dolor p2x7 2013

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published online 22 January 2013J DENT RESK. Murasaki, M. Watanabe, K. Takahashi, G. Ito, Y. Suekawa, T. Inubushi, N. Hirose, T. Uchida and K. Tanne

Receptor and Cytokines Contribute to Extra-territorial Facial Pain7P2X

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RESEARCH REPORTSBiological

DOI: 10.1177/0022034512474668

Received September 11, 2012; Last revision December 18, 2012; Accepted December 18, 2012

A supplemental appendix to this article is published elec-tronically only at http://jdr.sagepub.com/supplemental.


K. Murasaki1#, M. Watanabe2#*, K. Takahashi1, G. Ito1, Y. Suekawa1, T. Inubushi1, N. Hirose1, T. Uchida2, and K. Tanne1

1Department of Orthodontics, Applied Life Sciences, Hiroshima University Institute of Biomedical & Health Sciences, Minami-ku, Hiroshima, Japan; 2Department of Oral Biology, Basic Life Sciences, Hiroshima University Institute of Biomedical & Health Sciences, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan; and #authors con-tributing equally to this work; *corresponding author, [email protected]

J Dent Res X(X):xx-xx, XXXX

ABSTRACTThe whisker pad area (WP) is innervated by the second branch of the trigeminal nerve and experi-ences allodynia and hyperalgesia following tran-section of the mental nerve (MN; the third branch of the trigeminal nerve). However, the mecha-nisms of this extra-territorial pain remain unclear. The ionotropic P2X7 ATP receptor (P2X7) in microglia is known to potentiate, via cytokines, the perception of noxious stimuli, raising the possibil-ity that P2X7 and cytokines are involved in this extra-territorial pain. One day after MN transec-tion (MNT), WP allodynia/hyperalgesia devel-oped, which lasted for > 8 wks. Activation of microglia and up-regulation of P2X7, membrane-bound tumor necrosis factor (TNF)-α (mTNF-α), and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC) were evident for up to 6 wks after MNT. Allodynia/hyperalgesia after MNT was blocked by intracisternal adminis-tration of etanercept, a recombinant TNF-α recep-tor (p75)-Fc fusion protein. Intracisternal A438079, a P2X7 antagonist, also attenuated allodynia/hyperalgesia and blocked up-regulation of mTNF-α and sTNF-α in the TNC. We conclude that sTNF-α released by microglia following P2X7 activation may be important in both the initiation and maintenance of extra-territorial pain after MNT.

KEY WORDS: trigeminal nerve, behavioral sci-ence, neuropharmacology, neuroscience, nervous system.

INTRODUCTION

Several studies have implicated ATP-gated P2X7 ion channels (P2X7) in pain hypersensitivity (Honore et al., 2006; Ito et al., 2013). The distribu-

tion of abnormal pain that accompanies peripheral neuropathies often does not coincide with nerve territories (Tal and Bennett, 1994). This “extra-terri-torial pain” (Tal and Bennett, 1994) has been reported in the trigeminal sys-tem. After transection of the inferior alveolar nerve (IAN) or mental nerve (MN), the whisker pad area (WP), which is innervated by the maxillary nerve, displayed mechanical allodynia (Iwata et al., 2001; Piao et al., 2006; Takahashi et al., 2011). In this model, allodynia/hyperalgesia was not induced in the injured dermatome region, i.e., the lower lip (Takahashi et al., 2011), and because the MN was completely transected, perception of stimulus to the lower lip was completely inhibited. Therefore, MN transection (MNT) causes extra-territorial, but not intra-territorial, pain (Takahashi et al., 2011). We have suggested that IL-1b released from astrocytes in the dorsal portion of the trigeminal subnucleus caudalis (Vc, where the third trigeminal branch terminates) in response to MNT acts on nociceptive neurons in the medial portion of Vc (where the second trigeminal branch terminates) to affect pain perception in non-injured skin territory (Takahashi et al., 2011). Furthermore, minocycline (a microglia inhibitor) reduces cell activation and inhibits phos-phorylation of p38 mitogen-activated protein kinase (p38 MAPK) in microg-lia and attenuates the development of pain hypersensitivity following IAN transection (Piao et al., 2006). Microglia are probably involved in the onset of allodynia in intact skin territories adjacent to the denervated region follow-ing MNT. Indeed, our recent study reports that phosphorylated p38 (p-p38 MAPK) may, via P2X7, induce intra-territorial pain in the same nerve territory following chronic constriction injury of the infraorbital nerve (CCI-IoN) in a mechanism most likely involving tumor necrosis factor (TNF)-α release from microglia (Ito et al., 2013).

Several reports support the concept that microglia participate in the establishment, but not maintenance, of chronic pain (Raghavendra et al., 2003; Zhuang et al., 2005), although this paradigm remains unproven (Stuesse et al., 2001; Winkelstein and DeLeo, 2002). In the present study, we examined the role of P2X7 and inflammatory cytokines pro-duced by microglia in orofacial pain mechanisms at the intact facial region dermatomes following MNT and tested whether microglial activa-tion mediates the initiation and/or maintenance of chronic pain in a MNT model.

P2X7 Receptor and Cytokines Contribute to Extra-territorial Facial Pain




2 Murasaki et al. J Dent Res X(X) XXXX

MATERIALS & METHODS

Adult male Sprague-Dawley rats (wt, 200-250 g; Charles River Laboratories, Yokohama, Japan) were used. Animals were kept in separate cages in a controlled environment (lights on 08:00–20:00, 22°C) with food and water freely available. The animal model adhered to the International Association for the Study of Pain ethical guidelines (Zimmermann, 1983) and ARRIVE guidelines for animal research (Kilkenny et al., 2012) and was approved by the Institutional Animal Care and Use Committee of Hiroshima University (Approval number A09-88).

MNT was performed by a method described previously (Takahashi et al., 2011). Briefly, the MN was exposed, ligated with 4-0 silk, and sectioned distal to the ligation. Sham-operated controls were rats whose MN were exposed but not transected.

Tactile allodynia/hyperalgesia was assessed as described previously (Takahashi et al., 2011). Briefly, the rats were habitu-ated to stand on a soft pad and lean against the experimenter’s hand. A series of calibrated von Frey filaments with bending forces ranging from 0.4 to 60 g was applied to the WP. Active withdrawal of the head from the probing filament was defined as a response to a stimulus. Response frequencies [(number of responses/number of stimuli) × 100%] to a range of von Frey filament forces were determined to construct a stimulus-response (S-R) curve. Non-linear regression analysis of this curve pro-duced an EF50 value, defined as the von Frey filament force (g) that produces a 50% response frequency. Drug effects on this EF50 were quantified in the ipsilateral, but not contralateral, WP, given our previous demonstration that allodynia/hyperalgesia was not detected in the contralateral WP following MNT (Takahashi et al., 2011) and that these drugs had no influence on normal pain responses (Ito et al., 2013).

Drugs were delivered into the cerebrospinal fluid space around the medullary dorsal horn as described previously (Takahashi et al., 2011). Briefly, PE10 tubing (Intramedic, Hellerup, Denmark) was inserted into the subarachnoid space with the tip located at the level of the Vc and fastened by silk sutures (5-0). A P2X7-selective antagonist (A438079, 3.5 mg/mL, 10 μL; TOCRIS Bioscience, Ellisville, MO, USA), a recombinant TNF-α receptor (p75)-Fc fusion protein (etaner-cept, 1 or 10 μg/mL, 5 μL; Enbrel™, Immunex, Seattle, WA, USA), or recombinant rat TNF (0.1 pg/μL, 10 μL; PeproTech EC Ltd., London, UK) was administered as a bolus via this PE10 catheter. Determination of A438079 concentration by CCI-IoN is described in our previous study (Ito et al., 2013). All drugs were dissolved in 0.15 M NaCl.

Immunofluorescence studies were performed as described previously (Takahashi et al., 2011). Briefly, 30-μm-thick sec-tions of the trigeminal sensory nuclear complex (TNC) were incubated overnight with anti-P2X7 antibodies (rabbit, 1:1,000; Alomone Labs Ltd., Jerusalem, Israel) or anti-Iba1 (goat, 1:400; Abcam, Cambridge, MA, USA), anti-GFAP (mouse, 1:10,000; Millipore, Bedford, MA, USA), or anti-neuronal nuclei (NeuN mouse, 1:3,000; Chemicon International, Temecula, CA, USA).

Figure 1. Up-regulation of P2X7 in microglia after MNT. (a) TNC tissue was punched out and total protein isolated. Western blotting shows immunoreactive bands at 75 kDa (P2X7) and 45 kDa (β-actin). Blots are representative of 5 independent repeats. Graph shows quantification of P2X7 protein expression at each time point relative to that in naive rats. Data are mean ± SEM; n = 5 for each time point. **A statistically significant difference (p < 0.01) between the groups indicated by the bracket, as determined by one-way ANOVA with the Dunn-Bonferroni post-test. (b-j) Double-immunofluorescence labeling with antibodies against Iba1, P2X7, GFAP, and NeuN in the ipsilateral Vc at 3 days after MNT. (d) Superimposition of P2X7 (b; green) and Iba1 (c; red) images indicates areas of co-localization (yellow/orange), illustrating the induction of P2X7 in microglia. (g, j) The absence of double-labeling for P2X7 (e and h; green) and GFAP (f; red), an astrocyte marker, or NeuN (i; red), a neuronal marker.




J Dent Res X(X) XXXX P2X7 Receptor and Cytokines in Extra-territorial Facial Pain 3

After being washed with 0.1 M PBS, sections were incubated for 2 hrs with Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 568-conjugated goat anti-mouse IgG, Alexa Fluor 568-conjugated donkey anti-goat IgG, or Alexa Fluor 488- conjugated donkey anti-rabbit IgG (1:400; Invitrogen, San Diego, CA, US). Digital images were captured by means of a confocal laser-scanning microscope (Lsm5 Pascal; Carl Zeiss, Oberkochen, Germany) or fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

Preparation of spinal samples and western blotting were per-formed as described previously (Ito et al., 2013). Briefly, a block of caudal brainstem tissue was excised, punched out, and homogenized in solubilization buffer. Protein (15 μg) was sepa-rated on 15% (w/v) SDS-polyacrylamide gels and blotted onto nitrocellulose membranes (BIO-RAD Lab., Hercules, CA, USA). Blots were incubated with anti-P2X7 antibody (rabbit, 1:500; Alomone Labs Ltd), anti-TNF-α (rabbit, 1:500; Cell Signaling Technology), anti-p-p38 MAPK (rabbit, 1:1,000; Cell Signaling Technology), or anti-Iba1 (goat, 1:1,000, Abcam). Membranes were washed with TBS and incubated with horseradish-peroxidase-conjugated anti-rabbit IgG (1:2,000; Amersham Biosciences, Piscataway, NJ, USA) or anti-goat IgG

(1:2,000; BETHYL Lab. Inc., Montgomery, TX, USA), and immunoreactivity was detected by enhanced chemilumines-cence (ECL Plus kit; GE Healthcare, Hertfordshire, UK). We quantified protein in each lane by re-probing the membrane with anti-β-actin antibody (1:2,000; Sigma Chemical Co., St. Louis, MO, USA) and by Coomassie blue staining. Processed films were digitized and immunoreactive bands densitometrically quantified with Image J software (National Institutes of Health, Bethesda, MD, USA). Pixel density in the relevant protein band is presented as a percentage of pixel density in the control band.

Data are presented as the mean ± SEM. Statistical compari-sons of multiple groups were made by one- or two-way analysis of variance (ANOVA) with the Dunn-Bonferroni post-test. Any p < 0.05 was considered significant in all cases. For animals subjected to repeated tests, ANOVA with repeated measures was used, with time representing a within-animal effect. p < 0.05 was considered significant in all cases.

RESULTS

As previously reported (Takahashi et al., 2011), mechanical stimuli elicited increased responses and reduced response

Figure 2. Morphological patterns of microglial activation in response to (a) MNT and (b) up-regulation of Iba1, p-p38 MAPK, mTNF-α, and sTNF-α in the TNC after MNT. Iba1 immunofluorescence in Vc at 1,800-2,160 µm caudal to the obex at 24 hrs, 3 days, and 1, 3, and 6 wks after MNT. Scale bar, 500 µm. Sham operation was performed as a control. TNC tissue was punched out and total protein isolated. Blots shown are representative of 5 independent repeats and show immunoreactive bands at 45 kDa (β-actin), 38 kDa (p-p38 MAPK), 25 kDa (mTNF-α), and 17 kDa (sTNF-α and Iba1). Graphs show quantification of protein levels relative to those in naive rats. Data are mean ± SEM; n = 5 for each time point. **A statistically significant difference (p < 0.01) relative to naive control, as determined by one-way ANOVA with the Dunn-Bonferroni post-test.





thresholds in the ipsilateral, but not contralateral, WP from 24 hrs to 8 wks after MNT, a characteristic of tactile hyperalge-sia and allodynia in intact facial region dermatomes following MNT (data not shown).

Western blotting demonstrated a significant increase in P2X7 protein levels at all measured time points after MNT compared with naive rats (n = 5 for each) (Fig. 1a), but no difference between naive and sham-operated groups at 24 hrs and 3 days (n = 5). P2X7 immunofluorescence was co-localized in the Vc with the microglial marker, Iba1, suggesting that these microglia express P2X7. Neither astrocytes nor neurons expressed P2X7 (Fig. 1b).

Fig. 2a shows representative photo-micrographs of Iba1 immunofluores-cence in the Vc following MNT at 1,800-2,160 μm caudal to the obex. Naive rats exhibited no overt signs of microglial activation in the Vc. However, on the ipsilateral side of the Vc after MNT, microglia had profound Iba1 immunoreactivity and were hyper- activated. After MNT, activated microg-lia were spread diffusely throughout the dorsal third of the Vc at 24 hrs before forming dense clusters focused within the ipsilateral dorsal portion of the Vc at 3 days and lasting over 3 wks. At 6 wks after MNT, activated microglia had once more spread diffusely across the Vc.

We then quantified Iba1 protein lev-els, which are determined by both the number of activated microglial cells and their degree of activation (Scholz et al., 2008) and are thus excellent markers of microglial activation. As shown in Fig. 2b, western blotting showed a signifi-cant increase in Iba1 protein levels at all measured time points after MNT com-pared with naive rats (n = 5 for each time point).

The TNF-α-converting enzyme (TACE/ADAM-17) cleaves membrane-bound TNF-α (mTNF-α) to generate soluble TNF-α (sTNF-α). Recently, we showed that p-p38 MAPK may, via P2X7, induce sTNF-α release by microg-lia (Ito et al., 2013), so we also exam-ined p-p38 MAPK, mTNF-α and sTNF-α protein levels in the Vc following MNT. We found a significant increase in p-p38 MAPK, mTNF-α, and sTNF-α proteins at all measured time points after MNT compared with naive rats (n = 5 for each time point) (Fig. 2b). There was no sig-nificant difference between the levels of Iba1, p-p38 MAPK, mTNF-α, and sTNF-α in naive and sham-operated groups at 24 hrs and 3 days after sham operation (n = 5).

We next discovered that intracisternal treatment of rats with the P2X7 antagonist, A438079, starting at 30 min before MNT, completely prevented allodynia/hyperalgesia on the ipsilateral WP following MNT (Fig. 3a). After these behavioral tests, expression of P2X7, Iba1, p-p38 MAPK, and TNF-α in the TNC was examined by immunohistochemistry or western blotting. In both saline- and A438079-treated rats, we found that microglia in the ipsilateral Vc had profound Iba1 immunoreactivity and were in an activated state (data not shown). Similarly, P2X7 and Iba1 proteins increased significantly in both saline- and A438079-treated rats compared with naive rats (n = 4 for each) (Fig. 3b), indicating that A438079 did not affect microglial

Figure 3. Effects of the P2X7-selective antagonist, A438079, on (a) allodynia/hyperalgesia in the WP and (b) on Iba1, P2X7, mTNF-α, sTNF-α and p-p38 MAPK levels at 24 hrs after MNT. A438079 (35 μg/rat) was injected intracisternally via a cannula implanted at the level of the TNC. Drug infusion started at 30 min before MNT. Saline was infused as a vehicle control. Data are EF50 values (mean ± SEM, n = 4) derived from stimulus–response frequency curves in behavioral tests. Statistically significant differences (p < 0.01, determined by two-way ANOVA with the Dunn-Bonferroni post-test) relative to baseline and A438079 are denoted by ** and ##, respectively. A438079 (35 μg/rat) was injected intracisternally via a cannula implanted at the level of the TNC. Drug infusion started at 30 min before MNT. Saline was infused as a vehicle control. At 24 hrs after MNT, TNC tissues were punched out, and total protein was isolated and separated by western blotting. Blots shown are representative of 5 independent repeats and show immunoreactive bands at 75 kDa (P2X7), 45 kDa (β-actin), 38 kDa (p-p38 MAPK), 25 kDa (mTNF-α), and 17 kDa (sTNF-α and Iba1). Graphs show quantification of protein expression levels relative to those in naive rats. Data are mean ± SEM; n = 5 for each time point. *p < 0.05 and **p < 0.01 denote statistically significant differences between the groups indicated, as determined by one-way ANOVA with the Dunn-Bonferroni post-test.




J Dent Res X(X) XXXX P2X7 Receptor and Cytokines in Extra-territorial Facial Pain 5

activation or induction of P2X7 follow-ing MNT. In contrast, A438079 signifi-cantly inhibited the up-regulation of mTNF-α, sTNF-α, and p-p38 MAPK levels following MNT (Fig. 3b).

To clarify the functional relevance of TNF-α in prolonged extra-territorial allodynia/hyperalgesia following trigem-inal nerve injury in the rat WP, we exam-ined the effects of intracisternal administration of etanercept. In pre-drug experiments, we confirmed a significant decrease in EF50 in the ipsilateral WP at all measured time points after MNT compared with baseline. At each time point, saline (20 μL/rat, n = 5) or etaner-cept (5 or 50 ng/rat, n = 5 for each) was injected intracisternally. Administration of 50 (but not 5) ng etanercept signifi-cantly increased EF50 on the ipsilateral WP relative to saline control rats and pre-drug values in the same group. These effects of etanercept were evident 2 to 48 hrs after administration and indicate that etanercept dose-dependently allevi-ates extra-territorial tactile allodynia/hyperalgesia (Fig. 4).

Next, we examined whether etaner-cept could prevent MNT-induced up-regulation of TNF-α in the TNC. At 24 hrs after MNT, saline (20 μL/rat; n = 4) or etanercept (50 ng/rat; n = 4) was injected intracisternally, and after a fur-ther 24 hrs, TNC tissue was punched out and total protein isolated. Etanercept, but not saline, prevented the up-regulation of sTNF-α but not that of mTNF-α (see Appendix Fig. 1).

Finally, we confirmed that intracister-nal infusion of recombinant TNF induced tactile allodynia/hyperalgesia in the WP (Appendix Fig. 2, Appendix Results).

DISCUSSION

We aimed to investigate the functional role of P2X7 and cytokines in trigeminal neuropathic pain in the WP (innervated by the maxillary nerve), following transection of the MN (a branch of the mandibular nerve). We demonstrated that activa-tion of P2X7 in microglia facilitates perception of noxious stimuli via TNF-α in the TNC and is essential for both the ini-tiation and maintenance of extra-territorial allodynia/hyperalge-sia following MNT.

Minocycline reduced microglial activation, inhibited p-p38 MAPK activation in microglia, and attenuated pain hypersensi-tivity in the WP following IAN transection (Piao et al., 2006). Therefore, hyperactive microglia could facilitate the onset of allodynia in intact skin territories adjacent to the denervated

region following IAN transection. The middle and dorsal por-tions of the Vc receive afferents from the second and third branches of the trigeminal nerve, respectively (Noma et al., 2008). Following MNT, microglial activation, which may induce TNF-α via P2X7, was induced in the dorsal Vc, where MN afferents terminate. Alternatively, our recent study demon-strated Fos activation evoked within the medial portion of Vc following application of non-noxious mechanical stimulation of the WP after MNT (Takahashi et al., 2011). In addition, as shown in Appendix Fig. 1, etanercept prevents both the up- regulation of sTNF-α and the allodynia/hyperalgesia

Figure 4. Effects of etanercept on allodynia/hyperalgesia in the WP at various time points after MNT. Etanercept (5 or 50 ng/rat; n = 5) was injected intracisternally via a cannula implanted at the level of the TNC. Drug infusion started at 24 hrs, 3 days, and 1, 3, and 6 wks after MNT. Saline was infused as a vehicle control. Data are mean ± SEM; n = 5 for each time point. Data were analyzed statistically by two-way ANOVA with the Dunn-Bonferroni post-test for investigation of differences between groups and between time points within groups. Statistically significant differences between pre- and post-drug responses are denoted by **p < 0.01 and *p < 0.05. Differences between corresponding time points in the drug and saline groups are shown by ##p < 0.01 and #p < 0.05.





experienced following MNT, and thus may act as a scavenger that sequesters sTNF-α and precludes its visualization in west-ern blotting. Treatment of rats with 3´-O-(4-benzoylbenzoyl) adenosine 5´-triphosphate, a P2X7 agonist, induced allodynia/hyperalgesia and up-regulation of sTNF-α and p-p38 MAPK in the TNC, and these responses (except p38 up-regulation) were inhibited by etanercept (Ito et al., 2013). Analysis of these data suggests that, following MNT, sTNF-α released from microglia via P2X7 activation at the dorsal portion of the Vc produces paracrine signaling, acting on nociceptive neurons in the Vc to affect pain perception in non-injured skin territories.

Our previous study (Takahashi et al., 2011), with an identical MNT model, concluded that IL-1β released from astrocytes enhanced NMDA receptor phosphorylation on secondary neurons, which contributes to extra-territorial tactile allodynia/hyperalgesia. Our previous and present studies clearly demonstrated that there may be different pathways contributing to extra-territorial tactile allodynia/hyperalgesia via astrocytes or microglia. Although the priority of these different pathways remains to be established, intrathecal injection of astrocytes, which were prepared from cerebral cortexes of neonatal mice and briefly stimulated by TNF-α, induced mechanical allodynia in the paw (Gao et al., 2010). Therefore, there is a possibility that sTNF-α released from microglia following P2X7 activation may activate astro-cytes and induce the release of IL-1β in this MNT model.

In this study, the P2X7 antagonist, A438079, was able to block changes in TNF-α, p-p38 MAPK, and behavior without altering Iba1 protein levels, suggesting that microglial activation during extra-territorial pain does not depend on P2X7 and posing the interesting question of what does activate these microglia. Several endogenous molecules (e.g., ATP and fibronectin) induce both morphological microglial activation and allodynia (Nakagawa et al., 2007; Tsuda et al., 2008). Microglia can also, in response to extracellular ligand binding, secrete pro-inflammatory cytokines that modulate neuropathic pain (Inoue and Tsuda, 2009), although mice deficient in the neuronal chemokine, CCL21, developed no signs of neuropathic pain after spinal nerve injury, despite evident morphological microglial activation (Biber et al., 2011). Based on these recent studies and our present study, microglial morphology may be insufficient to predict function in pathological pain sce-narios. Nevertheless, our study clearly suggests that P2X7 activa-tion in microglia causes p38 MAPK phosphorylation and subsequent sTNF-α release, which may be involved in extra- territorial allodynia/hyperalgesia, but not in the morphological activation of microglia.

We conclude that sTNF-α produced by microglia following P2X7 activation appears to be involved in both the initiation and maintenance of extra-territorial tactile allodynia/hyperalgesia following MNT (see also Appendix Discussion).

ACKNOWLEDGMENTS

This study was supported by a Grant-in-Aid for Scientific Research (22592039) from the Japanese Ministry of Education, Science and Culture. The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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