Exploring The Role Of Aquaporin4in The Neuropathic Pain And The Possible Mechanisms

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.