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1.
PLoS One ; 15(8): e0237156, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780756

RESUMO

Ischemic neuropathy is common in subjects with critical limb ischemia, frequently causing chronic neuropathic pain. However, neuropathic pain caused by ischemia is hard to control despite the restoration of an adequate blood flow. Here, we used a rat model of ischemic-reperfusion nerve injury (IRI) to investigate possible effects of hepatocyte growth factor (HGF) against ischemic neuropathy. Hemagglutinating virus of Japan (HVJ) liposomes containing plasmids encoded with HGF was delivered into the peripheral nervous system by retrograde axonal transport following its repeated injections into the tibialis anterior muscle in the right hindlimb. First HGF gene transfer was done immediately after IRI, and repeated at 1, 2 and 3 weeks later. Rats with IRI exhibited pronounced mechanical allodynia and thermal hyperalgesia, decreased blood flow and skin temperature, and lowered thresholds of plantar stimuli in the hind paw. These were all significantly improved by HGF gene transfer, as also were sciatic nerve conduction velocity and muscle action potential amplitudes. Histologically, HGF gene transfer resulted in a significant increase of endoneurial microvessels in sciatic and tibial nerves and promoted nerve regeneration which were confirmed by morphometric analysis. Neovascularization was observed in the contralateral side of peripheral nerves as well. In addition, IRI elevated mRNA levels of P2X3 and P2Y1 receptors, and transient receptor potential vanilloid receptor subtype 1 (TRPV1) in sciatic nerves, dorsal root ganglia and spinal cord, and these elevated levels were inhibited by HGF gene transfer. In conclusion, HGF gene transfer is a potent candidate for treatment of acute ischemic neuropathy caused by reperfusion injury, because of robust angiogenesis and enhanced nerve regeneration.


Assuntos
Terapia Genética/métodos , Fator de Crescimento de Hepatócito/genética , Neuralgia/terapia , Traumatismo por Reperfusão/terapia , Animais , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Hiperalgesia/metabolismo , Lipossomos/metabolismo , Masculino , Ratos , Ratos Wistar , Nervo Isquiático/metabolismo , Vírus Sendai/genética , Resultado do Tratamento
2.
J Headache Pain ; 21(1): 72, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522232

RESUMO

BACKGROUND: Vestibular migraine has recently been recognized as a novel subtype of migraine. However, the mechanism that relate vestibular symptoms to migraine had not been well elucidated. Thus, the present study investigated vestibular dysfunction in a rat model of chronic migraine (CM), and to dissect potential mechanisms between migraine and vertigo. METHODS: Rats subjected to recurrent intermittent administration of nitroglycerin (NTG) were used as the CM model. Migraine- and vestibular-related behaviors were analyzed. Immunofluorescent analyses and quantitative real-time polymerase chain reaction were employed to detect expressions of c-fos and calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis (TNC) and vestibular nucleus (VN). Morphological changes of vestibular afferent terminals was determined under transmission electron microscopy. FluoroGold (FG) and CTB-555 were selected as retrograde tracers and injected into the VN and TNC, respectively. Lentiviral vectors comprising CGRP short hairpin RNA (LV-CGRP) was injected into the trigeminal ganglion. RESULTS: CM led to persistent thermal hyperalgesia, spontaneous facial pain, and prominent vestibular dysfunction, accompanied by the upregulation of c-fos labeling neurons and CGRP immunoreactivity in the TNC (c-fos: vehicle vs. CM = 2.9 ± 0.6 vs. 45.5 ± 3.4; CGRP OD: vehicle vs. CM = 0.1 ± 0.0 vs. 0.2 ± 0.0) and VN (c-fos: vehicle vs. CM = 2.3 ± 0.8 vs. 54.0 ± 2.1; CGRP mRNA: vehicle vs. CM = 1.0 ± 0.1 vs. 2.4 ± 0.1). Furthermore, FG-positive neurons was accumulated in the superficial layer of the TNC, and the number of c-fos+/FG+ neurons were significantly increased in rats with CM compared to the vehicle group (vehicle vs. CM = 25.3 ± 2.2 vs. 83.9 ± 3.0). Meanwhile, CTB-555+ neurons dispersed throughout the VN. The structure of vestibular afferent terminals was less pronounced after CM compared with the peripheral vestibular dysfunction model. In vivo knockdown of CGRP in the trigeminal ganglion significantly reduced the number of c-fos labeling neurons (LV-CGRP vs. LV-NC = 9.9 ± 3.0 vs. 60.0 ± 4.5) and CGRP mRNA (LV-CGRP vs. LV-NC = 1.0 ± 0.1 vs. 2.1 ± 0.2) in the VN, further attenuating vestibular dysfunction after CM. CONCLUSIONS: These data demonstrates the possibility of sensitization of vestibular nucleus neurons to impair vestibular function after CM, and anti-CGRP treatment to restore vestibular dysfunction in patients with CM.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transtornos de Enxaqueca/fisiopatologia , Núcleos Vestibulares/metabolismo , Animais , Hiperalgesia/metabolismo , Masculino , Nitroglicerina/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Gânglio Trigeminal/metabolismo
3.
Nat Commun ; 11(1): 2293, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385249

RESUMO

The sodium channels Nav1.7, Nav1.8 and Nav1.9 are critical for pain perception in peripheral nociceptors. Loss of function of Nav1.7 leads to congenital insensitivity to pain in humans. Here we show that the spider peptide toxin called HpTx1, first identified as an inhibitor of Kv4.2, restores nociception in Nav1.7 knockout (Nav1.7-KO) mice by enhancing the excitability of dorsal root ganglion neurons. HpTx1 inhibits Nav1.7 and activates Nav1.9 but does not affect Nav1.8. This toxin produces pain in wild-type (WT) and Nav1.7-KO mice, and attenuates nociception in Nav1.9-KO mice, but has no effect in Nav1.8-KO mice. These data indicate that HpTx1-induced hypersensitivity is mediated by Nav1.9 activation and offers pharmacological insight into the relationship of the three Nav channels in pain signalling.


Assuntos
Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/metabolismo , Peptídeos/efeitos adversos , Venenos de Aranha/efeitos adversos , Sequência de Aminoácidos , Animais , Feminino , Gânglios Espinais/patologia , Humanos , Hiperalgesia/complicações , Masculino , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.7/química , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/química , Neurônios/efeitos dos fármacos , Neurônios/patologia , Dor/complicações , Dor/fisiopatologia , Ratos
4.
J Neurosci ; 40(19): 3707-3719, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32269108

RESUMO

Calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, are widely used as standard immunosuppressants in organ transplantation recipients. However, these drugs can cause severe pain in patients, commonly referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin inhibition increases NMDAR activity in the spinal cord, the underlying mechanism remains enigmatic. Using an animal model of CIPS, we found that systemic administration of FK506 in male and female mice significantly increased the amount of α2δ-1-GluN1 complexes in the spinal cord and the level of α2δ-1-bound GluN1 proteins in spinal synaptosomes. Treatment with FK506 significantly increased the frequency of mEPSCs and the amplitudes of monosynaptic EPSCs evoked from the dorsal root and puff NMDAR currents in spinal dorsal horn neurons. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide completely reversed the effects of FK506. In α2δ-1 gene KO mice, treatment with FK506 failed to increase the frequency of NMDAR-mediated mEPSCs and the amplitudes of evoked EPSCs and puff NMDAR currents in spinal dorsal horn neurons. Furthermore, systemic administration of gabapentin or intrathecal injection of α2δ-1Tat peptide reversed thermal and mechanical hypersensitivity in FK506-treated mice. In addition, genetically deleting GluN1 in dorsal root ganglion neurons or α2δ-1 genetic KO similarly attenuated FK506-induced thermal and mechanical hypersensitivity. Together, our findings indicate that α2δ-1-bound NMDARs mediate calcineurin inhibitor-induced tonic activation of presynaptic and postsynaptic NMDARs at the spinal cord level and that presynaptic NMDARs play a prominent role in the development of CIPS.SIGNIFICANCE STATEMENT Calcineurin inhibitors are immunosuppressants used to prevent rejection of transplanted organs and tissues. However, these drugs can cause severe, unexplained pain. We showed that calcineurin inhibition enhances physical interaction between α2δ-1 and NMDARs and their synaptic trafficking in the spinal cord. α2δ-1 is essential for calcineurin inhibitor-induced aberrant activation of presynaptic and postsynaptic NMDARs in the spinal cord. Furthermore, inhibiting α2δ-1 or disrupting α2δ-1-NMDAR interaction reduces calcineurin inhibitor-induced pain hypersensitivity. Eliminating NMDARs in primary sensory neurons or α2δ-1 KO also attenuates calcineurin inhibitor-induced pain hypersensitivity. This new information extends our mechanistic understanding of the role of endogenous calcineurin in regulating synaptic plasticity and nociceptive transmission and suggests new strategies for treating this painful condition.


Assuntos
Inibidores de Calcineurina/toxicidade , Canais de Cálcio/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Feminino , Masculino , Camundongos , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo
5.
Pain Res Manag ; 2020: 4807674, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32190166

RESUMO

Vesicular glutamate transporter type 2 (VGLUT2) is known to play an important role in mediating heat hyperalgesia induced by inflammation. However, the underlying mechanism for this activity is poorly understood. Cyclin-dependent kinase 5 (Cdk5), serving as a key regulator in modulating release of glutamate, acted a key player in the formation of heat hyperalgesia of inflammatory pain. However, it remains unknown whether there is a bridge between Cdk5 and VGLUT2 for mediating inflammatory pain. Therefore, we designed the experiment to determine whether VGLUT2 signaling pathway is involved in inflammatory pain mediated by Cdk5 in the inflammatory pain model induced by complete Freund's adjuvant (CFA). Our results showed that the coexpression of Cdk5/VGLUT2 in small- and medium-sized neuronal cells of the dorsal root ganglion (DRG) and spinal cord between days 1 and 3 following subcutaneous injection of CFA was significantly increased. Moreover, our study revealed that the expression of VGLUT2 protein in the DRG and spinal cord was remarkably increased between days 1 and 3 following CFA injection and was significantly reduced by roscovitine, a selective antagonist of Cdk5. Additionally, p25 but not p35, an activator of Cdk5, protein was significantly increased by CFA and reduced by roscovitine. Our findings suggested that VGLUT2/Cdk5 signaling pathway contributes to inflammatory pain mediated by Cdk5/p25.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Inflamação/metabolismo , Dor/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Animais , Adjuvante de Freund/farmacologia , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Inflamação/induzido quimicamente , Inflamação/complicações , Masculino , Neurônios/metabolismo , Dor/etiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Medula Espinal/metabolismo
6.
Anesthesiology ; 132(4): 867-880, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32011337

RESUMO

BACKGROUND: Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. METHODS: In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. RESULTS: In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). CONCLUSIONS: Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.


Assuntos
Citocromo P-450 CYP11B2/biossíntese , Hiperalgesia/metabolismo , Medição da Dor/métodos , Células Receptoras Sensoriais/metabolismo , Adjuvantes Imunológicos/toxicidade , Aldosterona/biossíntese , Animais , Adjuvante de Freund/toxicidade , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Medição da Dor/efeitos dos fármacos , Estimulação Física/efeitos adversos , Ratos , Ratos Wistar , Células Receptoras Sensoriais/efeitos dos fármacos
7.
Muscle Nerve ; 61(5): 662-670, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32083755

RESUMO

INTRODUCTION: We investigated the mechanisms underlying immobilization-induced muscle pain in rats. METHODS: In rat skeletal muscle, pressure pain threshold (PPT) of the gastrocnemius muscle was measured, and nerve growth factor (NGF) level, peripheral nerve fiber density, macrophage number, and interleukin-1ß (IL-1ß) mRNA expression were examined. An NGF receptor inhibitor was injected intramuscularly to assess the relationship between PPT and NGF levels. RESULTS: Immobilization resulted in a decrease in PPT and increases in NGF level, C-fiber density, M1 macrophage number, and IL-1ß mRNA expression. Injection of NGF receptor inhibitor reversed the decrease in PPT. DISCUSSION: NGF upregulation may be a major contributor to immobilization-induced muscle pain. The increases in C-fiber density, M1 macrophage number, and IL-1ß mRNA expression may be related to immobilization-induced muscle pain.


Assuntos
Hiperalgesia/metabolismo , Imobilização , Interleucina-1beta/genética , Macrófagos/patologia , Músculo Esquelético/metabolismo , Fator de Crescimento Neural/metabolismo , Limiar da Dor/fisiologia , RNA Mensageiro/metabolismo , Animais , Carbazóis/farmacologia , Moldes Cirúrgicos , Inibidores Enzimáticos/farmacologia , Membro Posterior , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Alcaloides Indólicos/farmacologia , Masculino , Músculo Esquelético/inervação , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Fibras Nervosas/patologia , Fibras Nervosas Amielínicas/patologia , Limiar da Dor/efeitos dos fármacos , Pressão , Distribuição Aleatória , Ratos , Ratos Wistar , Receptor trkA/antagonistas & inibidores
8.
J Headache Pain ; 21(1): 4, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937253

RESUMO

BACKGROUND: According to our previous study, microglia P2X4 receptors (P2X4Rs) play a pivotal role in the central sensitization of chronic migraine (CM). However, the molecular mechanism that underlies the crosstalk between microglia P2X4Rs and neurons of the trigeminal nucleus caudalis (TNC) is not fully understood. Therefore, the aim of this study is to examine the exact P2X4Rs signalling pathway in the development of central sensitization in a CM animal model. METHODS: We used an animal model with recurrent intermittent administration of nitroglycerin (NTG), which closely mimics CM. NTG-induced basal mechanical and thermal hypersensitivity were evaluated using a von Frey filament test and an increasing-temperature hot plate apparatus (IITC). We detected P2X4Rs, brain-derived neurotrophic factor (BDNF) and phosphorylated p38 mitogen-activated protein kinase (p-p38-MAPK) expression profiles in the TNC. We investigated the effects of a P2X4R inhibitor (5-BDBD) and an agonist (IVM) on NTG-induced hyperalgesia and neurochemical changes as well as on the expression of p-p38-MAPK and BDNF. We also detected the effects of a tropomyosin-related kinase B (TrkB) inhibitor (ANA-12) on the CM animal model in vivo. Then, we evaluated the effect of 5-BDBD and SB203580 (a p38-MAPK inhibitors) on the release and synthesis of BDNF in BV2 microglia cells treated with 50 µM adenosine triphosphate (ATP). RESULTS: Chronic intermittent administration of NTG resulted in chronic mechanical and thermal hyperalgesia, accompanied by the upregulation of P2X4Rs and BDNF expression. 5-BDBD or ANA-12 prevented hyperalgesia induced by NTG, which was associated with a significant inhibition of the NTG-induced increase in phosphorylated extracellular regulated protein kinases (p-ERK) and calcitonin gene related peptide (CGRP) release in the TNC. Repeated administration of IVM produced sustained hyperalgesia and significantly increased the levels of p-ERK and CGRP release in the TNC. Activating P2X4Rs with ATP triggered BDNF release and increased BDNF synthesis in BV2 microglia, and these results were then reduced by 5-BDBD or SB203580. CONCLUSIONS: Our results indicated that the P2X4R contributes to the central sensitization of CM by releasing BDNF and promoting TNC neuronal hyper-excitability. Blocking microglia P2X4R-BDNF signalling may have an effect on the prevention of migraine chronification.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Sensibilização do Sistema Nervoso Central/fisiologia , Microglia/fisiologia , Transtornos de Enxaqueca/fisiopatologia , Receptores Purinérgicos P2X4/fisiologia , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Sensibilização do Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , Hiperalgesia/metabolismo , Masculino , Microglia/metabolismo , Transtornos de Enxaqueca/metabolismo , Nitroglicerina/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Phytomedicine ; 67: 153166, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31955133

RESUMO

BACKGROUND: Peripheral nerve injury can produce chronic and ultimately neuropathic pain. The chronic constriction injury (CCI) model has provided a deeper understanding of nociception and chronic pain. Loganin is a well-known herbal medicine with glucose-lowering action and neuroprotective activity. PURPOSE: This study investigated the molecular mechanisms by which loganin reduced CCI-induced neuropathic pain. METHODS: Sprague-Dawley rats were randomly divided into four groups: sham, sham+loganin, CCI and CCI+loganin. Loganin (1 or 5 mg/kg/day) was injected intraperitoneally once daily for 14 days, starting the day after CCI. For behavioral testing, mechanical and thermal responses were assessed before surgery and on d1, d3, d7 and d14 after surgery. Sciatic nerves (SNs) were collected to measure proinflammatory cytokines. Proximal and distal SNs were collected separately for Western blotting and immunofluorescence studies. RESULTS: Thermal hyperalgesia and mechanical allodynia were reduced in the loganin-treated group as compared to the CCI group. Loganin (5 mg/kg/day) prevented CCI from inducing proinflammatory cytokines (TNF-α, IL-1ß), inflammatory proteins (TNF-α, IL-1ß, pNFκB, pIκB/IκB, iNOS) and receptor (TNFR1, IL-1R), adaptor protein (TRAF2) of TNF-α, and Schwann cell demyelination and axonal damage. Loganin also blocked IκB phosphorylation (p-IκB). Double immunofluorescent staining further demonstrated that pNFκB/pIκB protein was reduced by loganin in Schwann cells on d7 after CCI. In the distal stumps of injured SN, Schwann cell demyelination was correlated with pain behaviors in CCI rats. CONCLUSION: Our findings indicate that loganin improves CCI-induced neuroinflammation and pain behavior by downregulating TNF-α/IL-1ß-dependent NF-κB activation.


Assuntos
Analgésicos não Entorpecentes/farmacologia , Iridoides/farmacologia , NF-kappa B/metabolismo , Neuralgia/tratamento farmacológico , Células de Schwann/efeitos dos fármacos , Animais , Dor Crônica/tratamento farmacológico , Dor Crônica/metabolismo , Dor Crônica/patologia , Constrição , Citocinas/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Interleucina-1beta/metabolismo , Masculino , Neuralgia/metabolismo , Neuralgia/patologia , Ratos Sprague-Dawley , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
J Neuroimmunol ; 340: 577145, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31945593

RESUMO

Lavandula angustifolia (LaEO) essential oil has been widely used by aromatherapy in the treatment of various clinical conditions, with evidence of its analgesic and anti-inflammatory potential. Our results demonstrate that sixty-five substances were identified in LaEO. Among the compounds found, the major ones were linalool (30.61%) and linalyl acetate (20.36%). We found that LaEO inhalation reduces mechanical hyperalgesia in conditions of chronic inflammatory and neuropathic pain. Furthermore, this effect seems to be mediated by peripheral and central opioid and cannabinoid 2 receptors. The findings of the present study suggests that the LaEO inhalation is effective on the chronic pain treatment.


Assuntos
Hiperalgesia , Óleos Voláteis/farmacologia , Receptores de Canabinoides/metabolismo , Receptores Opioides/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Hiperalgesia/metabolismo , Inflamação/metabolismo , Lavandula , Camundongos , Neuralgia/metabolismo , Extratos Vegetais/farmacologia
11.
Anesth Analg ; 131(1): 298-306, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31990732

RESUMO

BACKGROUND: Anti-inflammatory corticosteroids are a common treatment for different conditions involving chronic pain and inflammation. Clinically used steroids target the glucocorticoid receptor (GR) for its anti-inflammatory effects. We previously reported that GR in sensory neurons may play central roles in some pain models and that GR immunoreactivity signal in dorsal root ganglia (DRG) decreased after local inflammation of the DRG (a model of low back pain). In the current study, we aimed to determine if similar changes in GR signal also exist in a skin inflammation model, the complete Freund's adjuvant (CFA) model (a model of peripheral inflammatory pain), in which the terminals of the sensory neurons rather than the somata are inflamed. METHODS: A low dose of CFA was injected into the hind paw to establish the peripheral inflammation model in Sprague-Dawley rats of both sexes, as confirmed by measurements of behavior and paw swelling. Immunohistochemical and western blotting techniques were used to determine the expression pattern of the GR in the inflamed hind paw and the DRGs. Plasma corticosterone levels were measured with radioimmunoassay. RESULTS: The immunohistochemical staining revealed that GR is widely expressed in the normal DRG and skin tissues. Paw injection with CFA caused upregulation of the GR in the skin tissue on postinjection day 1, mostly detected in the dermis area. However, paw inflammation significantly reduced the GR signal in the L5 DRG 1 day after the injection. The GR downregulation was still evident 14 days after CFA inflammation. On day 1, western blotting confirmed this downregulation and showed that it could also be observed in the contralateral L5 DRG, as well as in the L2 DRG (a level which does not innervate the paw). Plasma corticosterone levels were elevated in both sexes on day 14 after CFA compared to day 1, suggesting autologous downregulation of the GR by corticosterone may have contributed to the downregulation observed on day 14 but not day 1. CONCLUSIONS: There are distinctive patterns of GR activation under different pain conditions, depending on the anatomical location. The observed downregulation of the GR in sensory neurons may have a significant impact on the use of steroids as treatment in these conditions and on the regulatory effects of endogenous glucocorticoids.


Assuntos
Modelos Animais de Doenças , Hiperalgesia/metabolismo , Receptores de Glucocorticoides/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Feminino , Adjuvante de Freund/toxicidade , Hiperalgesia/induzido quimicamente , Inflamação/induzido quimicamente , Inflamação/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos
12.
Chin Med J (Engl) ; 133(2): 190-197, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31929370

RESUMO

BACKGROUND: Pulsed radiofrequency (PRF), as a non-invasive treatment of neuropathic pain (NP), has been widely administered clinically. Previous studies have shown that PRF has the potential to improve hyperalgesia in animal models of NP. However, there have been few reports to clarify whether the mechanism of PRF treatment of NP involves intervention in the expression of substance P (SP). Therefore, this study administered PRF treatment to chronic constriction injury (CCI) model rats and observed the sciatic nerve mechanical pain threshold and SP expression in the spinal cord to explore the mechanism of PRF treatment. METHODS: A total of 96 Sprague-Dawley rats were randomly divided into the sham-surgery-sham-treatment group (S-S group), the sham-surgery-PRF group (S-P group), the CCI-sham-treatment group (C-S group), and the CCI-PRF group (C-P group). The C-S group and the C-P group underwent sciatic nerve CCI, while the other groups received a sham operation. At 14 days after the operation, the C-P group and the S-P group were treated with PRF for 300 s. We recorded the hindpaw withdrawal threshold (HWT) and the thermal withdrawal latency (TWL) of rats in the various groups at baseline, before treatment (0 days), and at 1, 7, 14, and 28 days after treatment. L4 to L6 spinal cord tissues were taken before treatment (0 days) and 1, 7, 14, and 28 days after treatment. The transcription and translation of SP were measured by quantitative polymerase chain reaction and Western blotting, respectively. RESULTS: The HWT and the TWL in the C-P group 28 days after PRF treatment were significantly higher than those in the C-S group (95% confidence interval [CI]: 5.84-19.50, P < 0.01; 95% CI: 2.58-8.69, P = 0.01). The expression of SP in the C-P group 28 days after PRF treatment was significantly lower than that in the C-S group (95% CI: 1.17-2.48, P < 0.01). CONCLUSIONS: PRF may alleviate CCI-induced NP by down-regulating the expression of SP in the spinal cord of CCI model rats.


Assuntos
Constrição Patológica/terapia , Neuralgia/terapia , Tratamento por Radiofrequência Pulsada/métodos , Substância P/metabolismo , Animais , Constrição Patológica/metabolismo , Modelos Animais de Doenças , Hiperalgesia/metabolismo , Hiperalgesia/terapia , Masculino , Neuralgia/metabolismo , Ratos , Ratos Sprague-Dawley
13.
Am J Physiol Renal Physiol ; 318(2): F298-F314, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31790304

RESUMO

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a common chronic pelvic disorder with sensory symptoms of urinary urgency, frequency, and pain, indicating a key role for hypersensitivity of bladder-innervating sensory neurons. The inflammatory mast cell mediator histamine has long been implicated in IC/BPS, yet the direct interactions between histamine and bladder afferents remain unclear. In the present study, we show, using a mouse ex vivo bladder afferent preparation, that intravesical histamine enhanced the mechanosensitivity of subpopulations of afferents to bladder distension. Histamine also recruited "silent afferents" that were previously unresponsive to bladder distension. Furthermore, in vivo intravesical histamine enhanced activation of dorsal horn neurons within the lumbosacral spinal cord, indicating increased afferent signaling in the central nervous system. Quantitative RT-PCR revealed significant expression of histamine receptor subtypes (Hrh1-Hrh3) in mouse lumbosacral dorsal root ganglia (DRG), bladder detrusor smooth muscle, mucosa, and isolated urothelial cells. In DRG, Hrh1 was the most abundantly expressed. Acute histamine exposure evoked Ca2+ influx in select populations of DRG neurons but did not elicit calcium transients in isolated primary urothelial cells. Histamine-induced mechanical hypersensitivity ex vivo was abolished in the presence of the histamine H1 receptor antagonist pyrilamine and was not present in preparations from mice lacking transient receptor potential vanilloid 1 (TRPV1). Together, these results indicate that histamine enhances the sensitivity of bladder afferents to distension via interactions with histamine H1 receptor and TRPV1. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS.


Assuntos
Cistite Intersticial/metabolismo , Histamina/administração & dosagem , Hiperalgesia/metabolismo , Mecanorreceptores/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Receptores Histamínicos H1/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Bexiga Urinária/inervação , Administração Intravesical , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cistite Intersticial/fisiopatologia , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Hiperalgesia/fisiopatologia , Masculino , Mecanorreceptores/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Limiar da Dor/efeitos dos fármacos , Pressão , Receptores Histamínicos H1/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Urotélio/efeitos dos fármacos , Urotélio/metabolismo
14.
Prostate ; 80(1): 28-37, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573117

RESUMO

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a disorder that is characterized by persistent pelvic pain in men of any age. Although several studies suggest that the transient receptor potential vanilloid 1 (TRPV1) channel is involved in various pathways of chronic pain, the TRPV1 channel has not been implicated in chronic pelvic pain associated with CP/CPPS. METHODS: Male C57BL/6J (B6) and TRPV1 knockout (TRPV1 KO) mice (5-7 weeks old) were used to study the development of pelvic allodynia in a murine model of CP/CPPS called experimental autoimmune prostatitis (EAP). The prostate lobes, dorsal root ganglia (DRG), and spinal cord were excised at day 20. The prostate lobes were assessed for inflammation, TRPV1 expression, and mast cell activity. DRG and spinal cord, between the L6-S4 regions, were analyzed to determine the levels of phosphorylated ERK1/2 (p-ERK 1/2). To examine the therapeutic potential of TRPV1, B6 mice with EAP received intraurethral infusion of a TRPV1 antagonist at day 20 (repeated every 2 days) and pelvic pain was evaluated at days 20, 25, 30, and 35. RESULTS: Our data showed that B6 mice with EAP developed pelvic tactile allodynia at days 7, 14, and 20. In contrast, TRPV1 KO mice with EAP do not develop pelvic tactile allodynia at any time point. Although we observed no change in the levels of TRPV1 protein expression in the prostate from B6 mice with EAP, there was evidence of significant inflammation and elevated mast cell activation. Interestingly, the prostate from TRPV1 KO mice with EAP showed a lack of mast cell activation despite evidence of prostate inflammation. Next, we observed a significant increase of p-ERK1/2 in the DRG and spinal cord from B6 mice with EAP; however, p-ERK1/2 expression was unaltered in TRPV1 KO mice with EAP. Finally, we confirmed that intraurethral administration of a TRPV1 antagonist peptide reduced pelvic tactile allodynia in B6 mice with EAP after day 20. CONCLUSIONS: We demonstrated that in a murine model of CP/CPPS, the TRPV1 channel is key to persistent pelvic tactile allodynia and blocking TRPV1 in the prostate may be a promising strategy to quell chronic pelvic pain.


Assuntos
Doenças Autoimunes/metabolismo , Prostatite/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Arginina/análogos & derivados , Arginina/farmacologia , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/imunologia , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/farmacologia , Dor Pélvica/tratamento farmacológico , Dor Pélvica/imunologia , Dor Pélvica/metabolismo , Dor Pélvica/patologia , Fosforilação , Prostatite/tratamento farmacológico , Prostatite/imunologia , Prostatite/patologia , Medula Espinal/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiência
15.
J Neurochem ; 152(5): 570-584, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31853976

RESUMO

Local microvascular dysfunction and consequent tissue ischemia/hypoxia contribute to the symptoms of complex regional pain syndrome (CRPS) and peripheral neuropathic pain. As nitric oxide (NO) is a key regulator of microvascular blood flow, compounds that increase it are potentially therapeutic for these pain conditions. This led us to hypothesize that the topical administration of drugs that modulate local tissue NO levels can alleviate the pain of CRPS and peripheral neuropathic pain. We investigated the anti-allodynic effect of a combination of two NO-modulating drugs: meldonium and N-acetylcysteine (NAC). An equimolar topical formulation of the two drugs was tested on chronic post-ischemic pain (CPIP), a rat model of CRPS, as well as chronic constriction injury (CCI) of the sciatic nerve and chemotherapy-induced painful neuropathy (CIPN), rat models of peripheral neuropathic pain. Topical meldonium-NAC produced significant anti-allodynia in CPIP, CCI, and CIPN rats. Moreover repeated application of topical meldonium-NAC produced an increase in the duration of anti-allodynia in the CPIP and CCI rats. While pre-treatment with an NO synthase inhibitor attenuated the anti-allodynic effects of meldonium-NAC, 30-min hyperbaric oxygen treatment combined with a non-effective dose of meldonium-NAC produced significant anti-allodynic effects in CPIP rats. Both experiments implicated NO in the drug combination's anti-allodynic effects. To ascertain the role played by changes in local tissue NO, we performed a quantification of plantar muscle NO in CPIP rats after hind paw topical treatment with meldonium-NAC and revealed significantly increased plantar muscle NO levels in drug-treated rats. The drug combination also reversed the reduction in tissue oxygenation normally observed in CPIP hind paws. In addition to introducing a novel topical treatment for mechanical allodynia in CRPS and peripheral neuropathic pain, this work showcases the analgesic potential of locally targeting microvascular dysfunction and tissue ischemia/hypoxia in these conditions, with emphasis on the role of NO.


Assuntos
Acetilcisteína/administração & dosagem , Metilidrazinas/administração & dosagem , Neuralgia/metabolismo , Óxido Nítrico/metabolismo , Distrofia Simpática Reflexa/metabolismo , Administração Tópica , Animais , Modelos Animais de Doenças , Hiperalgesia/metabolismo , Masculino , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley
16.
Eur J Pharmacol ; 869: 172882, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31863769

RESUMO

Remifentanil, ultra-short-acting µ-opioid receptor agonist, has the greatest advantage in analgesia but could increase postoperative pain scores and induces postoperative hyperalgesia. Dezocine is a mixed opioid receptor partial agonist/antagonist and has been used for postoperative hyperalgesia management in clinical patients,but the potential molecular mechanism is still unclear. Ca2+/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ) has been reported involved in remifentanil-induced hyperalgesia (RIH) in previous studies, but the relationship between CaMKⅡ and dezocine in RIH is still unclear. To investigate the mechanism of dezocine in RIH, we used a remifentanil induced postoperative hyperalgesia (RIPH) in incisional pain model of mouse. We subcutaneously infused remifentanil (40 µg/kg) to induce postoperative hyperalgesia. Dezocine (1.5 mg/kg, 3.0 mg/kg, and 6.0 mg/kg) was infused subcutaneously with remifentanil using the apparatus pump for 30 min. Paw withdrawal thermal latency (PWTL) and paw withdrawal mechanical threshold (PWMT) were used to assess thermal hyperalgesia and mechanical allodynia. Western blotting analysis and immunohistochemistry analysis were used to assess the expression of phosphorylated CaMKⅡα (p-CaMKⅡα) in somatosensory cortex, hippocampus and spinal cord. Subcutaneous infusion of remifentanil enhanced postoperative pain induced by surgical incision and increased PWTL and PWMT. Dezocine dose-dependently decreased the PWTL and PWMT in RIPH model. Correlating with behavioral effects, dezocine inhibited remifentanil-induced up-regulation of p-CaMKⅡα expression in somatosensory cortex, hippocampus and spinal cord. Dezocine could attenuate RIPH by suppressing p-CaMKⅡα.


Assuntos
Analgésicos Opioides/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Hiperalgesia/metabolismo , Dor Pós-Operatória/metabolismo , Tetra-Hidronaftalenos/farmacologia , Analgésicos Opioides/uso terapêutico , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Masculino , Camundongos Endogâmicos ICR , Dor Pós-Operatória/induzido quimicamente , Dor Pós-Operatória/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Período Pós-Operatório , Remifentanil , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Tetra-Hidronaftalenos/uso terapêutico
17.
Nat Commun ; 10(1): 5678, 2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31831729

RESUMO

As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8+ cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8+ neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8+ cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8+ sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8+ cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.


Assuntos
Córnea/metabolismo , Nociceptividade/fisiologia , Células Receptoras Sensoriais/metabolismo , Substância P/metabolismo , Canais de Cátion TRPV/metabolismo , Sensação Térmica/fisiologia , Animais , Temperatura Baixa , Síndromes do Olho Seco , Regulação da Expressão Gênica , Hiperalgesia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPV/genética , Tamoxifeno/farmacologia
18.
Int J Mol Sci ; 20(24)2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861182

RESUMO

Insulin-like growth factor-1 (IGF-1) is upregulated in the injured peripheral nerve bundle and controls nociceptive neuronal excitability associated with peripheral nerve injury. Here, we examined the involvement of IGF-1 signaling in orofacial neuropathic pain following infraorbital nerve injury (IONI) in rats. IONI promoted macrophage accumulation in the injured ION, as well as in the ipsilateral trigeminal ganglion (TG), and induced mechanical allodynia of the whisker pad skin together with the enhancement of neuronal activities in the subnucleus caudalis of the spinal trigeminal nucleus and in the upper cervical spinal cord. The levels of IGF-1 released by infiltrating macrophages into the injured ION and the TG were significantly increased. The IONI-induced the number of transient receptor potential vanilloid (TRPV) subfamily type 4 (TRPV4) upregulation in TRPV subfamily type 2 (TRPV2)-positive small-sized, and medium-sized TG neurons were inhibited by peripheral TRPV2 antagonism. Furthermore, the IONI-induced mechanical allodynia was suppressed by TRPV4 antagonism in the whisker pad skin. These results suggest that IGF-1 released by macrophages accumulating in the injured ION binds to TRPV2, which increases TRPV4 expression in TG neurons innervating the whisker pad skin, ultimately resulting in mechanical allodynia of the whisker pad skin.


Assuntos
Dor Facial/metabolismo , Hiperalgesia/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Neuralgia/metabolismo , Traumatismos do Nervo Trigêmeo/metabolismo , Animais , Dor Facial/fisiopatologia , Hiperalgesia/fisiopatologia , Macrófagos/metabolismo , Masculino , Neuralgia/fisiopatologia , Neurônios/metabolismo , Limiar da Dor , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Canais de Cátion TRPV/metabolismo , Gânglio Trigeminal , Traumatismos do Nervo Trigêmeo/fisiopatologia , Vibrissas/inervação , Vibrissas/metabolismo
19.
Molecules ; 24(23)2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31766439

RESUMO

Interstitial cystitis (IC) is a chronic bladder disorder with unclear etiology. The endocannabinoid system has been identified as a key regulator of immune function, with experimental evidence for the involvement of cannabinoid receptors in bladder inflammation. This study used intravital microscopy (IVM) and behavioral testing in lipopolysaccharide-induced IC, to investigate the anti-inflammatory analgesic effects of a natural dietary sesquiterpenoid, beta-caryophyllene (BCP), which is present in cannabis among other plants, and has reported agonist actions at the cannabinoid 2 receptor (CB2R). BCP's anti-inflammatory actions were compared to the synthetic CB2R-selective cannabinoid, HU308, and to an FDA-approved clinical treatment (dimethyl sulfoxide: DMSO). IVM data revealed that intravesical instillation of BCP and/or HU308 significantly reduces the number of adhering leukocytes in submucosal bladder venules and improves bladder capillary perfusion. The effects of BCP were found to be comparable to that of the selective CB2R synthetic cannabinoid, HU308, and superior to intravesical DMSO treatment. Oral treatment with BCP was also able to reduce bladder inflammation and significantly reduced mechanical allodynia in experimental IC. Based on our findings, we believe that CB2R activation may represent a viable therapeutic target for IC, and that drugs that activate CB2R, such as the generally regarded as safe (GRAS) dietary sesquiterpenoid, BCP, may serve as an adjunct and/or alternative treatment option for alleviating symptoms of inflammation and pain in the management of IC.


Assuntos
Canabinoides/farmacologia , Cistite Intersticial/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Inflamação/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Sesquiterpenos Policíclicos/farmacologia , Receptor CB2 de Canabinoide/metabolismo , Animais , Comportamento Animal/efeitos dos fármacos , Cistite Intersticial/induzido quimicamente , Cistite Intersticial/metabolismo , Cistite Intersticial/patologia , Modelos Animais de Doenças , Feminino , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C
20.
Elife ; 82019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31742556

RESUMO

Neuropathic pain is a debilitating condition caused by the abnormal processing of somatosensory input. Synaptic inhibition in the spinal dorsal horn plays a key role in that processing. Mechanical allodynia - the misperception of light touch as painful - occurs when inhibition is compromised. Disinhibition is due primarily to chloride dysregulation caused by hypofunction of the potassium-chloride co-transporter KCC2. Here we show, in rats, that excitatory neurons are disproportionately affected. This is not because chloride is differentially dysregulated in excitatory and inhibitory neurons, but, rather, because excitatory neurons rely more heavily on inhibition to counterbalance strong excitation. Receptive fields in both cell types have a center-surround organization but disinhibition unmasks more excitatory input to excitatory neurons. Differences in intrinsic excitability also affect how chloride dysregulation affects spiking. These results deepen understanding of how excitation and inhibition are normally balanced in the spinal dorsal horn, and how their imbalance disrupts somatosensory processing.


Assuntos
Cloretos/metabolismo , Neurônios/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Animais , Feminino , Hiperalgesia/metabolismo , Masculino , Modelos Animais , Modelos Biológicos , Fenômenos Fisiológicos do Sistema Nervoso , Neuralgia/metabolismo , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Simportadores/metabolismo
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