RESUMEN
Peripheral nerve injury-induced mechanical allodynia is often accompanied by abnormalities in the higher cortical regions, yet the mechanisms underlying such maladaptive cortical plasticity remain unclear. Here, we show that in male mice, structural and functional changes in the primary somatosensory cortex (S1) caused by peripheral nerve injury require neuron-microglial signaling within the local circuit. Following peripheral nerve injury, microglia in the S1 maintain ramified morphology and normal density but up-regulate the mRNA expression of brain-derived neurotrophic factor (BDNF). Using in vivo two-photon imaging and Cx3cr1CreER;Bdnfflox mice, we show that conditional knockout of BDNF from microglia prevents nerve injury-induced synaptic remodeling and pyramidal neuron hyperactivity in the S1, as well as pain hypersensitivity in mice. Importantly, S1-targeted removal of microglial BDNF largely recapitulates the beneficial effects of systemic BDNF depletion on cortical plasticity and allodynia. Together, these findings reveal a pivotal role of cerebral microglial BDNF in somatosensory cortical plasticity and pain hypersensitivity.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Encéfalo/metabolismo , Hiperalgesia/fisiopatología , Microglía/metabolismo , Plasticidad Neuronal/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Factor Neurotrófico Derivado del Encéfalo/genética , Ratones , Ratones Noqueados , Traumatismos de los Nervios Periféricos/fisiopatologíaRESUMEN
OBJECTIVES: Spinal cord ischemia secondary to trauma or a vascular occlusive event is a threatening phenomenon. The neuroprotective properties of minocycline have been shown in several models of central nervous system diseases and after spinal cord ischemia; however, the benefit of using the drug requires additional confirmation in different animal models. Astrocytes are essential as regulators of neuronal functions and for providing nutrients. The authors hypothesized that astrocytes in the spinal cord may be an important target for minocycline action after ischemia and thus in the prevention of secondary spreading damage. DESIGN: A prospective, randomized animal study. SETTING: University research laboratory, single institution. PARTICIPANTS: Adult male Sprague Dawley rats, weighing between 400 and 450 g. INTERVENTIONS: A model of spinal cord ischemia in the rat was used for this study to determine whether a single, high-dose (10 mg/kg) of minocycline protects against damage to the neuronal cytoskeleton, both in the white and gray matter, and whether it reduces glial fibrillary acidic protein levels, which is an index for prevention of astrocyte activation during ischemia. Thirty minutes before thoracic aorta occlusion, minocycline was administered for 18 minutes using a 2 F Fogarty catheter. MEASUREMENTS AND MAIN RESULTS: Minocycline given prophylactically significantly mitigated severe hindlimb motor impairment and reduced glial fibrillary acidic protein plus astrocytosis in both the white and gray matter of the spinal cord, caudal to the occlusion. Neuronal histologic cytoarchitecture, which was severely and significantly compromised in control animals, was preserved in the minocycline-treated animals. CONCLUSIONS: This study's data imply that minocycline may attenuate reactive astrocytosis in response to injury with better neurologic outcome in a model of spinal cord ischemia in rats. The data suggest that future use of minocycline, clinically, might be advantageous in surgeries with a potential risk for paraplegia due to spinal cord ischemia.
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Arteriopatías Oclusivas/prevención & control , Gliosis/tratamiento farmacológico , Miembro Posterior/irrigación sanguínea , Minociclina/administración & dosificación , Paraplejía/tratamiento farmacológico , Isquemia de la Médula Espinal/tratamiento farmacológico , Animales , Arteriopatías Oclusivas/patología , Gliosis/patología , Miembro Posterior/efectos de los fármacos , Miembro Posterior/patología , Masculino , Neuronas/efectos de los fármacos , Neuronas/patología , Paraplejía/patología , Profilaxis Pre-Exposición/métodos , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Isquemia de la Médula Espinal/patologíaRESUMEN
In DRG an increase in miR-133b-3p, miR-143-3p, and miR-1-3p correlates with the lack of development of neuropathic pain following a peripheral nerve injury. Using lentiviral (LV) vectors we found that a single injection of LV-miR-133b-3p or LV-miR-143-3p immediately after a peripheral nerve injury prevented the development of sustained mechanical and cold allodynia. Injection of LV-miR-133b-3p or LV-miR-143-3p by themselves or in combination, on day 3 post-injury produced a partial and transient reduction in mechanical allodynia and a sustained decrease in cold allodynia. Injection of LV-miR-1-3p has no effect. Co-injection of LV-miR-1a with miR-133b-3p or miR-143-3p on day 3 post-injury produced a sustained decrease in mechanical and cold allodynia. In DRG cultures, miR-133b-3p and miR-143-3p but not miR-1-3p, enhanced the depolarization-evoked cytoplasmic calcium increase. Using 3'UTR target clones containing a Gaussian luciferase reporter gene we found that with the 3'UTR-Scn2b, miR-133-3p and miR-143-3p reduced the expression while miR-1-3p enhanced the expression of the reporter gene. With the 3'UTR-TRPM8, miR-133-3p and miR-143-3p reduced the expression and miR-1-3p had no effect. With the 3'UTR-Piezo2, miR-133-3p increased the expression while miR-143-3p and miR-1-3p had no effect. LV-miR133b-3p, LV-miR-143-3p and LV-miR1a-3p reduced Scn2b-mRNA and Piezo2-mRNA. LV-miR133b-3p and LV-miR-143-3p reduced TRPM8-mRNA. LV-miR-133b-3p and LV-miR-143-3p prevent the development of chronic pain when injected immediately after the injury, but are only partially effective when injected at later times. LV-miR-1a-3p had no effect on pain, but complemented the actions of LV-miR-133b-3p or LV-miR-143-3p resulting in a sustained reversal of pain when co-injected 3â¯days following nerve injury.
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Frío/efectos adversos , Hiperalgesia/prevención & control , MicroARNs/administración & dosificación , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Tacto/fisiología , Animales , Células HEK293 , Humanos , Hiperalgesia/metabolismo , Inyecciones Espinales , Masculino , MicroARNs/biosíntesis , Traumatismos de los Nervios Periféricos/metabolismo , Ratas , Ratas Sprague-Dawley , Tacto/efectos de los fármacosRESUMEN
Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-ß-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.
Asunto(s)
Ganglios Espinales/metabolismo , Región Lumbosacra , Neuroglía/metabolismo , Células Satélites Perineuronales/metabolismo , Animales , Conexinas/metabolismo , Inmunohistoquímica , Región Lumbosacra/lesiones , Masculino , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteína delta-6 de Union ComunicanteRESUMEN
BACKGROUND: The GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18. RESULTS: The effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a-GluN2A or GluN1a-GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a-GluN2B (84%) than those mediated by the GluN1a-GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a-GluN2B (62%) and the GluN1a-GluN2A (55%) subunits. CONCLUSIONS: The newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.
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Antagonistas de Aminoácidos Excitadores/farmacología , Neuronas/efectos de los fármacos , Péptidos/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Conotoxinas/farmacología , Antagonistas de Aminoácidos Excitadores/síntesis química , Células HEK293 , Hipocampo/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Péptidos/síntesis química , RatasRESUMEN
Different classes of Kv1 potassium channels have different trafficking patterns despite having very similar amino acid sequences. Two amino acids responsible for these differences have been identified in the outer pore turret region of Kv1.1 and Kv1.4. Here we tested a series of substitutions at these two determinants on Kv1.4. All P506 substitutions tested resulted in a significant decrease in surface protein, total protein, and protein half-life, indicating that proline is required at 506 to stabilize protein conformation and increase trafficking to the cell surface. All K533 substitutions tested had no effect on total protein, suggesting that the lysine at 533 is not important for maintaining Kv1.4 protein conformation. However, a basic or long polar amino acid, such as K, R, or Q, at this position favored high surface protein and efficient trafficking of Kv1.4, whereas an acidic or short amino acid, such as D, E, S, L, N, or H, at this position induced partial high endoplasmic reticulum-retention. This intracellular retention was not due to protein misfolding. We propose that these four prolines and four lysines in a Kv1.4 homotetramer might provide a binding site for a putative endoplasmic reticulum-export molecule to ensure high cell surface protein expression of the channel.
Asunto(s)
Canal de Potasio Kv1.4/metabolismo , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Células CHO , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Semivida , Canal de Potasio Kv1.4/química , Canal de Potasio Kv1.4/genética , Dominios Proteicos , Transporte de Proteínas , RatasRESUMEN
Following injury, primary sensory neurons undergo changes that drive central sensitization and contribute to the maintenance of persistent hypersensitivity. NR2B expression in the dorsal root ganglia (DRG) has not been previously examined in neuropathic pain models. Here, we investigated if changes in NR2B expression within the DRG are associated with hypersensitivities that result from peripheral nerve injuries. This was done by comparing the NR2B expression in the DRG derived from two modalities of the spared nerve injury (SNI) model, since each variant produces different neuropathic pain phenotypes. Using the electronic von Frey to stimulate the spared and non-spared regions of the hindpaws, we demonstrated that sural-SNI animals develop sustained neuropathic pain in both regions while the tibial-SNI animals recover. NR2B expression was measured at Day 23 and Day 86 post-injury. At Day 23 and 86 post-injury, sural-SNI animals display strong hypersensitivity, whereas tibial-SNI animals display 50 and 100% recovery from post-injury-induced hypersensitivity, respectively. In tibial-SNI at Day 86, but not at Day 23 the perinuclear region of the neuronal somata displayed an increase in NR2B protein. This retention of NR2B protein within the perinuclear region, which will render them non-functional, correlates with the recovery observed in tibial-SNI. In sural-SNI at Day 86, DRG displayed an increase in NR2B mRNA which correlates with the development of sustained hypersensitivity in this model. The increase in NR2B mRNA was not associated with an increase in NR2B protein within the neuronal somata. The latter may result from a decrease in kinesin Kif17, since Kif17 mediates NR2B transport to the soma's plasma membrane. In both SNIs, microglia/macrophages showed a transient increase in NR2B protein detected at Day 23 but not at Day 86, which correlates with the initial post-injury induced hypersensitivity in both SNIs. In tibial-SNI at Day 86, but not at Day 23, satellite glia cells (SGCs) displayed an increase in NR2B protein. This study is the first to characterize of cell-specific changes in NR2B expression within the DRG following peripheral nerve injury. We discuss how the observed NR2B changes in DRG can contribute to the different neuropathic pain phenotypes displayed by each SNI variant.
RESUMEN
BACKGROUND: AMPAkines augment the function of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the brain to increase excitatory outputs. These drugs are known to relieve persistent pain. However, their role in acute pain is unknown. Furthermore, a specific molecular and anatomic target for these novel analgesics remains elusive. METHODS: The authors studied the analgesic role of an AMPAkine, CX546, in a rat paw incision (PI) model of acute postoperative pain. The authors measured the effect of AMPAkines on sensory and depressive symptoms of pain using mechanical hypersensitivity and forced swim tests. The authors asked whether AMPA receptors in the nucleus accumbens (NAc), a key node in the brain's reward and pain circuitry, can be a target for AMPAkine analgesia. RESULTS: Systemic administration of CX546 (n = 13), compared with control (n = 13), reduced mechanical hypersensitivity (50% withdrawal threshold of 6.05 ± 1.30 g [mean ± SEM] vs. 0.62 ± 0.13 g), and it reduced depressive features of pain by decreasing immobility on the forced swim test in PI-treated rats (89.0 ± 15.5 vs. 156.7 ± 18.5 s). Meanwhile, CX546 delivered locally into the NAc provided pain-relieving effects in both PI (50% withdrawal threshold of 6.81 ± 1.91 vs. 0.50 ± 0.03 g; control, n = 6; CX546, n = 8) and persistent postoperative pain (spared nerve injury) models (50% withdrawal threshold of 3.85 ± 1.23 vs. 0.45 ± 0.00 g; control, n = 7; CX546, n = 11). Blocking AMPA receptors in the NAc with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione inhibited these pain-relieving effects (50% withdrawal threshold of 7.18 ± 1.52 vs. 1.59 ± 0.66 g; n = 8 for PI groups; 10.70 ± 3.45 vs. 1.39 ± 0.88 g; n = 4 for spared nerve injury groups). CONCLUSIONS: AMPAkines relieve postoperative pain by acting through AMPA receptors in the NAc.
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Analgésicos/farmacología , Dioxoles/farmacología , Núcleo Accumbens/efectos de los fármacos , Dolor Postoperatorio/tratamiento farmacológico , Piperidinas/farmacología , Receptores AMPA/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Depresión/prevención & control , Modelos Animales de Enfermedad , Masculino , Neuralgia/tratamiento farmacológico , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. RESULTS: At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. CONCLUSION: Cold allodynia and significant recovery from spared-nerve injury-induced mechanical hypersensitivity are two novel phenotypes which characterize the global CB1R-/- mice. An increase in transient receptor potential channel of melastatin 8 channel function in DRG neurons may underlie the cold phenotype. Recovery of mechanical thresholds in the CB1R knockouts was independent of motor function. These results indicate that CB1R expression contributes to the development of persistent mechanical hypersensitivity, protects against the development of robust cold allodynia but is not involved in motor impairment following spared-nerve injury in mice.
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Frío , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Receptor Cannabinoide CB1/deficiencia , Recuperación de la Función , Nervio Ciático/lesiones , Nervio Ciático/fisiopatología , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Masculino , Mentol/farmacología , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Receptor Cannabinoide CB1/metabolismo , Recuperación de la Función/efectos de los fármacos , Nervio Ciático/efectos de los fármacosRESUMEN
BACKGROUND: Volatile anesthetics decrease Ca²âº entry through voltage-dependent Ca²âº channels. Ca influences neurotransmitter release and neuronal excitability. Because volatile anesthetics act specifically on the spinal cord to produce immobility, we examined the effect of isoflurane and the nonimmobilizers F6 (1, 2-dichlorohexafluorocyclobutane) and F8 (2, 3-dichlorooctafluorobutane) on CaV1 and CaV2 Ca²âº channels in spinal cord motor neurons and dorsal root ganglion neurons. METHODS: Using patch clamping, we compared the effects of isoflurane with those of F6 and F8 on CaV1 and CaV2 channels in isolated, cultured adult rat spinal cord motor neurons and on CaV1 and CaV2 channels in adult rat dorsal root ganglion sensory neurons. RESULTS: In spinal cord motor neurons, isoflurane, but not F6 or F8, inhibited currents through CaV1 channels. Isoflurane and at least one of the nonimmobilizers inhibited currents through CaV1 and CaV2 channels in dorsal root ganglion neurons and CaV2 in spinal cord motor neurons. CONCLUSIONS: The findings that isoflurane, but not nonimmobilizers, inhibited CaV1 Ca²âº channels in spinal cord motor neurons are consistent with the notion that spinal cord motor neurons might mediate isoflurane-induced immobility. Additional studies are required to examine whether inhibition of CaV1 calcium currents in spinal cord motor neurons is sufficient or whether actions on other channels/proteins contribute to isoflurane-induced immobility.
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Anestésicos por Inhalación/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Clorofluorocarburos/farmacología , Ciclobutanos/farmacología , Isoflurano/farmacología , Neuronas Motoras/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Animales , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Técnicas de Placa-Clamp , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Médula Espinal/citologíaRESUMEN
Kv1.4 potassium channels are heavily glycosylated proteins involved in shaping action potentials and in neuronal excitability and plasticity. Kv1.4 N354Q, without an N-glycan, exhibited decreased protein stability and trafficking to the cell surface (Watanabe et al. in J Biol Chem 279:8879-8885, 2004). Here we investigated whether the composition of the N-glycan affected Kv1.4 cell surface expression. Kv1.4 proteins carrying N-glycans with different compositions were generated by adding glycosidase inhibitors or using N-glycosylation-deficient mutant cell lines. We found that oligomannose-type, hybrid-type, or incomplete complex-type N-glycans had a negative effect on surface protein expression of Kv1.4 compared with complex-type N-glycans. The decrease in surface protein level of Kv1.4 was mainly due to a reduction in total protein level, induced by altered N-glycan composition. Kv1.4 in CSTP-treated cells carried a unique oligomannose-type N-glycan that contains three glucose residues. This N-glycan had the most negative effect on cell surface expression of Kv1.4. It decreased Kv1.4 surface protein level by a combined mechanism of reducing total protein level and increasing ER-retention. Our data suggest that composition of the N-glycan plays an important role in protein stability and trafficking, and a sialylated complex-type N-glycan promoted high cell surface expression of Kv1.4.
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Membrana Celular/metabolismo , Canal de Potasio Kv1.4/metabolismo , Animales , Células CHO , Cricetulus , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Expresión Génica , Glicósido Hidrolasas/antagonistas & inhibidores , Glicosilación/efectos de los fármacos , Canal de Potasio Kv1.4/química , Canal de Potasio Kv1.4/genética , Manosa/química , Manosa/metabolismo , Mutación , Polisacáridos/química , Polisacáridos/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Transporte de ProteínasRESUMEN
Peripheral nerve injury alters the expression of hundreds of proteins in dorsal root ganglia (DRG). Targeting some of these proteins has led to successful treatments for acute pain, but not for sustained post-operative neuropathic pain. The latter may require targeting multiple proteins. Since a single microRNA (miR) can affect the expression of multiple proteins, here, we describe an approach to identify chronic neuropathic pain-relevant miRs. We used two variants of the spared nerve injury (SNI): Sural-SNI and Tibial-SNI and found distinct pain phenotypes between the two. Both models induced strong mechanical allodynia, but only Sural-SNI rats maintained strong mechanical and cold allodynia, as previously reported. In contrast, we found that Tibial-SNI rats recovered from mechanical allodynia and never developed cold allodynia. Since both models involve nerve injury, we increased the probability of identifying differentially regulated miRs that correlated with the quality and magnitude of neuropathic pain and decreased the probability of detecting miRs that are solely involved in neuronal regeneration. We found seven such miRs in L3-L5 DRG. The expression of these miRs increased in Tibial-SNI. These miRs displayed a lower level of expression in Sural-SNI, with four having levels lower than those in sham animals. Bioinformatic analysis of how these miRs could affect the expression of some ion channels supports the view that, following a peripheral nerve injury, the increase of the seven miRs may contribute to the recovery from neuropathic pain while the decrease of four of them may contribute to the development of chronic neuropathic pain. The approach used resulted in the identification of a small number of potentially neuropathic pain relevant miRs. Additional studies are required to investigate whether manipulating the expression of the identified miRs in primary sensory neurons can prevent or ameliorate chronic neuropathic pain following peripheral nerve injuries.
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BACKGROUND: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. METHODS: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. RESULTS: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. CONCLUSIONS: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.
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Envejecimiento/fisiología , Anestésicos Locales/toxicidad , Calcio/metabolismo , Citosol/metabolismo , Ganglios Espinales/patología , Lidocaína/toxicidad , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Microscopía Fluorescente , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Proliferation of endogenous neural stem/progenitor cells (NSPCs) has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2). We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2) over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK) kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2) or the phosphoinositide 3-kinase (PI3K)/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase Cγ (PLCγ) pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCγ signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.
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Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury. Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately, the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol 220:303-315, 2009). Here, we now describe in detail the steps that we used for the optimization of the process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.
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Células del Asta Anterior/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Médula Espinal/citología , Animales , AMP Cíclico , Factores de Crecimiento Nervioso , RatasRESUMEN
In contrast to the adult brain, the adult spinal cord is a non-neurogenic environment. Understanding how to manipulate the spinal cord environment to promote the formation of new neurons is an attractive therapeutic strategy for spinal cord injury and disease. The cannabinoid 1 receptor (CB1R) has been implicated as a modulator of neural progenitor cell proliferation and fate specification in the brain; however, no evidence exists for modulation of adult spinal cord progenitor cells. Using adult rat spinal cord primary cultures, we demonstrated that CB1R antagonism with AM251 significantly decreased the number of Nestin(+) cells, and increased the number of ßIII tubulin(+) and DCX(+) cells, indicative of neuronal differentiation. AM251's effect was blocked by co-application of the CB1R agonists, WIN 55, 212-2, or ACEA. Consistent with our hypothesis, cultures, and spinal cord slices derived from CB1R knock-out (CB1-/-) mice had significantly higher levels of DCX(+) cells compared to those derived from wild type (CB1+/+) mice, indicative of enhanced neuronal differentiation in CB1-/- spinal cords. Moreover, AM251 promoted neuronal differentiation in CB1+/+, but not in CB1-/- cultures. Since CB1R modulates synaptic transmission, and synaptic transmission has been shown to influence progenitor cell fate, we evaluated whether AM251-induced neuronal differentiation was affected by chronic inactivity. Either the presence of the voltage-dependent sodium channel blocker tetrodotoxin (TTX), or the removal of mature neurons, inhibited the AM251-induced increase in DCX(+) cells. In summary, antagonism or absence of CB1R promotes neuronal differentiation in adult spinal cords, and this action appears to require TTX-sensitive neuronal activity. Our data suggest that the previously detected elevated levels of endocannabinoids in the injured adult spinal cord could contribute to the non-neurogenic environment and CB1R antagonists could potentially be used to enhance replacement of damaged neurons.
RESUMEN
Sialylation is an important carbohydrate modification of glycoconjugates that has been shown to modulate many cellular/molecular interactions in vertebrates. In Drosophila melanogaster (Dm), using sequence homology, several enzymes of the sialylation pathway have been cloned and their function tested in expression systems. Here we investigated whether sialic acid incorporation in cultured Dm central nervous system (CNS) neurons required endogenously expressed Dm sialic acid synthase (DmSAS). We compared neurons derived from wild type Dm larvae with those containing a DmSAS mutation (148 bp deletion). The ability of these cells to produce Sia5NAz (sialic acid form) from Ac(4)ManNAz (azide-derivatized N-acetylmannosamine) and incorporate it into their glycoconjugates was measured by tagging the azide group of Sia5NAz with fluorescent agents via Click-iT chemistry. We found that most of the wild type Dm CNS neurons incorporated Sia5NAz into their glycoconjugates. Sialic acid incorporation was higher at the soma than at the neurite and could also be detected at perinuclear regions and the plasma membrane. In contrast, neurons from the DmSAS mutant did not incorporate Sia5NAz unless DmSAS was reintroduced (rescue mutant). Most of the neurons expressed α2,6-sialyltransferase. These results confirm that the mutation was a null mutation and that no redundant sialic acid biosynthetic activity exists in Dm cells, i.e., there is only one DmSAS. They also provide the strongest proof to date that DmSAS is a key enzyme in the biosynthesis of sialic acids in Dm CNS neurons, and the observed subcellular distribution of the newly synthesized sialic acids offers insights into their biological function.
Asunto(s)
Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Ácido N-Acetilneuramínico/biosíntesis , Neuronas/metabolismo , Oxo-Ácido-Liasas/metabolismo , Animales , Células Cultivadas , Drosophila melanogaster/enzimología , Drosophila melanogaster/crecimiento & desarrollo , Células HEK293 , Humanos , Larva/citología , Larva/enzimología , Larva/metabolismo , Neuronas/citología , Neuronas/enzimologíaRESUMEN
Embryonic spinal cord motor neurons (MNs) can be maintained in vitro for weeks with a cocktail of trophic factors and muscle-derived factors under serum-containing conditions. Here we investigated the beneficial effects of muscle-derived factors in the form of muscle-conditioned medium (MCM) on the survival and neurite outgrowth of adult rat spinal cord MNs under serum-free conditions. Ventral horn dissociated cell cultures from the cervical enlargement were maintained in the presence of one or more of the following factors: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), a cell permeant cyclic adenosine-3',5'-monophosphate (cAMP) analog and MCM. The cell cultures were immunostained with several antibodies recognizing a general neuronal marker the microtubule-associated protein 2 (MAP2) and either one or more motor neuronal markers: the non-phosphorylated neurofilament heavy isoform (SMI32), the transcription factors HB9 and Islet-1 and the choline acetyl transferase. We found that treatment with MCM together with the cAMP analog was sufficient to promote selective survival and neurite outgrowth of adult spinal cord MNs. These conditions can be used to maintain adult spinal cord MNs in dissociated cultures for several weeks and may have therapeutic potential following spinal cord injury or motor neuropathies. More studies are necessary to evaluate how MCM and the cAMP analog act in synergy to promote the survival and neurite outgrowth of adult MNs.
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AMP Cíclico/farmacología , Neuronas Motoras/efectos de los fármacos , Músculo Esquelético/fisiología , Neuritas/efectos de los fármacos , Neuronas/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados , Medio de Cultivo Libre de Suero , Electrofisiología , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citologíaRESUMEN
BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels.
Asunto(s)
Anestésicos por Inhalación/farmacología , Calmodulina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Isoflurano/farmacología , Neuronas/efectos de los fármacos , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Calcio/metabolismo , Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Humanos , Potenciales de la Membrana , Neuroblastoma/metabolismo , Neuronas/metabolismo , Nitrendipino/farmacología , Fosforilación , Cloruro de Potasio/farmacología , Factores de Tiempo , omega-Conotoxina GVIA/farmacologíaRESUMEN
BACKGROUND: In addition to inhibiting the excitation conduction process in peripheral nerves, local anesthetics (LAs) cause toxic effects on the central nervous system, cardiovascular system, neuromuscular junction, and cell metabolism. Different postoperative neurological complications are ascribed to the cytotoxicity of LAs, but the underlying mechanisms remain unclear. Because the clinical concentrations of LAs far exceed their EC(50) for inhibiting ion channel activity, ion channel block alone might not be sufficient to explain LA-induced cell death. However, it may contribute to cell death in combination with other actions. In this study, we compared the cytotoxicity of six frequently used LAs and will discuss the possible mechanism(s) underlying their toxicity. METHODS: In human SH-SY5Y neuroblastoma cells, viability upon exposure to six LAs (bupivacaine, ropivacaine, mepivacaine, lidocaine, procaine, and chloroprocaine) was quantitatively determined by the MTT-(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-odium bromide) colorimetry assay and qualitatively confirmed by fluorescence imaging, using the LIVE/DEAD assay reagents (calcein/AM and ethidium homodimer-1). In addition, apoptotic activity was assessed by measuring the activation of caspase-3/-7 by imaging using a fluorescent caspase inhibitor (FLICA). Furthermore, LA effects on depolarization- and carbachol-stimulated intracellular Ca(2+)-responses were also evaluated. RESULTS: 1) After a 10-min treatment, all six LAs decreased cell viability in a concentration-dependent fashion. Their killing potency was procaine < or = mepivacaine < lidocaine < chloroprocaine < ropivacaine < bupivacaine (based on LD(50), the concentration at which 50% of cells were dead). Among these six LAs, only bupivacaine and lidocaine killed all cells with increasing concentration. 2) Both bupivacaine and lidocaine activated caspase-3/-7. Caspase activation required higher levels of lidocaine than bupivacaine. Moreover, the caspase activation by bupivacaine was slower than by lidocaine. Lidocaine at high concentrations caused an immediate caspase activation, but did not cause significant caspase activation at concentrations lower than 10 mM. 3) Procaine and chloroprocaine concentration-dependently inhibited the cytosolic Ca(2+)-response evoked by depolarization or receptor-activation in a similar manner as a previous observation made with bupivacaine, ropivacaine, mepivacaine, and lidocaine. None of the LAs caused a significant increase in the basal and Ca(2+)-evoked cytosolic Ca(2+)-level. CONCLUSION: LAs can cause rapid cell death, which is primarily due to necrosis. Lidocaine and bupivacaine can trigger apoptosis with either increased time of exposure or increased concentration. These effects might be related to postoperative neurologic injury. Lidocaine, linked to the highest incidence of transient neurological symptoms, was not the most toxic LA, whereas bupivacaine, a drug causing a very low incidence of transient neurological symptoms, was the most toxic LA in our cell model. This suggests that cytotoxicity-induced nerve injury might have different mechanisms for different LAs and different target(s) other than neurons.