos Animais de Doenças , GABAérgicos/metabolismo , Glutamatos/administração & dosagem , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Microdiálise , Pentilenotetrazol/efeitos adversos , Pilocarpina/efeitos adversos , Convulsões/induzido quimicamente , Chá/químicaRESUMO
Hypothermia is a promising neuroprotective therapy that has been shown to reduce apoptosis after an ischemic insult. This study evaluated the effect of mild hypothermia on activated caspase-3 up to 1 week after the induction of a stroke. Endothelin-1 (Et-1) was used to elicit transient focal cerebral ischemia in rats. Twenty minutes after the ischemic insult, a state of mild hypothermia (33°C) was imposed for a duration of 2h. The functional outcome, infarct volume and activated caspase-3 immunoreactivity (IR) were assessed at 8, 24 and 72h, and one week after the insult. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry. Hypothermia improved the neurological outcome at all of the time points studied compared to the normothermic group, and was associated with a reduction in infarct volume. In both groups, activated caspase-3 IR peaked 24h after the Et-1 induced insult and hypothermia significantly reduced the number of apoptotic cells at 8h, 24h and 1 week after ischemia. Furthermore, the hypothermic treatment did not affect the CBF in the Et-1 model. These findings indicate that in the Et-1 model, hypothermia exerts a long lasting effect on stroke-induced apoptosis.
Assuntos
Isquemia Encefálica/metabolismo , Caspase 3/metabolismo , Córtex Cerebral/metabolismo , Hipotermia Induzida , Neurônios/metabolismo , Animais , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/patologia , Córtex Cerebral/patologia , Endotelina-1 , Masculino , Neurônios/patologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologiaRESUMO
Oxytocin is a neuromodulator with antidepressant-like effects. In vitro, oxytocin is rapidly cleaved by insulin-regulated aminopeptidase (IRAP). Oxytocin metabolites are known to exert strong central activities that are different from the effects of the parent molecule. Our goal is to investigate in vivo whether IRAP deletion modifies the antidepressant-like effects of oxytocin. Male and female C57Bl/6 mice, IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice were injected subcutaneously with saline, oxytocin or oxytocin combined with angiotensin IV. One hour after injection, immobility was timed during a 5 min forced swim that was preceded by an open field to study locomotor behaviour. Oxytocin induced antidepressant-like effects in male (0.25 mg/kg oxytocin) and female (0.15 mg/kg oxytocin) C57Bl/6 mice subjected to the forced swim test. Oxytocin did not influence locomotor behaviour in mice, as shown with the open field. These findings were reproduced in transgenic male (aged 3-6 months) and female (aged 12-18 months) IRAP(+/+) mice. However, the major findings of our study were that the antidepressant-like effect was reversed in angiotensin IV treated IRAP(+/+) mice and was completely absent in age- and gender-matched IRAP(-/-) mice. The lack of an antidepressant-like effect of oxytocin in young male and middle-aged female IRAP(-/-) mice attributes an important role to IRAP in mediating this effect.
Assuntos
Aminopeptidases/metabolismo , Antidepressivos/uso terapêutico , Depressão/tratamento farmacológico , Ocitocina/uso terapêutico , Fatores Etários , Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Animais , Antidepressivos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cistinil Aminopeptidase/deficiência , Cistinil Aminopeptidase/genética , Depressão/sangue , Depressão/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Feminino , Imipramina/uso terapêutico , Resposta de Imobilidade Tônica/efeitos dos fármacos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Ocitocina/sangue , Ocitocina/farmacologiaRESUMO
BACKGROUND: Stroke remains one of the most common diseases with a serious impact on quality of life but few effective treatments exist. Mild hypothermia (33°C) is a promising neuroprotective therapy in stroke management. This study investigated whether a delayed short mild hypothermic treatment is still beneficial as neuroprotective strategy in the endothelin-1 (Et-1) rat model for a transient focal cerebral ischemia. Two hours of mild hypothermia (33°C) was induced 20, 60 or 120 minutes after Et-1 infusion. During the experiment the cerebral blood flow (CBF) was measured via Laser Doppler Flowmetry in the striatum, which represents the core of the infarct. Functional outcome and infarct volume were assessed 24 hours after the insult. In this sub-acute phase following stroke induction, the effects of the hypothermic treatment on apoptosis, phagocytosis and astrogliosis were assessed as well. Apoptosis was determined using caspase-3 immunohistochemistry, phagocytic cells were visualized by CD-68 expression and astrogliosis was studied by glial fibrillary acidic protein (GFAP) staining. RESULTS: Cooling could be postponed up to 1 hour after the onset of the insult without losing its positive effects on neurological deficit and infarct volume. These results correlated with the caspase-3 staining. In contrast, the increased CD-68 expression post-stroke was reduced in the core of the insult with all treatment protocols. Hypothermia also reduced the increased levels of GFAP staining, even when it was delayed up to 2 hours after the insult. The study confirmed that the induction of the hypothermia treatment in the Et-1 model does not affect the CBF. CONCLUSIONS: These data indicate that in the Et-1 rat model, a short mild hypothermic treatment delayed for 1 hour is still neuroprotective and correlates with apoptosis. At the same time, hypothermia also establishes a lasting inhibitory effect on the activation of astrogliosis.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica , Endotelina-1/efeitos adversos , Gliose/etiologia , Hipotermia Induzida/métodos , Estatística como Assunto , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apoptose/fisiologia , Infarto Encefálico/etiologia , Isquemia Encefálica/induzido quimicamente , Isquemia Encefálica/complicações , Isquemia Encefálica/terapia , Caspase 3/metabolismo , Contagem de Células , Circulação Cerebrovascular/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/terapia , Fluxometria por Laser-Doppler , Masculino , Transtornos dos Movimentos/etiologia , Exame Neurológico , Distribuição Aleatória , Ratos , Ratos Wistar , Fatores de TempoRESUMO
There is extensive evidence in Parkinson's disease of a link between oxidative stress and some of the monogenically inherited Parkinson's disease-associated genes. This paper focuses on the importance of this link and potential impact on neuronal function. Basic mechanisms of oxidative stress, the cellular antioxidant machinery, and the main sources of cellular oxidative stress are reviewed. Moreover, attention is given to the complex interaction between oxidative stress and other prominent pathogenic pathways in Parkinson's disease, such as mitochondrial dysfunction and neuroinflammation. Furthermore, an overview of the existing genetic mouse models of Parkinson's disease is given and the evidence of oxidative stress in these models highlighted. Taken into consideration the importance of ageing and environmental factors as a risk for developing Parkinson's disease, gene-environment interactions in genetically engineered mouse models of Parkinson's disease are also discussed, highlighting the role of oxidative damage in the interplay between genetic makeup, environmental stress, and ageing in Parkinson's disease.
Assuntos
Modelos Animais de Doenças , Estresse Oxidativo/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Interação Gene-Ambiente , Camundongos , Camundongos Transgênicos , Modelos BiológicosRESUMO
Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.
Assuntos
Anticonvulsivantes/uso terapêutico , Grelina/uso terapêutico , Sistema Límbico/efeitos dos fármacos , Receptores de Grelina/metabolismo , Convulsões/tratamento farmacológico , Convulsões/patologia , Análise de Variância , Animais , Anticonvulsivantes/farmacologia , Cálcio/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Humanos , Técnicas In Vitro , Sistema Límbico/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Agonistas Muscarínicos/toxicidade , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Pilocarpina/toxicidade , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Ratos , Ratos Wistar , Receptores de Grelina/agonistas , Receptores de Grelina/deficiência , Convulsões/genética , Índice de Gravidade de Doença , Especificidade da Espécie , Transfecção , Ácido gama-Aminobutírico/metabolismoRESUMO
BACKGROUND: For successful translation to clinical stroke studies, the Stroke Therapy Academic Industry Round Table criteria have been proposed. Two important criteria are testing of therapeutic interventions in conscious animals and the presence of a co-morbidity factor. We chose to work with hypertensive rats since hypertension is an important modifiable risk factor for stroke and influences the clinical outcome. We aimed to compare the susceptibility to ischemia in hypertensive rats with those in normotensive controls in a rat model for induction of ischemic stroke in conscious animals. METHODS: The vasoconstrictor endothelin-1 was stereotactically applied in the vicinity of the middle cerebral artery of control Wistar Kyoto rats (WKYRs) and spontaneously hypertensive rats (SHRs) to induce a transient decrease in striatal blood flow, which was measured by the laser Doppler technique. Infarct size was assessed histologically by cresyl violet staining. Sensory-motor functions were measured at several time points using the neurological deficit score. Activation of microglia and astrocytes in the striatum and cortex was investigated by immunohistochemistry using antibodies against CD68/Iba-1 and glial fibrillary acidic protein. RESULTS AND CONCLUSIONS: The SHRs showed significantly larger infarct volumes and more pronounced sensory-motor deficits, compared to the WKYRs at 24 h after the insult. However, both differences disappeared between 24 and 72 h. In SHRs, microglia were less susceptible to activation by lipopolysaccharide and there was a reduced microglial activation after induction of ischemic stroke. These quantitative and qualitative differences may be relevant for studying the efficacy of new treatments for stroke in accordance to the Stroke Therapy Academic Industry Round Table criteria.
Assuntos
Isquemia Encefálica/metabolismo , Regulação para Baixo/fisiologia , Hipertensão/metabolismo , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Microglia/patologia , Acidente Vascular Cerebral/metabolismo , Animais , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Regulação para Baixo/efeitos dos fármacos , Endotelina-1/toxicidade , Predisposição Genética para Doença/etiologia , Hipertensão/complicações , Hipertensão/patologia , Lipopolissacarídeos/administração & dosagem , Masculino , Microglia/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologiaRESUMO
A liquid chromatographic method with fluorescence detection was developed and validated for the quantification of the antidepressant reboxetine (RBX), a selective noradrenalin reuptake inhibitor, in rat brain microdialysates. After modification of the method in terms of sample preparation and sensitivity, it was also validated for the quantification of RBX in rat plasma samples. To enable fluorescence detection, a pre-column derivatization step with 9-fluorenylmethyl chloroformate was included. Separations were performed on a reversed phase C18 column using gradient elution. The retention time for RBX was found to be 8.8 min. The assay of RBX in brain microdialysis samples showed a linear relationship in the calibration curve from 2 to 200 ng/mL, with a correlation coefficient ≥0.999. The limit of detection (LOD) and the lower limit of quantification (LLOQ) were 0.6 and 2.0 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.5% and 11.7% with an average recovery of 101.2±8.2% (mean±SD, n=40). For the analysis of plasma samples, the calibration curve was linear between 20 and 700 ng/mL with a correlation coefficient ≥0.999. LOD and LLOQ were 6 and 20 ng/mL respectively. The intra-day and the inter-day precision (RSD %) ranged between 1.7% and 11.5% with an average recovery of 98.5±7.3% (mean±SD, n=40). We demonstrated the applicability of the method to determine the concentration-time profiles of RBX in brain and plasma following systemic administration.
Assuntos
Química Encefálica , Cromatografia Líquida/métodos , Microdiálise/métodos , Morfolinas/análise , Morfolinas/sangue , Animais , Modelos Lineares , Masculino , Ratos , Ratos Wistar , Reboxetina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodosRESUMO
Neuropeptides appear to be of importance when the central nervous system (CNS) is challenged, such as during high-frequency firing and pathologic conditions. Potential advantages of treatments that target neuropeptide systems in comparison to classical neurotransmitter systems and ion channels revolve around the subject of efficacy as well as the reduced likelihood of side effects, thus making them attractive candidates for the development of new clinical applications for various disorders. The number of neuropeptides linked to epilepsy is on the rise, reflecting the increased interest of researchers in this domain. Ghrelin has only very recently been introduced into the field of epilepsy, and has already led to contradictory clinical publications. There is a great paucity with regard to what mechanism of action is utilized by ghrelin to inhibit seizures. In this review we disclose how we can better understand the mechanism ghrelin uses to prevent seizures, which indirectly could give an insight to researchers who are studying ghrelin in other fields of research.