| Objective:Inflammatory pain is a common clinical symptom that widely exists in various diseases.Generally,inflammatory pain is a response to tissue injury that caused by trauma,bacteria or viral infection.Inflammatory pain can be characterized by spontaneous pain,allodynia and hyperalgesia,which adversely affects patients’ daily life.At present,inflammatory pain is mainly treated with drugs,however,the outcomes are not satisfactory.Moreover,a long-term use of these agents has been associated with severe side effects.Therefore,developing new analgesic drugs with fewer side effects to control inflammatory pain is of great significance to improve the quality of patients’life.The sodium leak channel(NALCN)is widely distributed in neurons of the brain and the spinal cord which can regulate neuronal excitability.The abnormal level of NALCN expression contributes to pain-insensitivity of African highveld mole-rat.Moreover,the NALCN regulate thermal sensitivity in elegans’ thermoreceptors.All these studies suggest that NALCN may be involved in the transmission of sensation and pain.Currently,no study has elucidated the relation between NALCN and inflammatory pain in vivo.We put forward the hypothesis that the expression and function regulation of NALCN in DRG and spinal dorsal horn is an important mechanism of pathogenesis of inflammatory pain.In this study,research methods including animal behavioral models,molecular biological techniques,RNA interference and electrophysiological patch-clamp recording were used to confirm:the increased expression and elevated function of NALCN contribute to the development of inflammatory pain.Our findings may provide insights into potential therapeutic targets for the treatment of inflammatory pain.Chapter 1 The Correlation between expression of NALCN in DRG and spinal dorsal horn and pain behavior in inflammatory pain in ratsExperiment 1 Expression of NALCN in DRG and spinal dorsal horn increased in inflammatory pain in ratsMaterials and methods:(1)The adult Sprague-Dawley rats(weight 180～200 g)were used for behavioral tests.After measuring the baseline pain threshold,the rats were subcutaneously injected with 100 μL complete Freund’s adjuvant(CFA)into the left footpad(CFAipsilateral).The right footpad(CFA-contralateral)was served as control.After injection of CFA,nociceptive behaviors were recorded in the first 30 minutes as paw licking,lifting,and shaking.The mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)were performed at 2 h,8 h,day 1 to day 12 after injection of CFA(n=10);(2)To determine the change of NALCN mRNA expression after injection of CFA,lumbar 4-6 DRG and the lumbar enlargement of spinal cord from both CFA-ipsilateral and CFA-contralateral sides were extracted for RT-PCR at 2 h,8 h,day 1,day 3,day 7 and day 12 after injection of CFA(n=10);(3)To determine the change of NALCN protein expression after injection of CFA,lumbar 4-6 DRG of both CFA-ipsilateral and CFA-contralateral sides were extracted for western blot at 8 h and day 12 after injection of CFA(n=5).The Lumbar enlargement of spinal cord from both CFA-ipsilateral and CFA-contralateral sides were extracted for western blot at day 3 and day 12 after injection of CFA(n=5);(4)To determine the expression patterns of NALCN,lumbar 4-6 DRG and the lumbar enlargement of spinal cord from both CFA-ipsilateral and CFA-contralateral sides were extracted for immunofluorescence staining at 8 h(n=5)and day 3(n=5)after injection of CFA,respectively.Results:(1)We recorded spontaneous pain-related behaviors for 30 min after injection of CFA,including lifting,licking and shaking of the rear foot(n=10,P<0.05).Compared with CFA-contralateral side,thermal hyperalgesia and mechanical allodynia were developed in the CFA-ipsilateral side from 2 h to day 10 after CFA injection(n=10,P<0.05,respectively).The most pronounced mechanical allodynia and thermal hyperalgesia were detected at day 1 after CFA injection;(2)Expression of NALCN mRNA in the DRG from the CFA-ipsilateral side were higher at 2 h(n=10,P=0.011)and 8 h(n=10,P=0.001)after CFA injection compared with the CFA-contralateral side.In the spinal dorsal horn,expression of NALCN mRNA were higherat2 h(n=6,P=0.026),8 h(n=10,P=0.015),day 1(n=10,P<0.001)and day 3(n=10,P<0.001)after CFA injection;(3)Compared with the CFA-contralateral side,expression of NALCN protein was greater in DRG at 8 h after CFA injection(n=5,P<0.001)and greater in spinal dorsal horn at day 3 after CFA injection(n=5,P=0.01)from the CFA-ipsilateral side;(4)NALCN was widely expressed in nearly all neurons of DRG and spinal dorsal horn by fluorescence staining.NALCN was widely merged with SP,NF200,IB4 and TRPV1 in DRG.We quantified representative DRG sections from five rats.The percent of SP neurons(P=0.032)and TRPV1 neurons(P=0.015)were higher in CFAipsilateral side than CFA-contralateral side.In the spinal dorsal horn,NALCN was widely expressed in SP positive neurons.Experiment 2 knockdown NALCN in DRG and spinal dorsal horn alleviated inflammatory pain in ratsMaterials and methods:(1)siRNA-NALCN or siRNA-control was injected into the left sciatic nerve and subarachnoid space.3 days later,L4-L6 DRG(n=8)and the lumbar enlargement of spinal cord(n=6)from both CFA-ipsilateral and CFA-contralateral side were extracted for RT-qPCR to detected NALCN mRNA in siRNA-control and siRNA-NALCN group;(2)After measuring the baseline pain threshold of adult rats(n=10),siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space.MWT and TWL were measured 3 days later;(3)The adult rats were randomly divided into siRNA-control(n=10)and siRNANALCN group(n=10).After measuring the baseline pain threshold in rats,siRNAcontrol or siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space.CFA model was established 2 days later.After CFA injection,the durations of nociceptive behaviors were recorded for 30 min,including paw liking,lifting,and shaking.MWT and TWL were tested at 2 h,day 1 to day 12 after injection of CFA(n=10).Results:(1)NALCN mRNA was decreased in DRG with 20.97%±15.95%(n=8,P=0.022)and in spinal dorsal horn with 27.46%± 11.99%(n=6,P=0.008)at 3 days after injection of siRNA-NALCN;(2)The rats received injection of siRNA-NALCN did not display any difference in MWT(n=10,P=0.707 for contralateral side;P=0.856 for ipsilateral side)and TWL(n=10,P=0.999 for contralateral side;P=0.833 for ipsilateral side)between the both sides;(3)Spontaneous pain within the first 30 min after CFA injection(n=10,P<0.05),thermal hyperalgesia(n=10,P<0.05),and mechanical allodynia(n=10,P<0.05)of CFA-ipsilateral side were alleviated by pre-injection of siRNA-NALCN.The therapeutic effects of siRNA-NALCN lasted until day 3 after CFA injection.Chapter 2 Elevated function of NALCN in DRG and spinal dorsal horn enhanced neuronal excitabilityExperiment 1 Function of NALCN in DRG and spinal dorsal horn elevated Materials and methods(1)The adult SD rats were randomly divided into siRNA-control group(n=10)and siRNA-NALCN group(n=10).siRNA-control or siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space.3 days later,CFA was injected into the left footpad.We performed whole-cell patch-clamp recordings on acutely isolated small and medium neurons of DRG at 8 h after CFA injection.The Na+-mediated holding current(Iholding)was recorded at a holding potential of-60 mV by replacing normal extracellular Na+ with NMDG and Gadolinium(Gd3+non-selective blocker of NALCN);(2)The PD7 rats were subcutaneously injected with 10 μL CFA into the left footpad.To determine the change of NALCN mRNA expression after CFA,lumbar 4-6 DRG and the lumbar enlargement of spinal cord of both CFA-ipsilateral and CFAcontralateral side were extracted for RT-qPCR at 8 h and day 3 after CFA injection,respectively;(3)The PD7 rats were randomly divided into siRNA-control group(n=10)and siRNA-NALCN group(n=10).siRNA-control or siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space.3 days later,L4-L6 DRG and the lumbar enlargement of spinal cord of both CFA-ipsilateral and CFA-contralateral side were extracted for RT-qPCR to detected the expression level of NALCN mRNA in siRNAcontrol and siRNA-NALCN group;(4)The PD7 rats were randomly divided into siRNA-control group(n=10)and siRNA-NALCN group(n=10).siRNA-control or siRNA-NALCN were injected into the left sciatic nerve and the subarachnoid space.3 days later,CFA was injected into the left footpad.We performed whole-cell patch-clamp recordings on acutely isolated neurons with small and medium diameters of DRG at 8 h after CFA injection and acutely isolated spinal cord slices at day 1 after CFA injection.The Na+-mediated holding current(Iholding)was recorded at a holding potential of-60 mV by replacing normal extracellular Na+with NMDG and Gadolinium(Gd3+,non-selective blocker of NALCN).Results:(1)For adult rats that received siRNA-control,the change in Iholding was larger in the neurons of DRG from CFA-ipsilateral side compared with the CFA-contralateral side(n=9,P=0.001).siRNA-NALCN treatment diminished the difference of Na+-mediated holding currents in small and medium DRG neurons between the CFA-ipsilateral and CFA-contralateral sides(n=9,P=0.865).The change of Gd3+-mediated inhibition of Iholding was greater in small and medium DRG neurons of the CFA-ipsilateral side compared with the CFA-contralateral side in the rats that received siRNA-control(n=9,P<0.001).Treatment with siRNA-NALCN did not produce any difference in Gd3+inhibited Iholding between the CFA-ipsilateral and CFA-contralateral sides in small and medium DRG neurons(n=9,P=0.984);(2)Compared with CFA-contralateral side,the level of NALCN mRNA was higher from the CFA-ipsilateral side at 8 h in DRG of neonatal rats after injection of CFA(n=9,P=0.040).The level of NALCN mRNA was higher in spinal dorsal cord of neonatal rats of the CFA-ipsilateral side compared to the CFA-contralateral side on day 1 after injection of CFA(n=9,P=0.003);(3)The level of NALCN mRNA was decreased with 33.42%±16.35%in DRG at day 3 after injection of siRNA-NALCN(n=12,P<0.001).Injection of siRNA-NALCN led to a decreased level of NALCN mRNA with 47.93%±18.53%at day 3 in spinal dorsal cord of neonatal rats(n=11,P<0.001);(4)For the PD7 rats that received siRNA-control,the change in Iholding was larger in the neurons of DRG from CFA-ipsilateral side compared with the CFA-contralateral side(n=9,P<0.001).siRNA-NALCN treatment diminished the difference of Na+mediated holding currents between the CFA-ipsilateral and the CFA-contralateral side in DRG neurons(n=9,P=0.997).The change of Gd3+-mediated inhibition of Iholding was greater in small and medium DRG neurons of the CFA-ipsilateral side compared with the CFA-contralateral side in the rats that received control-siRNA(n=9,P<0.001).Treatment with siRNA-NALCN did not produce any difference in Gd3+-inhibited Iholding between the CFA-ipsilateral and CFA-contralateral sides in small and medium DRG neurons(n=9-8,P=0.952).With control-siRNA,the change of Na+-mediated Iholding was larger in spinal dorsal cord neurons(n=9,P<0.001)of CFA-ipsilateral side compared with the CFA-contralateral side.siRNA-NALCN treatment diminished the difference of Na+-mediated holding currents between the CFA-ipsilateral and CFAcontralateral side in spinal dorsal horn neurons(n=9,P=0.957).The Gd3+-mediated inhibition of the Iholding was greater in spinal dorsal cord neurons on the CFA-ipsilateral side for rats treated with siRNA-control(n=9,P<0.001).For the rats treated with siRNA-NALCN,we did not find any difference in Gd3+-inhibited Iholding between CFAipsilateral and CFA-contralateral side in spinal dorsal cord neurons(n=9,P=0.919).Experiment 2 Increased expression and elevated function of NALCN in DRG and spinal dorsal horn enhanced neuronal excitabilityMaterials and methods:(1)The adult rats were randomly divided into siRNA-control group(n=10)and siRNA-NALCN group(n=10),siRNA-control or siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space.3 days later,CFA was injected into the left footpad.We performed whole-cell patch-clamp recordings on acutely isolated small and medium neurons of DRG 8 h after CFA injection to compare neuronal excitability between the CFA-ipsilateral and CFA-contralateral side,including RMP,rheobase,and AP;(2)PD7 SD rats were randomly divided into siRNA-control group(n=10)and siRNA-NALCN group(n=10).siRNA-control or siRNA-NALCN was injected into the left sciatic nerve and subarachnoid space 3 days later,CFA was injected into the left footpad.We performed whole-cell patch-clamp recordings on acutely isolated small and medium neurons of DRG at 8 h after CFA injection and acutely isolated spinal cord slices at day 1 after CFA inj ection to compare neuronal excitability between the CFA-ipsilateral and CFA-contralateral side,including RMP,rheobase,and AP.Results:(1)For adult rats with siRNA-control treatment,small and medium neurons in DRG from the CFA-ipsilateral side were hyperactive,as evidenced by depolarized RMP(n=15,P=0.001)and decreased rheobase(n=15,P<0.001).With siRNA-NALCNtreatment,the RMP(n=14-15,P=0.417)and rheobase(n=14-15,P=0.940)did not differ in small and medium DRG neurons between the CFA-ipsilateral side and the CFA-contralateral side;(2)For neonatal rats with siRNA-control treatment,small and medium neurons in DRG from the CFA-ipsilateral side were hyperactive,as evidenced by depolarized RMP(n=12,P<0.001;n=12-14,P=0.002)and decreased rheobase(n=12,P=0.015;n=12-14,P<0.001).With siRNA-NALCN treatment,no difference was found in the RMP and rheobase between CFA-ipsilateral and CFA-contralateral sides for small(n=14,P=0.184;P=0.171,respectively)or medium(n=13,P=0.538;P=0.912,respectively)DRG neurons.In the spinal dorsal horn,the neurons from the CFAipsilateral side were hyperactive in rats treated with siRNA-control(depolarized RMP,n=11,P<0.001;decreased rheobase,n=11,P=0.002).However,the RMP(n=10-11,P=0.654)and rheobase(n=10-11,P=0.902)did not differ between CFA-ipsilateral and CFA-contralateral sides in siRNA-NALCN-treated rats.Conclusion:(1)Expression levels of mRNA and protein for NALCN channels in L4-L6 DRG and the lumbar enlargement of spinal cord were increased in CFA-induced inflammatory pain in rats;(2)Knockdown the expression of NALCN in L4-L6 DRG and the lumbar enlargement of spinal cord significantly alleviated CFA-induced inflammatory pain in rats;(3)Function of NALCN channels in small and medium DRG neurons(L4-L6)and in spinal dorsal horn neurons(Lamina Ⅰ-Ⅱ)elevated in CFA-induced inflammatory pain in rats;(4)Increased expression and elevated function in small and medium DRG neurons(L4-L6)and lumbar enlargement neurons of spinal cord(Lamina Ⅰ-Ⅱ)enhanced neuronal excitability within DRG and dorsal spinal horn neurons.In conclusion,our results indicated that increased expression and elevated function in small and medium DRG neurons(L4-L6)and neurons of spinal cord(Lamina I-II)contribute to the development of CFA-induced inflammatory pain.NALCN may be a novel therapeutic target for inflammatory pain. |
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