Study On The Role Of G Protein-coupled Estrogen Receptor In Regulation Of Pain And Itch

As two distinct sensory experiences,there is a close relationship between pain and itch.Itch and pain are generally considered antagonistic as painful stimuli generated by scratching relieves itch whereas suppression of pain perception by opioids may produce itchy sensation,which suggests that there was a close relation in neuronal and molecular signaling pathways between pain and itch.The transmission pain and itch signals from the periphery to the dorsal horn of spinal cord is under modulation from the brainstem descending system.However the role of this descending system on the interaction of pain and itch remains unknown.There is a significant gender difference in pain and itch,implying that sex hormone especially estrogen may play an important role in regulation of pain and itch.We have detected high expression of the G protein-coupled estrogen receptors(GPER,also called GPR30)in Rostral Ventromedial Medulla(RVM)of rats and activation of GPER facilitates pain.The purpose of this study is to explore the pain and itch phenotypes of GPER gene conventional knockout animals,and to study the effect of itchy stimulus on neuronal activity in RVM by electrophysiology in vivo.The following are main findings of this study:1.GPER gene knockout rats and mice show attenuated pain but elevated itchChronic inflammatory pain model was established by single intraplantar injection of CFA and paw withdrawal threshold was assessed for mechanical pain sensitivity,which was measured by von Frey Filament.We carried out this experiment in Male,Female and ovariectomized rats independently,comparing phenotypic difference of pain between Wide Type(WT)and GPER knockout(KO)rats.Results showed that ipsilateral PWT of WT rats was significantly decreased after CFA injection and this decrease lasted for over 2 weeks,whereas contralateral PWT did not change significantly.Compared with the WT rats,there were less decrease and faster recovery of ipsilateral PWT in KO rats after CFA injection and contralateral PWT were significantly increased.These phenomena were particularly significant in OVX female KO rats.These results suggest that inflammatory pain was attenuated in GPER gene conventional knockout rats.To our surprise,the baseline PWT of KO rats was significantly lower than that of WT rats,suggesting hypersensitivity to mechanical stimuli in KO rats under physiological status.Capsaicin-induced acute pain was investigated by single intraplantar injection of capsaicin and paw lifting time was assessed for pain.We carried out this experiment in Male and Female rats independently and results showed that the paw lifting time of KO rats was shorter than that of WT rats regardless of gender,suggesting KO rats were less sensitive to acute pain.However,in another acute inflammatory pain model of Formalin intraplantar injection,the number of ipsilateral hindpaw flinches has no significance between WT and KO rats,whereas the duration of ipsilateral hindpaw biting&liking of KO rats was increased compared to WT ratsTo verify whether the decrease of baseline PWT in KO rats was due to alloknesis,we used low grams of Filament to stimulate the nape of animals to examine the sensitivity of mechanical itch.The results showed that KO rats exhibited significant mechanical alloknesis,manifested as scratching towards the site of stimulation with hind limbs under weak mechanical stimuli.Similarly,GPER KO mice also exhibited a phenotype of mechanical alloknesis.In order to test whether the behaviors(paw-flinching,licking/biting)evoked by formalin intraplantar injection also include inthy sensation,a cheek model was established to distinguish pain and itch.After formalin injected into the cheek,forelimb wiping(reflecting pain)and hindlimb scratching(reflecting itch)were observed and measured.Results showed that rats exhibited alternating behaviors of forelimb wiping and hind limbs scratching after formalin cheek injection,and the number of scratching in GPER KO rats was increased compared to WT rats.A cheek model was further used to investigate the response of GPER KO mice to pruritogens.After pruritogen compound 48/80 injection in the cheek,both male and female KO mice had significantly more hind limbs scratching than WT mice,as well as intraperitoneal injection of G15(GPER antagonist)in WT mice also significantly increased the number of hind limbs scratching evoked by compound 48 /80.Imiquimod was applied to the nape of mice for 6 days consecutively to establish a psoriasis-like chronic itch model.It was found that the GPER KO mice had significantly more scratching than WT mice.Above results revealed that the GPER gene conventional knockout rats and mice exhibited phenotype of pain attenuation and itch sensitization,suggesting that GPER plays an important regulatory role in pain and itch and interaction of each other.2.Investigate the mechanism of GPER regulating pain and itchPreliminary work of our laboratory found that GPER was highly expressed in rostral ventromedial medulla(RVM)of brain stem descending system on rats.Activation of GPER facilitates phosphorylation of mu-opioid receptor(MOR)and weakens morphine analgesia.Therefore,we conducted experiments to further examine the relationship between the phenotype of pain and itch in GPER knockout animals and MOR in RVM.Immunohistochemistry of RVM showed that GPER and MOR were co-expressed on non-serotoninergic neurons and gastrin-releasing peptide(GRP)-positive nerve fibers and gastrin-releasing peptide receptor(GRPR)-positive neurons also existed in RVM.In chronic inflammatory pain models established by CFA,the analgesic effect of morphine on GPER knockout rats was much more pronounced than WT rats and the alloknesis in GPER knockout rats and mice could be reversed by mu opioid receptor(MOR)antagonist naltrexone or naloxone.Above results supported that RVM plays an important role in regulation of itch,and the alloknesis in GPER knockout animals is associated with enhanced function of mu opiate receptors(MOR).3.Explore the response of neuronal activity in RVM to pruritogensPrevious studies have shown that there are at least three different functional neurons in the RVM,which are ON cells activated by painful stimuli,OFF cells inhibited by pain,and Neutral cells whose firing activity is not affected by peripheral painful stimuli.ON cells and OFF cells function as facilitation and inhibition separately in brainstem descending pain modulation system,affecting the transmission of pain information in the dorsal horn of spinal cord.However the role of these two neurons in regulation of itch remains unknown.Previous work in our laboratory showed that GPER was only expressed in ON cells.This study found the phenotype of alloknesis and pain inhibition appeared in GPER knockout rats and mice,and the presence of GRP-positive nerve fibers and GRPR-positive neurons in RVM,which suggested that GPER plays a role in regulation of pain and itch in the opposite direction –pain facilitation and itch inhibition.In this part of the experiment,we examined the response of ON cells and OFF cells to pruritogens in WT and GPER KO rats by electrophysiology in vivo.Under a steady mild anesthesia by iso-aneflurane,the extracellular discharge activity of RVM neurons was recorded and was identified as ON,OFF,and Neutral cells according to its response to pain stimuli(pinch: pinch the hind paw;thermal tail-flick: thermal stimulation at 48°C causing tail-flick reflexes)and then observed the changes of neuronal firing activity following pruritogen injection.In female WT rats,pruritogens significantly slowed the firing frequency of ON cells,whereas,the discharge frequency of ON cells did not change significantly after subcutaneous injection of pruritogens in female GPER knockout rats.In these two genotypes rats(WT/GPER-KO),the discharge frequency of most OFF cells was accelerated meanwhile a part of which was slowed after pruritogens injection,but there was no significance between these two genotypes rats.The discharge activity of Neutral cells was not affected by pruritogens.The opposite effect of painful and itchy stimuli on RVM neurons suggest that RVM may play an important role in the interaction of painful sensation and itchy sensation such as pain suppress itch.In summary,we found that GPER knockout rats and mice exhibit phenotypes of pain suppression and itchy sensitization,which may be related to the enhanced function of mu-opioid receptor(MOR)in RVM.It has been supported by a large number of studies that the ON cells excited by pain and OFF cells inhibited by painful stimulation function as facilitating and inhibiting pain separately.The present study found that the activity of ON and OFF cells is also affected by pruritogens,which provides clues for further exploration of mutual molecular mechanisms and neural circuit of pain and itch.