Astrocyte MeCP2 Regulates Kir4.1 In Neuropathic Pain

Objective:Neuropathic pain is widely recognized as one of the most difficult pain syndromes to treat,and has become an important factor in the global burden of disease.The unclear pathogenesis is an important factor limiting its treatment.Therefore,it is an important scientific problem to expound the mechanism of neuropathic pain and to find effective drugs for clinical treatment.Astrocyte activation in spinal dorsal horn plays an important role in chronic pain.Inward rectifying potassium channel protein 4.1(Kir4.1)is an important K+channel in astrocytes,which mediates many functions of astrocytes.Methyl-CpG-binding protein 2(MeCP2)regulates gene activity by binding methylated and unmethylated DNA,mediating multiple functions of neurons and astrocytes.In recent years,studies have found that the expression and function of Kir4.1 and MeCP2 are changed in neuropathic pain.Moreover,the expression of Kir4.1 decreased in MeCP2 deficient mice.Therefore,we will combine pain behavior,molecular biology,transgenic animals,gene knockout technology,gene overexpression technology,electrophysiological patch clamp recording to confirm:(1)the expression changes of Kir4.1 in spinal cord of chronic constriction injury(CCI)mice,and the therapeutic effects of Kir4.1 overexpression for neuropathic pain;(2)the expression changes of Kir4.1 in spinal dorsal horn is an important mechanism of neuropathic pain;(3)the expression changes of MeCP2 in spinal cord of CCI mice,and whether MeCP2 in astrocytes or MeCP2 in neurons regulates Kir4.1.This study may provide a new theory for the mechanism and treatment of neuropathic pain.Chapter 1 The expression of Kir4.1 in spinal dorsal horn of mice with chronic constriction injury and its therapeutic target1.1 The expression changes of Kir4.1 in spinal cord of mice with chronic constriction injuryMethods:(1)After measuring the basic mechanical pain threshold and thermal pain threshold,adult C57BL/6 mice were performed with CCI on the left hind limb(CCIipsilateral,CCI-ipsi)and the right hind limb as the control side(CCI-contralateral,CCI-contra).The mechanical pain threshold and thermal pain threshold were measured on the day 1,day 3,day 7 and day 14 after CCI.(2)On the 1st,3rd,7th and 14th day after CCI,the lumbar spinal cords of mice were taken for quantitative real time polymerase chain reaction(qRT-PCR),Western blot(WB)and immunofluorescence staining to compare the expression of Kir4.1 between CCI-contra and CCI-ipsi.Results:(1)Compared with the CCI-contra side,the CCI-ipsi hind limb developed mechanical allodynia and thermal hyperalgesia beginning on the first day after CCI(P<0.0001,n=20).(2)The results of qRT-PCR and WB showed that the expression of Kir4.1 mRNA and protein in CCI-ipsi side of lumbar spinal cords were significantly decreased on the 7th and 14th day after CCI,compared with the CCI-contra side(P<0.05,n=10).(3)Immunofluorescence staining showed that Kir4.1 was widely expressed in astrocytes(n=4).1.2 The therapeutic effects of Kir4.1 overexpression in spinal cord for neuropathic painMethods:(1)The distribution of Kir4.1 overexpression virus in the spinal cord was detected by immunofluorescence staining at 4 weeks after intrathecal injection.(2)After measuring the basic mechanical pain threshold and thermal pain threshold,adult C57BL/6 mice were performed with CCI on the left hind limb(CCI-ipsi)and the right hind limb as the control side(CCI-contra).The mechanical pain threshold and thermal pain threshold were measured daily after CCI for 7 days.On day 7 after CCI,Kir4.1 overexpressed virus or control virus was injected intrathecally,and the changes of mechanical and thermal pain thresholds were measured weekly for 4 weeks.(3)The overexpression rate of Kir4.1 mRNA in the spinal cord was detected by qRTPCR at 4 weeks after intrathecal injection of Kir4.1 overexpression virus or control virus.Results:(1)Immunofluorescence staining showed that Kir4.1 overexpression virus was specifically expressed in astrocytes(n=4).(2)The mechanical pain threshold and thermal pain threshold of CCI mice returned to its basic level in the 3rd and 4th week after intrathecal injection of Kir4.1 overexpressing virus(P=0.9999,n=10).(3)After intrathecal injection of Kir4.1 overexpression virus in CCI mice,the overexpression rate of Kir4.1 mRNA in spinal cord was 20.3%±14.9%,compared with that of control mice(P=0.0085,n=8).Chapter 2 The effects of Kir4.1 knockdown in spinal cord on pain behavior and electrophysiological functions of astrocytes and neurons2.1 The effects of Kir4.1 knockdown on pain behavior in miceMethods:(1)The distribution of Kir4.1 knockout virus in the spinal cord was detected by immunofluorescence staining at 4 weeks after intrathecal injection.(2)Kir4.1 cKO mice and control mice were cultured and their mechanical and thermal pain thresholds were measured.(3)After measuring the basic mechanical and thermal pain thresholds of Kir4.1 floxed/floxed mice,Kir4.1 knockout virus or control virus was intrathecally injected.Then the mechanical pain threshold and thermal pain threshold were measured every week for 6 weeks.(4)qRT-PCR was used to measure the knockout rate of Kir4.1 mRNA in the spinal cord of Kir4.1 cKO mice and Kir4.1 floxed/floxed mice intrathecally injected with Kir4.1 knockout virus.Results:(1)Immunofluorescence staining showed that Kir4.1 knockout virus was specifically expressed in astrocytes(n=4).(2)The mechanical and thermal pain thresholds of Kir4.1 cKO mice were significantly lower than those of control mice(P<0.0001,n=10).(3)From the third week after intrathecal injection,the mechanical and thermal pain thresholds of Kir4.1 knockout virus group were significantly lower than those of control virus group,and lasted until the sixth week(P<0.0001,n=10).(4)Compared with the control mice,the expression of Kir4.1 mRNA decreased 39.0%±16.1%in spinal cord of Kir4.1 cKO mice(P=0.0043,n=6).(5)Compared with the control virus group,the expression of Kir4.1 mRNA decreased 24.0%±12.6%in spinal cord of Kir4.1 knockout virus group(P=0.0167,n=9).2.2 The effects of Kir4.1 knockdown on electrophysiological functions of spinal astrocytes and neuronsMethods:C57BL/6 mice on postnatal day 7(P7)were intrathecally injected with Kir4.1siRNA or control-siRNA.After 3～5 days(P10～P12),the following operations were performed.(1)The knockout rate of Kir4.1 in spinal cord was detected by qRT-PCR.(2)The distribution of Kir4.1 in spinal cord were detected by immunofluorescence staining.(3)The above mice were selected for electrophysiological patch clamp recording.Under isoflurane anesthesia,the lumbar enlargement of spinal cord was taken out,and the transverse spinal cord slices(300 μm)were cut.The changes of resting membrane potential(RMP),membrane input resistance(Rinput),action potential(AP)and firing pattern of neurons,the changes of RMP,holding current and conductance of astrocytes were recorded.The current voltage relationship(I-V relationship)was also drawn.Results:(1)The expression of Kir4.1 mRNA in spinal cord of Kir4.1-siRNA group was decreased 51.6%±20.2%than that of control-siRNA group(P=0.0023,n=5).(2)According electrophysiological patch clamp recording,we found that compared with the control-siRNA group,the I-V relationship of Ba2+-sensitive(P<0.0001),the holding current(P=0.0072)and conductance(P=0.0059)of spinal astrocytes in Kir4.1-siRNA group were significantly decreased,while RMP was increased(P=0.0484).(3)By electrophysiological patch clamp recording,we found that compared with the control-siRNA group,Kir4.1-siRNA increased RMP of spinal neurons(P=0.0187),the AP at I=0 pA(P=0.03 52)and the spinal neurons are more likely to sustain tonic firing(P=0.001).Chapter 3 The role of MeCP2 in spinal cord regulating Kir4.1 for neuropathic pain3.1 The expression changes of Kir4.1 in spinal cord of mice with chronic constriction injuryMethods:(1)After measuring the basic mechanical pain threshold and thermal pain threshold,adult C57BL/6 mice were performed with CCI on the left hind limb(CCI-ipsi)and the right hind limb as the control side(CCI-contra).The mechanical pain threshold and thermal pain threshold of bilateral hind limbs were continuously measured on the 1st,3rd,7th and 14th day after CCI.(2)On the 1st,3rd,7th and 14th day after CCI,the lumbar spinal cords of mice were taken for qRT-PCR,WB and immunofluorescence staining to compare the expression of MeCP2 between CCI-ipsi and CCI-contra.Results:(1)Compared with the CCI-contra side,the CCI-ipsi side developed mechanical allodynia and thermal hyperalgesia beginning on the first day after CCI(P<0.0001,n=12).(2)The results of qRT-PCR and WB showed that the expression of MeCP2 mRNA and protein in CCI-ipsi of lumbar spinal cords were significantly decreased on the day 3,day 7 and day 14 after CCI(P<0.05,n=10).(3)MeCP2 was widely expressed in both neurons and astrocytes of spinal cord by immunofluorescence staining(n=4).3.2 The changes of pain behavior and Kir4.1 after MeCP2 knockdown in spinal cordMethods:(1)The distribution of virus in the spinal cord was detected by immunofluorescence staining at 4 weeks after intrathecal injection.(2)After measuring the basic mechanical pain threshold and thermal pain threshold,adult C57BL/6 mice were performed intrathecal injection of MeCP2 knockout virus/control virus in neurons or MeCP2 knockout virus/control virus in astrocyte.Then the mechanical pain threshold and thermal pain threshold were measured weekly for 5 weeks.(3)The knockout rates of MeCP2 mRNA in spinal cord were measured by qRT-PCR,after intrathecal injection virus.(4)The expression of Kir4.1 mRNA was measured by qRT-PCR after after intrathecal injection virus.(5)qRT-PCR was used to detect the expression changes of MeCP2 mRNA in spinal cord:Kir4.1 cKO mice compared with control mice,and Kir4.1 knockout virus group compared with control virus group.Results:(1)By immunofluorescence staining,we found that MeCP2 knockout virus in neurons was specifically expressed in neurons,while MeCP2 knockout virus in astrocytes was specifically expressed in astrocytes(n=4).(2)The mechanical pain threshold and thermal pain threshold of the MeCP2 knockout virus in neurons group were not significantly different from those of control virus group(P>0.05,n=10).However,the mechanical and thermal pain thresholds of the MeCP2 knockout virus in astrocytes group were significantly lower than those of control virus group at 4th and 5th week(P<0.0001,n=10).(3)According to the results of qRT-PCR,the knockout rates of MeCP2 mRNA in spinal were 30.0%±9.1%(P<0.0001,n=10)and 17.3%±9.6%(P=0.0033,n=10),after injected MeCP2 knockout virus in neurons/astrocytes.(4)Compared with the control virus group,there was no significant difference in the expression of Kir4.1 mRNA after knockout MeCP2 in spinal neurons(P=0.4967,n=10).But,after knockout MeCP2 in spinal astrocytes,the expression of Kir4.1 mRNA in spinal cord increased 23.7%±12.2%compared with that in control virus group(P=0.0011,n=10).(5)According to the results of qRT-PCR,there was no significant difference in the expression of MeCP2 mRNA in spinal cord between Kir4.1 cKO mice and control mice(P=0.5552,n=6),either in Kir4.1 knockout virus group and control virus group(P=0.6469,n=7).3.3 The prevention of neuropathic pain and regulation of Kir4.1 by overexpression of MeCP2 in astrocytesMethods:(1)The distribution of MeCP2 overexpression virus in astrocyte in the spinal cord was detected by immunofluorescence staining at 4 weeks after intrathecal injection.(2)After measuring the basic mechanical pain threshold and thermal pain threshold,adult C57BL/6 mice were intrathecally injected with MeCP2 overexpression virus/control virus in astrocyte.After 4 weeks of virus injection,mice were given 2%doxycycline and 1%sucrose to induce virus for 7 days.Then the mice were performed with CCI on the left hind limb(CCI-ipsi)and the right hind limb as the control side(CCI-contra).The mechanical pain threshold and thermal pain threshold of bilateral hind limbs were continuously measured on the 1st,3rd,7th and 14th day after CCI.(3)The expression of MeCP2 mRNA and Kir4.1 mRNA in spinal cord was measured by qRT-PCR.(4)qRT-PCR was used to measure the changes of MeCP2 mRNA in spinal cord of CCI mice after intrathecal injection of Kir4.1 overexpression virus.Results:(1)By immunofluorescence staining,we found that MeCP2 overexpression virus in astrocytes was specifically expressed in astrocytes(n=4).(2)After intrathecal injected MeCP2 overexpression virus in astrocyte into CCI mice,the mechanical pain threshold and thermal pain threshold of mice returned to the basic level before CCI(P>0.9999,n=8).(3)According to the results of qRT-PCR,the overexpression rate of MeCP2 mRNA in spinal cord was 22.6%±11.4%(P=0.0066,n=6),the expression of Kir4.1 mRNA increased 46.4%±14.4%compared with that in control group(P=0.0057,n=6).(4)There was no significant difference in the expression of MeCP2 mRNA in spinal cord between Kir4.1 overexpression virus group and the control virus group(P=0.3544,n=8).Conclusions:(1)The decrease of Kir4.1 in spinal dorsal horn mediate the increase of neuronal excitability and the changes of firing pattern,which is the key mechanism of neuropathic pain.(2)Kir4.1 is a potential target for the treatment of neuropathic pain.(3)Spinal astrocyte MeCP2 regulates Kir4.1,which mediates the occurrence and development of neuropathic pain.