RESUMO
Inflammation is an important event in ischemic injury. These immune responses begin with the expression of pro-inflammatory genes modulating transcription factors, such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and signal transducers and activator of transcription-1 (STAT-1). The 70-kDa heat shock protein (Hsp70) can both induce and arrest inflammatory reactions and lead to improved neurological outcome in experimental brain injury and ischemia. Since Hsp70 are induced under heat stress, we investigated the link between Hsp70 neuroprotection and phosphorylation of inhibitor of κB (IκB), c-Jun N-terminal kinases (JNK) and p38 through co-immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) assay. Transcription factors and pro-inflammatory genes were quantified by immunoblotting, electrophoretic-mobility shift assay and reverse transcription-polymerase chain reaction assays. The results showed that heat stress led to Hsp70 overexpression which rendered neuroprotection after ischemia-like injury. Overexpression Hsp70 also interrupts the phosphorylation of IκB, JNK and p38 and blunts DNA binding of their transcription factors (NF-κB, AP-1 and STAT-1), effectively downregulating the expression of pro-inflammatory genes in heat-pretreated astrocytes. Taken together, these results suggest that overexpression of Hsp70 may protect against brain ischemia via an anti-inflammatory mechanism by interrupting the phosphorylation of upstream of transcription factors.
Assuntos
Astrócitos/metabolismo , Isquemia Encefálica/metabolismo , Encefalite/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição AP-1/metabolismo , Animais , Glucose , Resposta ao Choque Térmico , Proteínas I-kappa B/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos ICR , Oxigênio , Fosforilação , Cultura Primária de CélulasRESUMO
The 70-kDa heat shock protein (HSP70) is known to protect the brain from injury through multiple mechanisms. We investigated the effect of pharmacological HSP70 induction in experimental traumatic brain injury (TBI). 3-month-old male C57/B6 mice were given 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) intraperitoneally (IP, 2 mg/kg) or intracerebroventricularly (ICV, 1 µg/kg) to determine whether HSP70 could be induced in the brain. Mice were subjected to TBI via cortical controlled impact, and were treated with 17-AAG (or vehicle) IP according to one of two treatment regimens: (1) 2 mg/kg at the time of injury, (2) a total of three doses (4 mg/kg) at 2 and 1d prior to TBI and again at the time of injury. Brains were assessed for HSP70 induction, hemorrhage volume at 3 d, and lesion size at 14 d post-injury. Immunohistochemistry showed that both IP and ICV administration of 17-AAG increased HSP70 expression primarily in microglia and in a few neurons by 24 h but not in astrocytes. 17-AAG induced HSP70 in injured brain tissue as early as 6 h, peaking at 48 h and largely subsiding by 72 h after IP injection. Both treatment groups showed decreased hemorrhage volume relative to untreated mice as well as improved neurobehavioral outcomes. These observations indicate that pharmacologic HSP70 induction may prove to be a promising treatment for TBI.
Assuntos
Benzoquinonas/administração & dosagem , Lesões Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Lactamas Macrocíclicas/administração & dosagem , Fármacos Neuroprotetores/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Hemorragia Encefálica Traumática/tratamento farmacológico , Hemorragia Encefálica Traumática/metabolismo , Hemorragia Encefálica Traumática/patologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Stroke is a frequent cause of long-term disability and death worldwide. Ischemic stroke is more commonly encountered compared to hemorrhagic stroke, and leads to tissue death by ischemia due to occlusion of a cerebral artery. Inflammation is known to result as a result of ischemic injury, long thought to be involved in initiating the recovery and repair process. However, work over the past few decades indicates that aspects of this inflammatory response may in fact be detrimental to stroke outcome. Acutely, inflammation appears to have a detrimental effect, and anti-inflammatory treatments have been been studied as a potential therapeutic target. Chronically, reports suggest that post-ischemic inflammation is also essential for the tissue repairing and remodeling. The majority of the work in this area has centered around innate immune mechanisms, which will be the focus of this review. This review describes the different key players in neuroinflammation and their possible detrimental and protective effects in stroke. A better understanding of the roles of the different immune cells and their temporal profile of damage versus repair will help to clarify more effective modulation of inflammation post stroke.
Assuntos
Imunidade Inata , Acidente Vascular Cerebral/imunologia , Animais , Barreira Hematoencefálica , Humanos , Inflamação/imunologia , Transdução de Sinais , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
The protective effect of therapeutic hypothermia in cerebral ischemia is well accepted in experimental models, and some clinical studies show that there is benefit in humans as well. Long-term observations in animal and clinical studies have documented recovery of neurological function following hypothermia treatment. Diminished damage by hypothermic protection should contribute to the recovery in many ways, but hypothermia appears to enhance regeneration of brain tissue as well. Since regeneration of the brain after damage initiates within hours and is active days and weeks after stroke, prolonged hypothermia might affect regenerative processes which have been documented to occur in these time frames. As there is a lack of data at the basic and clinical levels, the mechanism of neuroregeneration by hypothermia is unclear. Yet, we speculate that hypothermia enhances regeneration by positively influencing neurogenesis, angiogenesis, gliogenesis and synapse/circuit formation after stroke. In this chapter, we will provide up-to-date data from experimental studies and clinical reports on the effect of therapeutic hypothermia on neuroregeneration, with perspectives on future research.
Assuntos
Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , Hipotermia Induzida/métodos , Regeneração Nervosa/fisiologia , Animais , Isquemia Encefálica/patologia , Humanos , Neurogênese/fisiologia , Resultado do TratamentoRESUMO
Inflammation following ischemic stroke is known to contribute to injury. NADPH oxidase (NOX) is a major enzyme system originally studied in immune cells that leads to superoxide (O.*) generation. Apocynin is a NOX inhibitor that has been studied as a potential treatment in experimental stroke. Here we explored the effect of different doses of apocynin in a mouse model of 2 h transient middle cerebral artery occlusion (tMCAO) followed by 22 h reperfusion. Apocynin, given i.v. at a dose of 2.5 mg/kg 30 min before reperfusion, improved neurological function (P<0.01), reduced infarct volume (P<0.05), and reduced the incidence of cerebral hemorrhage (P<0.05), but not at higher doses of 3.75 and 5 mg/kg, where it actually increased brain hemorrhage. Apocynin also tended to reduce mortality at the lower dose, but not at higher doses. Using hydroethine fluorescence to delineate O.* in the brain, neurons and some microglia/macrophages, but not vascular endothelial cells were found to contain O.*. Apocynin at protective doses markedly prevented ischemia-induced increases in O.*. Our data suggested that apocynin can protect against experimental stroke, but with a narrow therapeutic window.
Assuntos
Acetofenonas/farmacologia , Inibidores Enzimáticos/farmacologia , Fármacos Neuroprotetores , Acidente Vascular Cerebral/tratamento farmacológico , Acetofenonas/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/fisiologia , Antígeno CD11b/metabolismo , Infarto Cerebral/patologia , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Hemorragias Intracranianas/complicações , Hemorragias Intracranianas/tratamento farmacológico , Hemorragias Intracranianas/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , NADPH Oxidases/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/patologia , Superóxidos/metabolismo , Resultado do TratamentoRESUMO
Statins are increasingly being used for the treatment of a variety of conditions beyond their original indication for cholesterol lowering. We previously reported that simvastatin affected the dopaminergic system in the rat brain. This study aims to investigate regional changes of muscarinic M1/4 receptors in the rat brain after 4-week administration of simvastatin (1 or 10 mg/kg/day). M1/4 receptor distribution and alterations in the post-mortem rat brain were detected by [(3)H]pirenzepine binding autoradiography. Simvastatin (1 mg/kg/day) increased [(3)H]pirenzepine binding, predominantly in the prefrontal cortex (171%, P<0.001), primary motor cortex (153%, P=0.001), cingulate cortex (109%, P<0.001), hippocampus (138%, P<0.001), caudate putamen (122%, P=0.002) and nucleus accumbens (170%, P<0.001) compared with controls; while lower but still significant increases of [(3)H]pirenzepine binding were observed in the examined regions following simvastatin (10 mg/kg/day) treatment. Our results also provide strong evidence that chronic simvastatin administration, especially at a low dosage, up-regulates M1/4 receptor binding, which is likely to be independent of its muscarinic agonist-like effect. Alterations in [(3)H]pirenzepine binding in the examined brain areas may represent the specific regions that mediate the clinical effects of simvastatin treatment on cognition and memory via the muscarinic cholinergic system. These findings contribute to a better understanding of the critical roles of simvastatin in treating neurodegenerative disorders, via muscarinic receptors.
Assuntos
Química Encefálica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Receptor Muscarínico M1/biossíntese , Receptor Muscarínico M4/biossíntese , Sinvastatina/farmacologia , Animais , Autorradiografia , Relação Dose-Resposta a Droga , Masculino , Antagonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/farmacologia , Pirenzepina/metabolismo , Pirenzepina/farmacologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
Agmatine is a primary amine formed by the decarboxylation of L-arginine synthesized in mammalian brain. In this study, we investigated the neuroprotective effect of agmatine on ischemic and ischemia-like insults. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation (OGD), a model of ischemia-like injury, and treated with agmatine before or at the start of OGD, or upon reperfusion. Neuronal death was reduced when agmatine was present during OGD, and this protection was associated with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not inducible NOS (iNOS). Protection by agmatine was also studied at the in vivo level using a model of middle cerebral artery occlusion (MCAO) in mice. Mice were subjected to 2 h MCAO. Agmatine was administered either 30 min before ischemia, at the start of MCAO, at the start of reperfusion, or 2 or 5 h into reperfusion. Agmatine markedly reduced infarct area in all treatment groups except when treatment was delayed 5 h. The number of nNOS immunopositive cells was correlated with neuroprotection. Interestingly, immunoreactivity for iNOS was reduced only when agmatine was administered before and at the onset of MCAO. Our study suggests that agmatine may be a novel therapeutic strategy to reduce cerebral ischemic injury, and may act by inhibiting the detrimental effects of nNOS.
Assuntos
Agmatina/uso terapêutico , Infarto Encefálico/prevenção & controle , Ataque Isquêmico Transitório/complicações , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Análise de Variância , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Infarto Encefálico/etiologia , Infarto Encefálico/patologia , Contagem de Células/métodos , Hipóxia Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/deficiência , Hipóxia/tratamento farmacológico , Imuno-Histoquímica/métodos , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Nitritos/metabolismo , Sais de Tetrazólio , Fatores de TempoRESUMO
We characterize the survival, migration, and differentiation of human neurospheres derived from CNS stem cells transplanted into the ischemic cortex of rats 7 days after distal middle cerebral artery occlusion. Transplanted neurospheres survived robustly in naive and ischemic brains 4 wk posttransplant. Survival was influenced by proximity of the graft to the stroke lesion and was negatively correlated with the number of IB4-positive inflammatory cells. Targeted migration of the human cells was seen in ischemic animals, with many human cells migrating long distances ( approximately 1.2 mm) predominantly toward the lesion; in naive rats, cells migrated radially from the injection site in smaller number and over shorter distances (0.2 mm). The majority of migrating cells in ischemic rats had a neuronal phenotype. Migrating cells between the graft and the lesion expressed the neuroblast marker doublecortin, whereas human cells at the lesion border expressed the immature neuronal marker beta-tubulin, although a small percentage of cells at the lesion border also expressed glial fibrillary acid protein (GFAP). Thus, transplanted human CNS (hCNS)-derived neurospheres survived robustly in naive and ischemic brains, and the microenvironment influenced their migration and fate.
Assuntos
Isquemia Encefálica/terapia , Movimento Celular , Neurônios/citologia , Transplante de Células-Tronco , Animais , Arteriopatias Oclusivas/terapia , Biomarcadores/análise , Isquemia Encefálica/patologia , Diferenciação Celular , Sobrevivência Celular , Córtex Cerebral/patologia , Proteína Duplacortina , Feto/citologia , Humanos , Ratos , Transplante HeterólogoRESUMO
BACKGROUND AND PURPOSE: Ischemic injury and reperfusion increases superoxide (O2-) production and reduces the ability of neurons to scavenge free radicals, leading to the release of cytochrome c and apoptosis. Here we test whether overexpression with the use of gene therapy of the antioxidant glutathione peroxidase (Gpx), delivered before or after experimental stroke, is protective against ischemic injury. METHODS: Sixty-two rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral vectors expressing Gpx/lacZ or lacZ alone (control) were delivered into each striatum 12 hours before or 2 or 5 hours after ischemia onset. RESULTS: Striatal neuron survival at 2 days was improved by 36% when Gpx was delivered 12 hours before ischemia onset, 26% with a 2-hour delay, and 25% when delayed 5 hours. After ischemia, Gpx overexpression significantly reduced cytosolic translocation of cytochrome c and increased the proportion of Bcl-2-positive cells compared with cells transfected with control vector. Bax and activated caspase-3, while present in control-transfected neurons after ischemia, were rarely noted in Gpx-transfected cells. CONCLUSIONS: Expression from these herpes simplex viral vectors begins 4 to 6 hours after injection, which suggests a 9- to 11-hour temporal therapeutic window for Gpx. This is the first study to show that overexpression of Gpx with the use of gene therapy protects against experimental stroke, even with postischemic transfection, and the neuroprotective mechanism involves attenuation of apoptosis-related events.
Assuntos
Apoptose/fisiologia , Grupo dos Citocromos c/metabolismo , Glutationa Peroxidase/biossíntese , Neurônios/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Grupo dos Citocromos c/efeitos dos fármacos , Citoproteção/genética , Modelos Animais de Doenças , Esquema de Medicação , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Glutationa Peroxidase/administração & dosagem , Glutationa Peroxidase/genética , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Simplexvirus/genética , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Fatores de Tempo , Proteína X Associada a bcl-2RESUMO
Significant advances have been made over the past few years concerning the cellular and molecular events underlying ischemic cell death. The brain succumbs to ischemic injury as a result of loss of metabolic stores, excessive intracellular calcium accumulation, oxidative stress, and potentiation of the inflammatory response. Neurons can also die via necrotic or apoptotic mechanisms, depending on the nature and severity of the insult. While it has been widely held that ischemia is notable for cessation of protein synthesis, brain regions with marginal reduction in blood supply are especially capable of expressing a variety of genes, the functions of many of which are only beginning to be understood. Gene expression is also upregulated upon reperfusion and reoxygenation. As a result, a number of signaling pathways have been identified and are now known to contribute to ischemic progression or, in some cases, attempts at self preservation. This review will focus on the roles of stress genes, apoptosis-related genes, and inflammation. Knowledge of such molecular events has fueled interest in developing specific molecular targets with the hope of someday affecting outcome in clinical stroke.
Assuntos
Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Animais , Apoptose , Regulação Enzimológica da Expressão Gênica , Humanos , Inflamação , Metaloproteinases da Matriz/metabolismo , Modelos Biológicos , Necrose , Neurônios/citologia , Estresse Oxidativo , Oxigênio/metabolismo , Acidente Vascular Cerebral/terapia , Regulação para CimaRESUMO
Mild hypothermia is an established neuroprotectant against cerebral ischemic injury. Studies have shown that inflammation potentiates cerebral ischemic injury, particularly in the setting of reperfusion. To further elucidate the mechanism by which mild hypothermia attenuates the inflammatory response, we assessed endothelial intercellular adhesion molecule-1 (ICAM-1) expression, neutrophil and monocyte infiltration, and microglial activation following 2 h of transient focal cerebral ischemia under normothermic and mildly hypothermic conditions. Ischemia was induced using the intraluminal suture method in Sprague-Dawley rats. Immunohistochemistry was used to detect endothelial ICAM-1, infiltrating neutrophils and monocytes, and microglia at 1, 3, and 7 days post-ischemia. Immunopositive cell and vessel densities were measured in the peri-infarct region. Mild hypothermia was associated with decreased neutrophils at 1 and 3 days post-ischemia, decreased ICAM-1-positive vessels at 1, 3, and 7 days, and decreased monocytes/activated microglia at 3 and 7 days, but not at 1 day. These data demonstrate that mild hypothermia significantly reduces endothelial adhesion molecule expression, acute (neutrophil) and subacute (monocyte) leukocyte infiltration, and microglial activation up to 7 days following insult in a rodent model of transient focal cerebral ischemia.
Assuntos
Hipotermia Induzida , Molécula 1 de Adesão Intercelular/metabolismo , Microglia/patologia , Neutrófilos/patologia , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/terapia , Animais , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/terapia , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/terapia , Masculino , Monócitos/patologia , Ratos , Ratos Sprague-DawleyRESUMO
The 72-kD inducible heat shock protein (HSP72) can attenuate cerebral ischemic injury when overexpressed before ischemia onset. Whether HSP72 overexpression is protective when applied after ischemia onset is not known, but would have important clinical implications. Fifty-seven rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral (HSV) vectors expressing hsp72 with lacZ as a reporter were delivered 0.5, 2, and 5 hours after ischemia onset into each striatum. Control animals received an identical vector containing only lacZ. Striatal neuron survival at 2 days was improved by 23% and 15% when HSP72 vectors were delayed 0.5 and 2 hours after ischemic onset, respectively ( P < 0.05). However, when delayed by 5 hours, HSP72 overexpression was no longer protective. This is the first demonstration that HSP72 gene transfer even after ischemia onset is neuroprotective. Because expression from these HSV vectors begins 4 to 6 hours after injection, this suggests that the temporal therapeutic window for HSP72 is at least 6 hours after ischemia onset. Future strategies aimed at enhancing HSP72 expression after clinical stroke may be worth pursuing. The authors suggest that in the future HSP72 may be an effective treatment for stroke.
Assuntos
Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , Proteínas de Choque Térmico/genética , Neurônios/fisiologia , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Animais , Isquemia Encefálica/patologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Terapia Genética , Proteínas de Choque Térmico HSP72 , Marcação In Situ das Extremidades Cortadas , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/terapia , Óperon Lac , Masculino , Camundongos , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologiaRESUMO
Mild hypothermia protects the brain against experimental ischemia, but the reasons are not well known. We examined whether the protective effects of mild hypothermia could be correlated with alterations in expression of Bcl-2, an anti-apoptotic protein in a rat model of transient global ischemia. Following 10 min of forebrain ischemia, hippocampal neurons were examined 72 h later for survival, expression of Bcl-2 family proteins and apoptosis. Intraischemic mild hypothermia was applied for 3 h (33 degrees C, isch-33) or normal body temperature was maintained (37 degrees C, isch-37). Survival of CA1 neurons was significantly improved in the isch-33 group compared to the isch-37 group (90 vs. 53% survival; P<0.01). The proportion of Bcl-2-positive cells among surviving CA1 neurons in the isch-33 group was increased compared to that of sham and isch-37 groups (P<0.01). Bax expression in CA1 was no different between sham and isch-33 groups, but was significantly decreased in isch-37 (P<0.05). TUNEL staining was positive in many isch-37 CA1 neurons, but absent in isch-33. Utilizing electron microscopy, more cells meeting criteria for apoptosis were observed in the isch-37 than isch-33. These data suggest that mild hypothermia attenuates apoptotic death, and that this protection may be related to increases in Bcl-2.
Assuntos
Temperatura Corporal , Ciclina D1/metabolismo , Ataque Isquêmico Transitório/metabolismo , Animais , Calefação , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Diffusion-weighted MRI (DWI) can detect early ischemic changes and is sometimes used as a surrogate neurological end point in clinical trials. Recent experimental stroke studies have shown that with brief periods of ischemia, some DWI lesions transiently reverse, only to recur later. This study examined the histological condition of the tissue during the period of DWI reversal. METHODS: Rats underwent 30 minutes of middle cerebral artery occlusion followed by reperfusion. DWI images were obtained during ischemia and 3 to 5 hours, 1 day, and 7 days later. MRI scans were compared with histology (5 hours, n=5; 7 days, n=5) with the use of neuronal (microtubule-associated protein 2 [MAP2]) and astrocytic (glial fibrillary acidic protein [GFAP]) markers and heat-shock protein 72 (HSP72). RESULTS: DWI abnormalities reversed 3 to 5 hours after ischemia onset but recurred at 1 day. Four animals showed complete reversal of the initial DWI hyperintensity, and 6 showed partial reversal. When the 5-hour DWI was completely normal, there was significant loss of MAP2 immunoreactivity, comprising approximately 30% of the initial DWI lesion. However, GFAP staining revealed morphologically normal astrocytes. HSP72 immunoreactivity at 5 hours was extensive and corresponded to the initial DWI lesion. CONCLUSIONS: After brief ischemic periods, normalization of the DWI does not necessarily imply that the tissue is normal. Neurons already exhibit evidence of structural damage and stress. Normal GFAP staining suggests that other nonneuronal cell populations may partially compensate for altered fluid balances at the time of DWI reversal despite the presence of neuronal injury. These observations suggest that caution is warranted when relying solely on DWI for assessment of ischemic damage.
Assuntos
Isquemia Encefálica/diagnóstico , Isquemia Encefálica/patologia , Imageamento por Ressonância Magnética , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/metabolismo , Difusão , Modelos Animais de Doenças , Progressão da Doença , Proteína Glial Fibrilar Ácida/biossíntese , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico/biossíntese , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética/métodos , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Neurônios/metabolismo , Neurônios/patologia , Valor Preditivo dos Testes , Ratos , Ratos Sprague-DawleyRESUMO
Significant advances have been made over the past few years concerning the cellular and molecular events underlying neuron death. Recently, it is becoming increasingly clear that some of genes induced during cerebral ischemia may actually serve to rescue the cell from death. However, the injured cell may not be capable of expressing protein at high enough levels to be protective. One of the most exciting arenas of such interventions is the use of viral vectors to deliver potentially neuroprotective genes at high levels. Neurotropic herpes simplex viral (HSV) strains are an obvious choice for gene therapy to the brain, and we have used bipromoter vectors that are capable of transferring various genes to neurons. Using this system in experimental models of stroke, cardiac arrest, and excitotoxicity, we have found that it is possible to enhance neuron survival against such cerebral insults by overexpressing genes that target various facets of injury. These include energy restoration by the glucose transporter (GLUT-1), buffering calcium excess by calbindin, preventing protein malfolding or aggregation by stress proteins and inhibiting apoptotic death by BCL-2. We show that in some cases, gene therapy is also effective after the onset of injury, and also address whether successful gene therapy necessarily spares function. Although gene therapy is limited to the few hundred cells the vector is capable of transfecting, we consider the possibility of such gene therapy becoming relevant to clinical neurology in the future.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Isquemia Encefálica/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Proteína G de Ligação ao Cálcio S100/metabolismo , Simplexvirus , Sistema X-AG de Transporte de Aminoácidos , Animais , Isquemia Encefálica/metabolismo , Calbindinas , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Genes bcl-2/fisiologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , RatosRESUMO
Significant advances have been made over the past few years concerning the cellular and molecular events underlying neuron death. Recently, it is becoming increasingly clear that some of the genes induced during cerebral ischemia may actually serve to rescue the cell from death. However, the injured cell may not be capable of expressing protein at levels high enough to be protective. One of the most exciting arenas of such interventions is the use of viral vectors to deliver potentially neuroprotective genes at high levels. Neurotrophic herpes simplex viral strains are an obvious choice for gene therapy to the brain, and we have utilized bipromoter vectors that are capable of transferring various genes to neurons. Using this system in experimental models of stroke, cardiac arrest and excitotoxicity, we have found that it is possible to enhance neuron survival against such cerebral insults by over-expressing genes that target various facets of injury. These include energy restoration by the glucose transporter (GLUT-1), buffering calcium excess by calbindin, preventing protein malfolding or aggregation by stress proteins and inhibiting apoptotic death by BCL-2. We show that in some cases, gene therapy is also effective after the onset of injury, and also address whether successful gene therapy necessarily spares function. Although gene therapy is limited to the few hundred cells the vector is capable of transfecting, we consider the possibility of such gene therapy becoming relevant to clinical neurology in the future.
Assuntos
Isquemia Encefálica/genética , Isquemia Encefálica/terapia , Morte Celular/genética , Regulação da Expressão Gênica/genética , Terapia Genética , Vetores Genéticos/uso terapêutico , Degeneração Neural/genética , Degeneração Neural/terapia , Simplexvirus/genética , Animais , HumanosRESUMO
We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.
Assuntos
Isquemia Encefálica/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Genótipo , Glucose/deficiência , Glucose/metabolismo , Glutationa/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/farmacologia , Células HeLa , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Hipoglicemia/metabolismo , Hipóxia Encefálica/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal/genética , Oxidantes/farmacologia , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND AND PURPOSE: Increased intracellular calcium accumulation is known to potentiate ischemic injury. Whether endogenous calcium-binding proteins can attenuate this injury has not been clearly established, and existing data are conflicting. Calbindin D28K (CaBP) is one such intracellular calcium buffer. We investigated whether CaBP overexpression is neuroprotective against transient focal cerebral ischemia. METHODS: Bipromoter, replication-incompetent herpes simplex virus vectors that encoded the genes for cabp and, as a reporter gene, lacZ were used. Sprague-Dawley rats received bilateral striatal injections of viral vector 12 to 15 hours before ischemia onset. With the use of an intraluminal occluding suture, animals were subjected to 1 hour of middle cerebral artery occlusion followed by 47 hours of reperfusion. Brains were harvested and stained with X-gal (to visualize beta-galactosidase, the gene product of lacZ). The number of remaining virally transfected, X-gal-stained neurons in both the ischemic and contralateral striata were counted and expressed as the percentage of surviving neurons in the ischemic striatum relative to the contralateral nonischemic striatum. RESULTS: Striatal neuron survivorship among cabp-injected animals was 53.5+/-4.1% (n=10) versus 26.8+/-5.4% among those receiving lacZ (n=9) (mean+/-SEM; P<0.001). CONCLUSIONS: We conclude that viral vector-mediated overexpression of CaBP leads to neuroprotection in this model of central nervous system injury. This is the first demonstration that CaBP overexpression protects neurons in a focal stroke model.
Assuntos
Corpo Estriado/metabolismo , Ataque Isquêmico Transitório/metabolismo , Neurônios/metabolismo , Proteína G de Ligação ao Cálcio S100/biossíntese , Animais , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Calbindina 1 , Calbindinas , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Ataque Isquêmico Transitório/genética , Ataque Isquêmico Transitório/patologia , Masculino , Microinjeções , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/farmacologiaRESUMO
If neuronal gene therapy is to be clinically useful, it is necessary to demonstrate neuroprotection when the gene is introduced after insult. We now report equivalent neuronal protection if calbindin D(28K) gene transfer via herpes simplex virus amplicon vector occurs immediately, 30 min, or 1 h after an excitotoxic insult, but not after a 4 h delay. Behavioral performance was evaluated for immediate and 1 h delay groups using a hippocampal-dependent task. Despite equivalent magnitude and pattern of sparing of neurons with the immediate and 1 h delay approaches, the delay animals took a significantly longer time after insult to return to normal performance.
Assuntos
Comportamento Animal , Lesões Encefálicas/terapia , Terapia Genética/métodos , Neurônios/patologia , Proteína G de Ligação ao Cálcio S100/genética , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/psicologia , Calbindinas , Sobrevivência Celular/genética , Genes Reporter , Vetores Genéticos , Hipocampo/patologia , Ácido Caínico , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Ratos , Ratos Sprague-Dawley , Proteína G de Ligação ao Cálcio S100/metabolismo , Simplexvirus/genética , Fatores de TempoRESUMO
In this study we investigated the utility of different MRI techniques for the detection and predictability of hemorrhagic transformation (HT) in a rat model of transient focal cerebral ischemia. Hemorrhagic infarction was reliably identified with gradient-echo sequences and developed between 2 and 7 days following the insult. None of the investigated early MRI features of the ischemic lesions (including the apparent diffusion coefficient and post-reperfusion blood-brain barrier damage) was a good predictor of HT severity at 7 days. This indicates that subacute HT at 2-7 days occurs independently of the severity of acute tissue and BBB damage.