RESUMO
Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in >90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.
Assuntos
Cálcio , Gânglio Trigeminal , Ratos , Camundongos , Animais , Células Receptoras Sensoriais , Corpo Celular , CraniotomiaRESUMO
Different peripheral nerve injuries cause neuropathic pain through distinct mechanisms. Even the site of injury may impact underlying mechanisms, as indicated by the clinical finding that the antiseizure drug carbamazepine (CBZ) relieves pain because of compression injuries of trigeminal but not somatic nerves. We leveraged this observation in the present study hypothesizing that because CBZ blocks voltage-gated sodium channels (VGSCs), its therapeutic selectivity reflects differences between trigeminal and somatic nerves with respect to injury-induced changes in VGSCs. CBZ diminished ongoing and evoked pain behavior in rats with chronic constriction injury (CCI) to the infraorbital nerve (ION) but had minimal effect in rats with sciatic nerve CCI. This difference in behavior was associated with a selective increase in the potency of CBZ-induced inhibition of compound action potentials in the ION, an effect mirrored in human trigeminal versus somatic nerves. The increase in potency was associated with a selective increase in the efficacy of the NaV1.1 channel blocker ICA-121431 and NaV1.1 protein in the ION, but no change in NaV1.1 mRNA in trigeminal ganglia. Importantly, local ICA-121431 administration reversed ION CCI-induced hypersensitivity. Our results suggest a novel therapeutic target for the treatment of trigeminal neuropathic pain.SIGNIFICANCE STATEMENT This study is based on evidence of differences in pain and its treatment depending on whether the pain is above (trigeminal) or below (somatic) the neck, as well as evidence that voltage-gated sodium channels (VGSCs) may contribute to these differences. The focus of the present study was on channels underlying action potential propagation in peripheral nerves. There were differences between somatic and trigeminal nerves in VGSC subtypes underlying action potential propagation both in the absence and presence of injury. Importantly, because the local block of NaV1.1 in the trigeminal nerve reverses nerve injury-induced mechanical hypersensitivity, the selective upregulation of NaV1.1 in trigeminal nerves suggests a novel therapeutic target for the treatment of pain associated with trigeminal nerve injury.
Assuntos
Analgésicos não Narcóticos/uso terapêutico , Carbamazepina/uso terapêutico , Neuralgia/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Neuralgia do Trigêmeo/tratamento farmacológico , Analgésicos não Narcóticos/farmacologia , Animais , Carbamazepina/farmacologia , Feminino , Masculino , Canal de Sódio Disparado por Voltagem NAV1.1/biossíntese , Neuralgia/metabolismo , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Neuralgia do Trigêmeo/metabolismoRESUMO
BACKGROUND: Peripheral diabetic neuropathy can be painful and its symptoms include hyperalgesia, allodynia and spontaneous pain. Hydrogen sulfide (H2S) is involved in diabetes-induced hyperalgesia and allodynia. However, the molecular target through which H2S induces hyperalgesia in diabetic animals is unclear. The aim of this study was to determine the possible involvement of transient receptor potential (TRP) channels in H2S-induced hyperalgesia in diabetic rats. RESULTS: Streptozotocin (STZ) injection produced hyperglycemia in rats. Intraplantar injection of NaHS (an exogenous donor of H2S, 3-100 µg/paw) induced hyperalgesia, in a time-dependent manner, in formalin-treated diabetic rats. NaHS-induced hyperalgesia was partially prevented by local intraplantar injection of capsazepine (0.3-3 µg/paw), HC-030031 (100-316 µg/paw) and SKF-96365 (10-30 µg/paw) blockers, at 21 days post-STZ injection. At the doses used, these blockers did not modify formalin-induced nociception. Moreover, capsazepine (0.3-30 µg/paw), HC-030031 (100-1000 µg/paw) and SKF-96365 (10-100 µg/paw) reduced formalin-induced nociception in diabetic rats. Contralateral injection of the highest doses used did not modify formalin-induced flinching behavior. Hyperglycemia, at 21 days, also increased protein expression of cystathionine-ß-synthase enzyme (CBS) and TRPC6, but not TRPA1 nor TRPV1, channels in dorsal root ganglia (DRG). Repeated injection of NaHS enhanced CBS and TRPC6 expression, but hydroxylamine (HA) prevented the STZ-induced increase of CBS protein. In addition, daily administration of SKF-96365 diminished TRPC6 protein expression, whereas NaHS partially prevented the decrease of SKF-96365-induced TRPC6 expression. Concordantly, daily intraplantar injection of NaHS enhanced, and HA prevented STZ-induced intraepidermal fiber loss, respectively. CBS was expressed in small- and medium-sized cells of DRG and co-localized with TRPV1, TRPA1 and TRPC6 in IB4-positive neurons. CONCLUSIONS: Our data suggest that H2S leads to hyperalgesia in diabetic rats through activation of TRPV1, TRPA1 and TRPC channels and, subsequent intraepidermal fibers loss. CBS enzyme inhibitors or TRP-channel blockers could be useful for treatment of painful diabetic neuropathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Sulfeto de Hidrogênio/metabolismo , Hiperalgesia/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Acetanilidas/farmacologia , Analgésicos/farmacologia , Animais , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Cistationina beta-Sintase/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Feminino , Formaldeído , Hidroxilamina/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/patologia , Imidazóis/farmacologia , Nociceptividade/efeitos dos fármacos , Nociceptividade/fisiologia , Purinas/farmacologia , Ratos Wistar , Pele/inervação , Pele/metabolismo , Raízes Nervosas Espinhais/efeitos dos fármacos , Raízes Nervosas Espinhais/metabolismo , Raízes Nervosas Espinhais/patologia , SulfitosRESUMO
BACKGROUND: The purpose of this study was to evaluate the participation of satellite glial cells (SGC), microglia and astrocytes in a model of streptozotocin-induced diabetes initiated in neonatal rats (nSTZ) and to determine the pharmacological profile for pain relief. METHODS: nSTZ was used to induce experimental diabetes. Von Frey filaments were used to assess tactile allodynia. Drugs were given by systemic administration. Western blotting and immunohistochemistry were used to determine protein expression and cellular localization. RESULTS: nSTZ produced mild hyperglycemia, weight loss, glucose intolerance, and reduction of nerve conduction velocity of C fibers. Moreover, nSTZ enhanced activating transcription factor 3 (ATF3) immunoreactivity in dorsal root ganglia (DRG) and sciatic nerve of adult rats. ATF3 was found in SGC (GFAP+ cells) surrounding DRG at week 16. Late changes in ATF3 immunoreactivity in DRG correlated with up-regulation of ATF3 and GFAP protein expression. nSTZ increased GFAP and OX-42 immunoreactivity and percentage of hypertrophied and ameboid microglia in the spinal dorsal horn. These changes correlated with the presence of mechanical hypersensitivity (tactile allodynia). Administration of gabapentin (30-100mg/kg, po) and metformin (200mg/kg/day, po for 2 weeks) alleviated tactile allodynia, whereas morphine (1-3mg/kg, ip) had a modest effect. CONCLUSIONS: Results suggest that nSTZ leads to activation of SGC, microglia and astrocytes in DRG and spinal cord. Pharmacological profile in the nSTZ model resembles diabetic neuropathic pain in humans. Our findings support the conclusion that the nSTZ rat model has utility for the study of a long-lasting diabetic neuropathic pain.
Assuntos
Diabetes Mellitus Experimental/patologia , Neuropatias Diabéticas/patologia , Neuralgia/patologia , Estreptozocina/farmacologia , Fator 3 Ativador da Transcrição , Aminas/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Ácidos Cicloexanocarboxílicos/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Neuropatias Diabéticas/tratamento farmacológico , Neuropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Gabapentina , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Masculino , Metformina/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/patologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Medição da Dor/métodos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
BACKGROUND: Nerve injury promotes release of 5-HT at the spinal cord. Once released, 5-HT may produce antinociceptive or pronociceptive effects depending of the nature of 5-HT receptors. The purpose of this study was to investigate the participation of spinal 5-HT4 and 5-HT6 receptors in the maintenance of neuropathic pain in rats. METHODS: Tactile allodynia was measured using von Frey hairs in male Wistar rats subjected to L5-L6 spinal nerve injury. Selective 5-HT4 (GR-113808, 0.01-10nmol/rat) and 5-HT6 (SB-258585, 1-1000nmol/rat) receptor antagonists were administered intrathecally to nerve injured rats. Likewise, the most effective dose of 5-HT4 (1nmol/rat) and 5-HT6 (100 nmol/rat) antagonists were co-administered with their respective agonists (ML-10302, 10-100nmol/rat and WAY-208466, 100-1000nmol/rat, respectively). Spinal cord protein expression of both receptors was determined by western blot. RESULTS: Intrathecal administration of 5-HT4 or 5-HT6 receptor antagonists, but not vehicle, decreased in a dose-dependent manner tactile allodynia in neuropathic rats. Moreover, intrathecal co-administration with the agonists prevented in a dose-dependent manner the antagonists-induced antiallodynic effect. Both 5-HT4 and 5-HT6 receptors were expressed in the spinal cord of naïve, sham and neuropathic rats. Nerve injury did not modify expression of any receptor. CONCLUSIONS: Data suggests that spinal 5-HT4 and 5-HT6 receptors are expressed in dorsal spinal cord and they participate in the maintenance of neuropathic pain in rats. In this regard, blockade of these receptors could be a useful strategy to treat neuropathic pain states.
Assuntos
Neuralgia/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Nervos Espinhais/patologia , Animais , Indóis/farmacologia , Masculino , Piperazinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Antagonistas do Receptor 5-HT4 de Serotonina , Sulfonamidas/farmacologiaRESUMO
The purpose of this study was to investigate whether 1%, 2% or 5% formalin injection produce hypersensitivity with characteristics of the neuropathic pain induced by spinal nerve injury. Formalin injection (1%, 2% and 5%) produced concentration-dependent long-lasting (at least 14 days) mechanical allodynia and hyperalgesia in both paws. Likewise, L5/L6 spinal nerve ligation induced allodynia and hyperalgesia in both paws. The intensity of hypersensitivity was greater in the ipsilateral than in the contralateral paw in all models. Systemic gabapentin or morphine completely reduced 1% formalin-induced hypersensitivity. In contrast, both drugs were not able to fully diminish 2-5% formalin- and nerve injury-induced hypersensitivity. Indomethacin produced a significant effect in the chronic 1% formalin test. Conversely, this drug did not modify 2 or 5% formalin- and nerve injury-induced hypersensitivity. Spinal nerve injury and 2-5%, but not 1%, formalin injection enhanced ATF3 protein expression and immunofluorescence in dorsal root ganglia (DRG) in a time-dependent manner. Furthermore, 2-5%, but not 1%, formalin injection or spinal nerve injury also enhanced α2δ-1 subunit protein levels in DRG. Our results suggest that 5% and, at lesser extent, 2% formalin injection produces long-lasting hypersensitivity with a pharmacological and molecular pattern that resembles neuropathic pain induced by spinal nerve ligation.
Assuntos
Formaldeído/farmacologia , Neuralgia/induzido quimicamente , Fator 3 Ativador da Transcrição/metabolismo , Aminas/farmacologia , Animais , Ácidos Cicloexanocarboxílicos/farmacologia , Feminino , Formaldeído/administração & dosagem , Gabapentina , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Indometacina/farmacologia , Injeções , Ligadura/efeitos adversos , Morfina/farmacologia , Neuralgia/metabolismo , Ratos , Fatores de Tempo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Essential fatty acids have an important effect on oxidative stress-related diseases. The Huntington's disease (HD) is a hereditary neurologic disorder in which oxidative stress caused by free radicals is an important damage mechanism. The HD experimental model induced by quinolinic acid (QUIN) has been widely used to evaluate therapeutic effects of antioxidant compounds. The aim of this study was to test whether the fatty acid content in olive- or fish-oil-rich diet prevents against QUIN-related oxidative damage in rats. Rats were fed during 20 days with an olive- or a fish-oil-rich diet (15% w/w). Posterior to diet period, rats were striatally microinjected with QUIN (240â nmol/µl) or saline solution. Then, we evaluated the neurological damage, oxidative status, and gamma isoform of the peroxisome proliferator-activated receptor (PPARγ) expression. Results showed that fatty acid-rich diet, mainly by fish oil, reduced circling behavior, prevented the fall in GABA levels, increased PPARγ expression, and prevented oxidative damage in striatal tissue. In addition none of the enriched diets exerted changes neither on triglycerides or cholesterol blood levels, nor or hepatic function. This study suggests that olive- and fish-oil-rich diets exert neuroprotective effects.
Assuntos
Corpo Estriado/efeitos dos fármacos , Ácidos Graxos Essenciais/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Quinolínico/toxicidade , Animais , Peso Corporal , Colesterol/sangue , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Óleos de Peixe/farmacologia , Doença de Huntington/tratamento farmacológico , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Fármacos Neuroprotetores/farmacologia , Azeite de Oliva/farmacologia , Ratos , Ratos Wistar , Triglicerídeos/sangue , Ácido gama-Aminobutírico/metabolismoRESUMO
The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1-3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1-3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.
Assuntos
Neuralgia/patologia , Receptores Purinérgicos P2Y1/metabolismo , Medula Espinal/metabolismo , Regulação para Cima/fisiologia , Animais , Axotomia/efeitos adversos , Nucleotídeos de Desoxiadenina/uso terapêutico , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Ligadura/efeitos adversos , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Ratos , Ratos Wistar , Receptores Purinérgicos P2Y1/genética , Medula Espinal/efeitos dos fármacos , Nervos Espinhais/lesões , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Serotonin (5-HT) participates in pain modulation by interacting with different 5-HT receptors. The role of 5-HT5A receptor in neuropathic pain has not previously studied. The purpose of this study was to investigate: A) the role of 5-HT5A receptors in rats subjected to spinal nerve injury; B) the expression of 5-HT5A receptors in dorsal spinal cord and dorsal root ganglia (DRG). Neuropathic pain was induced by L5/L6 spinal nerve ligation. Tactile allodynia in neuropathic rats was assessed with von Frey filaments. Western blot methodology was used to determine 5-HT5A receptor protein expression. Intrathecal administration (on day 14th) of 5-HT (10-100 nmol) or 5-carboxamidotryptamine (5-CT, 0.03-0.3 nmol) reversed nerve injury-induced tactile allodynia. Intrathecal non-selective (methiothepin, 0.1-0.8 nmol) and selective (SB-699551, 1-10 nmol) 5-HT5A receptor antagonists reduced, by ~60% and ~25%, respectively, the antiallodynic effect of 5-HT (100 nmol) or 5-CT (0.3 nmol). Moreover, both selective 5-HT1A and 5-HT1B/1D receptor antagonists, WAY-100635 (0.3-1 nmol) and GR-127935 (0.3-1 nmol), respectively, partially diminished the antiallodynic effect of 5-HT or 5-CT by about 30%. Injection of antagonists, by themselves, did not affect allodynia. 5-HT5A receptors were expressed in the ipsilateral dorsal lumbar spinal cord and DRG and L5/L6 spinal nerve ligation did not modify 5-HT5A receptor protein expression in those sites. Results suggest that 5-HT5A receptors reduce pain processing in the spinal cord and that 5-HT and 5-CT reduce neuropathic pain through activation of 5-HT5A and 5-HT1A/1B/1D receptors. These receptors could be an important part of the descending pain inhibitory system.
Assuntos
Neuralgia/metabolismo , Receptores 5-HT1 de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Medula Espinal/metabolismo , Nervos Espinhais/lesões , Analgésicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Vértebras Lombares , Metiotepina/farmacologia , Neuralgia/tratamento farmacológico , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Ratos Wistar , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Serotonina/análogos & derivados , Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Medula Espinal/efeitos dos fármacos , TatoRESUMO
BACKGROUND: Calcium-activated chloride channels (CaCCs) activation induces membrane depolarization by increasing chloride efflux in primary sensory neurons that can facilitate action potential generation. Previous studies suggest that CaCCs family members bestrophin-1 and anoctamin-1 are involved in inflammatory pain. However, their role in neuropathic pain is unclear. In this investigation we assessed the involvement of these CaCCs family members in rats subjected to the L5/L6 spinal nerve ligation. In addition, anoctamin-1 and bestrophin-1 mRNA and protein expression in dorsal root ganglion (DRG) and spinal cord was also determined in the presence and absence of selective inhibitors. RESULTS: L5/L6 spinal nerve ligation induced mechanical tactile allodynia. Intrathecal administration of non-selective CaCCs inhibitors (NPPB, 9-AC and NFA) dose-dependently reduced tactile allodynia. Intrathecal administration of selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) also dose-dependently diminished tactile allodynia and thermal hyperalgesia. Anoctamin-1 and bestrophin-1 mRNA and protein were expressed in the dorsal spinal cord and DRG of naïve, sham and neuropathic rats. L5/L6 spinal nerve ligation rose mRNA and protein expression of anoctamin-1, but not bestrophin-1, in the dorsal spinal cord and DRG from day 1 to day 14 after nerve ligation. In addition, repeated administration of CaCCs inhibitors (T16Ainh-A01, CaCCinh-A01 or NFA) or anti-anoctamin-1 antibody prevented spinal nerve ligation-induced rises in anoctamin-1 mRNA and protein expression. Following spinal nerve ligation, the compound action potential generation of putative C fibers increased while selective CaCCs inhibitors (T16Ainh-A01 and CaCCinh-A01) attenuated such increase. CONCLUSIONS: There is functional anoctamin-1 and bestrophin-1 expression in rats at sites related to nociceptive processing. Blockade of these CaCCs suppresses compound action potential generation in putative C fibers and lessens established tactile allodynia. As CaCCs activity contributes to neuropathic pain maintenance, selective inhibition of their activity may function as a tool to generate analgesia in nerve injury pain states.
Assuntos
Canais de Cloreto/metabolismo , Neuralgia/metabolismo , Nervos Espinhais/patologia , Animais , Anoctamina-1 , Bestrofinas , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Feminino , Hiperalgesia/complicações , Hiperalgesia/patologia , Hiperalgesia/fisiopatologia , Injeções Espinhais , Ligadura , Atividade Motora , Condução Nervosa , Neuralgia/complicações , Neuralgia/patologia , Neuralgia/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Nervos Espinhais/lesões , Nervos Espinhais/fisiopatologiaRESUMO
Metabotropic P2Y receptors subfamily consists of eight functional mammalian receptors. Specifically, P2Y1, P2Y6 and P2Y11 receptors have been described in the sensory nervous system, but their participation, at peripheral level, in behavioral pain models is scarcely understood. This study assessed the role of peripheral P2Y1, P2Y6 and P2Y11 receptors in formalin-induced inflammatory pain. Ipsilateral, but not contralateral peripheral pre-treatment with the endogenous P2Y1 (ADP, 100-1000nmol/paw), P2Y6 (UDP, 180-300nmol/paw) and P2Y11 (ATP, 100-1000nmol/paw), or selective P2Y1 (MRS2365, 0.1-10nmol/paw), P2Y6 (PSB0474, 0.1-0.10pmol/paw) and P2Y11 (NF546, 0.3-3nmol/paw) receptor agonists increased 0.5% formalin-induced flinching behavior. Concordantly, peripheral pre-treatment with the selective P2Y1 (MRS2500, 0.01-10pmol/paw), P2Y6 (MRS2578, 3-30nmol/paw) and P2Y11 (NF340, 1-10nmol/paw) receptor antagonists significantly decreased 1% formalin-induced flinching behavior. Furthermore, the pronociceptive effect of ADP (100nmol/paw) or MRS2365 (10nmol/paw), UDP (300nmol/paw) or PSB0474 (10pmol/paw) and ATP (1000nmol/paw) or NF546 (3nmol/paw) was blocked by the selective P2Y1 (MRS2500, 0.01nmol/paw), P2Y6 (MRS2578, 3nmol/paw), and P2Y11 (NF340, 1nmol/paw) receptor antagonists, respectively. Western blot analysis confirmed the presence of P2Y1 (66kDa), P2Y6 (36kDa) and P2Y11 (75kDa) receptors in dorsal root ganglia (DRG) and sciatic nerve. Results suggest that peripheral activation of P2Y1, P2Y6 and P2Y11 receptors plays a pronociceptive role in formalin-induced pain.
Assuntos
Dor Nociceptiva/fisiopatologia , Receptores Purinérgicos P2Y1/fisiologia , Receptores Purinérgicos P2/fisiologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Formaldeído/toxicidade , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/fisiopatologia , Inflamação/fisiopatologia , Isotiocianatos/farmacologia , Nociceptividade/efeitos dos fármacos , Nociceptividade/fisiologia , Dor Nociceptiva/induzido quimicamente , Nociceptores/efeitos dos fármacos , Nociceptores/fisiologia , Agonistas do Receptor Purinérgico P2/farmacologia , Antagonistas do Receptor Purinérgico P2/farmacologia , Ratos , Ratos Wistar , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/fisiopatologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Difosfato de Uridina/análogos & derivados , Difosfato de Uridina/metabolismo , Difosfato de Uridina/farmacologiaRESUMO
We determined the role of chloride-bicarbonate anion exchanger 3 in formalin-induced acute and chronic rat nociception. Formalin (1%) produced acute (first phase) and tonic (second phase) nociceptive behaviors (flinching and licking/lifting) followed by long-lasting evoked secondary mechanical allodynia and hyperalgesia in both paws. Local peripheral pre-treatment with the chloride-bicarbonate anion exchanger inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulfonic acid prevented formalin-induced nociception mainly during phase 2. These drugs also prevented in a dose-dependent fashion long-lasting evoked secondary mechanical allodynia and hyperalgesia in both paws. Furthermore, post-treatment (on day 1 or 6) with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid reversed established hypersensitivity. Anion exchanger 3 was expressed in dorsal root ganglion neurons and it co-localized with neuronal nuclei protein (NeuN), substance P and purinergic P2X3 receptors. Furthermore, Western blot analysis revealed a band of about 85 kDa indicative of anion exchanger 3 protein expression in dorsal root ganglia of naïve rats, which was enhanced at 1 and 6 days after 1% formalin injection. On the other hand, this rise failed to occur during 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid exposure. These results suggest that anion exchanger 3 is present in dorsal root ganglia and participates in the development and maintenance of short and long-lasting formalin-induced nociception.
Assuntos
Antiportadores de Cloreto-Bicarbonato/metabolismo , Formaldeído/efeitos adversos , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Dor/induzido quimicamente , Dor/metabolismo , Animais , Feminino , Formaldeído/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Dor/fisiopatologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Ácidos Sulfônicos/farmacologiaRESUMO
BACKGROUND: The participation of spinal P2X receptors in neuropathic pain is well recognized. However, the role of P2Y receptors has been less studied. The purpose of this study was to investigate the contribution of spinal P2Y6,11 receptors following peripheral nerve damage induced by spinal nerve ligation. In addition, we determined the expression of P2Y6,11 receptors in the dorsal spinal cord in presence of the selective P2Y6,11 receptors antagonists. Furthermore, we evaluated the participation of spinal microglia and astrocytes in the pronociceptive role of P2Y6,11 receptors. RESULTS: Spinal administration of the selective P2Y6 (MRS2578, 10-100 µM) and P2Y11 (NF340, 0.3-30 µM) receptor antagonists reduced tactile allodynia in spinal nerve ligated rats. Nerve injury increased the expression of P2Y6,11 receptors at 7, 14 and 21 days after injury. Furthermore, intrathecal administration of MRS2578 (100 µM/day) and NF340 (30 µM/day) for 3 days significantly reduced spinal nerve injury-induced increase in P2Y6,11 receptors expression, respectively. Spinal treatment (on day 14 after injury) with minocycline (100 µg/day) or fluorocitrate (1 nmol/day) for 7 days reduced tactile allodynia and spinal nerve injury-induced up-regulation in Iba-1 and GFAP, respectively. In addition, minocycline reduced nerve injury-induced up-regulation in P2Y6,11 receptors whereas that fluorocitrate diminished P2Y11, but not P2Y6, receptors up-regulation. Intrathecal treatment (on day 21 after injury) with the selective P2Y6 (PSB0474, 3-30 µM) and P2Y11 (NF546, 1-10 µM) receptor agonists produced remarkable tactile allodynia in nerve ligated rats previously treated with minocycline or fluorocitrate for 7 days. CONCLUSIONS: Our data suggest that spinal P2Y6 is present in spinal microglia while P2Y11 receptors are present in both spinal microglia and astrocytes, and both receptors are up-regulated in rats subjected to spinal nerve injury. In addition, our data suggest that the spinal P2Y6 and P2Y11 receptors participate in the maintenance of neuropathic pain.
Assuntos
Neuralgia/patologia , Neuroglia/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Medula Espinal/patologia , Animais , Citratos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Lateralidade Funcional , Expressão Gênica/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Isotiocianatos/farmacologia , Minociclina/farmacologia , Neuralgia/complicações , Medição da Dor , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ratos , Ratos Wistar , Medula Espinal/metabolismo , Nervos Espinhais/lesões , Tioureia/análogos & derivados , Tioureia/farmacologia , Regulação para CimaRESUMO
Neuropathic pain is a serious physical disabling condition resulting from lesion or dysfunction of the peripheral sensory nervous system. Despite the fact that the mechanisms underlying neuropathic pain are poorly understood, the involvement of voltage-gated calcium (Ca(V)) channels in its pathophysiology has justified the use of drugs that bind the Ca(V) channel α2δ auxiliary subunit, such as gabapentin (GBP), to attain analgesic and anti-allodynic effects in models involving neuronal sensitization and nerve injury. GBP binding to α2δ inhibits nerve injury-induced trafficking of the α1 pore forming subunits of Ca(V) channels, particularly of the N-type, from the cytoplasm to the plasma membrane of pre-synaptic terminals in dorsal root ganglion neurons and dorsal horn spinal neurons. In the search for alternative forms of treatment, in this study we describe the synthesis and pharmacological profile of a GABA derivative, 2-aminoadamantane-1-carboxylic acid (GZ4), which displays a close structure-activity relationship with GBP. Behavioral assessment using von Frey filament stimuli showed that GZ4 treatment reverted mechanical allodynia/hyperalgesia in an animal model of spinal nerve ligation-induced neuropathic pain. In addition, using the patch clamp technique we show that GZ4 treatment significantly decreased whole-cell currents through N-type Ca(V) channels heterologously expressed in HEK-293 cells. Interestingly, the behavioral and electrophysiological time course of GZ4 actions reflects that its mechanism of action is similar but not identical to that of GBP. While GBP actions require at least 24 h and imply uptake of the drug, which suggests that the drug acts mainly intracellularly affecting channels trafficking to the plasma membrane, the faster time course (1-3 h) of GZ4 effects suggests also a direct inhibition of Ca(2+) currents acting on cell surface channels.
Assuntos
Adamantano/análogos & derivados , Analgésicos/farmacologia , Canais de Cálcio Tipo N/metabolismo , Neuralgia/tratamento farmacológico , Adamantano/síntese química , Adamantano/química , Adamantano/farmacologia , Analgésicos/síntese química , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Estrutura Molecular , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
The present study aimed to evaluate the antinociceptive activity (in inflammatory and neuropathic pain models) and gastroprotective effect of the 3,5-diprenyl-4-hydroxyacetophenone (HYDP), isolated from Ageratina pichinchensis. The gastroprotective activity of this plant was previously reported by our workgroup, finding encesanescin to be one active compound. The present results show that HYDP reduced nociception in a dose-dependent manner in carrageenan and L5/L6 spinal nerve ligation, with efficacies of 72.6 and 57.1%, respectively, at doses of 100 and 562 mg/kg. HYDP also showed gastroprotective activity in the model of ethanol-induced gastric lesion, with a 75.59% maximum inhibition of ulcers at a dose of 100mg/kg. This gastroprotective effect was attenuated by N(G)-nitro-L-arginine methyl ester, indomethacin and N-ethylmaleimide, indicating that NO, prostaglandins and sulfhydryl groups are involved in the mechanisms of action. This is the first evidence, to our knowledge, of the antinociceptive and gastroprotective activities of HYDP.
Assuntos
Acetofenonas/uso terapêutico , Ageratina/química , Analgésicos/uso terapêutico , Inflamação/tratamento farmacológico , Neuralgia/tratamento farmacológico , Fitoterapia , Úlcera Gástrica/tratamento farmacológico , Acetofenonas/isolamento & purificação , Acetofenonas/farmacologia , Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Animais , Carragenina , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Etanol , Fármacos Gastrointestinais/isolamento & purificação , Fármacos Gastrointestinais/farmacologia , Fármacos Gastrointestinais/uso terapêutico , Inflamação/metabolismo , Ligadura , Masculino , Neuralgia/metabolismo , Óxido Nítrico/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Prostaglandinas/metabolismo , Ratos , Ratos Wistar , Nervos Espinhais , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologia , Compostos de Sulfidrila/metabolismoRESUMO
The role of peripheral and spinal 5-HT(3) receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia in both paws. In experiments where the test drug was anticipated to augment or antagonize the response, 0.5 or 1% formalin, respectively, was used for injection. Peripheral ipsilateral, but not contralateral, pre-treatment (-10 min) with serotonin (5-HT, 10-100 nmol/paw) and the selective 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG, 10-300 nmol/paw) increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. Moreover, spinal pre-treatment with m-CPBG (10-300 nmol/rat) increased 0.5% formalin-induced secondary hyperalgesia but not allodynia in both paws. Accordingly, peripheral ipsilateral (30-300 nmol/paw), but not contralateral (300 nmol/paw), and spinal (10-100 nmol) pre-treatment with the selective 5-HT(3) receptor antagonist ondansetron prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. The peripheral pronociceptive effects of 5-HT (100 nmol/paw) and m-CPBG (300 nmol/paw) as well as the spinal effect of m-CPBG (300 nmol/rat) were completely prevented by the peripheral (10 nmol/paw) and spinal (1 nmol/rat) injection, respectively, of ondansetron. At these doses, ondansetron did not modify per se formalin-induced nociceptive behaviors. Spinal (30-300 nmol/rat), but not peripheral (300 nmol/paw), post-treatment (on day 6) with ondansetron reversed established formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT(3) receptors participates in the development of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. In addition, our data suggest that spinal 5-HT(3) receptors play an important role during development and maintenance of these evoked long-term behaviors.