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Neuroimmunologic Mechanism of Spinal MCP-1Modulating the Release of Fractalkine in Rat Model of Bone Cancer Pain

Author: HuJi
Tutor: YangJianPing
School: Suzhou University
Course: Anesthesiology
Keywords: MCP-1/CCR2 Fractalkine/CX3CR1 Bone cancer pain Nerve ImmunityMCP-1/CCR2 Spinal cord Microglia RatsCathepsin S Ratsp38MAPK Rats
CLC: R738.1
Type: PhD thesis
Year: 2013
Downloads: 70
Quote: 0
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Abstract


We have described our mimic rat model of bone cancer pain. Based on that, weprevious studies have demonstrated that fractalkine, a newly discovered chemokine, isimplicated in spinal cord neuron-to-microglia activation signaling as well as mediation ofbone cancer pain via its sole receptor CX3CR1, which is specifically expressed onmicroglia. Recently, it has been demonstrated that chemokine Monocyte chemoattractantprotein-1(MCP-1,CCL2)/CCR2plays a critical role in pathological pain via neuron–gliacommunication, resulting in central sensitization; and regulates circulating monocytesFractalkine/CX3CR1expression and function. However, whether it is involved in bonecancer pain which is unique and distinct from neuropathic pain, and the underlyingmechanisms have not been elucidated. Therefore, we speculated that MCP-1/CCR2activated microglia and modulated Fractalkine/CX3CR1signaling in relation tocancer-induced bone pain, in the dorsal horn of a rat model of bone cancer pain. Thetechniques used in our studies include morphology, ethology and molecule biology. Theobservations will include technique of ethological test, real-time PCR, western blot, ELISAand immuno-histochemistry. We believe that the present research will primarilydemonstrate the neuro-immunological mechanism of bone cancer pain through a bran-newpoint based on the “cross talk” between CCR2and CX3CR1receptor (molecular level),microglia and neuron (celluar level), or immune and sensory system (systematic level). PartⅠ The Role of MCP-1/CCR2in Pain Facilitation and SpinalMechanisms in Rat Model of Bone Cancer PainObjective To investigate the role of MCP-1/CCR2signaling in bone cancer painand its spinal mechanism. Methods A rat model of bone cancer pain was developed byintra-tibial inoculations of Walker256breast cancer cells in female Sprague-Dawley rats(weight150-180g). The following study was conducted in accordance with thecorresponding experimental grouping. And the observations will include technique ofethological test (n=12)、 Real-time PCR (n=4)、 Western-Blot (n=4)、 immuno-histochemistry (n=4) and ELISA (n=4):1、To observe the changes in expression anddistribution of MCP-1and its receptor CCR2in L4-6spinal cord in a rat model of bonecancer pain.2、To observe the effect of intrathecal injection of MCP-1neutralizingantibody on behavioral responses of bone cancer pain rats and the expression changes ofspinal CCR2、OX-42(microglial marker) and cytokines (IL-1β、TNF-α).3、To observe theeffect of intrathecal injection of exogenous recombinant MCP-1on behavioral responses ofna ve rats. Results1、There is a significant increase in MCP-1and CCR2expression in the spinal cord from day6to day18following aninoculation with Walker256cells utilizing RT-PCR and Western-Blot (p<0.01);CCR2was expressed by both microglia and neurons in the spinal cord of bone cancer pain rats.2、During4~6days after operation, intrathecal anti-MCP-1antibody delays initiation ofmechanical allodynia of bone cancer pain (p<0.01), and attenuates CCR2、OX-42、IL-1β、TNF-α expression in the spinal cord (p<0.01); Intrathecal anti-MCP-1antibody wasadministered once a day from day10to day12attenuates established mechanical allodyniaof bone cancer pain (p<0.01).3、Intrathecal injection of exogenous rmMCP-1produced a striking mechanical allodynia in a dose-dependent manner whenadministered to na ve rats (p<0.01). Conclusion MCP-1/CCR2signaling might beinvolved in the initiation and maintenance of BCP via crosstalk between these neurons andglial cells. PartⅡ Involvement of MCP-1in bone cancer pain through theactivation of microglia Cathepsin S in the spinal cordObjective To investigate whether MCP-1could modulate activation of microgliaCathepsin S (CatS) in the spinal cord of bone cancer pain rats. Methods A rat model ofbone cancer pain was developed by intra-tibial inoculations of Walker256breast cancercells in female Sprague-Dawley rats (weight150-180g). The following study wasconducted in accordance with the corresponding experimental grouping. And theobservations will include technique of ethological test (n=8)、Western-Blot (n=4) andimmuno-histochemistry (n=4):1、To observe the changes in expression and distribution ofCatS in L4-6spinal cord in a rat model of bone cancer pain.2、To observe the effect ofintrathecal injection of MCP-1neutralizing antibody on the expression changes of spinalCatS in bone cancer pain rats.3、To observe the effect of intrathecal injection of exogenousrecombinant MCP-1on the expression changes of spinal CatS in na ve rats. Results1、Western-Blot analysis revealed that the CatS expression significantly increased in thespinal cord from day6to day18following inoculation with Walker256cells (p<0.01);CatS was expressed by microglia in the spinal cord of bone cancer pain rats.2、Intrathecal anti-MCP-1antibody attenuates established mechanical allodynia of bonecancer pain (p<0.01) and enhancement of microglia CatS expression in the spinal cord(p<0.01).3、Intrathecal injection of exogenous rmMCP-1induced a significant increase inCatS expression in the spinal cord of na ve rats (p<0.01). Conclusion MCP-1might beinvolved in the bone cancer pain through the activation of microglia Cathepsin S in thespinal cord. Part Ⅲ Cathepsin S modulate the liberation of Fractalkine in relation tothe bone cancer painObjective To investigate whether Cathepsin S could modulate the liberation ofFractalkine in relation to the bone cancer pain in rats. Methods A rat model of bonecancer pain was developed by intra-tibial inoculations of Walker256breast cancer cells infemale Sprague-Dawley rats (weight150-180g). Rats were randomly divided into5groups (n=10). The observations will include technique of ethological test (n=8)、immuno-histochemistry (n=4) and ELISA(n=10): To observe the effect of intrathecalinjection of an irreversible CatS inhibitor, morpholinurea-leucinehomophenylalanine-vinylphenyl sulfone (LHVS) on behavioral responses of bone cancer pain rats and theexpression changes of both spinal OX-42(microglial marker) and FKN, and the levels ofFKN in cerebrospinal fluid (CSF). Results Intrathecal LHVS was administered once a dayfrom day10to day12attenuates established mechanical allodynia of bone cancer pain(p<0.01) and enhancement of OX-42expression in the spinal cord (p<0.01) as well as thelevels of FKN in CSF (p<0.01). Conclusion Cathepsin S might be involved in thedevelopment of bone cancer pain through modulating the liberation of Fractalkine. Part Ⅳ MCP-1modulating the release of Fractalkine through theactivation of p38MAPK pathway in the spinal cord in rat model of bonecancer painObjective To investigate whether MCP-1could modulate the liberation ofFractalkine through the activation of p38MAPK pathway in the spinal cord in rat model ofbone cancer pain. Methods A rat model of bone cancer pain was developed by intra-tibialinoculations of Walker256breast cancer cells in female Sprague-Dawley rats (weight150-180g).The following study was conducted in accordance with the correspondingexperimental grouping. And the observations will include technique of ethological test(n=8)、Real-time PCR (n=4)、Western-Blot (n=4)、immuno-histochemistry (n=4) andELISA (n=4):1、To observe the effect of intrathecal injection of MCP-1neutralizingantibody on the changes of spinal p38、CX3CR1expression and the levels of FKN in CSF.2、To observe the effect of intrathecal injection of exogenous rmMCP-1on the expressionchanges of spinal p38、 CX3CR1in na ve rats.3、To observe the effect of intrathecalinjection of p38MAPK inhibitor4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) on behavioral responses of bone cancer pain ratsand the expression changes of both spinal CCR2and CX3CR1, and the levels of FKN in cerebrospinal fluid(CSF). Results Intrathecal anti-MCP-1antibody was administered oncea day from day10to day12attenuates p38and CX3CR1expression in the spinal cord ofbone cancer pain (p<0.01), and the levels of FKN in CSF (p<0.01).3、Intrathecal injectionof exogenous rmMCP-1induced a significant increase in p38and CX3CR1expressionexpression in the spinal cord of na ve rats (p<0.01).4、Intrathecal SB203580wasadministered once a day from day4to day6delays initiation of mechanical allodynia ofbone cancer pain (p<0.01) and attenuates the expression of CCR2and CX3CR1in thespinal cord (p<0.01) as well as the levels of FKN in CSF of bone cancer pain (p<0.01).Conclusion MCP-1might be modulate the liberation of Fractalkine through theactivation of p38MAPK pathway and further act on microglial CX3CR1participate in the

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