| Zinc,an essential trace element for mammals,can take important roles in the course of development,reproduction,immune,endocrine and nervous activities.In the nervous system,about 15%of zinc ions are free,and accumulate in the presynaptic vesicles.Free zinc ions can be released together with neurotransmitters into the synaptic cleft,and then react with postsynaptic neurons to implement their neuroregulation.In the recent years,the studies on the zinc transporters help people to reveal the biological functions of zinc.Zinc transporter 7(ZNT7) is an important part and its function is to transporting zinc ions into Golgi apparatus from cytoplasm to maintain the zinc homeostasis in cells.Neuropathic pain(NPP) is a chronic disease,induced by nervous system injury and clinically characterized by hyperpathia,allodynia,partly sensory loss and spontaneous pain.During the past few years,following the ageing of population,the attack rate of NPP increased year by year.However,for the defect of effective therapeutic tools,it has become one of the diseases which harm mankind body health.Foundational studies indicate that zinc ions can effect the expression of NMDA and un-NMDA receptor and their functions with an important role in the conduction of nociceptive stimulus.Additionally,the great quantity of metallothionein-Ⅲ,zinc ions and zinc containing primary afferent neurons in the spinal cord and dorsal root ganglion (DRG) reveals that zinc ions may take part in the conduction of pain.Animal experiments show that the thresholds of pain in NPP model mice decreased after accepting dorsal rhizotomy,accompanying with the decrease of zinc ions in spinal and then reveal the role of zinc in the neuropathic pain.In this study,we examined the distribution of zinc and zinc transporters in the normal spinal cord and DRG,using the autometallography technology(AMG), immunohistochemistry and immunofluorescence methods.Additionally,the effect of zinc pretreatment on the pain praxiology changes,the distribution of zinc, neuropeptides,apoptosis and horizontal cell in spinal cord and DRG were observed using spared nerve injury(SNI) modle.Then the metabolism of zinc in spinal cord and DRG and the role and mechanism of zinc in the development and conduction of NPP were studied.MethodsNormal CD-1 mice and SNI model were used for the present study.The distribution of free zinc ions and ZNT7 in normal spinal cord and DRG were detected by AMG,immunohistochemistry and Western Blot.The distribution patterns of ZNT7 in these organs and the positional relation between ZNT7 and TGN38,a maker of Golgi apparatus,were detected by double immunofluorescence and confocal laser scanning microscopy.The SNI models were used and evaluated by observing the changes of independent behaviour of mice and praxiology detecting technique.Then the effect of high or low-zinc pretreatment on the distribution of zinc in spinal cord and DRG of SNI mice was detected by AMG and TSQ(N-(6-methoxy-8-quinolyl)-para -toluenesulfonamide) staining technique and the effect on the praxiology changes were observed too.Additionally,The effect of high or low-zinc pretreatment on the expression of neuropeptides,calcitonin gene related protein(CGRP),neuropeptide Y (NPY),apoptosis factors and horizontal cell in spinal cord and DRG of SNI mice were analyzed by immunohistochemistry,westein blot and immunofluorescence.Results1.Distribution of zinc ions in CD-1 mice spinal cord and DRGAMG staining results showed that AMG-positive granules were widely distributed in the axon terminals of neurons and the areas nearing nuclear of DRG neurons.2.Abundant expression of ZnT7 in the spinal cord and DRGImmunohistochemimistry results revealed that ZNT7 immunopositive products were brown.ZNT7-positive structures were round or irregular and distributed in the perinuclear regions of neurons throughout the spinal cord and DRG.Double-immunofluorescence staining for ZNT7 and TGN38 showed that the ZNT7-positive structures were widely distributed in the spinal cord and DRG neurons, being co-expressed with TGN38 in the perinuclear areas.3.Expression of ZnT7 in the spinal cord of Western blot detectionWestern blot results showed that there were abundant expressions of ZNT7 in spinal cord in normal CD-1 mice.4.Evaluation of SNI modelPraxiological observation revealed that the sham operated mice(SHAM group) were recovered soon.Whereas the SNI model mice begun to limp,daring of weight bearing,discarding foot and licking foot.Mechanical pain threshold detection revealed that,comparing with preoperative pain threshold,the postoperative pain threshold of operated side in SHAM mice were unchanged(P＞0.05),while that of SNI mice were decreased significantly(P＜0.05). Additionally,the postoperative pain threshold of SNI were decreased significantly than that of SHAM mice(P＜0.05),revealing the success of model establishment.5.Effect of low or high-zinc pretreatment on the distribution of zinc in spinal cord and DGR.AMG and TSQ staining revealed that,comparing with SHAM group,the distribution of zinc in operated spinal dorsal horn superficial layer and DRG of normal SNI group(N-SNI) and low-zinc pretreatment SNI group(L-SNI) were decreased significantly(P＜0.05).The distribution of zinc in L-SNI group were decreased than that in N-SNI group(P＜0.05).Additionally,the distribution in high-zinc pretreatment SNI group(H-SNI) was significantly increased than that of SHAM group(P＜0.05).6.Effect of zinc on the praxiology changes of SNI mice.Independent behaviour observation revealed that the SHAM group were recovered soon,whereas the SNI group,including N-SNI,L-SNI and H-SNI group,begun to limp, daring of weight bearing,discarding foot and licking foot.Additionally,the independent behaviour changes in L-SNI were the most significant and the H-SNI group mice are relieved than N-SNI and L-SNI groups.Mechanical pain threshold detection revealed that,comparing with preoperative pain threshold,the postoperative pain threshold of operated side in three SNI groups were all decreased(P＜0.05).Comparing with SHAM group,the mechanical pain threshold in three SNI groups were all decreased(P＜0.05).Among the total,the L-SNI group was lower than N-SNI(P＜0.05) and the H-SNI group was higher than N-SNI(P＜0.05).7.Effect of zinc ions on the expression of CGRP and NPY in spinal dorsal horn and DRG of SNI modelImmunohistochemistry revealed that CGRP and NPY positive granules were located in the spinal dorsal horn superficial layer and the DRG neuron of operated side. Comparing with SHAM group,the expression of three SNI groups were all increased (P＜0.05).Among the total,the L-SNI group was higher than N-SNI(P＜0.05) and the H-SNI group was lower than N-SNI(P＜0.05).8.Effect of zinc ions on the apoptotisis of spinal dorsal horn of SNI modelImmunofluorescence detection revealed that caspases-3,Fas and FasL positive cells were mainly localized in the superficial layer of spinal dorsal horn of operated side.Both immunofluorescence and westein blot detection revealed that,comparing with SHAM group,the expression of caspases 3,Fas and FasL in three SNI groups were all increased(P＜0.01).Among the total,the L-SNI group was higher than N-SNI (P＜0.01) and the H-SNI group was lower than N-SNI(P＜0.01).9.Effect of zinc ions on the expression of GFAP in spinal dorsal horn of SNI model miceImmunofluorescence detection revealed that GFAP positive cells were located in the spinal dorsal horn of operated side.Comparing with SHAM mice,the number of GFAP positive cells in three SNI groups were all increased(P＜0.05).Among the total, the L-SNI group was higher than N-SNI(P＜0.05) and the H-SNI group was lower than N-SNI(P＜0.05).Conclusion1.Zinc ions and ZNT7 were enriched in the spinal cord and DRG neurons,with the co-localization of perineclear area with TGN38.2.Low-zinc pretreatment can reduce the distribution of zinc in the superficial layer of spinal dorsal horn and DGR neurons in SNI group and then mechanical pain threshold decreased,while the high-zinc pretreatment functions oppositely.3.Low-zinc pretreatment can increase the expression of CGRP,N-PY,apoptosis factors and horizontal cells in spinal dorsal horn,as well as that of CGRP and NPY in DRG of SNI mice,while high-zinc pretreatment functions oppositively.
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