| BackgroundThe majority of patients with metastatic bone cancer will experience moderate to severe pain. Bone pain is one of the most common types of chronic pain in these patients.Bone cancer pain is usually progressiveas the disease advances, and is particularly difficult to treat. The mechanisms responsible for bone pain are poorly understood, but bone cancer pain seems to be enhanced by a state of spinal sensitization. Previous studies indicate that spinal sensitization of bone cancer pain appears unique when compared to changes that occur in persistent inflammatory or neuropathic pain states. It has been hypothesized that one such sensitization mechanism is the regulation of gene expression. Thus, identification of unique gene expression profiles in the spinal cord from animals with bone cancer pain may provide insight into the widespread factors that drive spinal cord plasticity. This, in turn, will contribute to the development of more effective treatments for bone cancer pain.Genechip of full gene probe sets instead of traditional approach and coulddescribe all of the potential genes that may contribute to generation and maintenance of chronic pain.In the present study, whole-genome mRNA expression profiles in spinal cords of rats with bone cancer pain, inflammatory pain and neuropathic pain were examined to identify target genes that may contribute to spinal sensitization of chronic pain. The mRNA expression was examined using Affymetrix Rat Genome230.2microarrays that include30,000probe sets. The function and cross-effect of the pain-related genes were analysised with cluster and molecule annotation system to reveal the molecule mechanism in development of chronic pain.Spinal sensitization is the main mechanism of chronic pain.Plasticity changes of synapseincluding modifications of existing synapses and formation or loss of synaptic connections contribute to sensitization. Previousstudies demonstrated that bone cancer pain resulted from an augmentation of excitatory synaptic transmission between neurons over a wide area of spinal lumbar segments, but whether the synaptic plasticity is due to modifications of existing synapses or the formation of new synaptic connections is still unknown. Here we have shown that sarcoma implantation induced excitability synaptogenesis which drives the development of bone cancer pain. But the mechanisms of synapse formation induced by sarcoma inoculation are still unknown.New synapse establishment requires an interaction between axons and dendrites, accompanied by the appositional organization of pre-and postsynaptic specializations. Thus, the axon guidance cues are required to help establish specific connections between a neuron and its multiple cellular targets. Slit is a largeextracellular matrix protein that was identified and implicated in axonal guidance and branching during the development of nervous system. Roundabout (Robo) was found to encode a protein which is a transmembrane receptor of slit and a member of the immunoglobulin superfamily. Slits act through receptors of the Robo in axon chemorepulsion and guidance cues during the development of the central nervous system. The small GTPase RhoA, a signaling protein regulating neuronal morphogenesis, plays a critical role in regulationof axonal re-generation.In our chip array experiments, we have identified tSlit2upregulation and Robo1and RhoA downregulation in rats with bone cancer pain. Thus, it seems reasonable to hypothesize that Slit2/Robo1via RhoA mediate the synaptogenesis and contribute to the spinal sensitization of bone cancer pain. To test this notion, siRNA was used to knock down Slit2and Robol in vivo and in vitro, a carcinoma tibia implantation rat model was used to test the bone cancer-related pain behaviors, and synaptogenesis was examined in vivo and vitro. We have shown thatcarcinoma inoculation induces excitatory synaptogenesis and bone cancer pain behaviors which are reversed by Slit2knockdown but aggravated by Robol knockdown. In vitro experiments, neurite outgrowth and synaptogenesis of cultured neurons are inhibited by knockdown Slit2, but enhanced by Robo1knockdown. And that carcinoma implantation induces an increase of Slit2and decrease Robol and RhoA.whileSlit2knockdown results in increase of Robol and RhoA via N-terminal of Slit2directly bounding to Robo1and Robo1knockdown decreased RhoA with Slit2uneffected. These results indicate thatSlit2inhibiting Robo1and then RhoA promotessynaptogenesis and contributes to bone cancer pain. Our findingssuggest a new spinal sensitization mechanism underlying the development of bone cancer pain.Methods and results1. Unique gene expression profiles in spinal cord of rats with bone cancer painMethods To identify altered genes that might contribute to spinal sensitization of chronic pain, we conducted a time-course mRNA profiling experiment on carcinoma tibia implantation, L5spinal nerve ligation (SNL), and intraplantar complete Freund’s adjuvant (CFA) injection rat models. The mRNA expression in lumbar spinal cord of respective animals was examined using Affymetrix Rat Genome230.2microarrays that include30,000probe sets.The gene expression profiles in the chronic pain rats were compared with the corresponding data of sham.Results We identified2245,1989and1679probe sets that were upregulated respectively on days3,7,14post-CFA injection,1570,1560and1494probe sets that were upregulated respectively on days7,14,21post-SNL, and1381,1740and1908probe sets that were upregulated respectively on days7,14,21post-carcinoma implantation.These genes may involve the onset or maintenance of chronic pain. By comparing the microarray results among the three rat models, we were able to identify a large set of genes whose expressions were upregulated at each time point as a unique result of CFA injection, SNL and carcinoma implantation (352,196and216probe sets identified respectively). These genes may be involved in the maintenance of inflammatory, neuropathic and bone cancer pain, respectively. When the genes whose expressions were upregulated were pooled together,1093,884and344genes overlapped among three time points and344gene sets were overlap among three models,.This suggests that these three types of pain on the one hand shared a similar mechanisms, but on the other hand have a special gene expression profile themselves.2. Slit2/Robol promotes synaptogenesis and contributes to bone cancer painMethods A Local injection of carcinoma cells directly into rat tibia were used to mimic clinical bone cancer pain and the siRNA lentivirus were injected into the ipsilateral dorsal horn to specially knock down Slit2or Robol expression. Coimmunoprecipitation assay using L3-5spinal cord of carcinoma implantation rats and cultured neurons was used to test whether functions of Slit2in bone cancer pain was mediated through direct interaction with Robo1. Immunoprecipitation was performed using antibody to Slit2and Robo1, Slit2were detectable by western blotting using antibodies to Robo1, Slit2. Paw withdrawal threshold was measurement of mechanical allodynia.Tthe subcellular distribution and expression of Slit2, Robo1and RhoA were tested by mmunofluorescent stain, western blot and RT-PCR. Synapse was examined with Transmission Electron Microscopy and excitatory synapse was examined using double immunofluorescent labeling with the anti-Synaptophysin (Syn), a presynaptic vesicle protein and anti-PSD95antibody, a major scaffolding protein in the excitatory postsynaptic density (PSD).Results Immunofluorescence stain indicated that Slit2, Robo1and RhoA were colocalized in neuron of dorsal spinal cord. Carcinoma implantation resulted in an increase of Slit2, but decrease of Robo1and RhoA in ipsilateral dorsal horn. Transfection of dorsal horn neurons with siRNAto slit2reduced the level of endogenous slit2protein, but increased Robol and RhoA, while knockdown of Robol decreased RhoA and had no effect on Slit2. These Immunohistochemical studies were confirmed by RT-PCR and immunoblot analysis. The results of immunoprecipitation suggested that Slit2could bind to Robol directly in vivo and vitro.Taken together, carcinoma implantation upregulated Slit2which inhibited the expression of Robol and subsequent RhoA via Slit2binding to Robol. We had showed that carcinoma inoculation resulted in significant bone cancer-related pain behaviors on the ipsilateral paw, but not on the contralateral side. Bone cancer pain were attenuated by Slit2knockdown but aggravated by Robol knockdown. These results indicated that upregulation of Slit2and thereby downregulation of Robol and then RhoA were necessary, but not sufficient for the development of bone cancer pain. Quantitative analysis synapse by Transmission Electron Microscopyshowed that synapse number broadly increased in cancer bearing rats and knockdown of slit2resulted in a significant reduction in the synapse number.Moreover, the opposite effect was observed upon Robol knockdown, which resulted in a significant increase in the synapse number. Together with the quantitative experiments of slit2, Robol and RhoA expression, the results suggested that upregulation of slit2induced by carcinoma implantation resulted in an increase of synapse in dorsal horn by inhibition of Robol and RhoA. Using confocal microscopy, we showed that both the PSD95and Syn expression increased in ipsilateral dorsal hornbut not in contralateral of cancer bearing rats. Quantified analysis of the magnified images showed that synapse number was significantly higher in cancer bearing rats compared with sham. Moreover, injection of siSlit2-LV into dorsal horn resulted in decreasing synaptic formation, but siRobo1-LV led to an opposite effects.RT-PCR and immunoblot analysis revealed that carcinoma implantation induced a prominent increase both in the levels of PSD95and Synwhich were reversed by knockdown of Slit2, but further increased by Robo1knockdown. These results indicated that upregulation of Slit2inhibited Robol and promoted the excitatory synaptogenesis in dorsal horn post-carcinoma implantation.3. Slit2/Robol promotesneurite outgrowth and contributes to synaptogenesisMethods To test whether upregulation of Slit2promote excitability synapse formation through inhibiting Robo1and subsequent removing the RhoA-inhibiting effect on the processes of axon enlongation and arborization, we first investigate the subcellular distribution and interaction among Slit2, Robo1and RhoA. Cortial neurons from embryos (E17-E19) of pregnant rats were cultured, transfectedat5day in vitro (DIV)with a plasmid encoding green fluorescent protein(GFP) together with anRNAi to Slit2or Robol or a control RNAi, and examined at10DIV. To unambiguously visualize multiple branches from a single neuronal cell body, neurons were cultured at low density. We used immunocytochemistry to investigate the subcellular distribution of Slit2, Robo1and RhoA. Neuronal branches were examined using immunohistochemistrical staining of microtubule-associated protein2(MAP2), a neuronal marker. We next examined theexcitability synapse formation of cortial neurons cocultured with astroglia from embryos of pregnant rats. The siRNA lentivires transfected cells at5DIV and and fixed10days later for staining with antibodies that recognize Synand PSD95and Syn.To quantify the number of synapses formed on the normal or transfected neuron, we counted the number of apposed Syn/PSD95puncta along dendrites of normal or GFP-expressing neurons.Results We used immunocytochemistry to investigate the subcellular distribution of Slit2, Robo1and RhoA and found that they were broadly co-expressed onneuronal soma and dendritesas well as axons. Knockdown Slit2inhibited Robol and RhoA, while knockdown Robol resulted in decrease of RhoA but not Slit2. The results were further confirmed by RT-PCR and western blot. Together with immunoprecipitation study descripted above using cultured-neurons, these results indicated that Slit2bound to and inhibited Robol, which in turn inhibited RhoA in vitro. These results were agreed with those of animal experiment upon. Using immunohistochemistrical staining of microtubule-associated protein2(MAP2), we showed that Slit2knockdown strongly reduced neurite number and length, while knockdown of Robo1enhanced neurite outgrowth. Together with quantitative analysis of Slit2, Robol and RhoA expression in neuron, the results indicated that Slit2promoted axon elongationand branching probably due to inhibiting Robo1and sequent blocking RhoA-inhibition on neurite extension. Thus we next examined the synapse formation of neurons cocultured with astroglia from embryos of pregnant rats. Quantify the number of synapses showed that knockdown of Slit2resulted in a significant decrease inexcitatory synaptic numbe. Conversely, knockdown of Robo1induced an increase of excitatory synapse.4. Statistical analysisAll data are presented as means±SD. The statistical significance of difference between values was determined by analysis of variance (ANOVA). For all analyses, significance was set at P<0.05.Conclusion1. We have identified hundreds of altered genes that may mediate the onset or maintenance of inflammatory, neuropathy. and bone cancer pain respectively. When the genes whose expressions were upregulated were pooled together, lots of gene sets were overlap among three models.Thissuggests that these three types of pain on the one hand shared a similarmechanisms, but on the other hand have a special gene expression profile themselves.2. Slit2mediates axon guidance and branch and promotes excitatory synaptogenesis via inhibition of Robol and subsequent RhoA-inhibition of axon remodeling and contributes bone cancer pain. SignificanceIn the present study, we have identified the unique gene expression profiles in the spinal cord from animals with bone cancer pain may provide insight into the widespread factors that drive spinal cord sensitivity of pain. This, in turn, will contribute to the development of more effective treatments for bone cancer pain.Synaptic plasticity is fundamental to spinal sensitivity of bone cancer pain. Here we have shown that excitatory synaptogenesis contributes to bone cancer pain and described that Slit2, Robo1and RhoA act as such cues that promote neurite outgrowth and guides the axon for synapse formation. These results havedemonstrated a molecular mechanism of synaptogenesis in bone cancer pain.This maybe lead to a new treatment target for bone cancer pain. |
