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1.
Life Sci ; 245: 117351, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981629

RESUMO

AIMS: To study the specific therapeutic effect of zinc on spinal cord injury (SCI) and its specific protective mechanism. MAIN METHODS: The effects of zinc ions on neuronal cells were examined in a mouse SCI model and in vitro. In vivo, neurological function was assessed by Basso Mouse Scaleat (BMS) at 1, 3, 5, 7, 10, 14, 21, and 28 days after spinal cord injury. The number of neurons and histomorphology were observed by nissl staining and hematoxylin-eosin staining (HE). The chromatin and mitochondrial structure of neurons were detected by transmission electron microscopy (TEM). The expression of nuclear factor erythroid 2 related factor 2 (Nrf2)-related antioxidant protein and NLRP3 inflammation-related protein were detected in vivo and in vitro by western blot (WB) and immunofluorescence (IF), respectively. KEY FINDINGS: Zinc treatment promoted motor function recovery on days 3, 5, 7, 14, 21 and 28 after SCI. In addition, zinc reduces the mitochondrial void rate in spinal neuronal cells and promotes neuronal recovery. At the same time, zinc reduced the levels of reactive oxygen species (ROS) and malondialdehyde in spinal cord tissue after SCI, while increasing superoxide dismutase activity and glutathione peroxidase production. Zinc treatment resulted in up-regulation of Nrf2/Ho-1 levels and down-regulation of nlrp3 inflammation-associated protein expression in vitro and in vivo. SIGNIFICANCE: Zinc has a protective effect on spinal cord injury by inhibiting oxidative damage and nlrp3 inflammation. Potential mechanisms may include activation of the Nrf 2/Ho-1 pathway to inhibit nlrp3 inflammation following spinal cord injury. Zinc has the potential to treat SCI.


Assuntos
Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , Zinco/uso terapêutico , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Imunofluorescência , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Zinco/farmacologia
2.
Nat Genet ; 51(12): 1691-1701, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740836

RESUMO

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation.


Assuntos
Caderinas/genética , Cromatina/genética , Síndrome de Down/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/fisiologia , Adulto , Animais , Astrócitos/citologia , Astrócitos/fisiologia , Encéfalo/citologia , Encéfalo/embriologia , Diferenciação Celular , Linhagem Celular , Síndrome de Down/genética , Regulação da Expressão Gênica , Histonas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Pessoa de Meia-Idade , Neurônios/citologia , Regiões Promotoras Genéticas , Ratos , Análise de Célula Única , Medula Espinal/citologia , Medula Espinal/transplante , Transplante Heterólogo
3.
Nat Commun ; 10(1): 4830, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645570

RESUMO

Central nervous system (CNS) injuries persist for years, and currently there are no therapeutics that can address the complex injury cascade that develops over this time-scale. 17ß-estradiol (E2) has broad tropism within the CNS, targeting and inducing beneficial phenotypic changes in myriad cells following injury. To address the unmet need for vastly prolonged E2 release, we report first-generation poly(pro-E2) biomaterial scaffolds that release E2 at nanomolar concentrations over the course of 1-10 years via slow hydrolysis in vitro. As a result of their finely tuned properties, these scaffolds demonstrate the ability to promote and guide neurite extension ex vivo and protect neurons from oxidative stress in vitro. The design and testing of these materials reported herein demonstrate the first step towards next-generation implantable biomaterials with prolonged release and excellent regenerative potential.


Assuntos
Astrócitos/efeitos dos fármacos , Materiais Biocompatíveis , Estradiol/farmacologia , Estrogênios/farmacologia , Gânglios Espinais/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Fármacos do Sistema Nervoso Central/administração & dosagem , Fármacos do Sistema Nervoso Central/química , Fármacos do Sistema Nervoso Central/farmacologia , Implantes de Medicamento/química , Estradiol/administração & dosagem , Estradiol/química , Estrogênios/administração & dosagem , Estrogênios/química , Técnicas In Vitro , Macrófagos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Polímeros/química , Cultura Primária de Células , Pró-Fármacos/administração & dosagem , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Ratos , Medula Espinal/citologia
4.
Nat Commun ; 10(1): 4197, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519892

RESUMO

In all vertebrates, excitatory spinal interneurons execute dynamic adjustments in the timing and amplitude of locomotor movements. Currently, it is unclear whether interneurons responsible for timing control are distinct from those involved in amplitude control. Here, we show that in larval zebrafish, molecularly, morphologically and electrophysiologically distinct types of V2a neurons exhibit complementary patterns of connectivity. Stronger higher-order connections from type I neurons to other excitatory V2a and inhibitory V0d interneurons provide timing control, while stronger last-order connections from type II neurons to motor neurons provide amplitude control. Thus, timing and amplitude are coordinated by distinct interneurons distinguished not by their occupation of hierarchically-arranged anatomical layers, but rather by differences in the reliability and probability of higher-order and last-order connections that ultimately form a single anatomical layer. These findings contribute to our understanding of the origins of timing and amplitude control in the spinal cord.


Assuntos
Interneurônios/metabolismo , Locomoção/fisiologia , Animais , Interneurônios/citologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Peixe-Zebra
5.
BMC Genomics ; 20(1): 619, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362699

RESUMO

BACKGROUND: The regenerative ability of severed axons in the central nervous system is limited in mammals. However, after central nervous system injury, neural function is partially recovered by the formation of a compensatory neural circuit. In a mouse pyramidotomy model, axonal sprouting of the intact side of the corticospinal tract is observed in the spinal cord, and the axons make new synapses with the denervated side of propriospinal neurons. Moreover, this sprouting ability is enhanced in neonatal mice compared to that in adult mice. Myelin-associated molecules in the spinal cord or intrinsic factors in corticospinal neurons have been investigated in previous studies, but the factors that determine elevated sprouting ability in neonatal mice are not fully understood. Further, in the early phase after pyramidotomy, glial responses are observed in the spinal cord. To elucidate the basal difference in the spinal cord, we compared gene expression profiles of entire C4-7 cervical cord tissues between neonatal (injured at postnatal day 7) and adult (injured at 8 weeks of age) mice by RNA-sequencing. We also tried to identify discordant gene expression changes that might inhibit axonal sprouting in adult mice at the early phase (3 days) after pyramidotomy. RESULTS: A comparison of neonatal and adult sham groups revealed remarkable basal differences in the spinal cord, such as active neural circuit formation, cell proliferation, the development of myelination, and an immature immune system in neonatal mice compared to that observed in adult mice. Some inflammation-related genes were selectively expressed in adult mice after pyramidotomy, implying the possibility that these genes might be related to the low sprouting ability in adult mice. CONCLUSIONS: This study provides useful information regarding the basal difference between neonatal and adult spinal cords and the possible differential response after pyramidotomy, both of which are necessary to understand why sprouting ability is increased in neonatal mice compared to that in adult mice.


Assuntos
Envelhecimento/genética , Envelhecimento/fisiologia , Perfilação da Expressão Gênica , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Camundongos , Medula Espinal/citologia
6.
Int J Mol Med ; 44(4): 1585-1593, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364720

RESUMO

Neuropathic pain is one of most intense types of chronic pain. Numerous studies investigating neuropathic pain have described the critical involvement of microglia in the spinal cord. Previous studies have indicated that activation of large conductance Ca2+­activated K+ (BK) channels contributes to microglial activation in the spinal dorsal horn (SDH) and the generation of neuropathic pain. However, the specific role of BK channels in spinal microglia in neuropathic pain has not been fully addressed in previous studies, as BK channel inhibitors were used to inhibit microglial BK channel based on their inhibitory kinetics. We previously identified that Ca2+­activated K+ channel ß3 auxiliary subunit (KCNMB3), which is an auxiliary subunit of BK channels and regulates gating properties of the channel, is exclusively expressed in microglia in the spinal cord. The present study analyzed the role of BK channels in spinal microglia in neuropathic pain using a spinal microglia­specific BK channel knockdown method, with intrathecal injection of KCNMB3 small interfering RNA. Neuropathic pain was significantly attenuated in KCNMB3 knockdown mice. Increases in the number of microglia in the SDH following nerve injury were attenuated by KCNMB3 knockdown. Furthermore, increased levels of pain­associated molecules in the SDH were attenuated in KCNMB3 knockdown mice. Attempts were also made to analyze the effects of KCNMB3 knockdown on chronic pain. KCNMB3 knockdown ameliorated chronic pain and inhibited the expression levels of pain­associated molecules in the SDH. The results from the present study suggested that BK channels modulated the activation state of spinal microglia, and that KCNMB3 is a potential therapeutic target for neuropathic pain.


Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Microglia/metabolismo , Neuralgia/etiologia , Neuralgia/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Animais , Modelos Animais de Doenças , Inativação Gênica , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Masculino , Camundongos , Camundongos Knockout
7.
Adv Exp Med Biol ; 1155: 875-887, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31468454

RESUMO

Diabetes mellitus (DM) is a condition characterized by chronic hyperglycemia, which leads to diabetic neuropathy and apoptosis in the spinal cord. Taurine has been found to ameliorate the diabetic neuropathy and control apoptosis in various tissues. However, there are few reports that discuss the direct relationship between spinal cord and anti-apoptotic effect of taurine. In this study, DM was induced in male SD rats with STZ @ 25 mg/Kg of body weight in combination with high fat diet. After 2 weeks, they were divided into four groups as DM: diabetic rats, T1 (0.5%), T2 (1%) and T3 (2%) taurine solution, while control group was non-diabetic rats (no treatment). The results showed that DM increased apoptosis, decreased phosphorylated Akt and Bad. DM decreased expression of Bcl-2 and increased the Bax. Moreover, the release of cytochrome c into cytosol was increased in DM and activation of caspase-3 was also increased. However, taurine reversed all these abnormal changes in a dose dependent manner. Our results suggested the involvement of Akt/Bad signaling pathway and mitochondrial apoptosis pathway in protective effect of taurine against apoptosis in the spinal cord of diabetic rats. Therefore, taurine may be a potential medicine against diabetic neuropathy by controlling apoptosis.


Assuntos
Apoptose , Neuropatias Diabéticas , Medula Espinal/efeitos dos fármacos , Taurina/farmacologia , Animais , Diabetes Mellitus Experimental , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Medula Espinal/citologia
8.
Nat Commun ; 10(1): 3784, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439839

RESUMO

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Anexina A2/metabolismo , Neurônios Motores/patologia , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas S100/metabolismo , Fator de Transcrição Sp1/metabolismo , Esclerose Amiotrófica Lateral/etiologia , Animais , Membrana Celular/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Neurônios Motores/citologia , Degeneração Neural/etiologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição Sp1/genética , Medula Espinal/citologia , Medula Espinal/patologia
9.
J Integr Neurosci ; 18(2): 163-172, 2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31321957

RESUMO

In this paper, the modulation of ascending commissural interneurons by N-methyl-D-aspartate was investigated in neonatal rats by using retrograde labeling and whole-cell patch clamp. Data shows these interneurons can be divided into three types (single spike, phasic, and tonic) based on their firing patterns. A hyperpolarization-activated nonselective cation current and persistent inward current are expressed in these interneurons. The parameters studied (n = 48) include: resting membrane potential (-59.2 ± 0.8 mV), input resistance (964.4 ± 49.3 MΩ), voltage threshold (-39.5 ± 0.6 mV), rheobase (13.5 ± 0.7 pA), action potential height (55.6 ± 2.2 mV), action potential half-width (2.8 ± 0.1 ms), afterhyperpolarization magnitude (16.1 ± 1.2 mV) and half-decay (217.9 ± 10.7 ms). 10 µM N-methyl-D-aspartate increases excitability of ascending commissural interneurons by depolarizing the membrane potential, hyperpolarizing voltage threshold, reducing rheobase, and shifting the frequency-current relationship to the left. N-methyl-Daspartate enhances persistent inward currents but reduces hyperpolarization-activated nonselective cation currents. This research uncovers unique ionic and intrinsic properties of ascending commissural interneurons which can be modulated by major excitatory neurotransmitters such as N-methyl-D-aspartate to potentially facilitate left-right alternation during locomotion.


Assuntos
Interneurônios Comissurais/fisiologia , Potenciais da Membrana , N-Metilaspartato/fisiologia , Medula Espinal/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Interneurônios Comissurais/citologia , Interneurônios Comissurais/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/administração & dosagem , Potenciais da Membrana/efeitos dos fármacos , N-Metilaspartato/administração & dosagem , Ratos Wistar , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos
10.
Elife ; 82019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31355747

RESUMO

The spinal cord contains a diverse array of interneurons that govern motor output. Traditionally, models of spinal circuits have emphasized the role of inhibition in enforcing reciprocal alternation between left and right sides or flexors and extensors. However, recent work has shown that inhibition also increases coincident with excitation during contraction. Here, using larval zebrafish, we investigate the V2b (Gata3+) class of neurons, which contribute to flexor-extensor alternation but are otherwise poorly understood. Using newly generated transgenic lines we define two stable subclasses with distinct neurotransmitter and morphological properties. These V2b subclasses synapse directly onto motor neurons with differential targeting to speed-specific circuits. In vivo, optogenetic manipulation of V2b activity modulates locomotor frequency: suppressing V2b neurons elicits faster locomotion, whereas activating V2b neurons slows locomotion. We conclude that V2b neurons serve as a brake on axial motor circuits. Together, these results indicate a role for ipsilateral inhibition in speed control.


Assuntos
Interneurônios/fisiologia , Neurônios Motores/fisiologia , Medula Espinal/citologia , Animais , Movimento (Física) , Contração Muscular , Peixe-Zebra
11.
Elife ; 82019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31355748

RESUMO

Formation of long-range axons occurs over multiple stages of morphological maturation. However, the intrinsic transcriptional mechanisms that temporally control different stages of axon projection development are unknown. Here, we addressed this question by studying the formation of mouse serotonin (5-HT) axons, the exemplar of long-range profusely arborized axon architectures. We report that LIM homeodomain factor 1b (Lmx1b)-deficient 5-HT neurons fail to generate axonal projections to the forebrain and spinal cord. Stage-specific targeting demonstrates that Lmx1b is required at successive stages to control 5-HT axon primary outgrowth, selective routing, and terminal arborization. We show a Lmx1b→Pet1 regulatory cascade is temporally required for 5-HT arborization and upregulation of the 5-HT axon arborization gene, Protocadherin-alphac2, during postnatal development of forebrain 5-HT axons. Our findings identify a temporal regulatory mechanism in which a single continuously expressed transcription factor functions at successive stages to orchestrate the progressive development of long-range axon architectures enabling expansive neuromodulation.


Assuntos
Axônios/fisiologia , Proteínas com Homeodomínio LIM/metabolismo , Neurônios Serotoninérgicos/fisiologia , Fatores de Transcrição/metabolismo , Animais , Perfilação da Expressão Gênica , Proteínas com Homeodomínio LIM/deficiência , Camundongos , Prosencéfalo/citologia , Medula Espinal/citologia , Fatores de Transcrição/deficiência
12.
Curr Opin Anaesthesiol ; 32(5): 668-673, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31343465

RESUMO

PURPOSE OF REVIEW: Persistent postoperative pain (PPP) is a significant source of morbidity in our population. An excellent opportunity to understand the transition from acute to chronic pain states. Understanding the mechanisms that drive this and modulators that influence this transition is essential to both prevent and manage this condition. RECENT FINDINGS: Although the exact mechanism for the development of PPP is still poorly understood, hypotheses abound. Basic science research with animal models implicates nociceptive and neuropathic pain signals leading to pain sensitization due to persistent noxious signaling. Effects on the inhibitory modulation of noxious signaling in medullary-spinal pathways and descending modulation have also been implicated. SUMMARY: Persistent maladaptive neuroplastic changes secondary to neurotrophic factors and interactions between neurons and microglia may well explain the phenomenon. This article reviews the current thought processes on mechanisms and modulators from a basic science and epidemiological perspective.


Assuntos
Dor Crônica/etiologia , Neuralgia/etiologia , Nociceptividade/fisiologia , Dor Pós-Operatória/etiologia , Animais , Dor Crônica/fisiopatologia , Dor Crônica/terapia , Modelos Animais de Doenças , Humanos , Bulbo/citologia , Bulbo/fisiopatologia , Microglia/fisiologia , Vias Neurais/citologia , Vias Neurais/fisiopatologia , Neuralgia/fisiopatologia , Neuralgia/terapia , Neurônios/fisiologia , Dor Pós-Operatória/fisiopatologia , Dor Pós-Operatória/terapia , Medula Espinal/citologia , Medula Espinal/fisiopatologia , Fatores de Tempo
13.
Dev Cell ; 49(6): 907-919.e10, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31211994

RESUMO

During early spinal cord development, neurons of particular subtypes differentiate with a sparse periodic pattern while later neurons differentiate in the intervening space to eventually produce continuous columns of similar neurons. The mechanisms that regulate this spatiotemporal pattern are unknown. In vivo imaging in zebrafish reveals that differentiating spinal neurons transiently extend two long protrusions along the basal surface of the spinal cord before axon initiation. These protrusions express Delta protein, consistent with the hypothesis they influence Notch signaling at a distance of several cell diameters. Experimental reduction of Laminin expression leads to smaller protrusions and shorter distances between differentiating neurons. The experimental data and a theoretical model support the proposal that neuronal differentiation pattern is regulated by transient basal protrusions that deliver temporally controlled lateral inhibition mediated at a distance. This work uncovers a stereotyped protrusive activity of newborn neurons that organize long-distance spatiotemporal patterning of differentiation.


Assuntos
Padronização Corporal , Diferenciação Celular , Embrião não Mamífero/citologia , Laminina/metabolismo , Neurônios Motores/citologia , Medula Espinal/citologia , Peixe-Zebra/embriologia , Animais , Comunicação Celular , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Laminina/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Neurogênese , Transdução de Sinais , Análise Espaço-Temporal , Medula Espinal/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Neuroscience ; 411: 255-269, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31163207

RESUMO

Repeated stress induces systemic elevations in glucocorticoid levels. Stress is also associated with alterations in central nervous system astrocytes and oligodendrocytes that involve connexins and myelin proteins. Corticosteroid elevation seems a major factor in stress-induced neuropathology. Changes in astrocyte connexins and myelin components may be important mediators for the neurological effects of corticosteroid elevations. Two primary cell culture models, myelination culture from rat embryonic spinal cord (SC) or cerebral cortex (CC) consisting of neurons and glial cells (oligodendrocytes, microglia and astrocytes), and mixed astrocyte-and-oligodendrocyte culture prepared from postnatal rat CC, were used in this study. Cell cultures were treated with either vehicle, corticosterone (CORT) with or without glucocorticoid receptor antagonist mifepristone, or dexamethasone (DEX) during the period of in vitro myelination. Immunoreactivity of astrocyte connexin 43 (Cx43) and oligodendrocyte myelin basic protein (MBP), or the myelination index (co-localization of MBP and phosphorylated neurofilament) was determined by double immunofluorescent labeling. Oligodendrocyte morphology was evaluated by Sholl analysis. Prolonged exposure to CORT or DEX induced dose-dependent reduction of the myelination index, and of immunostaining for MBP and Cx43 in SC and CC myelination cultures, which was prevented by mifepristone. In glial cultures single CORT or DEX exposure caused shrinkage and simplification of/' MBP- or CNPase-positive oligodendrocyte processes. The results support that concurrent effects of glucocorticoids on myelination and astrocyte Cx43 immunoreactivity are mediated by glucocorticoid receptors and may partially account for the involvement of CNS glia in the pathological effects of prolonged stress.


Assuntos
Conexina 43/metabolismo , Dexametasona/farmacologia , Mifepristona/farmacologia , Bainha de Mielina/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Bainha de Mielina/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ratos , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo
15.
Phys Ther ; 99(9): 1211-1223, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31158282

RESUMO

BACKGROUND: Transcutaneous electrical nerve stimulation (TENS) is commonly used for pain control. However, the effects of TENS on osteoarthritis (OA) pain and potential underlying mechanisms remain unclear. OBJECTIVE: The objective of this study was to investigate the effect of TENS on OA pain treatment and underlying mechanisms related to glial cell inhibition. DESIGN: This was an experimental study. METHODS: OA was induced by injection of monosodium iodoacetate into the synovial space of the right knee joint of rats. High-frequency (HF) TENS (100 Hz), low-frequency (LF) TENS (4 Hz), or sham TENS was applied to the ipsilateral knee joint for 20 minutes. Paw withdrawal threshold (PWT), weight bearing, and knee bend score (KBS) were measured. Immunohistochemistry for microglia and astrocytes was performed with L3 to L5 spinal segment samples. To investigate the effects of glial inhibition on OA pain, minocycline, l-α-aminoadipate, or artificial cerebrospinal fluid was injected intrathecally, and PWT and KBS were measured. RESULTS: Compared with sham TENS, both HF TENS and LF TENS significantly increased PWT, decreased KBS, and inhibited activated microglia in the L3 to L5 segments but did not decrease the total number of microglia, except in the L4 segment (HF TENS). Astrocyte expression was significantly decreased in the L3 to L5 segments following LF TENS and in the L3 segment following HF TENS. Compared with artificial cerebrospinal fluid, both minocycline and l-α-aminoadipate increased PWT and decreased KBS. LIMITATIONS: These results cannot be generalized to humans. CONCLUSIONS: TENS alleviates OA pain in rats by inhibiting activated microglia and reducing astrocyte expression in the spinal cord. Although these results may not be generalizable to chronic pain in patients with OA, within the limitation of the experimental animal model used in the present study, they suggest a possible mechanism and preclinical evidence supporting further experimentation or clinical use of TENS in humans.


Assuntos
Artralgia/terapia , Neuroglia/citologia , Osteoartrite do Joelho/terapia , Medula Espinal/citologia , Estimulação Elétrica Nervosa Transcutânea/métodos , Animais , Astrócitos/citologia , Contagem de Células , Hiperalgesia/induzido quimicamente , Hiperalgesia/terapia , Ácido Iodoacético , Articulação do Joelho , Masculino , Osteoartrite do Joelho/induzido quimicamente , Medição da Dor/métodos , Ratos , Ratos Sprague-Dawley , Suporte de Carga
16.
J Pharmacol Exp Ther ; 370(3): 472-479, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31235533

RESUMO

Celecoxib is a nonsteroidal anti-inflammatory drug (NSAID) commonly used to treat pain conditions in humans. In addition to its blocking activity on cyclooxygenase (COX) enzymes, several other targets could contribute to its analgesic activity. Here we explore the spinal antinociceptive actions of celecoxib and the potential implication of Kv7 channels in mediating its effects. Spinal cord in vitro preparations from hind paw-inflamed animals were used to assess the segmental sensory-motor and the early sensory processing of nociceptive information. Electrophysiological recordings of ventral roots and dorsal horn neurones were obtained, and the effects of celecoxib and Kv7 modulators on responses to repetitive dorsal root stimulation at C-fiber intensity were assessed. Celecoxib applied at clinically relevant concentrations produced depressant effects on responses to dorsal root stimulation recorded from both ventral roots and individual dorsal horn neurones; by contrast, the non-nociceptive monosynaptic reflex was unaffected. The NSAID indomethacin had no effect on spinal reflexes, but further coapplication of celecoxib still produced depressant effects. The depressant actions of celecoxib were abolished after Kv7 channel blockade and mimicked by its structural analog dimethyl-celecoxib, which lacks COX-blocking activity. The present results identify Kv7 channels as novel central targets for celecoxib, which may be relevant to its analgesic effect. This finding contributes to better understand the pharmacology of celecoxib and reinforces both the role of Kv7 channels in modulating the excitability of central pain pathways and its validity as target for the design of analgesics.


Assuntos
Analgésicos/farmacologia , Celecoxib/farmacologia , Canais de Potássio KCNQ/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Células do Corno Posterior/citologia , Células do Corno Posterior/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/fisiologia
17.
Neuron ; 103(1): 102-117.e5, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31103358

RESUMO

Spinal transmission of pruritoceptive (itch) signals requires transneuronal signaling by gastrin-releasing peptide (GRP) produced by a subpopulation of dorsal horn excitatory interneurons. These neurons also express the glutamatergic marker vGluT2, raising the question of why glutamate alone is insufficient for spinal itch relay. Using optogenetics together with slice electrophysiology and mouse behavior, we demonstrate that baseline synaptic coupling between GRP and GRP receptor (GRPR) neurons is too weak for suprathreshold excitation. Only when we mimicked the endogenous firing of GRP neurons and stimulated them repetitively to fire bursts of action potentials did GRPR neurons depolarize progressively and become excitable by GRP neurons. GRPR but not glutamate receptor antagonism prevented this action. Provoking itch-like behavior by optogenetic activation of spinal GRP neurons required similar stimulation paradigms. These results establish a spinal gating mechanism for itch that requires sustained repetitive activity of presynaptic GRP neurons and postsynaptic GRP signaling to drive GRPR neuron output.


Assuntos
Peptídeo Liberador de Gastrina/genética , Prurido/fisiopatologia , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Neurônios , Optogenética , Bloqueadores dos Canais de Potássio/farmacologia , Prurido/genética , Prurido/psicologia , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/genética , Receptores de Glutamato/fisiologia , Receptores Pré-Sinápticos/metabolismo , Medula Espinal/citologia
18.
Nucleic Acids Res ; 47(12): 6208-6224, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31081019

RESUMO

Differentiating oligodendrocytes generate myelin to ensure rapid saltatory conduction in the vertebrate central nervous system. Although oligodendroglial differentiation and myelination are accompanied by dramatic chromatin reorganizations, previously studied chromatin remodelers had only limited direct effects on the process. To study the functional significance of chromatin changes for myelination and identify relevant remodelers, we deleted Ep400, the central ATP-hydrolyzing subunit of the TIP60/EP400 complex, at defined times of mouse oligodendrocyte development. Whereas Ep400-deficient oligodendrocyte precursors develop normally, terminal differentiation and myelination are dramatically impaired. Mechanistically, Ep400 interacts with transcription factor Sox10, binds to regulatory regions of the Myrf gene and is required to induce this central transcriptional regulator of the myelination program. In addition to reduced and aberrant myelin formation, oligodendrocytes exhibit increased DNA damage and apoptosis so that numbers never reach wildtype levels during the short lifespan of Ep400-deficient mice. Ep400 deletion in already mature oligodendrocytes remains phenotypically inapparent arguing that Ep400 is dispensable for myelin maintenance. Given its essential function in myelin formation, modulation of Ep400 activity may be beneficial in conditions such as multiple sclerosis where this process is compromised.


Assuntos
DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Bainha de Mielina/fisiologia , Oligodendroglia/citologia , Medula Espinal/citologia , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Camundongos Transgênicos , Bainha de Mielina/ultraestrutura , Oligodendroglia/metabolismo , Ratos , Ratos Wistar , Medula Espinal/embriologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismo
19.
Cells ; 8(5)2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31117227

RESUMO

Lesions to the CNS/PNS interface are especially severe, leading to elevated neuronal degeneration. In the present work, we establish the ventral root crush model for mice, and demonstrate the potential of such an approach, by analyzing injury evoked motoneuron loss, changes of synaptic coverage and concomitant glial responses in ß2-microglobulin knockout mice (ß2m KO). Young adult (8-12 weeks old) C57BL/6J (WT) and ß2m KO mice were submitted to a L4-L6 ventral roots crush. Neuronal survival revealed a time-dependent motoneuron-like cell loss, both in WT and ß2m KO mice. Along with neuronal loss, astrogliosis increased in WT mice, which was not observed in ß2m KO mice. Microglial responses were more pronounced during the acute phase after lesion and decreased over time, in WT and KO mice. At 7 days after lesion ß2m KO mice showed stronger Iba-1+ cell reaction. The synaptic inputs were reduced over time, but in ß2m KO, the synaptic loss was more prominent between 7 and 28 days after lesion. Taken together, the results herein demonstrate that ventral root crushing in mice provides robust data regarding neuronal loss and glial reaction. The retrograde reactions after injury were altered in the absence of functional MHC-I surface expression.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Microglia/metabolismo , Neurônios Motores/metabolismo , Medula Espinal/citologia , Raízes Nervosas Espinhais/patologia , Animais , Axotomia , Sobrevivência Celular , Feminino , Técnicas de Inativação de Genes , Gliose/metabolismo , Laminectomia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Raízes Nervosas Espinhais/cirurgia , Sinapses/patologia , Microglobulina beta-2/genética
20.
J Vis Exp ; (145)2019 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-30958458

RESUMO

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preBötzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.


Assuntos
Tronco Encefálico/fisiologia , Eletrofisiologia/métodos , Medula Espinal/fisiologia , Animais , Animais Recém-Nascidos , Tronco Encefálico/citologia , Neurônios Motores/citologia , Ratos , Medula Espinal/citologia
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