Netrin-1 And UNC5H2 Contribute To Electroacupuncture Improving Neuropathic Pain Of Postherpetic Neuralgia

Postherpetic neuralgia(PHN,Shingles)is a neuropathic pain condition characterized by the presence of profound mechanical allodynia and typically occurs in elderly but healthy individuals,which lowers the quality of life,causes great pain to the patient.Clinical practice of electroacupuncture(EA)treatment show that EA has many significant therapeutic effects,such as dredging the channel,promoting blood circulation,shorting the duration of symptoms,and reducing the recurrence rate.And EA has the characteristics of easy operation,safety,and no side effect.But little is known about the mechanism of how EA treats PHN because of lacking appropriate animal models,We have shown that depletion of capsaicin-sensitive afferents by systemic treatment with an ultrapotent transient receptor potential vanilloid 1(TRPV1)agonist,resiniferotoxin(RTX),in adult rats produce long-lasting paradoxical changes in mechanical and thermal sensitivities,which resembled the unique clinical features of patients with PHN.RTX depleted TRPV1 positive small and medium-sized DRG neurons and unmyelinated C fibers,thus induced the thermal impairment.The delayed tactile allodynia is likely attributable to damage of myelinated afferent fibers and their abnormal sprouting in lamina II of the spinal dorsal horn.The RTX induced PHN model has been accepted in the world,which provide a new opportunity to study the mechanism of how EA produces antinociceptive effects in PHN.Netrin-1 is a neuronal guidance molecule implicated in the development of embryonic spinal neurons and cortical neurons.Netrin-1 expresses on neurons and mature oligodendrocytes formed myelin sheath,and it is rich in membrane of myelin sheath near the axon,which may prompt the growth of myelinated nerve fibers in adult rat spinal cord.In mammals,several families of netrin receptors have been identified:the Deleted in Colorectal Cancer(DCC)family,and the UNC-5 homologue(UNC5H)family.DCC is required for cell and axon migration toward sources of netrin-1,which signals a chemoattractive response to netrin-1.Homologues of UNC5 mediate repellent or growth cone collapse responses to netrin-1.Furthermore,the repertoire of netrin-1 receptors expressed by an individual neuron in the spinal cord may change from DCC into UNC5 as these neurons mature,and UNC5H2 is the most abundant netrin-1 receptor expressed in adult spincal cord.The function of netrin-1 and its receptor UNC5H2 on tactile allodynia and sprouting of myelinated afferent fibers of this new RTX induced PHN model remains elusive.Moreover,vascular endothelial growth factor(VEGF)was previously identified as one of the contributors to abnormal sprouting of myelinated afferent fibers in injured spinal cord tissue from adult rats that developed allodynia.We determined whether EA regulated these factors associated with sprouting of myelinated fibers in RTX induced PHN model.Above all,we firstly tested whether EA attenuated RTX-induced damage to sensory nerves to prevent the sprouting of myelinated fibers and improved mechanical sensitivities.Secondly,we studied whether netrin-1 and its receptor UNC5H2 participated in the sprouting of myelinated nerve fibers and the mechanical hypersensitivity.Finally,we determined whether EA regulated the expression of netrin-1 and VEGF associated with sprouting of myelinated fibers and contributed to myelinated afferent fiber sprouting and neuropathic pain in this rat model of PHN.PARTIElectroacupuncture improves thermal and mechanical sensitivities in a rat model of postherpetic neuralgiaOBJECTIVESystemic injection of resiniferatoxin(RTX),an ultrapotent analog of TRPV1 agonist,can reproduce the clinical symptoms of PHN by ablating TRPV1-expressing sensory neurons in adult rats.EA is effective in relieving pain in patients with postherpetic neuralgia(PHN).However,the mechanism underlying the therapeutic effect of EA in PHN is still unclear.In this study,we determined the beneficial effects of EA improving thermal and mechanical sensitivities and the potential mechanisms in this rat model of PHN.METHODS(1)PHN was induced in rats by a single intraperitoneal injection of RTX(200 μg/kg).(2)In the EA treatment group,rats received EA administration on the left "Huantiao"(GB30)and "Yanglingquan"(GB34)once every other day,starting from 1 week after RTX injection for 5 weeks,EA(1 mA and 0.1 ms)was administered at different frequency(2 Hz,15 Hz or 100 Hz)for 30 min.Current was delivered with a modified current-constant Han’s Acupoint Nerve Stimulator(LH202,Huawei Co.Ltd,Beijing,China).For sham control,acupuncture needles were inserted ipsilaterally nto GB30 and GB34 for 30 min without electrical stimulation or manual needle manipulation.(3)Thermal sensitivity was tested with radiant heat stimulus,and mechanical allodynia was quantified with von Frey filaments.(4)TRPV1-expressing DRG neurons and afferent terminals were shown by using immunofluorescence labeling.(5)The ultrastrural changes of the sciatic nerve were assessed by electron microscopic examination.(6)The sprouting of myelinated primary afferent terminals into the spinal dorsal horn was mapped by using the transganglionic tracer cholera toxin B-subunit(CTB).RESULTS(1)A single RTX injection diminished thermal sensitivity and gradually induced tactile allodynia within 3 weeks.EA applied to GB30 and GB34 at 2 and 15 Hz,but not 100 Hz,significantly increased the thermal sensitivity 4 weeks after treatment and decreased the tactile allodynia 2 weeks after treatment in RTX-treated rats.(2)To examine the effect of EA on TRPV 1-expressing primary afferent neurons,TRPV1-immunofluorescent labeling of primary afferent neurons was conducted in the DRG from different groups 5 weeks after EA treatment.EA with 2 and 15 Hz,but not 100 Hz,recovered the loss of TRPV 1-positive DRG neurons induced by RTX injection(3)To determine the effect of EA on TRPV1-expressing central terminals of primary afferents,we also examined TRPV1 immunoreactivity in the spinal dorsal horn.EA at 2 and 15 Hz,but not 100 Hz,significantly attenuated the loss of TRPV1-positive central terminals of afferent fibers in the superficial dorsal horn induced by RTX injection(4)Electron microscopic examination of the sciatic nerve sections revealed the characteristic appearance of myelinated and unmyelinated fibers.RTX induced substantial depletion of unmyelinated fibers and evident damage to myelinated fibers in the sciatic nerve,including axonal swelling,altered myelination,and the appearance of loose and breakdown of the myelin sheath.Compared with the RTX group,the damaged myelinated fibers appeared less swollen and convoluted in shape,and were bounded by a more compact wall of the myelin sheath in 2 Hz and 15 Hz,but not in 100 Hz EA group.The unmyelinated fibers were recovered after 5 weeks of EA treatment,more unmyelinated fibers can be found in 2 Hz and15 Hz,but not 100 Hz EA group.(5)Preferential labeling of myelinated fibers by transganglionic transporting CTB allowed us to determine whether EA had any effect on the sprouting of myelinated afferent fibers in the spinal dorsal horn induced by RTX.Some CTB-labeled myelinated afferent fibers sprouted into lamina II in RTX-treated rats.EA at 2 and 15 Hz significantly attenuated the extension of CTB-labeled terminals into lamina II.However,the RTX-induced sprouting of myelinated afferent fibers in lamina II remained evident and unchanged in rats treated with 100 Hz EA.CONCLUSIONSEA with 2 and 15Hz,but not 100 Hz,improve thermal perception by recovering TRPV 1-positive sensory neurons and nerve terminals damaged by RTX.EA with 2 and 15 Hz,but not 100 Hz,also reduce RTX-induced tactile allodynia by attenuating the damage of myelinated afferent nerves and their abnormal sprouting into the spinal lamina Ⅱ.Our study provides new information about the mechanisms of the therapeutic actions of EA in the treatment of PHN.PART IIThe mechanism of netrin-1 and UNC5H2 involved in sprouting of myelinated fibers and mechanical allodynia in PHN model OBJECTIVEWe aim to demonstrate whether netrin-1 and UNC5H2 contribute to myelinated afferent fiber sprouting and neuropathic pain in RTX induced PHN model.METHODS(1)Mechanical allodynia was quantified with von Frey filaments.(2)The expression of mRNA and protein of netrin-1 were shown by qPCR(Quantitative polymerase chain reaction,PCR)and western blotting.(3)The distribution of netrin-1 in the spinal dorsal horn was shown by double-immunofluorescence labeling.(4)To determine whether netrin-1 and UNC5H2 contributed to the sprouting of myelinated afferent fibers and mechanical allodynia,we specifically silenced netrin-1 and UNC5H2 in the spinal dorsal horn.(5)To observe netrin-1 on the effect of the axon growth by pretreated RTX and blocking effect of TRPV1 channel antagonist capsazepine(CZP),primary DRG neurons were cultured.(6)Specific ELISA kit was used for determining changes of cAMP,and Ca2+fluorescence ratio Imaging was used for determining changes the intracelluar Ca2+concentration.RESULTS(1)Single intraperitoneal injection of RTX in adult rats induced long-lasting tactile allodynia and RTX treatment significantly increased the level of mRNA and protein of netrin-1 in dorsal spinal cord.(2)Using double immunolabeling staining,we showed netrin-1 was distributed in many NeuN-positive neurons and NK-1-positive noxious specific projection neurons in lamina I-II.Consistently,we detect UNC5H2 immunoreactivity was present in CTB-labeled myelinated fibers from laminas II in RTX-treated but not vehicle-treated rats.(3)RTX treatment increased netrin-1 expression in dorsal horn neurons and UNC5H2 expression in myelinated fibers.Silencing netrin-1 or UNC5H2 significantly attenuated RTX-induced allodynia and sprouting of myelinated fibers into spinal lamina Ⅱ.(4)In cultured adult rat DRG neurons,treatment with recombinant netrin-1 protein inhibited neurite outgrowth and elongation.Remarkably,in DRG neurons pretreated with RTX,subsequent netrin-1 treatment promoted neurite growth.In DRG neurons pretreated with specific TRPV1 antoganist CZP before RTX,netrin-1 produced an inhibitory effect on neurite growth.These results suggest that activation of TRPV1 by RTX converts the effect of netrin-1 on neurite growth from inhibition to stimulation.(5)DRG neurons incubated with DMSO or RTX for 1h or 24 h,forskolin for 30 min or forskolin stimulation followed by RTX didn’t increase intracellular cAMP level.In cultured adult rat DRG neurons,treatment with RTX significantly increased intracellular Ca2+ concentration in the remaining survival DRG neurons.To confirm the role of TRPV1 channels in RTX induced intracellular Ca2+ increase,we further treated DRG neurons with CZP 30 min before RTX,and we found CZP diminished RTX-induced Ca2+ mobilization.CONCLUSIONIn cultured adult rat DRG neurons,RTX-induced Ca2+ signaling through TRPV1 receptors can switch netrin-1 and UNC5H2 mediated-effect from inhibition to facilitation,which contributes to abnormal sprouting of myelinated afferent fibers and neuropathic pain development in RTX-induced PHN model.Together,our study provides novel evidence that netrin-1 and UNC5H2 from the adult dorsal spinal cord regulate the formation of abnormal neuronal circuit and may be targeted for reducing primary afferent sprouting and mechanical allodynia in neuropathic pain.PART IIIEffect of EA on RTX-induced expression of netrin-1 and VEGF in spinal cordOBJECTIVEWe aim to demonstrate the effect of EA on RTX induced expression of netrin-1 and VEGF.Furthermore,morphine was used to simulate EA and we observe its regulation of RTX induced netrin-1 expression in vitro.METHODS:(1)The expression of mRNA and protein of netrin-1 and VEGF in dorsal spinal cord were shown by qPCR and western blotting.(2)To observe the effect of morphine and CZP on RTX induced netrin-1 expression,SH-SY5Y cells were cultured.RESULTS(1)RTX treatment increased the mRNA and protein expression of netrin-1 and VEGF in the adult spinal cord.Moreover,2 Hz,but not 15 Hz and 100 Hz EA significantly reduced netrin-1 and VEGF expression.(2)We found that RTX treatment upregulated expression of netrin-1 in SH-SY5Y cells by western blotting.CZP also prevented expression of netrin-1.Thus,RTX-induced expression of netrin-1 is through TRPV1 channels.Moreover,we found morphine prevent RTX induced netrin-1 expression.CONCLUSIONRTX treatment increased netrin-1 expression in the adult dorsal spinal cord,Moreover,2Hz significantly reduced netrin-1 expression.In vitro,CZP inhibited RTX induced upregulation of netrin-1.Thus,RTX induced expression of netrin-1 through TRPV1 channels.Moreover,morphine was used to simulate EA,which inhibited RTX-induced upregulation of netrin-1.It will help to explain the mechanism of EA reducing RTX induced netrin-1 expression in spinal dorsal horn.SUMMARYThe neuronal guidance molecule netrin-1 may play a role in sprouting of myelinated afferent fibers and allodynia in RTX-induced PHN model,which is a new perspective in the research of the mechanism of pain and EA analgesia.We have proved that 2 Hz and 15 Hz EA significantly attenuated the loss of central terminals of afferent fibers,inhibited the sprouting of myelinated fibers and improved mechanical allodynia and thermal induced by RTX injection.EA at 2 Hz and 15 Hz are effective in the treatment of PHN.Moreover,netrin-1 and UNC5H2 are involved in sprouting of myelinated fibers and mechanical allodynia in PHN model.EA at 2 Hz,but not 15 Hz inhibited RTX-induced expression of netrin-1 and VEGF in spinal dorsal horn.It suggested that 2 Hz EA is the best frequency which should be chosen to inhibit myelinated afferent fibers sprouting and mechanical allodynia in the clinical treatment of PHN.Our study not only helps to clarify the mechanism of EA treating mechanical allodynia of PHN,but also provides new insights and strategies to enhance the clinical curative effect of EA analgesia.