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SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats
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NEUROSCIENCE 1
RESEARCH ARTICLE J. Wang et al. / Neuroscience xxx (2018) xxx–xxx
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SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats
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Jiang Wang, a Wei Xu, b Yan Kong, a Jiangju Huang, a Zhuofeng Ding, a Meiling Deng, a Qulian Guo a,c and Wangyuan Zou a,c*
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Department of Anesthesiology, Hunan Provincial Maternal and Child Health Care Hospital, China
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National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
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Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
Abstract—Synaptosomal-associated protein 25 (SNAP-25) plays an important role in neuropathic pain. However, the underlying mechanism is largely unknown. Vesicular glutamate transporter 2 (VGluT2) is an isoform of vesicular glutamate transporters that controls the storage and release of glutamate. In the present study, we found the expression levels of VGluT2 correlated with the upregulation of SNAP-25 in the spinal cord of rats following chronic constriction injury (CCI)-induced neuropathic pain. Cleavage of SNAP-25 by Botulinum toxin A (BoNT/A) attenuated mechanical allodynia, downregulated the expression of VGluT2 and reduced glutamate release. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. Upregulation of SNAP-25 in naive rats increased VGluT2 expression and induced pain-responsive behaviors. In pheochromocytoma (PC12) cells, the expression of VGluT2 was also depended on SNAP-25 dysregulation. Moreover, we found VGluT2 was involved in SNAP-25-mediated regulation of astrocyte expression and activation of the PKA/p-CREB pathway mediated the upregulation of SNAP-25 in neuropathic pain. The findings of our study indicate that VGluT2 contributes to the effect of SNAP-25 in maintaining the development of neuropathic pain and suggests a novel mechanism underlying SNAP-25 regulation of neuropathic pain. Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: SNAP-25, VGluT2, neuropathic pain, PKA.
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INTRODUCTION
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Neuropathic pain is a difficult clinical puzzle and remains a major burden on both individuals and society. Actually, neuropathic pain is resistant to conventional therapy and no definitive solutions have been discovered. Therefore, there is a great interest in exploring the mechanism of neuropathic pain and developing new therapies. In recent years, particular interest has been devoted to the application of Botulinum toxin A (BoNT/A) in the treatment of painful conditions including neuropathic pain (Pavone and Luvisetto, 2010, Matak et al., 2014). The antinociceptive effect of BoNT/A relies on its ability to selectively cleave Synaptosomal-associated protein 25 (SNAP-25), which makes SNAP-25 to be a promising target to understand the development of neuropathic pain. SNAP-25 is a component of the SNARE-complex, which is involved in the process of cell exocytosis during
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synaptic transmission (Jahn and Scheller, 2006). Dysregulation of SNAP-25 has been associated with neurological disorders and different pain conditions (Antonucci et al., 2013, Liu et al., 2013, Olbrich et al., 2017). Inhibiting the vesicle release of pro-nociceptive neurotransmitters including glutamate from presynaptic terminals accounts for the SNAP-25-mediated analgesic effects (Durham et al., 2004, Carmichael et al., 2010, Bittencourt et al., 2014). Glutamate is the major excitatory neurotransmitter in the central nervous system involved in the pain process (Peirs et al., 2015). The transport of glutamate into synaptic vesicles is a prerequisite for glutamate release, and VGluT is responsible for this process (Weston et al., 2011). Mammals express three Vesicular glutamate transporter (VGluT) isoforms, namely, VGluT1, VGluT2 and VGluT3, which exhibit a nonoverlapping distribution in the CNS (Morris et al., 2005, Moechars et al., 2006). Among these three VGluTs, VGluT2 is proposed as the major regulator of neuropathic pain (Moechars et al., 2006, Liu et al., 2010). Previous study suggested the expression of SNAP-25 and VGluT2 correlated in several neurological diseases (Piroli et al., 2013, Glazova et al., 2015). However, the specific relationship between SNAP-25 and VGluT2 in neuropathic pain has not been
*Correspondence to: Wangyuan Zou, Department of Anesthesiology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, China. Fax: +86-731-84327413. E-mail address: [email protected] (W. Zou). Abbreviations: BoNT/A, botulinum toxin A; CCI, chronic constriction injury; PC12, pheochromocytoma cells; SNAP-25, synaptosomalassociated protein 25; VGluT2, vesicular glutamate transporter 2. https://doi.org/10.1016/j.neuroscience.2019.10.007 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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studied yet. We, therefore, performed this study to test the hypothesis that VGluT2 is involved in SNAP-25-mediated regulation of neuropathic pain. Hence, the present study was designed to investigate (i) effect of dysregulation of SNAP-25 on VGluT2 expression in a rat model of chronic constriction injury (CCI)-induced neuropathic pain and PC12 cells; (ii) the role of VGluT2 in the SNAP-25-mediated regulation of astrocyte activation; and (iii) whether the PKA/p-CREB pathway was involved in the dysregulation process of SNAP-25 in neuropathic pain. Our findings may provide a novel mechanism underlying SNAP-25-mediated regulation of the development of neuropathic pain.
EXPERIMENTAL PROCEDURES
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Animals
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Male Sprague-Dawley rats weighting 180–220 g were used in this experiment. Rats were housed in groups of six per cage and maintained on a 12-h light/dark cycle with free access to food and water. Each experimental procedure was performed in accordance with the Administrative Committee of Experimental Animal Care and Use of Central South University and was approved by the Central South University Ethical Committee for Animal Research. The study was in agreement with the Ethical Guidelines of the International Association for the Study of Pain (guidance). An observer who was blinded to the animal treatment performed all behavioral tests, and all efforts were made to minimize suffering and reduce the number of animals used to the minimum required for statistical accuracy in the study.
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Surgery
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CCI surgery was performed as previously described (Zou et al., 2012). In brief, rats were anesthetized using isoflurane (4% to induce, 2% to maintain), and the left sciatic nerves were exposed at the mid-thigh level by blunt dissection. Four ligatures (4-0 chromic gut suture) were loosely tied around the nerve at 1-mm spacing. The sciatic nerves in the Sham group were only exposed without ligation.
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Behavioral studies
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Von Frey test. Mechanical paw withdrawal threshold was determined using von Frey filaments ranging from 0.4 g to 26 g. Rats were placed in plastic chambers on metal mesh for 15 min to acclimate to the environment before testing. Von Frey filaments (UGO, Italy) were applied to the mid-plantar surface of the hind paw for 2–3 s, with a 5 min interval between each test.
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Withdrawal or licking of the paw was considered a positive response. Each test was repeated three times, and the lowest filament that elicited a positive response was considered the threshold stimulus.
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Rotarod test. Locomotor function was evaluated using rotarod testing, as previously described (Zychowska et al., 2016). In brief, rats were acclimated to revolving drums for 5 min before testing. A training trial on the revolving drums (15 rpm) was performed prior to the actual day of testing. Rats that remained on the drum for more than 150 s were selected for testing. The rotarod performance time of each rat was recorded. The cut-off time was set as 300 s. Each test was repeated three times at five-minute intervals. The mean values of three tests were compared.
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Intrathecal drug delivery
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Intrathecal catheter surgery was performed as previously described (Loram et al., 2013). Rats were anesthetized with isoflurane. An 18-gauge guide needle was inserted into the L5/6 intervertebral space, and a PE-10 catheter was inserted into the subarachnoid space through the guide needle. A marker was preset to ensure that the end of the catheter rested over the lumbar enlargement. Drugs and lentivirus were injected over 30 s with a 1 min delay before removing the catheter or needle. Rats with neurological deficits were excluded from the experiment.
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Drugs administration
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The BoNT/A (BOTOXÒ, Allergan, USA) was dissolved in 0.9% saline. BoNT/A (20 ll of 20 U/kg) or saline was injected intraplantar into the left hind paw while the rats were under 2% isoflurane anesthesia 5 days after CCI surgery. The concentration of BoNT/A was based on a previous study (Favre-Guilmard et al., 2009). The PKA inhibitor H-89 (Sigma, USA) was dissolved in 1% DMSO and diluted to the appropriate concentration insterile saline. H-89 (10 ll of 10 lM) or vehicle was intrathecally injected 7 days after CCI. The concentration of H-89 was based on a previous study (Sun et al., 2004).
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Lentivirus injection
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Lentiviral vector-mediated VGluT2 (LV-VGluT2), SNAP25 (LV-SNAP-25) and scrambled control (LV-NC) were purchased from Genepharma. Lentivirus (10 ll) was intrathecally injected on the day of CCI surgery for in vivo testing. The transfection efficiency was evaluated 10 days after lentivirus injection by monitoring GFP
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" Fig. 1. SNAP-25 and VGLUT2 are upregulated during CCI-induced neuropathic pain. (A) Robust mechanical allodynia was observed in the ipsilateral hindpaw of CCI rats. n = 5, ***P < 0.001, versus Sham. (B) Western blot analysis revealed a gradual increase in SNAP-25 and VGluT2 in the spinal cord after CCI. n = 3, **P < 0.01, ***P < 0.001, versus Sham. (C) Immunofluorescence shows the expression pattern of SNAP-25 (green) and VGluT2 (red) in the spinal cord 10 days after surgery. SNAP-25 and VGluT2 were upregulated in the ipsilateral dorsal horn of CCI rats. n = 3, *P < 0.05, versus contralateral side of spinal dorsal horn. Arrows indicate the expression of SNAP-25 and VGluT2 in the dorsal horn. D. Double immunofluorescence revealed that SNAP-25 colocalized with VGluT2 in the dorsal horn of spinal cord. Contra = contralateral side, ipsi = ipsilateral side. Scale bar = 100 lm.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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expression in rat spinal cords with a fluorescence microscope (Leica, Germany).
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Cell culture and treatment
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Differentiated pheochromocytoma (PC12) cells were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Roswell Park Memorial Institute (PRMI) 1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 50 U/ml penicillin and 50 lg/ml streptomycin under humidified conditions in 5% CO2 at 37 °C. To examine the correlation between SNAP-25 and VGluT2 in PC12 cells, cells were first pretreated with BoNT/A (33 U/ml) for 24 h and collected for Western blot or immunochemistry analysis. The concentration of BoNT/A was determined as previously described (Jenkinson et al., 2017). Cells for lentivirus transfection were transfected with lentivirus (LV-SNAP-25 or LV-NC) at 100 multiplicity of infection (MOI) for 24 h, followed by Western blot analysis. GFP fluorescence in PC12 cells was monitored to evaluate transfection efficiency.
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Immunochemistry
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Immunochemistry was performed as previously described (Wang et al., 2016, Huang et al., 2019). Rats were transcardially perfused with 4% paraformaldehyde, and the lumbar spinal cord enlargements were dissected and transferred to 25% sucrose-PBS overnight. The spinal cord was sectioned at a thickness of 10 lm in a cryostat. Sections were rinsed in 0.1% PBS twice and were permeabilized for 10 min with phosphate-buffered saline (PBS) containing 0.1% Triton X-100. 10% donkey serum was used to block nonspecific binding for 1 h. Sections were incubated overnight with the following primary antibodies: rabbit anti-SNAP-25 (1:200, Abcam, USA); mouse antiVGluT2 (1:200, Abcam, USA); rabbit anti-p-CREB (1:200, Abcam, USA); mouse anti-GFAP (1:500, Abcam, USA); and rabbit anti-GFP (1:200, Cell Signal Technology, USA) overnight. Sections were rinsed in PBS three times and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG or Alexa Fluor 594 conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, USA) for 2 h. A Leica Observer Microscope was used to visualize the sections. PC12 cells for in vitro immunochemistry were seeded in 24-well plates and fixed with 1% paraformaldehyde for 10 min. All subsequent steps were performed as described above. All the sections were processed under standardized conditions in every experiment, tissues and cells from control and experimental groups were collected, and then processed for immunohistochemistry simultaneously (Taylor and Levenson, 2006, Glazova et al., 2015). All images were captured in a constant condition. Quantification was performed by using the Image-Pro Plus 6.0 (Media Cybernetics, USA).
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Western blot
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Western blots were performed as previously described (Wang et al., 2017). Rat lumbar spinal enlargements and cells were homogenized in Radio Immunoprecipita-
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tion Assay (RIPA) lysis buffer containing a protease inhibitor cocktail. The bicinchoninic acid (BCA) Protein Assay kit (Beyotime, China) was used to determine the concentrations of total protein. Equal amounts of protein (50 lg) were loaded in 10% dodecyl sulfate, sodium salt–Polyacrylamide gel electrophoresis (SDS–PAGE) and separated at 80–100 V for 90 min. Proteins at the desired molecular weights were transferred to polyvinylidene difluoride membranes (Merck Millipore, USA) at 300 mA for 60 min. Membranes were blocked with 5% nonfat milk and probed with the following antibodies: rabbit anti-SNAP-25 (1:2000, Abcam, USA); mouse antiVGluT2 (1:2000, Abcam, USA); mouse anti-VGluT1 (1:2000, Abcam, USA); mouse anti-VGluT3 (1:2000, Abcam, USA); rabbit anti-Tublin (1:5000, Abcam, USA); rabbit anti-p-CREB (1:2000, Abcam, USA); and rabbit anti-GAPDH (1:5000, Merck Millipore, USA). Primary antibodies were detected with rabbit or mouse Horseradish Peroxidase (HRP)-conjugated secondary IgG (1:20,000, Jackson ImmunoResearch, USA). Signals were visualized using an enhanced chemiluminescence plus system (Merck Millipore, USA). Each sample was run in duplicate. The densities of specific bands were analyzed using the Image Lab 3.0 system (Bio-Rad, USA) for quantification.
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Glutamate concentration measurement
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The Glutamate Colorimetric Assay Kit (K629, BioVision, USA) was used to measure glutamate concentrations. Spinal cord enlargements were collected and homogenized in 100 lL assay buffer. Homogenates were centrifuged at 13,000g for 10 min, and the supernatants were isolated and transferred to 96-well plates. Assay reagents were added, and the optical density (OD) at 450 nm was measured in the microplate reader. Glutamate concentrations were calculated according to the manufacturer’s instructions.
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Statistical analysis
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No statistical analyses were used to predetermine the sample size. Our sample sizes were based on previous similar publications (Fan et al., 2017). All data are expressed as the means ± SD. Behavioral tests were analyzed using two-way repeated measures ANOVA, followed by post hoc Bonferroni’s test. One-way ANOVA test was used for multiple comparisons. Bonferroni tests were performed for post hoc comparisons. Unpaired Student’s t test was used for comparisons of two groups. All statistical tests were performed in GraphPad prism 6.0. P < 0.05 was considered statistically significant.
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RESULTS
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Expression changes in SNAP-25 correlated with VGluT2 in the spinal dorsal horn of CCI rats
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We first examined the changes in expression of SNAP-25 and VGluT2 in the spinal cord in a CCI-induced neuropathic pain model. After CCI induction (Fig. 1A), western blots showed that the expression of SNAP-25 in the spinal cord gradually increased, and this increase
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Fig. 2. VGLUT2 is involved in SNAP-25-mediated development of neuropathic pain. (A) BoNT/A attenuated CCI-induced mechanical allodynia. BoNT/A (20 U/kg, 20 lL) or saline was intraplantarly injected into the ipsilateral hind paw of rats on day 5 postsurgery. The withdrawal threshold was measured 60 min after BoTN/A injection. n = 6, *P < 0.05, **P < 0.01, versus CCI + saline, ##P < 0.01, ###P < 0.001, versus Sham + saline. (B) Rotarod test showed that intraplantar injection of BoNT/A did not affect the motor function of rats. The rotarod test was measured 30 min after BoNT/A injection. n = 5. *P < 0.05, vs Sham + saline. (C) Western blot analysis showed BoNT/A reduced expression levels of SNAP-25 and VGluT2 proteins in the spinal cord 10 days after CCI induction. n = 4, *P < 0.05, **P < 0.01, versus Sham + saline. (D) Immunofluorescent staining for SNAP-25 (green) and VGluT2 (red) in the spinal cord of rats 10 days after CCI, n = 4. (E) The protein expression levels of VGluT1 and VGluT3 were not altered after BoNT/A injection. The proteins were extracted from rats 10 days after CCI induction. n = 3. (F) BoNT/A injection ameliorated the upregulation of glutamate concentrations induced by CCI. n = 3, *P < 0.05, versus Sham + saline, #P < 0.05, versus CCI + saline.
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was maintained up to 14 days. Notably, the expression levels of VGluT2 was also increased significantly after CCI treatment and exhibited the same expression pattern as SNAP-25 (Fig. 1B). Immunostaining results showed that positive SNAP-25 staining was primarily detected in the superficial laminae of the spinal dorsal horn, and VGluT2 was expressed in the superficial and deeper laminae. CCI treatment significantly increased SNAP-25- and VGluT2-positive staining in the ipsilateral spinal dorsal horn (Fig. 1C). Double-staining revealed that SNAP-25 mainly colocalized with VGluT2 in the dorsal horn (Fig. 1D).
Downregulation of SNAP-25 by BoNT/A reduced the expression of VGluT2
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After BoNT/A, which cleaves SNAP-25 (Frick et al., 2007), was administered to CCI rats, the mechanical sensitivity using von Frey test and spinal VGluT2 expression levels were examined. Intraplantar administration of BoNT/A significantly decreased CCI-induced mechanical allodynia 5 days after CCI, and the antinociceptive effect of BoNT/A was maintained 14 days after CCI (Fig. 2A). Rotarod test showed that the latencies of CCI rats were lower than sham rats, however, rat performances were
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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not different between the BoNT/A-treated and salinetreated rats (Fig. 2B). Western blots showed that BoNT/A administration significantly suppressed CCI-induced upregulation of SNAP-25 and VGluT2 in the spinal cord (Fig. 2C). Immunochemistry results showed that the expression levels of SNAP-25 and VGluT2 were reduced in the spinal cord after BoNT/A administration (Fig. 2D). We found the other two isoforms of VGluTs, namely, VGluT1 and VGluT3, were not significantly changed after BoNT/A injection (Fig. 2E). VGluT2 exerts its function mainly via regulating glutamate release between synaptic clefts. Therefore, the concentration of glutamate in the spinal cord was also examined. We found that the spinal glutamate concentrations were significantly elevated after CCI injury, and intraplantar injection of BoNT/A blocked the upregulation of glutamate (Fig. 2F). To further identify whether SNAP-25 targeted VGluT2 to regulate CCI-induced neuropathic pain, we intrathecally injected LV-VGluT2 to overexpress VGluT2 on the same day as CCI surgery. Immunochemistry results showed obvious GFP in the spianl cords of LVVGluT2 and LV-NC treated rats (Fig. 3A). After BoNT/A injection, LV-VGluT2 rats showed a small but significant decrease in withdrawal thresholds in response to von Frey filaments 10 days after CCI surgery compared to LV-NC rats (Fig. 3B).
Fig.3. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. (A) GFP was expressed in the lumbar spinal cord (L3–L4) 10 days after intrathecal injection of LV-VGluT2 or LV-NC in CCI rats. Scale bar = 100 lm. (B) Paw withdraw threshold of CCI rats after LV-VGluT2 or LV-NC injection. LV-VGluT2 or LV-NC at a titer of 1 109 TU/ml was intrathecally injected on the same day as CCI surgery. BoNT/A (20 U/kg, 20 lL) was intraplantarly injected into the ipsilateral hind paw of rats on day 8 postsurgery. n = 6, **P < 0.01, versus CCI + botox + LV-NC.
Overexpression of SNAP-25 increased VGluT2 expression and induced mechanical allodynia in naive rats
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To further explore the relationship between SNAP-25 and VGluT2 in neuropathic pain, naive rats were intrathecally injected with LV-SNAP-25, and behavior test and western blot were performed. We found that overexpression of SNAP-25 induced mechanical allodynia in naive rats 5 days after lentivirus injection (Fig. 4A). Following western blot tests showed that upregulated SNAP-25 also increased VGluT2 expression level in the spinal cord (Fig. 4B).
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SNAP-25 regulated the expression of VGluT2 in PC12 cells
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Differentiated PC12 cells were used to further evaluate the regulator role of SNAP-25 in VGluT2 expression in vitro. Immunochemistry found that SNAP-25 and VGluT2 were expressed abundantly in the cytoplasms of PC12 cells (Fig. 5A). Next, we treated PC12 cells with BoNT/A to downregulate SNAP-25 expression.
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Fig. 4. Upregulation of SNAP-25 induced mechanical allodynia in naive rats. (A) Intrathecal injection of LV-SNAP-25 produced mechanical allodynia in naive rats. LV-SNAP-25 or LV-NC at a titer of 2 109 TU/ml were intrathecally injected on day 1. n = 6, ***P < 0.001, versus LV-NC. (B) Western blot results show that the expression of SNAP-25 and VGluT2 was upregulated 5 days after lentivirus injection. n = 4, *P < 0.05, versus LV-NC.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Western blots and immunochemistry showed that the expressions of SNAP-25 and VGluT2 were both downregulated in PC12 cells (Fig. 5B, C), which is consistent with the in vivo test. To further unravel the relationship between SNAP-25 and VGluT2 in vitro, we transfected PC12 cells with lentiviral-mediated SNAP-25 to overexpress SNAP-25. GFP was visualized by microscopy in PC12 cells after lentivirus infection, and the results indicated successful lentivirus delivery. Western blot analysis showed that overexpression of SNAP-25 increased the protein level of VGluT2 (Fig. 5D).
VGluT2 was involved in SNAP-25-mediated regulation of astrocyte activation Previous studies suggested that SNAP-25 participated in the modulation of the astrocyte activation in chronic pain (Marinelli et al., 2012). To further determine whether VGluT2 was also involved in SNAP-25-mediated regulation of astrocyte activation, we first examined the spatial localization of VGluT2 and the astrocyte marker GFAP in the spinal cord. Double immunostaining revealed that
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VGluT2 colocalized with GFAP in the spinal cord (Fig. 6A). By western blot and immunostaining, we found GFAP expression increased significantly in the ipsilateral dorsal horns after CCI injury, which is consistent with the expression changes of SNAP-25 and VGluT2. And downregulation of SNAP-25 following intraplantar injection of BoNT/A, not only reduced VGluT2 expression, but also significantly inhibited the upregulation of GFAP. Moreover, we found VGluT2 overexpression via intrathecal injection of LV-VGluT2 rescued the CCI-induced upregulation of GFAP (Fig. 6B, C).
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The PKA/p-CREB pathway was involved in SNAP-25mediated regulation of neuropathic pain
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Previous studies identified SNAP-25 mRNA has a binding site for p-CREB and recognized that SNAP-25 was a known target of the PKA/p-CREB signal pathway (Hang et al., 2013). However, the specific role of PKA/p-CREB in the regulation of SNAP-25 expression in CCI-induced neuropathic pain has not been fully studied.
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Fig. 5. SNAP-25 regulates VGluT2 expression in PC12 cell. (A) Immunochemistry revealed the localization of SNAP-25 and VGluT2 in PC12 cells. SNAP-25 (green) and VGluT2 (red) are expressed in the cytoplasm of PC12 cells. (B, C) Suppression of SNAP-25 by BoNT/A downregulated the expression of VGluT2 in PC12 cells. PC12 cells were treated with 33 U/ml BoNT/A (botox group) or vehicle (control group) for 24 h. (B) Western blot showed that the protein expression of SNAP-25 and VGluT2 in PC12 cells were downregulated after BoNT/A administration. n = 4, *P < 0.05, ***P < 0.001, versus control. (C) Immunofluorescent staining revealed that the expression levels of SNAP-25 and VGluT2 were reduced after BoNT/A treatment. n = 3, **P < 0.01, versus control, scale bar = 100 lm. (D) Overexpression of LV-SNAP-25 upregulated the expression of VGluT2 in PC12 cells. Up: Green fluorescent protein (GFP) was visualized in PC12 cells after transfection with LV-SNAP-25 and LV-NC. Scale bar = 100 lm. Down: Western blot results showed that the expression levels of SNAP-25 and VGluT2 were increased after LV-SNAP-25 infection. n = 3, *P < 0.05, versus LV-NC.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Fig. 6. VGluT2 is involved in the SNAP-25-mediated activation of astrocyte. (A) Double immunofluorescence showed that VGluT2 was expressed in the astrocyte. Arrow indicates that GFAP colocalized with VGluT2 in the spinal cord. Scale bar = 100 lm. (B) Western blot results showed downregulated SNAP-25 by BoNT/A decreased the expression of GFAP, and intrathecal injection of LV-VGluT2 abolished the effect of BoNT/A. ***P < 0.001, vs Sham, ##P < 0.01, vs CCI, aP < 0.05, vs CCI + botox + LV-NC, n = 3. C. Immunofluorescent staining showed that overexpression of VGluT2 restored the activation of GFAP in the spinal cord of CCI rats that were treated with BoNT/A. LV-VGluT2 or LV-NC was intrathecally injected on the same day as CCI surgery. BoNT/A (20 U/kg, 20 lL) was intraplantarly injected into the ipsilateral hind paw of rats on day 8 postsurgery. Western blot and immunofluorescent staining for GFAP in the spinal cord was performed 14 days after CCI. n = 3, scale bar = 100 lm.
" Fig. 7. PKA/p-CREB signaling is involved in SNAP-25 dysregulation in CCI-induced neuropathic pain. (A) Blockade of PKA activation in the spinal cord attenuated CCI-induced mechanical allodynia. The PKA inhibitor H-89 or vehicle was intrathecally injected in the spinal cord 7 days after CCI. n = 6, *P < 0.05, ***P < 0.001, versus CCI + Vehicle. (B) CCI-induced p-CREB upregulation was blocked by intrathecal injection of H-89. The proteins were extracted from rats 60 min after H-89 injection. n = 3, *P < 0.05, versus CCI + Vehicle, #P < 0.05, versus Sham + Vehicle. (C) Downregulation of p-CREB was confirmed by immunochemistry after H-89 injection. Scale bar = 100 lm. (D) The protein levels of SNAP-25 and VGluT2 were reduced after H-89 injection. n = 3, *P < 0.05, versus CCI + Vehicle, #P < 0.05, versus Sham + Vehicle. The samples for immunohistochemistry and western blot were harvested at 7 days after CCI.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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To investigate whether CREB activation is involved in SNAP-25 upregulation in neuropathic pain, we intrathecally injected an inhibitor of the PKA/p-CREB pathway, H-89, to CCI rats. Behavioral tests showed that intrathecal administration of H-89 significantly reduced the mechanical allodynia of CCI rats 10 min after drug administration, and this effect lasted for 60 min (Fig. 7A). Western blot analysis showed that pCREB expression was upregulated in the spinal cord of CCI rats, and intrathecal injection of H-89 significantly reduced the upregulation of p-CREB (Fig. 7B). Immunochemistry results showed that the number of pCREB-positive cells increased in the ipsilateral spinal cord after CCI injury, which was blocked by H-89 administration (Fig. 7C). In addition, CCI induced upregulation of SNAP-25 and VGluT2 were also inhibited by the PKA/p-CREB pathway inhibitor H-89 (Fig. 7D).
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Recent studies showed that SNAP-25 was involved in the regulation of neuropathic pain. However, the precise mechanisms underlying this regulation remain elusive. The present study found that SNAP-25 targeted VGluT2 to regulate the development of neuropathic pain and astrocyte activation, and PKA/p-CREB pathway was involved in the dysregulation process of SNAP-25. These findings suggest novel mechanisms underlying SNAP-25-mediated neuropathic pain. Previous studies demonstrated that the expression of SNAP-25 and VGluT2 correlated in several neurological diseases. Glazova et al. found the alleviation of seizures by ERK1/2 inhibition led to an accumulation of SNAP-25 and VGluT2 in the hippocampus and temporal cortex (Glazova et al., 2015). Piroli et al. showed reduced glutamate efflux and VGluT2 expression in the central amygdala complex of animals subjected to repeated stress, and administration of the antidepressant tianeptine reversed stress-induced decreases in amygdala VGluT2 and increased the expression of SNAP-25 (Piroli et al., 2013). However, no study ever showed that there was a correlation between SNAP-25 and VGluT2 in neuropathic pain. The present study found a gradual upregulation of SNAP-25 and VGluT2 after nerve injury. Immunochemistry results showed that SNAP-25 was mainly expressed in the superficial laminae of the spinal dorsal horn, and colocalized with VGluT2. Moreover, SNAP-25 and VGluT2 were both upregulated in the ipsilateral side of spinal cord after nerve injury. The parallel expression changes and colocalization of SNAP-25 and VGluT2 suggest that the upregulation of VGluT2 is involved in the upregulation of SNAP-25 in CCI-induced neuropathic pain. In consistent with previous study (Marinelli et al., 2010), we found blockade of SNAP-25 upregulation by BoNT/A prevented the development of neuropathic pain, and rats perfomances in Rotarod test were not significantly changed after BoNT/A injection, indicating antinociceptive effects of the downregulation of SNAP-25 by BoNT/A were independent of muscle relaxation. Interest-
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ingly, the expression level of VGluT2 and glutamate concentration were also reduced after injection of BoNT/A. The overexpression of VGluT2 in the spinal cord abolished the antinociceptive effect of BoNT/A, which indicates that VGluT2 is required in the spinal cord for the antinociceptive effect of SNAP-25 downregulation. There are two more isoforms in the VGluT family, namely, VGluT1 and VGluT3. Previous study showed that the distribution pattern of VGluT1 and VGluT3 was not colocalized with SNAP-25 in the spinal cord (Alvarez et al., 2004). Our study showed that the protein levels of VGLuT1 and VGluT3 were not changed after BoNT/A injection, indicating the VGluT2 downregulation especially depended on the cleavage of SNAP-25. Therefore, there may be a lower correlation between SNAP-25 and VGluT1 or VGluT3 in the development of neuropathic pain. To further explore the relationship between SNAP25 and VGluT2 in neuropathic pain, we overexpressed SNAP-25 in naive rats, and found overexpression of SNAP-25 induced mechanical allodynia in naive rats, and upregulated the expression of VGluT2. Moreover, we found SNAP-25 also regulated VGluT2 expression in PC12 cells. Taken together, these results suggested that SNAP-25 may target VGluT2 to regulate the development of neuropathic pain. Mounting evidence demonstrated the contribution of astrocytes to the induction and maintenance of chronic pain (Scholz and Woolf, 2007). Our present study demonstrated that the astrocyte marker GFAP was upregulated in the spinal cord 14 days after CCI induction, which paralleled SNAP-25 expression. Intraplantar injection of BoNT/A reduced SNAP-25 expression, alleviated neuropathic pain, and reduced GFAP expression, which suggests that SNAP-25 regulates astrocyte activation in neuropathic pain. Vacca et al. (2013) found that BoNT/A reduced astrocyte activation and alleviated morphine tolerance, which was likely consequences of reduced release of glutamate. VGluT2 regulates glutamate release, and it was regulated by SNAP-25 in neuropathic pain. Therefore, we hypothesized that VGluT2 was also involved in the regulation of astrocyte expression. Double immunochemistry found that VGluT2 colocalized with GFAP in the dorsal horn of the spinal cord, and VGluT2 overexpression restored the upregulation of GFAP in the spinal cord, which indicates that VGluT2 plays a role in the SNAP-25-mediated regulation of astrocyte activation. A previous study demonstrated that activation of PKA in the spinal cord was required for the development of neuropathic pain (Zhou et al., 2015). Activation of PKA was implicated in CREB-mediated gene transcription (Hang et al., 2013). SNAP-25 is one of the downstream targets of CREB (Han et al., 2014). It is possible that SNAP-25 functions as a PKA-regulated CREB target in CCI-induced neuropathic pain. Our present results showed that the upregulation of SNAP-25 correlated with p-CREB activation in CCI-induced neuropathic pain. When PKA/p-CREB activation was inhibited, the CCIinduced upregulation of SNAP-25 and VGluT2 were significantly suppressed. Moreover, we found that the PKA inhibitor H-89 attenuated CCI-induced allodynia to a sim-
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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ilar level as SNAP-25 cleavage. Taken together, these findings suggest the CCI-induced upregulation of SNAP25 may depend on CREB activation, and SNAP-25 may be a downstream target in the PKA/p-CREB pathway in neuropathic pain. However, there are several limitations in our study. First, we used total protein from spinal cord to evaluate the expression levels of SNAP-25 and VGluT2. SNAP25 and VGluT2 are expressed abundantly in vesicle fractions, and it is better to evaluate the expression changes of these two proteins using homogenates from vesicle fractions. Second, the precise mechanisms underlying SNAP-25 mediated regulation of VGluT2 are not clear. We postulate that BDNF may play a role in this regulatory process. BDNF is an important neurotrophic factor in neurological process, the activation of the BDNF/TrkB signal pathway modulates the expression of various genes, and it is involved in numerous neurological processes (Martinowich et al., 2007, Sikandar et al., 2018). Recent studies revealed that presynaptic BDNF regulated the expression of VGluTs in vitro and in vivo (Melo et al., 2013, Azogu and Plamondon, 2017), which suggests that VGluTs are downstream targets of the BDNF pathway. Administration of BoNT/A induced a robust downregulation of BDNF in vivo (Pinto et al., 2010), Other study found that SNAP-25 and Syb2 mediated the vesicular release of BDNF in axons and dendrites of cortical neurons (Shimojo et al., 2015). These results suggest that SNAP-25 regulates the presynaptic release of BDNF. Therefore, there is a possibility that SNAP-25 may regulate VGluT expression by the modulation of BDNF expression. However, further studies are required to support this hypothesis. In conclusion, we identified SNAP-25 is a potential downstream target of PKA and p-CREB, and it contributes to the CCI-induced neuropathic pain by regulating the expression of VGluT2 and astrocyte activation. This study provides a potential mechanism underlying SNAP-25-mediated neuropathic pain.
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COMPETING INTERESTS
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The authors declare that they have no competing interests.
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FUNDING
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This study was supported by grants from the National Natural Science Foundation of China (81471135, 81771206 and 81974172 to Dr. Zou) and the Natural Science Funds for Distinguished Young Scholar of Hunan Province (2017JJ1036 to Dr. Zou).
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AUTHORS’ CONTRIBUTIONS
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Jian Wang: this author helped perform the experiments and write the manuscript. Wei Xu, Yan Kong and Jiangju Huang: these authors helped perform the experiments. Zhuofeng Ding and Meiling Deng: these authors helped performed the data analyses.
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Qulian Guo: this author helped perform the analysis with constructive discussions. Wangyuan Zou: this author helped conceive and design the experiment, approve the final version.
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ACKNOWLEDGEMENT
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(Received 13 July 2019, Accepted 3 October 2019) (Available online xxxx)
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats
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Jiang Wang, a Wei Xu, b Yan Kong, a Jiangju Huang, a Zhuofeng Ding, a Meiling Deng, a Qulian Guo a,c and Wangyuan Zou a,c*
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Department of Anesthesiology, Hunan Provincial Maternal and Child Health Care Hospital, China
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National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
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Department of Anesthesiology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China
Abstract—Synaptosomal-associated protein 25 (SNAP-25) plays an important role in neuropathic pain. However, the underlying mechanism is largely unknown. Vesicular glutamate transporter 2 (VGluT2) is an isoform of vesicular glutamate transporters that controls the storage and release of glutamate. In the present study, we found the expression levels of VGluT2 correlated with the upregulation of SNAP-25 in the spinal cord of rats following chronic constriction injury (CCI)-induced neuropathic pain. Cleavage of SNAP-25 by Botulinum toxin A (BoNT/A) attenuated mechanical allodynia, downregulated the expression of VGluT2 and reduced glutamate release. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. Upregulation of SNAP-25 in naive rats increased VGluT2 expression and induced pain-responsive behaviors. In pheochromocytoma (PC12) cells, the expression of VGluT2 was also depended on SNAP-25 dysregulation. Moreover, we found VGluT2 was involved in SNAP-25-mediated regulation of astrocyte expression and activation of the PKA/p-CREB pathway mediated the upregulation of SNAP-25 in neuropathic pain. The findings of our study indicate that VGluT2 contributes to the effect of SNAP-25 in maintaining the development of neuropathic pain and suggests a novel mechanism underlying SNAP-25 regulation of neuropathic pain. Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: SNAP-25, VGluT2, neuropathic pain, PKA.
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INTRODUCTION
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Neuropathic pain is a difficult clinical puzzle and remains a major burden on both individuals and society. Actually, neuropathic pain is resistant to conventional therapy and no definitive solutions have been discovered. Therefore, there is a great interest in exploring the mechanism of neuropathic pain and developing new therapies. In recent years, particular interest has been devoted to the application of Botulinum toxin A (BoNT/A) in the treatment of painful conditions including neuropathic pain (Pavone and Luvisetto, 2010, Matak et al., 2014). The antinociceptive effect of BoNT/A relies on its ability to selectively cleave Synaptosomal-associated protein 25 (SNAP-25), which makes SNAP-25 to be a promising target to understand the development of neuropathic pain. SNAP-25 is a component of the SNARE-complex, which is involved in the process of cell exocytosis during
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synaptic transmission (Jahn and Scheller, 2006). Dysregulation of SNAP-25 has been associated with neurological disorders and different pain conditions (Antonucci et al., 2013, Liu et al., 2013, Olbrich et al., 2017). Inhibiting the vesicle release of pro-nociceptive neurotransmitters including glutamate from presynaptic terminals accounts for the SNAP-25-mediated analgesic effects (Durham et al., 2004, Carmichael et al., 2010, Bittencourt et al., 2014). Glutamate is the major excitatory neurotransmitter in the central nervous system involved in the pain process (Peirs et al., 2015). The transport of glutamate into synaptic vesicles is a prerequisite for glutamate release, and VGluT is responsible for this process (Weston et al., 2011). Mammals express three Vesicular glutamate transporter (VGluT) isoforms, namely, VGluT1, VGluT2 and VGluT3, which exhibit a nonoverlapping distribution in the CNS (Morris et al., 2005, Moechars et al., 2006). Among these three VGluTs, VGluT2 is proposed as the major regulator of neuropathic pain (Moechars et al., 2006, Liu et al., 2010). Previous study suggested the expression of SNAP-25 and VGluT2 correlated in several neurological diseases (Piroli et al., 2013, Glazova et al., 2015). However, the specific relationship between SNAP-25 and VGluT2 in neuropathic pain has not been
*Correspondence to: Wangyuan Zou, Department of Anesthesiology, Xiangya Hospital, Central South University, 87 Xiangya Road, Changsha, Hunan 410008, China. Fax: +86-731-84327413. E-mail address: [email protected] (W. Zou). Abbreviations: BoNT/A, botulinum toxin A; CCI, chronic constriction injury; PC12, pheochromocytoma cells; SNAP-25, synaptosomalassociated protein 25; VGluT2, vesicular glutamate transporter 2. https://doi.org/10.1016/j.neuroscience.2019.10.007 0306-4522/Ó 2019 IBRO. Published by Elsevier Ltd. All rights reserved. 1
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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studied yet. We, therefore, performed this study to test the hypothesis that VGluT2 is involved in SNAP-25-mediated regulation of neuropathic pain. Hence, the present study was designed to investigate (i) effect of dysregulation of SNAP-25 on VGluT2 expression in a rat model of chronic constriction injury (CCI)-induced neuropathic pain and PC12 cells; (ii) the role of VGluT2 in the SNAP-25-mediated regulation of astrocyte activation; and (iii) whether the PKA/p-CREB pathway was involved in the dysregulation process of SNAP-25 in neuropathic pain. Our findings may provide a novel mechanism underlying SNAP-25-mediated regulation of the development of neuropathic pain.
EXPERIMENTAL PROCEDURES
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Animals
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Male Sprague-Dawley rats weighting 180–220 g were used in this experiment. Rats were housed in groups of six per cage and maintained on a 12-h light/dark cycle with free access to food and water. Each experimental procedure was performed in accordance with the Administrative Committee of Experimental Animal Care and Use of Central South University and was approved by the Central South University Ethical Committee for Animal Research. The study was in agreement with the Ethical Guidelines of the International Association for the Study of Pain (guidance). An observer who was blinded to the animal treatment performed all behavioral tests, and all efforts were made to minimize suffering and reduce the number of animals used to the minimum required for statistical accuracy in the study.
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CCI surgery was performed as previously described (Zou et al., 2012). In brief, rats were anesthetized using isoflurane (4% to induce, 2% to maintain), and the left sciatic nerves were exposed at the mid-thigh level by blunt dissection. Four ligatures (4-0 chromic gut suture) were loosely tied around the nerve at 1-mm spacing. The sciatic nerves in the Sham group were only exposed without ligation.
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Von Frey test. Mechanical paw withdrawal threshold was determined using von Frey filaments ranging from 0.4 g to 26 g. Rats were placed in plastic chambers on metal mesh for 15 min to acclimate to the environment before testing. Von Frey filaments (UGO, Italy) were applied to the mid-plantar surface of the hind paw for 2–3 s, with a 5 min interval between each test.
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Withdrawal or licking of the paw was considered a positive response. Each test was repeated three times, and the lowest filament that elicited a positive response was considered the threshold stimulus.
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Rotarod test. Locomotor function was evaluated using rotarod testing, as previously described (Zychowska et al., 2016). In brief, rats were acclimated to revolving drums for 5 min before testing. A training trial on the revolving drums (15 rpm) was performed prior to the actual day of testing. Rats that remained on the drum for more than 150 s were selected for testing. The rotarod performance time of each rat was recorded. The cut-off time was set as 300 s. Each test was repeated three times at five-minute intervals. The mean values of three tests were compared.
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Intrathecal drug delivery
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Intrathecal catheter surgery was performed as previously described (Loram et al., 2013). Rats were anesthetized with isoflurane. An 18-gauge guide needle was inserted into the L5/6 intervertebral space, and a PE-10 catheter was inserted into the subarachnoid space through the guide needle. A marker was preset to ensure that the end of the catheter rested over the lumbar enlargement. Drugs and lentivirus were injected over 30 s with a 1 min delay before removing the catheter or needle. Rats with neurological deficits were excluded from the experiment.
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Drugs administration
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The BoNT/A (BOTOXÒ, Allergan, USA) was dissolved in 0.9% saline. BoNT/A (20 ll of 20 U/kg) or saline was injected intraplantar into the left hind paw while the rats were under 2% isoflurane anesthesia 5 days after CCI surgery. The concentration of BoNT/A was based on a previous study (Favre-Guilmard et al., 2009). The PKA inhibitor H-89 (Sigma, USA) was dissolved in 1% DMSO and diluted to the appropriate concentration insterile saline. H-89 (10 ll of 10 lM) or vehicle was intrathecally injected 7 days after CCI. The concentration of H-89 was based on a previous study (Sun et al., 2004).
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Lentivirus injection
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Lentiviral vector-mediated VGluT2 (LV-VGluT2), SNAP25 (LV-SNAP-25) and scrambled control (LV-NC) were purchased from Genepharma. Lentivirus (10 ll) was intrathecally injected on the day of CCI surgery for in vivo testing. The transfection efficiency was evaluated 10 days after lentivirus injection by monitoring GFP
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" Fig. 1. SNAP-25 and VGLUT2 are upregulated during CCI-induced neuropathic pain. (A) Robust mechanical allodynia was observed in the ipsilateral hindpaw of CCI rats. n = 5, ***P < 0.001, versus Sham. (B) Western blot analysis revealed a gradual increase in SNAP-25 and VGluT2 in the spinal cord after CCI. n = 3, **P < 0.01, ***P < 0.001, versus Sham. (C) Immunofluorescence shows the expression pattern of SNAP-25 (green) and VGluT2 (red) in the spinal cord 10 days after surgery. SNAP-25 and VGluT2 were upregulated in the ipsilateral dorsal horn of CCI rats. n = 3, *P < 0.05, versus contralateral side of spinal dorsal horn. Arrows indicate the expression of SNAP-25 and VGluT2 in the dorsal horn. D. Double immunofluorescence revealed that SNAP-25 colocalized with VGluT2 in the dorsal horn of spinal cord. Contra = contralateral side, ipsi = ipsilateral side. Scale bar = 100 lm.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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expression in rat spinal cords with a fluorescence microscope (Leica, Germany).
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Cell culture and treatment
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Differentiated pheochromocytoma (PC12) cells were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Roswell Park Memorial Institute (PRMI) 1640 medium (Invitrogen, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA), 50 U/ml penicillin and 50 lg/ml streptomycin under humidified conditions in 5% CO2 at 37 °C. To examine the correlation between SNAP-25 and VGluT2 in PC12 cells, cells were first pretreated with BoNT/A (33 U/ml) for 24 h and collected for Western blot or immunochemistry analysis. The concentration of BoNT/A was determined as previously described (Jenkinson et al., 2017). Cells for lentivirus transfection were transfected with lentivirus (LV-SNAP-25 or LV-NC) at 100 multiplicity of infection (MOI) for 24 h, followed by Western blot analysis. GFP fluorescence in PC12 cells was monitored to evaluate transfection efficiency.
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Immunochemistry
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Immunochemistry was performed as previously described (Wang et al., 2016, Huang et al., 2019). Rats were transcardially perfused with 4% paraformaldehyde, and the lumbar spinal cord enlargements were dissected and transferred to 25% sucrose-PBS overnight. The spinal cord was sectioned at a thickness of 10 lm in a cryostat. Sections were rinsed in 0.1% PBS twice and were permeabilized for 10 min with phosphate-buffered saline (PBS) containing 0.1% Triton X-100. 10% donkey serum was used to block nonspecific binding for 1 h. Sections were incubated overnight with the following primary antibodies: rabbit anti-SNAP-25 (1:200, Abcam, USA); mouse antiVGluT2 (1:200, Abcam, USA); rabbit anti-p-CREB (1:200, Abcam, USA); mouse anti-GFAP (1:500, Abcam, USA); and rabbit anti-GFP (1:200, Cell Signal Technology, USA) overnight. Sections were rinsed in PBS three times and incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG or Alexa Fluor 594 conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, USA) for 2 h. A Leica Observer Microscope was used to visualize the sections. PC12 cells for in vitro immunochemistry were seeded in 24-well plates and fixed with 1% paraformaldehyde for 10 min. All subsequent steps were performed as described above. All the sections were processed under standardized conditions in every experiment, tissues and cells from control and experimental groups were collected, and then processed for immunohistochemistry simultaneously (Taylor and Levenson, 2006, Glazova et al., 2015). All images were captured in a constant condition. Quantification was performed by using the Image-Pro Plus 6.0 (Media Cybernetics, USA).
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Western blots were performed as previously described (Wang et al., 2017). Rat lumbar spinal enlargements and cells were homogenized in Radio Immunoprecipita-
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tion Assay (RIPA) lysis buffer containing a protease inhibitor cocktail. The bicinchoninic acid (BCA) Protein Assay kit (Beyotime, China) was used to determine the concentrations of total protein. Equal amounts of protein (50 lg) were loaded in 10% dodecyl sulfate, sodium salt–Polyacrylamide gel electrophoresis (SDS–PAGE) and separated at 80–100 V for 90 min. Proteins at the desired molecular weights were transferred to polyvinylidene difluoride membranes (Merck Millipore, USA) at 300 mA for 60 min. Membranes were blocked with 5% nonfat milk and probed with the following antibodies: rabbit anti-SNAP-25 (1:2000, Abcam, USA); mouse antiVGluT2 (1:2000, Abcam, USA); mouse anti-VGluT1 (1:2000, Abcam, USA); mouse anti-VGluT3 (1:2000, Abcam, USA); rabbit anti-Tublin (1:5000, Abcam, USA); rabbit anti-p-CREB (1:2000, Abcam, USA); and rabbit anti-GAPDH (1:5000, Merck Millipore, USA). Primary antibodies were detected with rabbit or mouse Horseradish Peroxidase (HRP)-conjugated secondary IgG (1:20,000, Jackson ImmunoResearch, USA). Signals were visualized using an enhanced chemiluminescence plus system (Merck Millipore, USA). Each sample was run in duplicate. The densities of specific bands were analyzed using the Image Lab 3.0 system (Bio-Rad, USA) for quantification.
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Glutamate concentration measurement
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The Glutamate Colorimetric Assay Kit (K629, BioVision, USA) was used to measure glutamate concentrations. Spinal cord enlargements were collected and homogenized in 100 lL assay buffer. Homogenates were centrifuged at 13,000g for 10 min, and the supernatants were isolated and transferred to 96-well plates. Assay reagents were added, and the optical density (OD) at 450 nm was measured in the microplate reader. Glutamate concentrations were calculated according to the manufacturer’s instructions.
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Statistical analysis
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No statistical analyses were used to predetermine the sample size. Our sample sizes were based on previous similar publications (Fan et al., 2017). All data are expressed as the means ± SD. Behavioral tests were analyzed using two-way repeated measures ANOVA, followed by post hoc Bonferroni’s test. One-way ANOVA test was used for multiple comparisons. Bonferroni tests were performed for post hoc comparisons. Unpaired Student’s t test was used for comparisons of two groups. All statistical tests were performed in GraphPad prism 6.0. P < 0.05 was considered statistically significant.
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Expression changes in SNAP-25 correlated with VGluT2 in the spinal dorsal horn of CCI rats
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We first examined the changes in expression of SNAP-25 and VGluT2 in the spinal cord in a CCI-induced neuropathic pain model. After CCI induction (Fig. 1A), western blots showed that the expression of SNAP-25 in the spinal cord gradually increased, and this increase
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Fig. 2. VGLUT2 is involved in SNAP-25-mediated development of neuropathic pain. (A) BoNT/A attenuated CCI-induced mechanical allodynia. BoNT/A (20 U/kg, 20 lL) or saline was intraplantarly injected into the ipsilateral hind paw of rats on day 5 postsurgery. The withdrawal threshold was measured 60 min after BoTN/A injection. n = 6, *P < 0.05, **P < 0.01, versus CCI + saline, ##P < 0.01, ###P < 0.001, versus Sham + saline. (B) Rotarod test showed that intraplantar injection of BoNT/A did not affect the motor function of rats. The rotarod test was measured 30 min after BoNT/A injection. n = 5. *P < 0.05, vs Sham + saline. (C) Western blot analysis showed BoNT/A reduced expression levels of SNAP-25 and VGluT2 proteins in the spinal cord 10 days after CCI induction. n = 4, *P < 0.05, **P < 0.01, versus Sham + saline. (D) Immunofluorescent staining for SNAP-25 (green) and VGluT2 (red) in the spinal cord of rats 10 days after CCI, n = 4. (E) The protein expression levels of VGluT1 and VGluT3 were not altered after BoNT/A injection. The proteins were extracted from rats 10 days after CCI induction. n = 3. (F) BoNT/A injection ameliorated the upregulation of glutamate concentrations induced by CCI. n = 3, *P < 0.05, versus Sham + saline, #P < 0.05, versus CCI + saline.
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was maintained up to 14 days. Notably, the expression levels of VGluT2 was also increased significantly after CCI treatment and exhibited the same expression pattern as SNAP-25 (Fig. 1B). Immunostaining results showed that positive SNAP-25 staining was primarily detected in the superficial laminae of the spinal dorsal horn, and VGluT2 was expressed in the superficial and deeper laminae. CCI treatment significantly increased SNAP-25- and VGluT2-positive staining in the ipsilateral spinal dorsal horn (Fig. 1C). Double-staining revealed that SNAP-25 mainly colocalized with VGluT2 in the dorsal horn (Fig. 1D).
Downregulation of SNAP-25 by BoNT/A reduced the expression of VGluT2
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After BoNT/A, which cleaves SNAP-25 (Frick et al., 2007), was administered to CCI rats, the mechanical sensitivity using von Frey test and spinal VGluT2 expression levels were examined. Intraplantar administration of BoNT/A significantly decreased CCI-induced mechanical allodynia 5 days after CCI, and the antinociceptive effect of BoNT/A was maintained 14 days after CCI (Fig. 2A). Rotarod test showed that the latencies of CCI rats were lower than sham rats, however, rat performances were
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Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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not different between the BoNT/A-treated and salinetreated rats (Fig. 2B). Western blots showed that BoNT/A administration significantly suppressed CCI-induced upregulation of SNAP-25 and VGluT2 in the spinal cord (Fig. 2C). Immunochemistry results showed that the expression levels of SNAP-25 and VGluT2 were reduced in the spinal cord after BoNT/A administration (Fig. 2D). We found the other two isoforms of VGluTs, namely, VGluT1 and VGluT3, were not significantly changed after BoNT/A injection (Fig. 2E). VGluT2 exerts its function mainly via regulating glutamate release between synaptic clefts. Therefore, the concentration of glutamate in the spinal cord was also examined. We found that the spinal glutamate concentrations were significantly elevated after CCI injury, and intraplantar injection of BoNT/A blocked the upregulation of glutamate (Fig. 2F). To further identify whether SNAP-25 targeted VGluT2 to regulate CCI-induced neuropathic pain, we intrathecally injected LV-VGluT2 to overexpress VGluT2 on the same day as CCI surgery. Immunochemistry results showed obvious GFP in the spianl cords of LVVGluT2 and LV-NC treated rats (Fig. 3A). After BoNT/A injection, LV-VGluT2 rats showed a small but significant decrease in withdrawal thresholds in response to von Frey filaments 10 days after CCI surgery compared to LV-NC rats (Fig. 3B).
Fig.3. Overexpression of VGluT2 abolished the antinociceptive effect of BoNT/A. (A) GFP was expressed in the lumbar spinal cord (L3–L4) 10 days after intrathecal injection of LV-VGluT2 or LV-NC in CCI rats. Scale bar = 100 lm. (B) Paw withdraw threshold of CCI rats after LV-VGluT2 or LV-NC injection. LV-VGluT2 or LV-NC at a titer of 1 109 TU/ml was intrathecally injected on the same day as CCI surgery. BoNT/A (20 U/kg, 20 lL) was intraplantarly injected into the ipsilateral hind paw of rats on day 8 postsurgery. n = 6, **P < 0.01, versus CCI + botox + LV-NC.
Overexpression of SNAP-25 increased VGluT2 expression and induced mechanical allodynia in naive rats
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To further explore the relationship between SNAP-25 and VGluT2 in neuropathic pain, naive rats were intrathecally injected with LV-SNAP-25, and behavior test and western blot were performed. We found that overexpression of SNAP-25 induced mechanical allodynia in naive rats 5 days after lentivirus injection (Fig. 4A). Following western blot tests showed that upregulated SNAP-25 also increased VGluT2 expression level in the spinal cord (Fig. 4B).
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SNAP-25 regulated the expression of VGluT2 in PC12 cells
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Differentiated PC12 cells were used to further evaluate the regulator role of SNAP-25 in VGluT2 expression in vitro. Immunochemistry found that SNAP-25 and VGluT2 were expressed abundantly in the cytoplasms of PC12 cells (Fig. 5A). Next, we treated PC12 cells with BoNT/A to downregulate SNAP-25 expression.
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Fig. 4. Upregulation of SNAP-25 induced mechanical allodynia in naive rats. (A) Intrathecal injection of LV-SNAP-25 produced mechanical allodynia in naive rats. LV-SNAP-25 or LV-NC at a titer of 2 109 TU/ml were intrathecally injected on day 1. n = 6, ***P < 0.001, versus LV-NC. (B) Western blot results show that the expression of SNAP-25 and VGluT2 was upregulated 5 days after lentivirus injection. n = 4, *P < 0.05, versus LV-NC.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Western blots and immunochemistry showed that the expressions of SNAP-25 and VGluT2 were both downregulated in PC12 cells (Fig. 5B, C), which is consistent with the in vivo test. To further unravel the relationship between SNAP-25 and VGluT2 in vitro, we transfected PC12 cells with lentiviral-mediated SNAP-25 to overexpress SNAP-25. GFP was visualized by microscopy in PC12 cells after lentivirus infection, and the results indicated successful lentivirus delivery. Western blot analysis showed that overexpression of SNAP-25 increased the protein level of VGluT2 (Fig. 5D).
VGluT2 was involved in SNAP-25-mediated regulation of astrocyte activation Previous studies suggested that SNAP-25 participated in the modulation of the astrocyte activation in chronic pain (Marinelli et al., 2012). To further determine whether VGluT2 was also involved in SNAP-25-mediated regulation of astrocyte activation, we first examined the spatial localization of VGluT2 and the astrocyte marker GFAP in the spinal cord. Double immunostaining revealed that
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VGluT2 colocalized with GFAP in the spinal cord (Fig. 6A). By western blot and immunostaining, we found GFAP expression increased significantly in the ipsilateral dorsal horns after CCI injury, which is consistent with the expression changes of SNAP-25 and VGluT2. And downregulation of SNAP-25 following intraplantar injection of BoNT/A, not only reduced VGluT2 expression, but also significantly inhibited the upregulation of GFAP. Moreover, we found VGluT2 overexpression via intrathecal injection of LV-VGluT2 rescued the CCI-induced upregulation of GFAP (Fig. 6B, C).
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The PKA/p-CREB pathway was involved in SNAP-25mediated regulation of neuropathic pain
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Previous studies identified SNAP-25 mRNA has a binding site for p-CREB and recognized that SNAP-25 was a known target of the PKA/p-CREB signal pathway (Hang et al., 2013). However, the specific role of PKA/p-CREB in the regulation of SNAP-25 expression in CCI-induced neuropathic pain has not been fully studied.
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Fig. 5. SNAP-25 regulates VGluT2 expression in PC12 cell. (A) Immunochemistry revealed the localization of SNAP-25 and VGluT2 in PC12 cells. SNAP-25 (green) and VGluT2 (red) are expressed in the cytoplasm of PC12 cells. (B, C) Suppression of SNAP-25 by BoNT/A downregulated the expression of VGluT2 in PC12 cells. PC12 cells were treated with 33 U/ml BoNT/A (botox group) or vehicle (control group) for 24 h. (B) Western blot showed that the protein expression of SNAP-25 and VGluT2 in PC12 cells were downregulated after BoNT/A administration. n = 4, *P < 0.05, ***P < 0.001, versus control. (C) Immunofluorescent staining revealed that the expression levels of SNAP-25 and VGluT2 were reduced after BoNT/A treatment. n = 3, **P < 0.01, versus control, scale bar = 100 lm. (D) Overexpression of LV-SNAP-25 upregulated the expression of VGluT2 in PC12 cells. Up: Green fluorescent protein (GFP) was visualized in PC12 cells after transfection with LV-SNAP-25 and LV-NC. Scale bar = 100 lm. Down: Western blot results showed that the expression levels of SNAP-25 and VGluT2 were increased after LV-SNAP-25 infection. n = 3, *P < 0.05, versus LV-NC.
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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Fig. 6. VGluT2 is involved in the SNAP-25-mediated activation of astrocyte. (A) Double immunofluorescence showed that VGluT2 was expressed in the astrocyte. Arrow indicates that GFAP colocalized with VGluT2 in the spinal cord. Scale bar = 100 lm. (B) Western blot results showed downregulated SNAP-25 by BoNT/A decreased the expression of GFAP, and intrathecal injection of LV-VGluT2 abolished the effect of BoNT/A. ***P < 0.001, vs Sham, ##P < 0.01, vs CCI, aP < 0.05, vs CCI + botox + LV-NC, n = 3. C. Immunofluorescent staining showed that overexpression of VGluT2 restored the activation of GFAP in the spinal cord of CCI rats that were treated with BoNT/A. LV-VGluT2 or LV-NC was intrathecally injected on the same day as CCI surgery. BoNT/A (20 U/kg, 20 lL) was intraplantarly injected into the ipsilateral hind paw of rats on day 8 postsurgery. Western blot and immunofluorescent staining for GFAP in the spinal cord was performed 14 days after CCI. n = 3, scale bar = 100 lm.
" Fig. 7. PKA/p-CREB signaling is involved in SNAP-25 dysregulation in CCI-induced neuropathic pain. (A) Blockade of PKA activation in the spinal cord attenuated CCI-induced mechanical allodynia. The PKA inhibitor H-89 or vehicle was intrathecally injected in the spinal cord 7 days after CCI. n = 6, *P < 0.05, ***P < 0.001, versus CCI + Vehicle. (B) CCI-induced p-CREB upregulation was blocked by intrathecal injection of H-89. The proteins were extracted from rats 60 min after H-89 injection. n = 3, *P < 0.05, versus CCI + Vehicle, #P < 0.05, versus Sham + Vehicle. (C) Downregulation of p-CREB was confirmed by immunochemistry after H-89 injection. Scale bar = 100 lm. (D) The protein levels of SNAP-25 and VGluT2 were reduced after H-89 injection. n = 3, *P < 0.05, versus CCI + Vehicle, #P < 0.05, versus Sham + Vehicle. The samples for immunohistochemistry and western blot were harvested at 7 days after CCI.
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To investigate whether CREB activation is involved in SNAP-25 upregulation in neuropathic pain, we intrathecally injected an inhibitor of the PKA/p-CREB pathway, H-89, to CCI rats. Behavioral tests showed that intrathecal administration of H-89 significantly reduced the mechanical allodynia of CCI rats 10 min after drug administration, and this effect lasted for 60 min (Fig. 7A). Western blot analysis showed that pCREB expression was upregulated in the spinal cord of CCI rats, and intrathecal injection of H-89 significantly reduced the upregulation of p-CREB (Fig. 7B). Immunochemistry results showed that the number of pCREB-positive cells increased in the ipsilateral spinal cord after CCI injury, which was blocked by H-89 administration (Fig. 7C). In addition, CCI induced upregulation of SNAP-25 and VGluT2 were also inhibited by the PKA/p-CREB pathway inhibitor H-89 (Fig. 7D).
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DISCUSSION
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Recent studies showed that SNAP-25 was involved in the regulation of neuropathic pain. However, the precise mechanisms underlying this regulation remain elusive. The present study found that SNAP-25 targeted VGluT2 to regulate the development of neuropathic pain and astrocyte activation, and PKA/p-CREB pathway was involved in the dysregulation process of SNAP-25. These findings suggest novel mechanisms underlying SNAP-25-mediated neuropathic pain. Previous studies demonstrated that the expression of SNAP-25 and VGluT2 correlated in several neurological diseases. Glazova et al. found the alleviation of seizures by ERK1/2 inhibition led to an accumulation of SNAP-25 and VGluT2 in the hippocampus and temporal cortex (Glazova et al., 2015). Piroli et al. showed reduced glutamate efflux and VGluT2 expression in the central amygdala complex of animals subjected to repeated stress, and administration of the antidepressant tianeptine reversed stress-induced decreases in amygdala VGluT2 and increased the expression of SNAP-25 (Piroli et al., 2013). However, no study ever showed that there was a correlation between SNAP-25 and VGluT2 in neuropathic pain. The present study found a gradual upregulation of SNAP-25 and VGluT2 after nerve injury. Immunochemistry results showed that SNAP-25 was mainly expressed in the superficial laminae of the spinal dorsal horn, and colocalized with VGluT2. Moreover, SNAP-25 and VGluT2 were both upregulated in the ipsilateral side of spinal cord after nerve injury. The parallel expression changes and colocalization of SNAP-25 and VGluT2 suggest that the upregulation of VGluT2 is involved in the upregulation of SNAP-25 in CCI-induced neuropathic pain. In consistent with previous study (Marinelli et al., 2010), we found blockade of SNAP-25 upregulation by BoNT/A prevented the development of neuropathic pain, and rats perfomances in Rotarod test were not significantly changed after BoNT/A injection, indicating antinociceptive effects of the downregulation of SNAP-25 by BoNT/A were independent of muscle relaxation. Interest-
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ingly, the expression level of VGluT2 and glutamate concentration were also reduced after injection of BoNT/A. The overexpression of VGluT2 in the spinal cord abolished the antinociceptive effect of BoNT/A, which indicates that VGluT2 is required in the spinal cord for the antinociceptive effect of SNAP-25 downregulation. There are two more isoforms in the VGluT family, namely, VGluT1 and VGluT3. Previous study showed that the distribution pattern of VGluT1 and VGluT3 was not colocalized with SNAP-25 in the spinal cord (Alvarez et al., 2004). Our study showed that the protein levels of VGLuT1 and VGluT3 were not changed after BoNT/A injection, indicating the VGluT2 downregulation especially depended on the cleavage of SNAP-25. Therefore, there may be a lower correlation between SNAP-25 and VGluT1 or VGluT3 in the development of neuropathic pain. To further explore the relationship between SNAP25 and VGluT2 in neuropathic pain, we overexpressed SNAP-25 in naive rats, and found overexpression of SNAP-25 induced mechanical allodynia in naive rats, and upregulated the expression of VGluT2. Moreover, we found SNAP-25 also regulated VGluT2 expression in PC12 cells. Taken together, these results suggested that SNAP-25 may target VGluT2 to regulate the development of neuropathic pain. Mounting evidence demonstrated the contribution of astrocytes to the induction and maintenance of chronic pain (Scholz and Woolf, 2007). Our present study demonstrated that the astrocyte marker GFAP was upregulated in the spinal cord 14 days after CCI induction, which paralleled SNAP-25 expression. Intraplantar injection of BoNT/A reduced SNAP-25 expression, alleviated neuropathic pain, and reduced GFAP expression, which suggests that SNAP-25 regulates astrocyte activation in neuropathic pain. Vacca et al. (2013) found that BoNT/A reduced astrocyte activation and alleviated morphine tolerance, which was likely consequences of reduced release of glutamate. VGluT2 regulates glutamate release, and it was regulated by SNAP-25 in neuropathic pain. Therefore, we hypothesized that VGluT2 was also involved in the regulation of astrocyte expression. Double immunochemistry found that VGluT2 colocalized with GFAP in the dorsal horn of the spinal cord, and VGluT2 overexpression restored the upregulation of GFAP in the spinal cord, which indicates that VGluT2 plays a role in the SNAP-25-mediated regulation of astrocyte activation. A previous study demonstrated that activation of PKA in the spinal cord was required for the development of neuropathic pain (Zhou et al., 2015). Activation of PKA was implicated in CREB-mediated gene transcription (Hang et al., 2013). SNAP-25 is one of the downstream targets of CREB (Han et al., 2014). It is possible that SNAP-25 functions as a PKA-regulated CREB target in CCI-induced neuropathic pain. Our present results showed that the upregulation of SNAP-25 correlated with p-CREB activation in CCI-induced neuropathic pain. When PKA/p-CREB activation was inhibited, the CCIinduced upregulation of SNAP-25 and VGluT2 were significantly suppressed. Moreover, we found that the PKA inhibitor H-89 attenuated CCI-induced allodynia to a sim-
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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ilar level as SNAP-25 cleavage. Taken together, these findings suggest the CCI-induced upregulation of SNAP25 may depend on CREB activation, and SNAP-25 may be a downstream target in the PKA/p-CREB pathway in neuropathic pain. However, there are several limitations in our study. First, we used total protein from spinal cord to evaluate the expression levels of SNAP-25 and VGluT2. SNAP25 and VGluT2 are expressed abundantly in vesicle fractions, and it is better to evaluate the expression changes of these two proteins using homogenates from vesicle fractions. Second, the precise mechanisms underlying SNAP-25 mediated regulation of VGluT2 are not clear. We postulate that BDNF may play a role in this regulatory process. BDNF is an important neurotrophic factor in neurological process, the activation of the BDNF/TrkB signal pathway modulates the expression of various genes, and it is involved in numerous neurological processes (Martinowich et al., 2007, Sikandar et al., 2018). Recent studies revealed that presynaptic BDNF regulated the expression of VGluTs in vitro and in vivo (Melo et al., 2013, Azogu and Plamondon, 2017), which suggests that VGluTs are downstream targets of the BDNF pathway. Administration of BoNT/A induced a robust downregulation of BDNF in vivo (Pinto et al., 2010), Other study found that SNAP-25 and Syb2 mediated the vesicular release of BDNF in axons and dendrites of cortical neurons (Shimojo et al., 2015). These results suggest that SNAP-25 regulates the presynaptic release of BDNF. Therefore, there is a possibility that SNAP-25 may regulate VGluT expression by the modulation of BDNF expression. However, further studies are required to support this hypothesis. In conclusion, we identified SNAP-25 is a potential downstream target of PKA and p-CREB, and it contributes to the CCI-induced neuropathic pain by regulating the expression of VGluT2 and astrocyte activation. This study provides a potential mechanism underlying SNAP-25-mediated neuropathic pain.
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COMPETING INTERESTS
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The authors declare that they have no competing interests.
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FUNDING
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This study was supported by grants from the National Natural Science Foundation of China (81471135, 81771206 and 81974172 to Dr. Zou) and the Natural Science Funds for Distinguished Young Scholar of Hunan Province (2017JJ1036 to Dr. Zou).
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AUTHORS’ CONTRIBUTIONS
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Jian Wang: this author helped perform the experiments and write the manuscript. Wei Xu, Yan Kong and Jiangju Huang: these authors helped perform the experiments. Zhuofeng Ding and Meiling Deng: these authors helped performed the data analyses.
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Qulian Guo: this author helped perform the analysis with constructive discussions. Wangyuan Zou: this author helped conceive and design the experiment, approve the final version.
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ACKNOWLEDGEMENT
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Not applicable.
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(Received 13 July 2019, Accepted 3 October 2019) (Available online xxxx)
Please cite this article in press as: Wang J et al. SNAP-25 Contributes to Neuropathic Pain by Regulation of VGLuT2 Expression in Rats. Neuroscience (2019), https://doi.org/10.1016/j.neuroscience.2019.10.007
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