| Background:Itch(pruritus)is an unpleasant sensation experienced in daily life,which evoke a desire to scratch.Chronic itch is clinically defined in human as itch that lasts for greater than 6 weeks.The incidence of chronic itch is high and the symptoms are stubborn.Especially the western medicine treatment is not effective,which seriously reduces the quality of life of patients.The descending projection of 5-HTergic neurons in the medulla is an analgesic pathway and a descending pathway for itch facilitation.The RVM(rostral ventromedial medulla,RVM)is an important downstream target of the PAG.Under pathological conditions of chronic itch,the balance of the local microcircuit composed of glutamatergic and γ-aminobutyric ergic(γaminobutyric ergic,GABAergic)neurons in PAG is disrupted,leading to the activation of the descending facilitating pathway of itch in the 5-HT system in medulla.Further studies have shown that the differential regulation of Cannabinoid type 1 receptors(CB1R)on glutamatergic and GABAergic neurons is also an important reason for affecting this local circuit.Therefore,the overall functional output of PAG is important for the regulation of descending pathway of pain and itch.Clinical studies have shown that electroacupuncture can significantly treat chronic itch that is ineffective with antihistamines medicine,and achieve long-lasting effects.The peripheral mechanism of electroacupuncture to relieve itch has been reported,but the central mechanism of electroacupuncture to relieve itch remains to be studied.The previous work of our research group proved that one of the central analgesic mechanisms of electroacupuncture is achieved by regulating the CB1 receptor in vlPAG.Based on the complex interaction between pain and itch and the important role of PAG in the information processing and descending pathways of pain and itch,this study assume that CB1 receptor in PAG is likely to be involved in the regulation of itching information.Object:This study employed a chronic dry skin model,combined with immunohistochemistry,in situ hybridization,virus injection and other research methods to explore the central mechanism of electroacupuncture to play an antipruritic effect by regulating the CB1 receptors in the PAG brain area.Method:(1)The itch model is divided into acute itch and chronic dry skin itch.To perform acute itch,mice received subcutaneous injection of Chloroquine or saline in the nape.And to establish a dry skin model,mice were treated with a mixture of acetone-ether on their shaved back of neck,twice a day for 9 consecutive days.Then,using c-Fos immunohistochemistry staining method to study whether acute itch and chronic itch activate neurons in PAG;(2)The parameter of electroacupuncture is 3 mA、100 Hz.The sham electroacupuncture treatment only shallowly insert into the skin of acupoint without electrical current stimulus.Electroacupuncture treatment was from the first day of employing AEW treatment,once every other day,a total of 5 times.And acupoints "Quchi"(LI11)and "Hegu"(LI14)were chosen.(3)A video recording device was used to record the spontaneous scratching behavior of experimental mice,in which acute itch was recorded for 0.5h and chronic itch was recorded for 1 hour.(4)Western blot was used to detect the expression of endocannabinoid receptors in midbrain vlPAG.(5)In situ hybridization and immunofluorescence double-labeling techniques were used to detect the co-labeling of the GAB Aergic neurons marker GAD67 and CB1 receptors in vlPAG.(4)CB 1R-flox mice were bilaterally inj ected with anterograde AAV-mdlx-Cre virus in the vlPAG.(5)Fluorescence spectrophotometer was used to detect the content of 5-HT neurotransmitter in the cervical spinal cord of C57 mice.(6)Employing GRPR-eGFP transgenic mice,immunofluorescence labeling method was used to detect the number of GRPR+neurons in the spinal dorsal horn.Results:(1)The immunofluorescence staining showed that compared with the control group,the number of c-Fos positive neurons in the PAG brain area was significantly increased in the acute itch and chronic models.(2)The scratch behavior results of C57 mice showed that compared with the control group,the spontaneous itch behavior of chronic itch mice was significantly increased.After electroacupuncture treatment,the spontaneous itch behavior of chronic itch model decreased significantly.(3)The results of HE staining showed that compared with the control group,the epidermal thickness of the modeled area of the neck of the chronic itch model was significantly increased;after electroacupuncture treatment,the epidermal thickness of the modeled area of the chronic itch model was significantly reduced.(4)Western blot results showed that compared with the control group,the expression of CB1 receptor in PAG of chronic itch model was up-regulated;after electroacupuncture treatment,the expression of CB1 receptor in chronic itch mice was down-regulated.There was no significant difference in the expression of CB2 receptor in each group of mice.(5)In situ hybridization and immunofluorescence double labeling results showed that compared with the control group,the expression of CB1 receptors on GABAergic neurons in vlPAG of chronic itch model was up-regulated;after electroacupuncture treatment,the expression of CB1 receptors on GABAergic neurons in vlPAG of chronic itch model was down-regulated.(6)The immunofluorescence staining of GAD67 and CB1R double-labeling results showed that CB1 receptors on GABAergic neurons in vlPAG were specifically knocked out after AAV-mdlxCre virus were bilaterally injected into vlPAG of CB1Rf/f mice.The statistical results of spontaneous itch behavior showed that in chronic itch group,compared with the non-knockout mice,the spontaneous itch behavior of the specific knockout CB1R mice was significantly reduced;in the electroacupuncture group,Compared with the non-knockout mice,the spontaneous itch behavior of the specific knockout CB1R mice were significantly reduced.In the specific knock-out CB1R mice,compared with the control group,the spontaneous itch behavior of chronic itch group was significantly increased;in the specific knock-out CB1R mice,compared with chronic itch group,the spontaneous itch behavior of electroacupuncture treatment group was significantly reduced.(7)Fluorescence spectrophotometer measurement results showed that compared with the control group,the 5-HT content in the cervical spinal cord of chronic itch model was increased;after electroacupuncture treatment,the 5-HT content in the cervical spinal cord of chronic itch model was decreased.(8)The immunofluorescence staining of GRPR-eGFP mice showed that the number of GRPR+neurons in the cervical spinal dorsal horn increased significantly in the chronic itch model.After electroacupuncture treatment,the number of GRPR+neurons in the the cervical spinal dorsal horn decreased significantly.Conclusions:Taken togehter,this study shows that electroacupuncture down-regulated the expression of CB 1 receptors on GABAergic neurons in vlPAG and inhibited the facilitation of itch descending pathway composed of 5-HT descending projection neurons,which resulted in a decrease of the release of 5-HT into the cervical spinal cord.The decreased content of 5-HT in the cervical spinal cord inhibited the expression of GRPR receptors,which in turn inhibits the transmission of itch information in the cervical spinal cord to the brain,thereby producing a central antipruritic effect. |
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