水通道4在神经病理性疼痛中的作用及可能机制
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摘要
国际疼痛研究协会将神经病理性疼痛定义为在外周或中枢水平由躯体感觉神经系统的损伤或疾病而直接造成的疼痛。神经病理性疼痛严重影响患者的生活质量，治疗困难，给社会带来巨大的卫生资源压力和沉重的经济负担。神经病理性疼痛的发病机制复杂，目前尚未完全阐明。近年来国内外对神经病理性疼痛的机制已进行了大量研究并取得了一定的进展。目前学术界普遍认为神经病理性疼痛的产生及维持涉及神经系统的可塑性，包括外周及中枢机制，但是确切机制尚未阐明。越来越多的证据表明，包括星形胶质细胞和小胶质细胞在内的脊髓胶质细胞在慢性疼痛特别是神经病理性疼痛的发生、发展、维持过程中发挥着重要的作用，因此研究和发现影响胶质细胞激活的分子对于解释神经病理性疼痛的生物学机制以及发现新的干预靶点和治疗措施具有十分重要的意义。
水通道4（aquaporin4, AQP4）是参与水转运的水通道家族中的一员，是中枢神经系统表达最丰富的水通道。在脑内AQP4呈典型的极性分布，主要表达于星形胶质细胞与血管相连的终足上，同时在室管膜细胞和内皮细胞上也有表达；在神经元中几乎不表达；在静息态的小胶质细胞上不表达，激活态的小胶质细胞上是否表达尚存在争议。在脊髓中，AQP4主要分布于围绕兴奋性和抑制性突触结构的星形胶质细胞突起部位，提示其可能参与突触传递功能。AQP4在脊髓背角中的分布从浅层到深层逐渐降低，在背角浅层（I、II层）表达最高，提示可能与脊髓背角浅层的功能密切相关。文献调研显示，AQP4是星形胶质细胞上最重要的水通道，能够影响星形胶质细胞的细胞骨架结构和细胞间隙连接；能够调节星形胶质细胞的功能，影响星形胶质细胞的迁移、增殖以及瘢痕的形成。此外，AQP4与钾通道4.1（Kir4.1）和谷氨酸转运体1（glutamate transporter1, GLT1）共存于星形胶质细胞中，在功能上存在偶联，对于细胞外K+稳态的维持以及突触间隙谷氨酸的清除非常重要。AQP4还通过影响星形胶质细胞的功能来调节神经元的功能，包括调节神经元的兴奋性、多巴胺（dopamine，DA）、谷氨酸（glutamate，Glu）等神经递质的含量及释放和某些病理条件下神经元存活和神经再生，但详细机制有待进一步研究。近年来大量研究报道星形胶质细胞参与神经病理性疼痛。最近研究显示，损伤、缺血诱发的脑内AQP4表达上调与星形胶质细胞的特异性标志物胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）表达上调相平行，而下调AQP4表达则影响碱性成纤维细胞生长因子刺激的星形胶质细胞的增殖；AQP4基因敲除能够降低脑内注射脂多糖（lipopolysaccharide,LPS）引起的神经炎症，也能够降低培养的星形胶质细胞中LPS刺激的前炎性细胞因子的释放。以上研究提示，AQP4可能与星形胶质细胞的功能密切相关，但是AQP4是否参与神经病理性疼痛目前尚未见报道。因此，本课题的目的是研究AQP4在神经病理性疼痛中的作用及可能机制。
本课题的研究内容主要包括以下三部分：第一部分，在小鼠急性疼痛模型和大鼠神经病理性疼痛模型中给予AQP4非选择性抑制剂乙酰唑胺（acetazolamide,AZA），初步研究AQP4在急性疼痛和神经病理性疼痛中的作用。结合本课题组在AQP4基因敲除动物中的研究结果，在明确了AQP4在神经病理性疼痛中的作用后，第二部分，应用野生型和AQP4基因敲除小鼠构建坐骨神经分支损伤模型(spared nerve injury, SNI）模型，研究神经病理性疼痛发生、发展进程中脊髓AQP4表达的时程特征及其与星形胶质细胞和小胶质细胞激活的关系。明确AQP4与胶质细胞激活的关系后，第三部分研究哪些刺激胶质细胞激活的因素依赖于AQP4，即在细胞水平从胶质细胞激活方面探讨AQP4影响了哪些刺激胶质细胞激活的因素。通过以上研究，探讨AQP4参与神经病理性疼痛的作用机制。具体研究结果如下：
一、AQP4非选择性抑制剂乙酰唑胺对急性疼痛和神经病理性疼痛的作用：AQP4非选择性抑制剂AZA对热刺激（热辐射甩尾和热板模型）诱发的小鼠急性疼痛无显著影响，但是AZA可剂量依赖的减轻大鼠坐骨神经慢性压迫性损伤（chronic constriction injury, CCI）模型机械刺激痛觉超敏。本课题组尚未发表的实验结果表明AQP4基因敲除对热刺激（热板和热辐射甩尾模型）、化学刺激（福尔马林和辣椒素致痛模型）和机械刺激（von Frey hair模型）诱发的急性疼痛均无影响，但是能显著减轻神经病理性疼痛诱发的机械刺激痛觉超敏。这些实验结果提示AQP4不影响急性疼痛，可能参与神经病理性疼痛。
二、在神经病理性疼痛中AQP4表达的时程特征及其与胶质细胞激活的关系：行为学测试结果显示，在小鼠SNI模型中，从术后1d到术后28d，AQP4基因敲除（knockout, KO）小鼠与野生型（wild-type, WT）小鼠相比机械刺激痛觉超敏的程度显著减轻，提示AQP4在神经病理性疼痛中发挥了作用。在此模型中，我们研究了脊髓中的AQP4在神经病理性疼痛中表达的时程特征及其与胶质细胞激活的关系。蛋白免疫印迹分析结果显示，在WT小鼠中，与假手术组相比，SNI手术组小鼠脊髓（L4-L6节段）中AQP4的表达在术后1d和3d无显著变化，而在术后7d开始出现显著升高，该现象至少持续至术后28d；GFAP（星形胶质细胞的特异性标志物，其表达上调反映了星形胶质细胞的激活）的表达和表达的时程改变与AQP4完全一致；簇分化抗原11b（Cluster differentiation antigen11b, CD11b,小胶质细胞的标志物，其表达上调反映了小胶质细胞的激活）的表达则在术后1d就出现明显升高并持续至术后7d，之后开始降低，术后28d时已恢复至正常水平。在AQP4KO小鼠中，与假手术组相比，SNI手术组小鼠脊髓中GFAP的表达在术后1d到术后28d均未出现明显改变；CD11b的表达在术后1d和3d未出现显著性升高。术后7d开始降低，术后14d时已恢复至正常水平。这些结果提示，在神经病理性疼痛早期，AQP4的表达没有改变，星形胶质细胞也没有激活，但此时小胶质细胞已经激活；而在神经病理性疼痛中晚期，AQP4表达增强，此时星形胶质细胞也处于激活状态，但小胶质细胞的激活开始降低并恢复至正常水平。以上结果说明星形胶质细胞的激活和AQP4的表达升高具有相关性，而小胶质细胞的激活和AQP4的表达升高的相关性还有待进一步研究。因此，AQP4基因敲除减轻神经病理性疼痛的机制可能与其抑制脊髓中星形胶质细胞的激活有关。
在神经病理性疼痛中，激活的星形胶质细胞和小胶质细胞均能够释放前炎性细胞因子，如白介素1beta（interleukin-1β, IL-1β）、白介素6（interleukin-6, IL-6）和肿瘤坏死因子alpha（tumor necrosis factor-, TNF-），这些因子反映了胶质细胞激活后的功能变化。在WT小鼠中，与假手术组相比，SNI手术组小鼠脊髓（L4-L6节段）中IL-1β、IL-6和TNF-的含量在术后1、3、7、14和28d均显著升高，与胶质细胞的激活相一致；在AQP4KO小鼠中，与假手术组相比，SNI手术组小鼠脊髓中上述3种因子的含量在术后1d和3d未出现显著性升高，从术后7d开始3种因子的含量恢复至正常水平。结果提示，在神经病理性疼痛早期，3种因子的来源主要是激活的小胶质细胞；而在神经病理性疼痛中晚期，3种因子的来源主要是激活的星形胶质细胞。因此，在神经病理性疼痛中晚期，AQP4基因敲除能够降低脊髓中前炎性细胞因子的含量。
综上所述，本部分研究发现AQP4基因敲除减轻神经病理性疼痛的机制可能与其抑制脊髓星形胶质细胞的激活、降低前炎性细胞因子的含量有关；与小胶质细胞的关系有待进一步有研究。
三、AQP4对胶质细胞激活的调节及机制：本课题的第二部分在整体水平上观察到了AQP4与星形胶质细胞激活的关系，而AQP4又表达于星形胶质细胞上，那么AQP4是否直接影响了星形胶质细胞的激活？因此本部分研究用体外实验来回答这个问题。神经病理性疼痛中（如SNI模型），前炎性细胞因子和致痛递质是两个很重要的引起胶质细胞激活的刺激因素，在本部分的研究中，用LPS刺激胶质细胞模拟前炎性细胞因子引起的胶质细胞的激活，用谷氨酸（glutamicacid, Glu）刺激胶质细胞是致痛递质导致胶质细胞激活的刺激因素。在原代培养的星形胶质细胞模型上，通过免疫荧光实验发现LPS（100ng/ml）能够激活体外培养的大鼠乳鼠脊髓星形胶质细胞；而AQP4非选择性抑制剂AZA（0.1μM、0.3μM、1μM、3μM和10μM）可以剂量依赖的抑制LPS诱导的星形胶质细胞的激活。此外，LPS（100ng/ml）可以引起AQP4表达阳性的WT乳鼠脊髓星形胶质细胞的激活，而不引起AQP4表达阴性的KO乳鼠脊髓星形胶质细胞的激活。这些实验结果说明LPS诱导的星形胶质细胞的激活依赖于AQP4的存在。同时还发现Glu（10μM、100μM和1mM）能够激活体外培养的大鼠乳鼠脊髓星形胶质细胞；而AZA（10μM）可以抑制上述3个浓度的Glu诱导的星形胶质细胞的激活。此外，Glu（1mM）可以引起AQP4表达阳性的WT乳鼠脊髓星形胶质细胞的激活，而不引起AQP4表达阴性的KO乳鼠脊髓星形胶质细胞的激活。这些实验结果表明Glu诱导的星形胶质细胞的激活依赖于AQP4的存在。因此，体外实验直接证实了AQP4的存在是星形胶质细胞激活所必需的。
因此，本论文研究首次发现，AQP4不影响急性疼痛，但参与神经病理性疼痛；其可能机制是通过直接影响脊髓星形胶质细胞的激活以及前炎性细胞因子的释放实现的。
According to the International Association for the Study of Pain (IASP),Neuropathic pain (NPP) is defned as the pain arising as a direct consequence of alesion or disease affecting the somatosensory system either at peripheral or centrallevel. NPP is difficult to be interrupted and it seriously affects patients’ quality of lifeand brings enormous economic burden to the society. Pathogenesis of NPP is acomplicated process and it has not been fully understood. Recently, the mechanismsof NPP have been intensively studied and certain progress has been achieved. It isgenerally believed that the plasticity of the nervous system is involved in thegeneration and the maintenance of NPP, including peripheral and central mechanisms,but the exact mechanism has not been elucidated. More and more evidence indicatesthat spinal cord glial cells, such as, astrocytes and microglia, play an important role inchronic pain, especially in the generation and maintenance of NPP. Thus, themolecular research of glial cells activation is very important to understand thebiological mechanisms of NPP and discover new intervention targets and therapeuticmeasures.
Aquaporin4(AQP4) blongs to the family of water channels that are in charge ofwater transport. AQP4is the predominant water channel existing in the centralnervous system. In the brain, AQP4mainly expresses in astrocytes terminal feet thatconnects to blood vessels, and it also expresses in ependymal cells and endothelialcells. AQP4barely expresses in neurons. Interestingly, in microglia, AQP4does notexpress in the resting state, and the expression of AQP4in the activation state is stillin controversial. In the spinal cord, AQP4expresses mainly on the membrane ofastrocytes wraping symmetric and asymmetric synapses. This reveals that AQP4mayparticipate in synaptic transmission. The distribution of AQP4in the spinal corddorsal horn decreases gradually from shallow layer to deep layer. And the expressionof AQP4is mainly in the superficial dorsal horn (I, II layer) suggesting that thefunction of AQP4may be associated with spinal cord dorsal horn. AQP4is the most important water channel in astrocytes which affects the structure of the cytoskeletonand gap junctional of astrocytes, regulates the function of astrocytes, and affectsastrocytes migration, proliferation, and scar formation. AQP4coexists with potassiumchannel4.1(Kir4.1) and glutamate transporter1(GLT1) in astrocytes. It is knownthat Kir4.1is important for the maintenance of extracellular K+homeostasis, andGLT1plays a critical role for the clearance of synaptic gap glutamic acid (Glu).Through affecting the functions of astrocytes, AQP4regulates the functions ofneurons: it regulates the excitability of neurons, controls the content and release ofneurotransmitters, such as DA and Glu, and coordinates nerve neuron survival andnerve regeneration in some pathological conditions. However, the exact mechanismremains unclear. Recently, a number of studies reported that astrocytes participate inthe NPP. It was reported that AQP4upregulation induced by injury and ischemia wasparallel with the activation of astrocytes. While, downregulation of AQP4or AQP4gene knockout significantly affected the response of astrocytes. AQP4gene knockoutwas able to reduce nerve inflammation caused by intracerebral injection oflipopolysaccharide (LPS), and decrease the release of LPS-stimulatedproinflammatory cytokines in cultured astrocytes. These studies indicate that AQP4may be closely related to the activation of astrocytes. But no report has yet beenpresented about the participation of AQP4in NPP.
The aim of this study is to investigate the role of AQP4in NPP and to understandthe possible mechanisms.
In order to answer these questions, three parts of experiment were designed:(1)to confirm the role of AQP4in acute and chronic pain;(2) to study the time course ofAQP4expression in the spinal cord and the relationship between AQP4expressionand glia cells activation during the generation and development processes of NPP inwild-type (WT) and AQP4gene knockout (KO) mice SNI models;(3) to explore thepossible mechanisms of AQP4in glial cells activation involved in neuropathic pain incellular levels.
Result1, the role of AQP4in acute and chronic pain. AZA had no effect onacute pain induced by thermal stimulation (hot-plate and tail-flick tests) but candose-dependently reduce the mechanical allodynia in rat sciatic nerve chronicconstriction injury (CCI) model. These results were consistent with our lab studies using WT and KO mice. All of these results confirmed that AQP4does not involve inthe activation of acute pain but it does act on neuropathic pain.
Result2, the time course of AQP4expression in the spinal cord and therelationship between AQP4expression and glial cells activation in NPP. In theSNI model, mechanical allodynia was significantly decreased in KO-SNI groupcompared with WT-SNI group from1d to28d after injury, suggesting that AQP4hadeffect on NPP. Nest, we studied the time course of AQP4expression in the spinalcord and the relationship between AQP4expression and glial cells activation in NPP.Western blot resultes showed that, in WT mice spinal cord, compared with shamgroup, AQP4expression significantly increased from7d after injury and at leastlasted until28d after injury in SNI group; meanwhile, glial fibrillary acidic protein(GFAP, specific marker of astrocyte, can reflect the activation of astrocytes)expression was the same as that of AQP4in SNI group; cluster differentiation antigen11b (CD11b, marker of microglia, can reflect the activation of microglia) expressiondramatically increased from1d to7d after injury and decreased to the normal level at14d after injury in SNI group. In KO mice spinal cord, compared with sham group,GFAP expression did not change significantly from1d to28d after injury in SNIgroup; CD11b expression appeared as an increased trend at1d after injury anddecreased to the normal level at7d after injury in SNI group. These results suggestedthat AQP4expression had no changes and astrocytes were not activated but microgliawas activated during the early period of NPP. In the late period of NPP, AQP4expression increased significantly and astrocytes were activated at the same time,whereas microglia was not activated. These findings demonstrated that the activationof astrocytes is associated with increased expression of AQP4and the activation ofmicroglia may be not obviously associated with increased expression of AQP4. So thesuppression of astrocytes activation might manipulate the attenuation of NPP withAQP4deficiency.
In NPP, activated astrocyes and microglia can release proinflammatory cytokinessuch as, interleukin-1beta (IL-1β), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-), which can reflect the functions of the activated glia cells. The resultsof ELISA showed that, in WT mice spinal cord, compared with sham group, IL-1β,IL-6and TNF-were elevated at d1,3d,7d,14d and28d after injury in SNI group,which was consistent with glia cells activation. In KO mice spinal cord, compared with sham group, the IL-1β, IL-6and TNF-appeared as an increased trend at1d and3d after injury and decreased to normal level at7d after injury in SNI group. Theseresults indicated that three cytokines were mainly released by activated microgliaduring the early period of NPP, whereas during the late period of NPP, thesecytokines were mainly released by the activated astrocytes. So the suppression ofproinflammatory cytokines release might mediate the attenuation of NPP with AQP4deficiency.
In conclusion, the suppression of astrocytes activation and proinflammatorycytokines release might mediate the attenuation of NPP with AQP4deficiency. Therelationship between AQP4and microglia activation is interested to be furtherstudied.
Result3, the regulation and possible mechanisms of AQP4in glial cellsactivation. In NPP (such as SNI model), inflammatory factors and neurotransmittersare the two important stimuli that can cause glia cells activation. In this part of thestudy, LPS mimics the stimulation factors inducing the activation of glia cells causedby inflammatory cytokines; and Glu is the stimulating factor that induces theactivation of glia cells caused by neurotransmitters. In primary cultured astrocytes, itwas found that LPS (100ng/ml) was able to activate the neonatal rat spinal cordastrocytes by immunofluorescence assay. Meanwhile, non-selective AQP4inhibitorAZA (0.1μM,0.3μM,1μM,3μM and10μM) can dose-dependently inhibit theLPS-induced astrocytes activation. In addition, LPS (100ng/ml) can induce WT(AQP4positive expression) neonatal mice spinal cord astrocytes activation but cannotinduce KO (AQP4negative expression) neonatal mice spinal cord astrocytesactivation. These results demonstrated that LPS-induced astrocytes activation wasdependent on the expression or the function of AQP4. Meanwhile, it was also foundthat Glu (10μM,100μM and1mM）was able to activate the cultured neonatal ratspinal cord astrocytes. Meanwhile, AZA (10μM) can inhibit the Glu-inducedastrocytes activation. In addition, Glu (1mM) can induce WT (AQP4positiveexpression) neonatal mice spinal cord astrocytes activation but cannot induce KO(AQP4negative expression) neonatal mice spinal cord astrocytes activation. Theseresults indicated that Glu-induced astrocytes activation was dependent on theexpression or function of AQP4. So the experiments in vitro confirmed directly thatAQP4expression or function is necessary for astrocyte activation.
In conclusion, AQP4involves in neuropathic pain, but not acute pain. Thepossible mechanism is that AQP4directly affects on spinal cord astrocytes activationand manipulates proinflammatory cytokines release.
水通道4（aquaporin4, AQP4）是参与水转运的水通道家族中的一员，是中枢神经系统表达最丰富的水通道。在脑内AQP4呈典型的极性分布，主要表达于星形胶质细胞与血管相连的终足上，同时在室管膜细胞和内皮细胞上也有表达；在神经元中几乎不表达；在静息态的小胶质细胞上不表达，激活态的小胶质细胞上是否表达尚存在争议。在脊髓中，AQP4主要分布于围绕兴奋性和抑制性突触结构的星形胶质细胞突起部位，提示其可能参与突触传递功能。AQP4在脊髓背角中的分布从浅层到深层逐渐降低，在背角浅层（I、II层）表达最高，提示可能与脊髓背角浅层的功能密切相关。文献调研显示，AQP4是星形胶质细胞上最重要的水通道，能够影响星形胶质细胞的细胞骨架结构和细胞间隙连接；能够调节星形胶质细胞的功能，影响星形胶质细胞的迁移、增殖以及瘢痕的形成。此外，AQP4与钾通道4.1（Kir4.1）和谷氨酸转运体1（glutamate transporter1, GLT1）共存于星形胶质细胞中，在功能上存在偶联，对于细胞外K+稳态的维持以及突触间隙谷氨酸的清除非常重要。AQP4还通过影响星形胶质细胞的功能来调节神经元的功能，包括调节神经元的兴奋性、多巴胺（dopamine，DA）、谷氨酸（glutamate，Glu）等神经递质的含量及释放和某些病理条件下神经元存活和神经再生，但详细机制有待进一步研究。近年来大量研究报道星形胶质细胞参与神经病理性疼痛。最近研究显示，损伤、缺血诱发的脑内AQP4表达上调与星形胶质细胞的特异性标志物胶质纤维酸性蛋白（glial fibrillary acidic protein, GFAP）表达上调相平行，而下调AQP4表达则影响碱性成纤维细胞生长因子刺激的星形胶质细胞的增殖；AQP4基因敲除能够降低脑内注射脂多糖（lipopolysaccharide,LPS）引起的神经炎症，也能够降低培养的星形胶质细胞中LPS刺激的前炎性细胞因子的释放。以上研究提示，AQP4可能与星形胶质细胞的功能密切相关，但是AQP4是否参与神经病理性疼痛目前尚未见报道。因此，本课题的目的是研究AQP4在神经病理性疼痛中的作用及可能机制。
本课题的研究内容主要包括以下三部分：第一部分，在小鼠急性疼痛模型和大鼠神经病理性疼痛模型中给予AQP4非选择性抑制剂乙酰唑胺（acetazolamide,AZA），初步研究AQP4在急性疼痛和神经病理性疼痛中的作用。结合本课题组在AQP4基因敲除动物中的研究结果，在明确了AQP4在神经病理性疼痛中的作用后，第二部分，应用野生型和AQP4基因敲除小鼠构建坐骨神经分支损伤模型(spared nerve injury, SNI）模型，研究神经病理性疼痛发生、发展进程中脊髓AQP4表达的时程特征及其与星形胶质细胞和小胶质细胞激活的关系。明确AQP4与胶质细胞激活的关系后，第三部分研究哪些刺激胶质细胞激活的因素依赖于AQP4，即在细胞水平从胶质细胞激活方面探讨AQP4影响了哪些刺激胶质细胞激活的因素。通过以上研究，探讨AQP4参与神经病理性疼痛的作用机制。具体研究结果如下：
一、AQP4非选择性抑制剂乙酰唑胺对急性疼痛和神经病理性疼痛的作用：AQP4非选择性抑制剂AZA对热刺激（热辐射甩尾和热板模型）诱发的小鼠急性疼痛无显著影响，但是AZA可剂量依赖的减轻大鼠坐骨神经慢性压迫性损伤（chronic constriction injury, CCI）模型机械刺激痛觉超敏。本课题组尚未发表的实验结果表明AQP4基因敲除对热刺激（热板和热辐射甩尾模型）、化学刺激（福尔马林和辣椒素致痛模型）和机械刺激（von Frey hair模型）诱发的急性疼痛均无影响，但是能显著减轻神经病理性疼痛诱发的机械刺激痛觉超敏。这些实验结果提示AQP4不影响急性疼痛，可能参与神经病理性疼痛。
二、在神经病理性疼痛中AQP4表达的时程特征及其与胶质细胞激活的关系：行为学测试结果显示，在小鼠SNI模型中，从术后1d到术后28d，AQP4基因敲除（knockout, KO）小鼠与野生型（wild-type, WT）小鼠相比机械刺激痛觉超敏的程度显著减轻，提示AQP4在神经病理性疼痛中发挥了作用。在此模型中，我们研究了脊髓中的AQP4在神经病理性疼痛中表达的时程特征及其与胶质细胞激活的关系。蛋白免疫印迹分析结果显示，在WT小鼠中，与假手术组相比，SNI手术组小鼠脊髓（L4-L6节段）中AQP4的表达在术后1d和3d无显著变化，而在术后7d开始出现显著升高，该现象至少持续至术后28d；GFAP（星形胶质细胞的特异性标志物，其表达上调反映了星形胶质细胞的激活）的表达和表达的时程改变与AQP4完全一致；簇分化抗原11b（Cluster differentiation antigen11b, CD11b,小胶质细胞的标志物，其表达上调反映了小胶质细胞的激活）的表达则在术后1d就出现明显升高并持续至术后7d，之后开始降低，术后28d时已恢复至正常水平。在AQP4KO小鼠中，与假手术组相比，SNI手术组小鼠脊髓中GFAP的表达在术后1d到术后28d均未出现明显改变；CD11b的表达在术后1d和3d未出现显著性升高。术后7d开始降低，术后14d时已恢复至正常水平。这些结果提示，在神经病理性疼痛早期，AQP4的表达没有改变，星形胶质细胞也没有激活，但此时小胶质细胞已经激活；而在神经病理性疼痛中晚期，AQP4表达增强，此时星形胶质细胞也处于激活状态，但小胶质细胞的激活开始降低并恢复至正常水平。以上结果说明星形胶质细胞的激活和AQP4的表达升高具有相关性，而小胶质细胞的激活和AQP4的表达升高的相关性还有待进一步研究。因此，AQP4基因敲除减轻神经病理性疼痛的机制可能与其抑制脊髓中星形胶质细胞的激活有关。
在神经病理性疼痛中，激活的星形胶质细胞和小胶质细胞均能够释放前炎性细胞因子，如白介素1beta（interleukin-1β, IL-1β）、白介素6（interleukin-6, IL-6）和肿瘤坏死因子alpha（tumor necrosis factor-, TNF-），这些因子反映了胶质细胞激活后的功能变化。在WT小鼠中，与假手术组相比，SNI手术组小鼠脊髓（L4-L6节段）中IL-1β、IL-6和TNF-的含量在术后1、3、7、14和28d均显著升高，与胶质细胞的激活相一致；在AQP4KO小鼠中，与假手术组相比，SNI手术组小鼠脊髓中上述3种因子的含量在术后1d和3d未出现显著性升高，从术后7d开始3种因子的含量恢复至正常水平。结果提示，在神经病理性疼痛早期，3种因子的来源主要是激活的小胶质细胞；而在神经病理性疼痛中晚期，3种因子的来源主要是激活的星形胶质细胞。因此，在神经病理性疼痛中晚期，AQP4基因敲除能够降低脊髓中前炎性细胞因子的含量。
综上所述，本部分研究发现AQP4基因敲除减轻神经病理性疼痛的机制可能与其抑制脊髓星形胶质细胞的激活、降低前炎性细胞因子的含量有关；与小胶质细胞的关系有待进一步有研究。
三、AQP4对胶质细胞激活的调节及机制：本课题的第二部分在整体水平上观察到了AQP4与星形胶质细胞激活的关系，而AQP4又表达于星形胶质细胞上，那么AQP4是否直接影响了星形胶质细胞的激活？因此本部分研究用体外实验来回答这个问题。神经病理性疼痛中（如SNI模型），前炎性细胞因子和致痛递质是两个很重要的引起胶质细胞激活的刺激因素，在本部分的研究中，用LPS刺激胶质细胞模拟前炎性细胞因子引起的胶质细胞的激活，用谷氨酸（glutamicacid, Glu）刺激胶质细胞是致痛递质导致胶质细胞激活的刺激因素。在原代培养的星形胶质细胞模型上，通过免疫荧光实验发现LPS（100ng/ml）能够激活体外培养的大鼠乳鼠脊髓星形胶质细胞；而AQP4非选择性抑制剂AZA（0.1μM、0.3μM、1μM、3μM和10μM）可以剂量依赖的抑制LPS诱导的星形胶质细胞的激活。此外，LPS（100ng/ml）可以引起AQP4表达阳性的WT乳鼠脊髓星形胶质细胞的激活，而不引起AQP4表达阴性的KO乳鼠脊髓星形胶质细胞的激活。这些实验结果说明LPS诱导的星形胶质细胞的激活依赖于AQP4的存在。同时还发现Glu（10μM、100μM和1mM）能够激活体外培养的大鼠乳鼠脊髓星形胶质细胞；而AZA（10μM）可以抑制上述3个浓度的Glu诱导的星形胶质细胞的激活。此外，Glu（1mM）可以引起AQP4表达阳性的WT乳鼠脊髓星形胶质细胞的激活，而不引起AQP4表达阴性的KO乳鼠脊髓星形胶质细胞的激活。这些实验结果表明Glu诱导的星形胶质细胞的激活依赖于AQP4的存在。因此，体外实验直接证实了AQP4的存在是星形胶质细胞激活所必需的。
因此，本论文研究首次发现，AQP4不影响急性疼痛，但参与神经病理性疼痛；其可能机制是通过直接影响脊髓星形胶质细胞的激活以及前炎性细胞因子的释放实现的。
According to the International Association for the Study of Pain (IASP),Neuropathic pain (NPP) is defned as the pain arising as a direct consequence of alesion or disease affecting the somatosensory system either at peripheral or centrallevel. NPP is difficult to be interrupted and it seriously affects patients’ quality of lifeand brings enormous economic burden to the society. Pathogenesis of NPP is acomplicated process and it has not been fully understood. Recently, the mechanismsof NPP have been intensively studied and certain progress has been achieved. It isgenerally believed that the plasticity of the nervous system is involved in thegeneration and the maintenance of NPP, including peripheral and central mechanisms,but the exact mechanism has not been elucidated. More and more evidence indicatesthat spinal cord glial cells, such as, astrocytes and microglia, play an important role inchronic pain, especially in the generation and maintenance of NPP. Thus, themolecular research of glial cells activation is very important to understand thebiological mechanisms of NPP and discover new intervention targets and therapeuticmeasures.
Aquaporin4(AQP4) blongs to the family of water channels that are in charge ofwater transport. AQP4is the predominant water channel existing in the centralnervous system. In the brain, AQP4mainly expresses in astrocytes terminal feet thatconnects to blood vessels, and it also expresses in ependymal cells and endothelialcells. AQP4barely expresses in neurons. Interestingly, in microglia, AQP4does notexpress in the resting state, and the expression of AQP4in the activation state is stillin controversial. In the spinal cord, AQP4expresses mainly on the membrane ofastrocytes wraping symmetric and asymmetric synapses. This reveals that AQP4mayparticipate in synaptic transmission. The distribution of AQP4in the spinal corddorsal horn decreases gradually from shallow layer to deep layer. And the expressionof AQP4is mainly in the superficial dorsal horn (I, II layer) suggesting that thefunction of AQP4may be associated with spinal cord dorsal horn. AQP4is the most important water channel in astrocytes which affects the structure of the cytoskeletonand gap junctional of astrocytes, regulates the function of astrocytes, and affectsastrocytes migration, proliferation, and scar formation. AQP4coexists with potassiumchannel4.1(Kir4.1) and glutamate transporter1(GLT1) in astrocytes. It is knownthat Kir4.1is important for the maintenance of extracellular K+homeostasis, andGLT1plays a critical role for the clearance of synaptic gap glutamic acid (Glu).Through affecting the functions of astrocytes, AQP4regulates the functions ofneurons: it regulates the excitability of neurons, controls the content and release ofneurotransmitters, such as DA and Glu, and coordinates nerve neuron survival andnerve regeneration in some pathological conditions. However, the exact mechanismremains unclear. Recently, a number of studies reported that astrocytes participate inthe NPP. It was reported that AQP4upregulation induced by injury and ischemia wasparallel with the activation of astrocytes. While, downregulation of AQP4or AQP4gene knockout significantly affected the response of astrocytes. AQP4gene knockoutwas able to reduce nerve inflammation caused by intracerebral injection oflipopolysaccharide (LPS), and decrease the release of LPS-stimulatedproinflammatory cytokines in cultured astrocytes. These studies indicate that AQP4may be closely related to the activation of astrocytes. But no report has yet beenpresented about the participation of AQP4in NPP.
The aim of this study is to investigate the role of AQP4in NPP and to understandthe possible mechanisms.
In order to answer these questions, three parts of experiment were designed:(1)to confirm the role of AQP4in acute and chronic pain;(2) to study the time course ofAQP4expression in the spinal cord and the relationship between AQP4expressionand glia cells activation during the generation and development processes of NPP inwild-type (WT) and AQP4gene knockout (KO) mice SNI models;(3) to explore thepossible mechanisms of AQP4in glial cells activation involved in neuropathic pain incellular levels.
Result1, the role of AQP4in acute and chronic pain. AZA had no effect onacute pain induced by thermal stimulation (hot-plate and tail-flick tests) but candose-dependently reduce the mechanical allodynia in rat sciatic nerve chronicconstriction injury (CCI) model. These results were consistent with our lab studies using WT and KO mice. All of these results confirmed that AQP4does not involve inthe activation of acute pain but it does act on neuropathic pain.
Result2, the time course of AQP4expression in the spinal cord and therelationship between AQP4expression and glial cells activation in NPP. In theSNI model, mechanical allodynia was significantly decreased in KO-SNI groupcompared with WT-SNI group from1d to28d after injury, suggesting that AQP4hadeffect on NPP. Nest, we studied the time course of AQP4expression in the spinalcord and the relationship between AQP4expression and glial cells activation in NPP.Western blot resultes showed that, in WT mice spinal cord, compared with shamgroup, AQP4expression significantly increased from7d after injury and at leastlasted until28d after injury in SNI group; meanwhile, glial fibrillary acidic protein(GFAP, specific marker of astrocyte, can reflect the activation of astrocytes)expression was the same as that of AQP4in SNI group; cluster differentiation antigen11b (CD11b, marker of microglia, can reflect the activation of microglia) expressiondramatically increased from1d to7d after injury and decreased to the normal level at14d after injury in SNI group. In KO mice spinal cord, compared with sham group,GFAP expression did not change significantly from1d to28d after injury in SNIgroup; CD11b expression appeared as an increased trend at1d after injury anddecreased to the normal level at7d after injury in SNI group. These results suggestedthat AQP4expression had no changes and astrocytes were not activated but microgliawas activated during the early period of NPP. In the late period of NPP, AQP4expression increased significantly and astrocytes were activated at the same time,whereas microglia was not activated. These findings demonstrated that the activationof astrocytes is associated with increased expression of AQP4and the activation ofmicroglia may be not obviously associated with increased expression of AQP4. So thesuppression of astrocytes activation might manipulate the attenuation of NPP withAQP4deficiency.
In NPP, activated astrocyes and microglia can release proinflammatory cytokinessuch as, interleukin-1beta (IL-1β), interleukin-6(IL-6) and tumor necrosis factor-alpha (TNF-), which can reflect the functions of the activated glia cells. The resultsof ELISA showed that, in WT mice spinal cord, compared with sham group, IL-1β,IL-6and TNF-were elevated at d1,3d,7d,14d and28d after injury in SNI group,which was consistent with glia cells activation. In KO mice spinal cord, compared with sham group, the IL-1β, IL-6and TNF-appeared as an increased trend at1d and3d after injury and decreased to normal level at7d after injury in SNI group. Theseresults indicated that three cytokines were mainly released by activated microgliaduring the early period of NPP, whereas during the late period of NPP, thesecytokines were mainly released by the activated astrocytes. So the suppression ofproinflammatory cytokines release might mediate the attenuation of NPP with AQP4deficiency.
In conclusion, the suppression of astrocytes activation and proinflammatorycytokines release might mediate the attenuation of NPP with AQP4deficiency. Therelationship between AQP4and microglia activation is interested to be furtherstudied.
Result3, the regulation and possible mechanisms of AQP4in glial cellsactivation. In NPP (such as SNI model), inflammatory factors and neurotransmittersare the two important stimuli that can cause glia cells activation. In this part of thestudy, LPS mimics the stimulation factors inducing the activation of glia cells causedby inflammatory cytokines; and Glu is the stimulating factor that induces theactivation of glia cells caused by neurotransmitters. In primary cultured astrocytes, itwas found that LPS (100ng/ml) was able to activate the neonatal rat spinal cordastrocytes by immunofluorescence assay. Meanwhile, non-selective AQP4inhibitorAZA (0.1μM,0.3μM,1μM,3μM and10μM) can dose-dependently inhibit theLPS-induced astrocytes activation. In addition, LPS (100ng/ml) can induce WT(AQP4positive expression) neonatal mice spinal cord astrocytes activation but cannotinduce KO (AQP4negative expression) neonatal mice spinal cord astrocytesactivation. These results demonstrated that LPS-induced astrocytes activation wasdependent on the expression or the function of AQP4. Meanwhile, it was also foundthat Glu (10μM,100μM and1mM）was able to activate the cultured neonatal ratspinal cord astrocytes. Meanwhile, AZA (10μM) can inhibit the Glu-inducedastrocytes activation. In addition, Glu (1mM) can induce WT (AQP4positiveexpression) neonatal mice spinal cord astrocytes activation but cannot induce KO(AQP4negative expression) neonatal mice spinal cord astrocytes activation. Theseresults indicated that Glu-induced astrocytes activation was dependent on theexpression or function of AQP4. So the experiments in vitro confirmed directly thatAQP4expression or function is necessary for astrocyte activation.
In conclusion, AQP4involves in neuropathic pain, but not acute pain. Thepossible mechanism is that AQP4directly affects on spinal cord astrocytes activationand manipulates proinflammatory cytokines release.
引文
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[19] DeLeo JA, Rutkowski MD, Stalder AK, et al. Transgenic expression of TNF byastrocytes increases mechanical allodynia in a mouse neuropathy model. Neuroreport.2000;11(3):599-602.
[20] Madiai F, Hussain SR, Goettl VM, et al. Upregulation of FGF-2in reactivespinal cord astrocytes following unilateral lumbar spinal nerve ligation. Exp BrainRes.2003;148(3):366-76.
[21] Ma W, Quirion R. Partial sciatic nerve ligation induces increase in thephosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminalkinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus.Pain.2002;99(1-2):175-84.
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[25] Zhuang ZY, Gerner P, Woolf CJ, et al. ERK is sequentially activated in neurons,microglia, and astrocytes by spinal nerve ligation and contributes to mechanicalallodynia in this neuropathic pain model. Pain.2005;114(1-2):149-59.
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[28] Ji RR, Strichartz G. Cell signaling and the genesis of neuropathic pain. Sci STKE.2004;2004(252): reE14.
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[33] Gao YJ, Zhang L, Samad OA, et al. JNK-induced MCP-1production in spinalcord astrocytes contributes to central sensitization and neuropathic pain. J Neurosci.2009;29(13):4096-108.
[34] Gao YJ, Ji RR.Chemokines, neuronal–glial interactions, and central processingof neuropathic pain. Pharmacol Ther.2010;126(1):56-68.
[35] Tanga FY, Raghavendra V, DeLeo JA.Quantitative real-time RT-PCRassessment of spinal microglial and astrocytic activation markers in a rat model ofneuropathic pain. Neurochem Int.2004;45(2-3):397-407.
[36] Mika J. Modulation of microglia can attenuate neuropathic pain symptoms andenhance morphine effectiveness. Pharmacol Rep.2008;60(3):297-307.
[37] Obata K, Katsura H, Miyoshi K, et al. Toll-like receptor3contributes to spinalglial activation and tactile allodynia after nerve injury. J Neurochem.2008;105(6):2249-59.
[38] Wen YR, Tan PH, Cheng JK, et al. Microglia: a promising target for treatingneuropathic and postoperative pain, and morphine tolerance. J Formos Med Assoc.2011;110(8):487-94.
[39] Clark AK, Malcangio M. Microglial signalling mechanisms: cathepsin s andfractalkine. Exp Neurol.2012;234(2):283-92.
[40] Zhuo M, Wu G, Wu LJ. Neuronal and microglial mechanisms of neuropathic pain.Mol Brain.2011;4:31.
[41] Zhang J, De Koninck Y. Spatial and temporal relationship between monocytechemoattractant protein-1expression and spinal glial activation following peripheralnerve injury. J Neurochem.2006;97(3):772-83.
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[45] Guo LH, Schluesener HJ. The innate immunity of the central nervous system inchronic pain: The role of Toll-like receptors. Cell Mol Life Sci.2007;64(9):1128-36.
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[1] Haanpaa M, Attal N, Backonja M, et al. NeuPSIG guidelines on neuropathic painassessment. Pain.2001,152:14-27.
[2] Cavenagh J, Good P, Ravenscroft P. Neuropathic pain: are we out of the woods yet?Intern Med J.2006;36(4):251-5.
[3] Watkins LR, Hutchinson MR, Ledeboer A, et al. Glia as the “bad guys”:Implications for improving clinical pain control and the clinical utility of opioids.Brain Behav Immun.2007;21(2):131-46.
[4] Bradesi S. Role of spinal cord glia in the central processing of peripheral painperception. Neurogastroenterol Motil.2010;22(5):499-511.
[5] Coyle DE. Partial peripheral nerve injury leads to activation of astroglia andmicroglia which parallels the development of allodynic behavior. Glia.1998;23:75-83
[6] Raghavendra V, Tanga F, DeLeo JA. Inhibition of microglial activation attenuatesthe development but not existing hypersensitivity in a rat model of neuropathy. JPharmacol Exp Ther.2003,306:624-30
[7] Zhuang ZY, Gerner P, Woolf CJ, et al. ERK is sequentially activated in neurons,microglia, and astrocytes by spinal nerve ligation and contributes to mechanicalallodynia in this neuropathic pain model. Pain.2005,114:149-59
[8] Allen NJ, Barres BA. Neuroscience: Glia—more than just brain glue. Nature.2009;457(7230):675-7.
[9] Chen JJ, Lue JH, Lin LH, et al. Effects of pre-emptive drug treatment on astrocyteactivation in the cuneate nucleus following rat median nerve injury. Pain.2010;148(1):158-66.
[10] Tsuda M, Kohro Y, Yano T, et al. JAK-STAT3pathway regulates spinal astrocyteproliferation and neuropathic pain maintenance in rats. Brain.2011;134(Pt4):1127-39.
[11] Zhang X, Wang J, Zhou Q, et al. Brain-derived neurotrophic factor–activatedastrocytes produce mechanical allodynia in neuropathic pain. Neuroscience.2011;199:452-60.
[12] Kim D, Kim MA, Cho IH, et al. A Critical Role of Toll-like Receptor2in NerveInjury-induced Spinal Cord Glial Cell Activation and Pain Hypersensitivity. J BiolChem.2007;282(20):14975-83.
[13] Xia Y, Liu H, Shen A, et al. A Critical Role of Src-Suppressed C KinaseSubstrate in Rat Astrocytes After Chronic Constriction Injury. Neuromolecular Med.2010;12(3):205-16.
[14] Guillota X, Semeranoa L, Deckera P, et al. Pain and immunity. Joint Bone Spine.2012;79(3):228-36.
[15] Ohtori S, Takahashi K, Moriya H, et al. TNF-and TNF-Receptor Type1Upregulation in Glia and Neurons After Peripheral Nerve Injury. Spine (Phila Pa1976).2004;29(10):1082-8.
[16] DeLeo JA, Rutkowski MD, Stalder AK, et al. Transgenic expression of TNF byastrocytes increases mechanical allodynia in a mouse neuropathy model. Neuroreport.2000;11(3):599-602.
[17] Madiai F, Hussain SR, Goettl VM, et al. Upregulation of FGF-2in reactivespinal cord astrocytes following unilateral lumbar spinal nerve ligation. Exp BrainRes.2003;148(3):366-76.
[18] Ma W, Quirion R. Partial sciatic nerve ligation induces increase in thephosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminalkinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus.Pain.2002;99(1-2):175-84.
[19] Zhuang ZY, Wen YR, Zhang DR, et al. A Peptide c-Jun N-Terminal Kinase (JNK)Inhibitor Blocks Mechanical Allodynia after Spinal Nerve Ligation: Respective Rolesof JNK Activation in Primary Sensory Neurons and Spinal Astrocytes for NeuropathicPain Development and Maintenance. J Neurosci.2006;26(13):3551-60.
[20] Ji RR, Kawasaki Y, Zhuang ZY, et al. Possible role of spinal astrocytes inmaintaining chronic pain sensitization: review of current evidence with focus onbFGF/JNK pathway. Neuron Glia Biol.2006;2(4):259-69.
[21] Crown ED, Gwak YS, Ye Z, et al. Activation of p38MAP kinase is involved incentral neuropathic pain following spinal cord injury. Exp Neurol.2008;213(2):257-67.
[22] Zhuang ZY, Gerner P, Woolf CJ, et al. ERK is sequentially activated in neurons,microglia, and astrocytes by spinal nerve ligation and contributes to mechanicalallodynia in this neuropathic pain model. Pain.2005;114(1-2):149-59.
[23] Ji RR, Gereau RW4th, Malcangio M, et al. MAP kinase and pain. Brain Res Rev.2009;60(1):135-48.
[24] Crown ED. The role of mitogen activated protein kinase signaling inmicrogliaand neurons in the initiation and maintenance of chronic pain. Exp Neurol.2012;234(2):330-9.
[25] Ji RR, Strichartz G. Cell Signaling and the Genesis of Neuropathic Pain. SciSTKE.2004;2004(252): reE14.
[26] Tanga FY, Raghavendra V, Nutile-McMenemy N, et al. Role of astrocytic s100βin behavioral hypersensitivity in rodent models of neuropathic pain. Neuroscience.2006;140(3):1003-10.
[27] Werry EL, Liu GJ, Bennett MR. Glutamate-stimulated ATP release from spinalcord astrocytes is potentiated by substance P. J Neurochem.2006;99(3):924-36.
[28] Hulsebosch CE, Hains BC, Crown ED, et al. Mechanisms of chronic centralneuropathic pain after spinal cord injury. Brain Res Rev.2009;60(1):202-13.
[29] Gao YJ, Ji RR. Targeting Astrocyte Signaling for Chronic Pain.Neurotherapeutics.2010;7(4):482-93.
[30] Gao YJ, Zhang L, Samad OA, et al. JNK-induced MCP-1production in spinalcord astrocytes contributes to central sensitization and neuropathic pain. J Neurosci.2009;29(13):4096-108.
[31] Gao YJ, Ji RR.Chemokines, neuronal–glial interactions, and central processingof neuropathic pain. Pharmacol Ther.2010;126(1):56-68.
[32] Tanga FY, Raghavendra V, DeLeo JA.Quantitative real-time RT-PCRassessment of spinal microglial and astrocytic activation markers in a rat model ofneuropathic pain. Neurochem Int.2004;45(2-3):397-407.
[33] Mika J. Modulation of microglia can attenuate neuropathic pain symptoms andenhance morphine effectiveness. Pharmacol Rep.2008;60(3):297-307.
[34] Obata K, Katsura H, Miyoshi K, et al. Toll-like receptor3contributes to spinalglial activation and tactile allodynia after nerve injury. J Neurochem.2008;105(6):2249-59.
[35] Wen YR, Tan PH, Cheng JK, et al. Microglia: A Promising Target for TreatingNeuropathic and Postoperative Pain, and Morphine Tolerance. J Formos Med Assoc.2011;110(8):487-94.
[36] Clark AK, Malcangio M. Microglial signalling mechanisms: Cathepsin S andFractalkine. Exp Neurol.2012;234(2):283-92.
[37] Zhuo M, Wu G, Wu LJ. Neuronal and microglial mechanisms of neuropathic pain.Mol Brain.2011;4:31.
[38] Zhang J, De Koninck Y. Spatial and temporal relationship between monocytechemoattractant protein-1expression and spinal glial activation following peripheralnerve injury. J Neurochem.2006;97(3):772-83.
[39] Inoue K, Tsuda M, Koizumi S. ATP-and Adenosine-Mediated Signaling in theCentral Nervous System: Chronic Pain and Microglia: Involvement of the ATPReceptor P2X4. J Pharmacol Sci.2004;94(2):112-4.
[40] Tsuda M, Inoue K, Salter MW.Neuropathic pain and spinal microglia: a bigproblem from molecules in‘small’glia. Trends Neurosci.2005;28(2):101-7.
[41] Inoue K. Purinergic systems in microglia. Cell Mol Life Sci.2008;65(19):3074-80.
[42] Guo LH, Schluesener HJ. The innate immunity of the central nervous system inchronic pain: The role of Toll-like receptors. Cell Mol Life Sci.2007;64(9):1128-36.
[43] Trang T. Beggs S. Salter MW. Purinoceptors in microglia and neuropathic pain.Pflugers Arch-Eur J Physiol.2006;452:645–652.
[2] Haanpaa M, Attal N, Backonja M, et al. NeuPSIG guidelines on neuropathic painassessment. Pain.2001,152:14-27.
[3] Cavenagh J, Good P, Ravenscroft P. Neuropathic pain: are we out of the woods yet?Intern Med J.2006;36(4):251-5.
[4] Baron R, Binder A, Wasner G. Neuropathic pain: diagnosis, pathophysiologicalmechanisms, and treatment. Lancet Neurol.2010;9(8):807-19.
[5] Thacker MA, Clark AK, Marchand F, et al. Pathophysiology of peripheralneuropathic pain: immune cells and molecules. Anesth Analg.2007;105(3):838-47.
[6] Watkins LR, Hutchinson MR, Ledeboer A, et al. Glia as the “bad guys”:implications for improving clinical pain control and the clinical utility of opioids.Brain Behav Immun.2007;21(2):131-46.
[7] Bradesi S. Role of spinal cord glia in the central processing of peripheral painperception. Neurogastroenterol Motil.2010;22(5):499-511.
[8] Coyle DE. Partial peripheral nerve injury leads to activation of astroglia andmicroglia which parallels the development of allodynic behavior. Glia.1998;23:75-83
[9] Raghavendra V, Tanga F, DeLeo JA. Inhibition of microglial activation attenuatesthe development but not existing hypersensitivity in a rat model of neuropathy. JPharmacol Exp Ther.2003,306:624-30
[10] Zhuang ZY, Gerner P, Woolf CJ, et al. ERK is sequentially activated in neurons,microglia, and astrocytes by spinal nerve ligation and contributes to mechanicalallodynia in this neuropathic pain model. Pain.2005,114:149-59
[11] Allen NJ, Barres BA. Neuroscience: Glia—more than just brain glue. Nature.2009;457(7230):675-7.
[12] Chen JJ, Lue JH, Lin LH, et al. Effects of pre-emptive drug treatment onastrocyte activation in the cuneate nucleus following rat median nerve injury. Pain.2010;148(1):158-66.
[13] Tsuda M, Kohro Y, Yano T, et al. JAK-STAT3pathway regulates spinal astrocyteproliferation and neuropathic pain maintenance in rats. Brain.2011;134(Pt4):1127-39.
[14] Zhang X, Wang J, Zhou Q, et al. Brain-derived neurotrophic factor–activatedastrocytes produce mechanical allodynia in neuropathic pain. Neuroscience.2011;199:452-60.
[15] Kim D, Kim MA, Cho IH, et al. A critical role of toll-like receptor2in nerveinjury-induced spinal cord glial cell activation and pain hypersensitivity. J Biol Chem.2007;282(20):14975-83.
[16] Xia Y, Liu H, Shen A, et al. A critical role of src-suppressed c kinase substrate inrat astrocytes after chronic constriction injury. Neuromolecular Med.2010;12(3):205-16.
[17] Guillota X, Semeranoa L, Deckera P, et al. Pain and immunity. Joint Bone Spine.2012;79(3):228-36.
[18] Ohtori S, Takahashi K, Moriya H, et al. TNF-and TNF-receptor type1upregulation in glia and neurons after peripheral nerve injury. Spine (Phila Pa1976).2004;29(10):1082-8.
[19] DeLeo JA, Rutkowski MD, Stalder AK, et al. Transgenic expression of TNF byastrocytes increases mechanical allodynia in a mouse neuropathy model. Neuroreport.2000;11(3):599-602.
[20] Madiai F, Hussain SR, Goettl VM, et al. Upregulation of FGF-2in reactivespinal cord astrocytes following unilateral lumbar spinal nerve ligation. Exp BrainRes.2003;148(3):366-76.
[21] Ma W, Quirion R. Partial sciatic nerve ligation induces increase in thephosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminalkinase (JNK) in astrocytes in the lumbar spinal dorsal horn and the gracile nucleus.Pain.2002;99(1-2):175-84.
[22] Zhuang ZY, Wen YR, Zhang DR, et al. A peptide c-Jun N-Terminal Kinase (JNK)inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles ofJNK activation in primary sensory neurons and spinal astrocytes for neuropathic paindevelopment and maintenance. J Neurosci.2006;26(13):3551-60.
[23] Ji RR, Kawasaki Y, Zhuang ZY, et al. Possible role of spinal astrocytes inmaintaining chronic pain sensitization: review of current evidence with focus onbFGF/JNK pathway. Neuron Glia Biol.2006;2(4):259-69.
[24] Crown ED, Gwak YS, Ye Z, et al. Activation of p38MAP kinase is involved incentral neuropathic pain following spinal cord injury. Exp Neurol.2008;213(2):257-67.
[25] Zhuang ZY, Gerner P, Woolf CJ, et al. ERK is sequentially activated in neurons,microglia, and astrocytes by spinal nerve ligation and contributes to mechanicalallodynia in this neuropathic pain model. Pain.2005;114(1-2):149-59.
[26] Ji RR, Gereau RW4th, Malcangio M, et al. MAP kinase and pain. Brain Res Rev.2009;60(1):135-48.
[27] Crown ED. The role of mitogen activated protein kinase signaling in microgliaand neurons in the initiation and maintenance of chronic pain. Exp Neurol.2012;234(2):330-9.
[28] Ji RR, Strichartz G. Cell signaling and the genesis of neuropathic pain. Sci STKE.2004;2004(252): reE14.
[29] Tanga FY, Raghavendra V, Nutile-McMenemy N, et al. Role of astrocytic s100βin behavioral hypersensitivity in rodent models of neuropathic pain. Neuroscience.2006;140(3):1003-10.
[30] Werry EL, Liu GJ, Bennett MR. Glutamate-stimulated ATP release from spinalcord astrocytes is potentiated by substance P. J Neurochem.2006;99(3):924-36.
[31] Hulsebosch CE, Hains BC, Crown ED, et al. Mechanisms of chronic centralneuropathic pain after spinal cord injury. Brain Res Rev.2009;60(1):202-13.
[32] Gao YJ, Ji RR. Targeting astrocyte signaling for chronic pain. neurotherapeutics.2010;7(4):482-93.
[33] Gao YJ, Zhang L, Samad OA, et al. JNK-induced MCP-1production in spinalcord astrocytes contributes to central sensitization and neuropathic pain. J Neurosci.2009;29(13):4096-108.
[34] Gao YJ, Ji RR.Chemokines, neuronal–glial interactions, and central processingof neuropathic pain. Pharmacol Ther.2010;126(1):56-68.
[35] Tanga FY, Raghavendra V, DeLeo JA.Quantitative real-time RT-PCRassessment of spinal microglial and astrocytic activation markers in a rat model ofneuropathic pain. Neurochem Int.2004;45(2-3):397-407.
[36] Mika J. Modulation of microglia can attenuate neuropathic pain symptoms andenhance morphine effectiveness. Pharmacol Rep.2008;60(3):297-307.
[37] Obata K, Katsura H, Miyoshi K, et al. Toll-like receptor3contributes to spinalglial activation and tactile allodynia after nerve injury. J Neurochem.2008;105(6):2249-59.
[38] Wen YR, Tan PH, Cheng JK, et al. Microglia: a promising target for treatingneuropathic and postoperative pain, and morphine tolerance. J Formos Med Assoc.2011;110(8):487-94.
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