The Mechanisms Of Extracellular Signal-regulated Kinase Activation In Insular Cortex Regulating Orofacial Pain

Trigeminal neuralgia(TN)is a recurrent paroxysmal,severe facial pain in the distribution of one or more branches of the trigeminal nerve.The etiology and pathogenesis of TN has not been completely elucidated,and the currently available therapies for orofacial neuropathic pain remain inadequate.Orofacial nociceptive information is preliminarily transmitted to the trigeminal caudal subnucleus(Vc),and then ascends to higher brain regions.Trigeminal nerve injury can produce a sensitisation of the Vc neurons.Thus,the Vc plays a pivotal role in orofacial nociceptive transmission.Recently,the insular cortex(IC)has been increasingly reported to play important roles in pain processing.Directly electrical stimulation of the IC elicits painful sensations.Patients who undergone IC resection suffered from glioma or cerebral infarction show reduction in nociception.It is reported that bilateral IC lesions significantly alleviate inflammatory and neuropathic pain behaviors in rats.These findings support the critical roles of the IC in pain perception.Based on its cytoarchitecture,the IC has been artificially divided into three main subregions: the dysgranular insular cortex(DI),the granular insular cortex(GI)and the agranular insular cortex(AI).Long-term alleviation of allodynic manifestations is produced bydiscreetly lesioning the caudal granular insular cortex(CGIC)in the rat,either before or after a chronic constriction injury of the sciatic nerve.However,CGIC lesions alone have no effect on normal mechanical stimulus thresholds.In addition,the electrophysiological study reveal a corticospinal loop could be the anatomical source of CGIC’s influence on allodynia.DI at the level that coincides with the crossing of the anterior commissure sends efferent fibers directly to the contralateral Vc.The dorsomedial part of the Vc receives direct,contralateral projections from the GI and the DI.It is suggested this top–down corticotrigeminal projection from the GI / DI to the Vc is likely involved in orofacial nociceptive processing.Pain has been defined by the International Association for the Study of Pain(IASP)as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage,or described in terms of such damage." Chronic pain is usually described as pain that has persisted for 3 months and/or beyond the expected time of healing.In a chronic pain state,the negative affect including anxiety and depression is well known to accompany the pain perception during disease progression.The IC receives afferent projections from thalamic nuclei and forms reciprocal connections with the amygdala,prefrontal cortex(PFC),and limbic system.Therefore,the IC is a complicated integration site for sensations,attention,memory and affections,and may be involved in both the sensory and affective dimensions of pain.Extracellular signal-regulated kinase(ERK)plays important roles in synaptic plasticity and remodeling during long-term potentiation(LTP),learning,memory consolidation,the induction and maintenance of chronic pain.According to previous studies,inflammation or nerve injury activates the ERK pathway not only in the spinal dorsal horn but also in supraspinal structures such as the medial prefrontal cortex(mPFC),anterior cingulate cortex(ACC),amgydala,and IC.For example,inhibiting ERK activation in the ACC in the early phase attenuated postoperative pain-related anxiety and mechanical hypersensitivity whereas inhibiting ERK activation in the late phase only reduced the anxiety-like behavior.U0126,a potent and selective MEK/ERK inhibitor,could inhibit ERK pathway activation and limitthe subsequent synaptic plasticity.The current study aims to explore ERK activity in the IC and its role in the modulation of anxiety and hypersensitivity in orofacial pain.Usually,cortical and subcortical sites can influence nociception in the spinal cord.The corticotrigeminal descending pathway from the IC to the Vc support that Vc could be activated by orofacial nociceptive transmission,but also could receive descending modulations from the IC.In the current study,we hypothesized that ERK activity in the IC exerted a potentiation effect on the top-down corticotrigeminal pathway,which exaggerated nociceptive transmission and led to orofacial pain maintenance.ERK inhibition via U0126 may inhibit the activity of the Vc-projecting neurons in the IC,directly regulate neurons of the Vc,thus could effectively alleviate neuropathic pain as well as pain-related negative emotions in IoN-CCI rats.These findings may indicate a novel mechanism of ERK inhibitor-induced analgesia.Materials and methods1.The anterograde tracer Phaseolusvulgaris-leukoagglutinin(PHA-L)was iontophoretically injected into the anterior IC(AIC)or posterior IC(PIC).The efferent connections of the IC were observed.To precisely determine the projections from the IC to the Vc,the retrograde tracer Fluoro-gold(FG)was next injected into dorsalmedial Vc and ventrallateral Vc,respectively.FG retrogradely-labelled neurons were detected in the IC.2.Infraorbital nerve chronic constriction injury(IoN-CCI)-induced orofacial pain in rats was performed.Mechanical allodynia was assessed by measuring the50 % head withdrawal threshold(50 % HWT).The asymmetrical unilateral face-grooming actions were calculated as a spontaneous nociceptive behavioral response.The open field(OF)test and the elevated plus maze(EPM)test were conducted.The percentage of time spent in the center area(center time %)was used as a parameter for evaluating anxiety/depression levels in the OF test.The percent of time spent in open arms(OA time %)and the percent of open arm entries(OA entries %)were considered measurements of general anxiety/depression levels in theEPM test.3.Immunohistochemistry staining and Western blot assay were used to measure the expression of ERK signaling pathway members,including ERK and CREB in the contralateral IC after IoN-CCI surgery.To further investigate the role of ERK activation,we investigated the effects of U0126 on the established nociceptive behaviors as well as pain-related negative emotions in IoN-CCI rats after U0126 microinjection into the IC.4.By combining retrograde tract tracing and electrophysiological techniques,we injected tetramethylrhodamine(TMR)into the Vc to label Vc-projecting neurons in the IC.Whole-cell patch-clamp recordings were made from TMR-containing Vc-projecting neurons.The effects of U0126 on the spontaneous excitatory postsynaptic currents(sEPSCs)and the excitability of Vc-projecting neurons in the IC slices of IoN-CCI rats were studied after U0126 perfusion.Finally,the immunofluorescent staining of Fos was investigated to reveal the effect of ERK activation on neuronal activation in the Vc after intra-IC U0126 injection in IoN-CCI rats.Results1.The anterograde tracer PHA-L was iontophoretically injected into the AIC or PIC.As a result,we detected that PHA-L labeled fibers and terminals were mainly found in the following: pyramidal tract(py),contralateral raphe magnus nucleus(RMg),both the superficial laminae(I / II)and the deeper laminae(III-V)of the contralateral Vc,basolateral amygdaloid nucleus(BLA),lateral hypothalamic area(LH),dorsal raphe nucleus(DR),accumnens nucleus core(AcbC),central medial thalamic nucleus(CM),ventral posterior thalamic nucleus,parvicellular part(VPpc),and nucleus of the solitary tract(Sol).No PHA-L labeled fibers and terminals were observed in the trigeminal interpolar subnucleus(Vi)and the trigeminal oral subnucleus(Vo).FG injections into the superficial laminae(I / II)of the dorsalmedial Vc and theventrallateral Vc produced dense retrograde labeling of a band of pyramidal cells located in rostral(from 3.0 mm anterior to Bregma to 1.0 mm posterior to Bregma)and caudal(between Bregma and 2.0 mm posterior to Bregma)levels of the GI and DI,respectively.However,no FG-labeled neurons were observed in the AI.2.There was a significant mechanical allodynia in the IoN-CCI rats compared with the sham-operated animals.This lasted for over 21 days after the surgery.The asymmetrical unilateral face-grooming actions were calculated as a spontaneous nociceptive behavioral response.IoN-CCI rats showed significant increase in face grooming from day 3 to day 21 after surgery compared with the sham group.Compared with the sham group,the IoN-CCI group showed significantly reduced center time % in the OF test and reduced OA Time % and OA Entries % in the EPM test.3.The number of p-ERK-immunoreactive(p-ERK-IR)neurons was significantly increased in the IC from day 3 through day 21 in the IoN-CCI group compared with that in the sham group.Increased p-ERK expression was observed in the apical dendrites of neurons in the IC,especially on day 21 after IoN-CCI surgery(in the late phase of neuropathic pain).Western blot was conducted to evaluate the expression of ERK and CREB after IoN-CCI surgery.Compared with naive control rats,the expression of both phosphorylated ERK and phosphorylated CREB(p-ERK and p-CREB)was not significantly altered in the sham rats.p-ERK and p-CREB were significantly elevated in the IC 3 days after IoN-CCI compared with their levels in the control group,and phosphorylation was maintained for at least 21 days.Total ERK and total CREB(t-ERK and t-CREB)levels were not changed in any of the groups.4.Intra-IC U0126 microinjection significantly reversed the upregulation of p-ERK and its downstream molecule,p-CREB in the IoN-CCI rats.t-ERK and t-CREB levels were not changed in any of the groups.Compared with the vehicle control group,the mechanical allodynia revealed by 50 % HWT was significantly reduced after U0126 microinjection into the IC on day 14 after IoN-CCI.Similarly,the number of face-grooming actions was also deceased after U0126 microinjection in IoN-CCI rats compared with vehicle-treated rats.Compared with vehicle,U0126 significantly increased the center time % in the OF test as well as the OA Entries %and OA Time % in the EPM test.5.To further investigate the effect of U0126 on the excitability of Vc-projecting neurons in the IC,we made whole-cell patch-clamp recordings from the TMRlabeled Vc-projecting neurons.Perfusion of U0126 for 1 h considerably reduced the frequency and amplitude of sEPSCs.The paired-pulse ratio(PPR)of Vc-projecting neurons was significantly increased.The spike numbers of the recorded neurons in the IC slices of IoN-CCI rats were significantly reduced in the presence of U0126 compared to vehicle.The number of Fos-IR neurons in the Vc was significantly decreased following intra-IC U0126 administration compared with that following vehicle treatment.Conclusions1.Orofacial nociceptive primary afferents terminated in the superficial laminae of the Vc.The Vc,especially the superficial laminae,also received direct descending projections from the GI and DI.These projections were bilateral,with obvious predominance in the contralateral side.The Vc received no projections from the AI.Rostral GI / DI and caudal GI / DI sent projections to the dorsalmedial Vc and the ventrallateral Vc,respectively.The IC also sent projecting fibers to amygdala,Sol,Acb and brainstem.It was suggested that top-down corticotrigeminal projection from the GI / DI to the laminae I / II of the Vc were likely involved in orofacial nociceptive processing.However,the AI may not be involved in orofacial pain processing,at least in this top-down direct modulation pathway.2.The ERK pathway was significantly activated in the IC after IoN-CCI.Intra-IC microinjection of U0126,a potent and selective MEK / ERK inhibitor not only alleviated nociceptive behaviors but also decreased negative emotions in the IoN-CCI rats,indicating that ERK pathway was involved in the regulation of pain sensations as well as pain-related affections.3.The inhibition of ERK led to both pre-and postsynaptic plasticity changes inthe Vc-projecting neurons,and decreased the excitability of the Vc-projecting neurons in the IC.ERK deactivation reduced the elevated neuronal activation in the Vc via projections from the IC to the Vc after IoN-CCI.Thus nociceptive behaviors were attenuated and anxiety/depression-like negative emotions were improved.