RESUMO
Ischemic stroke causes secondary neurodegeneration in the thalamus ipsilateral to the infarction site and impedes neurological recovery. Axonal degeneration of thalamocortical fibers and autophagy overactivation are involved in thalamic neurodegeneration after ischemic stroke. However, the molecular mechanisms underlying thalamic neurodegeneration remain unclear. Sterile /Armadillo/Toll-Interleukin receptor homology domain protein (SARM1) can induce Wallerian degeneration. Herein, we aimed to investigate the role of SARM1 in thalamic neurodegeneration and autophagy activation after photothrombotic infarction. Neurological deficits measured using modified neurological severity scores and adhesive-removal test were ameliorated in Sarm1-/- mice after photothrombotic infarction. Compared with wild-type mice, Sarm1-/- mice exhibited unaltered infarct volume; however, there were markedly reduced neuronal death and gliosis in the ipsilateral thalamus. In parallel, autophagy activation was attenuated in the thalamus of Sarm1-/- mice after cerebral infarction. Thalamic Sarm1 re-expression in Sarm1-/- mice increased thalamic neurodegeneration and promoted autophagy activation. Auotophagic inhibitor 3-methyladenine partially alleviated thalamic damage induced by SARM1. Moreover, autophagic initiation through rapamycin treatment aggravated post-stroke neuronal death and gliosis in Sarm1-/- mice. Taken together, SARM1 contributes to secondary thalamic neurodegeneration after cerebral infarction, at least partly through autophagy inhibition. SARM1 deficiency is a potential therapeutic strategy for secondary thalamic neurodegeneration and functional deficits after stroke.
Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Camundongos , Animais , Gliose , Infarto Cerebral/metabolismo , Acidente Vascular Cerebral/metabolismo , AVC Isquêmico/metabolismo , Tálamo/metabolismo , Axônios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismoRESUMO
Cerebral infarction induces angiogenesis in the thalamus and influences functional recovery. The mechanisms underlying angiogenesis remain unclear. This study aimed to investigate the role of RTN4/Nogo-A in mediating macroautophagy/autophagy and angiogenesis in the thalamus following middle cerebral artery occlusion (MCAO). We assessed secondary neuronal damage, angiogenesis, vascular autophagy, RTN4 and S1PR2 signaling in the thalamus. The effects of RTN4-S1PR2 on vascular autophagy and angiogenesis were evaluated using lentiviral and pharmacological approaches. The results showed that RTN4 and S1PR2 signaling molecules were upregulated in parallel with angiogenesis in the ipsilateral thalamus after MCAO. Knockdown of Rtn4 by siRNA markedly reduced MAP1LC3B-II conversion and levels of BECN1 and SQSTM1 in vessels, coinciding with enhanced angiogenesis in the ipsilateral thalamus. This effect coincided with rescued neuronal loss of the thalamus and improved cognitive function. Conversely, activating S1PR2 augmented vascular autophagy, along with suppressed angiogenesis and aggravated neuronal damage of the thalamus. Further inhibition of autophagic initiation with 3-methyladenine or spautin-1 enhanced angiogenesis while blockade of lysosomal degradation by bafilomycin A1 suppressed angiogenesis in the ipsilateral thalamus. The control of autophagic flux by RTN4-S1PR2 was verified in vitro. Additionally, ROCK1-BECN1 interaction along with phosphorylation of BECN1 (Thr119) was identified in the thalamic vessels after MCAO. Knockdown of Rtn4 markedly reduced BECN1 phosphorylation whereas activating S1PR2 increased its phosphorylation in vessels. These results suggest that blockade of RTN4-S1PR2 interaction promotes angiogenesis and secondary neural repair in the thalamus by suppressing autophagic activation and alleviating dysfunction of lysosomal degradation in vessels after cerebral infarction.Abbreviations: 3-MA: 3-methyladenine; ACTA2/É-SMA: actin alpha 2, smooth muscle, aorta; AIF1/Iba1: allograft inflammatory factor 1; BafA1: bafilomycin A1; BMVECs: brain microvascular endothelial cells; BrdU: 5-bromo-2'-deoxyuridine; CLDN11/OSP: claudin 11; GFAP: glial fibrillary acidic protein; HUVECs: human umbilical vein endothelial cells; LAMA1: laminin, alpha 1; MAP2: microtubule-associated protein 2; MBP2: myelin basic protein 2; MCAO: middle cerebral artery occlusion; PDGFRB/PDGFRß: platelet derived growth factor receptor, beta polypeptide; RECA-1: rat endothelial cell antigen-1; RHOA: ras homolog family member A; RHRSP: stroke-prone renovascular hypertensive rats; ROCK1: Rho-associated coiled-coil containing protein kinase 1; RTN4/Nogo-A: reticulon 4; RTN4R/NgR1: reticulon 4 receptor; S1PR2: sphingosine-1-phosphate receptor 2; SQSTM1: sequestosome 1.
Assuntos
Autofagia , Infarto da Artéria Cerebral Média , Proteínas Nogo , Receptores de Esfingosina-1-Fosfato , Animais , Humanos , Ratos , Autofagia/fisiologia , Células Endoteliais/metabolismo , Infarto da Artéria Cerebral Média/complicações , Neovascularização Patológica/metabolismo , Proteínas Nogo/metabolismo , Proteínas Nogo/farmacologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/farmacologia , Proteína Sequestossoma-1/metabolismo , Tálamo/metabolismoRESUMO
Focal cerebral infarction leads to autophagic activation, which contributes to secondary neuronal damage in the ipsilateral thalamus. Although Nogo-A deactivation enhances neuronal plasticity, its role in autophagic activation in the thalamus after ischemic stroke remains unclear. This study aimed to investigate the potential roles of Nogo-A/Nogo-66 receptor 1 (NgR1) in autophagic activation in the ipsilateral thalamus after cerebral infarction. Focal neocortical infarction was established using the middle cerebral artery occlusion (MCAO) method. Secondary damage in the ipsilateral thalamus was assessed by Nissl staining and immunostaining. The expression of Nogo-A, NgR1, Rho-A and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) as well as autophagic flux were evaluated by immunofluorescence and immunoblotting. The roles of Nogo-A-NgR1 signaling in autophagic activation were determined by intraventricular delivery of an NgR1 antagonist peptide, NEP1-40, at 24â¯h after MCAO. The results showed that Nogo-A and NgR1 overexpression temporally coincided with marked increases in the levels of Beclin1, LC3-II and sequestosome 1 (SQSTM1)/p62 in the ipsilateral thalamus at seven and fourteen days after MCAO. In contrast, NEP1-40 treatment significantly reduced the expression of Rho-A and ROCK1 which was accompanied by marked reductions of LC3-II conversion as well as the levels of Beclin1 and SQSTM1/p62. Furthermore, NEP1-40 treatment significantly reduced neuronal loss and gliosis in the ipsilateral thalamus, and accelerated somatosensory recovery at the observed time-points after MCAO. These results suggest that blockade of Nogo-A-NgR1 signaling inhibits autophagic activation, attenuates secondary neuronal damage in the ipsilateral thalamus, and promotes functional recovery after focal cerebral cortical infarction.
Assuntos
Neurônios , Tálamo , Animais , Infarto da Artéria Cerebral Média/tratamento farmacológico , Proteínas Nogo , Receptor Nogo 1 , Ratos , Ratos Sprague-DawleyRESUMO
Background. Subcortical infarcts can result in verbal memory impairment, but the potential underlying mechanisms remain unknown. Objective. We investigated the spatiotemporal deterioration patterns of brain structures in patients with subcortical infarction and identified the regions that contributed to verbal memory impairment. Methods. Cognitive assessment and structural magnetic resonance imaging were performed 1, 4, and 12 weeks after stroke onset in 28 left-hemisphere and 22 right-hemisphere stroke patients with subcortical infarction. Whole-brain volumetric analysis combined with a further-refined shape analysis was conducted to analyze longitudinal morphometric changes in brain structures and their relationship to verbal memory performance. Results. Between weeks 1 and 12, significant volume decreases in the ipsilesional basal ganglia, inferior white matter, and thalamus were found in the left-hemisphere stroke group. Among those 3 structures, only the change rate of the thalamus volume was significantly correlated with that in immediate recall. For the right-hemisphere stroke group, only the ipsilesional basal ganglia survived the week 1 to week 12 group comparison, but its change rate was not significantly correlated with the verbal memory change rate. Shape analysis of the thalamus revealed atrophies of the ipsilesional thalamic subregions connected to the prefrontal, temporal, and premotor cortices in the left-hemisphere stroke group and positive correlations between the rates of those atrophies and the change rate in immediate recall. Conclusions. Secondary damage to the thalamus, especially to the left subregions connected to specific cortices, may be associated with early verbal memory impairment following an acute subcortical infarct.
Assuntos
Infarto Cerebral , Transtornos da Memória , Memória de Curto Prazo/fisiologia , Rememoração Mental/fisiologia , Tálamo/patologia , Aprendizagem Verbal/fisiologia , Adulto , Idoso , Atrofia/patologia , Gânglios da Base/diagnóstico por imagem , Gânglios da Base/patologia , Infarto Cerebral/complicações , Infarto Cerebral/diagnóstico por imagem , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Feminino , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Pessoa de Meia-Idade , Tálamo/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Substância Branca/patologia , Adulto JovemRESUMO
Cerebral infarction causes secondary neurodegeneration and angiogenesis in thalamus, which impacts functional recovery after stroke. Here, we hypothesize that activation of ephrinB2 could stimulate angiogenesis and restore the secondary neurodegeneration in thalamus after cerebral infarction. Focal cerebral infarction was induced by middle cerebral artery occlusion (MCAO). Secondary damage, angiogenesis, amyloid-ß (Aß) deposits, levels of ephrinB2 and receptor for advanced glycation end product (RAGE) in the ipsilateral thalamus were determined by immunofluorescence and immunoblot. The contribution of ephrinB2 to angiogenesis was determined by siRNA-mediated knockdown of ephrinB2 and pharmacological activation of ephrinB2. The results showed that formation of new vessels and ephrinB2 expression was markedly increased in the ipsilateral thalamus at seven days after MCAO. EphrinB2 knockdown markedly suppressed angiogenesis coinciding with increased Aß accumulation, neuronal loss and gliosis in the ipsilateral thalamus. In contrast, clustered EphB2-Fc significantly enhanced angiogenesis, alleviated Aß accumulation and the secondary thalamic damage, which was accompanied by accelerated function recovery. Additionally, activation of ephrinB2 significantly reduced RAGE levels in the ipsilateral thalamus. Our findings suggest that activation of ephrinB2 promotes angiogenesis, ameliorates Aß accumulation and the secondary thalamic damage after cerebral infarction. Additionally, RAGE might be involved in Aß clearance by activating ephrinB2 in the thalamus.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Efrina-B2/metabolismo , Hipertensão/complicações , Infarto da Artéria Cerebral Média/complicações , Neovascularização Patológica/complicações , Animais , Hipertensão/metabolismo , Hipertensão/patologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ratos , Ratos Sprague-Dawley , Tálamo/metabolismo , Tálamo/patologiaRESUMO
Focal cerebral cortical infarction causes secondary neurodegeneration in the remote regions, such as the ventroposterior nucleus of the thalamus. Retrograde degeneration of thalamocortical fibers is considered as the principle mechanism, but the exact molecular events remain to be elucidated. This study aimed to investigate whether unfolded protein response (UPR) is activated in thalamic neurons following distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats. Immunostaining and immunoblotting were performed to evaluate the expression of Grp78 and its downstream effectors in the thalamus at 3, 7 and 14 days after MCAO. Secondary thalamic degeneration was assessed with Nissl staining and NeuN immunostaining. Neuronal death was not apparent at 3 days post-ischaemia but was evident in the thalamus at 7 and 14 days after MCAO. Grp78 level was reduced in the ipsilateral thalamus at 3 and 7 days after MCAO. In parallel, phosphorylated eIF2α and ATF4 levels were elevated, indicating the activation of UPR. In contrast, ATF6α and CHOP levels were not changed. These results suggest that UPR is activated before neuronal death in the ipsilateral thalamus after MCAO and may represent a key early event in the secondary thalamic degeneration.
Assuntos
Infarto Cerebral/metabolismo , Hipertensão/metabolismo , Tálamo/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Animais , Infarto Cerebral/patologia , Hipertensão/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Tálamo/patologiaRESUMO
Previous studies have demonstrated that both oxidative stress and autophagy play important roles in secondary neuronal degeneration in the ipsilateral thalamus after distal middle cerebral artery occlusion (MCAO). This study aimed to investigate whether oxidative stress is associated with autophagy activation within the ipsilateral thalamus after distal MCAO. Sixty stroke-prone renovascular hypertensive rats were subjected to distal MCAO or sham operation, and were killed at 14 days after MCAO. Mn-SOD, LC3-II, Beclin-1 and p62 expression were evaluated by immunostaining and immunoblotting. Secondary damage in the thalamus was assessed with Nissl staining and immunostaining. The association of oxidative stress with autophagy activation was investigated by the antioxidant, ebselen. We found that treatment with ebselen at 24h after MCAO significantly reduced the expression of Mn-SOD in the ipsilateral thalamus at 14 days following focal cerebral infarction. In parallel, it prevented the elevation of LC3-II and Beclin-1, and the reduction of p62. Furthermore, ebselen attenuated the neuronal loss and gliosis in the ipsilateral thalamus. These results suggested that ebselen reduced oxidative stress, autophagy activation and secondary damage in the ipsilateral thalamus following MCAO. There are associations between oxidative stress, autophagy activation and secondary damage in the thalamus after MCAO.
Assuntos
Antioxidantes/farmacologia , Azóis/farmacologia , Morte Celular/efeitos dos fármacos , Infarto Cerebral/tratamento farmacológico , Compostos Organosselênicos/farmacologia , Tálamo/efeitos dos fármacos , Animais , Antioxidantes/uso terapêutico , Autofagia/efeitos dos fármacos , Azóis/uso terapêutico , Infarto Cerebral/etiologia , Infarto Cerebral/metabolismo , Infarto Cerebral/patologia , Gliose/patologia , Infarto da Artéria Cerebral Média/complicações , Isoindóis , Masculino , Neurônios/efeitos dos fármacos , Neurônios/patologia , Compostos Organosselênicos/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Superóxido Dismutase/metabolismo , Tálamo/metabolismo , Tálamo/patologiaRESUMO
PURPOSE: Tongxinluo, a well-known traditional Chinese medicine complex, has been widely used for the treatment of cerebrovascular diseases in China. The present study was to explore whether treatment with tongxinluo could improve neurological function and alleviate secondary damage in the ipsilateral thalamus after focal cortical infarction in hypertensive rats. METHODS: Tongxinluo was given through oral gavage starting 24 h after distal middle cerebral artery occlusion (MCAO). Neurological function was assessed and then rats were sacrificed 7 and 14 days after MCAO. Brains were harvested for examining infarction volume, Nissl staining and immunofluorescence analysis. RESULTS: Compared with vehicle treatment, tongxinluo remarkably improved neurological function without reducing infarction volume, attenuated neuronal loss and astrocyte activation in the ipsilateral thalamus 7 and 14 days after MCAO (all p < 0.05). Also, tongxinluo markedly increased the number of BrdU+/nestin+ and BrdU+/NeuN+ cells 14 days after MCAO. Moreover, vascular density, the number of BrdU+ vascular endothelial cells, and vascular perimeter in the ipsilateral thalamus were markedly increased in the tongxinluo group relative to that of the vehicle group (all p < 0.05). CONCLUSION: Administration of tongxinluo 24 h after cortical infarction may promote neurogenesis and angiogenesis in the ipsilateral thalamus and improves neurological function after cortical infarction in rats.
Assuntos
Infarto Cerebral/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Hipertensão , Neovascularização Patológica/tratamento farmacológico , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Tálamo/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Infarto Cerebral/etiologia , Modelos Animais de Doenças , Lateralidade Funcional/efeitos dos fármacos , Hipertensão/patologia , Hipertensão/fisiopatologia , Infarto da Artéria Cerebral Média/complicações , Masculino , Neovascularização Patológica/etiologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Tálamo/patologia , Fatores de TempoRESUMO
Focal cerebral infarction causes amyloid-ß (Aß) deposits and secondary thalamic neuronal degeneration. The present study aimed to determine the protective effects of Cerebrolysin on Aß deposits and secondary neuronal damage in thalamus after cerebral infarction. At 24h after distal middle cerebral artery occlusion (MCAO), Cerebrolysin (5 ml/kg) or saline as control was once daily administered for consecutive 13 days by intraperitoneal injection. Sensory function and secondary thalamic damage were assessed with adhesive-removal test, Nissl staining and immunofluorescence at 14 days after MCAO. Aß deposits, activity of ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1), apoptosis and autophagy were determined by TUNEL staining, immunofluorescence and immunoblot. The results showed that Cerebrolysin significantly improved sensory deficit compared to controls (p<0.05). Aß deposits and BACE1 were obviously reduced by Cerebrolysin, which was accompanied by decreases in neuronal loss and astroglial activation compared to controls (all p < 0.05). Coincidently, Cerebrolysin markedly inhibited cleaved caspase-3, conversion of LC3-II, downregulation of Bcl-2 and upregulation of Bax in the ipsilateral thalamus compared to controls (all p<0.05). These findings suggest that Cerebrolysin reduces Aß deposits, apoptosis and autophagy in the ipsilateral thalamus, which may be associated with amelioration of secondary thalamic damage and functional recovery after cerebral infarction.
Assuntos
Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose/efeitos dos fármacos , Ácido Aspártico Endopeptidases/metabolismo , Córtex Cerebral/patologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , Recuperação de Função Fisiológica/efeitos dos fármacos , Tálamo/patologia , Aminoácidos/farmacologia , Aminoácidos/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Modelos Animais de Doenças , Lateralidade Funcional , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Endogâmicos SHR , Tálamo/efeitos dos fármacosRESUMO
Abnormal diffusion tensor imaging (DTI) results have been observed in the periventricular white matter in patients with ischemic leukoaraiosis (ILA). However, the underlying pathological changes and their relationship to cognitive impairments are obscure. In addition, damage in the thalamus, an important structure in the executive function network, has been suggested in ILA, but is poorly understood. Twenty patients with ILA and 20 healthy volunteers with similar ages and educational histories underwent DTI, magnetic resonance spectroscopy (MRS) and a neuropsychological assessment. In patients with ILA, we observed an increased mean diffusivity (MD) and decreased levels of N-acetylaspartate (NAA)/creatine (Cr) in the anterior and posterior periventricular region and the thalamus, as well as decreased fractional anisotropy (FA) in the anterior and posterior periventricular regions. MD and NAA/Cr levels in the anterior and posterior periventricular white matter and NAA/Cr levels in the thalamus were correlated with executive function. DTI and MRS abnormalities were consistent with axonal and/or neuronal loss and dysfunction in the anterior and posterior periventricular white matter and the thalamus. This study demonstrates that DTI and MRS techniques can be used to investigate pathological changes in the anterior and posterior periventricular white matter and the thalamus; these changes may be correlated with executive functional changes in patients with ILA.
Assuntos
Isquemia Encefálica/patologia , Transtornos Cognitivos/patologia , Leucoaraiose/patologia , Fibras Nervosas Mielinizadas/patologia , Tálamo/patologia , Idoso , Isquemia Encefálica/complicações , Isquemia Encefálica/fisiopatologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Feminino , Humanos , Leucoaraiose/complicações , Leucoaraiose/fisiopatologia , Masculino , Pessoa de Meia-Idade , Tálamo/fisiopatologiaRESUMO
Focal cerebral cortical infarction after distal middle cerebral artery occlusion causes ß-amyloid deposition and secondary neuronal degeneration in the ipsilateral ventroposterior nucleus of the thalamus. Several studies suggest that autophagy is an active pathway for ß-amyloid peptide generation. This study aimed to investigate the role of autophagy in thalamic ß-amyloid deposition and neuronal degeneration after cerebral cortical infarction in hypertensive rats. At 7 and 14days after middle cerebral artery occlusion, neuronal death and ß-amyloid deposits were evident in the ipsilateral ventroposterior nucleus, and the activity of ß-site amyloid precursor protein (APP)-cleaving enzyme 1, required for ß-amyloid peptide generation, was elevated in the thalamus. In correlation, both the number of cells showing punctate microtubule-associated protein 1A light chain 3 fluorescence and levels of light chain 3-II protein, an autophagosome marker, were markedly increased. Notably, most of the cells that over-expressed ß-site APP-cleaving enzyme 1 displayed punctate light chain 3 staining. Furthermore, the inhibition of autophagy with 3-methyladenine significantly reduced the thalamic neuronal damage, ß-amyloid deposits, and ß-site APP-cleaving enzyme 1 activity. These results suggest that autophagosomes accumulate within thalamic cells after cerebral cortical infarction, which is associated with thalamic ß-amyloid deposition and secondary neuronal degeneration via elevation of ß-site APP-cleaving enzyme 1 level.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Autofagia/fisiologia , Infarto Cerebral/patologia , Hipertensão/patologia , Fagossomos/patologia , Placa Amiloide/patologia , Tálamo/patologia , Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/fisiologia , Peptídeos beta-Amiloides/fisiologia , Animais , Ácido Aspártico Endopeptidases/biossíntese , Ácido Aspártico Endopeptidases/fisiologia , Infarto Cerebral/enzimologia , Infarto Cerebral/metabolismo , Hipertensão/enzimologia , Hipertensão/metabolismo , Masculino , Degeneração Neural/enzimologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Fagossomos/enzimologia , Fagossomos/metabolismo , Placa Amiloide/enzimologia , Placa Amiloide/metabolismo , Ratos , Ratos Sprague-Dawley , Tálamo/enzimologia , Tálamo/metabolismoRESUMO
Cerebral infarction can cause secondary degeneration of thalamus and delay functional recovery. However, the mechanisms underlying secondary degeneration are unclear. The present study aimed to determine the occurrence and contribution of autophagy to the thalamic degeneration after cerebral infarction. Focal cerebral infarction was induced by distal middle cerebral artery occlusion (MCAO). Autophagic activation, Beclin 1 expression and amyloid ß (Aß) deposits were determined by immunofluorescence, immunoblot and electron microscopy. Secondary damage to thalamus was assessed with Nissl staining and immunofluorescence analysis. Apoptosis was determined using TUNEL staining. The contribution of autophagy to the secondary damage was evaluated by shRNA-mediated downregulation of Beclin 1 and the autophagic inhibitor, 3-methyladenine (3-MA). The potential role of Aß in autophagic activation was determined with N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The results showed that the conversion of LC3-II, the formation of autophagosomes, and the levels of activated cathepsin B and Beclin 1 were significantly increased in the ipsilateral thalamus at 7 and 14 days after MCAO (p < 0.05 or 0.01). Both Beclin 1 knockdown and 3-MA treatment significantly reduced LC3-II conversion and autophagosome formation, which were accompanied by obvious decreases in neuronal loss, gliosis and apoptosis in the ipsilateral thalamus (p < 0.05 or 0.01). Additionally, DAPT treatment markedly reduced Aß deposits, which coincided with decreases in LC3-II conversion and autophagosome formation (p < 0.01). These results suggest that inhibition of autophagy by Beclin 1 knockdown can attenuate the secondary thalamic damage after focal cerebral infarction. Furthermore, Aß deposits may be involved in the activation of autophagy.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Infarto Cerebral/complicações , Técnicas de Silenciamento de Genes , Degeneração Neural/etiologia , Degeneração Neural/prevenção & controle , Tálamo/patologia , Adenina/análogos & derivados , Adenina/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1 , Infarto Cerebral/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Tálamo/efeitos dos fármacos , Tálamo/ultraestrutura , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
1. Whether damage to the blood-brain barrier (BBB) occurs in remote areas after a focal cortical lesion remains unknown. The present study investigated tight junction-related proteins and tight junction microstructure in the ipsilateral thalamus during the acute stage after middle cerebral artery occlusion (MCAO) and cortical aspiration lesion (CAL) in rats. 2. Thirty-six hypertensive and normotensive rats were subjected to MCAO or CAL; another 18 rats in each group were submitted to sham operation. Zonula Occluden (ZO)-1, occludin and albumin were detected by western blotting 12 and 24 h after surgery. Tight junction microstructure was evaluated using electron microscopy, whereas albumin location in the ipsilateral thalamus was determined using double immunostaining for albumin and occludin or albumin and neuronal nuclei (NeuN) 24 h after surgery. 3. Twenty-four hours after MCAO or CAL, occludin expression was reduced to 78.4% and 81.3%, respectively, compared with control. A reduction in ZO-1 expression in the ipsilateral thalamus (to 79%) was seen only after CAL (P < 0.05). Membrane contact at the tight junction was discontinuous in the ipsilateral thalamus in both MCAO and CAL rats. Albumin levels were 23.2% and 82.5% higher in the ipsilateral thalamus after MCAO and CAL, respectively (P < 0.05). The percentage of the albumin-positive area that coincided with the occludin-positive area in the MCAO and CAL groups was 76.8% and 64.6%, respectively, indicating that albumin was mainly localized around the microvessels. 4. The results of the present study suggest that tight junction integrity decreases during the acute stage in the ipsilateral thalamus after MCAO and CAL in rats.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Córtex Cerebral/fisiopatologia , Infarto Cerebral/fisiopatologia , Infarto da Artéria Cerebral Média/fisiopatologia , Tálamo/fisiopatologia , Junções Íntimas/patologia , Albuminas/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Córtex Cerebral/metabolismo , Infarto Cerebral/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microvasos/metabolismo , Microvasos/fisiopatologia , Ocludina , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Tálamo/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1RESUMO
BACKGROUND: Traditional magnetic resonance (MR) imaging can identify abnormal changes in ipsilateral thalamus in patients with unilateral middle cerebral artery (MCA) infarcts. However, it is difficult to demonstrate these early changes quantitatively. Diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy (MRS) are potentially sensitive and quantitative methods of detection in examining changes of tissue microstructure and metabolism. In this study, We used both DTI and MRS to examine possible secondary damage of thalamus in patients with corona radiata infarction. METHODS: Twelve patients with unilateral corona radiata infarction underwent MR imaging including DTI and MRS at one week (W1), four weeks (W4), and twelve weeks (W12) after onset of stroke. Twelve age-matched controls were imaged. Mean diffusivity (MD), fractional anisotropy (FA), N-acetylaspartate (NAA), choline(Cho), and creatine(Cr) were measured in thalami. RESULTS: T1-weighted fluid attenuation inversion recovery (FLAIR), T2-weighted, and T2-FLAIR imaging showed an infarct at unilateral corona radiate but no other lesion in each patient brain. In patients, MD was significantly increased at W12, compared to W1 and W4 (all P< 0.05). NAA was significantly decreased at W4 compared to W1, and at W12 compared to W4 (all P< 0.05) in the ipsilateral thalamus. There was no significant change in FA, Cho, or Cr in the ipsilateral thalamus from W1 to W12. Spearman's rank correlation analysis revealed a significant negative correlation between MD and the peak area of NAA, Cho, and Cr at W1, W4, and W12 and a significant positive correlation of FA with NAA at W1. CONCLUSIONS: These findings indicate that DTI and MRS can detect the early changes indicating secondary damage in the ipsilateral thalamus after unilateral corona radiata infarction. MRS may reveal the progressive course of damage in the ipsilateral thalamus over time.
Assuntos
Infarto Cerebral/complicações , Infarto Cerebral/diagnóstico , Imagem de Difusão por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Tálamo/irrigação sanguínea , Tálamo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Abnormal ß-amyloid (Aß) deposits in the thalamus have been reported after cerebral cortical infarction. In this study, we investigated the association of Aß deposits, with the secondary thalamic damage after focal cortical infarction in rats. Thirty-six stroke-prone renovascular hypertensive rats were subjected to distal middle cerebral artery occlusion (MCAO) and then randomly divided into MCAO, vehicle, and N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) groups and 12 sham-operated rats as control. The DAPT was administered orally at 72 hours after MCAO. Seven days after MCAO, sensory function, neuron loss, and glial activation and proliferation were evaluated using adhesive removal test, Nissl staining, and immunostaining, respectively. Thalamic Aß accumulation was evaluated using immunostaining and enzyme-linked immunosorbent assay (ELISA). Compared with vehicle group, the ipsilateral thalamic Aß, neuronal loss, glial activation and proliferation, and the mean time to remove the stimulus from right forepaw significantly decreased in DAPT group. The mean time to remove the stimulus from the right forepaw and thalamic Aß burden were both negatively correlated with the number of thalamic neurons. These findings suggest that Aß deposits are associated with the secondary thalamic damage. Reduction of thalamic Aß by γ-secretase inhibitor may attenuate the secondary damage and improve sensory function after cerebral cortical infarction.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Infarto Cerebral/patologia , Inibidores de Proteases/farmacologia , Sensação/fisiologia , Tálamo/patologia , Peptídeos beta-Amiloides/análise , Animais , Proliferação de Células , Infarto Cerebral/psicologia , Corantes , Dipeptídeos , Ensaio de Imunoadsorção Enzimática , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Infarto da Artéria Cerebral Média/psicologia , Neuroglia/fisiologia , Neurônios/patologia , Neurônios/fisiologia , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Tálamo/químicaRESUMO
OBJECTIVE: To investigate the damage within the ventroposterior nucleus (VPN) of the thalamus after focal cortical infarction and its mechanism, and explore the effect of ebselen on the oxidative damage after cerebral cortex infarction in hypertensive rats. METHODS: Middle cerebral artery occlusion (MCAO) was induced in stroke-prone renovascular hypertensive rats (RHRSP), and the rats were divided into four groups by table of random number: sham operation group, model group, vehicle group and ebselen group, each group consisted of 8 rats. In animals subjected to sham surgery the middle cerebral artery was exposed only. Ebselen (5 ml/kg) or vehicle (a mixed solvent consisting of 0.5% carboxymethyl cellulose and 0.02% Tween 20, 5 ml/kg) was given by gastric gavage starting 24 hours after cerebral cortical infarction. Two weeks after the MCAO, the rats were sacrificed, and VPN from each group was sectioned and stained with hematoxylin-eosin (HE), and apurinic/apyrimidinic endonuclease (APE) and Escherichia coli MutY DNA glycosylase (MYH) were determined by immunohistochemistry. RESULTS: HE staining showed that ebselen ameliorated the VPN damage induced by ischemia. Immunohistochemical imaging analysis revealed a distinct nuclear staining of APE and nuclear and cytoplasm distribution of MYH in the entire region of the VPN. Compared with sham operation group, the number of APE and MYH positive cells decreased in model group and vehicle group (APE: 57.0±14.7, 49.4±12.5 vs. 101.0±13.6, MYH: 15.0±4.7, 10.4±2.5 vs. 56.0±13.2, all P<0.05). Compared with model group and vehicle group, the number of APE and MYH positive cells increased significantly in ebselen group (APE: 72.2±7.6 vs. 57.0±14.7, 49.4±12.5, MYH: 32.2±7.6 vs. 15.0±4.7, 10.4±2.5, all P<0.05); the difference of the number of APE and MYH positive cells between model group and vehicle group showed no statistical significance. CONCLUSION: After 2 weeks of MCAO, there is a marked decrease of APE and MYH in VPN; ebselen can obviously increase the level of APE and MYH, and ebselen may protect the VPN of the thalamus from damage after focal cortical infarction in rats.
Assuntos
Córtex Cerebral/metabolismo , Infarto Cerebral/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Tálamo/metabolismo , Animais , Córtex Cerebral/patologia , Infarto Cerebral/patologia , Hipertensão , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Neurogenesis and angiogenesis in the subventricular zone and peri-infarct region have been confirmed. However, newly formed neuronal cells and blood vessels that appear in the nonischemic ipsilateral ventroposterior nucleus (VPN) of the thalamus with secondary damage after stroke has not been previously studied. Twenty-four stroke-prone renovascular hypertensive rats were subjected to distal right middle cerebral artery occlusion (MCAO) or sham operation. 5'-Bromo-2'-deoxyuridine (BrdU) was used to label cell proliferation. Rats were killed at 7 or 14 days after the operation. Neuronal nuclei (NeuN), OX-42, BrdU, nestin, laminin(+), BrdU(+)/nestin(+), BrdU(+)/NeuN(+), nestin(+)/GFAP(+)(glial fibrillary acidic protein), and BrdU(+)/laminin(+) immunoreactive cells were detected within the ipsilateral VPN. The primary infarction was confined to the right somatosensory cortex. Within the ipsilateral VPN of the ischemic rats, the number of NeuN(+) neurons decreased, the OX-42(+) microglia cells were activated, and BrdU(+) and nestin(+) cells were detected at day 7 after MCAO and increased in number at day 14. Moreover, BrdU(+)/nestin(+) cells and BrdU(+)/NeuN(+) cells were detected at day 14 after MCAO. In addition, the ischemic rats showed a significant increase in vascular density in the ipsilateral VPN compared with the sham-operated rats. These results suggest that secondary damage with neurogenesis and angiogenesis of the ipsilateral VPN of the thalamus occurs after focal cortical infarction.
Assuntos
Córtex Cerebral , Infarto Cerebral , Hipertensão , Neovascularização Fisiológica/fisiologia , Neurogênese/fisiologia , Tálamo , Animais , Antimetabólitos/metabolismo , Bromodesoxiuridina/metabolismo , Proliferação de Células , Córtex Cerebral/anatomia & histologia , Córtex Cerebral/patologia , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Hipertensão/patologia , Hipertensão/fisiopatologia , Infarto da Artéria Cerebral Média , Proteínas de Filamentos Intermediários/metabolismo , Laminina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Ratos , Ratos Sprague-Dawley , Tálamo/anatomia & histologia , Tálamo/patologia , Tálamo/fisiologiaRESUMO
Oxidative DNA damage has been proposed to be a major contributor to focal cerebral ischemic injury. However, little is known about the role of oxidative DNA damage in remote damage secondary to the primary infarction. In the present study, we investigated oxidative damage within the ventroposterior nucleus (VPN) after distal middle cerebral artery occlusion (MCAO) in hypertensive rats. We also examined the possible protective effect of ebselen, one glutathione peroxidase mimic, on delayed degeneration in the VPN after distal MCAO. Neuronal damage in the ipsilateral VPN was examined by Nissl staining. Oxidative DNA damage and base repair enzyme activity were assessed by analyzing immunoreactivity of 8-hydroxy-2'-deoxyguanosine (8-ohdG) and 8-oxoguanine DNA glycosylase (OGG1), respectively. The number of intact neurons in the ipsilateral VPN decreased by 52% compared to the contralateral side in ischemia group 2 weeks after distal cerebral cortical infarction. The immunoreactivity of 8-ohdG significantly increased while OGG1 immunoreactivity significantly decreased in the ipsilateral VPN 2 weeks after distal cortical infarction (all p<0.01). Compared with vehicle treatment, ebselen significantly attenuated the neuron loss, ameliorated ischemia-induced increase in 8-ohdG level as well as decrease in OGG1 level within the ipsilateral VPN (all p<0.01). OGG1 was further demonstrated to mainly express in neurons. These findings strongly suggest that oxidative DNA damage may be involved in the delayed neuronal death in the VPN region following distal MCAO. Furthermore, ebselen protects against the delayed damage in the VPN when given at 24 h following distal MCAO.
Assuntos
Azóis/farmacologia , Infarto Cerebral/enzimologia , Reparo do DNA/efeitos dos fármacos , Hipertensão Renal/complicações , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Tálamo/efeitos dos fármacos , Análise de Variância , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Infarto Cerebral/complicações , Infarto Cerebral/patologia , Dano ao DNA/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Hipertensão Renal/enzimologia , Hipertensão Renal/patologia , Imuno-Histoquímica , Isoindóis , Masculino , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Tálamo/metabolismo , Tálamo/patologiaRESUMO
We investigate whether Nogo-A is involved in the secondary axonal degeneration in the thalamus after distal middle cerebral artery occlusion (MCAO) in stroke-prone renovascular hypertensive rats (RHRSP). The expression of Nogo-A in ipsilateral ventroposterior nucleus (VPN) of the thalamus in RHRSP was observed at 1, 2 and 4 weeks after distal MCAO. In addition, intracerebroventricular infusion of NEP1-40, a Nogo-66 receptor (NgR) antagonist peptide, was administered starting 24 h after MCAO and continued for 1, 2 and 4 weeks, respectively. Axonal damage and regeneration were evaluated by analysis of the immunoreactivity (IR) of amyloid betaA4 precursor protein (APP), growth associated protein 43 (GAP-43) and microtubule associated protein 2 (MAP-2) in ipsilateral VPN of the thalamus at 1, 2 and 4 weeks after distal MCAO. Following ischemia, the expression of Nogo-A in oligodendrocytes increased persistently and its localization became redistributed around damaged axons and dendrites. Administration of NEP1-40 downregulated the expression of Nogo-A, reduced axonal injury and enhanced axonal regeneration. Our data suggest that Nogo-A is involved in secondary axonal degeneration and that inhibition of Nogo-A can reduce neuronal damage in the thalamus after distal MCAO.