RESUMO
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
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
BACKGROUND: Stroke is an important cause of morbidity and mortality and few therapies exist thus far. Mild hypothermia (33°C) is a promising neuroprotective strategy to improve outcome after ischemic stroke. However, its complete mechanism of action has not yet been fully elaborated. This study is the first to investigate whether this neuroprotection occurs through modulation of the neuroinflammatory response after stroke in a time-dependent manner. METHODS: The Endothelin-1 (Et-1) model was used to elicit a transient focal cerebral ischemia in male Wistar rats. In this model, the core and penumbra of the insult are represented by the striatum and the cortex respectively. We assessed the effects of 2 hours of hypothermia, started 20 minutes after Et-1 injection on neurological outcome and infarct volume. Furthermore, pro- and anti-inflammatory cytokine expression was determined using ELISA. Microgliosis and astrogliosis were investigated using CD-68 and GFAP staining respectively. All parameters were determined 8, 24, 72 hours and 1 week after the administration of Et-1. RESULTS: Et-1 infusion caused neurological deficit and a reproducible infarct size which increased up to 3 days after the insult. Both parameters were significantly reduced by hypothermia. The strongest reduction in infarct volume with hypothermia, at 3 days, corresponded with increased microglial activation. Reducing the brain temperature affected the stroke induced increase in interleukin-1ß and tumor necrosis factor α in the striatum, 8 hours after its induction, but not at later time points. Transforming growth factor ß increased as a function of time after the Et-1-induced insult and was not influenced by cooling. Hypothermia reduced astrogliosis at 1 and 3 days after stroke onset. CONCLUSIONS: The beneficial effects of hypothermia after stroke on infarct volume and functional outcome coincide with a time-dependent modulation of the cytokine expression and gliosis.
Assuntos
Citocinas/metabolismo , Endotelina-1/farmacologia , Gliose , Hipotermia/metabolismo , Ataque Isquêmico Transitório/induzido quimicamente , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Gliose/induzido quimicamente , Gliose/metabolismo , Gliose/patologia , Interleucina-1beta/metabolismo , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Noradrenaline exerts inhibitory effects on seizure susceptibility. Subtype selective agonists and antagonists were used to identify the anticonvulsant hippocampal adrenoreceptors. Intrahippocampal dialysis was used for administration of all compounds, including pilocarpine for limbic seizure induction, and as the neurotransmitter sampling tool. The noradrenaline reuptake inhibitor maprotiline mediated anticonvulsant effects, associated with dose-dependent increases in extracellular hippocampal noradrenaline, dopamine and GABA levels. At high concentrations, maprotiline produced proconvulsant effects associated with high levels of noradrenaline, dopamine and glutamate. Maprotiline's anticonvulsant effect was blocked by administration of either a selective α(2) - and ß(2) -antagonist. α(2) -Antagonist administration with maprotiline was associated with a further increase in noradrenaline and dopamine from maprotiline alone; whereas ß(2) -antagonist administered with maprotiline inhibited the dopamine increases produced by maprotiline. α(1A) -Antagonism blocked the GABA-ergic but not the anticonvulsive effect of maprotiline. These results were confirmed as combined but not separate α(2) - and ß(2) -adrenoreceptor stimulation, using selective agonists, inhibited limbic seizures. Interestingly, α(1A) -receptor stimulation and α(1D) -antagonism alone also inhibited seizures associated with respectively significant hippocampal GABA increases and glutamate decreases. The main findings of this study are that (i) increased hippocampal noradrenergic neurotransmission inhibits limbic seizures via combined α(2) - and ß(2) -receptor activation and (ii) α(1A) - and α(1D) -adrenoreceptors mediate opposite effects on hippocampal excitability.
Assuntos
Agonistas Adrenérgicos/farmacologia , Antagonistas Adrenérgicos/farmacologia , Hipocampo/metabolismo , Sistema Límbico/metabolismo , Receptores Adrenérgicos/metabolismo , Convulsões/metabolismo , Agonistas Adrenérgicos/uso terapêutico , Antagonistas Adrenérgicos/uso terapêutico , Animais , Hipocampo/química , Hipocampo/efeitos dos fármacos , Sistema Límbico/química , Sistema Límbico/efeitos dos fármacos , Masculino , Subunidades Proteicas/agonistas , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores Adrenérgicos alfa 1/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Convulsões/tratamento farmacológicoRESUMO
Neuroinflammation is a key element in the ischemic cascade after cerebral ischemia that results in cell damage and death in the subacute phase. However, anti-inflammatory drugs do not improve outcome in clinical settings suggesting that the neuroinflammatory response after an ischemic stroke is not entirely detrimental. This review describes the different key players in neuroinflammation and their possible detrimental and protective effects in stroke. Because of its inhibitory influence on several pathways of the ischemic cascade, hypothermia has been introduced as a promising neuroprotective strategy. This review also discusses the influence of hypothermia on the neuroinflammatory response. We conclude that hypothermia exerts both stimulating and inhibiting effects on different aspects of neuroinflammation and hypothesize that these effects are key to neuroprotection.
Assuntos
Isquemia Encefálica/patologia , Hipotermia Induzida , Inflamação/patologia , Inflamação/terapia , Acidente Vascular Cerebral/patologia , Animais , Anti-Inflamatórios/uso terapêutico , Isquemia Encefálica/complicações , Isquemia Encefálica/imunologia , Isquemia Encefálica/fisiopatologia , Quimiocinas/imunologia , Citocinas/imunologia , Proteínas HMGB/imunologia , Humanos , Inflamação/etiologia , Inflamação/imunologia , Integrinas/imunologia , Metaloproteinases da Matriz/imunologia , Fármacos Neuroprotetores/uso terapêutico , Espécies Reativas de Oxigênio/imunologia , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/fisiopatologiaRESUMO
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
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
Stroke remains a disease with a serious impact on quality of life but few effective treatments exist. There is an urgent need to develop and/or improve neuroprotective strategies to combat this. Many drugs proven to be neuroprotective in experimental models fail to improve patient outcome in a clinical setting. An emerging treatment, therapeutic hypothermia (TH), is a promising neuroprotective therapy in stroke management. Several studies with TH in experimental models and small clinical trials have shown beneficial effects. Despite this, implementation into the clinical setting is still lacking due to methodological considerations as well as hypothermia-related complications. This paper discusses the possible opportunities and limitations of the use of TH in animal models and the translation into the clinic.
RESUMO
UNLABELLED: In this study, we validated the use of (99m)Tc-hexamethylpropyleneamine oxime ((99m)Tc-HMPAO) micro-SPECT combined with micro-CT for semiquantification of the infarct size after an experimental stroke in rats and compared our observations with those obtained from histology. This imaging strategy was applied to measure the longitudinal effect of mild hypothermia on the progression of brain damage after stroke in rats. METHODS: The endothelin-1 model was used to elicit a transient focal cerebral ischemia in rats. This resulted in a reproducible insult in which the core is represented by the striatum and the penumbra by the cortex. Micro-SPECT and micro-CT images were taken at 1, 3, and 7 d after infusion of endothelin-1 and compared with those taken before the insult. After the last acquisition, rats were sacrificed and the infarct volume was determined via Nissl staining. The results obtained with micro-SPECT and micro-CT were compared with histology at the same time points. Mild hypothermia (33°C) was induced for 2 h, starting 20 min after the insult. RESULTS: Brain damage was estimated using micro-SPECT and micro-CT and was reproducible with minimal interobserver variability. Normothermic stroke rats had reduced (99m)Tc-HMPAO uptake at 1 and 3 d after the insult, whereas hypothermia improved damage after stroke. These findings corroborate with histology at the same time points. At 1 wk after the insult, no reduction of radioactive uptake was observed in any treatment group. CONCLUSION: Micro-SPECT and micro-CT allow quick and reproducible semiquantification of brain damage as an interesting alternative to histology to measure the extent of infarcted tissue in small animals after stroke.