Investigating The Role Of Dorsal Root Ganglion And Spinal TRPM3 In Inflammatory Pain And Neuropathic Pain

IntroductionChronic pain is a common clinical symptom that affects about 20%of the gross population and has been a major public health problem in recent decades.However,current therapies for this disorder have limited effects,in part due to the associated severe side effects,for example,opioid addiction.Therefore,exploring effective and non-addictive treatments is one of the main research directions in the development of new analgesics.Transient Receptor Potential(TRP)channels are considered as prominent drug targets,but by far no clinically useful medication targeting TRP channels has been developed.Most efforts focused on the heat-and capsaicin-sensitive Transient Receptor Potential Vanilloid 1(TRPV1),but they failed because of the severe,often life-threatening hyperthermia caused by TRPV1 antagonists.In this study,we evaluated the potency of heat-sensitive channel TRPM3 as an alternative drug target for the treatment of evoked and spontaneous pain after inflammation and peripheral nerve injury.TRPM3 is a Ca2+ permeable non-selective cation channel expressed in the nociceptive neurons of the dorsal root ganglia(DRG)and trigeminal ganglia(TG).It is activated by heat,and chemical compounds,such as endogenous neurosteroid pregnenolone sulfate(PregS)and synthetic compound CIM0216.Genetic deletion of TRPM3 in mice decreased sensitivity to noxious heat,as well as reduced inflammatory thermal hyperalgesia.Furthermore,it has been shown that the response to noxious heat completely disappeared in a trio of TRP(Trpal/Trpv1/Trpm3)channels combined deletion mice.While genetic deletion of TRPM3 impairs the noxious heat sensation,evidence showed that this channel could be activated by the temperature well below the noxious temperatures.For example,TRPM3 currents in HEK293 cells were shown to increase when the temperature was raised from 15 to 26℃,with a further increase at 35℃.TRPM3 expressed in Xenopus oocytes showed an obvious increase in currents when the temperature was raised from room temperature to 38℃.This relatively low temperature threshold makes TRPM3 an ideal candidate for contributing to spontaneous pain in pathological conditions,but this possibility has not yet been experimentally addressed.It is well documented that skin temperatures are sensed by the free nerve endings of the peripheral processes of DRG neurons.The temperature stimuli were converted into electrical signals and then transmitted to the central nerve system.The mechanisms by which thermo-sensitive TRP channels in DRGs and TGs implicated in pain have been extensively studied.In recent reports,TRP channels have also been shown to exist in the central processes of DRG neurons that form synapses with the secondary neurons in the dorsal horn,and they can modulate the transmission of painful stimuli.Lee et al detected the expression of TRPM3 mRNA in the spinal cord.However,the role of TRPM3 expressed in the spinal cord,DRG cell bodies and the central processes of DRG neurons in nociceptive processing has not yet been experimentally studied.TRPM3 inhibitors have shown significant analgesic effects in some rodent pain models,and unlike TRPV1 inhibitors,they do not increase body temperatures.Although the role of TRPM3 in noxious heat sensation is well supported,its role in mechanical and cold sensation is still controversial.More importantly,to date,none of the effects of TRPM3 inhibitors have been validated in Trpm3-/-mice,therefore it is unclear whether the reported effects are mediated by TRPM3 inhibition.AimsHere we used Trpm3-/-mice and two structurally different TRPM3 inhibitors to investigate the role of TRPM3 in evoked pain(such as mechanical,thermal and cold hypersensitivity)and spontaneous pain in different pain models(including carrageenan-induced inflammatory pain and sciatic nerve injury induced-neuropathic pain).The effects of intrathecal administration of TRPM3 agonists and inhibitors on nociceptive behaviors and neuronal activity in DRG and spinal dorsal horn(SDH)were examined to clarify whether TRPM3 located in the spinal cord,DRG cell bodies and the central processes of DRG neurons participated in the transmission of nociceptive information.The present study investigated the role of endogenous heat-sensitive TRPM3 in pain processing,which might provide the theoretical basis for the development of a new generation of analgesic drugs.Methods1.To validate the deficiency of functional TRPM3 channels in Trpm3-/mice via calcium imaging assay.Calcium imaging assay was carried out to measure the responses of cultured DRG neurons from wild-type and Trpm3-/-mice to the TRPM3 agonists PregS and CIM0216.In addition,the responses of DRG neurons to the TRPV1 agonist capsaicin and the TRPM8 agonist menthol were examined.2.To investigate the role of TRPM3 in carrageenan-induced inflammatory pain(1)To establish carraggenan-induced inflammatory painBoth wild-type and Trpm3-/-mice were subjected to carrageenan-induced inflammatory pain.Briefly,20 μL 1%λ-carrageenan solution was injected subcutaneously to the dorsal part of the left hind paw to produce paw inflammation.(2)To detect the nociceptive behaviors and neuronal activity after carrageenan injectionWe first assessed whether the model was established successfully by measuring the evoked behavioral responses including mechanical paw withdrawal frequencies(PWFs)and thermal paw withdrawal latency(PWLs)at different time points during a 2-day period.The spontaneous pain was also measured at 1 h after carrageenan injection.Finally,immunofluorescence assay was carried out to detect the expression of c-Fos and pERK in DRGs and SDH in each group.The role of TRPM3 in different pain modalities and neuronal activity in inflammatory pain was investigated.3.To investigate the role of TRPM3 in sciatic nerve injury inducedneuropathic pain(1)To establish sciatic nerve injury-induced neuropathic painThe CCI model was established in wild-type and Trpm3-/-mice,respectively.In brief,the left sciatic nerve trunk was exposed by blunt dissection at mid-thigh level.The 3 ligatures were tied loosely with 7-0 silk thread around the nerve with 1 mm interval.The sham groups underwent procedures identical to that in the CCI group,but without the ligature of the respective nerve.(2)To measure the effect of TRPM3 inhibitors via systemic injectionSix days post the surgery,TRPM3 inhibitor isosakuranetin at different doses of 2 mg/kg and 0.5 mg/kg,and another inhibitor primidone at the dose of lmg/kg were intraperitoneally injected,the behaviors including mechanical paw withdrawal frequencies(PWFs),thermal paw withdrawal latency(PWLs),paw withdrawal responses to cold stimuli(PWLs&Numbers of paw lifting)and spontaneous pain were measured,respectively.Immunofluorescence staining was performed to detect the expression of c-Fos and pERK in the DRGs and SDH from each treated group.We assess the role of TRPM3 in neuropathic pain by evaluating the effects of TRPM3 deficiency and TRPM3 inhibitors on nociceptive behaviors and neuronal activity in DRGs and SDH after CCI.4.To investigate the effect of local or intrathecal TRPM3 inhibitor on neuropathic pain(1)To examine the peripheral effect of the TRPM3 inhibitors via plantar injectionSix days after CCI or sham operation,primidone(0.5 μg/5 μL)was injected into the plantar side of the ipsilateral hind paw,mechanical paw withdrawal frequencies(PWFs)and thermal paw withdrawal latencies(PWLs)were measured to evaluate the analgesic effect of TRPM3 local inhibition.(2)To detect the expression and distribution of TRPM3 in DRGs and SDHIn situ hybridization RNAscope assay was used to detect the co-localization of TRPM3 with NeuN(neuronal marker)and GFAP(astrocyte marker)in DRGs and SDH from sham-operated groups and CCI groups.(3)To examine the effect of intrathecal TRPM3 inhibitors or agonistsSix days after CCI or sham operation,primidone(0.5 μg/5 μL)was injected intrathecally.Mechanical and thermal tests were carried out to assess the effect of intrathecal TRPM3 inhibitor.Additionally,the TRPM3 agonist CIM0216(25 nmol/5μL)was injected into the subarachnoid space in wild-type and Trpm3-/-na(?)ve mice.Thermal paw withdrawal latencies(PWLs)and the expression of c-Fos and pERK in DRGs and SDH were observed to further clarify whether TRPM3 in the spinal cord and central processes and bodies of DRG neurons implicates in pain processing.Results1.Calcium imaging validated the lack of TRPM3 function in Trpm3-/-mice.TRPM3 agonists PregS and CIM0216 triggered the robust Ca2+ increase in DRG neurons of wild-type mice.In contrast,the response of DRG neurons from Trpm3-/mice was significantly reduced.DRG neurons from Trpm3-/-mice,like those from wild-type mice,displayed normal responses to TRPV1 agonist capsaicin and TRPM8 agonist menthol.2.TRPM3 is involved in the carrageenan-induced thermal hyperalgesia and spontaneous pain.(1)Carrageenan injection successfully induced inflammatory pain.Compared to the basal threshold,carrageenan injection led to mechanical hypersensitivity and thermal hyperalgesia on the ipsilateral side of wild-type mice,as indicated by the increases in paw withdrawal frequencies and the decreases in paw withdrawal latencies,respectively.In contrast,Trpm3-/-mice exhibited the carrageenan-induced mechanical hypersensitivity,but not thermal hyperalgesia.Compared with wild-type mice,Trpm3-/-mice displayed prolonged basal paw withdrawal latencies in response to thermal stimulation.Moreover,carrageenan injection produced spontaneous pain in both wild-type and Trpm3-/-mice at 1 h after injection,as indicated by the increased duration of paw shrinking,licking and lifting within 10 min.The duration of paw shrinking,licking and lifting in Trpm3-/-mice was greatly reduced when compared to that in wild-type mice.These data demonstrated that TRPM3 deficiency impaired the sensitivity to thermal stimuli and spontaneous pain,but not to mechanical stimuli.(2)TRPM3 deficiency inhibited the increased neuronal activity induced by carrageenan.Immunofluorescence staining data showed that carrageenan injection induced the elevated expression of c-Fos and pERK in DRGs and SDH both in wild-type and Trpm3-/-mice.However,the levels of c-Fos and pERK in DRGs and SDH of Trpm3-/mice were much lower than those in wild-type mice,suggesting that TRPM3 deficiency inhibits the increased neuronal activity in DRGs and SDH after carrageenan injection.3.TRPM3 is involved in thermal hyperalgesia and spontaneous pain after sciatic nerve injury.(1)CCI produced significant pain hypersensitivities.Six days post-surgery,CCI,but not sham surgery,led to mechanical allodynia,thermal hyperalgesia and cold hyperalgesia in wild-type mice on the ipsilateral side.Trpm3-/-mice displayed intact mechanical allodynia and cold hyperalgesia,but failed to develop thermal hyperalgesia after CCI.TRPM3 deficiency likely impaired the sensitivity to thermal stimuli but didn’t affect the responses to mechanical or cold stimuli.(2)Systemic TRPM3 inhibition attenuated CCI-induced evoked and spontaneous pain.Systemic injection of TRPM3 inhibitors isosakuranetin(2 mg/kg,0.5 mg/kg)and primidone(1 mg/kg)reduced the CCI-induced thermal hyperalgesia in wild-type mice,but had no effect in Trpm3-/-mice.Systemic isosakuranetin at 2 mg/kg alleviated cold hypoeralgesia,mechanical allodynia and spontaneous pain both in wild-type mice and Trpm3-/-mice after CCI.The lower dose of isosakuranetin(0.5 mg/kg)inhibited thermal hyperalgesia and spontaneous pain,but didn’t affect mechanical allodynia or cold hyperalgesia in wild-type mice following CCI.(3)TRPM3 deficiency and pharmacological inhibition reversed the increased neuronal activity following nerve injury.The levels of c-Fos and pERK were largely elevated in DRGs and SDH both in wild-type and Trpm3-/-mice after CCI.However,the levels of c-Fos and pERK in Trpm3-/-mice were conspicuously reduced as compared with wild-type mice.Compared with vehicle groups,systemic isosakuranetin or primidone inhibited the elevated expression of spinal c-Fos and pERK from wild-type mice or Trpm3-/-mice after nerve injury.4.Effect of local or intrathecal injection of TRPM3 inhibitors on neuropathic pain(1)Intra-plantar injection of TRPM3 inhibitor attenuated thermal hyperalgesia in wild-type mice after nerve injuryIntraplantar injection of primidone(0.5 μg/5 μL)was effective in alleviating thermal hyperalgesia after nerve injury in wild-type mice,but not in Trpm3-/-mice.Intraplantar injection of primidone at this dosage had no effect on CCI-induced mechanical allodynia or clod hyperalgesia in mice of either genotypes.(2)TRPM3 is co-localized with NeuN and GFAP in DRGs and SDH.In situ hybridization RNAscope assay demonstrated that TRPM3 mRNA was co-expressed with NeuN or GFAP in the individual cells from both DRGs and SDH.(3)Intrathecal injection of TRPM3 inhibitor or agonist altered the sensitivity to thermal stimuli in wild-type mice.① Six days after CCI or sham operation,intrathecal injection of primidone(0.5μg/5μL)significantly alleviated thermal hyperalgesia in wild-type mice,but not in Trpm3-/-mice.Intrathecal injection of primidone at this dosage did not affect CCI-induced mechanical allodynia in mice with either genotypes.②Compared with the vehicle-treated group,intrathecal administration of the TRPM3 agonist CIM0216 significantly reduced paw withdrawal latencies and increased the sensitivity to thermal stimuli in wild-type mice,but not in Trpm3-/mice.③ Compared with the vehicle-treated group,intratehcal CIM0216 markedly increased the expression of c-Fos and pERK in SDH in both wild-type mice and Trpm3-/-mice.However,the expression of c-Fos and pERK in these two regions of Trpm3-/-mice was remarkably lower than that in wild-type mice.ConclusionThe present study carried out the combined approaches of genetic knockout strategy with pharmacological inhibitors and examined the role of TRPM3 in two different pain models.Our data indicate that TRPM3 is required for heat hyperalgesia and spontaneous pain,but not in cold or mechanical hypersensitivity after peripheral inflammation or nerve injury.Our data also suggest that TRPM3 expressed not only in the peripheral processes but also in the spinal cord,DRG cell bodies and the central processes of DRG neurons is important for heat hypersensitivity.TRPM3 is likely a key player in noxious heat sensation and spontaneous pain.The current study may provide a new potential target and theoretical basis for prevention and treatment of chronic pain.