The Role Of Locus Coeruleus-spinal Dorsal Horn Noradrenergic Pathway In The Regulation Of The Neuropathic Pain

Objective:Locus coeruleus（LC）noradrenergic neurons project descending fibers to the spinal dorsal horn（DH）,releasing norepinephrine（NE）to inhibit pain transmission.This pathway has no obvious inhibitory effect on physiological pain.Under pathological conditions,the NE released to DH increases,which can obviously inhibit the transmission of pathological pain,however,its specific mechanism is unclear.This study is mainly to explore the effect of LC-noradrenergic pathway in spinal DH on neuropathic pain（NP）and its possible mechanism.Methods:1.Establishment of LC and spinal cord chemogenetics model:12 c57 mice（25-30g）were randomly selected and divided into designer receptors exclusively activated by designer drugs（DREADD）activation group（M3 group,n=12）and DREADD control group（n=12）.Mice were anesthetized and microinjected with adeno-associated virus（AAV）into the LC and spinal cord,DREADD group（LC:r AAV-Efla-DIO-h M3D（Gq）-m Cherry-WPREs,spinal DH:r AAV-TH-NLS-CRE-WPRE-h GH-p A AAV2/retro）,Control group（LC:r AAV-Efla-DIO-h M3D（Gq）-m Cherry-WPREs,spinal DH:r AAV-TH-NLS-CRE-WPRE-h GH-p A.AAV2/retro）.Immunofluorescence experiments were conducted to verify the transfection of AAV three weeks after microinjection.2.Establishment of NP model of peripheral nerve injury and determination of pain threshold Mouse chronic constriction injury（CCI）model was set to simulate NP.One week after the CCI surgery,the Sham group（n=12）,CCI group（n=12）,CCI+M3 group（n=12）and CCI+vector group（n=12）were tested for mechanical pain thr eshold and thermal pain threshold to confirm the success of CCI modeling.3.In vivo electrophysiological recording of LC neurons:To observe the extracellular currents,a metal electrode was placed into the LC of the CCI+M3 group mice（n=6）.The continuous and stable neuronal firing was explored and recorded for 5 minutes,and then the mice was given CNO（1 mg/kg）intraperitoneally,and then recorded for 60 minutes with the help of Spike2 software.4 Enzyme-linked immunosorbent assay（ELISA）was used to detect spinal cord（L3 and L4）NE content:The NE contents in the spinal cord tissue of Sham group（n=6）,CCI group（n=6）,CCI+M3+Saline group（n=6）,CCI+M3+CNO（n=6）,CCI+vector+Saline group（n=6）,and CCI+vector+CNO group（n=6）were tested by ELISA kit.5.The effect of LC neuron activation on the behavior of CCI mice:CCI+M3 group（n=6）and CCI+control group（CCI+vector）group（n=6）were given CNO（1mg/kg）intraperitoneally,then the threshold of mechanical pain and thermal pain in the CCI mice were detected.6.In vivo electrophysiological recording of WDR neurons in the spinal DH:The spontaneous and tactile-evoked firing of the WDR neurons in L3-L5 spinal cord was recorded after intraperitoneal administration of CNO（1mg/kg）or CNO+α₂ receptor blocker yohimbine（0.1μg/kg）in the DH.7.Determination of glial cell markers in the DH of the spinal cord:The expression of GFAP and Iba-1,the marker of astrocytes and microglia,respectively,were observed with immunofluorescence to make sure the effect of the activation of LC NE neurons in the spinal DH.8.Determination of inflammatory factors in the spinal DH:The expression levels of inflammatory factors in the spinal DH were detected by PCR and ELISA.Results:1.Twenty-one days after the establishment of the chemogenetics model,the M3-DREADD AAV was successfully transfected in the NE neurons in the LC.2.Compared with the sham group at the same time point,the mechanical pain and thermal pain thresholds of mice in the CCI group,CCI+M3 group,and CCI+vector group were significantly reduced on the 5th day after modeling（P<0.05）;Compared with the baseline of the same group,the mechanical pain and thermal pain thresholds of mice in above group decreased significantly on the 5th day after modeling,and reached the lowest value on the7th day（P<0.05）.3.Compared with the baseline value before CNO administration in the CCI+M3 group,the discharge frequency of the noradrenergic neurons in LC increased（P<0.05）.4.Compared with the CCI+M3+saline group,the NE contents in the spinal DH of mice in the CCI+M3+CNO group increased（P<0.05）,and there was no statistical difference between the other groups（P>0.05）.5.Compared with the CNO-solvent control group,the mechanical pain threshold of the mice in the CNO group increased（P<0.05）;Compared with the CNO-solvent control group,the thermal pain threshold of the mice in the CNO group increased（P<0.05）;There was no significant change in mechanical pain and thermal pain in the empty virus group（P>0.05）.6.When the mice were given brush stimulation,1g von Frey pressure,or light clamp stimulation,compared with the baseline value,the frequency of electric discharge in the M3activation group（CNO group）was reduced（P<0.05）;Compared with the CNO group,the frequency of electrical firing of yohimbine blocker group（CNO+Yohimbine group）increased（P<0.05）;Compared with the CNO+Yohimbine group,the frequency of electrical firing of yohimbine wash out group（Wash out group）decreased the（P<0.05）;There is no significant difference in the spontaneous firing between groups.7.Compared with the sham group,the expression of astrocytes and microglia markers（GFAP and Iba-1,respectively）in the spinal DH of the CCI group increased（P<0.05）;Compared with the CCI+M3+saline group,the number and the activation of astrocytes and microglia in the spinal DH in the CCI+M3+CNO group decreased（P<0.05）.8.Compared with the sham group,the m RNA expression of TNF-α,IL-1β,IL-6,i NOS,CD11b,and CD68 in the spinal DH of the CCI group increased（P<0.05）;Compared with the CCI+M3+saline group,the m RNA expression of these genes in the+M3+CNO group decreased（P<0.05）;Compared with the sham group,the m RNA expression of Arg1,IL-4,IL-10,and CD206 in the spinal DH of the CCI group decreased（P<0.05）;Compared with the M3+saline group,the above-mentioned m RNA expression in the CCI+M3+CNO group increased（P<0.05）;Compared with the sham group,the expression of TNF-αand IL-1βprotein in the spinal DH of the CCI group increased（P<0.05）;Compared with the CCI+M3+saline group,the expression of TNF-αand IL-1βprotein in the CCI+M3+CNO group reduced（P<0.05）.Conclusion:Selective activation of the locus coeruleus-spinal dorsal horn NE pathway can relieve pain in CCI mice.The possible mechanism is:（1）Activating this pathway inhibits the activation of microglia and astrocytes in the spinal dorsal horn;（2）Activating the locus coeruleus-spinal dorsal horn NE energy pathway inhibits the spinal dorsal horn pro-inflammatory cytokines TNF-α,IL-1β,etc.,promoting the expression of IL-4,IL-10 and other anti-inflammatory factors;（3）NE inhibits the electrical discharge of WDR neurons in the dorsal horn through inhibiting the upload of pain.