RESUMO
OBJECTIVE: The aim of this study was to investigate the effect and the mechanism of the NogoA/NgR1/RhoA signaling pathway on the apoptosis of neurons in cerebral infarction (CI) rats. Our findings might provide references for clinical prevention and treatment of CI. MATERIALS AND METHODS: A total of 60 adult male Wistar rats were randomly divided into 3 groups, including: Sham operation group (Sham group), CI group, and CI + NogoA gene knockout group (CI + NogoA KO group) using a random number table. The model of CI was successfully constructed using suture method in rats of CI group and CI + NogoA KO group. Only blood vessels were exposed in Sham group. At 2 days after CI operation, the rats were killed, and brain tissues were collected. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting were used to detect the messenger ribonucleic acid (mRNA) and protein expression levels of NogoA/NgR1/RhoA in brain lesion tissues of rats in the three groups, respectively. Subsequently, the pathological damage of brain tissues was detected via hematoxylin and eosin (H&E) staining. TTC staining was carried out to evaluate the infarction area in each group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted to measure the apoptosis level of neurons in brain tissues of rats in each group. Additionally, the level of Nissl's body in brain tissues of each group was examined by Nissl staining. Furthermore, the expression level of the platelet-derived growth factor (PDGF) in brain tissues of rats in the three groups was measured via immunohistochemistry. RESULTS: The mRNA and protein expression levels of the NogoA/NgR1/RhoA signaling pathway in brain tissues of rats in CI group increased significantly (p<0.05). NogoA KO could significantly reduce the infarction area of brain tissues in rats (p<0.05). H&E staining and Nissl's body staining revealed that neurons in the brain tissues of rats showed evident edema and disordered arrangement after CI. Meanwhile, the number of Nissl's body was remarkably reduced. However, after KO of NogoA, brain tissue damage was significantly alleviated in rats, and the number of Nissl's body increased remarkably at the same time (p<0.05). According to TUNEL staining results, inhibiting NogoA could notably reverse CI-induced apoptosis of neurons in brain tissues of rats (p<0.05). Immunohistochemical staining results demonstrated that the expression of PDGF in brain tissues of rats in CI group decreased markedly, whereas was significantly elevated in rats of CI + NogoA KO group (p<0.05). CONCLUSIONS: The expression of the NogoA/NgR1/RhoA signaling pathway was significantly elevated in brain tissues of CI rats. Furthermore, suppressing the NogoA/NgR1/RhoA signaling pathway could reduce CI-induced apoptosis of neurons in rats.
Assuntos
Córtex Cerebral/metabolismo , Infarto Cerebral/metabolismo , Neurônios/metabolismo , Receptor Nogo 1/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Apoptose , Córtex Cerebral/patologia , Infarto Cerebral/patologia , Modelos Animais de Doenças , Masculino , Neurônios/patologia , Receptor Nogo 1/deficiência , Receptor Nogo 1/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
A variety of conditions ranging from glaucoma to blunt force trauma lead to optic nerve atrophy. Identifying signaling pathways for stimulating axon growth in the optic nerve may lead to treatments for these pathologies. Inhibiting signaling by the nogo-66 receptor 1 (NgR1) promotes the re-extension of axons following a crush injury to the optic nerve, and while NgR1 mRNA and protein expression are observed in the retinal ganglion cell (RGC) layer and inner nuclear layer, which retinal cell types express NgR1 remains unknown. Here we determine the expression pattern of NgR1 in the mouse retina by co-labeling neurons with characterized markers of specific retinal neurons together with antibodies specific for NgR1 or Green Fluorescent Protein expressed under control of the ngr1 promoter. We demonstrate that more than 99% of RGCs express NgR1. Thus, inhibiting NgR1 function may ubiquitously promote the regeneration of axons by RGCs. These results provide additional support for the therapeutic potential of NgR1 signaling in reversing optic nerve atrophy.
Assuntos
Receptor Nogo 1/genética , Receptor Nogo 1/metabolismo , Células Ganglionares da Retina/metabolismo , Animais , Axônios/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Regeneração Nervosa/genética , Regeneração Nervosa/fisiologia , Receptor Nogo 1/deficiência , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/patologia , Transdução de SinaisRESUMO
Nogo receptor 1 (NgR1) is a high-affinity receptor of myelin-associated inhibitors (MAIs), and suppresses neurogenesis. Lentiviral vector are commonly used to alter the expression of targeted genes. However, little is known about the potential function of lentiviral vector harboring NgR1 shRNA (LV-NgR1 shRNA) on neurogenesis in spinal cord injury (SCI). In this study, the rats were randomly divided into three groups: including the LN (LV-NgR1 shRNA injection), LC (LV-control shRNA injection) and Sham (laminectomy only). Eight weeks post-injection of LV, spinal cords were examined by histology for changes in cavity size and by immunohistochemistry for changes in expression of NgR1, cell apoptosis, astrocytes, neurons and myelination. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Animals that received LV-NgR1 shRNA remarkably improved the motor function. These animals also showed an increase in levels of nerve fibers, synapses and myelination, a decrease in levels of lesion cavity and cell apoptosis at 8 weeks post-treatment. These findings give evidence that NgR1 may be a promising target for SCI treatment.
Assuntos
Lentivirus/genética , Locomoção/genética , Regeneração Nervosa/genética , Receptor Nogo 1/genética , RNA Interferente Pequeno/genética , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/terapia , Animais , Apoptose/genética , Astrócitos/patologia , Sobrevivência Celular , Regulação da Expressão Gênica/genética , Terapia Genética , Vetores Genéticos/genética , Sequências Repetidas Invertidas , Bainha de Mielina/fisiologia , Neurônios/patologia , Receptor Nogo 1/deficiência , Oligodendroglia/patologia , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Axo-glial units are highly organised microstructures propagating saltatory conduction and are disrupted during multiple sclerosis (MS). Nogo receptor 1 (NgR1) has been suggested to govern axonal damage during the progression of disease in the MS-like mouse model, experimental autoimmune encephalomyelitis (EAE). Here we have identified that adult ngr1 -/- mice, previously used in EAE and spinal cord injury experiments, display elongated paranodes, and nodes of Ranvier. Unstructured paranodal regions in ngr1 -/- mice are matched with more distributed expression pattern of Caspr. Compound action potentials of optic nerves and spinal cords from naïve ngr1 -/- mice are delayed and reduced. Molecular interaction studies revealed enhanced Caspr cleavage. Our data suggest that NgR1 may regulate axo-myelin ultrastructure through Caspr-mediated adhesion, regulating the electrophysiological signature of myelinated axons of central nervous system (CNS).