Study On Wnt/β-catenin Signaling In Satellite Glial Cells Contribute To Peripheral Nerve Injury-induced Neuropathic Pain

BackgroundIt is known that damage to the peripheral nerve often results in neuropathic pain. Theestablishment and maintenance of neuropathic pain involves not only neurons in neuronalpathways, but also glial cells and immune cells. The reaction of these cells in neuralsystem has formed a signal network for neural inflammation. In addition to alteringneuronal pathways, the activation of glial cells often results in increased production ofinflammation-and pain-related molecules, such as neuromediators, chemokines, andcytokines. These secreted molecules finally influence the excitement of neuron，so thatincreasing reports regard neuropathic pain as a kind of immune disorder in neural system.Besides, β-catenin, the central player of Wnt signaling that regulate repair andregeneration after neural injury, have been implied involved in the induction ofneuropathic pain. Wnt proteins is a secreted protein in neural system. And it plays a keyrole in cell proliferation and differentiation during neural development. Wnt signalingpathway involves proteins that directly participate in both gene transcription and celladhesion. And β-catenin is a transcription cofactor with T cell factor/lymphoid enhancerfactor (TCF/LEF) in the Wnt pathway. Increasing evidences indicate that β-catenin is apowerful transcription cofactor that regulate axon regeneration and myelination. Inaddition, β-catenin is reported to influence the expression of connexin43, NADPH oxidize,COX2, MMP-2, TNF-α in tumor cells or immune cells, which are important regulators forinflammation and neuropathic pain. The change of Wnt/β-catenin exppresion and itsfuction haved been studied in central nervous system. However, the Wnt/β-cateninsignaling in peripheral neural system, especially in DRG has not been formallycharacterized, nor has the role of β-catenin in the development of neuropathic pain beenaddressed. In this regard, we examined the expression of4representative Wnt proteins andβ-catenin after peripheral nerve injury and found that Wnt1, Wnt3a, Wnt5a, Wnt4transcript level in injured DRG were downregulated. The β-catenin which maily expressedin satellite glial cells were also decreased after SNL surgery. It is acknowledged thatsatellite glial cells which tightly surround primary sensory neurons can modulate the neuronal excitability. From these results, we deem that it is critical to clarify thesite-specific effects of Wnt/β-catenin to reveal the underlying mechanisms of neuropathicpain.Methods1. SNL surgeryThe spinal nerve ligation (SNL) model was performed. The rats were anesthetized by10%Chloralhydrate (0.4ml/100g body weight, i. p.) and placed in a prone position. Thenan incision was made into the left of the spine at the L3–S2level. After the left L6vertebral transverse process was removed, the left L5spinal nerve was exposed, and thentightly ligated with6-0silk thread. The wounds were closed. Sham-operated ratsunderwent an same surgical procedure as the SNL rats except that the left L5spinal nervewas not ligated.2. Implantation of the intrathecal catheter and drug deliveryFor spinal drug delivery, all animals received implanted intrathecal (i.t.) catheter atthe same time of SNL surgery. After an excision of S1processus spinalis, a polyethylenecatheter (PE-10;20.0cm) was inserted into the subarachnoid space at the level of theL6–S1vertebrae until the tip of the tube (1.5cm) reached the lumbar cistern between theL4and L5vertebrae. The catheters were fixed at the lumbar part and the back of the head.Animals that appeared motor dysfunction after intrathecal injection of small dose lidocaineindicated successful implantation of the intrathecal catheter. The applied chemicals werelimited at the L4–L5DRG levels by using a small volume (10μl) of the injectant andlimiting the rate of injection (3.3μl/min). The intrathecal application of drugs were done7days after SNL surgery. Wnt agonist thiophene pyrimidine derivative (Santa cruz) wasdissolved in DMSO at a final concentration of50μM,10μM and1μM, respectively.3. Behavioral tests for mechanical thresholdsBehavior tests were conducted after the SNL surgery on postoperative days1,3,5,7,14and after the drug delivery. Prior to the surgery, the rats were habituated to the testing apparatus and a baseline behavior was established. All tests were conducted by a personblinded to the treatment groups. The foot withdrawal threshold to mechanical stimuli wasassessed by von Frey hairs, which applied from underneath to the left hind-paw betweenthe third and fourth digits.(9calibrated von Frey hairs with bending forces of0.4-15.0g)For each test, the rats were placed in a plastic chamber (20×20×25cm) and habituated forat least30min. The chamber was placed on top of a mesh screen so that mechanicalstimuli could be administered to the plantar surface of left hind paw. The same set of vonFrey stimuli was given in an ascending order. Each von Frey hair was tested repetitively10times with2-3min interval between stimuli and the bending force being able to evoke50%of the paw withdrawal occurrence was deemed as mechanical threshold. If the15g failed toevoke withdrawal response, the value was recorded as15g. The decrease by7g in pawwithdrawal mechanical threshold indicated mechanical hyperalgesia.4. Cell culture and treatmentPrimary mixed neuron-SGC cultures were prepared from DRG of3day-oldSprague–Dawley rats. Briefly, after DRG excision and enzyme dissociation at37°C with0.25mg/ml trypsin,1mg/ml collagenase and0.2mg/ml DNase (Sigma-Aldrich), cellswere centrifuged, resuspended in culture medium (Ham’s F-12with10%fetal calf serumand1%penicillin/streptomycin), and plated onto poly-L-lysine-coated wells or glasscoverslips. SGC-purified cultures were prepared from mixed cultures at day5in culture,after replacing culture medium at days1and3. At day5, cells were detached from thePetri dish by a5min treatment with0.5%-trypsin/0.2%-EDTA (Sigma-Aldrich) at37°C,resuspended in fresh culture medium, and replated onto uncoated wells or24mm diametercoverslips. This procedure completely removed all neurons without affecting SGCadhesion and growth[England S et al.,2001; Capuano A et al.,2009](Fig.1A). The culturemedium was replaced24h later, and experiments were performed after additional24h.Wnt agonist thiophene pyrimidine derivative (Santa cruz) was dissolved in DMSO at afinal concentration of3μM and the culture medium was incubated for12h or24h. Wntagonist thiophene pyrimidine derivative (Santa cruz) was dissolved in DMSO at a finalconcentration of3μM. SGCs were infected with Lentivirus-β-catenin shRNA or NCvirus(Fig1C) for12h and then replace to normal medium and harvest in72h later. 5. Statistical AnalysisData are represented as mean±SEM. The statistical significance of differences wasanalyzed using the PASW statistical program (SPSS Inc.). Statistical analyses wereperformed using a Student t test and one-way ANOVA. For behavior responses, a two-wayANOVA with repeated measure analyses of variance were performed followed by theholm-sidak post hoc test or Student t test for multiple comparisons. P<0.05was consideredstatistically significant.Results1. SNL Downregulates Wnts Expression in Nerons of DRGWnt1, Wnt3a, Wnt5a, Wnt4mRNA expression in injured DRG were detected byreal-time PCR. Total RNA was isolated from L5injured and contralateral DRG at7d aftersurgery. The Wnt1, Wnt3a, Wnt5a, Wnt4transcript level in injured and contralateral DRGwas normalized to the level of GAPDH and is presented as fold induction, compared withthe mRNA level of contralateral DRG.(SNL/CON, Wnt1:17.2±5.21%;Wnt3a:17.9±5.18%;Wnt5a:16.9±7.91%; Wnt4:57.1±8.94%, each group, n=3，*P <0.05，**P <0.01). Wnt1expression in DRG after SNL was detected by Western. Theresult showed that SNL significantly reduced Wnt1expression in injured DRGs (SNL vs.CON, Wnt1:0.42±0.07vs.1.12±0.12, n=3,*P＜0.05). Then immunocytochemicalanalyses of Wnt1expression in DRGs showed that Wnt1were found only in NeuN-labeledneurons.2. SNL Downregulates β-catenin Expression in Satellite Glial Cells of DRGβ-catenin expression is reduced in injured DRG after SNL. Western analysis showedthat SNL significantly reduced β-catenin expression in injured DRG at7day and14dayafter surgery (SNL vs. CON,7d:0.18±0.03vs.0.34±0.03;14d:0.14±0.02vs.0.35±0.05, n=4,*P＜0.05). Immunocytochemical analyses of β-catenin expression inS100-labeled (green) satellite glial cells. 3. Intrathecal Injection of Wnt Agonist Thiophene Pyrimidine DerivativeAttenuates SNL-induced Neuropathic Pain Behavior and Enhances β-cateninExpression in DRG Satellite Glial CellsIntrathecal injection of Wnt agonist thiophene pyrimidine derivative attenuatesSNL-induced neuropathic pain behavior at7day after surgery. Mechanical allodynia weremeasured using the sham-operated rats with vehicle injection as the negative control(Veh+Sham, n=8), SNL-injured rats with vehicle injection (Veh+SNL, n=8),SNL-injured rats with the graded doses of thiophene pyrimidine derivative injection (Wntagonist+SNL, n=8, in50μM,10μM and1μM.), and sham-operated rats with thiophenepyrimidine derivative injection (Wnt agonist+Sham, n=8, in50μM.) as the positivecontrol (Veh+Sham, n=8). The paw withdrawal threshold to von Frey stimulation in SNLrats was much reduced. Compared with the group of Veh+SNL, the allodynia in SNL ratswhich was injected Wnt agonist thiophene pyrimidine derivative (50μM and10μM,10μl)was effectively reversed.(Wnt agonist+SNL vs Veh+SNL,*P＜0.05,**P＜0.01).β-catenin expression is enhanced in rats by intrathecal injection of Wnt agonist thiophenepyrimidine derivative. Then western result indicated that intrathecal injection of Wntagonist significantly increased β-catenin expression in injured DRGs at8hours aftersurgery (Wnt agonist vs Veh:0.39±0.04vs.0.24±0.03,n=4,**P＜0.01).4. Vitro Administration of Lentivirus Encoding β-catenin shRNA Induces COX2and iNOS Expression in Satellite Glial Cells.β-catenin mRNA expression were detected by real-time PCR3d after lentiviral vectortreatment. The β-catenin transcript level in satellite glial cells with β-catenin shRNAlentiviral vector and negative vector were normalized to the level of GAPDH and ispresented as fold induction, compared with the mRNA level of control. Vitro treatmentwith β-catenin shRNA lentiviral vector significantly reduces the expression β-catenin ofsatellite glial cells(β-catenin mRNA: KD/NC:28±8%, n=3,**P <0.01). Westernanalysis showed that β-catenin shRNA lentiviral vector significantly reduced β-cateninexpression in satellite glial cells（NC vs. KD,0.52±0.04vs.0.31±0.04，n=3,*P <0.05). Then iNOS and COX2mRNA expression were detected by real-time PCR3d afterlentiviral vector treatment. The iNOS and COX2transcript level in satellite glial cells withβ-catenin shRNA lentiviral vector and negative vector were normalized to the level ofGAPDH and is presented as fold induction, compared with the mRNA level of control. (COX2mRNA: KD/NC:187±30.7%; iNOS mRNA: KD/NC:1260±136%; n=3,**P <0.01). Western analysis showed that β-catenin shRNA lentiviral vector significantlyehanced iNOS and COX2expression in satellite glial cells (COX2: KD vs. NC,1.22±0.13vs.0.77±0.1; iNOS: KD vs. NC,0.32±0.02vs.0.15±0.01, n=3,*P <0.05).5. Vitro Administration of Wnt Agonists Thiophene Pyrimidine DerivativeReduces the COX2Expression in Satellite Glial Cells.Vitro treatment with Wnt agonist thiophene pyrimidine derivative significantlyincreases the expression of β-catenin of satellite glial cells. Western analysis showed thatWnt agonist significantly enhanced β-catenin expression in satellite glial cells in12hoursafter the treatment(β-catenin:12h vs CON:1.08±0.09vs0.53±0.03, n=3,*P <0.05).Vitro treatment with Wnt agonist thiophene pyrimidine derivative downregulates theexpression COX2of satellite glial cells. Western analysis showed that Wnt agonistsignificantly reduced COX2expression in satellite glial cells (12h vs CON:0.53±0.06vs.0.74±0.04;24h vs. CON:0.3±0.04vs.0.74±0.04n=3,*P <0.05,**P <0.01).COX2mRNA expression were detected by real-time PCR12hours after Wnt agonisttreatment The COX2transcript level in satellite glial cells with Wnt agonist werenormalized to the level of GAPDH and is presented as fold induction, compared with themRNA level of control.(COX2: Wnt agonist/CON:43.6±4.62%, n=3,*P <0.05).ConclusionIn conclusion, the present study revealed that SNL surgery can downregulates wntsexpression in nerons of DRG and decrease β-catenin Expression in Satellite Glial Cells.And intrathecal injection of wnt agonist thiophene pyrimidine derivative attenuatesSNL-induced neuropathic pain behavior and enhances β-catenin expression in DRGSatellite Glial Cells. In order to clarify the site-specific effects of Wnt/β-catenin to revealthe underlying mechanisms of neuropathic pain. We further certified that vitroadministration of lentivirus encoding β-catenin shRNA induces COX2and iNOSexpression in satellite glial cells. And vitro administration of wnt agonists thiophenepyrimidine derivative reduces the COX2expression in satellite glial cells. It has drawn aprimary relationship between Wnt/β-catenin and pain-related molecular in satellite glialcells.