| Galanin(Gal),a 29-amino-acid neuropeptide in most species(30-amino-acid in human),is widely distributed throughout the nervous system including sensory ganglion and sympathetic ganglion(SG)neurons and is involved in the regulation of manifold functions including nociception,developmental and trophic effects.Gal may play a role in the adaptive response of the peripheral nervous system to injury and modulate pain transmission.Gal is recognized as one of the dorsal root ganglion(DRG)injury markers.Increased Gal expression was used as a marker of the change of phenotype that occurs in sympathetic neuronal cell bodies when their axons are severely damaged.Gal is normally expressed at low levels in sensory neurons and SG neurons and is markedly up-regulated within these neurons following peripheral nerve injury and inflammation in the adult.Peripheral noradrenergic system is involved in intrinsic control of pain. Norepinephrine(NE)has little influence on pain in healthy tissues,whereas noradrenergic system is subject to various plastic changes that influence its antinociceptive efficacy after injury or inflammation.Distinct alpha-adrenoreceptors are expressed in sympathetic neurons.Activation of distinct alpha-adrenoreceptors influences tone of sympathetic nervous system or neurotransmitter synthesis or release and finally results in different effects.Interestingly,functional alpha-adrenoreceptors are expressed in primary sensory neurons and regulate neurogenic inflammation and nociceptive responses.Alpha-adrenoreceptors have a key role in mediating pain regulatory effects of NE.These adrenoreceptors are functionally active may vary with the presence of nerve injury,inflammation or other physiological and pathophysiological conditions.It is not known whether activation of alpha adrenoreceptors could affect Gal expression in DRG and SG neurons.Nerve growth factor(NGF)initially interested neurobiologists because of its effects in the developing nervous system of the survival,differentiation and maturation.In the course of the last years,several lines of evidence converged to indicate that NGF is a major regulator of inflammatory and homeostatic pain states, influencing both sensory neuron phenotype and physiologic responses.NGF could influence the expression of several neuropeptides including Gal in both DRG and SG neurons.It is not clear whether exogenous NGF could affect Gal expression in DRG or SG neurons.6-Hydroxydopamine(6-OHDA)and capsaicin(CAP)could cause neurotoxicity on SG neurons and DRG neurons,respectively.The highly potent and catecholamine selective neurotoxin 6-OHDA could causes changes in the expression of Gal mRNA in the SCG similar to those seen after axotomy.It is not known to what extent Gal expression is affected by 6-OHDA in cultured SCG neurons.CAP,the pungent component of hot peppers,elicits a sensation of burning pain,via activation of vanilloid receptor 1(VRI,CAP receptor)expressed in primary sensory neurons that convey information about noxious stimuli to the central nervous system.Large dosage of CAP could selectively destroy primary sensory neurons.Interestingly,co-expression of GalR2 and VR1 in DRG neurons suggested that Gal-induced effects are mediated by GalR2 on CAP-sensitive primary sensory neurons.Whether expression of Gal and GalR2 was affected by CAP should be further studied. Neuropeptide expression and VR1 expression may reflect nociceptive properties of DRG neurons.Substance P(SP),an 11-amino acid-long neuropeptide, is expressed in primary sensory neurons and plays an important role in nociception. Gal immunoreactive cells are colocalized with SP immunoreactive cells in single sensory neurons of the rat DRG suggesting the functional significance of these neurotransmitters in the modulation of sensory action and neuropathic pain transmission.Whether exogenous Gal could affect expression of VR1 and SP should be verified.Gal is thought to play only a minor role in nociception under normal conditions. However,it may have a critical role in modulation of nociception in neuropathic states.Several lines of evidence demonstrated that Gal was involved in peripheral pain processing.In both non-inflammatory stimulation and inflammatory conditions, Gal is one of the important mediators in the processing of pain sensation or hypersensitivity.However,the possible mechanisms by which Gal exerts an excitatory action are still unknown.Based on the above research backgrounds,we know that Gal was involved in so many physiological and pathophysiological conditions.Whereas whether NE, CAP and NGF influence Gal expression in DRG neurons and NGF,6-OHDA and adrenoreceptor agonists affect Gal expression in SG neurons in vitro needs to be clarified.The effects and mechanisms of Gal in formalin-induced nociception remain unknown.In the present study,both DRG and superior cervical ganglion (SCG)neuronal culture models were established.Gal expression in DRG neurons induced by NE,CAP and NGF and Gal expression in SCG neurons induced by NGF, 6-OHDA and alpha-adrenoreceptor agonists were investigated using these two culture models,respectively.The effects and mechanisms of Gal in formalin-induced inflammatory pain were also investigated.In addition,the relationship between NGF-induced axonal regeneration and Gal expression,the effect of CAP on GalR2 expression in DRG neurons and the effect of exogenous Gal on SP release and VR1 expression in DRG neurons were investigated in the present experiment. DRG and SCG culture experiments.DRG and SCG were dissected out from embryonic 15-day-old or newborn Wistar rats,respectively.DRG and SCG cells were cultured in Dulbecco's Modified Eagle Medium with F-12 supplement (DMEM/F-12)media at 37℃with 5%CO2 for 24 hours and then maintained in culture media containing cytarabine(ara-C)(5μg/ml)for another 24 hours to inhibit growth of non-neuronal cells.After that,DRG and SCG cells were maintained in different culture conditions for additional 4 days with media change every 2 days. Neurons were cultured continuously in culture media for 6 days as control.(1) Exposure of NE on DRG neurons:DRG neurons were exposed to NE(10-4mol/L) for 4 days.When requested,DRG neurons were pretreated with alpha 1-adrenoreceptor antagonist prazosin(10-6mol/L)or alpha 2-adrenoreceptor antagonist yohimbine(10-5mol/L),10 minutes prior to the NE challenge.(2) Exposure of NGF on DRG neurons:DRG neurons were exposed to NGF(10 ng/ml)for 4 days.When requested,DRG neurons we,re exposed to NE(10-4mol/L) during the 4 days NGF treatment.(3)Exposure of CAP on DRG neurons:DRG neurons were exposed to CAP(10-8,10-7,10-6mol/L)for 4 hours as the acute treatment and exposed to CAP(10-8,10-7,10-6mol/L)for 4 days as the chronic treatment.(4)Exposure of Gal on DRG neurons:DRG neurons were exposed to Gal(10-9,10-8,10-7mol/L)for 4 days.When requested,DRG neurons were pretreated with GalR antagonist M35(10-8mol/L)or PKC inhibitor(10-7mol/L),10 minutes prior to the Gal treatment.(5)Exposure of selective alpha-adrenoreceptor agonists on SCG neurons:SCG neurons were exposed to alpha 1-adrenoreceptor agonist phenylephrine(10-5mol/L)or alpha 2-adrenoreceptor agonist clonidine(10-5mol/L).(6)Exposure of NGF or/and 6-OHDA on SCG neurons:SCG neurons were exposed to NGF(10 ng/ml), 6-OHDA(10-5mol/L)and NGF(10 ng/ml)plus 6-OHDA(10-5mol/L)for 4 days.Animal behavioral experiments.Male Wistar rats weighing 220-250 g were used in this experiment.Animals were randomly divided into 5 groups(n=10 per group):Gal group,formalin group,formalin+Gal group,formalin+Gal+GalR antagonist group,and formalin+Gal+PKC inhibitor group.Inflammation in the formalin-induced arthritis of the left tibiotarsal joint of male Wistar rats was induced by subcutaneous injection of 20μl 2%formalin into the plantar surface of the left hind paw.20μl Gal(0.1 ng/μl)was injected into the left hind paw simultaneously and flinching animal behavior would be examined at this time.GalR antagonist M35 was injected 20 min prior to Gal injection.PKC inhibitor Calphostin C was injected 60 min prior to Gal injection.Animals in Gal or formalin group,only 20μl Gal(0.1 ng/μl)or 20μl 2%formalin was injected into the left hind paw, respectively.After animal behavior examination,the expression of Gal mRNA and peptide in lumbosacral DRG and SG were examined.The results are as follows:(1)NE(10-4mol/L)promoted Gal mRNA and Gal peptide expression in cultured DRG neurons after 4 days incubation.Pretreatment with alpha 1-adrenoreceptor antagonist prazosin(10-6mol/L)could block the effects caused by NE,whereas alpha 2-adrenoreceptor antagonist yohimbine(10-5mol/L) did not have the effects on NE induced elevation of Gal mRNA and Gal peptide levels.(2)NGF(10 ng/ml)inhibited Gal mRNA and Gal peptide expression as compared with control at the same time point.NGF also inhibited NE-induced the elevation Gal mRNA and Gal peptide expression in DRG cultures.NGF but not NE could promote axonal regeneration of DRG neurons.(3)Acute exposure(4 hours) of CAP(10-6mol/L)and chronic exposure(4 days)of CAP(10-8mol/L,10-7mol/L) promoted Gal mRNA and Gal peptide expression in DRG cultures.Chronic exposure(4 days)of 10-7mol/L CAP promoted GalR2 mRNA and GalR2 protein expression in DRG cultures.(4)Exogenous Gal sensitized CAP-evoked SP release, but did not have effects on SP mRNA,SP peptide expression and basal SP release in DRG cultures.Exogenous Gal promoted VR1 mRNA and VR1 protein expression in a dose-dependent manner in cultured DRG neurons after 4 days incubation.The elevation of VR1 expression could partially be inhibited by GalR antagonist M35 or PKC inhibitor Calphostin C.(5)The levels of Gal mRNA and Gal peptide expression in cultured SCG neurons decreased significantly after stimulation with alpha 2-adrenoreceptor agonist clonidine(10-5mol/L).Alpha 1-adrenoreceptor agonist phenylephrine(10-5mol/L)stimulation did not have effects on Gal mRNA and Gal peptide expression.(6)NGF(10 ng/ml)inhibited Gal mRNA and Gal peptide expression in cultured SCG neurons.Exposure of 6-OHDA(10-5mol/L) promoted Gal mRNA and Gal peptide expression in SCG cultures,whereas NGF had no effect on the increase of Gal mRNA and Gal peptide expression induced by 6-OHDA treatment.NGF could promote axonal regeneration of SCG neurons.(7) Intraplantar injection of 20μl Gal(0.1 ng/μl)to formalin-induced inflammation male Wistar rats produced spontaneous flinches of the injected hindpaw. Intraplantar administration of M35 or calphostin C partially reversed the Gal potentiation of formalin-induced nociception.(8)The levels of Gal mRNA in lumbosacral DRG were increased after intraplantar injection of formalin.The expression of Gal peptide was not increased.Both Gal mRNA and Gal peptide expression in lumbosacral SG were not affected by intraplantar injection of formalin.The results in the present study indicate that:(1)Gal expression in DRG neurons might be affected by different stimulators such as NE,NGF,or CAP.NE,due to action on alpha 1-adrenoreceptors but not alpha 2-adrenoreceptors,increases Gal expression in DRG neurons indicating that it is one of the pronociceptive noradrenergic mechanisms in the periphery.NGF inhibits Gal expression in the absence or presence of NE on cultured DRG neurons. Certain concentrations or exposure time of CAP stimulation may be relevant to up-regulation of Gal and its receptor GalR2 expression in DRG cultures.These results implicated the complexity of the mechanisms of Gal-related nociception.(2)Gal expression in SCG neurons might be modulated by different factors such as alpha-adrenoreceptor,NGF,or 6-OHDA.Gal may be regulated by activation alpha 2-adrenoreceptors,but not alpha 1-adrenoreceptors in sympathetic neurons suggested alpha 2-adrenoreceptors may be involved in the Gal related injury or inflammatory responses.Gal expression was attenuated by administration of exogenous NGF and enhanced by administration of exogenous 6-OHDA in SCG cultures.(3)Exogenous Gal could induce CAP-evoked SP release and increase VR1 expression suggested that Gal may be,at least in part,correlated with VR1-related nociception.Activation of GalR and PKC pathway may be involved in the enhancement of formalin-induced inflammatory pain caused by exogenous Gal.The use of GalR antagonists and PKC inhibitor in the periphery may have therapeutic value in the treatment of inflammatory pain.
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