锌与锌转运体在脊髓和DGR的分布以及锌在NPP中的作用和机制
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摘要
前言
锌是哺乳动物体内重要的微量元素之一,在机体的生长发育、生殖遗传、免疫、内分泌、神经活动等重要生理过程中发挥重要的作用。在中枢神经系统中,大约15%的锌离子处于游离状态,这些锌离子主要位于含锌神经元(zinc-enrichedneuron,ZEN)轴突末端的突触小泡内。当神经活动时,游离锌离子往往随着一些神经递质的释放而进入突触间隙并与突触后膜的某些受体结合或者穿越突触后膜进入到突触后神经元,从而发挥其神经调节作用。近年来人们对锌转运体(zinctransporters,ZNTs)的研究进一步揭示了锌的生物学功能,ZNT7就是其中一个重要组成成员,它的功能是将锌转运到高尔基体内,从而维持细胞内的锌稳态。
神经病理性疼痛(neuropathic pain,NPP)是一种由神经系统损伤而引起的慢性疾病,临床上以痛觉过敏、触诱发痛(或称痛觉超敏)、局部感觉缺失以及自发性疼痛等为特点。近年来,随着人口的老龄化,NPP的发病率呈逐年上升趋势,但是由于缺乏有效的治疗手段,已经成为危害人类健康的主要疾病之一。
基础研究表明,锌能影响NMDA和非NMDA受体的表达与功能,后者在伤害性刺激传导中起重要作用;金属硫蛋白-Ⅲ和游离锌离子以及含锌初级传入神经元在脊髓和背根神经节(dorsal root ganglion,DRG)的大量分布也提示锌可能参与痛觉的传导。动物实验显示,脊神经背根切断术后,小鼠痛阈明显降低,脊髓背侧角浅层灰质的锌离子明显减少,进一步证实锌可能参与痛觉的产生和传导。
因此,本研究利用金属自显影技术(autometallography,AMG)、免疫组织化学、免疫荧光双标记与共聚焦激光扫描显微镜技术、Westein blot技术研究锌及ZNTs在小鼠脊髓和DRG的分布。在此基础上,利用坐骨神经分支选择损伤模型(spared nerve injury,SNI)观察低锌和高锌预处理对SNI模型动物行为学变化以及脊髓和DRG中锌离子、神经肽、神经元凋亡和星形胶质细胞的影响,从而为探讨脊髓和DRG中的锌代谢以及锌在NPP产生与传导中的作用和机制研究提供重要的实验依据。
实验方法
应用AMG技术、免疫组织化学技术、Western Blot技术检测锌和ZNT7在CD-1小鼠脊髓和DRG的分布,应用免疫荧光双标记和共聚焦激光扫描显微镜技术检测ZNT7与TGN38在小鼠脊髓和DRG的定位分布及其位置关系。建立SNI动物模型,利用观察动物自主行为变化与行为学检测技术对模型进行鉴定。在此基础上,分别给予低锌或高锌预处理,观察锌预处理对SNI模型动物行为学的影响。同时,利用AMG技术、N-(6-甲基-8-喹啉)-甲苯磺酰胺荧光染色技术(N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide,TSQ)检测锌在各组模型动物脊髓和DRG的分布变化;利用免疫组织化学技术、Western Blot技术和免疫荧光技术检测锌预处理对SNI模型动物脊髓和DRG中的降钙素基因相关肽(calcitoningene related protein,CGRP)、神经肽Y(neuropeptide Y,NPY)、细胞凋亡因子和星形胶质细胞表达的影响。
实验结果
1、锌离子在CD-1小鼠脊髓和DRG的分布
AMG阳性反应产物为棕黑色颗粒,主要分布在小鼠脊髓的神经元轴突终末和DRG神经元细胞核的周围,呈斑块状或颗粒状围绕细胞核。
2、ZNT7在CD-1小鼠脊髓和DRG的分布
免疫组织化学结果显示:ZNT7免疫阳性反应产物呈棕黄色,在脊髓灰质的神经元以及DRG神经元的细胞核周围均可见ZNT7阳性产物分布,呈斑块状或颗粒状围绕在细胞核周围高尔基体的分布区域。
免疫荧光双标记和共聚焦激光扫描结果显示:在小鼠脊髓灰质和DRG的神经元细胞核周围均有ZNT7阳性产物的表达,其与TGN38的分布相同,均呈斑块状或颗粒状共分布于神经元细胞核周围。
3、ZNT7在CD-1小鼠脊髓内表达的Western Blot分析
应用Western Blot技术对小鼠脊髓内ZNT7在蛋白水平的表达进行检测。结果显示,小鼠的脊髓内有ZNT7的丰富表达。
4、SNI模型的鉴定
观察动物自主行为发现:假手术组(SHAM)术后第2天步态基本恢复正常,无甩足和舔足等自发痛行为;而SNI模型动物在术后第1天就开始跛行,术侧后足不敢承重,出现甩足和舔足等自发痛行为,一直持续到观察期末。
机械性痛阈检测表明:与术前基础痛阈值相比,SHAM组术后痛阈无明显下降,差异无显著性意义(P＞0.05);SNI组术后痛阈明显下降,差异有显著性意义(P＜0.05)。与SHAM组相比,SNI组术后机械痛阈显著降低,差异有显著性意义(P＜0.05),表明模型制备成功。
5、低锌或高锌预处理对SNI模型动物脊髓背侧角和DRG中锌离子的影响
AMG和TSQ检测结果显示:与SHAM组相比,正常动物模型组(N-SNI)与低锌预处理组(L-SNI)的术侧脊髓背侧角浅层和DRG中的锌离子均显著减少(P＜0.05),其中L-SNI组比N-SNI组减少更为显著(P＜0.05);而高锌预处理组(H-SNI)术侧脊髓背侧角浅层和DRG中的锌离子均显著增多(P＜0.05)。
6、锌对SNI模型动物行为学的影响
观察动物自主行为发现:SHAM组术后第2天步态基本恢复正常,无舔足和甩足等自发痛行为;而N-SNI、L-SNI和H-SNI组在术后第1天就开始跛行,术侧后足不敢承重,出现甩足、舔足等自发痛行为,一直持续到观察期末。此外,L-SNI组小鼠的自主行为最为显著,而H-SNI组的自主行为相对较轻。
机械性痛阈检测表明:与术前基础痛阈值相比,N-SNI、L-SNI和H-SNI组术后痛阈均明显下降,差异有显著性意义(P＜0.05);与SHAM组相比,N-SNI、L-SNI和H-SNI组术后机械性痛阈均降低,差异有显著性意义(P＜0.05)。其中,L-SNI组痛阈低于N-SNI组(P＜0.05),而H-SNI组痛阈高于N-SNI组(P＜0.05)。
7、锌对SNI模型脊髓背侧角和DRG中CGRP和NPY的影响
免疫组织化学结果显示:CGRP和NPY阳性产物为棕褐色细颗粒状。主要分布在术侧的脊髓背侧角浅层和DRG神经元细胞质中。与SHAM组相比,N-SNI、L-SNI和H-SNI组的阳性表达均显著增高(P＜0.05)。其中,L-SNI组的阳性表达高于N-SNI组(P＜0.05),而H-SNI组的阳性表达低于N-SNI组(P＜0.05)。
8、锌对SNI模型脊髓背侧角神经元凋亡的影响
免疫组织化学检测结果表明:Caspases-3、Fas和FasL阳性产物为棕黄色颗粒状,主要分布在脊髓背侧角浅层的神经元中。与SHAM组相比,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角Caspases-3、Fas和FasL阳性表达均增高(P＜0.01)。L-SNI组的表达高于N-SNI组(P＜0.01);H-SNI组的表达低于N-SNI组(P＜0.01)。
Westein blot检测结果表明:与SHAM组相比,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角Caspases-3、Fas、FasL的阳性表达均显著增高(P＜0.01)。与N-SNI组相比,L-SNI组的阳性表达增多(P＜0.01);H-SNI组的阳性表达减少(P＜0.01)。
9、锌对SNI模型脊髓背侧角GFAP表达的影响
免疫荧光染色结果显示,星形胶质酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞呈绿色荧光,细胞多突起,胞体较小。SHAM组术侧脊髓背侧角只有少量GFAP阳性细胞。与SHAM组相比较,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角的GFAP阳性细胞均增多(P＜0.05)。与N-SNI组相比,L-SNI组的阳性细胞增多(P＜0.05),H-SNI组阳性细胞减少(P＜0.05)。
结论
1、正常小鼠脊髓和DRG中富含游离锌离子和ZNT7。其中,ZNT7与高尔基复合体标记物TGN38的表达存在共区域性。
2、低锌预处理能使SNI模型脊髓背侧角浅层锌离子更加减少,加重自发性痛等行为学变化,机械性痛阈值降低更加显著;而高锌预处理能使脊髓背侧角锌离子增多,改善自发性痛等行为学变化,提高机械性痛阈值。
4、低锌预处理能使SNI模型脊髓背侧角CGRP、NPY、凋亡因子和星形胶质细胞的表达增加,使DRG中的CGRP和NPY表达增加;而高锌预处理能使表达降低。
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.
Methods
Normal 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.
Results
1.Distribution of zinc ions in CD-1 mice spinal cord and DRG
AMG 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 DRG
Immunohistochemimistry 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 detection
Western blot results showed that there were abundant expressions of ZNT7 in spinal cord in normal CD-1 mice.
4.Evaluation of SNI model
Praxiological 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 model
Immunohistochemistry 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 model
Immunofluorescence 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 mice
Immunofluorescence 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).
Conclusion
1.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.
锌是哺乳动物体内重要的微量元素之一,在机体的生长发育、生殖遗传、免疫、内分泌、神经活动等重要生理过程中发挥重要的作用。在中枢神经系统中,大约15%的锌离子处于游离状态,这些锌离子主要位于含锌神经元(zinc-enrichedneuron,ZEN)轴突末端的突触小泡内。当神经活动时,游离锌离子往往随着一些神经递质的释放而进入突触间隙并与突触后膜的某些受体结合或者穿越突触后膜进入到突触后神经元,从而发挥其神经调节作用。近年来人们对锌转运体(zinctransporters,ZNTs)的研究进一步揭示了锌的生物学功能,ZNT7就是其中一个重要组成成员,它的功能是将锌转运到高尔基体内,从而维持细胞内的锌稳态。
神经病理性疼痛(neuropathic pain,NPP)是一种由神经系统损伤而引起的慢性疾病,临床上以痛觉过敏、触诱发痛(或称痛觉超敏)、局部感觉缺失以及自发性疼痛等为特点。近年来,随着人口的老龄化,NPP的发病率呈逐年上升趋势,但是由于缺乏有效的治疗手段,已经成为危害人类健康的主要疾病之一。
基础研究表明,锌能影响NMDA和非NMDA受体的表达与功能,后者在伤害性刺激传导中起重要作用;金属硫蛋白-Ⅲ和游离锌离子以及含锌初级传入神经元在脊髓和背根神经节(dorsal root ganglion,DRG)的大量分布也提示锌可能参与痛觉的传导。动物实验显示,脊神经背根切断术后,小鼠痛阈明显降低,脊髓背侧角浅层灰质的锌离子明显减少,进一步证实锌可能参与痛觉的产生和传导。
因此,本研究利用金属自显影技术(autometallography,AMG)、免疫组织化学、免疫荧光双标记与共聚焦激光扫描显微镜技术、Westein blot技术研究锌及ZNTs在小鼠脊髓和DRG的分布。在此基础上,利用坐骨神经分支选择损伤模型(spared nerve injury,SNI)观察低锌和高锌预处理对SNI模型动物行为学变化以及脊髓和DRG中锌离子、神经肽、神经元凋亡和星形胶质细胞的影响,从而为探讨脊髓和DRG中的锌代谢以及锌在NPP产生与传导中的作用和机制研究提供重要的实验依据。
实验方法
应用AMG技术、免疫组织化学技术、Western Blot技术检测锌和ZNT7在CD-1小鼠脊髓和DRG的分布,应用免疫荧光双标记和共聚焦激光扫描显微镜技术检测ZNT7与TGN38在小鼠脊髓和DRG的定位分布及其位置关系。建立SNI动物模型,利用观察动物自主行为变化与行为学检测技术对模型进行鉴定。在此基础上,分别给予低锌或高锌预处理,观察锌预处理对SNI模型动物行为学的影响。同时,利用AMG技术、N-(6-甲基-8-喹啉)-甲苯磺酰胺荧光染色技术(N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide,TSQ)检测锌在各组模型动物脊髓和DRG的分布变化;利用免疫组织化学技术、Western Blot技术和免疫荧光技术检测锌预处理对SNI模型动物脊髓和DRG中的降钙素基因相关肽(calcitoningene related protein,CGRP)、神经肽Y(neuropeptide Y,NPY)、细胞凋亡因子和星形胶质细胞表达的影响。
实验结果
1、锌离子在CD-1小鼠脊髓和DRG的分布
AMG阳性反应产物为棕黑色颗粒,主要分布在小鼠脊髓的神经元轴突终末和DRG神经元细胞核的周围,呈斑块状或颗粒状围绕细胞核。
2、ZNT7在CD-1小鼠脊髓和DRG的分布
免疫组织化学结果显示:ZNT7免疫阳性反应产物呈棕黄色,在脊髓灰质的神经元以及DRG神经元的细胞核周围均可见ZNT7阳性产物分布,呈斑块状或颗粒状围绕在细胞核周围高尔基体的分布区域。
免疫荧光双标记和共聚焦激光扫描结果显示:在小鼠脊髓灰质和DRG的神经元细胞核周围均有ZNT7阳性产物的表达,其与TGN38的分布相同,均呈斑块状或颗粒状共分布于神经元细胞核周围。
3、ZNT7在CD-1小鼠脊髓内表达的Western Blot分析
应用Western Blot技术对小鼠脊髓内ZNT7在蛋白水平的表达进行检测。结果显示,小鼠的脊髓内有ZNT7的丰富表达。
4、SNI模型的鉴定
观察动物自主行为发现:假手术组(SHAM)术后第2天步态基本恢复正常,无甩足和舔足等自发痛行为;而SNI模型动物在术后第1天就开始跛行,术侧后足不敢承重,出现甩足和舔足等自发痛行为,一直持续到观察期末。
机械性痛阈检测表明:与术前基础痛阈值相比,SHAM组术后痛阈无明显下降,差异无显著性意义(P＞0.05);SNI组术后痛阈明显下降,差异有显著性意义(P＜0.05)。与SHAM组相比,SNI组术后机械痛阈显著降低,差异有显著性意义(P＜0.05),表明模型制备成功。
5、低锌或高锌预处理对SNI模型动物脊髓背侧角和DRG中锌离子的影响
AMG和TSQ检测结果显示:与SHAM组相比,正常动物模型组(N-SNI)与低锌预处理组(L-SNI)的术侧脊髓背侧角浅层和DRG中的锌离子均显著减少(P＜0.05),其中L-SNI组比N-SNI组减少更为显著(P＜0.05);而高锌预处理组(H-SNI)术侧脊髓背侧角浅层和DRG中的锌离子均显著增多(P＜0.05)。
6、锌对SNI模型动物行为学的影响
观察动物自主行为发现:SHAM组术后第2天步态基本恢复正常,无舔足和甩足等自发痛行为;而N-SNI、L-SNI和H-SNI组在术后第1天就开始跛行,术侧后足不敢承重,出现甩足、舔足等自发痛行为,一直持续到观察期末。此外,L-SNI组小鼠的自主行为最为显著,而H-SNI组的自主行为相对较轻。
机械性痛阈检测表明:与术前基础痛阈值相比,N-SNI、L-SNI和H-SNI组术后痛阈均明显下降,差异有显著性意义(P＜0.05);与SHAM组相比,N-SNI、L-SNI和H-SNI组术后机械性痛阈均降低,差异有显著性意义(P＜0.05)。其中,L-SNI组痛阈低于N-SNI组(P＜0.05),而H-SNI组痛阈高于N-SNI组(P＜0.05)。
7、锌对SNI模型脊髓背侧角和DRG中CGRP和NPY的影响
免疫组织化学结果显示:CGRP和NPY阳性产物为棕褐色细颗粒状。主要分布在术侧的脊髓背侧角浅层和DRG神经元细胞质中。与SHAM组相比,N-SNI、L-SNI和H-SNI组的阳性表达均显著增高(P＜0.05)。其中,L-SNI组的阳性表达高于N-SNI组(P＜0.05),而H-SNI组的阳性表达低于N-SNI组(P＜0.05)。
8、锌对SNI模型脊髓背侧角神经元凋亡的影响
免疫组织化学检测结果表明:Caspases-3、Fas和FasL阳性产物为棕黄色颗粒状,主要分布在脊髓背侧角浅层的神经元中。与SHAM组相比,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角Caspases-3、Fas和FasL阳性表达均增高(P＜0.01)。L-SNI组的表达高于N-SNI组(P＜0.01);H-SNI组的表达低于N-SNI组(P＜0.01)。
Westein blot检测结果表明:与SHAM组相比,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角Caspases-3、Fas、FasL的阳性表达均显著增高(P＜0.01)。与N-SNI组相比,L-SNI组的阳性表达增多(P＜0.01);H-SNI组的阳性表达减少(P＜0.01)。
9、锌对SNI模型脊髓背侧角GFAP表达的影响
免疫荧光染色结果显示,星形胶质酸性蛋白(glial fibrillary acidic protein,GFAP)阳性细胞呈绿色荧光,细胞多突起,胞体较小。SHAM组术侧脊髓背侧角只有少量GFAP阳性细胞。与SHAM组相比较,N-SNI、L-SNI和H-SNI组术侧脊髓背侧角的GFAP阳性细胞均增多(P＜0.05)。与N-SNI组相比,L-SNI组的阳性细胞增多(P＜0.05),H-SNI组阳性细胞减少(P＜0.05)。
结论
1、正常小鼠脊髓和DRG中富含游离锌离子和ZNT7。其中,ZNT7与高尔基复合体标记物TGN38的表达存在共区域性。
2、低锌预处理能使SNI模型脊髓背侧角浅层锌离子更加减少,加重自发性痛等行为学变化,机械性痛阈值降低更加显著;而高锌预处理能使脊髓背侧角锌离子增多,改善自发性痛等行为学变化,提高机械性痛阈值。
4、低锌预处理能使SNI模型脊髓背侧角CGRP、NPY、凋亡因子和星形胶质细胞的表达增加,使DRG中的CGRP和NPY表达增加;而高锌预处理能使表达降低。
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.
Methods
Normal 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.
Results
1.Distribution of zinc ions in CD-1 mice spinal cord and DRG
AMG 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 DRG
Immunohistochemimistry 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 detection
Western blot results showed that there were abundant expressions of ZNT7 in spinal cord in normal CD-1 mice.
4.Evaluation of SNI model
Praxiological 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 model
Immunohistochemistry 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 model
Immunofluorescence 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 mice
Immunofluorescence 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).
Conclusion
1.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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