The Underlying Mechanism Of The Role Of Acupuncture For Alleviating Migraine Of The Rat Through CGRP In The Trigeminal Pathway

Background Migraine is one of the most common disease of the primary headache,the incidence is about 15%aboard and 9.3%in home.Long-term migraine can lead to serious complications such as stroke and coronary heart disease,which not only affect patients’ health,but also bring them economic burden.However,the pathogenesis and mechanism of migraine is still poorly understood.Until now,the trigeminal vascular theory is supposed to be the major contributing pathophysiological theory.This theory suggests that neuropeptides play important roles in the trigeminal pathway,in particular the calcitonin gene-related peptides(CGRP).CGRP in the trigeminal pathway is a key mediator in migraine by affecting a variety of chemicals.Clinically,tradition Chinese medicine and western drugs are usually used to treat migraine,including acupuncture,CGRP antagonist and 5-hydroxytryptamine agonist.Although these drugs have good analgesic effect,there are also many side effects.Acupuncture can alleviate pain and improve patients’quality of life.The most relevant studies about the mechanism of acupuncture are conducted with animal experiments.Currently,many studies show that acupuncture can alleviate migraine by regulating the concentration of chemicals in the blood such as CGRP.However,there are few studies focus on the mechanism in the perspective of neuroscience.The correlation of acupuncture,the trigeminal pathway and CGRP also remains unknown.In this sense,the hypothesis of this study is that the mechanism of acupuncture for alleviating migraine may through CGRP in the trigeminal pathway.Objective First,the aim is to reveal the neural pathway of acupoints from periphery nervous system(PNS)to central nervous system(CNS)and to provide the evidence for the choosing of the acupoints for migraine.Second,the objective is to reveal the neurochemical characteristics,the innervations and the neural pathway of the rat dura mater,and also to provide the relevant indexes for detecting.Third,the purpose is to reveal the mechanism and to provide the relevant evidences of neuroanatomy and biochemistry for clinical treatment Methods To the first part of the study,the process is that the neural tracer cholera toxin subunit B(CTB)was injected into the tissues of acupuncture points GB14(Yangbai),ST2(Sibai)and Jiache(ST6).The neural labeling associated with each acupuncture point was revealed by fluorescent immunohistochemistry of the nervous system,including the trigeminal ganglion(TRG),cervical dorsal root ganglia(DRG),spinal cord and brainstem.Then the CTB and fluorogold(FG)were simutaneously injected into the GB14 and ventral posteromedial nucleus of thalamus(VPM),respectively.After three days,the brain would be dissected out and the correlation between the neural labeling in Sp5 associated with acupoints and the VPM were revealed by fluorescent immunohistochemistry.Besides,CTB was also injected into the VPM to observe the distribution of the transganglionic axonal terminals labeled with CTB in primary somatosensory cortex(S1).The second part of this study was mainly focused on the neurochemical characteristics,innervations and the neural pathway of the rat dura mater.Firstly,the distribution and the correlations of neurochemicals were observed,including CGRP and substance P(SP)positive sensory nerve fibers,tyrosine hydroxylase(TH)and neuropeptide Y(NPY)positive sympathetic nerve fibers,calcitonin-like receptors(CRLR)positive CGRP receptors.Besides,some immune cells and neurotransmitter also distributed on the dura mater,including tryptase positive mast cells(MC)and 5-hydroxyptamine(5-HT)positive cells.Secondly,the innervations of dura mater was observed by applying the fluorogold(FG)neural tracing technique.The distribution and neurochemical characteristics of FG labeled neurons were detected on the TRG,superior cervical ganglion(SCG),vagus ganglia(VAG),geniculate ganglia(GG),pterygopalatine ganglion(PG),Sp5 and DRG.Then,CTB and FG were also simutaneously injected into the GB14 and dura mater,respectively.The related tissues would be dissected out to observe the co-labeled neurons in TRG and to demonstrate the same characteristics of neural pathways for acupoints and dura mater,and to reveal its correlation with trigeminal pathway.Based on the previous two parts’ studies,the third part was related to analgesic effect of acupuncture intervention in rat migraine model and its correlation with CGRP-mediated chemical changes through trigeminal pathway.The migraine models were made under general anaesthesia with isoflurane.The normal group was injected with saline,and the other three groups were injected with nitroglycerin.Then,after 30 min,rats in the acupuncture group would be treated by acupuncture on GB14 and ST2.In contrast,rats in drug group were injected CGRP receptor antagonist intravenously.Meanwhile,behavior tests would be carried out,including the pain threshold and behaviors,such as scratching head and ear,climbing cages and shaking bodies.After the interventions and behavior tests,all the animals would be transcardially perfused and the dura mater would be dissected out.Then the chemical changes of CGRP,CRLR,tryptase,lysosomal-associated membrane protein 1(LAMP-1),5-HT were detected,in order to observe the correlation of the analgesic effect of acupuncture with the CGRP-related chemical changes through the trigeminal pathways.Results For the beginning results of the first part,in terms of the innervations of acupoints on rat head and face,the CTB labeling included sensory neurons and their transganglionic axonal terminals,as well as motor neurons.The labeled sensory neurons associated with GB14,ST2 and ST6 were distributed in both the TRG and cervical DRG,and their centrally projected axons terminated in an orderly fashion at their corresponding targets in the spinal trigeminal nucleus and cervical spinal dorsal horn.In addition,labeled motor neurons were observed in the facial motor nucleus,trigeminal motor nucleus and cervical spinal ventral horn,in which facial motor neurons projected to the tissues located at all three acupuncture points.Trigeminal motor neurons innervated both ST2 and ST6,while spinal motor neurons only correlated with ST6.CGRP positive sensory nerve fibers distributed on GB14、ST2 and ST6,while parts of the CTB labeled neurons in TRG,facial nucleus and trigeminal motor nucleus were also expressed with CGRP.At the same time,after CTB and FG tracing for GB14 and VPM,the CTB labeled terminals and FG labeled sensory neurons were both distributed on the Sp5,but their relationship need further study.After applying CTB neural tracing technique on VPM independently,the CTB labeled axon ternimals were found to be distributed on the third to fourth layer of S1.Therefore,the primary sensory pathway of GB14,ST2 and ST6 was from peripheral acupoints to Sp5,and their secondary and tertiary pathway was from Sp5 to S1.For the second part,in terms of the neurochemical characteristics,innervations and neural pathway of dura mater,the whole-mount dura mater had been observed.First,CGRP and SP positive sensory nerve fibers,TH and NPY positive sympathetic nerve fibers distributed on the dura mater,some of them were along the vessels,some were twining the vessels and some were on the connective tissues independently.CGRP and SP,TH and NPY positive nerve fibers were found to be co-expression,respectively.Meanwhile,there was no co-expression between CGRP and TH positive nerve fibers by 3D reconstruction.Additionally,other chemicals related to CGRP were also found in dura mater,such as MC and 5-HT.They were found to be closely related to each other as there were lots of co-expressions.Meanwhile,by combining tracing technique,CGRP-positive sensory nerve fibers were found mainly derived from the TRG,and TH-positive sympathetic nerve fibers were mainly from SCG.Second,after CTB tracing for dura mater,the CTB labeled axon terminals were distributed on Sp5 and ventral horn of cervical spinal cords.And the labeled sensory neurons were observed on TRG,mainly on the region related to the first and second branches of trigenimal nerves.Third,after the CTB and FG tracing techniques were applied for GB14 and dura mater,both CTB and FG labeled sensory neurons were found on TRG,and some of them were found to be co-expressed.The result of the third part was about the experiment of acupuncture on rat migraine model.Under NTG-induced migraine models,rats’ ears turned red,and the irritating behavioral activities increased obviously in model group,including the number of scratching head and ear,climbing cages and shaching bodies(P<0.05),while there was an obvious decrease after interventions in both acupuncture and CGRP receptors antagonist groups(P<0.05).But there was no obvious difference between these two intervention groups(P>0.05).On the pain threshold,there was an obvious decrease in model group(P<0.05),and there was an obvious increase after taking acupuncture and CGRP receptor antagonist(P<0.05).Compared with the control group,the numbers of tryptase positive activated MC,tryptase/LAMP-1 co-expression actived and degranulated MC,CRLR positive CGRP receptors,5-HT positive cells and tryptase/5-HT co-expressed cells were significantly increased in model group(P<0.05).After the acupuncture,the number of tryptase positive activated MC,tryptase/LAMP-1 co-expression activated and degranulated MC,CRLR positive CGRP receptors expressed on MC,and tryptase/5-HT co-expressed cells were significantly decreased by comparing with model group(P<0.05).After applying the CGRP receptor antagonist,the number of tryptase positive activated MC,tryptase/LAMP-1 co-expression activated and degranulated MC,CRLR positive CGRP receptors,CRLR positive CGRP receptors expressed on MC,5-HT positive cells and tryptase/5-HT co-expressed cells were significantly decreased compared with those in model group(P<0.05).There was no obvious difference between acupuncture and CGRP receptor antagonist groups(P>0.05).Conclusions These results indicate that the tissues located at GB14,ST2 and ST6 in the rat forehead and face has its own sensory connection with the Sp5 in a region-specific pattern through the trigeminal sensory pathway.As part of them co-expressed with CGRP positive neurons in TRG,indicating that the effect of acupunture may closely related to the CGRP through trigenimal pathway.The dura mater also establish the connection with Sp5 through trigenimal sensory pathway.The CGRP positive nerve fibers in dura mater mainly from the TRG,and dura mater mainly innervated by the first and second branches of the trigenimal nerves.There exist the same neural pathway of acupoints on face and head and the dura mater.Acupuncture has good analgesic effect for migraine by alleviating the behaviours and decreasing the pain threshold.Meanwhile,the CGRP positive nerve fibers derived from the CGRP positive neurons in TRG has closely relationship with the effect of acupuncture by participating in the synthesis and release of a variety of chemicals.These results demonstrate that the underlying mechanism of the role of acupuncture for alleviating migraine may through CGRP in the trigeminal pathway.