RESUMEN
Loss of calcineurin (protein phosphatase 3) activity and protein content in the postsynaptic density (PSD) of spinal dorsal horn neurons was associated with pain behavior after chronic constriction injury (CCI) of the rat sciatic nerve, and intrathecal administration of the phosphatase provided prolonged analgesia (Miletic et al. 2013). In this study, we examined whether one consequence of the loss of calcineurin was the persistent phosphorylation of the GluA1 subunit of α-amino-3-hydroxy-5-methyl-4-isoxazolepropioinic acid (AMPAR) receptors in the PSD. This would allow continual activation of AMPAR receptors at the synapse to help maintain a long-lasting enhancement of synaptic function, ie, neuropathic pain. We also investigated if the phosphorylation was mediated by protein kinase A (PKA), protein kinase C gamma (PKCγ), or calcium-calmodulin dependent kinase II (CaMKII), and if the prolonged calcineurin analgesia was associated with GluA1 dephosphorylation. Mechanical thresholds and thermal latencies were obtained before CCI. Seven days later, the behavioral testing was repeated before saline, calcineurin, or the specific peptide inhibitors of PKA (PKI-tide), PKCγ (PKC 19-31), or CaMKII (autocamtide-2-related inhibitory peptide) were injected intrathecally. The behavior was retested before the animals were euthanized and their PSD isolated. All CCI animals developed mechanical and thermal hypersensitivity. This was associated with phosphorylation of GluA1 in the ipsilateral PSD at Ser831 (but not Ser845) by PKCγ and not by PKA or CaMKII. Intrathecal treatment with calcineurin provided prolonged analgesia, and this was accompanied by GluA1 dephosphorylation. Therapy with calcineurin may prove useful in the prolonged clinical management of well-established neuropathic pain.
Asunto(s)
Analgesia , Calcineurina/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Neuralgia/metabolismo , Densidad Postsináptica/efectos de los fármacos , Células del Asta Posterior/efectos de los fármacos , Proteína Quinasa C/metabolismo , Receptores AMPA/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Inyecciones Espinales , Masculino , Fosforilación , Densidad Postsináptica/metabolismo , Células del Asta Posterior/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Nervio Ciático/lesionesRESUMEN
Calcineurin (protein phosphatase 3) regulates synaptic plasticity in the brain. The development of neuropathic pain appears dependent on some of the same mechanisms that underlie brain synaptic plasticity. In this study, we examined whether calcineurin regulates chronic constriction injury (CCI)-elicited plasticity in the spinal dorsal horn. CCI animals exhibited mechanical and thermal hypersensitivity 7 days after ligation of the sciatic nerve. Neither control uninjured nor sham-operated animals exhibited pain behavior. Calcineurin activity and content of its Aα isoform were significantly decreased in the ipsilateral postsynaptic density (PSD) of dorsal horn neurons in CCI animals. Calcineurin activity and content in the contralateral PSD of CCI animals or either side of the dorsal horn in sham animals were not modified. The pain behavior in CCI animals was attenuated by intrathecal application of exogenous calcineurin. The treatment was long-lasting as a single injection provided analgesia for 4 days by restoring the phosphatase's activity and Aα content in the PSD. No signs of toxicity were detected up to 14 days after the single intrathecal injection. Intrathecal application of the calcineurin inhibitor FK-506 elicited pain behavior in control uninjured animals and significantly reduced calcineurin activity in the PSD. CCI may elicit neuropathic pain at least in part as a result of the loss of calcineurin-mediated dephosphorylation in the dorsal horn. Addition of the phosphatase by intrathecal injection reverses the injury-elicited loss and provides prolonged pain relief. Clinical therapy with calcineurin may prove to be a novel, effective, and safe approach in the management of well-established neuropathic pain.
Asunto(s)
Analgesia/métodos , Calcineurina/administración & dosificación , Calcineurina/biosíntesis , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Células del Asta Posterior/metabolismo , Animales , Inyecciones Espinales , Masculino , Manejo del Dolor/métodos , Células del Asta Posterior/efectos de los fármacos , Distribución Aleatoria , Ratas , Ratas Sprague-DawleyRESUMEN
Plasticity in the spinal dorsal horn may contribute to the development of pain following peripheral nerve injury. Shank proteins are a constituent family of the post-synaptic density (PSD), and they may play a role in synaptic plasticity through activity-dependent synaptic remodeling and growth. In this study we examined the early consequences of the loose ligation of the sciatic nerve on Shank1 protein and message levels in the PSD of spinal dorsal horn neurons. Four hours after sciatic ligation, the protein levels of Shank1 increased in the ipsilateral PSD of ligated animals. In contrast, no changes were detected in the contralateral PSD of these ligated animals, or either the ipsilateral or contralateral PSD of sham-operated animals. Shank1 was linked to the PSD marker protein PSD-95 and the NR2B subunit of NMDA receptors. The ligated animals also exhibited two early signs of pain behavior, a shift in weight distribution and thermal hyperalgesia. There was no overall change in Shank1 message in either ligated or sham-operated animals. The accumulation of Shank1 in the PSD was abolished by intrathecal pre-treatment with anisomycin or Shank1 siRNA, but not with non-target siRNA. The same pre-treatment prevented both the early signs of pain behavior. Intrathecal pre-treatment with either MK-801 or U0126 similarly prevented the Shank1 accumulation and alleviated both the behavioral signs of pain. The early accumulation of Shank1 in the PSD of dorsal horn neurons may be a necessary step in the injury-associated plasticity that in time leads to the development of persistent pain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Hiperalgesia/fisiopatología , Células del Asta Posterior/metabolismo , Neuropatía Ciática/fisiopatología , Potenciales Sinápticos , Animales , Hiperalgesia/etiología , Hiperalgesia/patología , Ligadura , Masculino , Proteínas del Tejido Nervioso , Ratas , Ratas Sprague-Dawley , Neuropatía Ciática/complicaciones , Neuropatía Ciática/patologíaRESUMEN
BACKGROUND: Plasticity in the spinal dorsal horn is thought to underlie, at least in part, pain behavior after peripheral nerve injury. Homer1 proteins play an important role in synaptic plasticity through an activity-dependent remodeling of the postsynaptic density (PSD). In this study, we examined the early consequences of the loose ligation of the sciatic nerve on the levels of Homer1a and Homer1b/c proteins in the PSD of spinal dorsal horn neurons. METHODS: Male rats were randomly assigned to control, sham-operated, or ligated groups. Four hours after sciatic exposure or ligation, the animals were anesthetized and killed. Dorsal horn ipsilateral and contralateral quadrants were homogenized and centrifuged to obtain a PSD-containing LP1 fraction. Homer1 isoforms were identified in Western immunoblots. In some animals, Homer1 small interfering RNA (siRNA), nontarget siRNA, MK-801, or U01026 was injected intrathecally before surgery to assess the effects of this treatment on the levels of Homer1 isoforms and on 2 signs of injury-associated pain behavior, a shift in weight-bearing distribution and thermal hyperalgesia. RESULTS: In ligated animals, the protein levels of Homer1a increased and those of Homer1b/c decreased in the ipsilateral LP1 fraction of the spinal dorsal horn. In contrast, no changes were detected in the contralateral LP1 fraction of ligated animals or the ipsilateral or contralateral LP1 fraction of sham-operated animals. Intrathecal injections of Homer1 siRNA, but not nontarget siRNA, 2 h before the ligation prevented the accumulation of Homer1a and loss of Homer1b/c in the ipsilateral LP1 fraction. The same pretreatment with Homer1 siRNA also alleviated both a shift in weight-bearing behavior and thermal hyperalgesia in the ligated animals. Intrathecal injections of MK-801 or U0126 15 min before the ligation similarly prevented the injury-associated changes in Homer1 protein levels and the behavioral signs of pain. CONCLUSION: The ligation-associated changes in the protein levels of Homer1a and Homer1b/c in the ipsilateral PSD of spinal dorsal horn neurons may be an important early reflection of the injury-associated plasticity that in time leads to the development of persistent pain.
Asunto(s)
Proteínas Portadoras/metabolismo , Neuralgia/metabolismo , Plasticidad Neuronal , Células del Asta Posterior/metabolismo , Neuropatía Ciática/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Butadienos/administración & dosificación , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Maleato de Dizocilpina/administración & dosificación , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Proteínas de Andamiaje Homer , Inyecciones Espinales , Ligadura , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuralgia/genética , Neuralgia/fisiopatología , Neuralgia/prevención & control , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/genética , Nitrilos/administración & dosificación , Células del Asta Posterior/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Nervio Ciático/cirugía , Neuropatía Ciática/complicaciones , Neuropatía Ciática/genética , Neuropatía Ciática/fisiopatología , Neuropatía Ciática/terapia , Factores de TiempoRESUMEN
Regulation of load-induced bone formation is considered a local phenomenon controlled by osteocytes, although it has also been hypothesized that functional adaptation may be neuronally regulated. The aim of this study was to examine bone formation in multiple bones, in response to loading of a single bone, and to determine whether adaptation may be neuronally regulated. Load-induced responses in the left and right ulnas and humeri were determined after loading of the right ulna in male Sprague-Dawley rats (69 +/- 16 days of age). After a single period of loading at -760-, -2000-, or -3750-microepsilon initial peak strain, rats were given calcein to label new bone formation. Bone formation and bone neuropeptide concentrations were determined at 10 days. In one group, temporary neuronal blocking was achieved by perineural anesthesia of the brachial plexus with bupivicaine during loading. We found right ulna loading induces adaptive responses in other bones in both thoracic limbs compared with Sham controls and that neuronal blocking during loading abrogated bone formation in the loaded ulna and other thoracic limb bones. Skeletal adaptation was more evident in distal long bones compared with proximal long bones. We also found that the single period of loading modulated bone neuropeptide concentrations persistently for 10 days. We conclude that functional adaptation to loading of a single bone in young rapidly growing rats is neuronally regulated and involves multiple bones. Persistent changes in bone neuropeptide concentrations after a single loading period suggest that plasticity exists in the innervation of bone.
Asunto(s)
Adaptación Fisiológica , Huesos/fisiología , Neuronas/fisiología , Anestesia , Animales , Desarrollo Óseo , Remodelación Ósea , Plexo Braquial , Masculino , Neuropéptidos/metabolismo , Periostio/fisiología , Ratas , Ratas Sprague-Dawley , Cúbito/fisiología , Soporte de PesoRESUMEN
BACKGROUND: Loss of gamma-aminobutyric acid (GABA) inhibition in the spinal dorsal horn may contribute to neuropathic pain. Here we examined whether systemic administration of the benzodiazepine midazolam would alleviate thermal hyperalgesia due to chronic constriction injury (CCI) of the sciatic nerve. METHODS: Hyperalgesia was evaluated with the thermal paw withdrawal latency test before, and 3 and 7 days after CCI. Animals randomly received, via osmotic minipump infusion, midazolam (2.0 mg x kg(-1) x h(-1)), flumazenil (0.004 mg x kg(-1) x h(-1)), midazolam plus flumazenil at the same doses, or saline (0.01 mg x kg(-1) x h(-1)). Four groups of sham-operated rats (surgery without nerve ligation) received matched treatments. Levels of the GABA transporter 1 (GAT-1) in the lumbar spinal dorsal horn were estimated using western immunoblots 7 days after surgery. RESULTS: Saline-treated CCI rats developed thermal hyperalgesia on Day 3 with a more pronounced effect on Day 7. Continuous midazolam infusion prevented thermal hyperalgesia on both days. The antihyperalgesic effect of midazolam was reversed by the coadministration of flumazenil. Infusion of flumazenil alone had no effect on the thermal hyperalgesia in CCI rats. Sham-operated rats treated with saline, midazolam, or midazolam plus flumazenil exhibited no thermal hyperalgesia. Unexpectedly, thermal paw withdrawal latency in sham animals treated with flumazenil alone was significantly decreased. Changes in GAT-1 levels paralleled the behavior. Midazolam prevented the CCI-associated decreases, and flumazenil reversed midazolam's effect. Flumazenil alone did not modify GAT-1 levels in CCI animals but in sham animals the transporter levels were significantly reduced. CONCLUSIONS: GABA inhibition plays an important role in neuropathic pain. Continuous systemic benzodiazepine administration may prove effective in alleviating neuropathic pain.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Hiperalgesia/prevención & control , Midazolam/uso terapéutico , Neuritis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Calor , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Significant decreases in the protein levels of potassium-chloride co-transporter 2 (KCC2) were detected in the ipsilateral spinal dorsal horn 4h following loose ligation of the sciatic nerve. These decreases were associated with a change in hindlimb weight distribution suggestive of pain behavior. In contrast, no changes in GABA-A receptor subunit alpha-1 levels were detected. The decreases in KCC2 coincided with a significant ipsilateral increase in BDNF protein levels. Both the decreases in KCC2 levels and the early pain behavior were prevented by intrathecal pre-treatment with the BDNF-sequestering TrkB/Fc chimera protein or the tyrosine kinase blocker K252a. The ligation-associated decreases in KCC2 levels were transient. In the ipsilateral spinal dorsal horn of ligated animals exhibiting weight-bearing pain behavior 7 days after the ligation the KCC2 levels were identical to those in control or sham-operated animals. These data suggested that TrkB-dependent reduction in KCC2 protein levels in the spinal dorsal horn was an early consequence of peripheral nerve injury. This decrease in KCC2 may have elicited an early increase in overall dorsal horn neuronal excitability perhaps through a loss of GABA inhibition which is critically dependent on KCC2 activity. The increased neuronal excitability may in turn have caused enhanced and exaggerated communication between primary afferents and dorsal horn neurons to contribute to the early behavioral signs of pain.
Asunto(s)
Síndromes de Compresión Nerviosa/metabolismo , Neuralgia/metabolismo , Células del Asta Posterior/metabolismo , Receptor trkB/metabolismo , Neuropatía Ciática/metabolismo , Simportadores/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Cotransportadores de K ClRESUMEN
OBJECTIVE: To test the hypothesis that butorphanol or morphine induces antinociception with minimal respiratory depression in conscious red-eared slider turtles. DESIGN: Prospective crossover study. ANIMALS: 37 adult male and female red-eared slider turtles (Trachemys scripta). PROCEDURES: Antinociception (n = 27 turtles) and respiratory (10 turtles) experiments were performed. Infrared heat stimuli were applied to the plantar surface of turtle limbs. Thermal withdrawal latencies were measured before and at intervals after SC administration of physiologic saline (0.9% NaCl) solution, butorphanol tartrate (2.8 or 28 mg/kg [1.27 or 12.7 mg/lb]), or morphine sulfate (1.5 or 6.5 mg/kg [0.68 or 2.95 mg/lb]). Ventilation was assessed in freely swimming turtles before and after SC administration of saline solution, butorphanol (28 mg/kg), or morphine (1.5 mg/kg). RESULTS: For as long as 24 hours after injection of saline solution or either dose of butorphanol, thermal withdrawal latencies among turtles did not differ. Low- and high-dose morphine injections increased latencies significantly by 8 hours. Ventilation was not altered by saline solution administration, was temporarily depressed by 56% to 60% for 1 to 2 hours by butorphanol (28 mg/kg) administration, and was significantly depressed by a maximum of 83 +/- 9% at 3 hours after morphine (1.5 mg/kg) injection. Butorphanol and morphine depressed ventilation by decreasing breathing frequency. CONCLUSIONS AND CLINICAL RELEVANCE: Although widely used in reptile species, butorphanol may not provide adequate antinociception for invasive procedures and caused short-term respiratory depression in red-eared slider turtles. In contrast, morphine apparently provided antinociception but caused long-lasting respiratory depression.
Asunto(s)
Analgésicos Opioides/farmacología , Butorfanol/farmacología , Morfina/farmacología , Tortugas/fisiología , Analgésicos Opioides/efectos adversos , Animales , Butorfanol/efectos adversos , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Morfina/efectos adversos , Dolor/prevención & control , Dolor/veterinaria , Dimensión del Dolor/veterinaria , Estudios Prospectivos , Respiración/efectos de los fármacos , Factores de TiempoRESUMEN
STUDY DESIGN: Laboratory investigation of pain behavior following spinal cord injury. OBJECTIVE: To explore changes in the spinal cord expression of nociceptive genes following spinal cord injury (SCI) as they relate to the manifestation of pain behavior in rats. SUMMARY OF BACKGROUND DATA: Neuropathic pain following SCI is common, disabling, and largely untreatable. In peripheral nerve injury models, bradykinin B1 and vanilloid 1 (TRPV-1) receptor activity is associated with neuropathic pain behavior. We sought to examine the role of these gene products in SCI-mediated pain. METHODS: Rats were subjected to SCI using the MASCIS impactor. Animals were tested preinjury and at regular intervals postinjury for the appearance of thermal hyperalgesia using a hind limb withdrawal latency test. The expression of B1 and TRPV-1 genes was assessed using real-time polymerase chain reaction. Immunohistochemistry was used to localize the B1 and TRPV-1 receptors within the spinal cord. RESULTS: Greater than twofold increases in the expression of the B1 and TRPV-1 genes were detected in the injured region of the spinal cord in animals exhibiting hyperalgesia compared with animals with SCI that did not display hyperalgesia. Immunohistochemical staining revealed that both receptor types were largely localized to the dorsal horn. Staining for TRPV-1 receptors decreased while that for B1 receptors increased in all of the injured animals when compared with sham-operated controls. CONCLUSION: B1 and TRPV-1 receptor genes are overexpressed in the injured spinal cord of animals manifesting thermal hyperalgesia following SCI compared with similarly injured animals without hyperalgesia. This finding is consistent with past work regarding the role of these receptors in nociception and indicates that ongoing modifiable processes are occurring in the spinal cord that lead to clinical pain syndromes.
Asunto(s)
Hiperalgesia/etiología , Proteínas del Tejido Nervioso/biosíntesis , Nociceptores/fisiología , Receptor de Bradiquinina B1/biosíntesis , Traumatismos de la Médula Espinal/complicaciones , Canales Catiónicos TRPV/biosíntesis , Animales , Contusiones/complicaciones , Contusiones/fisiopatología , Regulación de la Expresión Génica , Miembro Posterior/inervación , Calor/efectos adversos , Hiperalgesia/genética , Hiperalgesia/fisiopatología , Inflamación , Laminectomía , Masculino , Modelos Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción , Receptor de Bradiquinina B1/genética , Receptor de Bradiquinina B1/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Traumatismos de la Médula Espinal/fisiopatología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/fisiologíaRESUMEN
Changes in the expression of many genes underlie injury-elicited plasticity in the spinal dorsal horn. Homer1 is a recently identified gene that appears to play a critical role in the expression of synaptic plasticity in several brain regions, including the hippocampus. In this study we investigated the early consequences of chronic constriction injury of the sciatic nerve on Homer1 gene expression in the spinal dorsal horn. Significant increases in Homer1a mRNA levels in the ipsilateral dorsal horn were detected at 4h post-ligation, and these levels remained elevated at 8h before returning to baseline values by 24h after the ligation. In contrast, the levels of Homer1b/c mRNA did not change at any of these selected post-ligation times. The ligation-associated induction of Homer1a was dependent on activation of NMDA receptors and the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway. The non-competitive NMDA-receptor antagonist, MK-801, and a specific inhibitor of the ERK1/2 pathway, U0126, significantly attenuated the injury-elicited increases in Homer1a mRNA when compared to saline-treated animals. These data provide the first evidence for a potential role of Homer1a in peripheral nerve injury-elicited plasticity in the spinal dorsal horn. These data also imply that the early and transient up-regulation of Homer1a gene expression may be an important contributor to the eventual development of neuropathic pain.
Asunto(s)
Proteínas Portadoras/biosíntesis , Dolor/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células del Asta Posterior/metabolismo , Animales , Butadienos/farmacología , Proteínas Portadoras/genética , Constricción Patológica , Maleato de Dizocilpina/farmacología , Proteínas de Andamiaje Homer , Ligadura , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Plasticidad Neuronal , Nitrilos/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Nervio Ciático/lesiones , Regulación hacia ArribaRESUMEN
Activity-dependent plasticity in the spinal dorsal horn may underlie the development of neuropathic pain following peripheral nerve injury. In this study we examined whether the expression and loss of behavioral signs of neuropathic pain were associated with changes in the content of the scaffolding proteins Homer and Shank in the post-synaptic density (PSD) of the spinal dorsal horn. In animals exhibiting thermal hyperalgesia and differential weight-bearing behavior 7 days after loose ligation of the sciatic nerve the levels of Homer1b/c and Shank1a were significantly greater than in control, uninjured or sham-operated animals. These greater levels were specifically a reflection of increases in the injured, ipsilateral and not contralateral dorsal horn. In contrast, there were no differences in the PSD content of Homer1b/c and Shank1a in the dorsal horn of control or sham-operated animals and ligated animals in which the thermal hyperalgesia and differential weight-bearing behavior had disappeared 28 days after the loose ligation. These data revealed a close association between the expression and loss of allodynia and hyperalgesia with changes in the levels of Homer1b/c and Shank1a in the spinal dorsal horn. The reversible shift in the content of scaffolding proteins in the PSD may have important implications for the development of injury-elicited neuropathic pain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Neuralgia/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células del Asta Posterior/metabolismo , Membranas Sinápticas/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas de Andamiaje Homer , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Ligadura , Masculino , Proteínas del Tejido Nervioso , Neuralgia/fisiopatología , Plasticidad Neuronal/fisiología , Neuronas Aferentes/metabolismo , Nociceptores/metabolismo , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Células del Asta Posterior/ultraestructura , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Neuropatía Ciática/metabolismo , Neuropatía Ciática/fisiopatología , Raíces Nerviosas Espinales/metabolismo , Membranas Sinápticas/ultraestructura , Transmisión Sináptica/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Phosphorylation of the cyclic AMP response element-binding protein (CREB) in the spinal dorsal horn may critically contribute to chronic pain following peripheral nerve injury. We employed inhibitors and activators of protein kinase A (PKA), protein kinase C (PKC), extracellular signal-regulated kinase 1 and 2 (ERK1/2) and calcium/calmodulin-dependent kinase II (CaMKII) to examine whether these kinases individually or in concert mediate the increase in CREB phosphorylation that is evident as early as 2 h after loose ligation of the sciatic nerve. Specific inhibitors of each kinase significantly attenuated the ligation-associated CREB phosphorylation when compared to saline-treated animals. Combined application of the ERK1/2 and CaMKII inhibitors also attenuated the ligation-associated CREB activation but not to a greater extent than either inhibitor alone. Specific activators of PKA, PKC and ERK1/2 elicited significant increases in CREB phosphorylation 2 h after drug application in the spinal dorsal horn of control, peripherally uninjured animals. Pre-treatment of animals with the ERK1/2 inhibitor abolished the increases elicited by either the PKA or the PKC activator. Significant increases in ERK1/2 phosphorylation were also detected 2 h after sciatic ligation confirming a role for the ERK pathway in injury-related responses in the dorsal horn. Each kinase inhibitor significantly attenuated the ligation-associated activation of ERK1/2 as well. These data suggest that early, sciatic ligation-elicited phosphorylation of CREB in the spinal dorsal horn is mediated by multiple kinase pathways, and that PKA, PKC and CaMKII activate CREB at least in part by way of the ERK pathway.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfotransferasas/metabolismo , Células del Asta Posterior/metabolismo , Neuropatía Ciática/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Proteína de Unión a CREB , Masculino , Complejos Multienzimáticos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Brain-derived neurotrophic factor (BDNF) and cyclic AMP response element binding protein (CREB) may critically contribute to injury-associated plasticity and thus to the development of persistent pain. In the present study we examined the potential interaction between CREB and BDNF in the spinal dorsal horn. Significant CREB phosphorylation was elicited by local application of BDNF (1 microg) onto the spinal dorsal horn of control, uninjured animals. The degree of phosphorylation was similar to that elicited by loose ligation of the sciatic nerve. The tyrosine kinase (Trk) blocker K252a (2 microg) significantly reduced the CREB phosphorylation elicited either by BDNF or the sciatic ligation. These data provided further support for the notion that at least some of the injury-associated activation of CREB in the spinal dorsal horn may be dependent upon BDNF-mediated activation of Trk receptors.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Neuralgia/metabolismo , Células del Asta Posterior/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Neuropatía Ciática/metabolismo , Vías Aferentes/efectos de los fármacos , Vías Aferentes/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Carbazoles/farmacología , Enfermedad Crónica , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Alcaloides Indólicos , Ligadura , Masculino , Neuralgia/fisiopatología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Nervios Periféricos/fisiopatología , Fosforilación/efectos de los fármacos , Células del Asta Posterior/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Neuropatía Ciática/fisiopatología , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/fisiologíaRESUMEN
We examined whether early injury-associated activation of cyclic AMP response element binding protein (CREB) in the spinal dorsal horn was mediated by the cyclic AMP-dependent protein kinase A (PKA) pathway. Significant increases in the levels of phosphorylated CREB (pCREB), phosphorylated PKAIIalpha regulatory subunit (pPKA), and PKAalpha catalytic subunit (PKAalpha cat) were elicited 2 h after loose ligation of the sciatic nerve. These injury-elicited increases were significantly reduced by dorsal horn application of the cell-permeable PKA inhibitor Rp-8-Br-cAMPS. The cell-permeable PKA activator Sp-8-Br-cAMPS significantly increased the levels of pCREB, pPKA and PKAalpha cat 2 h after application onto the dorsal horn of control, uninjured animals. Our data lent further support to the notion that activation of PKA may play an important role in the early stages of nerve injury-elicited plasticity in the dorsal horn.
Asunto(s)
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células del Asta Posterior/metabolismo , Nervio Ciático/lesiones , Médula Espinal/citología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Western Blotting/métodos , Dominio Catalítico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Laminectomía/métodos , Ligadura/métodos , Masculino , Fosforilación , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/enzimología , Ratas , Ratas Sprague-Dawley , Tionucleótidos/farmacologíaRESUMEN
The inhibitory activity of gamma-aminobutyric acid (GABA) is considered critical in setting the conditions for synaptic plasticity, and many studies support an important role of GABA in the suppression of nociceptive transmission in the dorsal horn. Consequently, any injury-induced modification of the GABA action has the potential to critically modify spinal synaptic plasticity. We have previously reported that chronic constriction injury of the sciatic nerve was accompanied by long-lasting potentiation of superficial spinal dorsal horn field potentials following high-frequency tetanus. In this study we examined whether the GABA-A receptor agonist muscimol would modify post-tetanic responses in rats with chronic constriction injury. In animals exhibiting maximal thermal hyperalgesia as one sign of neuropathic pain 7 days after loose ligation of the sciatic nerve, spinal application of muscimol (5, 10 or 20 microg) before the high-frequency (50 Hz) tetanus produced a long-lasting depression (rather than potentiation) of spinal dorsal horn field potentials. In separate but related Western immunoblot experiments, we also established that the chronic constriction injury was accompanied by significant decreases in the content of the GABA transporter GAT-1. These data demonstrated that GABA-A receptor agonists may effectively influence the expression of long-lasting synaptic plasticity in the spinal dorsal horn, and that an injury-induced loss in GABA transporter content may have contributed to a depletion of GABA from its terminals within the spinal dorsal horn. These data lent further support to the notion that the loss of GABA inhibition may have important consequences for the development of neuropathic pain.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Muscimol/farmacología , Transportadores de Anión Orgánico , Nervio Ciático/lesiones , Médula Espinal/fisiopatología , Potenciales de Acción/efectos de los fármacos , Animales , Enfermedad Crónica , Estimulación Eléctrica , Proteínas Transportadoras de GABA en la Membrana Plasmática , Calor , Hiperalgesia/etiología , Hiperalgesia/metabolismo , Ligadura , Masculino , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Heridas y Lesiones/complicaciones , Heridas y Lesiones/metabolismo , Heridas y Lesiones/fisiopatologíaRESUMEN
Plasticity in the spinal dorsal horn may underlie the development of chronic pain following peripheral nerve injury or inflammation. In this study, we examined whether chronic constriction injury of the sciatic nerve was associated with changes in the immunoreactive content of cyclic AMP response element binding protein (CREB), protein kinase A (PKA), and calcineurin Aalpha and Abeta in the spinal dorsal horn. In animals exhibiting thermal hyperalgesia as a behavioral sign of neuropathic pain 7 days after loose ligation of the sciatic nerve (chronic constriction injury), there was a significant increase in the content of phosphorylated (activated) CREB (pCREB). In contrast, following the typical disappearance of thermal hyperalgesia 28 days after loose ligation surgery, there were no differences in pCREB content between control and sciatic ligation animals. The increased CREB activation associated with thermal hyperalgesia was accompanied by significant decreases in the content of both calcineurin Aalpha and Abeta. In contrast, there were no differences in the content of non-phosphorylated CREB, and phosphorylated or non-phosphorylated PKA between control and sciatic ligation animals either 7 or 28 days after surgery. These data established a close association in the expression of thermal hyperalgesia with CREB activation and decreased calcineurin content in the spinal dorsal horn. The data revealed a significant but reversible shift in the manner in which spinal neurons processed sensory information following peripheral nerve injury, and lent further support to the notion that plasticity in the spinal dorsal horn may have contributed to the development of chronic pain.
Asunto(s)
Calcineurina/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Hiperalgesia/metabolismo , Neuropatía Ciática/metabolismo , Animales , Constricción Patológica/metabolismo , Regulación hacia Abajo , Masculino , Fosforilación , Células del Asta Posterior/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/fisiologíaRESUMEN
We examined whether continual constant-rate infusion of lidocaine would provide analgesia during the initial post-injury phase in the chronic constriction injury model of neuropathic pain. Male Sprague-Dawley rats were divided into control and ligated groups and infused with saline or lidocaine (0.15, 0.33, 0.67, and 1.3mg/kg/h) via subcutaneously implanted Alzet((R)) osmotic minipumps. Thermal withdrawal latencies were obtained prior (Day 0) and 3 days after loose sciatic ligation and pump implantation surgery. Ligated animals receiving lidocaine at 0.67 or 1.3mg/kg/h exhibited no change in withdrawal latency on Day 3 after surgery, indicating that lidocaine at these doses prevented the development of thermal hyperalgesia as a sign of neuropathic pain. In contrast, ligated animals treated with saline or lidocaine at 0.15 or 0.33mg/kg/h exhibited hyperalgesia on Day 3 after surgery, indicating that these lower doses of lidocaine failed to provide analgesia. Control animals treated with saline or any of the lidocaine doses exhibited no change in withdrawal latencies between Day 0 and Day 3. In a separate group of ligated animals, lidocaine infusion (0.67mg/kg/h) that was started 24h after sciatic ligation surgery reversed the already present thermal hyperalgesia. Average plasma lidocaine concentrations were 0.11, 0.36, and 0.45microg/ml for animals receiving 0.33, 0.67 and 1.3mg/kg/h of lidocaine, respectively. These results suggest that continual systemic infusion of lidocaine prevents or reverses the development of neuropathic pain following chronic constriction injury. These results add to the increasing body of evidence supporting the therapeutic value of preemptive and post-operative lidocaine administration for the relief of neuropathic pain.
Asunto(s)
Anestésicos Locales/farmacología , Lidocaína/farmacología , Ciática/tratamiento farmacológico , Anestésicos Locales/sangre , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hiperalgesia/tratamiento farmacológico , Bombas de Infusión Implantables , Lidocaína/sangre , Ligadura , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Animals exhibiting thermal hyperalgesia as a sign of neuropathic pain 7 days after loose ligation of the sciatic nerve exhibited a significant increase in the concentration of brain derived neurotrophic factor (BDNF) in their lumbar spinal dorsal horn. In contrast, following the disappearance of thermal hyperalgesia 28 days after loose ligation of the sciatic nerve, there were no differences in BDNF levels between control animals and those with sciatic ligations. These data suggest a close association in the timeline of the development and disappearance of behavioral signs of neuropathic pain with changes in BDNF levels in the lumbar spinal dorsal horn, and lend further support to the notion that plasticity in the processing of sensory information in the spinal dorsal horn may contribute to the development of persistent pain.