The Mechanisms Underlying The Effects Of Artemin-GFRα3 On The Trigeminal Pain Pathway Of Migraine

Part Ⅰ Study on the expression of Artemin and GFRα3 in a rat model of migraineObjectiveMigraine,a common neurovascular disorder in clinical,is mainly characterized by recurrent attacks of typically unilateral and throbbing headache.To date,the pathogenesis of migraine has not been fully elucidated and there is no radically effective treatment for migraine until now.It is widely accepted that the pathogenesis of migraine is closely related to the theory of trigeminovascular system.The theory includes three mechanisms:the dilatation of meningeal vascular,neurogenic inflammation caused by release of vasoactive peptides from peripheral nerve and reduced central descending inhibitory of nociceptive transmission.Among them,the dural neurogenic inflammation plays a key role.In recent years,studies have found that Artemin,a member of the glial cell line-derived neurotrophic factor family,exerts biological effects by binding to its specific receptor GFRα3.It is reported that Artemin is expressed in the blood vessels of the dura mater,and GFRα3 is mainly expressed in sensory neurons and sympathetic neurons of trigeminal ganglion(TG).However,the mechanisms of Artemin and GFRα3 involved in the pathogenesis of migraine has not yet been systematically investigated.In this regard,the purpose of this study is to further evaluate the expression of Artemin and GFRα3 in a rat migraine model induced by nitroglycerin,so as to explore the possible mechanisms of Artemin and GFRα3 underlying the pathogenesis of migraine,and provide new therapeutic targets for migraine.MethodsMale adult wistar rats were utilized in this study and divided into three groups:normal control group,normal saline(NS)control group and nitroglycerin model group.In the nitroglycerin group,rats were subcutaneously injected with nitroglycerin to set up the rat model of migraine.In the NS group,rats were subcutaneously injected with 0.9%NS using the same volume as nitroglycerin.The rats in the normal control group were untreated.Then the behaviors of the rats were observed and recorded in a quiet environment.The immunofluorescence staining was used to detect the expression of Artemin in the dura mater.We used the lipophilic dye tracing technique and immunofluorescence staining to detect the expression of GFRa3 in TG.The total RNA was extracted from the dura mater and TG after NTG or NS treatment for different time points(2 h,4 h,6 h).The mRNA levels of Artemin and GFRa3 were detected by means of quantitative reverse transcription-polymerase chain reaction(qRT-PCR).The total protein was extracted from the dura mater and TG after NTG or NS treatment for different time points(2 h,4 h,6 h,8 h,10 h).The protein levels of Artemin and GFRα3 were detected by means of western blot.In addition,the immunofluorescence staining was used to evaluate the expression of GFRa3 protein in the TG of migraine rats.ResultsIn the NTG group,the rats showed series of behaviors including constantly scratching head,frequent climbing cage,turning in circles,red ears and other symptoms of irritability after NTG treatment.The rats in NS group just showed occasional phenomenon of scratching head,while no crawling cage and other irritability behaviors.Immunofluorescence staining results showed that Artemin was expressed in the smooth muscle cells of the dural blood vessels and the expression of GFRa3 was co-localized with the TG neurons innervating the dura mater as evidenced by nerve tracing.By using the lipophilic dye tracing,immunofluorescence staining results showed that GFRa3 was mainly expressed in the cytoplasm of neurons with smaller diameter in the TG.Meanwhile,the expression of GFRa3 was co-localized with the TG neurons innervating the dura mater.The mRNA level of Artemin in the dura mater was significantly increased at 2 h and 4 h after NTG treatment than that in the normal control group(p<0.05).The mRNA level of GFRa3 in TG was significantly increased at 4 h time point after NTG treatment compared wih that in the normal control group(p<0.01).There was no significant difference in Artemin and GFRa3 mRNA expression between the NS control group and normal control group.Western blot results showed that the protein level of Artemin was dramatically elevated from 4 h and peaked at 8 h in the dura mater following NTG administration compared with the normal control group(p<0.05).And the protein level of GFRa3 was dramatically elevated at 4 h and continued up to 10 h in the TG following NTG administration compared with the normal control group(p<0.05).However,there was no significant difference in Artemin or GFRa3 protein expression between the NS control group and normal control group.The results of immunofluorescence staining showed that the GFRa3 protein was just expressed in small-diameter TG neurons in the NS control group.The number of GFRa3-positive neurons was significantly increased in the TG in NTG group than that in the NS group.In addition,the GFRa3 was not only expressed in small-diameter neurons,but also in large-diameter neurons.ConclusionsArtemin was found to be expressed in the smooth muscle cells of the dural blood vessels and the expression was upregulated in the dura mater of migraine model.In addition.GFRα3 was found to be co-expressed in trigeminal primary neurons innervating the dura and GFRa3 expression was increased in the migraine rats.Our results indicated that Artemin may contribute to the dural inflammation of migraine as a nociceptive signal.And the release of Artemin from the dura mater could be transferred to the TG through the dural peripheral nerve fibers and then activate the GFRα3 receptor,which probably be a critical process in migraine pathogenesis.Taken together,our studies suggested that Artemin and GFRa3 might be a novel target for therapeutic strategies of migraine.Part Ⅱ Study on the effect of Artemin in regulation of iNOS in cultured trigeminal ganglion neuronsObjectiveIn recent years,studies indicate that Artemin,a member of the glial cell line-derived neurotrophic factors(GDNF)family.not only plays protective roles on the development,growth and regeneration of the nerve.but also exerts important roles on the pathogenesis of inflammation and pain.In the first part of our study,Artemin was found to be elevated in the dura mater of migraine rats and the expression of GFRa3 was also increased in the TG of migraine rats.However,the specific mechanisms about how Artemin and GFRa3 involved in trigeminal pain pathways of migraine need to be further explored.It is reported that iNOS expression is increased in the dura mater of migraine rats and iNOS is active for extended periods yielding high outputs of NO that modulates the pathogenesis of migraine.Moreover,iNOS is a Ca2+-independent isoform and often expressed in response to painful and inflammatory stimuli including tumor necrosis factor and interferon.However,the mechanism of Artemin modulating the iNOS in TG neurons remains unclear.Therefore,the present study was designed to examine how Artemin regulates the iNOS expression in primary cultured TG neurons,so as to clarify the mechanisms underlying Artemin and its receptor GFRa3 involved in the migraine pathogenesis.MethodsPostnatal wistar rats were used in this study.After the cells were cultured in vitro for 48 h,the TG neurons were identified by anti-NeuN and anti-TUJ-1 antibody using immunofluorescence staining.In Artemin-treated groups,the primary cultured TG neurons were incubated with Artemin for different times(15 min,30 min,1 h,2 h and 4 h).In the normal control group,the TG neurons were treated with the same volume of medium without Artemin.Then the cell protein was extracted from each group.Western blot was used to detect the expression iNOS after Artemin treatment for different time points in the primary cultured TG neurons.Immunofluorescence staining was used to evaluate the expression of iNOS,GFRa3 and TUJ-lin primary cultured TG neurons and TG tissues.ResultsAfter being cultured in vitro for 48 h.the TG neurons were identified with the neuronal markers including anti-NeuN and anti-TUJ-1 antibody.The immunofluorescence staining results showed that the proportion of TG neurons in our study was very high.Immunofluorescence staining showed that NeuN was present in the neuronal nuclei and TUJ-1 was expressed in the neuronal cytoplasm and neuronal axon.The NeuN-positive neurons accounted for 91.8±2.7%of total cells and the TUJ-positive neurons accounted for 87.9±2.4%of total cells.Western blot results showed that the protein expression of iNOS was significantly increased in cultured TG neurons after Artemin treatment for 15 min compared to the control group(p<0.01),and then the protein levels of iNOS were decreased in the following time points.Immunofluorescence staining results showed that both the protein expressions of iNOS and GFRa3 were obviously elevated in Artemin-treated group compared with that of control group in primary cultured TG neurons and tissues.In addition,Artemin-induced iNOS expression was co-expressed with TUJ-1 and GFRa3 respectively in the TG neurons.ConclusionsIn summary,our study revealed that Artemin could induce iNOS protein expression quickly in TG neurons,suggesting that this process may be involved in early neurogenic inflammation and the transmission of pain signal of migraine.Part Ⅲ Study on the mechanisms of Artemin in regulation of TRPV1/TRPV4 in cultured trigeminal ganglion neuronsObjectiveIn recent years,it is reported that Artemin,one member of glial cell line-derived neurotrophic factor family,plays an important regulatory role in the pathogenesis of neuropathic and inflammatory pain.Overexpression of Artemin in the peripheral tissue could induce the increase of transient receptor potential(TRP)family channel expression in the primary afferent neurons and lead hyperalgesia in vivo.During the development of migraine,the TRP family channel is activated as nociceptive receptors in primary trigeminal afferent neurons,which contributes to the release of neuropeptides from the neurons.However,it remains unclear that the mechanism by which Artemin regulates TRP family channel members including TRPV1 and TRPV4.Therefore.the aim of this study is to further explore the modulatory effect of Artemin on the expression of TRPV1 and TRPV4 in primary cultured trigeminal ganglion neurons,so as to clarify the concrete mechanism of Artemin participating in the trigeminal pain pathway of migraine.MethodsAdult wistar rats were used in this study.The TG neurons were cultured in vitro for 48 h and then were incubated with Artemin for different times(15 min,30 min,1 h,2 h and 4 h)in Artemin-treated groups.In the normal control group,the TG cells were treated with the same volume of medium without Artemin.Western blot was used to detect the protein levels of TRPV1,TRPV4,p-p38 and p-ERKl/2 respectively after Artemin treatment for different time points in primary cultured TG neurons.Immunofluorescence staining was used to respectively evaluate the expression of TRPV1,TPPV4 and TUJ-1 in primary cultured TG neurons.In order to evaluate whether Artemin regulates the expression of TRPV1 and TRPV4 through ERK1/2 and p38MAPK signaling pathways,the TG neurons were incubated with the inhibitors of ERK1/2 and p38 signal pathways U0126 and SB202190 for 30 min,and then Artemin were added in the TG neurons and incubated for 4 h or 2 h.The expression of TRPV1 and TRPV4 protein was then detected by western blot.ResultsWestern blot results showed that the protein expression of TRPV1 was increased at 2 h time point after Artemin treatment and continued up to 4 h compared to the control group.The protein expression of TRPV4 was increased at 1 h and peaked at 2 h time point after Artemin treatment compared with the control group.The results of immunofluorescence staining also showed that the protein levels of TRPV1 and TRPV4 were elevated and co-expressed with TUJ-1 in the Artemin-treated group as compared to the control group in the cultured TG neurons.The results of western blot showed that the trends of p-ERKl/2 protein expression were not consistent with the expression of p-p38.The protein expression of p-ERK1/2 in cultured TG neurons was significantly increased after Artemin treatment for 30 min compared to the control group(p<0.01),and then decreased to the similar level as the control group in the following time point 1 h,and then elevated again at 2 h following Artemin treatment compared with that in the control group(p<0.01).However,the protein expression of p-p38 in cultured TG neurons was significantly increased following Artemin treatment for 30 min and continued up to 2 h compared with the control group.In addition.the Artemin-induced increase of TRPV1 and TRPV4 expression was inhibited by p38 and ERK1/2 signaling pathway inhibitors.ConclusionsIn summary,our study revealed that the protein expression of TRPV1 and TRPV4 could be up-regulated after Artemin treatment.and the increase of TRPV1 and TRPV4 could be suppressed by ERK1/2 and p38 MAPK inhibitors.These results suggested that Artemin might be involved in the migraine pathogenesis through up-regulating TRPV1 and TRPV4 expression via the ERK1/2 and p38 MAPK signaling pathways in the TG neurons.