Microglia Are Involved In The Regulation Of Acute And Chronic Itch

Part Ⅰ:Microglia regulate acute itchBackground:Although itch and pain have many similarities,they are completely different in perceptual experience and behavioral response.In recent years,we have a deep understanding of the neural pathway of itch sensation transmission.However,there are few reports on the role of non-neuronal cells in itch.A large amount of previous work,including ours,has shown that central microglia play a key role in the development of hyperalgesia and chronic pain.Then,are microglia also involved in the transmission of itch sensation?Objective:To investigate the role of spinal microglia on acute itch.Methods:Using CSF1Rf/f:CX3CR1reER/+and ROSAiDTR/+:CX3CR1CreER/+transgenic mice to specifically deplete central microglia and peripheral macrophages,and ROSAiDTR/+:P2Y12CreER/CreER transgenic mice to specifically deplete central microglia only,or minocycline to inhibit microglia activity,we observed the acute itch responses to histamine(HA),Compound 48/80(C48/80)chloroquine(CQ),β-alanine and mechanical stimuli in mice.CX3CR1 KO mice were used to observe the acute itch response induced by HA,C48/80 and CQ.Immunofluorescence was used to verify the microglia depletion efficiencies and the neuronal activity represented by c-Fos in spinal cord.RNAscope was used to detect which neurons in the itch circuit of spinal cord were regulated by microglia.Results:The experimental results showed that the elimination of central microglia and peripheral macrophages significantly reduced the acute itch responses to HA,C48/80 and CQ,but did not affect mechanical itch and β-alanine induced acute itch response.And the itch response in the mice with central microglia depleted only were also reduced in the similar way in the HA,C48/80 and CQ models.Inhibition of spinal microglia activity by minocycline also significantly reduced acute itch response in HA,Compound C48/80 and CQ models.CX3CR1 knockout significantly reduced acute pruritus induced by HA and C48/80,but did not affect acute pruritus induced by CQ.Microglia depletion significantly reduced overall c-Fos activation in spinal cord in HA,C48/80 and CQ models,respectively.While the reduced activity of npr 1 and sst neurons in spinal cord were only seen in the HA dependent models but not CQ model,with either microglia depletion or CX3CR1 KO.Conclusion:Central microglia were involved in HA,Compound C48/80 and CQ-induced acute itch transmission,but not in mechanical itch and β-alanine acute itch.Microglia may mediate HA dependent itch transmission through CX3CL1-CX3CR1 pathway,which activated the primary projection to spinal cord(nprl and sst neurons)from DRG.While the unspecific Microglial activation could enhance the itch transmission in both HA dependent and non-HA dependent(CQ)pathways.Part Ⅱ:Microglia regulate chronic itchBackground:Chronic pruritus is one of the common problems in clinic.At present,the research on chronic pruritus is relatively weak due to many reasons,such as complex pathogenesis and researching technology limitations.There are several chronic itch models for laboratorial study of the related mechanism.Among those,acetone-ether-water(AEW)model that induces skin dry and causes chronic itching is a commonly used one.Our above work has shown that microglia play an important role in acute itch transmission.Microglia have also been reported to be activated in chronic itch induced by psoriasis and 2,4-dinitro-1-fluorobenzene(DNFB).However,what is the role of microglia in chronic itch development is still unknown.Objective:To investigate the effect of spinal microglia on AEW chronic itch.Methods:We used CSF1Rf/f:CX3CR1CreER/+ and ROSAiDTR/+:CX3CR1CreER/+to specifically deplete central microglia and peripheral macrophage to observe whether AEW induced chronic itch response was affected.Chronic itch induced by AEW was observed in P2Y12f/f:CX3CR1CreER/+ transgenic mice,in which The P2Y12 receptor is knocked out in microglia.Immunofluorescence was used to verify the depletion efficiency of microglia and the knockout efficiency of P2Y12 receptor in the transgenic mice,and the expression of c-Fos in spinal cord of the modeled mice was observed.Results:The results showed that microglia marker CD11b expression was significantly up-regulated in AEW model.Elimination of central microglia and peripheral macrophages significantly reduced AEW induced itch response,and depleting central microglia only also significantly reduced AEW induced itch response.Knockout of P2Y12 receptor on microglia significantly reduced AEW chronic itch.Microglia depletion significantly reduced c-Fos expression in the spinal cord of AEW mice.Conclusion:Central microglia participate in AEW induced chronic itch development.Microglia may regulate chronic itch through P2Y12 receptor.