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1.
Chemosphere ; 238: 124602, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31545211

RESUMO

Polybrominated diphenyl ethers (PBDEs) have been known to exhibit neurotoxicity in rats; however, the underlying mechanism remains unknown and there is no available intervention. In this study, we aimed to investigate the role of oxidative and nitrosative stress in the neurotoxicity in the cerebral cortex and primary neurons in rats following the BDE-153 treatment. Compared to the untreated group, BDE-153 treatment significantly induced the neurotoxic effects in rats, as manifested by the increased lactate dehydrogenase (LDH) activities and cell apoptosis rates, and the decreased neurotrophic factor contents and cholinergic enzyme activities in rats' cerebral cortices and primary neurons. When compared to the untreated group, the oxidative and nitrosative stress had occurred in the cerebral cortex or primary neurons in rats following the BDE-153 treatment, as manifested by the increments in levels of reactive oxygenspecies (ROS), malondialdehyde (MDA), nitric oxide (NO), and neuronal nitric oxide synthase (nNOS) mRNA and protein expressions, along with the decline in levels of superoxide dismutase (SOD) activity, glutathione (GSH) content, and peroxiredoxin I (Prx I) and Prx II mRNA and protein expressions. In addition, the ROS scavenger N-acetyl-l-cysteine (NAC) or NO scavenger NG-Nitro-l-arginine (L-NNA) significantly rescued the LDH leakage and cell survival, reversed the neurotrophin contents and cholinergic enzymes, mainly via regaining balance between oxidation/nitrosation and antioxidation. Overall, our findings suggested that oxidative and nitrosative stresses are involved in the neurotoxicity induced by BDE-153, and that the antioxidation is a potential targeted intervention.


Assuntos
Córtex Cerebral/patologia , Éteres Difenil Halogenados/toxicidade , Síndromes Neurotóxicas/patologia , Estresse Nitrosativo/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Bifenil Polibromatos/toxicidade , Acetilcisteína/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Glutationa/metabolismo , Éteres Difenil Halogenados/metabolismo , Masculino , Malondialdeído/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/efeitos dos fármacos , Neurotrofina 3/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Estresse Nitrosativo/fisiologia , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
2.
J Stroke Cerebrovasc Dis ; 28(11): 104288, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31395423

RESUMO

PURPOSE: The present study was to observe the therapeutic efficiency of Clematichinenoside (AR) on cerebral ischemic injury in rats, especially on neurological and motor function recovery and to explore the underlying mechanism. METHODS: Following middle cerebral artery occlusion/reperfusion (MCAO/R) surgery, rats were treated orally with 32, 16, and 8 mg/kg AR respectively for 14 days during which cerebral injury was evaluated and proinflammatory factors tumor necrosis factor-α and interleukin-6 as well as neurotrophic factors brain-derived neurotrophic factor and Neurotrophin-3 levels were determined with ELISA kits. Immunohistochemical analysis on number of neurons and reactive astrocytes in the hippocampus was to demonstrate the effect of AR on neuronal survival. Motor, learning, and memory recovery were assessed by Morris water maze, passive avoidance experiment, and rotatory rod test. Neuroprotection and anti-inflammation-related Notch and nuclear factor-κB (NF-κB) signaling pathways were analyzed by PCR and Western blot techniques on mammalian achaete-scute homologs1, Notch-1, intracellular Notch receptor domain, Jagged-1, transcription factor hairy, enhancer of split1 (Hes1), as well as the nuclear import of NF-κB in hippocampus. RESULTS: AR administration reduced cerebral injury in rats exposed to MCAO/R and after treatment of AR for 14 days, proinflammatory reaction was inhibited, with neuronal survival rate raised and motor function recovery facilitated. PCR and WB analysis of Notch/NF-κB signaling pathway revealed the inhibitory effect of AR on pathway related components. CONCLUSIONS: AR is beneficial to recovery of neurological and motor function in rats after cerebral ischemic injury via inhibiting Notch/NF-κB pathway.


Assuntos
Anti-Inflamatórios/farmacologia , Comportamento Animal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Receptor Notch1/metabolismo , Saponinas/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Infarto da Artéria Cerebral Média/psicologia , Masculino , Memória/efeitos dos fármacos , Neurotrofina 3/metabolismo , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Transdução de Sinais
3.
J Parasitol ; 105(2): 313-320, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30995165

RESUMO

Neospora caninum is a parasite that infects many animal species and has tropism for various tissues, particularly the nervous system, where it generally remains in cysts. Under N. caninum infection, glial cells activate immune responses by a Th2 profile, suggesting an immunologically privileged environment that controls parasite proliferation, with neuronal preservation. In this study, we investigated the role of soluble neurotrophic factors released by glial cells on neuronal integrity during N. caninum infection in vitro. Primary cultures of rat glial cells enriched in astrocytes were infected with N. caninum tachyzoites (1:1) for 24 hr. Neuron-glia co-cultures were cultured for 24 hr with conditioned medium from glial cells infected with N. caninum (CMNc) and from uninfected cultures (control). Cell viability was determined through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test; astrocyte morphology and reactivity were determined through immunocytochemistry for glial fibrillar acid protein (GFAP) and the integrity of neurons through immunocytochemistry for ß-tubulin III. Expression of inflammatory cytokines and neurotrophic factors was determined through RT-qPCR. The MTT test demonstrated that 1:1 was the best parasite/host cell ratio, considering that it was enough to increase metabolism of glial cells when compared with control cultures and was not cytotoxic after 48 hr infection. N. caninum-infected glial cultures responded with astrogliosis characterized by an increase in GFAP expression and increase in IL-10 (2-fold), BDNF (1.6-fold), and NGF (1.7-fold) gene expression. In the neuron/glia co-cultures, it was observed that treatment with CMNc induced neuritis outgrowth without toxicity. Together, these results show that modulatory mechanisms by neurotrophic factors derived from glial cells, primarily astrocytes during the N. caninum infection, can favor neuroprotection.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neospora/fisiologia , Fator de Crescimento Neural/metabolismo , Neuroglia/parasitologia , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Técnicas de Cocultura , Meios de Cultivo Condicionados , DNA Complementar/biossíntese , Neospora/genética , Fatores de Crescimento Neural/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurotrofina 3/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Células Vero
4.
J Affect Disord ; 245: 1106-1113, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30699853

RESUMO

BACKGROUND: Bipolar disorder (BD) and substance use disorders share common symptoms, such as behavioral sensitization. Amphetamine-induced behavioral sensitization can serve as an animal model of BD. Neurotrophic factors have an important role in BD pathophysiology. This study evaluated the effects of amphetamine sensitization on behavior and neurotrophic factor levels in the brains of rats. METHODS: Wistar rats received daily intraperitoneal (i.p) injections of dextroamphetamine (d-AMPH) 2 mg/kg or saline for 14 days. After seven days of withdrawal, the animals were challenged with d-AMPH (0.5 mg/kg, i.p) and locomotor behavior was assessed. In a second protocol, rats were similarly treated with d-AMPH (2 mg/kg, i.p) for 14 days. After withdrawal, without d-AMPH challenge, depressive- and anxiety-like behaviors were evaluated through forced swimming test and elevated plus maze. Levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4/5 (NT-4/5) and glial-derived neurotrophic factor (GDNF) were evaluated in the frontal cortex, hippocampus, and striatum. RESULTS: D-AMPH for 14 days augmented locomotor sensitization to a lower dose of d-AMPH (0.5 mg/kg) after the withdrawal. d-AMPH withdrawal induced depressive- and anxious-like behaviors. BDNF, NGF, and GDNF levels were decreased, while NT-3 and NT-4 levels were increased in brains after d-AMPH sensitization. LIMITATIONS: Although d-AMPH induces manic-like behavior, the mechanisms underlying these effects can also be related to phenotypes of drug abuse. CONCLUSIONS: Together, vulnerability to mania-like behavior following d-AMPH challenge and extensive neurotrophic alterations, suggest amphetamine-induced behavioral sensitization is a good model of BD pathophysiology.


Assuntos
Ansiedade/metabolismo , Transtorno Bipolar/metabolismo , Encéfalo/metabolismo , Depressão/metabolismo , Dextroanfetamina/farmacologia , Fatores de Crescimento Neural/metabolismo , Animais , Ansiedade/induzido quimicamente , Comportamento Animal/efeitos dos fármacos , Transtorno Bipolar/induzido quimicamente , Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Depressão/induzido quimicamente , Modelos Animais de Doenças , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Fator de Crescimento Neural/efeitos dos fármacos , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/efeitos dos fármacos , Neurotrofina 3/efeitos dos fármacos , Neurotrofina 3/metabolismo , Ratos , Ratos Wistar
5.
Exp Neurol ; 312: 51-62, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30471251

RESUMO

It was previously reported that a tube holding chitosan carriers loaded with neurotrophin-3 (NT-3), after insertion into a 5 mm long transection gap in the adult rat spinal cord, triggered de novo neural tissue generation and functional recovery. Here, we report an effort to validate these findings using stringent blinding methodologies, which are crucial for robustness in reproducing biomedical studies. Radio frequency identification (RFID) chips were utilized to label rats that were randomly assigned into three experimental groups: transection with chitosan-NT-3 implant (C-NT3), transection only (T-controls), and laminectomy only (S-controls), blinding the experimenters to the treatments. Three months after surgery, animals only known by their RFID were functionally, electrophysiologically, and anatomically assessed. The data were then collected into the proper groups and statistically analyzed. Neural tissue with nestin-, Tuj1-, and NeuN-positive cells was found bridging the transection gap in C-NT3 rats, but not in T-controls. Motor- and somatosensory-evoked potentials were detected in C-NT3 rats and S-controls, but not in T-controls. Hind limb movement was significantly better in C-NT3 rats compared with T-controls. Our validation study indicates that C-NT3 implants facilitate neural tissue generation, at least in part, by eliciting endogenous neurogenesis. Our data support the use of C-NT3 implants for tissue remodeling in the injured spinal cord.


Assuntos
Quitosana/administração & dosagem , Regeneração Nervosa/fisiologia , Neurotrofina 3/administração & dosagem , Índice de Gravidade de Doença , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Quitosana/metabolismo , Implantes de Medicamento/administração & dosagem , Feminino , Regeneração Nervosa/efeitos dos fármacos , Neurotrofina 3/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia
6.
Neuroscience ; 400: 17-32, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30553796

RESUMO

Despite advances in technology and rehabilitation, no effective therapies are available for patients with SCI, which remains a major medical challenge. This study compared the efficacy of 3 different doses of mesenchymal stem cells (MSCs) administered by intraperitoneal injection as a therapeutic strategy for compressive SCI. We used adult female C57BL/6 mice that underwent laminectomy at the T9 level, followed by spinal-cord compression for 1 min with a 30-g vascular clip. The animals received an intraperitoneal (i.p.) injection of MSCs (8 × 104, 8 × 105 or 8 × 106 in 500 µl) or DMEM (500 µl), one week after SCI. The cells of the three MSC doses administered i.p. were able to migrate to the injury site, increase local expression of trophic factors, and enhance fiber sparing and/or regeneration, accompanied by substantial improvement in locomotor performance. Cell transplantation at 8 × 105 density showed the best therapeutic potential, leading to significant tissue and functional improvements compared to the other two doses. These findings indicate that i.p. application of MSCs at the density of 8 × 105 yielded the best results, suggesting that this dose is a good choice for SCI treatment.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Recuperação de Função Fisiológica , Compressão da Medula Espinal/fisiopatologia , Compressão da Medula Espinal/cirurgia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Gliose/etiologia , Locomoção , Camundongos Endogâmicos C57BL , Fibras Nervosas Mielinizadas/fisiologia , Neurotrofina 3/metabolismo , Compressão da Medula Espinal/complicações
7.
Biochem Biophys Res Commun ; 509(2): 429-434, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30594389

RESUMO

Appropriate synapse formation during development is necessary for normal brain function, and synapse impairment is often associated with brain dysfunction. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are key factors in regulating synaptic development. We previously reported that BDNF/NT-3 secretion was enhanced by calcium-dependent activator protein for secretion 2 (CADPS2). Although BDNF/NT-3 and CADPS2 are co-expressed in various brain regions, the effect of Cadps2-deficiency on brain region-specific BDNF/NT-3 levels and synaptic development remains elusive. Here, we show developmental changes of BDNF/NT-3 levels and we assess disruption of excitatory/inhibitory synapses in multiple brain regions (cerebellum, hypothalamus, striatum, hippocampus, parietal cortex and prefrontal cortex) of Cadps2 knockout (KO) mice compared with wild-type (WT) mice. Compared with WT, BDNF levels in KO mice were reduced in young/adult hippocampus, but increased in young hypothalamus, while NT-3 levels were reduced in adult cerebellum and young hippocampus, but increased in adult parietal cortex. Immunofluorescence of vGluT1, an excitatory synapse marker, and vGAT, an inhibitory synapse marker, in adult KO showed that vGluT1 was higher in the cerebellum and parietal cortex but lower in the hippocampus, whereas vGAT was lower in the hippocampus and parietal cortex compared with WT. Immunolabeling for both vGluT1 and vGAT was increased in the parietal cortex but vGAT was decreased in the cerebellum in adult KO compared with WT. These data suggest that CADPS2-mediated secretion of BDNF/NT-3 may be involved in development and maturation of synapses and in the balance between inhibitory and excitatory synapses.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Ligação ao Cálcio/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neurotrofina 3/genética , Sinapses/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/deficiência , Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Corpo Estriado/citologia , Corpo Estriado/crescimento & desenvolvimento , Corpo Estriado/metabolismo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Hipotálamo/citologia , Hipotálamo/crescimento & desenvolvimento , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Neurônios/citologia , Neurotrofina 3/metabolismo , Especificidade de Órgãos , Lobo Parietal/citologia , Lobo Parietal/crescimento & desenvolvimento , Lobo Parietal/metabolismo , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Córtex Pré-Frontal/metabolismo , Sinapses/classificação , Sinapses/metabolismo , Transmissão Sináptica/genética , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo
8.
J Gene Med ; 20(12): e3062, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30414229

RESUMO

BACKGROUND: Spinal cord injury (SCI) is a severe health problem worldwide, and efficacious strategies to properly repair SCI have not yet been developed. Recently, gene and cell therapies as alternative treatments for SCI have been proposed to comprise safe and promising strategies. METHODS: The present study investigated the therapeutic effects and underlying mechanisms of hypoxia-inducible factor-1α carried in recombinant adenovirus (Adv-HIF-1α), as administered immediately after SCI in adult rats. RESULTS: Adv-HIF-1α-treated animals showed better functional recovery and smaller cavity volume than those in the vehicle-treated control group. Both the numbers of green fluorescent protein-labeled bone marrow stromal cells (GFP-BMSCs) and cells double-positive for GFP and a cell lineage marker (NeuN) in the injured spinal cord were larger in the Adv-HIF-1α-treated group. The expression levels of neurotrophins such as neurotrophin-3 and brain-derived neurotrophic factor were also higher in the Adv-HIF-1α-treated group. CONCLUSIONS: Adv-HIF-1α improves functional recovery in rats with SCI, and the underlying mechanism may be related to the mobilization of BMSCs to the injured area and higher expression levels of neurotrophin-3 and brain-derived neurotrophic factor.


Assuntos
Células da Medula Óssea/metabolismo , Movimento Celular/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células-Tronco Mesenquimais/metabolismo , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/genética , Adenoviridae/genética , Animais , Células da Medula Óssea/citologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Mesenquimais/citologia , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia
9.
Elife ; 72018 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-30207538

RESUMO

Retrogradely-transported neurotrophin signaling plays an important role in regulating neural circuit specificity. Here we investigated whether targeted delivery of neurotrophin-3 (NT-3) to lumbar motoneurons (MNs) caudal to a thoracic (T10) contusive spinal cord injury (SCI) could modulate dendritic patterning and synapse formation of the lumbar MNs. In vitro, Adeno-associated virus serotype two overexpressing NT-3 (AAV-NT-3) induced NT-3 expression and neurite outgrowth in cultured spinal cord neurons. In vivo, targeted delivery of AAV-NT-3 into transiently demyelinated adult mouse sciatic nerves led to the retrograde transportation of NT-3 to the lumbar MNs, significantly attenuating SCI-induced lumbar MN dendritic atrophy. NT-3 enhanced sprouting and synaptic formation of descending serotonergic, dopaminergic, and propriospinal axons on lumbar MNs, parallel to improved behavioral recovery. Thus, retrogradely transported NT-3 stimulated remodeling of lumbar neural circuitry and synaptic connectivity remote to a thoracic SCI, supporting a role for retrograde transport of NT-3 as a potential therapeutic strategy for SCI.


Assuntos
Atividade Motora/fisiologia , Neurônios Motores/fisiologia , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/fisiopatologia , Animais , Células Cultivadas , Dendritos/fisiologia , Dependovirus/genética , Feminino , Masculino , Camundongos Endogâmicos C57BL , Neurônios Motores/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Vértebras Torácicas
10.
Bone ; 116: 232-247, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30125729

RESUMO

Faulty bony repair causes dysrepair of injured growth plate cartilage and bone growth defects in children; however, the underlying mechanisms are unclear. Recently, we observed the prominent induction of neurotrophin­3 (NT-3) and its important roles as an osteogenic and angiogenic factor promoting the bony repair. The current study investigated its roles in regulating injury site remodelling. In a rat tibial growth plate drill-hole injury repair model, NT-3 was expressed prominently in osteoblasts at the injury site. Recombinant NT-3 (rhNT-3) systemic treatment enhanced, but NT-3 immunoneutralization attenuated, expression of cartilage-removal proteases (MMP-9 and MMP-13), presence of bone-resorbing osteoclasts and expression of osteoclast protease cathepsin K, and remodelling at the injury site. NT-3 was also highly induced in cultured mineralizing rat bone marrow stromal cells, and the conditioned medium augmented osteoclast formation and resorptive activity, an ability that was blocked by presence of anti-NT-3 antibody. Moreover, NT-3 and receptor TrkC were induced during osteoclastogenesis, and rhNT-3 treatment activated TrkC downstream kinase Erk1/2 in differentiating osteoclasts although rhNT-3 alone did not affect activation of osteoclastogenic transcription factors NF-κB or NFAT in RAW264.7 osteoclast precursor cells. Furthermore, rhNT-3 treatment increased, but NT-3 neutralization reduced, expression of osteoclastogenic cytokines (RANKL, TNF-α, and IL-1) in mineralizing osteoblasts and in growth plate injury site, and rhNT-3 augmented the induction of these cytokines caused by RANKL treatment in RAW264.7 cells. Thus, injury site osteoblast-derived NT-3 is important in promoting growth plate injury site remodelling, as it induces cartilage proteases for cartilage removal and augments osteoclastogenesis and resorption both directly (involving activing Erk1/2 and substantiating RANKL-induced increased expression of osteoclastogenic signals in differentiating osteoclasts) and indirectly (inducing osteoclastogenic signals in osteoblasts).


Assuntos
Cartilagem Articular/patologia , Lâmina de Crescimento/metabolismo , Lâmina de Crescimento/patologia , Neurotrofina 3/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Lâmina de Crescimento/efeitos dos fármacos , Humanos , Masculino , Camundongos , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Células RAW 264.7 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptor trkC/metabolismo
11.
Cell Tissue Res ; 374(2): 251-262, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29974252

RESUMO

RNAscope® technology provided by Advanced Cell Diagnostics (ACD) allows the detection and evaluation of coinciding mRNA expression profiles in the same or adjacent cells in unprecedented quantitative detail using multicolor fluorescent in situ hybridization (FISH). While already extensively used in thinly sectioned material of various pathological tissues and, to a lesser extent, in some whole mounts, we provide here a detailed approach to use the fluorescent RNAscope method in the mouse inner ear and thick brain sections by modifying and adapting existing techniques of whole mount fluorescent in situ hybridization (WH-FISH). We show that RNAscope WH-FISH can be used to quantify local variation in overlaying mRNA expression intensity, such as neurotrophin receptors along the length of the mouse cochlea. We also show how RNAscope WH-FISH can be combined with immunofluorescence (IF) of some epitopes that remain after proteinase digestion and, to some extent, with fluorescent protein markers such as tdTomato. Our WH-FISH technique provides an approach to detect cell-specific quantitative differences in developing and mature adjacent cells, an emerging issue revealed by improved cellular expression profiling. Further, the presented technique may be useful in validating single-cell RNAseq data on expression profiles in a range of tissue known or suspected to have locally variable mRNA expression levels.


Assuntos
Imunofluorescência/métodos , RNA Mensageiro/genética , Animais , Cóclea/metabolismo , Regulação da Expressão Gênica , Imagem Tridimensional , Hibridização in Situ Fluorescente , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurotrofina 3/metabolismo , RNA Mensageiro/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismo
12.
Sci Rep ; 8(1): 10707, 2018 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013050

RESUMO

After injury to the mature central nervous system (CNS), myelin-derived inhibitory ligands bind to the Nogo-66 tripartite receptor complex expressed on axonal growth cones, comprised of LINGO-1 and p75NTR/TROY and induce growth cone collapse through the RhoA pathway. We have also shown that amphoterin-induced gene and open reading frame-3 (AMIGO3) substitutes for LINGO-1 and can signal axon growth cone collapse. Here, we investigated the regeneration of dorsal root ganglion neuron (DRGN) axons/neurites after treatment with a short hairpin RNA (sh) AMIGO3 plasmid delivered with a non-viral in vivo-jetPEI vector, and the pro-survival/axogenic neurotrophin (NT) 3 in vitro and in vivo. A bicistronic plasmid, containing both shAMIGO3 and NT3 knocked down >75% of AMIGO3 mRNA in cultured DRGN and significantly overexpressed NT3 production. In vivo, intra-DRG injection of in vivo-jetPEI plasmids containing shAMIGO3/gfp and shAMIGO3/nt3 both knocked down AMIGO3 expression in DRGN and, in combination with NT3 overexpression, promoted DC axon regeneration, recovery of conduction of compound action potentials across the lesion site and improvements in sensory and locomotor function. These findings demonstrate that in vivo-jetPEI is a potential non-viral, translatable DRGN delivery vehicle in vivo and that suppression of AMIGO3 disinhibits the growth of axotomised DRGN enabling NT3 to stimulate the regeneration of their DC axons and enhances functional recovery.


Assuntos
Terapia Genética/métodos , Proteínas de Membrana/genética , Neurotrofina 3/metabolismo , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal/genética , Medula Espinal/fisiologia , Animais , Axônios/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Técnicas de Silenciamento de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Injeções Espinhais , Proteínas de Membrana/metabolismo , Neurotrofina 3/genética , Plasmídeos/administração & dosagem , Plasmídeos/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Resultado do Tratamento
13.
Brain Res ; 1699: 19-33, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29883625

RESUMO

Transplanting stem cells engineered to overexpress trophic factors can improve motor abilities and facilitate axon regeneration following spinal cord injury. This study compared several transplantation paradigms using mesenchymal stem cells (MSCs) that overexpress the multi-neurotrophin, NT-3/D15A (NT-3-MSCs), to determine if different grafting strategies can elicit improved axon regeneration and/or behavioral outcomes following a complete T9 spinal transection. At one week post-transection, NT-3-MSCs were transplanted above, and at several locations below, the lesion site. A rostral-to-caudal gradient of NT-3-MSCs was produced by incrementally increasing the number of transplanted cells at locations distal to the transection. Motor function was analyzed using the Basso, Beattie, and Bresnahan scale for 7-weeks post-injury. The corticospinal tract was traced using biotinylated dextran amines, while raphespinal fibers were visualized using immunohistochemistry. Cell viability was assessed using transplants of NT-3-MSCs that express tdTomato. Retrograde tracing using fluorogold, as well as spinal re-transections, were performed to discriminate between a supra-spinal or reflexive influence of regained motor functions. NT-3-MSC transplants improved motor outcomes and tissue continuity at the transection site, however retrograde tracing using fluorogold revealed no evidence of axon regeneration. A spinal re-transection also failed to eliminate the improvement in motor outcomes produced by the transplant. We conclude that transplantation of NT-3-MSCs can improve motor function and morphological outcomes following a complete spinal transection without promoting axonal regeneration.


Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Transtornos dos Movimentos/terapia , Neurotrofina 3/metabolismo , Traumatismos da Medula Espinal/terapia , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Masculino , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/patologia , Transtornos dos Movimentos/fisiopatologia , Neurotrofina 3/genética , Ratos Sprague-Dawley , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Regeneração da Medula Espinal
14.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 32(4): 420-427, 2018 04 15.
Artigo em Chinês | MEDLINE | ID: mdl-29806299

RESUMO

Objective: To explore the feasibility of co-transduction and co-expression of Nogo extracellular peptide residues 1-40 (NEP1-40) gene and neurotrophin 3 (NT-3) gene into neural stem cells (NSCs). Methods: NSCs were derived from the cortex tissue of Sprague Dawley rat embryo. The experiment included 5 groups: no-load lentiviral vector transducted NSCs (group A), NEP1-40 transducted NSCs (group B), NT-3 transducted NSCs (group C), NEP1-40 and NT-3 corporately transducted NSCs (group D), and blank control (group E). Target genes were transducted into NSCs by lentiviral vectors of different multiplicity of infection (MOI; 5, 10, 15) for different time (24, 48, 72 hours). Fluorescent microscope was used to observe the expression of fluorescence protein and acquire the optimum MOI and optimum collection time. Real-time fluorescence quantitative PCR and Western blot tests were utilized to evaluate the gene expressions of NEP1-40 and NT-3 in NSCs and protein expressions of NEP1-40 and NT-3 in NSCs and in culture medium. Results: The optimum MOI for both target gene was 10 and the optimum collection time was 48 hours. The real-time fluorescence quantitative PCR and Western blot results showed that the mRNA and protein relative expressions of NEP1-40 in groups B and D were significantly higher than those in groups A and C ( P<0.05), but no significant difference was found between groups B and D, and between groups A and C ( P>0.05). The mRNA and protein relative expressions of NT-3 in groups C and D were significantly higher than those in groups A and B ( P<0.05), but no significant difference was found between groups A and B, and between groups C and D ( P>0.05). Conclusion: NEP1-40 and NT-3 gene can be successfully co-transducted into NSCs by the mediation of lentiviral vector. The expressions of the two target genes are stable and have no auxo-action or antagonism between each other.


Assuntos
Córtex Cerebral , Lentivirus/genética , Células-Tronco Neurais/citologia , Neurotrofina 3/genética , Fragmentos de Peptídeos/genética , Animais , Células Cultivadas , Lentivirus/metabolismo , Neurotrofina 3/metabolismo , Ratos , Ratos Sprague-Dawley
15.
J Neurosci ; 38(24): 5606-5619, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29789375

RESUMO

The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is upregulated, resulting in formation of TrkA-p75 complexes, which are high-affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 guanine nucleotide exchange factors. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth, whereas the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system.SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface availability may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Neurônios/metabolismo , Neurotrofina 3/metabolismo , Receptor trkA/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Neurogênese/fisiologia , Ratos , Ratos Sprague-Dawley , Sistema Nervoso Simpático/crescimento & desenvolvimento , Sistema Nervoso Simpático/metabolismo
16.
Mol Neurobiol ; 55(11): 8403-8413, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29549646

RESUMO

POU-homeodomain transcription factor POU3F2 is a critical transcription factor that participates in neuronal differentiation. However, little is known about its downstream mediators. Here genome-wide analyses of a human neuronal differentiation cell model, NT2D1, suggested neurotrophin-3 (NTF3), a key mediator of neuronal development during the early neurogenic period, as a putative regulatory target of POU3F2. Western blot, cDNA microarray, and real-time quantitative PCR analyses showed that POU3F2 and NTF3 were upregulated during neuronal differentiation. Next-generation-sequence-based POU3F2 chromatin immunoprecipitation-sequencing and genome-wide in silico prediction demonstrated that POU3F2 binds to the NTF3 promoter during neuronal differentiation. Furthermore, unidirectional deletion or mutation of the binding site of POU3F2 in the NTF3 promoter decreased promoter-driven luciferase activity, indicating that POU3F2 is a positive regulator of NTF3 promoter activity. While NTF3 knockdown resulted in decreased viability and differentiation of NT2D1 cells, and POU3F2 knockdown downregulated NTF3 expression, recombinant NTF3 significantly rescued viable neuronal cells from NTF3- or POU3F2-knockdown cell cultures. Moreover, immunostaining showed colocalization of POU3F2 and NTF3 in developing mouse neurons. Thus, our data suggest that NTF3 is a novel target gene of POU3F2 and that the POU3F2/NTF3 pathway plays a role in the process of neuronal differentiation.


Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurotrofina 3/genética , Fatores do Domínio POU/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Feminino , Inativação Gênica/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Neurotrofina 3/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Ativação Transcricional/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
17.
Sci Rep ; 8(1): 2003, 2018 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-29386650

RESUMO

Oxygen-induced retinopathy (OIR) upregulates Müller cell vascular endothelial growth factor A (VEGFA) that causes intravitreal neovascularization similar to severe retinopathy of prematurity (ROP). Safety concerns exist with anti-VEGF treatment for ROP. We evaluated long-term knockdown of Müller cell-VEGFA with short-hairpin RNAs to VEGFA or VEGF164 via subretinal lentivirus delivery (L-VEGFAshRNA, L-VEGF164shRNA) on retinal structure and function in a rat OIR model. Lectin-stained retinal flat mounts analyzed for areas of avascular/total retina (AVA) and intravitreal neovascular/total retina (IVNV) showed initial significantly reduced IVNV by L-VEGFAshRNA and L-VEGF164shRNA compared to control, luciferase-shRNA lentivirus, without late recurrence. Spectral-domain optical coherence tomography (OCT) and immunohistochemical sections (IHC) demonstrated changes in retinal layer thicknesses in L-VEGFAshRNA or L-VEGF164shRNA  compared to control. Ganzfeld electroretinograms were increased in L-VEGFAshRNA or L-VEGF164shRNA compared to control. Erythropoietin (EPO), brain-derived neurotrophic factor, glial-derived neurotrophic factor, nerve growth factor, neurotrophin-3 (NT-3) mRNAs were increased in L-VEGFAshRNA, but not L-VEGF164shRNA retinas. In cultured rat Müller cells, knockdown of VEGF upregulated NT-3 and EPO, whereas treatment with EPO activated neuroprotective signaling. Methods to reduce IVNV by selective knockdown of VEGFA, and particularly VEGF164, in Müller cells may have fewer deleterious effects than nonselective VEGFA inhibition to all cells in the retina.


Assuntos
Retina/metabolismo , Retinopatia da Prematuridade/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Células Cultivadas , Eritropoetina/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator de Crescimento Neural/metabolismo , Neurotrofina 3/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/patologia , Fator A de Crescimento do Endotélio Vascular/genética
18.
CNS Neurosci Ther ; 24(4): 353-363, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29453932

RESUMO

AIMS: Neurotrophin-3 (NT-3) is expressed in the mouse striatum; however, it is not clear the NT-3 role in striatal physiology. The expression levels of mRNAs and immune localization of the NT-3 protein and its receptor TrkC are altered in the striatum following damage induced by an in vivo treatment with 3-nitropropionic acid (3-NP), a mitochondrial toxin used to mimic the histopathological hallmarks of Huntington's disease (HD). The aim of this study was to evaluate the role of NT-3 on corticostriatal synaptic transmission and its plasticity in both the control and damaged striatum. METHODS: Corticostriatal population spikes were electrophysiologically recorded and striatal synaptic plasticity was induced by high-frequency stimulation. Further, the phosphorylation status of Trk receptors was tested under conditions that imitated electrophysiological experiments. RESULTS: NT-3 modulates both synaptic transmission and plasticity in the striatum; nonetheless, synaptic plasticity was modified by the 3-NP treatment, where instead of producing striatal long-term depression (LTD), long-term potentiation (LTP) was obtained. Moreover, the administration of NT-3 in the recording bath restored the plasticity observed under control conditions (LTD) in this model of striatal degeneration. CONCLUSION: NT-3 modulates corticostriatal transmission through TrkB stimulation and restores striatal LTD by signaling through its TrkC receptor.


Assuntos
Corpo Estriado/metabolismo , Doença de Huntington/metabolismo , Plasticidade Neuronal/fisiologia , Neurotrofina 3/metabolismo , Transmissão Sináptica/fisiologia , Animais , Corpo Estriado/efeitos dos fármacos , Modelos Animais de Doenças , Doença de Huntington/tratamento farmacológico , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Neurotrofina 3/administração & dosagem , Nitrocompostos , Propionatos , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Distribuição Aleatória , Receptor trkC/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Técnicas de Cultura de Tecidos
19.
Neurotox Res ; 34(2): 173-187, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29417440

RESUMO

In preterm infants, phenobarbital is the first-line antiepileptic drug for neonatal seizures while caffeine is used for the treatment of apnea. Data from experimental animals suggest that phenobarbital and other anticonvulsants are toxic for the developing brain, while neuroprotective effects have been reported for caffeine both in newborn rodents and preterm human infants. To characterize the interaction of phenobarbital and caffeine in the hippocampus of the developing rodent brain, we examined the effects of both drugs given separately or together on postnatal neurogenesis after administration to neonatal rats throughout postnatal day (P) 4 to P6. Phenobarbital treatment (50 mg/kg) resulted in a significant decrease of proliferative capacity in the dentate gyrus. Phenobarbital also reduced expression of neuronal markers (doublecortin (DCX), calretinin, NeuN), neuronal transcription factors (Pax6, Sox2, Tbr1/2, Prox1), and neurotrophins (NGF, BDNF, NT-3) up to 24 h after the last administration. The phenobarbital-mediated impairment of neurogenesis was largely ameliorated by preconditioning with caffeine (10 mg/kg). In contrast, caffeine alone reduced proliferative capacity and expression of the neuronal markers DCX and NeuN at 6 h, but increased expression of neurotrophins and neuronal transcription factors at 6 and 12 h. These results indicate that administration of phenobarbital during the vulnerable phase of brain development negatively interferes with neuronal development, which can be prevented in part by co-administration of caffeine.


Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Cafeína/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Anticonvulsivantes/toxicidade , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Masculino , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Fenobarbital/toxicidade , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo
20.
Exp Eye Res ; 166: 1-12, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29030174

RESUMO

There is increasing evidence to suggest a beneficial neuroprotective effect of growth hormone (GH) in the nervous system. While our previous studies have largely focused on retinal ganglion cells (RGCs), we have also found conclusive evidence of a pro-survival effect of GH in cells of the inner nuclear layer (INL) as well as a protective effect on the dendritic trees of the inner plexiform layer (IPL) in the retina. The administration of GH in primary neuroretinal cell cultures protected and induced neural outgrowths. Our results, both in vitro (embryo) and in vivo (postnatal), showed neuroprotective actions of GH against kainic acid (KA)-induced excitotoxicity in the chicken neuroretina. Intravitreal injections of GH restored brain derived neurotrophic factor (BDNF) expression in retinas treated with KA. In addition, we demonstrated that GH over-expression and exogenous administration increased BDNF and neurotrophin-3 (NT3) gene expression in embryonic neuroretinal cells. Thus, GH neuroprotective actions in neural tissues may be mediated by a complex cascade of neurotrophins and growth factors which have been classically related to damage prevention and neuroretinal tissue repair.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hormônio do Crescimento/farmacologia , Ácido Caínico/farmacologia , Fármacos Neuroprotetores/farmacologia , Neurotrofina 3/metabolismo , Retina/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Modelos Animais de Doenças , Neuroproteção/efeitos dos fármacos , Retina/embriologia , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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