RESUMEN
A hyperexcitable state and spontaneous activity of nociceptors have been suggested to play a critical role in the development of chronic neuropathic pain following spinal cord injury (SCI). In male rats, we employed the action potential-clamp technique to determine the underlying ionic mechanisms responsible for driving SCI-nociceptors to a hyperexcitable state and for triggering their spontaneous activity. We found that the increased activity of low voltage activated T-type calcium channels induced by the injury sustains the bulk (â¼60-70%) of the inward current active at subthreshold voltages during the interspike interval in SCI-nociceptors, with a modest contribution (â¼10-15%) from tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In current-clamp recordings, inhibition of T-type calcium channels with 1 µm TTA-P2 reduced both the spontaneous and the evoked firing in response to current injections in SCI-nociceptors to a level similar to sham-nociceptors. Electrophysiology in vitro was then combined with the conditioned place preference (CPP) paradigm to determine the relationship between the increased activity of T-type channels in SCI-nociceptors and chronic neuropathic pain following SCI. The size of the interspike T-type calcium current recorded from nociceptors isolated from SCI rats showing TTA-P2-induced CPP (responders) was â¼6 fold greater than the interspike T-type calcium current recorded from nociceptors isolated from SCI rats without TTA-P2-induced CPP (non-responders). Taken together, our data suggest that the increased activity of T-type calcium channels induced by the injury plays a primary role in driving SCI-nociceptors to a hyperexcitable state and contributes to chronic neuropathic pain following SCI.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major comorbidity of spinal cord injury (SCI), affecting up to 70-80% of patients. Anticonvulsant and tricyclic antidepressant drugs are first line analgesics used to treat SCI-induced neuropathic pain, but their efficacy is very limited. A hyperexcitable state and spontaneous activity of SCI-nociceptors have been proposed as a possible underlying cause for the development of chronic neuropathic pain following SCI. Here, we show that the increased activity of T-type calcium channels induced by the injury plays a major role in driving SCI-nociceptors to a hyperexcitable state and for promoting their spontaneous activity, suggesting that T-type calcium channels may represent a pharmacological target to treat SCI-induced neuropathic pain.
Asunto(s)
Potenciales de Acción , Canales de Calcio Tipo T/metabolismo , Nocicepción , Nociceptores/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Masculino , Nociceptores/fisiología , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
The dorsal horn of the spinal cord represents the first relay station in the pain pathway where primary nociceptive inputs are modulated by local circuits and by descending signals before being relayed to supraspinal nuclei. To determine whether dopamine can modulate primary nociceptive Aδ- and C-fiber signals, the effects of dopamine were tested on the excitatory postsynaptic currents (EPSCs) recorded from large lamina I neurons and from retrograde-labeled spinoparabrachial lamina I neurons upon stimulation of the L4/L5 dorsal root in horizontal spinal cord slices in vitro Dopamine inhibited the EPSCs in a dose-dependent manner, with substantial inhibition (33%) at 1 µm and maximum inhibition (â¼70%) at 10-20 µm Dopamine reduced the frequency of miniature EPSCs recorded from large lamina I neurons, increased the paired pulse depression ratio of paired EPSCs, and induced similar inhibition of EPSCs after dialysis of large lamina I neurons with GDP-ß-S, consistent with actions at presynaptic sites. Pharmacological experiments suggested that the inhibitory effects of dopamine were largely mediated by D4 receptors (53%). Similar inhibition (66%) by dopamine was observed on EPSCs recorded from ipsilateral large lamina I neurons 6 d after injection of complete Freund's adjuvant in the hindpaw, suggesting that dopamine downregulates primary nociceptive inputs to lamina I neurons during chronic inflammatory pain. We propose that presynaptic inhibition of primary nociceptive inputs to lamina I projection neurons is a mechanism whereby dopamine can inhibit incoming noxious stimuli to the dorsal horn of the spinal cord.SIGNIFICANCE STATEMENT Lamina I projection neurons represent the main output for the pain signals from the dorsal horn of the spinal cord to brainstem and thalamic nuclei. We found that dopamine inhibits the nociceptive Aδ- and C-fiber synaptic inputs to lamina I projection neurons via presynaptic actions. Similar inhibitory effects of dopamine on the EPSCs were observed in rats subjected to complete Freund's adjuvant to induce peripheral inflammation, suggesting that dopamine inhibits the synaptic inputs to lamina I neurons in the setting of injury. A better understanding of how primary nociceptive inputs to the dorsal horn of the spinal cord are modulated by descending monoaminergic signals may help in the development of new pharmacological strategies to selectively downregulate the output from lamina I projection neurons.
Asunto(s)
Dopamina/fisiología , Inhibición Neural , Nocicepción/fisiología , Células del Asta Posterior/fisiología , Terminales Presinápticos/fisiología , Animales , Dopamina/administración & dosificación , Potenciales Postsinápticos Excitadores , Femenino , Ganglios Espinales/fisiología , Masculino , Potenciales Postsinápticos Miniatura , Fibras Nerviosas Mielínicas/fisiología , Fibras Nerviosas Amielínicas/fisiología , Ratas Sprague-Dawley , Receptores de Dopamina D3/fisiología , Receptores de Dopamina D4/fisiologíaRESUMEN
BACKGROUND: The primary goal of this study was to determine whether administration of intrathecal morphine reduces postoperative pain. The secondary goal was to determine the effect of intrathecal morphine upon circulating levels of the weakly analgesic endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and the related lipids palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). METHODS: Forty two total knee arthroplasty (TKA) patients were enrolled in a prospective, double-blinded, randomized study. The intervention consisted of intrathecal morphine (200 µg) or placebo administered at the time of the spinal anesthesia. Postoperative pain was measured during the first 4 h after surgery while serum levels of AEA, 2-AG, PEA, OEA, and cortisol were measured at baseline and 4 h after surgery. RESULTS: Administration of intrathecal morphine reduced postoperative pain 4 h after TKA surgery compared to placebo (p = 0.005) and reduced postoperative systemic opioid consumption (p = 0.001). At baseline, intrathecal morphine led to a significant reduction in AEA, 2-AG, and OEA levels but did not affect PEA or cortisol levels. In patients administered intrathecal placebo, 2-AG levels were elevated 4 h after surgery; whereas patients receiving intrathecal morphine showed reductions in AEA, PEA, and OEA when compared to placebo. At 4 h after TKA surgery cortisol levels were significantly elevated in the placebo group and reduced in those receiving morphine. CONCLUSIONS: These results indicate that intrathecal morphine reduces postoperative pain in TKA patients. Furthermore, activation of central opioid receptors negatively modulates the endocannabinoid tone, suggesting that potent analgesics may reduce the stimulus for production of peripheral endocannabinoids. This study is the first to document the existence of rapid communication between the central opioid and peripheral endocannabinoid systems in humans. TRIAL REGISTRATION: This trial was registered retrospectively. TRIAL REGISTRY: NCT02620631 . Study to Examine Pain Relief With Supplemental Intrathecal Morphine in TKA Patients, NCT02620631 , 12/03/2015.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Anestesia Raquidea/métodos , Artroplastia de Reemplazo de Rodilla , Endocannabinoides/sangre , Morfina/uso terapéutico , Dolor Postoperatorio/tratamiento farmacológico , Anciano , Analgésicos Opioides/administración & dosificación , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Morfina/administración & dosificación , Estudios Prospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: Metabolism of the endocannabinoid 2-arachidonoylglycerol (2-AG) yields arachidonic acid (AA), the precursor to proalgesic eicosanoids including prostaglandin E2 (PGE2). Diacylglycerol lipase ß (DAGLß) is an enzyme that synthesizes 2-AG and its inhibition reduces eicosanoid levels and produces antinociceptive effects in models of inflammatory pain. Here we test whether inhibition of DAGLß produces antinociceptive effects in a model of postoperative pain. METHODS: Rats were administered the selective DAGLß inhibitor KT109 or vehicle and underwent plantar incision. Postsurgical pain/disability was examined using evoked (mechanical hyperalgesia), functional (incapacitance/weight bearing), and functional/spontaneous (locomotion) modalities. RESULTS: Activity-based protein profiling confirmed that KT109 inhibited DAGLß in the lumbar spinal cord (LSC) and brain, confirming that it is a systemically active DAGLß inhibitor. Treatment with KT109 reduced basal 2-AG, AA, and PGE2 levels in the liver but not the brain, indicating that DAGLß activity does not significantly contribute to basal PGE2 production within the central nervous system. Plantar incision elevated the levels of 2-AG and PGE2 in the LSC. Although KT109 did not alter postsurgical 2-AG levels in the LSC, it slightly reduced PGE2 levels. In contrast, the clinically efficacious cyclooxygenase inhibitor ketoprofen completely suppressed PGE2 levels in the LSC. Similarly, KT109 had no significant effect upon postsurgical 2-AG, AA, or PGE2 levels at the incision site, while ketoprofen abolished PGE2 production at this location. KT109 and ketoprofen reversed the weight bearing imbalance induced by plantar incision, yet neither KT109 nor ketoprofen had any significant effect on mechanical hyperalgesia. Treatment with ketoprofen partially but significantly rescued the locomotor deficit induced by incision while KT109 was without effect. CONCLUSION: DAGLß is not the principal enzyme that controls 2-AG derived AA and PGE2 production after surgery, and inhibitors targeting this enzyme are unlikely to be efficacious analgesics superior to those already approved to treat acute postoperative pain.
RESUMEN
BACKGROUND: Identifying drivers of pain that can serve as novel drug targets is important for improving perioperative analgesia. Total knee arthroplasty (TKA) is associated with significant postoperative pain. Cytokines contribute to the pathophysiology of osteoarthritis (OA) and associated pain. However, the influence of perioperative cytokine levels after TKA surgery upon postoperative pain remains unexplored. METHODS: We designed a prospective observational study to profile three proinflammatory cytokines, interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and leptin in serum, synovial, and cerebrospinal fluid of TKA patients perioperatively to determine associations between cytokine levels and pain. We characterized time-trajectories in cytokines pre- and post-surgery and explored their relationships to pain across gender. RESULTS: Preoperative pain, measured by functional pain disability scores (PDQ), was predictive of postoperative pain. There were no gender differences in severity of preoperative pain or acute postoperative pain. Serum IL-6, serum leptin, and synovial fluid leptin were positively correlated with body mass index and preoperative pain severity. Stratification of patients by gender revealed strong correlations between serum IL-6, leptin, and PDQ only in females, suggesting that females may be more sensitive to the nociceptive actions of these cytokines. Although serum IL-6 increased dramatically (and TNFα increased modestly) four hours after surgery and remained elevated at 72h; they were not associated with the severity of acute postoperative pain. CONCLUSIONS: Our data suggest that while preoperative chronic pain is predictive of the severity of acute postoperative pain in TKA patients, the pre- and post-operative inflammatory status does not predict postoperative pain.
Asunto(s)
Artralgia/fisiopatología , Artroplastia de Reemplazo de Rodilla/efectos adversos , Interleucina-6/metabolismo , Leptina/metabolismo , Osteoartritis de la Rodilla/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anciano , Artralgia/metabolismo , Líquido Cefalorraquídeo/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/fisiopatología , Osteoartritis de la Rodilla/cirugía , Dimensión del Dolor , Dolor Postoperatorio/metabolismo , Dolor Postoperatorio/fisiopatología , Estudios Prospectivos , Líquido Sinovial/metabolismoRESUMEN
BACKGROUND: We have previously reported elevated expression of multiple pro-inflammatory markers in the lumbar spinal cord (LSC) of middle-aged male rats compared to young adults suggesting a para-inflammatory state develops in the LSC by middle age, a time that in humans is associated with the greatest pain prevalence and persistence. The goal of the current study was to examine the transcriptome-wide gene expression differences between young and middle aged LSC. METHODS: Young (3 month) and middle-aged (17 month) naïve Fisher 344 rats (n = 5 per group) were euthanized, perfused with heparinized saline, and the LSC were removed. RESULTS: ~70% of 31,000 coding sequences were detected. After normalization, ~ 1100 showed statistically significant differential expression. Of these genes, 353 middle-aged annotated genes differed by > 1.5 fold compared to the young group. Nearly 10% of these genes belonged to the microglial sensome. Analysis of this subset revealed that the principal age-related differential pathways populated are complement, pattern recognition receptors, OX40, and various T cell regulatory pathways consistent with microglial priming and T cell invasion and modulation. Many of these pathways substantially overlap those previously identified in studies of LSC of young animals with chronic inflammatory or neuropathic pain. CONCLUSIONS: Up-modulation of complement pathway, microglial priming and activation, and T cell/antigen-presenting cell communication in healthy middle-aged LSC was found. Taken together with our previous work, the results support our conclusion that an incipient or para-inflammatory state develops in the LSC in healthy middle-aged adults.
RESUMEN
Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.
Asunto(s)
Analgésicos/uso terapéutico , Sistema Nervioso Central/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Dolor/tratamiento farmacológico , Nervios Periféricos/metabolismo , Analgésicos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Ciclobutanos/uso terapéutico , Ácidos Dicarboxílicos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Adyuvante de Freund/toxicidad , Ganglios Espinales/metabolismo , Hiperalgesia/etiología , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Dolor/complicaciones , Dolor/etiología , Umbral del Dolor/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción GenéticaRESUMEN
KEY POINTS: The transient receptor potential vanilloid type 1 (TRPV1) receptor is a polymodal molecular integrator in the pain pathway expressed in Aδ- and C-fibre nociceptors and is responsible for the thermal hyperalgesia associated with inflammatory pain. Noradrenaline strongly inhibited the activity of TRPV1 channels in dorsal root ganglia neurons. The effect of noradrenaline was reproduced by clonidine and antagonized by yohimbine, consistent with contribution of α2 adrenergic receptors. The inhibitory effect of noradrenaline on TRPV1 channels was dependent on calcium influx and linked to calcium/calmodulin-dependent protein kinase II. In spinal cord slices, clonidine reduced the frequency of capsaicin-induced miniature EPSCs in the presence of tetrodotoxin and ω-conotoxin-MVIIC, consistent with inhibition of presynaptic TRPV1 channels by α2 adrenergic receptors. We suggest that modulation of presynaptic TRPV1 channels in nociceptive neurons by descending noradrenergic inputs may constitute a mechanism for noradrenaline to modulate incoming noxious stimuli in the dorsal horn of the spinal cord. ABSTRACT: The transient receptor potential vanilloid type 1 (TRPV1) receptor is a well-known contributor to nociceptor excitability. To address whether noradrenaline can down-regulate TRPV1 channel activity in nociceptors and reduce their synaptic transmission, the effects of noradrenaline and clonidine were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27 µm) dorsal root ganglia (DRG) neurons and on miniature (m)EPSCs recorded from large lamina I neurons in horizontal spinal cord slices. Noradrenaline or clonidine inhibited the capsaicin-activated current by â¼60%, and the effect was reversed by yohimbine, confirming that it was mediated by activation of α2 adrenergic receptors. Similarly, clonidine reduced the frequency of capsaicin-induced mEPSCs by â¼60%. Inhibition of capsaicin-activated current by noradrenaline was mediated by GTP binding proteins, and was highly dependent on calcium influx. The inhibitory effect of noradrenaline on the capsaicin-activated current was not affected either by blocking the activity of protein kinase A with H89, or by blocking the activity of protein kinase C with bisindolylmaleimide II. In contrast, when the calcium/calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of noradrenaline on the capsaicin-activated current was greatly reduced, suggesting that activation of adrenergic receptors in DRG neurons is preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by noradrenaline in nociceptive neurons is a mechanism whereby noradrenaline may suppress incoming noxious stimuli at the primary synaptic afferents in the dorsal horn of the spinal cord.
Asunto(s)
Inhibición Neural/fisiología , Nociceptores/fisiología , Norepinefrina/farmacología , Terminales Presinápticos/fisiología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/fisiología , Animales , Capsaicina/farmacología , Relación Dosis-Respuesta a Droga , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Masculino , Inhibición Neural/efectos de los fármacos , Nociceptores/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
We previously reported that pretreatment with the potent antioxidant TEMPOL improves mitochondrial function and restores preconditioning in the aging heart. Because mitophagy is implicated in cardiac preconditioning and declines with age, this study was designed to investigate how age influences mitophagy in response to preconditioning and whether TEMPOL pretreatment improves it. Old (22-24 months) rats were pretreated with or without 4-week TEMPOL and compared with young (4-6 months) untreated rats. Cardioprotection induced by isoflurane (ISO) in vivo and in isolated cardiomyocytes in vitro was assessed following ischemia/reperfusion and simulated hypoxia/reoxygenation, respectively. Mitophagy was determined by comparing the levels/subcellular locations of key mitophagic markers using Western blotting and immunofluorescence techniques. ISO preconditioned the young but not old heart in vivo and in vitro. Aging impaired ISO-induced mitochondrial accumulation of PINK1 and Parkin, as well as mitochondrial ubiquitination, and baseline and ISO-induced autophagic flux assessed by LC3 puncta, membrane associated LC3-II and p62. Pretreatment with TEMPOL improved these processes and restored ISO preconditioning. Inhibition of autophagy abolished ISO-induced protection in cardiomyocytes from young and TEMPOL pretreated old rats. Thus, antioxidant pretreatment significantly improves mitophagic response to ISO in old myocardium, which may contribute to restoration of cardioprotection in senescent animals.
Asunto(s)
Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Anestésicos por Inhalación/farmacología , Antioxidantes/farmacología , Autofagia/fisiología , Óxidos N-Cíclicos/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Isoflurano/farmacología , Mitofagia/fisiología , Animales , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Medicación Preanestésica , Ratas , Ratas Endogámicas F344 , Marcadores de SpinRESUMEN
The transient receptor potential vanilloid type 1 (TRPV1) receptor plays a key role in the modulation of nociceptor excitability. To address whether dopamine can modulate the activity of TRPV1 channels in nociceptive neurons, the effects of dopamine and dopamine receptor agonists were tested on the capsaicin-activated current recorded from acutely dissociated small diameter (<27 µm) dorsal root ganglia (DRG) neurons. Dopamine or SKF 81297 (an agonist at D1/D5 receptors), caused inhibition of both inward and outward currents by â¼60% and â¼48%, respectively. The effect of SKF 81297 was reversed by SCH 23390 (an antagonist at D1/D5 receptors), confirming that it was mediated by activation of D1/D5 dopamine receptors. In contrast, quinpirole (an agonist at D2 receptors) had no significant effect on the capsaicin-activated current. Inhibition of the capsaicin-activated current by SKF 81297 was mediated by G protein coupled receptors (GPCRs), and highly dependent on external calcium. The inhibitory effect of SKF 81297 on the capsaicin-activated current was not affected when the protein kinase A (PKA) activity was blocked with H89, or when the protein kinase C (PKC) activity was blocked with bisindolylmaleimide II (BIM). In contrast, when the calcium-calmodulin-dependent protein kinase II (CaMKII) was blocked with KN-93, the inhibitory effect of SKF 81297 on the capsaicin-activated current was greatly reduced, suggesting that activation of D1/D5 dopamine receptors may be preferentially linked to CaMKII activity. We suggest that modulation of TRPV1 channels by dopamine in nociceptive neurons may represent a way for dopamine to modulate incoming noxious stimuli.
Asunto(s)
Agonistas de Dopamina/farmacología , Dopamina/metabolismo , Ganglios Espinales/metabolismo , Neuronas/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Benzazepinas/farmacología , Calcio/metabolismo , Células Cultivadas , Femenino , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Quinpirol/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Canales Catiónicos TRPV/efectos de los fármacosRESUMEN
BACKGROUND: Fatty acid binding proteins (FABPs) serve as intracellular carriers that deliver endocannabinoids and N-acylethanolamines to their catabolic enzymes. Inhibition of FABPs reduces endocannabinoid transport and catabolism in cells and FABP inhibitors produce antinociceptive and anti-inflammatory effects in mice. Potential analgesic effects in mice lacking FABPs, however, have not been tested. FINDINGS: Mice lacking FABP5 and FABP7, which exhibit highest affinities for endocannabinoids, possessed elevated levels of the endocannabinoid anandamide and the related N-acylethanolamines palmitoylethanolamide and oleoylethanolamide. There were no compensatory changes in the expression of other FABPs or in endocannabinoid-related proteins in the brains of FABP5/7 knockout mice. These mice exhibited reduced nociception in the carrageenan, formalin, and acetic acid tests of inflammatory and visceral pain. The antinociceptive effects in FABP5/7 knockout mice were reversed by pretreatment with cannabinoid receptor 1, peroxisome proliferator-activated receptor alpha, and transient receptor potential vanilloid 1 receptor antagonists in a modality specific manner. Lastly, the knockout mice did not possess motor impairments. CONCLUSIONS: This study demonstrates that mice lacking FABPs possess elevated levels of N-acylethanolamines, consistent with the idea that FABPs regulate the endocannabinoid and N-acylethanolamine tone in vivo. The antinociceptive effects observed in the knockout mice support a role for FABPs in regulating nociception and suggest that these proteins should serve as targets for the development of future analgesics.
Asunto(s)
Endocannabinoides/metabolismo , Etanolaminas/metabolismo , Proteínas de Unión a Ácidos Grasos/deficiencia , Inflamación/metabolismo , Proteínas de Neoplasias/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Dolor/metabolismo , Animales , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos/metabolismo , Inflamación/complicaciones , Inflamación/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nocicepción , Dolor/complicaciones , Dolor/fisiopatologíaRESUMEN
Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFß1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.
Asunto(s)
Envejecimiento/metabolismo , Ganglios Espinales/metabolismo , Neuralgia/metabolismo , Nocicepción/fisiología , Neuropatía Ciática/metabolismo , Médula Espinal/metabolismo , Factores de Edad , Envejecimiento/inmunología , Animales , Antígenos CD/metabolismo , Citocinas/metabolismo , Ganglios Espinales/inmunología , Masculino , Microglía/inmunología , Microglía/metabolismo , Neuralgia/inmunología , Ratas , Células Satélites Perineuronales/inmunología , Células Satélites Perineuronales/metabolismo , Neuropatía Ciática/inmunología , Médula Espinal/inmunologíaRESUMEN
BACKGROUND: There is compelling evidence in humans that peripheral endocannabinoid signaling is disrupted in obesity. However, little is known about the corresponding central signaling. Here, we have investigated the relationship between gender, leptin, body mass index (BMI) and levels of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in the serum and cerebrospinal fluid (CSF) of primarily overweight to obese patients with osteoarthritis. METHODOLOGY/PRINCIPAL FINDINGS: Patients (20 females, 15 males, age range 44-78 years, BMI range 24-42) undergoing total knee arthroplasty for end-stage osteoarthritis were recruited for the study. Endocannabinoids were quantified by liquid chromatography - mass spectrometry. AEA and 2-AG levels in the serum and CSF did not correlate with either age or BMI. However, 2-AG levels in the CSF, but not serum, correlated negatively with CSF leptin levels (Spearman's ρ -0.48, P=0.0076, n=30). No such correlations were observed for AEA and leptin. CONCLUSIONS/SIGNIFICANCE: In the patient sample investigated, there is a negative association between 2-AG and leptin levels in the CSF. This is consistent with pre-clinical studies in animals, demonstrating that leptin controls the levels of hypothalamic endocannabinoids that regulate feeding behavior.
Asunto(s)
Endocannabinoides/sangre , Endocannabinoides/líquido cefalorraquídeo , Leptina/sangre , Osteoartritis/sangre , Osteoartritis/líquido cefalorraquídeo , Adulto , Anciano , Ácidos Araquidónicos/sangre , Ácidos Araquidónicos/líquido cefalorraquídeo , Artroplastia de Reemplazo de Rodilla , Índice de Masa Corporal , Cromatografía Liquida , Femenino , Glicéridos/sangre , Glicéridos/líquido cefalorraquídeo , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Obesidad/sangre , Obesidad/líquido cefalorraquídeo , Osteoartritis/cirugía , Alcamidas Poliinsaturadas/sangre , Alcamidas Poliinsaturadas/líquido cefalorraquídeoRESUMEN
Osteoarthritis (OA) of the knee is a progressive disease that is associated with inflammation of the joints and lower extremity pain. Total knee arthroplasty (TKA) is a surgical procedure that aims to reduce pain and restore motor function in patients suffering from OA. The immediate postoperative period can be intensely painful leading to extended recovery times including persistent pain. The endocannabinoid system regulates nociception, and the activation of cannabinoid receptors produces antinociceptive effects in preclinical models of OA. To date, the influence of the endocannabinoid tone on pain and disability in OA patients and on acute postoperative pain in humans has not been explored. In this study, we provide the first comprehensive profile of endocannabinoids in serum, cerebrospinal fluid, and synovial fluid of patients with painful end-stage OA undergoing TKA and examine correlations between endocannabinoid levels, interleukin 6, functional disability, acute postoperative pain, and postoperative opioid use. Our results reveal that central (cerebrospinal fluid) and peripheral (synovial fluid) levels of the endocannabinoid 2-arachidonoyl glycerol were significantly elevated in patients who developed higher postoperative pain after TKA. In addition, synovial fluid 2-arachidonoyl glycerol levels were positively correlated with postoperative opioid use. Similarly, synovial fluid levels of the anti-inflammatory lipid palmitoylethanolamide correlated with functional disability in OA. Taken together, our results are the first to reveal associations between central and peripheral endocannabinoid levels and postoperative pain. This suggests that endocannabinoid metabolism may serve as a target for the development of novel analgesics both for systemic or local delivery into the joint.
Asunto(s)
Dolor Agudo/metabolismo , Artroplastia de Reemplazo de Rodilla/efectos adversos , Endocannabinoides/metabolismo , Dolor Postoperatorio/metabolismo , Líquido Sinovial/metabolismo , Dolor Agudo/líquido cefalorraquídeo , Dolor Agudo/diagnóstico , Anciano , Artroplastia de Reemplazo de Rodilla/tendencias , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Endocannabinoides/líquido cefalorraquídeo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Dolor Postoperatorio/líquido cefalorraquídeo , Dolor Postoperatorio/diagnóstico , Estudios ProspectivosRESUMEN
AIM: To investigate the role of extracellular signal-regulated kinases (ERKs) in sevoflurane post-conditioning induced cardioprotection in vitro. METHODS: Isolated rat hearts were subjected to 30 min ischemia followed by 120 min reperfusion (I/R). Sevoflurane post-conditioning was carried out by administration of O2-enriched gas mixture with 3% sevoflurane (SEVO) for 15 min from the onset of reperfusion. Cardiac functions, myocardial infarct size, myocardial ATP and NAD(+) contents, mitochondrial ultrastructure, and anti-apototic and anti-oncosis protein levels were measured. RESULTS: Sevoflurane post-conditioning significantly improved the heart function, decreased infarct size and mitochondria damage, and increased myocardial ATP and NAD(+) content in the I/R hearts. Furthermore, sevoflurane post-conditioning significantly increased the levels of p-ERK and p-p70S6K, decreased the levels of porimin, caspase-8, cleaved caspase-3, and cytosolic cytochrome c in the I/R hearts. Co-administration of the ERK1/2 inhibitor PD98059 (20 µmol/L) abolished the sevoflurane-induced protective effects against myocardial I/R. CONCLUSION: Sevoflurane post-conditioning protects isolated rat hearts against myocardial I/R injury and inhibits cell oncosis and apoptosis via activation of the ERK1/2 pathway.
Asunto(s)
Cardiotónicos/farmacología , Éteres Metílicos/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Infarto del Miocardio/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/enzimología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Citoprotección , Modelos Animales de Enfermedad , Activación Enzimática , Frecuencia Cardíaca/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/ultraestructura , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Contracción Miocárdica/efectos de los fármacos , Infarto del Miocardio/enzimología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/ultraestructura , NAD/metabolismo , Perfusión , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas Sprague-Dawley , Sevoflurano , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.
Asunto(s)
Analgesia , Analgésicos/farmacología , Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Endocannabinoides/metabolismo , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Alcamidas Poliinsaturadas/metabolismo , Analgésicos/química , Analgésicos/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , PPAR alfa/metabolismo , Unión Proteica , Ratas , Receptor Cannabinoide CB1/metabolismoRESUMEN
BACKGROUND: Dopaminergic fibers originating from area A11 of the hypothalamus project to different levels of the spinal cord and represent the major source of dopamine. In addition, tyrosine hydroxylase, the rate-limiting enzyme for the synthesis of catecholamines, is expressed in 8-10% of dorsal root ganglia (DRG) neurons, suggesting that dopamine may be released in the dorsal root ganglia. Dopamine has been shown to modulate calcium current in DRG neurons, but the effects of dopamine on sodium current and on the firing properties of small DRG neurons are poorly understood. RESULTS: The effects of dopamine and dopamine receptor agonists were tested on the tetrodotoxin-resistant (TTX-R) sodium current recorded from acutely dissociated small (diameter ≤ 25 µm) DRG neurons. Dopamine (20 µM) and SKF 81297 (10 µM) caused inhibition of TTX-R sodium current in small DRG neurons by 23% and 37%, respectively. In contrast, quinpirole (20 µM) had no effects on the TTX-R sodium current. Inhibition by SKF 81297 of the TTX-R sodium current was not affected when the protein kinase A (PKA) activity was blocked with the PKA inhibitory peptide (6-22), but was greatly reduced when the protein kinase C (PKC) activity was blocked with the PKC inhibitory peptide (19-36), suggesting that activation of D1/D5 dopamine receptors is linked to PKC activity. Expression of D1and D5 dopamine receptors in small DRG neurons, but not D2 dopamine receptors, was confirmed by Western blotting and immunofluorescence analysis. In current clamp experiments, the number of action potentials elicited in small DRG neurons by current injection was reduced by ~ 30% by SKF 81297. CONCLUSIONS: We conclude that activation of D1/D5 dopamine receptors inhibits TTX-R sodium current in unmyelinated nociceptive neurons and dampens their intrinsic excitability by reducing the number of action potentials in response to stimulus. Increasing or decreasing levels of dopamine in the dorsal root ganglia may serve to adjust the sensitivity of nociceptors to noxious stimuli.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D5/metabolismo , Sodio/metabolismo , Tetrodotoxina/farmacología , Anestésicos Locales/farmacología , Animales , Benzazepinas/farmacología , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Femenino , Masculino , Ratones , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D5/agonistasRESUMEN
Cardioprotective effects of anesthetic preconditioning and cyclosporine A (CsA) are lost with aging. To extend our previous work and address a possible mechanism underlying age-related differences, we investigated the role of oxidative stress in the aging heart by treating senescent animals with the oxygen free radical scavenger Tempol. Old male Fischer 344 rats (22-24 mo) were randomly assigned to control or Tempol treatment groups for 2 or 4 wk (T×2wk and T×4wk, respectively). Rats received isoflurane 30 min before ischemia-reperfusion injury or CsA just before reperfusion. Myocardial infarction sizes were significantly reduced by isoflurane or CsA in the aged rats treated with Tempol (T×4wk) compared with old control rats. In other experiments, young (4-6 mo) and old rats underwent either chronic Tempol or vehicle treatment, and the levels of myocardial protein oxidative damage, antioxidant enzymes, mitochondrial Ca(2+) uptake, cyclophilin D protein, and mitochondrial permeability transition pore opening times were measured. T×4wk significantly increased MnSOD enzyme activity, GSH-to-GSSH ratios, MnSOD protein level, mitochondrial Ca(2+) uptake capacity, reduced protein nitrotyrosine levels, and normalized cyclophilin D protein expression in the aged rat heart. T×4wk also significantly prolonged mitochondrial permeability transition pore opening times induced by reactive oxygen species in old cardiomyocytes. Our studies demonstrate that 4 wk of Tempol pretreatment restores anesthetic preconditioning and cardioprotection by CsA in the old rat and that this is associated with decreased oxidative stress and improved mitochondrial function. Our results point to a new protective strategy for the ischemic myocardium in the high-risk older population.
Asunto(s)
Envejecimiento/metabolismo , Antioxidantes/farmacología , Óxidos N-Cíclicos/farmacología , Precondicionamiento Isquémico Miocárdico/métodos , Isquemia Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Animales , Calcio/farmacocinética , Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Corazón/fisiología , Hemodinámica/fisiología , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/metabolismo , Infarto del Miocardio/patología , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patología , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas Endogámicas F344 , Marcadores de Spin , Superóxido Dismutasa/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Anesthetic preconditioning (APC) and ischemic preconditioning (IPC) are lost with normal aging. Here, we investigated age-related difference between phosphoglycogen synthase kinase-3beta (pGSK-3ß) and pGSK-3ß with modulators of mitochondrial permeability transition pore, including adenine nucleotide translocase (ANT), cyclophilin-D, or voltage-dependent anion channel. APC or IPC significantly increased pGSK-3ß in the young groups in both the cytosol and the mitochondria and also significantly increased pGSK-3ß in co-immunoprecipitates with ANT. Importantly, the level of cyclophilin-D in co-immunoprecipitates with ANT was significantly decreased in the young APC and IPC groups, but not in old rats. We also found that APC or IPC significantly prolonged mitochondrial permeability transition pore opening time in the young cardiomyocytes under oxidative stress, but not in the elderly. Attenuation of APC or IPC protection in the aging heart is associated with failure to reduce ANT-cyclophilin-D interactions and to decreased pGSK-3ß responsiveness of ANT, critical modulators of mitochondrial permeability transition pore.
Asunto(s)
Ciclofilinas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Precondicionamiento Isquémico Miocárdico/métodos , Mitocondrias Cardíacas/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Canales Aniónicos Dependientes del Voltaje/metabolismo , Factores de Edad , Animales , Senescencia Celular , Peptidil-Prolil Isomerasa F , Glucógeno Sintasa Quinasa 3 beta , Inmunoprecipitación , Masculino , Poro de Transición de la Permeabilidad Mitocondrial , Modelos Animales , Miocitos Cardíacos/metabolismo , Estrés Oxidativo , Ratas , Ratas Endogámicas F344RESUMEN
Phospholipase C-δ(1) (PLCδ(1)) is a widely expressed highly active PLC isoform, modulated by Ca(2+) that appears to operate downstream from receptor signaling and has been linked to regulation of cytokine production. Here we investigated whether PLCδ(1) modulated expression of the pro-inflammatory cytokines interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in rat C6 glioma cells. Expression of PLCδ(1) was specifically suppressed by small interfering RNA (siRNA) and the effects on cytokine mRNA expression, stimulated by the Toll-like receptor (TLR) agonist, lipopolysaccharide (LPS), were examined. Real-time polymerase chain reaction (RT-PCR) results showed that PLCδ(1) knockdown enhanced expression IL-1ß and tumor necrosis factor-α (TNF-α) mRNA by at least 100 fold after 4 h of LPS stimulation compared to control siRNA treatment. PLCδ(1) knock down caused persistently high Nfκb levels at 4 h of LPS stimulation compared to control siRNA-treated cells. PLCδ(1) knockdown was also associated with elevated nuclear levels of c-Jun after 30 min of LPS stimulation, but did not affect LPS-stimulated p38 or p42/44 MAPK phosphorylation, normally associated with TLR activation of cytokine gene expression; rather, enhanced protein kinase C (PKC) phosphorylation of cellular proteins was observed in the absence of LPS stimulation. An inhibitor of PKC, bisindolylmaleimide II (BIM), reversed phosphorylation, prevented elevation of nuclear c-Jun levels, and inhibited LPS-induced increases of IL-1ß and TNF-α mRNA's induced by PLCδ(1) knockdown. Our results show that loss of PLCδ(1) enhances PKC/c-Jun signaling and up-modulates pro-inflammatory cytokine gene transcription in concert with the TLR-stimulated p38MAPK/Nfκb pathway. Our findings are consistent with the idea that PLCδ(1) is a suppressor of PKC activity.