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ejp174-sup-0001-si.tif262K Figure S1. Validation test using western immunoblotting for TNF-α and P2X7R antibody specificity. Western blotting of TNC 3 days after CCI of IoN resulted in staining of a major band running at 75 kDa (P2X7R), 25 kDa (mTNF-α) and 17 kDa (sTNF-α) when detected with the anti-TNF-α or P2X7R antibody. Pre-absorption of antibodies with the recombinant peptide of TNF-α or P2X7R abolished staining of the membrane.
ejp174-sup-0002-si.tif358K Figure S2. Comparison of the expression of P2X7R between the CCI of the IoN and sham operation groups. Western blots analysis showing P2X7R protein expression 3 days after CCI and sham operation. Example blot and relative protein levels are shown. Western blots show immunoreactive bands at 45 kDa (β-actin) and 75 kDa (P2X7R). **p < 0.01, *p < 0.05 as indicated by the bracket. n = 4 for each group. Error bars represent SEM.
ejp174-sup-0003-si.tif6443K Figure S3. Immunofluorescence labelling with the antibody against CD45 (green, left lane) and DAPI (red, middle lane) in the spleen (upper row) and ipsilateral Vc (lower row) at 3 days after CCI. Overlap of CD45 and DAPI are shown in the right lane. Arrow indicates double-labelled CD45/DAPI cell in the Vc.
ejp174-sup-0004-si.tif2887K Figure S4. Effects of the P2X7R selective antagonist A438079 on tactile allodynia/hyperalgesia on the ipsilateral WP following CCI of the IoN. The histogram shows EF50 values derived from stimulus–response frequency curves in behavioural testing. A438079 (3.5 or 35 μg/rat) was injected intrathecally via a cannula implanted at the level of the TNC. Infusion of the drugs started 3 days after CCI of the IoN. Saline was infused as a vehicle control. **p < 0.01, versus CCI + saline group. n = 4 for each. Error bars represent SEM.
ejp174-sup-0005-si.tif509K Figure S5. Comparison of the levels of mTNF-α and sTNF-α between the CCI of the IoN and sham operation groups. Western blots analysis showing mTNF-α and sTNF-α protein expression 3 days after CCI and sham operation. Example blot and relative protein levels are shown. Western blots show immunoreactive bands at 45 kDa (β-actin), 25 kDa (mTNF-α) and 17 kDa (sTNF-α). **p < 0.01, *p < 0.05 as indicated by the bracket. n = 4 for each group. Error bars represent SEM.
ejp174-sup-0006-si.tif1985K Figure S6. Effects of etanercept on allodynia/hyperalgesia on the WP (A) and TNF-α levels (B) induced by BzATP. (A) A decrease in EF50 following intrathecal treatment of BzATP was prevented by pretreatment of rats with etanercept. This effect lasted 24 h. **p < 0.01, *p < 0.05 versus BzATP + saline. n = 4 for each group. Error bars represent SEM. (B) the increase in the level of sTNF-α protein was observed 1 day after intrathecal BzATP treatment. The inhibition of this increase in the level of sTNF-α protein by etanercept was confirmed. Example blots and relative protein levels are shown. Western blots show immunoreactive bands at 45 kDa (β-actin), 25 kDa (mTNF-α) and 17 kDa (sTNF-α). Quantification of Western blots showing mTNF-α and sTNF-α protein levels relative to those in naïve rats. **p < 0.01, *p < 0.05 as indicated by the bracket. n = 4 for each group. Error bars represent SEM.
ejp174-sup-0007-si.docx18K Appendix S1. All behavioural tests were conducted under blind conditions. The tactile allodynia/hyperalgesia induced by CCI of the IoN was assessed by a method described previously (Takahashi et al., 2011). Briefly, the rats were habituated to stand on a soft pad and lean against the experimenter's hand. Twelve stimuli had the following marking (force) values: 3.61 (0.4 g), 3.84 (0.6 g), 4.08 (1.0 g), 4.17 (1.4 g), 4.31 (2.0 g), 4.56 (4.0g), 4.74 (6.0 g), 4.93 (8.0 g), 5.07 (10.0 g), 5.18 (15.0 g), 5.46 (26.0 g), and 5.88 (60.0 g). An active withdrawal of the head from the probing filament was defined as a response to a stimulus. Each von Frey filament was applied 5 times in intervals of a few seconds. The response frequencies [(number of responses/number of stimuli) × 100%] to a range of von Frey filament forces were determined and a stimulus-response (S-R) curve was plotted. After a non-linear regression analysis, an EF50 value, defined as the von Frey filament force (g) that produces a 50% response frequency, was derived from the S-R curve. We used EF50 value as a measure of mechanical sensitivity. A cut-off value of the EF50 was defined at 91.08 g. All behavioural testing was performed between 9 a.m. and 4 p.m.
ejp174-sup-0008-si.docx18K Appendix S2. To avoid systemic effects of drugs, we delivered drugs into the cerebrospinal fluid space around the medullary dorsal horn as described previously (Takahashi et al., 2011). Briefly, BzATP (0.1 μg/μL, 5 μL; Sigma Chemical Co., St Louis, MO), A438079 (0.35 or 3.5 mg/mL, 10 μL; TOCRIS Bioscience, Ellisville, MO), SB203580 (0.1 or 1 mg/mL, 10 μL, Calbiochem, La Jolla, CA), or etanercept (10 μg/mL, 10 μL, Enbrel; Immunex, Seattle, WA) were delivered by bolus treatment via a PE10 catheter implanted into the cerebral spinal fluid space around the medullary dorsal horn. These drugs were dissolved in 0.15 M NaCl or 0.1 M PBS. Intrathecal microtreatments were performed using a 10-μL void volume. To ensure complete drug delivery, all catheter placements were verified on completion of experiments by visual inspection. Data were only analysed from animals with catheters verified as having the catheter tip at the medullary dorsal horn level.
ejp174-sup-0009-si.docx21K Appendix S3. For ABC method, free-floating sections were incubated in 0.1 M PBS containing 3% normal goat serum with 0.1% Triton X-100 for 1 h, and then incubated overnight at 4 °C with primary antibodies against CD11b (clone OX-42; mouse, 1:300,Gene Tex, Irvine, CA), or P2X7R (rabbit, 1:10000, Alomone Labs Ltd, Jerusalem, Israel). The diluents for the antibodies were 0.1 M PBS containing 3% normal goat serum. After repeated washes in PBS, sections were incubated for 1 h with biotinylated anti-mouse IgG antibody (Goat, 1:400; Vector Laboratories, Burlingame, CA) or biotinylated anti-rabbit IgG antibody (Goat, 1:400; Vector Laboratories). After repeated washes in PBS, sections were incubated for 1 h with avidin and biotinylated horseradish peroxidase complex (1:50; Vector Laboratories). After washes in PBS, tissue sections were finally reacted with DynaChrome DAB chromogen system (Thermo Electron, Pittsburgh, PA) for 5–6 min. These sections were mounted onto slides, dehydrated through an ascending series of ethanol, cleared with xylene, coverslipped with an Entellan (E. Merck, Darmstadt, Germany) mounting medium and observed under a light microscope (OLYMPUS). For immunofluorescence, the free-floating sections were incubated in 0.1 M PBS containing 3% normal goat serum with 0.1% Triton X-100 for 1 h and then, respective sections were incubated overnight at 4 °C with primary antibodies against CD11b (mouse, 1:100, GeneTex) and P2X7R (rabbit, 1:1000, Alomone Labs Ltd), or CD45 (mouse, 1:200, AbD Serotec, Raleigh, NC). The diluent for the antibodies was 0.1 M PBS containing 3% goat serum. Sections were washed with 0.1 M PBS, and then incubated for 2 h with anti-rabbit IgG antibody raised in goat conjugated to Alexa Fluor 488 and anti-mouse IgG antibody raised in goat conjugated to Alexa Fluor 568 (1:400; Invitrogen, San Diego, CA). Sections were then washed in 0.1 M PBS, mounted on slides and coverslipped with VECTASHIELD with DAPI (Vector Laboratories). Cover-slipped slides were inspected, and digital images were captured using a confocal laser-scanning microscope (Lsm5 Pascal. Carl Zeiss, Oberkochen, Germany). We also confirmed specificity of antibody of P2X7R by the validation test. To determine the specificity of the primary antibody, antibody was pre-adsorbed with the recombinant peptide of P2X7R (Alomone Labs Ltd). Molar ratio of antibody to recombinant peptide was 1 to 20. 50 μg of peptide was mixed with 25 μg of each antibody (2:1) and incubated on a shaker at room temperature overnight on a shaker at 4 °C, and then centrifuged at 2000 rpm for 2 min. Then supernatant was used for further immunostaining detection.
ejp174-sup-0010-si.docx17K Appendix S4. Tissues were homogenized in solubilization buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.5% (v/v) deoxycholic acid, 0.1% (w/v) SDS, 1 mM Na3VO4, 1 U/mL aprotinin, 20 μg/mL leupeptin, 20 μg/mL pepstatin A). The homogenates were centrifuged at 20,000 × g for 10 min at 4 °C, and supernatants were removed. The protein concentrations were determined using a detergent-compatible protein assay with a bovine serum albumin standard. Each sample contained proteins from one animal. Protein (50 μg) was separated on 7.5% (w/v) SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The blots were blocked with 5% (w/v) skim milk in Tris-buffered saline (TBS) and then incubated with the rabbit anti-P2X7R antibody (1:500; Alomone Labs Ltd, Jerusalem, Israel), TNF-α (1:1000; Cell Signaling Technology, Beverly, MA) or p-p38 (1:1000; Cell Signaling Technology). The membranes were washed with TBS and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:1000; Amersham Biosciences, NJ). Immunoreactivity was detected using enhanced chemiluminescence (ECL Plus kit; GE Healthcare). The amount of protein in each lane was verified by reprobing the membrane with anti-β-actin antiserum (1:1000, Sigma Chemical Co.) and Coomassie blue staining. For Western blot analysis, ECL-exposed films were digitized and densitometric quantification of immunoreactive bands was performed using Image J (National Institutes of Health, Bethesda, MD). The pixel density of the examined protein band is presented as a percentage of the pixel density of the naïve control band. These data were used for statistical comparisons. We also confirmed the specificity of TNF-α and P2X7R antibodies using the validation test. Antibodies were pre-adsorbed with the recombinant peptide of TNF-α (Cell Signaling Technology) or P2X7R (Alomone Labs Ltd, Jerusalem, Israel). The molar ratio of antibody to recombinant peptide was 1 to 20. Each peptide was mixed with each antibody and incubated overnight on a shaker at 4 °C, and then centrifuged at 2000 rpm for 2 min. The supernatant was used for further Western blotting detection.
ejp174-sup-0011-si.docx15K Appendix S5. Tissues were homogenized in TRIzol reagent (1000 μL, Invitrogen, Carlsbad, CA) and left on ice for 30 min. Total RNA was isolated from these samples using an RNeasy Mini kit (Qiagen, Tokyo, Japan), and reverse transcription was performed using ReverTraAce (Toyobo, Osaka, Japan). The RT reaction was carried out at 25 °C for 10 min, 37 °C for 120 min and 95 °C for 5 min in a PTC-200 thermal cycler (MJ Research, MA). We performed quantitative analysis of P2X7Rs by RT-PCR using an 18 s rRNA TaqMan probe (Applied Biosystems, Foster City, CA), a designed P2X7R primer, SYBR Green real-time master mix (Applied Biosystems) and TaqMan Universal PCR master mix (Applied Biosystems). The P2X7R primer sequences were as follows: forward, 5′-GCTTGGGAAAAGTCTGCAAG-3′; reverse, 5′-TAGTTGAGACGGGAGGCAGT-3′. RT-PCR analyses were carried out using the ABI Prism 7900HT Sequence Detection System instrument and software (Applied Biosystems). Data were normalized against 18S rRNA levels. RT-PCR reactions were carried out under the following reaction conditions: 40 cycles of denaturing at 95 °C for 15 s, annealing at 60 °C for 60 s and extension at 72 °C for 10 s.
ejp174-sup-0012-si.docx19K Appendix S6. Several microglia-specific molecules, including cell surface receptors, intracellular signalling molecules and diffusible factors, have been identified, and their roles in the development and maintenance of neuropathic pain behaviours have been demonstrated. p38 MAPK in spinal microglia was phosphorylated after spinal nerve injury and was shown to be involved in neuropathic pain (Jin et al., 2003; Tsuda et al., 2004; Piao et al., 2006). Notably, p-p38 MAPK in the spinal cord is induced in microglia, but not in astrocytes or neurons (Jin et al., 2003; Tsuda et al., 2004; Piao et al., 2006). Pharmacological inhibition of p-p38 MAPK by SB203580 reversed established tactile allodynia (Jin et al., 2003; Tsuda et al., 2004). On the other hand, microglia express a variety of receptors. Binding of ligands to these receptors on microglia results in signal transduction through intracellular signalling cascades. In fact, phosphorylation of p38 MAPK in microglia following nerve injury may result from activation of Toll-like receptor (TLR) 3 and P2Y12R (Kobayashi et al., 2008; Obata et al., 2008). Therefore, TLR3 and P2Y12R may be considered as candidate receptors upstream of p38 MAPK phosphorylation in microglia following nerve injury. Nevertheless, the participation of cell-surface receptors on microglia, which activate intracellular signalling pathways during neuropathic pain, has been unclear until now. Because P2X7R has a long C terminus with an extra 200 amino acid residues that interact with several proteins and lipids in the cytoplasm (North, 2002; Costa-Junior et al., 2011), extracellular ATP activates multiple intracellular signalling pathways in microglia (Ferrari et al., 1997; Suzuki et al., 2004; Takenouchi et al., 2010). BzATP has been reported to significantly increase the levels of p-p38 MAPK in hippocampal slices of wild-type mice, an effect that is absent in P2X7R (-/-) mice (Papp et al., 2007). Therefore, it remains possible that p38 MAPK may be regulated upon P2X7R activation in the TNC during neuropathic pain. Thus, this study was designed to explore whether the activation of P2X7R promotes p38 MAPK signalling in microglia. A very important finding in the present study was that BzATP treatment of naïve rats induced the up-regulation of p-p38 MAPK and allodynia/hyperalgesia. In addition, up-regulation of p-p38 MAPK and allodynia/hyperalgesia following BzATP treatment were blocked by pretreatment of rats with SB203580. Therefore, P2X7R/p38 MAPK activation in microglia may have an important role in the pathomechanisms underlying allodynia/hyperalgesia owing to neuron–glia interactions.

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