The Role Of Spinal Cord Neuron CB1 Receptors And Microglia In SCS For The Treatment Of Neuropathic Pain

BackgroundNeuropathic pain(NP)is defined as pain caused by damage or disease affecting the somatosensory nervous system.Up to 10% of the general population in the world is affected,and in 5% of persons it may be severe,that makes it a major global public health problem.The general effect of favored treatments is not ideal with only some 40-60% of people achieving partial relief,which means the majority of these patients still do not receive satisfactory relief with existing treatments.Spinal cord stimulation(SCS)provides important alternative strategies for treating neuropathic pain when other therapies have failed or when side effects associated with drug treatments become substantial.The technology of SCS has developed greatly with several novel devices and stimulation modalities in the past decades,the mechanisms of SCS for the treatment of pain is still in the dark.Differences in lead design,stimulation mode,and intensity-selecting criteria also present barriers to correlating previous findings in experimental animals with mechanisms underlying therapeutic effects in patients.Therefore,animal models that are closer to clinical practical applications will be more conducive to animal experiments to provide support for the mechanism research and clinical use of clinical applications.Therefore,this study intends to use Medtronic's custom-made four-level electrodes,with genetic tool mice,using animal behavior,immunohistochemistry,Real-time Polymerase Chain Reaction(PCR),Western blot analysis,and electrophysiology to study the neuropathic pain model of rodents,to study the role of spinal microglia and CB1 receptors in the SCS of rodent neuropathic pain model.Methods and resultsPart 1: Establishment of rat SCS analgesia model and evaluation of behavioral effectsMethods: Male SD rats were used to establish a neuropathic pain model of chronic sciatic nerve constriction injury(CCI).The baseline of paw withdrawal threshold(PWT)was measured one day before CCI,and electrode implantation was performed 7 days after CCI.15 days post-CCI,rats received 30 minutes of continuous SCS or sham SCS.PWT were tested pre-SCS,30 minutes intra-SCS and 30,60 minutes post-SCS.16 days post-CCI,rats received 3 hours of continuous SCS or sham SCS.PWT were tested pre-SCS,30,60,120,180 minutes intra-SCS and 30,60 minutes post-SCS.Day 18-20 post-CCI,rats received 3 days of SCS,PWT were measured post-CCI before,at 30,60,90 and 180 minutes intra-SCS on days 18 to 20 in the a.m.session.Another group of SD rats were taken to establish a CCI neuropathic pain animal model.Electrode implantation was performed 7 days after CCI,conditioned place preference(CPP)habituation was performed on 15-17 days,pre-conditioning baseline testing was done on day 18,day 19-21 was the conditioning session,then day 22 performed the post-conditioning test.Results:From the 3rd day post CCI,the ipsilateral PWT of CCI group decreased significantly,and showed a continuous downward trend till day 7 post-CCI.PWT increased significantly during 30 minutes and 3 hours of continuous SCS,And reach a peak between 60 minutes and 120 minutes.The differences between CCI and SCS group at post-SCS 30 minutes and 60 minutes was not significant.The PWT of the CCI+SCS group increased significantly at 30 minutes,60 minutes and 120 minutes intra-SCS,and then decreased at 180 minutes intra-SCS on the first day of 3 days continuously SCS.On the second and third days of SCS,The PWT of CCI+SCS group increased significantly at 3 minutes,60 minutes,120 minutes and 180 minutes intra-SCS.There was no statistically significant difference among PWTs of same SCS stimulation time in 3 days.Taking the average of the PWTs during the daily SCS stimulation in the CCI+SCS group,the average PWT on the second day was higher than that on the first day,and the average PWT on the third day was not statistically different from the first and second days.In the CPP experiment,there was no statistically significant difference of the rats' residence time in the SCS experiment box between Post-test and Pre-test;the difference of the rats' residence time in the Sham SCS experiment box between Post-test and Pre-test was also not statistically significant.The difference of the CPP score(post-test minus the pre-test)between SCS and sham group was not statistically significant.Part 2: The potential mechanism of microglia involved in SCS analgesiaMethods: After the first part of the experiment,the animals were sacrificed and tissue were tested by immunofluorescence reaction,Real-time PCR and Western Blot to detect CCI and SCS spinal cord astrocyte-specific markers(GFAP)and microglia-specific markers(OX-42)changes in protein and m RNA expression.Real-time PCR was used to detect the changes in the m RNA expression levels of M1 type activation markers(i NOS,CD16,CD32),M2 type activation markers(Arg1,CD163,TGF-?),pro-inflammatory cytokines(IL-1?,TNF-?)and anti-inflammatory cytokines(IL-4,IL10)in CCI rat spinal microglia induced by SCS.Western Blot was used to detect the changes of the protein expression levels of SCS-induced spinal cord microglia activation markers(i NOS,Arg1),cytokines(TNF-?,IL-10),and spinal cord nociceptive molecules(p-ERK1/2,c-Fos,BDNF,PKC-?,p-NR1,p-Glu R1ser831)in CCI rats.Finally,we tested the effect of intrathecal injection of different doses of microglia inhibitor minocycline on PWT in CCI rats,and observed the analgesic effect of low-dose intrathecal injection of minocycline preconditioning combined with SCS.Results:Immunofluorescence showed that the expressions of GFAP and OX-42 in the dorsal horns of the ipsilateral spinal cord of CCI rats were significantly increased.SCS significantly enhanced the expression of OX-42 in the ipsilateral and contralateral spinal cord dorsal horns of the CCI rats.SCS has no obvious effect on the expression of GFAP in both ipsilateral and contralateral dorsal horns of spinal cord.Real-time PCR and Western Blot data are in line with this.Real-time PCR results showed that the m RNA levels of CD16 and CD32(M1 type specific molecular markers)of CCI rats were significantly increased than those of Na(?)ve rats.SCS significantly increases the m RNA levels of CD16 and i NOS in CCI rats.The m RNA levels of pro-inflammatory cytokines IL-1? and TNF-? in CCI rats and Na(?)ve rats are similar,but IL-1? m RNA levels are significantly increased after SCS.The m RNA levels of M2 specific molecular markers Arg1,CD163 and TGF-? were not changed by CCI or SCS.The m RNA levels of anti-inflammatory cytokines IL-4 and IL-10 in CCI rats were significantly decreased than those in na(?)ve rats,and SCS did not change the levels of IL-4 or IL-10 m RNA in CCI rats.Western Blot data of the spinal dorsal horn tissue showed that compared with Sham SCS,the expression of i NOS of the ipsilateral spinal dorsal horn in SCS rats was up-regulated,but the expression of TNF-?,Arg1 and IL-10 remained unchanged.Western Blot results also showed that the level of p-ERK1/2 in the injured spinal cord of Sham SCS CCI rats was significantly increased than that of naive rats.However,there is no significant difference in protein levels of c-Fos,BDNF,PKC-?,p-NR1 and p-Glu R1ser831 between this two groups.SCS significantly reduced BDNF levels in CCI rats compared with Sham SCS.Intrathecal injection of microglia inhibitor minocycline can reduce the mechanical hypersensitivity of the ipsilateral hind paw in CCI rats dose-dependent;intrathecal injection of low-dose minocycline pretreatment can prolong the pain suppression effect of SCS.Part 3: CB1 receptor activation is involved in SCS analgesiaMethods: VGlut2-Cre:Rosa 26-Td Tomat and GAD1-GFP gene tool mice were used to make tissue sections,and immunofluorescence staining was used to detect the expression of CB1 receptors in glutamatergic excitatory neurons and GABAergic neurons.Patch-clamp recording of the evoked excitatory postsynaptic currents(e EPSCs)was used in mice 7 days after spinal nerve ligation(SNL)to detected the effect of electrical stimulation of Ab-fibers(A?-ES)using clinical SCS-like parameters(50 Hz,0.2 millisecond,10 m A)to the depression of e EPSCs to C-fiber inputs in Substantia Gelatinosa(SG)neurons.Subsequent pretreatment with CB1 receptor antagonist AM251(2?M,5min),and record the changes of e EPSCs caused by A?-ES.Finally,through the rat SNL animal model,observe the pretreatment of AM251(50?g),CB2 receptor antagonist AM630(100?g)and cannabinoid absorption blocker LY2183240(50?g).SCS(50 Hz,80% Mo T,0.2ms,60 minutes)on the behavior of SNL rats.Results:Double-immunofluorescence labeling showed that 52.3% of v Glut2-Td+ SG neurons were positive for CB1 receptor immunoreactivity.A?-ES induced prolonged depression of e EPSCs to C-fiber inputs in v Glut2-Td+ neurons.AM251 pretreatment blocked the inhibitory effect of A?-ES on C-e EPSCs and stabilized C-e EPSCs 5 minutes after administration.71.4% of GAD1-GFP+ cells in SG express CB1 receptor immunoreactivity.A?-ES induced prolonged depression of e EPSCs to C-fiber inputs in GAD1-GFP+ neurons,AM251 pretreatment can partially reduce the inhibitory effect of A?-ES.Behavior data showed that intrathecal injection of AM251 attenuated the increase in PWT induced by SCS,AM630 had no significant effect on the increase in PWT induced by SCS,while LY2183240 enhanced the increase in PWT induced by SCS.When combined AM251 with LY2183240 and pretreated SCS rats together,AM251 blocked the enhancement effect of LY2183240 on SCS analgesia.Conclusion1.SCS can produce sustained analgesic effect,but the analgesic effect disappears after the stimulation is stopped;CPP experiment results show that rats have no dependence on SCS treatment.2.SCS increases the activation of M1-like microglia in the spinal cord,thereby reducing its own analgesic effect.Minocycline pretreatment can prolong the analgesic effect of SCS by inhibiting microglia3.CB1 receptors play an important role in SCS-induced pain suppression.CB1 receptor antagonist can attenuate the analgesic effect of SCS,Endocannabinoid degradation inhibitors can enhance the efficacy of SCS.CB1 receptor may provide an effective target for improving the analgesic effect of SCS.