The Role Of Brain-derived Neurotrophic Factor In Pain Facilitation And Neuro-immunological Mechanism In Rat Model Of Bone Cancer Pain

We have described our mimic rat model of bone cancer pain. Based on that, we foundmicroglial P2X4purinoceptor (P2X4R) is associated with behavioral hypersensitivityinduced by bone cancer. Moreover, brain-derived neurotrophic factor (BDNF) has beenshown to be a key factor mediating the neuron-glia signaling and induce centralsensitization. It is evoked by the activation of microglial P2X4R that leading to the releaseof BDNF from microglia under neuropathic pain. However, mechanisms involved incentral sensitization of BDNF remain poorly understood, and little is known about its rolein bone cancer pain which is in some ways unique. Therefore,we speculated that activationof microglial P2X4R that leading to the release of BDNF from microglia in relation to theCancer induced bone pain, in the dorsal horn under a rat model of bone cancer pain. Weapplied the research to make our studies go further in a rat model of bone cancer pain andthe experiments in vivo and in vitro. The techniques used in the research includemorphology, ethology, molecule biology and radiology. The observations will includetechnique of small interfering RNA (siRNA),ethological test, real-time PCR, western Blot,ELISA and 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 P2X4R receptor and BDNF(molecular level),microglia and neuron (cellular level), or immune and sensory system (systematic level). Objective and Methods To investigate the possibility of brain-derived neurotrophicfactor (BDNF) are involved in the induction of bone cancer pain and its mechanism. Bonecancer pain model was created by receiving left superior extremity intra-tibialinoculations of Walker256syngeneic SD rat carcinoma cells in female SD rats weighing150-180g. The development of bone tumour and pain were observated by digitalradiology, histology, pain behavioural signs indicative of pain and analgesicpharmacodynamics. The following study was conducted in accordance with thecorresponding experimental grouping. And the observations will include technique ofethological test (n=10), Western blots (n=4), Real-time PCR (n=4),immuno-histochemistry(n=4), small interfering RNA (siRNA)(n=4) and SRB tes(tn=6):①To investigate if alterations in BDNF expression in the L4-6spinal cord and dorsalroot ganglion(DRG) following the bone cancer pain model.②To investigate whetherminocycline intrathecal injection could attenuated cancer induced bone pain andenhancement of BDNF expression;③To investigate the anti-nociceptive role anddownstream signaling (phosphorylation of extracellular signal-regulated proteinkinase1/2,p-ERK1/2) mechanism of intrathecal BDNF IgG administration under cancerinduced mechanical allodynia;④To pick out the siRNA which could most effectivelyinhibit the expression of BDNF in microglial cells and to detect the cytotoxicity of thetransfection complex;⑤To investigate whether intrathecal BDNF siRNA attenuates bonecancer pain. Results①Data from Western blots, real-time PCR andimmunohistochemistry all revealed a significant up-regulation of BDNF (n=4, P <0.01),and the increase all reached peak on day6after walker256inoculation;②The PWTs andthe ambulatory scores in group MC-E (injected early from day4to day6) increasedsignificantly post minocycline intrathecal injection compared with that in groups vehicleand control (n=10, P<0.01). But minocycline injection later from day10to day12(GroupMC-L) had no effect on nociceptive behaviour(n=10, P>0.05).In addition,immunohistochemistry experiments revealed a significant up-regulation of OX-42/BDNF double-immunoreactive products in the L4-6spinal cord compared with those in shamrats on day6after inoculation. Further, minocycline intrathecal injection early couldattenuate enhancement of OX-42or BDNF expression in the L4-6spinal dorsal horn inthe bone cancer pain model.③Intrathecal administration of BDNF IgG attenuated cancerinduced mechanical allodynia(n=10, P<0.01) and enhancement of BDNF or p-ERK1/2expression(n=6, P<0.01) in the L4-6spinal dorsal horn in the bone cancer pain model.④The highest transfection efficiency was achieved with the following optimized condition:at passage2-3,1×10~6cells,1:3ratio for liposome and siRNA,30min for complexformation(n=6,P<0.01). BDNF mRNA and protein levels in microglial cells line (HAPI),normalized to β-actin, were significantly reduced in BDNF siRNA versus mismatchsiRNA-and vehicle-treated cells. The BDNF siRNA1588was the most effective atknocking down BDNF expression.⑤In the siRNA-treated early CIBP group (From day4to6once daily, Group SIBDNF-E), the PWTs were significantly higher than thoseobserved in mismatch siRNA-and vehicle-treated rats(n=10),and there were nosignificant differences compared to normal rats. Meanwhile, the ambulatory scores ofGroup SIBDNF-E were significantly lower than those observed in vehicle-treated rats. Inthe siRNA-treated later group (From day10to12once daily,Group SIBDNF-L), thePWTs were significantly elevated1-24h post intrathecal injection compared with themismatch siRNA-and vehicle-treated rats(n=10), and the peak time of anti-nociceptiveeffect was at4h, but still lower than in normal rats. The same results were found in thedata of ambulatory scores. In addition, intrathecal delivery of trkB-IgG to the lumbardorsal horn30min before BDNF injection in these animals significantly attenuated theBDNF induced nociceptive response. Conclusions BDNF-trkB-ERK1/2-mediatedsignaling pathway maybe involved in the induction and development of bone cancer pain. Objective and Methods This study explored whether brain-derived neurotrophicfactor (BDNF) could modulate activation of N-methyl-D-aspartate (NMDA) receptors inrelation to the cancer induced bone pain in rats. Thereby desire to study the mechanism ofBDNF in the induction and development of bone cancer pain. Bone cancer pain model wascreated by receiving left superior extremity intra-tibial inoculations of Walker256syngeneic SD rat carcinoma cells in female SD rats. The following study was conducted inaccordance with the corresponding experimental grouping. And the observations willinclude technique of ethological tes(tn=8), Real-time PCR(n=4),immuno-histochemistry(n=4)and small interfering RNA (siRNA)(n=4):①To investigate if alterations inphosphorylation of NMDA receptor subunit NR1(p-NR1) expression in the L4-6spinalcord and dorsal root ganglion(DRG) following the bone cancer pain model.②Toinvestigate whether BDNF siRNA intrathecal injection could attenuate cancer inducedbone pain and enhancement of BDNF and p-NR1expression;③To investigate if BDNFcould enhance NMDA-induced allodynia in the rat spinal cord through TrkB signaling invivo. Results①Data from real-time PCR and immunohistochemistry all revealed asignificant up-regulation of NR1(n=4, P <0.01), and the increase all reached peak on day9after walker256inoculation;②Compared with rats treated with vehicle or the mismatchsiRNA, BDNF siRNA injection (intrathecal injection from day4to day6) significantlydecreased p-NR1in the spinal cord and DRG neurons was coincident with decreasedexpression of BDNF in the spinal cord on day9after inoculation (n=4, P <0.01);③BDNF enhances NMDA-induced allodynia in the rat spinal cord and intrathecal delivery oftrkB-IgG significantly attenuated the pain behaviour (n=8, P <0.01). ConclusionsThese results demonstrate that BDNF maybe could modulate activation of NMDAreceptors in relation to the cancer induced bone pain in rats through its receptor trkB. Objective and Methods To investigate the possibility of activation of microglialP2X4purinoceptor (P2X4R) leading to the release of brain-derived neurotrophic factor(BDNF) which are involved in the induction of bone cancer pain and its mechanism.Bone cancer pain model was created by receiving left superior extremity intra-tibialinoculations of Walker256syngeneic SD rat carcinoma cells in female SD rats. Thefollowing study was conducted in accordance with the corresponding experimentalgrouping. And the observations will include technique of ethological tes(tn=10), Westernblots(n=4), Real-time PCR(n=4),immuno-histochemistry(n=4)and small interferingRNA (siRNA)(n=4):①To determine the effects of siRNA suppressing the P2X4Rexpression in microglial cells in vitro;②To investigate if activation of microglial P2X4Rleading to the secretion and release of BDNF from microglia which dependent onp38MAPK signaling.③To investigate whether alterations in P2X4R, phosphorylatedp38(p-p38),BDNF, TNFα and IL-1βin the bone cancer pain model.④To investigate ifintrathecal P2X4R siRNA could attenuate bone cancer pain and enhancement of P2X4R,p-p38,BDNF, TNFα and IL-1β expression; Results①P2X4R mRNA and proteinlevels in microglial cells line (HAPI), normalized to β-actin, were significantly reduced inP2X4R siRNA versus mismatch siRNA-and vehicle-treated cells(n=4, P <0.01).②ATP causes an increase of BDNF protein in rat microglial cells and their supernatants,peaking60min after ATP addition, which could be blocked by P2X4R siRNA apply(n=4,P <0.01). Further, ATP causes an increase of p-p38protein which leading to the release of BDNF from microglia and dependent on P2X4receptor signaling in vitro (n=4, P <0.01).③The mRNA or protein expression levels of P2X4R were all increased in bonecancer pain rats respectively, and the highest levels were all at day18(n=4, P <0.01).There were significantly enhancement in the expression of p-p38, BDNF, TNFαandIL-1β in the dorsal horn after Walker256cells' inoculation, compared with those innormal and sham rats(n=4, P <0.01). In addition, on day6following bone cancer pain,P2X4R/BDNF or P2X4R/p-p38double labeled cells all revealed a heavier colocalizationthan sham group.④Intrathecal P2X4R siRNA attenuates bone cancer pain andenhancement of p-p38, BDNF, TNFαand IL-1βexpression in the L4-6spinal dorsal horn.Conclusions Activation of microglial P2X4R maybe lead to the release of BDNF frommicroglia and involved in the induction of bone cancer pain which dependent onp38MAPK signaling.