RESUMO
CRISPR-Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1-6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe 'verification of in vivo off-targets' (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR-Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR-Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Edição de Genes/normas , Genoma/genética , Mutação , Especificidade por Substrato/genética , Animais , Proteínas Associadas a CRISPR/genética , Feminino , Humanos , Mutação INDEL , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/genética , Transgenes/genéticaRESUMO
BACKGROUND: Plasma concentration of low-density lipoprotein (LDL) cholesterol is a well-established risk factor for cardiovascular disease. Inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9), which regulates cholesterol homeostasis, has recently emerged as an approach to reduce cholesterol levels. The development of humanized animal models is an important step to validate and study human drug targets, and use of genome and base editing has been proposed as a mean to target disease alleles. RESULTS: To address the lack of validated models to test the safety and efficacy of techniques to target human PCSK9, we generated a liver-specific human PCSK9 knock-in mouse model (hPCSK9-KI). We showed that plasma concentrations of total cholesterol were higher in hPCSK9-KI than in wildtype mice and increased with age. Treatment with evolocumab, a monoclonal antibody that targets human PCSK9, reduced cholesterol levels in hPCSK9-KI but not in wildtype mice, showing that the hypercholesterolemic phenotype was driven by overexpression of human PCSK9. CRISPR-Cas9-mediated genome editing of human PCSK9 reduced plasma levels of human and not mouse PCSK9, and in parallel reduced plasma concentrations of total cholesterol; genome editing of mouse Pcsk9 did not reduce cholesterol levels. Base editing using a guide RNA that targeted human and mouse PCSK9 reduced plasma levels of human and mouse PCSK9 and total cholesterol. In our mouse model, base editing was more precise than genome editing, and no off-target editing nor chromosomal translocations were identified. CONCLUSIONS: Here, we describe a humanized mouse model with liver-specific expression of human PCSK9 and a human-like hypercholesterolemia phenotype, and demonstrate that this mouse can be used to evaluate antibody and gene editing-based (genome and base editing) therapies to modulate the expression of human PCSK9 and reduce cholesterol levels. We predict that this mouse model will be used in the future to understand the efficacy and safety of novel therapeutic approaches for hypercholesterolemia.
Assuntos
Colesterol/sangue , Hipercolesterolemia/genética , Fígado/metabolismo , Pró-Proteína Convertase 9/genética , Animais , Modelos Animais de Doenças , Edição de Genes , Genoma , Humanos , Hipercolesterolemia/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: The proteasome system plays an important role in synaptic plasticity. Induction and maintenance of long term potentiation is directly dependent on selective targeting of proteins for proteasomal degradation. The 20S proteasome activator PA28αß activates hydrolysis of small nonubiquitinated peptides and possesses protective functions upon oxidative stress and proteinopathy. The effect of PA28αß activity on behavior and memory function is, however, not known. We generated a mouse model that overexpresses PA28α (PA28αOE) to understand PA28αß function during healthy adult homeostasis via assessment of physiological and behavioral profiles, focusing on female mice. RESULTS: PA28α and PA28ß protein levels were markedly increased in all PA28αOE tissues analyzed. PA28αOE displayed reduced depressive-like behavior in the forced swim test and improved memory/learning function assessed by intersession habituation in activity box and shuttle box passive avoidance test, with no significant differences in anxiety or general locomotor activity. Nor were there any differences found when compared to WT for body composition or immuno-profile. The cognitive effects of PA28αOE were female specific, but could not be explained by alterations in estrogen serum levels or hippocampal regulation of estrogen receptor ß. Further, there were no differences in hippocampal protein expression of neuronal or synaptic markers between PA28αOE and WT. Biochemical analysis of hippocampal extracts demonstrated that PA28α overexpression did not increase PA28-20S peptidase activity or decrease K48-polyubiquitin levels. Instead, PA28αOE exhibited elevated efficiency in preventing aggregation in the hippocampus. CONCLUSIONS: This study reveals, for the first time, a connection between PA28αß and neuronal function. We found that PA28α overexpressing female mice displayed reduced depressive-like behavior and enhanced learning and memory. Since the positive effects of PA28α overexpression arose without an activation of 20S proteasome capacity, they are likely independent of PA28αß's role as a 20S proteasome activator and instead depend on a recognized chaperone-like function. These findings suggest that proteostasis in synaptic plasticity is more diverse than previously reported, and demonstrates a novel function of PA28αß in the brain.
Assuntos
Aprendizagem/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Depressão/metabolismo , Receptor beta de Estrogênio/metabolismo , Estrogênios/sangue , Feminino , Expressão Gênica , Técnicas de Introdução de Genes , Hipocampo/metabolismo , Homeostase/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
BACKGROUND AND PURPOSE: Cortical ischemia induces neural progenitor cell migration toward the injury site; however, whether these cells are capable of maintaining the migratory response for a longer period after injury remains uncertain. METHODS: We analyzed progenitor migration up to 1 year after induction of photothrombotic stroke to the mouse neocortex. Migrating progenitors identified as doublecortin positive cells (DCX+) were assessed using the immunohistochemistry and immunofluorescence. The thymidine analogues chlorodeoxyuridine and iododeoxyuridine were used to birth-date the progenitor cells. RESULTS: In the striatum, we detected elevated numbers of DCX+ cells up to 6 weeks postlesion. In the corpus callosum and the peri-infarct cortex (Ctx), DCX+ cell numbers were increased up to 1 year. The orientation of the migrating progenitors was mostly aligned with the corpus callosum fiber tract at all time points; however, in the Ctx, they aligned parallel to the infarct border. The injured cortex continuously receives new progenitors up to 1 year after lesion. Cells born after lesion did not become mature neurons, although a portion of the migrating progenitors showed initial signs of differentiation into neurons. CONCLUSIONS: Neural progenitors might have a role in brain plasticity after cortical stroke, especially considering the prolonged window of migratory responses of up to 1 year after stroke lesion.
Assuntos
Isquemia Encefálica/patologia , Movimento Celular/fisiologia , Corpo Caloso/patologia , Corpo Estriado/patologia , Células-Tronco Neurais/patologia , Animais , Isquemia Encefálica/metabolismo , Corpo Caloso/metabolismo , Corpo Estriado/metabolismo , Proteína Duplacortina , Feminino , Camundongos , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson's disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks. 500 mg of frozen putamen (affected preclinically) and frontal cortex (affected late) was homogenized, fractionated and α-synuclein levels evaluated using specific antibodies (syn-1, BD Transduction Laboratories; S129P phospho-α-synuclein, Elan Pharmaceuticals) and quantitative western blotting. Statistical analyses assessed the relationship between the different forms of α-synuclein, compared levels between groups, and determined any changes over the disease course. Soluble S129P was detected in controls with higher levels in putamen compared with frontal cortex. In contrast, insoluble α-synuclein occurred in Parkinson's disease with a significant increase in soluble and lipid-associated S129P, and a decrease in soluble frontal α-synuclein over the disease course. Increasing soluble S129P in the putamen correlated with increasing S129P in other fractions and regions. These data show that soluble non-phosphorylated α-synuclein decreases over the course of Parkinson's disease, becoming increasingly phosphorylated and insoluble. The finding that S129P α-synuclein normally occurs in vulnerable brain regions, and in Parkinson's disease has the strongest relationships to the pathogenic forms of α-synuclein in other brain regions, suggests a propagating role for putamenal phospho-α-synuclein in disease pathogenesis.
Assuntos
Lobo Frontal/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Putamen/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Progressão da Doença , Feminino , Lobo Frontal/patologia , Humanos , Masculino , Doença de Parkinson/classificação , Fosforilação/fisiologia , Putamen/patologia , Serina/metabolismo , Estatísticas não ParamétricasRESUMO
Prokaryotic restriction enzymes, recombinases and Cas proteins are powerful DNA engineering and genome editing tools. However, in many primary cell types, the efficiency of genome editing remains low, impeding the development of gene- and cell-based therapeutic applications. A safe strategy for robust and efficient enrichment of precisely genetically engineered cells is urgently required. Here, we screen for mutations in the receptor for Diphtheria Toxin (DT) which protect human cells from DT. Selection for cells with an edited DT receptor variant enriches for simultaneously introduced, precisely targeted gene modifications at a second independent locus, such as nucleotide substitutions and DNA insertions. Our method enables the rapid generation of a homogenous cell population with bi-allelic integration of a DNA cassette at the selection locus, without clonal isolation. Toxin-based selection works in both cancer-transformed and non-transformed cells, including human induced pluripotent stem cells and human primary T-lymphocytes, as well as it is applicable also in vivo, in mice with humanized liver. This work represents a flexible, precise, and efficient selection strategy to engineer cells using CRISPR-Cas and base editing systems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Mutação , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Células HCT116 , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , CamundongosRESUMO
H. Bart van der Worp and colleagues discuss the controversies and possibilities of translating the results of animal experiments into human clinical trials.
Assuntos
Modelos Animais de Doenças , Pesquisa Translacional Biomédica , Animais , Humanos , Viés de Publicação , Reprodutibilidade dos TestesRESUMO
Cranial irradiation (IR) is commonly used to treat primary brain tumors and metastatic diseases. However, cranial IR-treated patients often develop vascular abnormalities later in life that increase their risk for cerebral ischemia. Studies in rodents have demonstrated that IR impairs maintenance of the neural stem/precursor cell (NSPC) pool and depletes neurogenesis. We and others have previously shown that stroke triggers NSPC proliferation in the subventricular zone and migration towards the stroke-injured neocortex. Whether this response is sustained in the irradiated brain remains unknown. Here, we demonstrate that cranial IR in mice at an early postnatal age significantly reduced the number to neuronal progenitors responding to cortical stroke in adults. This was accompanied by a reduced number of microglia/macrophages in the peri-infarct cortex; however, the astrocytic response was not altered. Our findings indicate that IR impairs the endogenous repair capacity in the brain in response to stroke, hence pointing to another side effect of cranial radiotherapy which requires further attention.
Assuntos
Envelhecimento , Isquemia Encefálica , Córtex Cerebral , Irradiação Craniana/efeitos adversos , Células-Tronco Neurais/metabolismo , Lesões Experimentais por Radiação , Acidente Vascular Cerebral , Animais , Animais Recém-Nascidos , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Feminino , Camundongos , Células-Tronco Neurais/patologia , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologiaRESUMO
The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.
Assuntos
Sistemas CRISPR-Cas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Descoberta de Drogas/métodos , Edição de Genes/métodos , Neoplasias Pulmonares/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína 9 Associada à CRISPR/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Doxiciclina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Células HEK293 , Ensaios de Triagem em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia de Cinetoplastídeos/genética , Recombinação Genética/efeitos dos fármacos , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacos , Transfecção/métodos , Transgenes/genéticaRESUMO
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Edição de Genes/métodos , Proteína Homóloga a MRE11/metabolismo , Análise de Sequência de DNA/métodos , Adenoviridae , Animais , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular , Imunoprecipitação da Cromatina , DNA/química , DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Proteína Homóloga a MRE11/genética , RNA Guia de CinetoplastídeosRESUMO
Liver X receptors limit cellular lipid uptake by stimulating the transcription of Inducible Degrader of the LDL Receptor (IDOL), an E3 ubiquitin ligase that targets lipoprotein receptors for degradation. The function of IDOL in systemic metabolism is incompletely understood. Here we show that loss of IDOL in mice protects against the development of diet-induced obesity and metabolic dysfunction by altering food intake and thermogenesis. Unexpectedly, analysis of tissue-specific knockout mice revealed that IDOL affects energy balance, not through its actions in peripheral metabolic tissues (liver, adipose, endothelium, intestine, skeletal muscle), but by controlling lipoprotein receptor abundance in neurons. Single-cell RNA sequencing of the hypothalamus demonstrated that IDOL deletion altered gene expression linked to control of metabolism. Finally, we identify VLDLR rather than LDLR as the primary mediator of IDOL effects on energy balance. These studies identify a role for the neuronal IDOL-VLDLR pathway in metabolic homeostasis and diet-induced obesity.
Assuntos
Metabolismo Energético/fisiologia , Neurônios/metabolismo , Receptores de LDL/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Glicemia/metabolismo , Dieta , Metabolismo Energético/genética , Hipotálamo/metabolismo , Resistência à Insulina , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Obesidade/prevenção & controle , Ubiquitina-Proteína Ligases/genéticaRESUMO
Inflammation in the CNS predominantly involves microglia and macrophages, and is believed to be a significant cause of secondary injury following trauma. This study compares the microglial and macrophage response in the rat brain and spinal cord following discrete mechanical injury to better appreciate the degree to which these cells could contribute to secondary damage in these areas. We find that, 1 week after injury, the microglial and macrophage response is significantly greater in the spinal cord compared to the brain. This is the case for injuries to both gray and white matter. In addition, we observed a greater inflammatory response in white matter compared to gray matter within both the brain and spinal cord. Because activated microglia and macrophages appear to be effectors of secondary damage, a greater degree of inflammation in the spinal cord is likely to result in more extensive secondary damage. Tissue saving strategies utilizing anti-inflammatory treatments may therefore be more useful in traumatic spinal cord than brain injury.
Assuntos
Lesões Encefálicas/imunologia , Lesões Encefálicas/patologia , Encefalite/imunologia , Encefalite/patologia , Mielite/imunologia , Mielite/patologia , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/fisiopatologia , Antígeno CD11b/análise , Antígeno CD11b/metabolismo , Modelos Animais de Doenças , Encefalite/fisiopatologia , Feminino , Gliose/imunologia , Gliose/patologia , Gliose/fisiopatologia , Macrófagos/imunologia , Macrófagos/patologia , Microglia/imunologia , Microglia/patologia , Mielite/fisiopatologia , Fibras Nervosas Mielinizadas/imunologia , Fibras Nervosas Mielinizadas/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Endogâmicos F344 , Medula Espinal/imunologia , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Fatores de TempoRESUMO
Traumatic injury to the CNS results in peri-wound sprouting without significant axonal growth beyond the lesion edge. We have previously demonstrated that dopaminergic sprouting in the injured striatum follows an increasing gradient of BDNF and GDNF expression, with sprouting ceasing at the point of maximal factor expression. Progressively more complicated associations of sprouting fibers with increasingly activated microglia and macrophages suggest these factors are localized to the cell surface. To establish whether an increased concentration of immobilized BDNF and GDNF could stimulate axonal growth beyond the lesion edge, both factors were covalently attached to 10 microm polycarbonate microspheres. These spheres were implanted into the site of striatal injury 1 week after lesioning. A profusion of axons grew from the region of the lesion edge across the surface of the spheres. Some axons traversed the entire site of injury. Ultrastructural examination demonstrated the juxtaposition of regenerating axons to the surface of implanted spheres. CSPG immunostaining demonstrated that, in animals implanted with neurotrophin-microspheres, axonal growth was induced beyond the area of maximal CSPG reactivity. Surprisingly however, CSPG production at the wound edge was greater in control animals than those implanted with neurotrophin-microspheres. Overall, we show that axonal growth can be encouraged beyond the wound edge by an elevated concentration of immobilized trophic factors. This growth occurs despite the presence of inhibitory CSPGs at the lesion edge. Axonal growth appears to be stimulated mainly via the direct effects of neurotrophins. However, there also appears to be an indirect mechanism whereby neurotrophins reduce the synthesis of CSPG at the wound edge, making the peri-wound environment more permissive.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Cones de Crescimento/efeitos dos fármacos , Microesferas , Fatores de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Lesões Encefálicas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator Neurotrófico Derivado do Encéfalo/uso terapêutico , Proteoglicanas de Sulfatos de Condroitina/análise , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/análise , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/uso terapêutico , Cones de Crescimento/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Crescimento Neural/uso terapêutico , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Polímeros/química , Resultado do Tratamento , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologiaRESUMO
α1-antitrypsin (AAT) is a circulating serine protease inhibitor secreted from the liver and important in preventing proteolytic neutrophil elastase associated tissue damage, primarily in lungs. In humans, AAT is encoded by the SERPINA1 (hSERPINA1) gene in which a point mutation (commonly referred to as PiZ) causes aggregation of the miss-folded protein in hepatocytes resulting in subsequent liver damage. In an attempt to rescue the pathologic liver phenotype of a mouse model of human AAT deficiency (AATD), we used adenovirus to deliver Cas9 and a guide-RNA (gRNA) molecule targeting hSERPINA1. Our single dose therapeutic gene editing approach completely reverted the phenotype associated with the PiZ mutation, including circulating transaminase and human AAT (hAAT) protein levels, liver fibrosis and protein aggregation. Furthermore, liver histology was significantly improved regarding inflammation and overall morphology in hSERPINA1 gene edited PiZ mice. Genomic analysis confirmed significant disruption to the hSERPINA1 transgene resulting in a reduction of hAAT protein levels and quantitative mRNA analysis showed a reduction in fibrosis and hepatocyte proliferation as a result of editing. Our findings indicate that therapeutic gene editing in hepatocytes is possible in an AATD mouse model.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Fenótipo , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/genética , Adenoviridae/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Expressão Gênica , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Transgênicos , Transdução Genética , Transgenes , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina/patologia , Deficiência de alfa 1-Antitripsina/terapiaRESUMO
BACKGROUND AND PURPOSE: The ischemic penumbra is a major focus of stroke research. 18F-fluoromisonidazole (18F-FMISO), a positron emission tomography (PET) marker of hypoxic cells, has shown promise as a technique to image the penumbra in humans. Our aim was to delineate the pattern of 18F-FMISO binding in a rat middle cerebral artery transient thread-occlusion model, and correlate this with tissue outcome at 24 hours. We hypothesized that the pattern of 18F-FMISO binding would mimic that seen in humans. METHODS: Thirty-eight rats underwent 2 hours transient middle cerebral artery (MCA) occlusion, and then received 18F-FMISO at time points from 0.5 to 22 hours post-MCA occlusion and were killed 2 hours later. Autoradiographic assessment of 18F-FMISO binding and assessment (triphenyltetrazolium chloride) of the area of infarction were performed on tissue slices. RESULTS: Until 1 hour after MCA occlusion, 18F-FMISO binding was increased in the entire MCA territory, with little or no infarction visible. Over the next 5 hours, the pattern of binding evolved to a small rim of intensely binding tissue surrounding the infarct core, which itself showed reduced binding compared with the contralateral hemisphere. By 24 hours, there was minimal accumulation of 18F-FMISO binding and a large area of infarction. CONCLUSIONS: The pattern of 18F-FMISO binding rats reproduced the pattern seen in humans, consistent with this tracer being a marker of the ischemic penumbra in both species. This technique may have application in studying the ischemic penumbra in animal models, and correlating this with similar studies in humans.
Assuntos
Isquemia Encefálica/diagnóstico por imagem , Infarto da Artéria Cerebral Média/diagnóstico por imagem , Misonidazol/análogos & derivados , Misonidazol/análise , Acidente Vascular Cerebral/diagnóstico por imagem , Animais , Autorradiografia , Isquemia Encefálica/patologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologia , Tomografia Computadorizada de EmissãoRESUMO
Sulforaphane-induced activation of the transcription factor NF-E2 related factor 2 (Nrf2 or the gene Nfe2l2) and subsequent induction of the phase II antioxidant system has previously been shown to exert neuroprotective action in a transient model of focal cerebral ischemia. However, its ability to attenuate functional and cellular deficits after permanent focal cerebral ischemia is not clear. We assessed the neuroprotective effects of sulforaphane in the photothrombotic model of permanent focal cerebral ischemia. Sulforaphane was administered (5 or 50 mg/kg, i.p.) after ischemic onset either as a single dose or as daily doses for 3 days. Sulforaphane increased transcription of Nrf2, Hmox1, GCLC and GSTA4 mRNA in the brain confirming activation of the Nrf2 system. Single or repeated administration of sulforaphane had no effect on the infarct volume, nor did it reduce the number of activated glial cells or proliferating cells when analyzed 24 and 72 h after stroke. Motor-function as assessed by beam-walking, cylinder-test, and adhesive test, did not improve after sulforaphane treatment. The results show that sulforaphane treatment initiated after photothrombosis-induced permanent cerebral ischemia does not interfere with key cellular mechanisms underlying tissue damage.
Assuntos
Infarto Cerebral/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/farmacologia , Tiocianatos/farmacologia , Animais , Infarto Cerebral/genética , Infarto Cerebral/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Gliose , Isotiocianatos , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/genética , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fármacos Neuroprotetores/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfóxidos , Tiocianatos/administração & dosagemRESUMO
BACKGROUND: Reactive gliosis and scar formation after brain injury can inhibit the recovery process. As many glial cells utilize gap junctions for intercellular signaling, this study investigated whether two commonly used gap junction blockers, octanol and carbenoxolone, could attenuate reactive gliosis following a minor traumatic brain injury. METHODS: Octanol (710 mg/kg) or carbenoxolone (90 mg/kg) was administered 30 minutes before or after a needle track injury in adult male Sprague-Dawley rats. To mark dividing cells, animals were injected with bromodeoxyuridine (BrdU; 150 mg/kg) intraperitoneally two times per day, 8 hours apart and killed 2 days later. Immunohistochemistry for BrdU and markers for reactive glial cells [glial fibrillary acidic protein (GFAP), ED1, and NG2] were investigated using immunohistochemistry and western blot techniques. RESULTS: Two days after injury, increased cellular proliferation, activated astrocytes and microglia, and upregulation of NG2 expression were observed surrounding the injury site. Octanol and carbenoxolone administrated prior to injury significantly decreased cell proliferation by 60 and 70% respectively. The distance of GFAP immunoreactive astrocytes from the wound margin was decreased by 32 and 18% when octanol was administrated prior to or post injury respectively. Treatment with octanol also decreased the number of reactive microglia by 55% and, when administrated prior to injury, octanol reduced the distance of NG2 expression from the wound by 48%. CONCLUSION: The present study demonstrates that two important components of reactive gliosis, cellular activation and proliferation, can be attenuated by octanol and carbenoxolone.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Carbenoxolona/uso terapêutico , Gliose/tratamento farmacológico , Octanóis/uso terapêutico , Animais , Animais Recém-Nascidos , Lesões Encefálicas/complicações , Lesões Encefálicas/patologia , Células Cultivadas , Gliose/etiologia , Gliose/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Angiotensin-converting enzyme (ACE) inhibition can reduce stroke risk by up to 43% in humans and reduce the associated disability, and hence understanding the mechanism of improvement is important. In animals and humans, these effects may be independent of the blood pressure-lowering effects of ACE inhibition. Normotensive (Wistar-Kyoto (WKY)) and hypertensive (spontaneously hypertensive rat (SHR)) animals were treated with the ACE inhibitors ramipril or lisinopril for 7 or 42 days before 2 hours of transient middle cerebral artery occlusion (MCAo). Blood pressure, serum ACE, and blood glucose levels were measured and stroke infarct volume was recorded 24 hours after stroke. Despite greater reductions in blood pressure, infarct size was not improved by ACE inhibition in hypertensive animals. Short-term ACE inhibition produced only a modest reduction in blood pressure, but WKY rats showed marked reductions in infarct volume. Long-term ACE inhibition had additional reductions in blood pressure; however, infarct volumes in WKY rats did not improve further but worsened. WKY rats differed from SHR in having marked cortical ACE activity that was highly sensitive to ACE inhibition. The beneficial effects of ACE inhibition on infarct volume in normotensive rats do not correlate with changes in blood pressure. However, WKY rats have ACE inhibitor-sensitive cortical ACE activity that is lacking in the SHR.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Hipertensão/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Lisinopril/uso terapêutico , Peptidil Dipeptidase A/metabolismo , Ramipril/uso terapêutico , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Hipertensão/enzimologia , Infarto da Artéria Cerebral Média/enzimologia , Masculino , Ratos , Ratos WistarRESUMO
No single animal model is able to encompass all of the variables known to affect human ischemic stroke. This review highlights the major strengths and weaknesses of the most commonly used animal models of acute ischemic stroke in the context of matching model and experimental aim. Particular emphasis is placed on the relationships between outcome and underlying vascular variability, physiologic control, and use of models of comorbidity. The aim is to provide, for novice and expert alike, an overview of the key controllable determinants of experimental stroke outcome to help ensure the most effective application of animal models to translational research.