RESUMEN
This study examined whether peripheral inflammatory injury increases the levels or changes the disposition of substance P (SubP) in the rostral ventromedial medulla (RVM), which serves as a central relay in bulbospinal pathways of pain modulation. Enzyme immunoassay and reverse transcriptase quantitative polymerase chain reaction were used to measure SubP protein and transcript, respectively, in tissue homogenates prepared from the RVM and the periaqueductal gray (PAG) and cuneiform nuclei of rats that had received an intraplantar injection of saline or complete Freund's adjuvant (CFA). Matrix-Assisted Laser Desorption/Ionization Time of Flight analysis confirmed that the RVM does not contain hemokinin-1 (HK-1), which can confound measurements of SubP because it is recognized equally well by commercial antibodies for SubP. Levels of SubP protein in the RVM were unchanged four hours, four days and two weeks after injection of CFA. Tac1 transcripts were similarly unchanged in the RVM four days or two weeks after CFA. In contrast, the density of SubP immunoreactive processes in the RVM increased 2-fold within four hours and 2.7-fold four days after CFA injection; it was unchanged at two weeks. SubP-immunoreactive processes in the RVM include axon terminals of neurons located in the PAG and cuneiform nucleus. SubP content in homogenates of the PAG and cuneiform nucleus was significantly increased four days after CFA, but not at four hours or two weeks. Tac1 transcripts in homogenates of these nuclei were unchanged four days and two weeks after CFA. These findings suggest that there is an increased mobilization of SubP within processes in the RVM shortly after injury accompanied by an increased synthesis of SubP in neurons that project to the RVM. These findings are consonant with the hypothesis that an increase in SubP release in the RVM contributes to the hyperalgesia that develops after peripheral inflammatory injury.
Asunto(s)
Inflamación/complicaciones , Bulbo Raquídeo/metabolismo , Dolor/etiología , Dolor/patología , Sustancia P/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Lateralidad Funcional , Regulación de la Expresión Génica , Hiperalgesia/fisiopatología , Inflamación/inducido químicamente , Masculino , Proteína Quinasa 6 Activada por Mitógenos/metabolismo , Dimensión del Dolor , Umbral del Dolor/fisiología , Sustancia Gris Periacueductal/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Taquicininas/genética , Taquicininas/metabolismoRESUMEN
The rostral ventromedial medulla (RVM), a central relay in the bulbospinal pathways that modulate nociception, contains high concentrations of substance P (Sub P) and neurokinin-1 (NK1) receptors. However, the function of Sub P in the RVM is poorly understood. This study characterized the actions of Sub P in the RVM in the absence of injury and then used two NK1 receptor antagonists, L-733,060 and L-703, 606, to probe the role of endogenously released Sub P in the development and maintenance of persistent inflammatory nociception of immune or neurogenic origin. In uninjured rats, microinjection of Sub P in the RVM produced a transient thermal antinociception that was attenuated by pretreatment with L-733,060 or L-703,606. It did not alter threshold to withdrawal from tactile stimulation with von Frey filaments. Microinjection of the antagonists alone did not alter paw withdrawal latency (PWL) or threshold suggesting that Sub P is not tonically released in the RVM in the absence of injury. However, microinjection of either antagonist in the RVM was sufficient to reverse heat hyperalgesia 4 h, 4 days or 2 weeks after intraplantar (ipl) injection of complete Freund's adjuvant (CFA). Antagonism of NK1 receptors in the RVM did not prevent or reverse tactile hypersensitivity induced by CFA, but did attenuate that produced by capsaicin. NK1 receptor antagonism did not prevent the development of thermal hyperalgesia, tactile hypersensitivity or spontaneous pain behaviors induced by mustard oil (MO). The results suggest that Sub P has bimodal actions in the RVM and that following inflammatory injury, it can play a critical role as a pronociceptive agent in the development and maintenance of hyperalgesia and tactile hypersensitivity. However, its actions are highly dependent on the stimulus modality and the type of injury, and this may be an additional basis for the poor efficacy of NK1 receptor antagonists in clinical trials.
Asunto(s)
Bulbo Raquídeo/efectos de los fármacos , Antagonistas del Receptor de Neuroquinina-1 , Dolor/tratamiento farmacológico , Receptores de Neuroquinina-1/agonistas , Animales , Capsaicina , Fármacos del Sistema Nervioso Central/farmacología , Adyuvante de Freund , Calor , Masculino , Bulbo Raquídeo/metabolismo , Planta de la Mostaza , Dolor/inducido químicamente , Dolor/metabolismo , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Estimulación Física , Piperidinas/farmacología , Aceites de Plantas , Quinuclidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Factores de TiempoRESUMEN
Chronic neuropathic pain caused by peripheral nerve injury is associated with global changes in gene expression in damaged neurons. To understand the molecular mechanisms underlying neuropathic pain, it is essential to elucidate how nerve injury alters gene expression and how the change contributes to the development and maintenance of chronic pain. MicroRNAs are non-protein-coding RNA molecules that regulate gene expression in a wide variety of biological processes mainly at the level of translation. This study investigated the possible involvement of microRNAs in gene regulation relevant to neuropathic pain. The analyses focused on a sensory organ-specific cluster of microRNAs that includes miR-96, -182, and -183. Quantitative real-time polymerase chain reaction (qPCR) analyses confirmed that these microRNAs were highly enriched in the dorsal root ganglion (DRG) of adult rats. Using the L5 spinal nerve ligation (SNL) model of chronic neuropathic pain, we observed a significant reduction in expression of these microRNAs in injured DRG neurons compared to controls. In situ hybridization and immunohistochemical analyses revealed that these microRNAs are expressed in both myelinated (N52 positive) and unmyelinated (IB4 positive) primary afferent neurons. They also revealed that the intracellular distributions of the microRNAs in DRG neurons were dramatically altered in animals with mechanical hypersensitivity. Whereas microRNAs were uniformly distributed within the DRG soma of non-allodynic animals, they were preferentially localized to the periphery of neurons in allodynic animals. The redistribution of microRNAs was associated with changes in the distribution of the stress granule (SG) protein, T-cell intracellular antigen 1 (TIA-1). These data demonstrate that SNL induces changes in expression levels and patterns of miR-96, -182, and -183, implying their possible contribution to chronic neuropathic pain through translational regulation of pain-relevant genes. Moreover, SGs were suggested to be assembled and associated with microRNAs after SNL, which may play a role in modification of microRNA-mediated gene regulation in DRG neurons.
Asunto(s)
Ganglios Espinales/metabolismo , MicroARNs/metabolismo , Neuralgia/metabolismo , Neuronas/metabolismo , Nervios Espinales/lesiones , Animales , Enfermedad Crónica , Modelos Animales de Enfermedad , Espacio Intracelular/metabolismo , Ligadura , Masculino , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Amielínicas/metabolismo , Neuralgia/etiología , Neuronas Aferentes/metabolismo , Estimulación Física , Ratas , Ratas Sprague-DawleyRESUMEN
Loss of GABA-mediated inhibition in the spinal cord is thought to mediate allodynia and spontaneous pain after nerve injury. Despite extensive investigation of GABA itself, relatively little is known about how nerve injury alters the receptors at which GABA acts. This study examined levels of GABA(B) receptor protein in the spinal cord dorsal horn, and in the L4 and L5 (lumbar designations) dorsal root ganglia one to 18 weeks after L5 spinal nerve ligation. Mechanical allodynia was maximal by 1 week and persisted at blunted levels for at least 18 weeks after injury. Spontaneous pain behaviors were evident for 6 weeks. Western blotting of dorsal horn detected two isoforms of the GABA(B(1)) subunit and a single GABA(B(2)) subunit. High levels of GABA(B(1a)) and low levels of GABA(B(1b)) protein were present in the dorsal root ganglia. However, GABA(B(2)) protein was not detected in the dorsal root ganglia, consistent with the proposed existence of an atypical receptor composed of GABA(B(1)) homodimers. The levels of GABA(B(1a)), GABA(B(1b)), and GABA(B(2)) protein in the ipsilateral dorsal horn were unchanged at any time after injury. Immunohistochemical staining also did not detect a change in GABA(B(1)) or GABA(B(2)) subunits in dorsal horn segments having a robust loss of isolectin B4 staining. The levels of GABA(B(1a)) protein were also unchanged in the L4 or L5 dorsal root ganglia at any time after spinal nerve ligation. Levels of GABA(B(2)) remained undetectable. Finally, baclofen-stimulated binding of guanosine-5'-(gamma-O-thio)triphosphate in dorsal horn did not differ between sham and ligated rats. Collectively, these results argue that a loss of GABA(B) receptor-mediated inhibition, particularly of central terminals of primary afferents, is unlikely to mediate the development or maintenance of allodynia or spontaneous pain behaviors after spinal nerve injury.
Asunto(s)
Ganglios Espinales/metabolismo , Neuralgia/metabolismo , Traumatismos de los Nervios Periféricos , Nervios Periféricos/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Células del Asta Posterior/metabolismo , Receptores de GABA-B/metabolismo , Animales , Baclofeno/farmacología , Desnervación , Modelos Animales de Enfermedad , Agonistas del GABA/farmacología , Ganglios Espinales/citología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Ligadura , Masculino , Inhibición Neural/fisiología , Neuralgia/fisiopatología , Nervios Periféricos/fisiopatología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Células del Asta Posterior/citología , Terminales Presinápticos/metabolismo , Subunidades de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/fisiología , Regulación hacia Arriba/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
This study examined the role of spinal GABAergic, serotoninergic and alpha(2) adrenergic receptors in the antinociception produced by the microinjection of equi-antinociceptive doses of selective opioid receptor agonists in the nucleus raphe magnus (NRM) or the nucleus reticularis gigantocellularis pars alpha (NGCpalpha) of the rat. Rats were pretreated with intrathecal administration of either the GABA(A) receptor antagonist bicuculline, the GABA(B) receptor antagonist CGP35348, the serotonin(1/2) receptor antagonist methysergide, the alpha(2) adrenergic receptor antagonist yohimbine or saline. Ten minutes later, either the delta(1) opioid receptor agonist [D-Pen(2,5)]enkephalin (DPDPE), delta(2) opioid receptor agonist [D-Ala(2),Glu(4)]deltorphin (DELT) or mu opioid receptor agonist [D-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) was microinjected into the NRM, NGCpalpha or sites in the medulla outside these two regions. The increase in tail-flick latency produced by microinjection of DPDPE into the NRM or NGCpalpha was antagonized by intrathecal pretreatment with either methysergide or yohimbine. Intrathecal pretreatment with CGP35348 antagonized the antinociception produced by microinjection of DPDPE in the NRM, whereas bicuculline antagonized the antinociception produced by microinjection of DPDPE in the NGCpalpha. The increase in tail-flick latency produced by microinjection of DELT into the NGCpalpha, but not the NRM was antagonized by intrathecal pretreatment with yohimbine or CGP35348. Intrathecal pretreatment with methysergide or bicuculline did not antagonize the antinociception produced by microinjection of DELT into either the NRM or the NGCpalpha. The increase in tail-flick latency produced by microinjection of DAMGO in the NRM was antagonized by intrathecal pretreatment with methysergide or CGP35348, but not by bicuculline or yohimbine. Taken together, these results support the hypothesis that the antinociception produced by activation of delta(1), delta(2) or mu opioid receptors in the rostral ventromedial medulla is mediated by different neural substrates.
Asunto(s)
Vías Eferentes/metabolismo , Bulbo Raquídeo/metabolismo , Neuronas/metabolismo , Nociceptores/metabolismo , Dolor/metabolismo , Receptores Opioides/metabolismo , Médula Espinal/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Interacciones Farmacológicas/fisiología , Vías Eferentes/citología , Vías Eferentes/efectos de los fármacos , Antagonistas del GABA/farmacología , Masculino , Bulbo Raquídeo/citología , Bulbo Raquídeo/efectos de los fármacos , Narcóticos/farmacología , Neuronas/efectos de los fármacos , Nociceptores/efectos de los fármacos , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores de GABA/efectos de los fármacos , Receptores de GABA/metabolismo , Receptores Opioides/agonistas , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/metabolismo , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Médula Espinal/citología , Médula Espinal/efectos de los fármacosRESUMEN
BACKGROUND: Possible utility of high-dose i.v. melatonin as an anaesthetic adjuvant has not been studied. This study compared its effects with thiopental and propofol. METHODS: Sprague Dawley rats were assigned to receive bolus or cumulative i.v. doses of melatonin, thiopental or propofol. Righting reflex, hindpaw withdrawal to a noxious stimulus, response to tail clamping and haemodynamic effects were assessed. RESULTS: Melatonin caused a dose-dependent increase in paw withdrawal threshold and the percent of rats displaying loss of the righting reflex. Melatonin was comparable to thiopental and propofol in terms of its rapid onset of hypnosis. The mean ED(50) values for loss of righting reflex were 5.4 (SEM 1.2), 12.5 (1.1) and 178 (1.1) mg kg(-1) for propofol, thiopental and melatonin, respectively. The percent of rats displaying loss of response to tail clamping was greater with propofol than with melatonin (P<0.05). Haemodynamic changes produced by melatonin or propofol were similar in onset and magnitude. CONCLUSIONS: I.V. melatonin can exert hypnotic effects similar to those observed with thiopental and propofol. Melatonin exhibited significant antinociceptive effects but was less effective in abolishing the response to tail clamping.
Asunto(s)
Adyuvantes Anestésicos/farmacología , Anestésicos Intravenosos/farmacología , Melatonina/farmacología , Umbral del Dolor/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Hemodinámica/efectos de los fármacos , Masculino , Dimensión del Dolor/métodos , Propofol/farmacología , Ratas , Ratas Sprague-Dawley , Reflejo/efectos de los fármacos , Tiopental/farmacologíaRESUMEN
Our understanding of the molecular, structural and pharmacological properties of the GABA(B) receptor has lagged behind that of the GABA(A) receptor for several decades. This situation has changed with the recent cloning of this receptor, revealing an unexpected and highly intriguing complexity of both structure and function.
Asunto(s)
Receptores de GABA-B/efectos de los fármacos , Analgésicos/farmacología , Animales , Sistema Nervioso Central/metabolismo , Humanos , Ligandos , Receptores de GABA-B/química , Transducción de Señal/efectos de los fármacosRESUMEN
Human immunodeficiency virus-1 (HIV-1) infection is associated with numerous effects on the nervous system, including pain and peripheral neuropathies. We now demonstrate that cultured rat dorsal root ganglion (DRG) neurons express a wide variety of chemokine receptors, including those that are thought to act as receptors for the HIV-1 coat protein glycoprotein120 (gp120). Chemokines that activate all of the known chemokine receptors increased [Ca(2+)](i) in subsets of cultured DRG cells. Many neurons responded to multiple chemokines and also to bradykinin, ATP, and capsaicin. Immunohistochemical studies demonstrated the expression of the CXCR4 and CCR4 chemokine receptors on populations of DRG neurons that also expressed substance P and the VR1 vanilloid receptor. RT-PCR analysis confirmed the expression of CXCR4, CX3CR1, CCR4, and CCR5 mRNAs in DRG neurons. Chemokines and gp120 produced excitatory effects on DRG neurons and also stimulated the release of substance P. Chemokines and gp120 also produced allodynia after injection into the rat paw. Thus these results provide evidence that chemokines and gp120 may produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons. Chemokine receptor antagonists may be important therapeutic interventions in the pain that is associated with HIV-1 infection and inflammation.
Asunto(s)
Quimiocinas/metabolismo , Proteína gp120 de Envoltorio del VIH/farmacología , Hiperalgesia/fisiopatología , Neuronas/metabolismo , Nociceptores/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Bradiquinina/farmacología , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Quimiocinas/farmacología , Colorantes Fluorescentes , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Hiperalgesia/inducido químicamente , Inflamación/metabolismo , Líquido Intracelular/metabolismo , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Nociceptores/citología , Nociceptores/efectos de los fármacos , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores CCR4 , Receptores CXCR4/biosíntesis , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Sustancia P/metabolismoRESUMEN
In vitro evidence indicates that gamma-aminobutyric acid (GABA), acting at GABA(A) receptors, exerts a positive trophic effect on monoaminergic neurons during embryogenesis. To determine whether in vivo antagonism of GABA(A) receptors during embryogenesis interferes with the development of monoaminergic neurons, we used mice in which the number of GABA(A) receptors was decreased by 50% by targeted deletion of the beta(3) subunit gene of the GABA(A) receptor. Levels of serotonin, dopamine, norepinephrine, and the metabolites 3,4-deoxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in the brainstem, cortex, striatum and spinal cord of female adult homozygous null (beta3-/-) and wild-type (beta3+/+) mice, as well as progenitor C57BL/6J and Strain 129/SvJ mice. The level of norepinephrine in the spinal cord of beta3-/- mice was 44% less than that of beta3+/+ mice, and did not differ in the brainstem, cortex or striatum. This finding suggests that beta3 subunit-containing GABA(A) receptors mediate the trophic effects of GABA on a subpopulation of spinally-projecting noradrenergic neurons. In contrast, the levels of serotonin, dopamine or their metabolites were unaffected, suggesting that the development of serotonergic and dopaminergic neurons may require activation of only a small fraction of GABA(A) receptors or may not be dependent on beta3 subunit-containing GABA(A) receptors. Finally, Strain 129/SvJ and C57BL/6J mice differed with respect to the levels of dopamine and its metabolites in the brainstem, spinal cord and cortex. These differences may need to be considered when assessing the phenotype of gene-targeted mice for which these mice serve as progenitor strains.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptores de GABA-A/deficiencia , Animales , Femenino , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de GABA-A/genéticaRESUMEN
This study examined a mechanism responsible for the enhanced antihyperalgesic and antinociceptive effects of the mu opioid receptor agonist (ORA) [D-Ala(2), NMePhe(4), Gly(5)-ol]enkephalin (DAMGO) microinjected in the rostroventromedial medulla (RVM) of rats with inflammatory injury induced by injection of complete Freund's adjuvant (CFA) in one hindpaw. In rats injected with CFA 4 hr earlier, microinjection of the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) in the RVM antagonized both the marginal enhancement of the potency of DAMGO and its antinociceptive effect. The delta opioid receptor antagonist naltriben (NTB) was without effect. In rats injected with CFA 2 weeks earlier, CTAP antagonized the effects of DAMGO to a lesser extent. However, NTB completely prevented the enhancement of the potency of DAMGO, whereas it did not antagonize DAMGO's antinociceptive effects. Microinjection of NTB alone, but not CTAP in the RVM of CFA-treated rats, enhanced the hyperalgesia present in the ipsilateral hindpaw and induced hyperalgesia in the contralateral, uninjured hindpaw. These results suggest that persistent inflammatory injury increased the release in the RVM of opioid peptides with preferential affinity for the delta opioid receptor, which can interact in a synergistic or additive manner with an exogenously administered mu opioid receptor agonist. Indeed, the levels of [Met(5)]enkephalin and [Leu(5)]enkephalin were increased in the RVM and in other brainstem nuclei in CFA-treated rats. This increase most likely presents a compensatory neuronal response of the CNS of the injured animal to mitigate the full expression of inflammatory pain and to enhance the antinociceptive and antihyperalgesic effects of exogenously administered mu opioid receptor analgesics.
Asunto(s)
Encéfalo/efectos de los fármacos , Encefalinas/metabolismo , Inflamación/metabolismo , Dolor/metabolismo , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/efectos de los fármacos , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5)/administración & dosificación , Masculino , Microinyecciones , Naltrexona/administración & dosificación , Naltrexona/análogos & derivados , Fragmentos de Péptidos , Péptidos/administración & dosificación , Ratas , Ratas Sprague-Dawley , SomatostatinaRESUMEN
A line of mice was recently created in which the gabrb3 gene, which encodes the beta3 subunit of the GABA(A) receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA(A) receptors in the CNS enabled an investigation of the role of GABA and GABA(A) receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA(A) and GABA(B) receptor agonists. Homozygous null (beta3-/-) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (beta3+/+) and heterozygous (beta3+/-) littermates. The beta3-/- mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their beta3+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in beta3-/- mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA(A) receptors in the spinal cord. As expected, subcutaneous administration of the GABA(A) receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in beta3-/- mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, beta3+/+ and beta3+/- mice. However, the antinociceptive effect of the GABA(B) receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in beta3-/- mice compared to the progenitor strains, beta3+/+ or beta3+/- mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA(A) receptors can affect the production of antinociception by other analgesic drugs as well.
Asunto(s)
Agonistas del GABA/farmacología , Nociceptores/efectos de los fármacos , Isoformas de Proteínas/deficiencia , Receptores de GABA-A/deficiencia , Umbral Sensorial/fisiología , Animales , Baclofeno/administración & dosificación , Baclofeno/farmacología , Agonistas del GABA/administración & dosificación , Calor , Inyecciones Espinales , Inyecciones Subcutáneas , Ácidos Isonicotínicos/administración & dosificación , Ácidos Isonicotínicos/farmacología , Isoxazoles/administración & dosificación , Isoxazoles/farmacología , Ratones , Ratones Endogámicos C57BL/genética , Ratones Endogámicos/genética , Estimulación Física , Isoformas de Proteínas/genética , Receptores de GABA-A/genética , Caracteres SexualesRESUMEN
Several lines of evidence indicate that the antinociception produced by intrathecal administration of the alpha(2)-adrenoceptor agonists dexmedetomidine or ST-91 is mediated by different subtypes of the alpha(2)-adrenoceptor. We recently provided additional pharmacologic evidence for this idea, as well as for differences in the function of these receptors between Harlan and Sasco rats, two widely-used outbred substrains of Sprague-Dawley rat. The present study used isobolographic analysis to further characterize the receptors at which intrathecally administered ST-91 and dexmedetomidine act in these two substrains. The rationale for these studies derives from the assumption that if dexmedetomidine and ST-91 act as agonists at the same receptor then they should interact in an additive manner. However, if they interact in a supra-additive manner, then they must act at different subtypes of the alpha(2)-adrenoceptor. In the tail-flick test, the dose-effect relationship for a 1:3 mixture of dexmedetomidine and ST-91 was shifted significantly to the left of the theoretical dose-additive line in both Harlan and Sasco Sprague-Dawley rats. A similar finding was made in the hot-plate test despite the fact that the dose-response characteristics of the agonists were different in this test. Thus, in Harlan rats, in which ST-91 is a full agonist and dexmedetomidine is essentially inactive, the dose-effect relationship for the mixture of dexmedetomidine and ST-91 was shifted far to the left of the dose-additive line. Similarly, in Sasco rats, in which ST-91 is a partial agonist and dexmedetomidine is inactive, co-administration of the two agonists also shifted the dose-response relationship to the left of the dose-additive line. The consistent finding that these two alpha(2)-adrenoceptor agonists interact in a supra-additive manner provides strong evidence that dexmedetomidine and ST-91 produce antinociception by acting at different alpha(2)-adrenoceptor subtypes in the spinal cord. This conclusion is consistent with the earlier proposal that dexmedetomidine acts predominantly at alpha(2A)-adrenoceptors whereas ST-91 acts predominantly at non-alpha(2A)-adrenoceptors. Recent anatomical evidence indicates that these non-alpha(2A) adrenoceptors may be of the alpha(2C) type. The synergistic combination of an alpha(2A)- and an alpha(2C)-adrenoceptor agonist may provide a unique and highly effective drug combination for the treatment of pain without the sedation produced by an equianalgesic dose of a single alpha(2)-adrenoceptor agonist.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2 , Agonistas alfa-Adrenérgicos/farmacología , Clonidina/análogos & derivados , Dexmedetomidina/farmacología , Animales , Clonidina/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Masculino , Actividad Motora/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Especificidad de la EspecieRESUMEN
This study examined the antihyperalgesic and antinociceptive effects of opioid receptor agonists microinjected in the rostral ventromedial medulla (RVM) of rats 4 hr, 4 d, and 2 weeks after the induction of an inflammatory injury by injection of complete Freund's adjuvant (CFA) in one hindpaw. Nociceptive sensitivity of the ipsilateral, inflamed and the contralateral, uninflamed hindpaws was determined by the radiant-heat paw withdrawal test. The antihyperalgesic potency of the mu opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), determined for the inflamed hindpaw, was enhanced 4 d and 2 weeks after injury. The antinociceptive potency of DAMGO, determined for the contralateral, uninflamed hindpaw, was also progressively enhanced 4 hr, 4 d, and 2 weeks after injury. The magnitude of enhancement paralleled the chronicity of the injury. The greatest potentiation occurred 2 weeks after injury when the ED(50) value of DAMGO in CFA-treated rats was one-tenth that in saline-treated rats. The antihyperalgesic and antinociceptive effects of the delta opioid receptor agonist [D-Ala(2),Glu(4)]deltorphin were also increased 2 weeks after injury. These results indicate that peripheral inflammatory injury alters the pharmacology of excitatory and inhibitory inputs that modulate the activity of RVM neurons in such a manner as to enhance the effects of opioid agonists in this region. These changes have ramifications not only for the alleviation of hyperalgesia at the site of injury but also for opioid-induced antinociception at sites remote to the injury as revealed by increases in the potency of opioid agonists to suppress nociceptive responses of the contralateral, uninflamed hindpaw.
Asunto(s)
Analgésicos Opioides/administración & dosificación , Encefalina Ala(2)-MeFe(4)-Gli(5)/administración & dosificación , Inflamación/fisiopatología , Oligopéptidos/administración & dosificación , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Analgésicos Opioides/farmacología , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Miembro Posterior/inervación , Hiperalgesia/fisiopatología , Inflamación/patología , Masculino , Bulbo Raquídeo/fisiología , Microinyecciones , Nociceptores/efectos de los fármacos , Oligopéptidos/farmacología , Ratas , Ratas Sprague-Dawley , Temperatura Cutánea/efectos de los fármacosRESUMEN
In the present study, we investigated the effects of intrathecal gabapentin on nociceptive behaviors and the numbers of spinal Fos-like immunoreactive (Fos-LI) neurons evoked by injection of 0.25 to 2.5% formalin in the hindpaw of the rat. Pretreatment with gabapentin dose dependently decreased flinches and weighted pain scores in phase 2, but not phase 1, at each concentration of formalin. The highest dose of gabapentin (100 microgram) shifted the EC(50) values of formalin for both flinches and weighted pain scores to the right by 2.5-fold, suggesting that formalin was perceived to be significantly less noxious. Gabapentin also decreased phase 2 behaviors when administered after formalin but was only one third as potent. Unlike its inhibition of formalin-evoked nociceptive behaviors, the effect of gabapentin on the expression of Fos-like immunoreactivity in the spinal cord was highly dependent on the concentration of formalin. Intrathecal pretreatment with 100 microgram of gabapentin did not decrease the numbers of Fos-LI neurons evoked by 0.5% formalin, yet this dose decreased the numbers of Fos-LI neurons in laminae I-II and VII-X of rats that received 1.25% formalin and uniformly decreased by 50% the numbers of Fos-LI neurons in all laminae of rats that received 2.5% formalin. These latter findings suggest that gabapentin neither nonselectively decreases the excitability of spinal cord neurons nor uniformly inhibits the release of all neurotransmitters from primary afferent terminals. Rather, its effects may be preferential for those neurotransmitters released by higher, more noxious concentrations of formalin and for conditions in which there is a greater induction of central sensitization.
Asunto(s)
Acetatos/farmacología , Aminas , Analgésicos/farmacología , Ácidos Ciclohexanocarboxílicos , Formaldehído/toxicidad , Nociceptores/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Ácido gamma-Aminobutírico , Acetatos/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Pie/fisiología , Gabapentina , Miembro Posterior/efectos de los fármacos , Inmunohistoquímica , Inyecciones Espinales , Masculino , Neuronas/efectos de los fármacos , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Factores de TiempoRESUMEN
Concurrent administration of low doses of [D-Ala(2), Glu(4)]deltorphin (DELT) in the spinal cord and rostral ventromedial medulla of the rat produces a synergistic antinociception in the tail-flick test. It was postulated that the synergistic antinociception results from an interaction of the intrathecally-administered DELT with norepinephrine released in the spinal cord as a result of the microinjection of DELT in the rostral ventromedial medulla. Three approaches were taken to test this hypothesis. The first experiment determined that microinjection of DELT in the rostral ventromedial medulla produced an increase in tail-flick latency that was partially attenuated by intrathecal administration of the alpha(2)-adrenoceptor antagonist yohimbine. These data indicated that microinjection of DELT in the medulla causes a release of norepinephrine in the spinal cord. The second experiment determined that intrathecal co-administration of DELT with the alpha(2)-adrenoceptor agonist dexmedetomidine in a 2:1 fixed dose ratio produced a synergistic antinociception in the tail-flick test. The final experiment determined that the antinociception produced by concurrent medullary and intrathecal administration of DELT was completely antagonized by intrathecal administration of yohimbine. Taken together, these findings support the hypothesis that the synergistic antinociception produced by concurrent activation of medullary and spinal delta(2) opioid receptors is mediated, in part, by endogenous norepinephrine release in the spinal cord. The norepinephrine, acting at alpha(2)-adrenoceptors, interacts in a synergistic manner with intrathecally administered DELT, acting at spinal delta(2) opioid receptors, to produce antinociception.
Asunto(s)
Analgésicos Opioides/farmacología , Nociceptores/efectos de los fármacos , Norepinefrina/fisiología , Oligopéptidos/farmacología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Receptores Opioides delta/efectos de los fármacos , Médula Espinal/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Analgésicos Opioides/administración & dosificación , Anestesia Raquidea , Animales , Dexmedetomidina/farmacología , Sinergismo Farmacológico , Inyecciones Espinales , Masculino , Nociceptores/fisiología , Norepinefrina/farmacología , Oligopéptidos/administración & dosificación , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/fisiología , Receptores Opioides delta/fisiología , Médula Espinal/efectos de los fármacos , Yohimbina/farmacologíaRESUMEN
Previous work supports the existence of two types of delta opioid receptor (delta1 and delta2) and a role of both subtypes in the spinal cord and the ventromedial medulla (VMM) in the production of antinociception. Although it is well established that spinal and supraspinal mu opioid receptors interact in a synergistic manner to produce antinociception, little is known about the interaction of delta opioid receptors. This study used isobolographic analysis to determine how delta1 and delta2 opioid receptors in the VMM interact with their respective receptors in the spinal cord to produce antinociception. Concurrent administration of the delta1 opioid receptor agonist [D-Pen2,D-Pen5]enkephalin at spinal and supraspinal sites in a fixed-dose ratio produced antinociception in an additive manner in the tail-flick test. In contrast, concurrent administration of very low doses of the delta2 opioid receptor agonist [D-Ala2,Glu4]deltorphin at spinal and medullary sites produced antinociception in a synergistic manner. However, as the total dose of [D-Ala2,Glu4]deltorphin increased, this interaction converted to additivity. These observations suggest that different mechanisms mediate the antinociceptive effects of different doses of delta2 opioid receptor agonists. The difference in the nature of the interaction produced by delta1 and delta2 opioid receptor agonists provides additional evidence for the existence of different subtypes of the delta opioid receptor. These results also suggest that delta2 opioid receptor agonists capable of crossing the blood-brain barrier will be more potent or efficacious analgesics than delta1 opioid receptor agonists after systemic administration.
Asunto(s)
Analgésicos Opioides/farmacología , Bulbo Raquídeo/metabolismo , Receptores Opioides delta/metabolismo , Médula Espinal/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Encefalina D-Penicilamina (2,5) , Encefalinas/farmacología , Masculino , Bulbo Raquídeo/anatomía & histología , Bulbo Raquídeo/efectos de los fármacos , Microinyecciones , Oligopéptidos/farmacología , Dimensión del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/efectos de los fármacos , Médula Espinal/anatomía & histología , Médula Espinal/efectos de los fármacosRESUMEN
Intrathecal (i.t.) administration of the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline enhances pain behaviors in the formalin test. This study examined whether bicuculline also increases the peripheral inflammation induced by formalin. Subcutaneous injection of 0.25 to 5.0% formalin in the plantar surface of one hindpaw of the rat produced a concentration-dependent increase in plasma extravasation as measured by the Evans Blue method. Pretreatment with 0.3 microg i.t. bicuculline neither enhanced nor suppressed formalin-induced plasma extravasation. This dose of bicuculline also did not affect plasma extravasation induced by injection of 3% kaolin/3% carrageenan in the knee of the rat. These data indicate that the enhancement of formalin-induced pain behaviors by i.t. bicuculline is not secondary to enhanced peripheral inflammation, but more likely reflects enhancement of nociceptive transmission in the spinal cord.
Asunto(s)
Bicuculina/farmacología , Antagonistas del GABA/farmacología , Inflamación/inducido químicamente , Animales , Artritis/inducido químicamente , Carragenina , Colorantes , Azul de Evans , Extravasación de Materiales Terapéuticos y Diagnósticos , Miembro Posterior , Inyecciones Espinales , Caolín , Articulación de la Rodilla , Masculino , Dimensión del Dolor , Ratas , Ratas Sprague-DawleyRESUMEN
Microinjection of baclofen, a gamma-aminobutyric acidB (GABA[B]) receptor agonist, in the nucleus raphe magnus (NRM) or nucleus reticularis gigantocellularis pars alpha (NGCpalpha) of the rat produces antinociception at doses of 0.1-1.0 ng and hyperalgesia at doses of 30-150 ng in the tail-flick test. The antinociception is proposed to result from disinhibition of spinally-projecting neurons in this region that contain serotonin. The hyperalgesia is proposed to result either from inhibition of these neurons or from disinhibition of a serotonergic pain facilitatory pathway that also originates in this area of the ventromedial medulla. To determine the involvement of bulbospinal serotonergic pathways in the biphasic effects of baclofen, rats were pretreated intrathecally with either 30 microg of methysergide or saline. Ten minutes later, either saline, 0.5 ng or 150 ng of baclofen was microinjected in the NRM and NGCpalpha, and alterations in nociceptive threshold were assessed by the tail-flick and hot-plate tests. Intrathecal pretreatment with methysergide prevented the increase in tail-flick latency produced by 0.5 ng of baclofen, but did not prevent the decrease in tail-flick latency produced by 150 ng of baclofen. Neither dose of baclofen altered hot-plate latency and this lack of effect was unchanged by methysergide. These data support the idea that the antinociceptive effect of low doses of baclofen in the tail-flick test is mediated by disinhibition of a bulbospinal serotonergic projection and release of serotonin in the spinal cord. These data also suggest that the hyperalgesia produced by high doses of baclofen does not result from disinhibition of a serotonergic pain facilitatory pathway, but rather from direct inhibition of tonically-active pain inhibitory neurons in the NRM and NGCpalpha.
Asunto(s)
Analgésicos/farmacología , Baclofeno/farmacología , Hiperalgesia , Bulbo Raquídeo/fisiología , Metisergida/farmacología , Dolor , Médula Espinal/fisiología , Analgésicos/administración & dosificación , Animales , Baclofeno/administración & dosificación , Antagonistas de Receptores de GABA-B , Inyecciones Espinales , Masculino , Bulbo Raquídeo/efectos de los fármacos , Metisergida/administración & dosificación , Microinyecciones , Núcleos del Rafe/efectos de los fármacos , Núcleos del Rafe/fisiología , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Factores de TiempoRESUMEN
This study examined the effects of intrathecally (i.t.) administered mu and delta opioid receptor agonists on the flinching behavior and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal cord elicited by s.c. injection of 5% formalin in one hindpaw of the rat. Intrathecal pretreatment with either the delta-1 opioid receptor agonist [D-Pen2,5]enkephalin (DPDPE) or the delta-2 opioid receptor agonist [D-Ala2,Glu4]deltorphin (DELT) produced a dose-dependent inhibition of flinching behavior in phase 1 and phase 2 that was antagonized by coadministration of the delta-1 opioid receptor antagonist 7-benzylidinenaltrexone or the delta-2 opioid receptor antagonist Naltriben, respectively. Although i.t. pretreatment with 60 micrograms of DPDPE produced a small decrease in the numbers of Fos-LI neurons in laminae I, IIi and IIo, as well as laminae V and VI and laminae VII-X, i.t. pretreatment with 30 micrograms of DELT did not decrease the number of Fos-LI neurons in any region of the spinal cord. In contrast, i.t. pretreatment with an equieffective dose of the mu opioid receptor agonist [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) not only significantly decreased the number of flinches in phase 1 and phase 2, but also nearly completely prevented the expression of Fos-LI in all regions of the spinal cord. These effects were antagonized by pretreatment with the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Phe-Thr-NH2. The efficacy of i.t. administered DAMGO suggests that a direct spinal action contributes to the inhibition of noxious stimulus-evoked Fos-LI in the spinal cord produced by systemically administered mu opioid receptor agonists such as morphine. The relative lack of effect of DPDPE or DELT suggests that delta opioid receptors do not modulate the early-immediate gene c-fos. Alternatively, because delta opioid receptor agonists inhibit synaptic transmission in the spinal cord by predominantly presynaptic mechanisms and do not hyperpolarize dorsal horn neurons, the excitatory inputs that persist in the presence of these agonists may be sufficient to activate the c-fos gene. Taken together, these results provide new evidence, at the level of a "third messenger," that the antinociception produced by i.t. administration of delta and mu opioid receptor agonists is mediated by different mechanisms.