The Roles Of Wnt Signaling In Brachial Plexus Avulsion Injury-induced Neuropathic Pain

BackgroundBachial plexus avulsion (BPA) is a special traumatic peripheral nerve injury complicated with high risk of neuropathic pain. Because the lesions are implicated in dorsal root ganglion (DRG) and spinal dorsal horn (SDH), BPA is a borderline injury that both central and peripheral nervous systems are involved. The pathogenesis and classification of BPA varied, therefore, it is difficult for the diagnosis. Causalgia and neuroma always occurred in high risk after BPA injury and no definite clinical therapy is to be recommended. Recent study found that Wnt signalling pathway was critical for the development of nervous system, which contributed to the synapses formation as well as its structural and functional plasticity in response to the activation of Wnt downstream cascades. Change of synaptic plasticity is closely associated with pathogenesis of neuropathic pain. However, the roles and underlying mechanisms of Wnt signalling in BPA-induced neuropathic pain are unrevealed. Therefore, our study aims to investigate the association between Wnt pathway and BPA-induced neuropathic pain. Objectives1. Estanblish rat model of BPA-induced neuropathic pain and identify the model by behavioral and molecular approaches.2. Evaluate the expression of Wnt pathway-associated molecules in BPA-NPP rat spinal dorsal horn and determin their cellular location, and furtherly observe the effects of this pathway on the neuroglia activation and neuron plasticity.3. Investigate the effects of Wnt signalling on neuropathic pain behavior and synaptic plasticity of the BPA-NPP rat.4. Determine the expression of Wnt pathway-associated molecules in DRG nurons and evaluate the underlying mechamisms of Wnt on Nav1.8.Methods1. Estanblish rat model of BPA-induced neuropathic pain by avulsion of C8 and T1 nerve roots. Von Frey filaments were used to measure the mechanical foot threshold (MWT) and Hargreaves thermal radiation method was applied to determine the thermal withdrawal latency (TWL) of BPA-NPP rat model. Acetone spray was used to evaluate he cold allodynia score and open-field test was employed to evaluate the locomotor activity of the animals. Western blotting was performed to detwermin the expression of p-ERK in rat DRG and SDH and immunofluorescence histochemistry staining was used to observe the expression of c-Fos.2. RT-PCR, Western blotting and immunofluorescence double staining were performed to detect the expression and distribution of Wnt pathway-related molecules (Wnt-3a, Frizzled 4 and β-catenin). The phosphorylation of synaptic plasticity-regulating molecules, Navl.8、CCL2 and CCR2 were determined by Western blotting.3. Different dosage of wnt inhibitor IWP-2 and niclosamide were delivered to spinal fluid )y intrathecal administration.4. Immunofluorescence histochemistry staining was performed to measure the expression of neuroglia activation marker GAFP and spinal superficial layer synapse formation related protein (PSD-95 and synaptophysin). Pro-inflammatory cytokines (IL-1β, IL-6 and TNF-a) were measured by RT-PCR. Whole-cell patch clamp record the miniatureexcitatory postsynaptic currents (mEPSCs) of rat spinal superficial layer neurons and Nav1.8 in DRG neurons.Results1. BPA-NPP rat model was successfully established and after the operation, the animal ipsilateral and contralateral MFT decreased while no significant change in TWL. Cold allodynia score elevated while locomotor activity remained unchanged in BPA animals. The neuropathic pain behavioral change lasted at least for 28 days. Injury-related pERK and c-Fos increased in SDH and DRG neurons in BPA-NPP animals.2. After BPA operation, Wnt-3a and the receptor Frizzled 4 as well as its downstream effector β-catenin were unregulated. Moreover, Wnt-3a was predominantly expressed in astrocytes and neurons in SDH, while Frizzled 4 and β-catenin were found only in neurons. BPA induced elevated phosphorylation of synaptic plasticity-regulating molecules (CaMK Ⅱ, NMDR isoform NR2B, CREB and PKC).3. After intrathecal administration of Wnt signals inhibitor IWP-2, MFT significantly increased for about nine days. After ntrathecal administration of Wnt signals inhibitor WP-2, astrocyte activation and generation of pro-inflammatory cytokines reduced, phosphorylation of synaptic plasticity-regulating molecules and spinal superficial layer synapse formation related protein (PSD-95 and synaptophysin) reduced and mEPSCs were inhibited.4. BPA operation caused Wnt-3a, Frizzled 4 and β-catenin as well as CCL2/CCR2 and Nav1.8 upregulation in DRG neurons. IWP inhibited the generation of CCR2 and Navl.8 rather than CCL2. CCR2 antagonist suppressed Nav1.8 expression and currents.Conclusions1. BPA injury induced Wnt/β-catenin signalling pathway activation in SDH and DRG (?)eurons.2. BPA injury induced animal spinal dorsal horn neuroglia activation and elevated secretion of proinflammatory cytokines. And the activated Wnt/β-catenin pathway lead to increased neuron synaptic plasticity and reduced sensory threshold in response to BPA lesions.3. CCL2/CCR2 and Navl.8 participated in nervous impulse transmission after BPA and Wnt/β-catenin signalling caused neuropathic pain by CCR2-induced Navl.8 expression and currents.4. Wnt/β-catenin signalling activation induced alternation of neuron synaptic plasticity in SDH and Nav1.8 excitation in DRG are critical to BPA caused neuropathic pain.