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Changes in protein expression and distribution of spinal CCR2 in a rat model of bone cancer pain
brain research 1509 (2013) 1–7
Available online at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Changes in protein expression and distribution of spinal CCR2 in a rat model of bone cancer pain Ji-Hua Hua,1, Meng-Yao Wub,1, Min Taob, Jian-Ping Yanga,n a
Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China
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art i cle i nfo
ab st rac t
Article history:
Accumulating evidence suggests that chemokine C–C motif receptor 2 (CCR2) plays an important
Accepted 3 March 2013
role in neuropathic pain. It has been shown that spinal CCR2 is upregulated in several
Available online 17 March 2013
neuropathic pain models and expressed by neuronal and glial cells in the spinal cord. In this
Keywords:
study, we investigated the expression changes and cellular localization of spinal CCR2 in a rat
Bone cancer pain
model of bone cancer induced by Walker 256 cell inoculation. The present results indicated that
Chemokine
mechanical allodynia progressively increased in bone cancer pain (BCP) rats. Western blot and
CCR2
immunohistochemical analysis demonstrated that the expression of CCR2 in the spinal cord was
Spinal cord
significantly increased on day 6, 12, and 18 in BCP rats, with a peak on day 6. Furthermore, double
Microglia
immunofluorescence labeling indicated that CCR2 was expressed by both microglia and neurons in the spinal cord. These results suggest that CCR2 may be involved in the development of BCP, and that targeting CCR2 may be a new strategy for the treatment of BCP. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Bone cancer pain (BCP), which seriously compromises patient quality of life (Coleman, 2006; Jimenez-Andrade et al., 2010), is the most common symptom detected in patients with advanced breast, prostate, and lung cancer. Therefore, understanding the underlying mechanisms related to the development of BCP is important for effectively treating these patients. Monocyte chemoattractant protein-1 (MCP-1, also named CCL2) has been reported to play a role in the pathophysiology of neuropathic pain by binding to its β- chemokine receptor 2 (CCR2) (Abbadie et al., 2009; White et al., 2005; White and Wilson, 2008). CCR2 is a G protein-coupled receptor (GPCR) that preferentially binds to the MCP-1 chemokine. Previous studies have demonstrated that mice lacking CCR2 fail to develop tactile
n
Corresponding author. Fax: þ86 512 677 80149. E-mail addresses: [email protected], [email protected] (J.-P. Yang). 1 These two authors contributed equally.
0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.03.002
allodynia after partial nerve ligation (Abbadie et al., 2003), and that mice overexpressing MCP-1 showed enhanced nociceptive behavior (Menetski et al., 2007). Blockade of CCR2 produced an attenuation of the nociceptive behavior (Bhangoo et al., 2007; Serrano et al., 2010) in neuropathic pain rats and prevented MCP1-induced nociceptive responses (Dansereau et al., 2008). In addition, the expression of CCR2 is upregulated in the spinal cord in several kinds of neuropathic pain models, including peripheral nerve injury (Abbadie et al., 2003), spinal nerve ligation (Gao et al., 2009), and spinal cord contusion injuries (KnerlichLukoschus et al., 2008). These results suggest that CCR2 activation plays an important role in neuropathic pain. Understanding the function of CCR2 in nociceptive responses requires a detailed knowledge of its distribution in the dorsal horn of the spinal cord. Previously, CCR2 expression was detected in spinal glial cells or
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neurons under neuropathic pain conditions (Abbadie et al., 2003; Gao et al., 2009; Knerlich-Lukoschus et al., 2008). MCP-1/CCR2 has also been identified as a signaling molecule in neural–glial communication in neuropathic pain (Abbadie et al., 2009). Bone cancer pain is unique and distinct from neuropathic pain (Honore et al., 2000; Mantyh, 2006). What is in some ways unique about bone cancer pain is that the inflammation, tumorreleased products, and tumor-induced injury to primary afferent neurons can simultaneously drive the chronic pain state. Rarely is CIBP purely neuropathic, inflammatory, ischemic, or visceral but rather is a combination. However, little is known about possible alterations in CCR2 expression in BCP rats. In this study, we examined CCR2 expression changes and the cellular localization of CCR2 in the spinal cord of BCP rats.
2.
Results
2.1.
Mechanical allodynia induced by bone cancer
All rat groups exhibited similar baseline hind paw withdrawal threshold (PWT) to mechanical stimulation (von Frey filaments). Bone cancer pain rats displayed a significant decrease in PWT of the ipsilateral hind paw compared with sham rats on day 6. With the progression of bone cancer, the PWT progressively decreased in the inoculated hind paw from days 6 to 18 (Fig. 1). Thus mechanical allodynia was induced in this rat model of bone cancer.
2.2.
Increase of CCR2 expression in BCP rats
As shown in Fig. 2A, CCR2 bands (42 kDa) indicated a significantly upregulation of CCR2 in the spinal cord of BCP rats compared with sham rats. The protein expression was increased at each time point investigated, with a maximum on day 6 following Walker 256 cells inoculation (Fig. 2A, B).
Fig. 2 – Time course of CCR2 expression in the spinal cord (L4–L5) of BCP rats. (A) Western blot analysis showed an increase in the expression of CCR2 in the spinal cord of BCP rats. β-actin served as a loading control. (B) Quantification of CCR2 expression in the spinal cord. n¼ 4; * Po0.05 vs. sham; ** Po0.01 vs. sham.
2.3.
Immunoreactivity of CCR2 in BCP rats
Immunohistochemistry data also demonstrated that compared with sham rats (Fig. 3A, E), the expression of CCR2 was strikingly increased on day 6, and maintained on day 12 and 18 in the ipsilateral spinal cord dorsal horn (Fig.3B, D). CCR2immunoreactive (IR) cells were detected throughout the spinal cord following Walker 256 cells inoculation (Fig. 3F, G). Although most positive cells were distributed in the ipsilateral superficial (laminae I–III) dorsal horn, a few positive cells were also found in the ventral horn (Fig. 3F, G). In addition, the expression of CCR2 was striking increased in the ipsilateral spinal cord compared with that in contralateral superficial dorsal horn (Fig. 3F–H).
2.4.
Cellular localization of CCR2 in BCP rats
Double immunofluorescence labeling indicated that CCR2 was expressed both on microglia and neurons in the ipsilateral dorsal horn on day 6 following Walker 256 cell inoculation, since CCR2IR cells were colocalized with OX-42 (microglial marker) (Fig. 4A, B) and NeuN (neuron marker) (Fig.4C, D) respectively. In contrast, CCR2 was not expressed on GFAP-IR astrocytes in the dorsal horn of BCP rats on day 6 (Fig. 4E). Fig. 1 – Mechanical allodynia is induced in BCP rats. The paw withdrawal threshold (PWT) to mechanical stimulation (von Frey filaments) was decreased in BCP rats compared with sham rats on days 6, 12 and 18. Results are expressed as mean7SEM. n¼ 10; ** Po0.01 vs. sham.
3.
Discussion
In the present study, we demonstrated that mechanical allodynia progressively increased in BCP rats from days 6 to 18, and that CCR2
brain research 1509 (2013) 1–7
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Fig. 3 – CCR2 like immunoreactivity in the spinal cord (L4–L5) of BCP rats. Immunofluorescence data showed that CCR2 expression was significant increased in the dorsal horn of BCP rats on day 6 (B, F), 12 (C) and 18 (D, G) compared with that in sham rats (A, C). CCR2 immunoreactivity cells were mainly presented in superficial layers of the ipsilateral dorsal horn (arrows). (H) Quantification of CCR2 immunoreactivity cells in the dorsal horn of spinal cord. n ¼ 6; ** Po0.01 vs. sham; ## Po0.01 vs. cont ( ipsi indicates ipsilateral; cont, contralateral). Scale bar: 100 μm in A–D, 200 μm in E–G.
expression was significantly upregulated in the ipsilateral spinal cord of BCP rats, peaking on day 6. Furthermore, double immunofluorescence labeling indicated that CCR2 was expressed by microglia and neurons in the dorsal horn of the spinal cord in BCP rats.
Accumulating evidence suggests that MCP-1 and its receptor CCR2 play important roles in the development of neuropathic pain (Gosselin et al., 2008; Old and Malcangio, 2012; White and Miller, 2010). For example, an increase in MCP-1 expression was
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Fig. 4 – Cellular type of CCR2 expression in the spinal cord (L4–L5) of BCP rats. (A and B) Double immunofluorescence data showed that CCR2 immunoreactivity cells were co-localized with OX-42 and (C and D) co-localized with NeuN in the dorsal horn of spinal cord on day 6 after Walker 256 cell inoculation. Yellow fluorescence showed colocalization. (E) No colocalization between CCR2 and GFAP was observed in the spinal cord. Scale bar: 100 μm in A and C, 25 μm in B, D and E.
brain research 1509 (2013) 1–7
in the spinal cord of BCP rats was found in our previous study (Hu et al., 2012b) and MCP-1 mediates pain via its cognate receptors CCR2 (Abbadie et al., 2009). The expression of CCR2 was significantly upregulated on day 6 and remained moderately increased on days 12 and 18, as shown by western blot and immunofluorescence analysis. Thus, we hypothesize that CCR2 might be involved in the initiation and maintenance of BCP and might play a more important role at the early stage of BCP than in its late stages. To confirm that CCR2 plays a more important role in the initiation of BCP, further investigation is required. In addition, immunofluorescence results showed that CCR2 could be found all over the dorsal horn, though it was mainly restricted to the ipsilateral superficial dorsal horn in BCP rats. Nociceptive afferent fibers terminate almost exclusively in the most superficial laminae of the dorsal horn. Consistent with our data, a previous study also demonstrated that CCR2 immunostaining was increased in the spinal cord following the inoculation of osteolytic sarcoma cells into the humeri of mice (Vit et al., 2006). Previous studies revealed that spinal CCR2 was expressed by various cell types, including microglia (Abbadie et al., 2003), astrocytes (Knerlich-Lukoschus et al., 2008) and neurons (Gao et al., 2009; Gosselin et al., 2005), during neuropathic pain. The present study showed the distinctive cellular expression of CCR2 in BCP rats: CCR2 was not only expressed by microglial cells but also by neurons in the dorsal horn of the spinal cord. A series of studies show that MCP-1/CCR2 signaling is involved in neuropathic pain via the activation of spinal microglia. For example, following partial sciatic nerve injury, an immunohistochemical study found that CCR2 was expressed by microglia in the dorsal horn of the spinal cord (Abbadie et al., 2003). The MCP1 expression in the spinal cord paralleled the microglial activation after nerve injury (Zhang and De Koninck, 2006). Intrathecal injection of MCP-1 neutralizing antibody and a CCR2 antagonist prevented the activation of microglia induced by neuropathic pain (Thacker et al., 2009; Van Steenwinckel et al., 2011; Zhang et al., 2007). In addition, MCP-1-induced microglial activation and nerve injury-induced p38 activation in spinal microglia were attenuated in CCR2 knockout mice (Abbadie et al., 2003; Zhang and De Koninck, 2006; Zhang et al., 2007). Moreover, we have previously demonstrated that bone cancer induced strong activation of spinal microglia, reaching a maximum on day 6 (Wang et al., 2011a); these findings are consistent with the changes in spinal CCR2 expression of BCP rats seen in the present results. Accordingly, we hypothesize that MCP-1 might act on microglial CCR2 receptors, thereby activating microglia in the spinal dorsal horn. Activated microglia can release a variety of substances, such as proinflammatory cytokines and chemokines, which lead to central sensitization and an exaggeration of pain responses (Cao and Zhang, 2008; O'Callaghan and Miller, 2010; Watkins et al., 2006). CCR2 has also been found in spinal cord neurons in other studies (Gao et al., 2009; Gosselin et al., 2005). Gao et al. reported that spinal injection of MCP-1 potentiated synaptic transmission and activated ERK in dorsal horn neurons, inducing hyperalgesia. MCP-1 perfusion also increased the activity of NMDA receptors in dorsal horn neurons. Furthermore, MCP-1 strongly inhibited GABAergic transmission in cultured spinal neurons (Gosselin et al., 2005). These data suggest that there are functional CCR2 receptors in dorsal horn neurons. MCP-1 could result in central sensitization and a nociceptive response via a direct action on
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spinal neurons (Gao et al., 2010). Consistent with previous reports on neuropathic pain (Gao et al., 2009), we observed that CCR2 was also expressed in spinal neurons of BCP rats. Therefore, we speculate that MCP-1 might activate CCR2 on spinal neurons to modulate pain sensitivity induced by bone cancer. In addition, CCR2 has also been shown to be upregulated in astrocytes in neuropathic pain caused by spinal cord contusion injuries (Knerlich-Lukoschus et al., 2008). However, CCR2 expression in astrocytes could not be observed in the present study. Moreover, because glial cells and neurons in the spinal cord also express MCP-1 and CCR2 receptors, MCP-1/CCR2 signaling might be involved in the regulation of BCP via crosstalk between these neurons and glial cells. To investigate the precise mechanism of action of CCR2 in BCP rats and verify this intriguing hypothesis, further studies that combine biochemical and pharmacological approaches are needed. In conclusion, the present study demonstrates that CCR2 expression is significantly increased in a time-dependent manner in the spinal cord of BCP rats and that CCR2 is expressed by spinal microglial cells as well as neurons in BCP rats. These findings suggest that CCR2 may be involved in the development of bone cancer pain.
4.
Experimental procedures
4.1.
Animals
Female adult Sprague-Dawley rats (150–180 g; Experimental Animal Center of Soochow University, China) were maintained under a 12 h/12 h light-dark cycle regime, with ad libitum access to food and water. All experiments were approved by the Animal Care and Use Committee of the Soochow University. Animals were treated in accordance with the Guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
4.2.
Bone cancer model
The Walker 256 mammary gland carcinoma cell line was prepared as previously described (Lan et al., 2010; Wang et al., 2011b). The cells were collected and diluted to a concentration of 2 107 cells/ ml. A rat model of bone cancer pain was established as described previously (Hu et al., 2012b; Wang et al., 2011b). In brief, under anesthesia with pentobarbital sodium (40 mg/kg, i.p.), Walker 256 cells (1 105) in 10 μl Hank's solution were injected into the cavity of the left tibia, whereas sham rats were injected with 10 μl Hank's solution alone. The injected site was sealed with medical glue to prevent the cells from leaking out at the injection site, and the wound was cleaned with sterile water.
4.3.
Mechanical allodynia
Mechanical allodynia of BCP rats (n ¼10) and sham rats (n ¼10) was tested before operation, and 6, 12, and 18 days after operation. Mechanical allodynia was measured using von Frey filaments (Stoelting, Wood Dale, IL) as described previously (Lan et al., 2010). Briefly, animals were placed in individual plastic boxes (20 cm 25 cm 15 cm) on a metal mesh floor and allowed to acclimatize for 30 min. The filaments perpendicular to the plantar surface exhibited sufficient force to cause slight bending
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against the paw when held in place for 6–8 s. Brisk withdrawal or paw flinching were considered as positive responses. The paw withdrawal threshold (PWT) was assessed by sequentially increasing and decreasing the stimulus strength (the“up-anddown” method) (Chaplan et al., 1994) as described previously (Lan et al., 2010; Yao et al., 2008).
4.4.
Western blot analysis
The Western Blot was performed on sample from BCP rats (6, 12, and 18 days after operation, n¼ 4 at each time point) and sham group (day 6, n ¼4) to analyze the protein expression of CCR2 in the spinal cord. Rats were killed at each time point and the tissue samples from the L4–L5 spinal cord segments were rapidly removed and immediately frozen in liquid nitrogen and stored at −80 1C until use. All tissue samples were homogenized in lysis buffer containing PMSF and 0.02% protease inhibitor cocktail. The homogenates were centrifuged at 14,000g for 15 min at 4 1C. The sample supernatants were used for Western blot analysis. Equivalent amounts of protein (30 μg) were separated using 10% SDS– PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% nonfat milk for 1 h at room temperature (RT) and incubated with primary antibodies for rabbit anti-CCR2 (1:3000, Abcam, Cambridge, MA) overnight at 4 1C. The membranes were incubated for 1 h with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Kangchen, Shanghai, China). Bands were finally revealed using an ECL kit (Kangchen, Shanghai, China). A rat monoclonal antibody against β-actin (1:10,000, Sigma, USA) was used as a loading control. Western blot analysis was performed as previously described (Zhuang et al., 2005). Scanning densitometry was used for semiquantitative analysis with a computer-assisted image analyzer (Image J).
4.5.
To quantify the numbers of immunoreactive staining of positive cells in the spinal cord, digital images were captured randomly from six non-adjacent spinal selected sections per animal and three fixed squares (250 250 μm) in the superficial spinal dorsal horn (I–IV layers) per section were chosen and observed using fluorescence microscopy (Zeiss Imager M1, Germany) and laser scanning confocal microscopy (TCS SP2 CLSM; Leica, Wetzlar, Germany). The stained sections were analyzed by Image Pro-plus 6.0 (Image Pro-Plus Kodak, USA).
4.6.
Statistical analysis
Statistical analysis was performed using software SPSS 13.0 (SPSS Inc). All data are expressed as mean7SEM. Data from immunohistochemical analysis and Western blot studies were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett post hoc testing. Data from the nociceptive tests were analyzed by two-way ANOVA followed by Bonferroni post hoc for mechanical allodynia testing. A value of Po0.05 was considered statistically significant. The investigator was blind to the tested groups.
Acknowledgments This study was supported by National Natural Science Fund of China (NSFC) (81171057, and 81000479), NSFC for Distinguished Young Scholars (30872442), Natural Science Fund of Jiangsu Province (SBK201121879), Department of Health Fund of Jiangsu Province (H200917), Science and Technology Fund of Suzhou City (ZS0901).
r e f e r e n c e s
Immunohistochemistry
The BCP rats (n ¼6 at each time point) were sacrificed on day 6, 12, and 18, and sham rats (n¼ 6) were killed on day 6 after operation. Rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4). L4–L5 spinal cords segments were excised and postfixed in the same fixative for 6 h at 4 1C, then placed in a 30% sucrose solution overnight at 4 1C. Immunohistochemistry was performed on 10 μm thick L4–L5 transverse spinal sections and processed as previously described (Hu et al., 2012a). Sections were incubated with primary antibodies for rabbit anti-CCR2 (1:250, Abcam, Cambridge, MA) overnight at 4 1C. Next, the sections were processed with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:250, Invitrogen, USA) for 2 h at RT. For double immunolabeling for CCR2/NeuN, CCR2/OX-42, CCR2/GFAP, sections were incubated with a mixture of CCR2 and NeuN (1:500, Chemicon, USA), OX-42 (1:250, Cedarlane, Canada) and GFAP (1:500, Sigma, USA) overnight at 4 1C. Sections were rinsed and incubated with a mixture of Alexa Fluor 488- and 594-conjugated secondary antibodies (1:250, Invitrogen, USA) for 2 h at RT. Control sections were similarly processed, except that the primary antisera were omitted. All antibodies were tested for sensitivity and specificity before the study. Immunohistochemistry analysis was performed as previously described (Wang et al., 2011a). These cells with distinct contrast to the background were marked as positive.
Abbadie, C., Lindia, J.A., Cumiskey, A.M., Peterson, L.B., Mudgett, J.S., Bayne, E.K., DeMartino, J.A., MacIntyre, D.E., Forrest, M.J., 2003. Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc. Natl. Acad. Sci. USA 100, 7947–7952. Abbadie, C., Bhangoo, S., De Koninck, Y., Malcangio, M., MelikParsadaniantz, S., White, F.A., 2009. Chemokines and pain mechanisms. Brain Res. Rev. 60, 125–134. Bhangoo, S., Ren, D., Miller, R.J., Henry, K.J., Lineswala, J., Hamdouchi, C., Li, B., Monahan, P.E., Chan, D.M., Ripsch, M.S., White, F.A., 2007. Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors. Mol. Pain 3, 38. Cao, H., Zhang, Y.Q., 2008. Spinal glial activation contributes to pathological pain states. Neurosci. Biobehav. Rev. 32, 972–983. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63. Coleman, R.E., 2006. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin. Cancer Res. 12, 6243s–6249s. Dansereau, M.A., Gosselin, R.D., Pohl, M., Pommier, B., Mechighel, P., Mauborgne, A., Rostene, W., Kitabgi, P., Beaudet, N., Sarret, P., Melik-Parsadaniantz, S., 2008. Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats. J. Neurochem. 106, 757–769. Gao, Y.J., Zhang, L., Samad, O.A., Suter, M.R., Yasuhiko, K., Xu, Z.Z., Park, J.Y., Lind, A.L., Ma, Q., Ji, R.R., 2009. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. J. Neurosci. 29, 4096–4108.
brain research 1509 (2013) 1–7
Gao, Y.J., Zhang, L., Ji, R.R., 2010. Spinal injection of TNF-alphaactivated astrocytes produces persistent pain symptom mechanical allodynia by releasing monocyte chemoattractant protein-1. Glia 58, 1871–1880. Gosselin, R.D., Varela, C., Banisadr, G., Mechighel, P., Rostene, W., Kitabgi, P., Melik-Parsadaniantz, S., 2005. Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones. J. Neurochem. 95, 1023–1034. Gosselin, R.D., Dansereau, M.A., Pohl, M., Kitabgi, P., Beaudet, N., Sarret, P., Melik Parsadaniantz, S., 2008. Chemokine network in the nervous system: a new target for pain relief. Curr. Med. Chem. 15, 2866–2875. Honore, P., Rogers, S.D., Schwei, M.J., Salak-Johnson, J.L., Luger, N.M., Sabino, M.C., Clohisy, D.R., Mantyh, P.W., 2000. Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience 98, 585–598. Hu, J.H., Yang, J.P., Liu, L., Li, C.F., Wang, L.N., Ji, F.H., Cheng, H., 2012a. Involvement of CX3CR1 in bone cancer pain through the activation of microglia p38 MAPK pathway in the spinal cord. Brain Res. 1465, 2012,1–9. Hu, J.H., Zheng, X.Y., Yang, J.P., Wang, L.N., Ji, F.H., 2012b. Involvement of spinal monocyte chemoattractant protein-1 (MCP-1) in cancerinduced bone pain in rats. Neurosci. Lett. 517, 60–63. Jimenez-Andrade, J.M., Mantyh, W.G., Bloom, A.P., Ferng, A.S., Geffre, C.P., Mantyh, P.W., 2010. Bone cancer pain. Ann. NY Acad. Sci. 1198, 173–181. Knerlich-Lukoschus, F., Juraschek, M., Blomer, U., Lucius, R., Mehdorn, H.M., Held-Feindt, J., 2008. Force-dependent development of neuropathic central pain and time-related CCL2/CCR2 expression after graded spinal cord contusion injuries of the rat. J. Neurotrauma. 25, 427–448. Lan, L.S., Ping, Y.J., Na, W.L., Miao, J., Cheng, Q.Q., Ni, M.Z., Lei, L., Fang, L.C., Guang, R.C., Jin, Z., Wei, L., 2010. Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model. Mol. Pain 6, 2. Mantyh, P.W., 2006. Cancer pain and its impact on diagnosis, survival and quality of life. Nat. Rev. Neurosci. 7, 797–809. Menetski, J., Mistry, S., Lu, M., Mudgett, J.S., Ransohoff, R.M., Demartino, J.A., Macintyre, D.E., Abbadie, C., 2007. Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses. Neuroscience. 149, 706–714. O'Callaghan, J.P., Miller, D.B., 2010. Spinal glia and chronic pain. Metabolism 59 (Suppl. 1), S21–S26. Old, E.A., Malcangio, M., 2012. Chemokine mediated neuron-glia communication and aberrant signalling in neuropathic pain states. Curr. Opin. Pharmacol. 12, 67–73. Serrano, A., Pare, M., McIntosh, F., Elmes, S.J., Martino, G., Jomphe, C., Lessard, E., Lembo, P.M., Vaillancourt, F., Perkins, M.N., Cao, C.Q., 2010. Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain. Mol. Pain 6, 90. Thacker, M.A., Clark, A.K., Bishop, T., Grist, J., Yip, P.K., Moon, L.D., Thompson, S.W., Marchand, F., McMahon, S.B., 2009. CCL2 is a key
7
mediator of microglia activation in neuropathic pain states. Eur. J. Pain 13, 263–272. Van Steenwinckel, J., Reaux-Le Goazigo, A., Pommier, B., Mauborgne, A., Dansereau, M.A., Kitabgi, P., Sarret, P., Pohl, M., Melik Parsadaniantz, S., 2011. CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury. J. Neurosci. 31, 5865–5875. Vit, J.P., Ohara, P.T., Tien, D.A., Fike, J.R., Eikmeier, L., Beitz, A., Wilcox, G.L., Jasmin, L., 2006. The analgesic effect of low dose focal irradiation in a mouse model of bone cancer is associated with spinal changes in neuro-mediators of nociception. Pain 120, 188–201. Wang, L.N., Yang, J.P., Zhan, Y., Ji, F.H., Wang, X.Y., Zuo, J.L., Xu, Q.N., 2011a. Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats. J. Neurosci. Res. Wang, L.N., Yao, M., Yang, J.P., Peng, J., Peng, Y., Li, C.F., Zhang, Y.B., Ji, F. H., Cheng, H., Xu, Q.N., Wang, X.Y., Zuo, J.L., 2011b. Cancer-induced bone pain sequentially activates the ERK/MAPK pathway in different cell types in the rat spinal cord. Mol. Pain 7, 48. Watkins, L.R., Wieseler-Frank, J., Milligan, E.D., Johnston, I., Maier, S.F., 2006. Chapter 22 Contribution of glia to pain processing in health and disease. Handb. Clin. Neurol. 81, 309–323. White, F.A., Sun, J., Waters, S.M., Ma, C., Ren, D., Ripsch, M., Steflik, J., Cortright, D.N., Lamotte, R.H., Miller, R.J., 2005. Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion. Proc. Natl. Acad. Sci USA 102, 14092–14097. White, F.A., Wilson, N.M., 2008. Chemokines as pain mediators and modulators. Curr. Opin. Anaesthesiol. 21, 580–585. White, F.A., Miller, R.J., 2010. Insights into the regulation of chemokine receptors by molecular signaling pathways: functional roles in neuropathic pain. Brain Behav. Immun. 24, 859–865. Yao, M., Yang, J.P., Wang, L.N., Cheng, H., Zhang, Y.B., Xu, Q.N., Wu, Y.W., 2008. Feasibility of establishment of rat model of bone cancer pain by using Walker 256 cells cultured in vitro or in vivo. Zhonghua Yi Xue Za Zhi. 88, 880–884. Zhang, J., De Koninck, Y., 2006. Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J. Neurochem. 97, 772–783. Zhang, J., Shi, X.Q., Echeverry, S., Mogil, J.S., De Koninck, Y., Rivest, S., 2007. Expression of CCR2 in both resident and bone marrowderived microglia plays a critical role in neuropathic pain. J. Neurosci. 27, 12396–12406. Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114, 149–159. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110.
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Research Report
Changes in protein expression and distribution of spinal CCR2 in a rat model of bone cancer pain Ji-Hua Hua,1, Meng-Yao Wub,1, Min Taob, Jian-Ping Yanga,n a
Department of Anesthesiology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, China
b
art i cle i nfo
ab st rac t
Article history:
Accumulating evidence suggests that chemokine C–C motif receptor 2 (CCR2) plays an important
Accepted 3 March 2013
role in neuropathic pain. It has been shown that spinal CCR2 is upregulated in several
Available online 17 March 2013
neuropathic pain models and expressed by neuronal and glial cells in the spinal cord. In this
Keywords:
study, we investigated the expression changes and cellular localization of spinal CCR2 in a rat
Bone cancer pain
model of bone cancer induced by Walker 256 cell inoculation. The present results indicated that
Chemokine
mechanical allodynia progressively increased in bone cancer pain (BCP) rats. Western blot and
CCR2
immunohistochemical analysis demonstrated that the expression of CCR2 in the spinal cord was
Spinal cord
significantly increased on day 6, 12, and 18 in BCP rats, with a peak on day 6. Furthermore, double
Microglia
immunofluorescence labeling indicated that CCR2 was expressed by both microglia and neurons in the spinal cord. These results suggest that CCR2 may be involved in the development of BCP, and that targeting CCR2 may be a new strategy for the treatment of BCP. & 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Bone cancer pain (BCP), which seriously compromises patient quality of life (Coleman, 2006; Jimenez-Andrade et al., 2010), is the most common symptom detected in patients with advanced breast, prostate, and lung cancer. Therefore, understanding the underlying mechanisms related to the development of BCP is important for effectively treating these patients. Monocyte chemoattractant protein-1 (MCP-1, also named CCL2) has been reported to play a role in the pathophysiology of neuropathic pain by binding to its β- chemokine receptor 2 (CCR2) (Abbadie et al., 2009; White et al., 2005; White and Wilson, 2008). CCR2 is a G protein-coupled receptor (GPCR) that preferentially binds to the MCP-1 chemokine. Previous studies have demonstrated that mice lacking CCR2 fail to develop tactile
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Corresponding author. Fax: þ86 512 677 80149. E-mail addresses: [email protected], [email protected] (J.-P. Yang). 1 These two authors contributed equally.
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allodynia after partial nerve ligation (Abbadie et al., 2003), and that mice overexpressing MCP-1 showed enhanced nociceptive behavior (Menetski et al., 2007). Blockade of CCR2 produced an attenuation of the nociceptive behavior (Bhangoo et al., 2007; Serrano et al., 2010) in neuropathic pain rats and prevented MCP1-induced nociceptive responses (Dansereau et al., 2008). In addition, the expression of CCR2 is upregulated in the spinal cord in several kinds of neuropathic pain models, including peripheral nerve injury (Abbadie et al., 2003), spinal nerve ligation (Gao et al., 2009), and spinal cord contusion injuries (KnerlichLukoschus et al., 2008). These results suggest that CCR2 activation plays an important role in neuropathic pain. Understanding the function of CCR2 in nociceptive responses requires a detailed knowledge of its distribution in the dorsal horn of the spinal cord. Previously, CCR2 expression was detected in spinal glial cells or
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neurons under neuropathic pain conditions (Abbadie et al., 2003; Gao et al., 2009; Knerlich-Lukoschus et al., 2008). MCP-1/CCR2 has also been identified as a signaling molecule in neural–glial communication in neuropathic pain (Abbadie et al., 2009). Bone cancer pain is unique and distinct from neuropathic pain (Honore et al., 2000; Mantyh, 2006). What is in some ways unique about bone cancer pain is that the inflammation, tumorreleased products, and tumor-induced injury to primary afferent neurons can simultaneously drive the chronic pain state. Rarely is CIBP purely neuropathic, inflammatory, ischemic, or visceral but rather is a combination. However, little is known about possible alterations in CCR2 expression in BCP rats. In this study, we examined CCR2 expression changes and the cellular localization of CCR2 in the spinal cord of BCP rats.
2.
Results
2.1.
Mechanical allodynia induced by bone cancer
All rat groups exhibited similar baseline hind paw withdrawal threshold (PWT) to mechanical stimulation (von Frey filaments). Bone cancer pain rats displayed a significant decrease in PWT of the ipsilateral hind paw compared with sham rats on day 6. With the progression of bone cancer, the PWT progressively decreased in the inoculated hind paw from days 6 to 18 (Fig. 1). Thus mechanical allodynia was induced in this rat model of bone cancer.
2.2.
Increase of CCR2 expression in BCP rats
As shown in Fig. 2A, CCR2 bands (42 kDa) indicated a significantly upregulation of CCR2 in the spinal cord of BCP rats compared with sham rats. The protein expression was increased at each time point investigated, with a maximum on day 6 following Walker 256 cells inoculation (Fig. 2A, B).
Fig. 2 – Time course of CCR2 expression in the spinal cord (L4–L5) of BCP rats. (A) Western blot analysis showed an increase in the expression of CCR2 in the spinal cord of BCP rats. β-actin served as a loading control. (B) Quantification of CCR2 expression in the spinal cord. n¼ 4; * Po0.05 vs. sham; ** Po0.01 vs. sham.
2.3.
Immunoreactivity of CCR2 in BCP rats
Immunohistochemistry data also demonstrated that compared with sham rats (Fig. 3A, E), the expression of CCR2 was strikingly increased on day 6, and maintained on day 12 and 18 in the ipsilateral spinal cord dorsal horn (Fig.3B, D). CCR2immunoreactive (IR) cells were detected throughout the spinal cord following Walker 256 cells inoculation (Fig. 3F, G). Although most positive cells were distributed in the ipsilateral superficial (laminae I–III) dorsal horn, a few positive cells were also found in the ventral horn (Fig. 3F, G). In addition, the expression of CCR2 was striking increased in the ipsilateral spinal cord compared with that in contralateral superficial dorsal horn (Fig. 3F–H).
2.4.
Cellular localization of CCR2 in BCP rats
Double immunofluorescence labeling indicated that CCR2 was expressed both on microglia and neurons in the ipsilateral dorsal horn on day 6 following Walker 256 cell inoculation, since CCR2IR cells were colocalized with OX-42 (microglial marker) (Fig. 4A, B) and NeuN (neuron marker) (Fig.4C, D) respectively. In contrast, CCR2 was not expressed on GFAP-IR astrocytes in the dorsal horn of BCP rats on day 6 (Fig. 4E). Fig. 1 – Mechanical allodynia is induced in BCP rats. The paw withdrawal threshold (PWT) to mechanical stimulation (von Frey filaments) was decreased in BCP rats compared with sham rats on days 6, 12 and 18. Results are expressed as mean7SEM. n¼ 10; ** Po0.01 vs. sham.
3.
Discussion
In the present study, we demonstrated that mechanical allodynia progressively increased in BCP rats from days 6 to 18, and that CCR2
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Fig. 3 – CCR2 like immunoreactivity in the spinal cord (L4–L5) of BCP rats. Immunofluorescence data showed that CCR2 expression was significant increased in the dorsal horn of BCP rats on day 6 (B, F), 12 (C) and 18 (D, G) compared with that in sham rats (A, C). CCR2 immunoreactivity cells were mainly presented in superficial layers of the ipsilateral dorsal horn (arrows). (H) Quantification of CCR2 immunoreactivity cells in the dorsal horn of spinal cord. n ¼ 6; ** Po0.01 vs. sham; ## Po0.01 vs. cont ( ipsi indicates ipsilateral; cont, contralateral). Scale bar: 100 μm in A–D, 200 μm in E–G.
expression was significantly upregulated in the ipsilateral spinal cord of BCP rats, peaking on day 6. Furthermore, double immunofluorescence labeling indicated that CCR2 was expressed by microglia and neurons in the dorsal horn of the spinal cord in BCP rats.
Accumulating evidence suggests that MCP-1 and its receptor CCR2 play important roles in the development of neuropathic pain (Gosselin et al., 2008; Old and Malcangio, 2012; White and Miller, 2010). For example, an increase in MCP-1 expression was
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Fig. 4 – Cellular type of CCR2 expression in the spinal cord (L4–L5) of BCP rats. (A and B) Double immunofluorescence data showed that CCR2 immunoreactivity cells were co-localized with OX-42 and (C and D) co-localized with NeuN in the dorsal horn of spinal cord on day 6 after Walker 256 cell inoculation. Yellow fluorescence showed colocalization. (E) No colocalization between CCR2 and GFAP was observed in the spinal cord. Scale bar: 100 μm in A and C, 25 μm in B, D and E.
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in the spinal cord of BCP rats was found in our previous study (Hu et al., 2012b) and MCP-1 mediates pain via its cognate receptors CCR2 (Abbadie et al., 2009). The expression of CCR2 was significantly upregulated on day 6 and remained moderately increased on days 12 and 18, as shown by western blot and immunofluorescence analysis. Thus, we hypothesize that CCR2 might be involved in the initiation and maintenance of BCP and might play a more important role at the early stage of BCP than in its late stages. To confirm that CCR2 plays a more important role in the initiation of BCP, further investigation is required. In addition, immunofluorescence results showed that CCR2 could be found all over the dorsal horn, though it was mainly restricted to the ipsilateral superficial dorsal horn in BCP rats. Nociceptive afferent fibers terminate almost exclusively in the most superficial laminae of the dorsal horn. Consistent with our data, a previous study also demonstrated that CCR2 immunostaining was increased in the spinal cord following the inoculation of osteolytic sarcoma cells into the humeri of mice (Vit et al., 2006). Previous studies revealed that spinal CCR2 was expressed by various cell types, including microglia (Abbadie et al., 2003), astrocytes (Knerlich-Lukoschus et al., 2008) and neurons (Gao et al., 2009; Gosselin et al., 2005), during neuropathic pain. The present study showed the distinctive cellular expression of CCR2 in BCP rats: CCR2 was not only expressed by microglial cells but also by neurons in the dorsal horn of the spinal cord. A series of studies show that MCP-1/CCR2 signaling is involved in neuropathic pain via the activation of spinal microglia. For example, following partial sciatic nerve injury, an immunohistochemical study found that CCR2 was expressed by microglia in the dorsal horn of the spinal cord (Abbadie et al., 2003). The MCP1 expression in the spinal cord paralleled the microglial activation after nerve injury (Zhang and De Koninck, 2006). Intrathecal injection of MCP-1 neutralizing antibody and a CCR2 antagonist prevented the activation of microglia induced by neuropathic pain (Thacker et al., 2009; Van Steenwinckel et al., 2011; Zhang et al., 2007). In addition, MCP-1-induced microglial activation and nerve injury-induced p38 activation in spinal microglia were attenuated in CCR2 knockout mice (Abbadie et al., 2003; Zhang and De Koninck, 2006; Zhang et al., 2007). Moreover, we have previously demonstrated that bone cancer induced strong activation of spinal microglia, reaching a maximum on day 6 (Wang et al., 2011a); these findings are consistent with the changes in spinal CCR2 expression of BCP rats seen in the present results. Accordingly, we hypothesize that MCP-1 might act on microglial CCR2 receptors, thereby activating microglia in the spinal dorsal horn. Activated microglia can release a variety of substances, such as proinflammatory cytokines and chemokines, which lead to central sensitization and an exaggeration of pain responses (Cao and Zhang, 2008; O'Callaghan and Miller, 2010; Watkins et al., 2006). CCR2 has also been found in spinal cord neurons in other studies (Gao et al., 2009; Gosselin et al., 2005). Gao et al. reported that spinal injection of MCP-1 potentiated synaptic transmission and activated ERK in dorsal horn neurons, inducing hyperalgesia. MCP-1 perfusion also increased the activity of NMDA receptors in dorsal horn neurons. Furthermore, MCP-1 strongly inhibited GABAergic transmission in cultured spinal neurons (Gosselin et al., 2005). These data suggest that there are functional CCR2 receptors in dorsal horn neurons. MCP-1 could result in central sensitization and a nociceptive response via a direct action on
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spinal neurons (Gao et al., 2010). Consistent with previous reports on neuropathic pain (Gao et al., 2009), we observed that CCR2 was also expressed in spinal neurons of BCP rats. Therefore, we speculate that MCP-1 might activate CCR2 on spinal neurons to modulate pain sensitivity induced by bone cancer. In addition, CCR2 has also been shown to be upregulated in astrocytes in neuropathic pain caused by spinal cord contusion injuries (Knerlich-Lukoschus et al., 2008). However, CCR2 expression in astrocytes could not be observed in the present study. Moreover, because glial cells and neurons in the spinal cord also express MCP-1 and CCR2 receptors, MCP-1/CCR2 signaling might be involved in the regulation of BCP via crosstalk between these neurons and glial cells. To investigate the precise mechanism of action of CCR2 in BCP rats and verify this intriguing hypothesis, further studies that combine biochemical and pharmacological approaches are needed. In conclusion, the present study demonstrates that CCR2 expression is significantly increased in a time-dependent manner in the spinal cord of BCP rats and that CCR2 is expressed by spinal microglial cells as well as neurons in BCP rats. These findings suggest that CCR2 may be involved in the development of bone cancer pain.
4.
Experimental procedures
4.1.
Animals
Female adult Sprague-Dawley rats (150–180 g; Experimental Animal Center of Soochow University, China) were maintained under a 12 h/12 h light-dark cycle regime, with ad libitum access to food and water. All experiments were approved by the Animal Care and Use Committee of the Soochow University. Animals were treated in accordance with the Guidelines of the International Association for the Study of Pain (Zimmermann, 1983).
4.2.
Bone cancer model
The Walker 256 mammary gland carcinoma cell line was prepared as previously described (Lan et al., 2010; Wang et al., 2011b). The cells were collected and diluted to a concentration of 2 107 cells/ ml. A rat model of bone cancer pain was established as described previously (Hu et al., 2012b; Wang et al., 2011b). In brief, under anesthesia with pentobarbital sodium (40 mg/kg, i.p.), Walker 256 cells (1 105) in 10 μl Hank's solution were injected into the cavity of the left tibia, whereas sham rats were injected with 10 μl Hank's solution alone. The injected site was sealed with medical glue to prevent the cells from leaking out at the injection site, and the wound was cleaned with sterile water.
4.3.
Mechanical allodynia
Mechanical allodynia of BCP rats (n ¼10) and sham rats (n ¼10) was tested before operation, and 6, 12, and 18 days after operation. Mechanical allodynia was measured using von Frey filaments (Stoelting, Wood Dale, IL) as described previously (Lan et al., 2010). Briefly, animals were placed in individual plastic boxes (20 cm 25 cm 15 cm) on a metal mesh floor and allowed to acclimatize for 30 min. The filaments perpendicular to the plantar surface exhibited sufficient force to cause slight bending
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against the paw when held in place for 6–8 s. Brisk withdrawal or paw flinching were considered as positive responses. The paw withdrawal threshold (PWT) was assessed by sequentially increasing and decreasing the stimulus strength (the“up-anddown” method) (Chaplan et al., 1994) as described previously (Lan et al., 2010; Yao et al., 2008).
4.4.
Western blot analysis
The Western Blot was performed on sample from BCP rats (6, 12, and 18 days after operation, n¼ 4 at each time point) and sham group (day 6, n ¼4) to analyze the protein expression of CCR2 in the spinal cord. Rats were killed at each time point and the tissue samples from the L4–L5 spinal cord segments were rapidly removed and immediately frozen in liquid nitrogen and stored at −80 1C until use. All tissue samples were homogenized in lysis buffer containing PMSF and 0.02% protease inhibitor cocktail. The homogenates were centrifuged at 14,000g for 15 min at 4 1C. The sample supernatants were used for Western blot analysis. Equivalent amounts of protein (30 μg) were separated using 10% SDS– PAGE and transferred onto a PVDF membrane. Membranes were blocked with 5% nonfat milk for 1 h at room temperature (RT) and incubated with primary antibodies for rabbit anti-CCR2 (1:3000, Abcam, Cambridge, MA) overnight at 4 1C. The membranes were incubated for 1 h with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Kangchen, Shanghai, China). Bands were finally revealed using an ECL kit (Kangchen, Shanghai, China). A rat monoclonal antibody against β-actin (1:10,000, Sigma, USA) was used as a loading control. Western blot analysis was performed as previously described (Zhuang et al., 2005). Scanning densitometry was used for semiquantitative analysis with a computer-assisted image analyzer (Image J).
4.5.
To quantify the numbers of immunoreactive staining of positive cells in the spinal cord, digital images were captured randomly from six non-adjacent spinal selected sections per animal and three fixed squares (250 250 μm) in the superficial spinal dorsal horn (I–IV layers) per section were chosen and observed using fluorescence microscopy (Zeiss Imager M1, Germany) and laser scanning confocal microscopy (TCS SP2 CLSM; Leica, Wetzlar, Germany). The stained sections were analyzed by Image Pro-plus 6.0 (Image Pro-Plus Kodak, USA).
4.6.
Statistical analysis
Statistical analysis was performed using software SPSS 13.0 (SPSS Inc). All data are expressed as mean7SEM. Data from immunohistochemical analysis and Western blot studies were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett post hoc testing. Data from the nociceptive tests were analyzed by two-way ANOVA followed by Bonferroni post hoc for mechanical allodynia testing. A value of Po0.05 was considered statistically significant. The investigator was blind to the tested groups.
Acknowledgments This study was supported by National Natural Science Fund of China (NSFC) (81171057, and 81000479), NSFC for Distinguished Young Scholars (30872442), Natural Science Fund of Jiangsu Province (SBK201121879), Department of Health Fund of Jiangsu Province (H200917), Science and Technology Fund of Suzhou City (ZS0901).
r e f e r e n c e s
Immunohistochemistry
The BCP rats (n ¼6 at each time point) were sacrificed on day 6, 12, and 18, and sham rats (n¼ 6) were killed on day 6 after operation. Rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS, pH 7.4). L4–L5 spinal cords segments were excised and postfixed in the same fixative for 6 h at 4 1C, then placed in a 30% sucrose solution overnight at 4 1C. Immunohistochemistry was performed on 10 μm thick L4–L5 transverse spinal sections and processed as previously described (Hu et al., 2012a). Sections were incubated with primary antibodies for rabbit anti-CCR2 (1:250, Abcam, Cambridge, MA) overnight at 4 1C. Next, the sections were processed with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:250, Invitrogen, USA) for 2 h at RT. For double immunolabeling for CCR2/NeuN, CCR2/OX-42, CCR2/GFAP, sections were incubated with a mixture of CCR2 and NeuN (1:500, Chemicon, USA), OX-42 (1:250, Cedarlane, Canada) and GFAP (1:500, Sigma, USA) overnight at 4 1C. Sections were rinsed and incubated with a mixture of Alexa Fluor 488- and 594-conjugated secondary antibodies (1:250, Invitrogen, USA) for 2 h at RT. Control sections were similarly processed, except that the primary antisera were omitted. All antibodies were tested for sensitivity and specificity before the study. Immunohistochemistry analysis was performed as previously described (Wang et al., 2011a). These cells with distinct contrast to the background were marked as positive.
Abbadie, C., Lindia, J.A., Cumiskey, A.M., Peterson, L.B., Mudgett, J.S., Bayne, E.K., DeMartino, J.A., MacIntyre, D.E., Forrest, M.J., 2003. Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2. Proc. Natl. Acad. Sci. USA 100, 7947–7952. Abbadie, C., Bhangoo, S., De Koninck, Y., Malcangio, M., MelikParsadaniantz, S., White, F.A., 2009. Chemokines and pain mechanisms. Brain Res. Rev. 60, 125–134. Bhangoo, S., Ren, D., Miller, R.J., Henry, K.J., Lineswala, J., Hamdouchi, C., Li, B., Monahan, P.E., Chan, D.M., Ripsch, M.S., White, F.A., 2007. Delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors. Mol. Pain 3, 38. Cao, H., Zhang, Y.Q., 2008. Spinal glial activation contributes to pathological pain states. Neurosci. Biobehav. Rev. 32, 972–983. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., Yaksh, T.L., 1994. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63. Coleman, R.E., 2006. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin. Cancer Res. 12, 6243s–6249s. Dansereau, M.A., Gosselin, R.D., Pohl, M., Pommier, B., Mechighel, P., Mauborgne, A., Rostene, W., Kitabgi, P., Beaudet, N., Sarret, P., Melik-Parsadaniantz, S., 2008. Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats. J. Neurochem. 106, 757–769. Gao, Y.J., Zhang, L., Samad, O.A., Suter, M.R., Yasuhiko, K., Xu, Z.Z., Park, J.Y., Lind, A.L., Ma, Q., Ji, R.R., 2009. JNK-induced MCP-1 production in spinal cord astrocytes contributes to central sensitization and neuropathic pain. J. Neurosci. 29, 4096–4108.
brain research 1509 (2013) 1–7
Gao, Y.J., Zhang, L., Ji, R.R., 2010. Spinal injection of TNF-alphaactivated astrocytes produces persistent pain symptom mechanical allodynia by releasing monocyte chemoattractant protein-1. Glia 58, 1871–1880. Gosselin, R.D., Varela, C., Banisadr, G., Mechighel, P., Rostene, W., Kitabgi, P., Melik-Parsadaniantz, S., 2005. Constitutive expression of CCR2 chemokine receptor and inhibition by MCP-1/CCL2 of GABA-induced currents in spinal cord neurones. J. Neurochem. 95, 1023–1034. Gosselin, R.D., Dansereau, M.A., Pohl, M., Kitabgi, P., Beaudet, N., Sarret, P., Melik Parsadaniantz, S., 2008. Chemokine network in the nervous system: a new target for pain relief. Curr. Med. Chem. 15, 2866–2875. Honore, P., Rogers, S.D., Schwei, M.J., Salak-Johnson, J.L., Luger, N.M., Sabino, M.C., Clohisy, D.R., Mantyh, P.W., 2000. Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons. Neuroscience 98, 585–598. Hu, J.H., Yang, J.P., Liu, L., Li, C.F., Wang, L.N., Ji, F.H., Cheng, H., 2012a. Involvement of CX3CR1 in bone cancer pain through the activation of microglia p38 MAPK pathway in the spinal cord. Brain Res. 1465, 2012,1–9. Hu, J.H., Zheng, X.Y., Yang, J.P., Wang, L.N., Ji, F.H., 2012b. Involvement of spinal monocyte chemoattractant protein-1 (MCP-1) in cancerinduced bone pain in rats. Neurosci. Lett. 517, 60–63. Jimenez-Andrade, J.M., Mantyh, W.G., Bloom, A.P., Ferng, A.S., Geffre, C.P., Mantyh, P.W., 2010. Bone cancer pain. Ann. NY Acad. Sci. 1198, 173–181. Knerlich-Lukoschus, F., Juraschek, M., Blomer, U., Lucius, R., Mehdorn, H.M., Held-Feindt, J., 2008. Force-dependent development of neuropathic central pain and time-related CCL2/CCR2 expression after graded spinal cord contusion injuries of the rat. J. Neurotrauma. 25, 427–448. Lan, L.S., Ping, Y.J., Na, W.L., Miao, J., Cheng, Q.Q., Ni, M.Z., Lei, L., Fang, L.C., Guang, R.C., Jin, Z., Wei, L., 2010. Down-regulation of Toll-like receptor 4 gene expression by short interfering RNA attenuates bone cancer pain in a rat model. Mol. Pain 6, 2. Mantyh, P.W., 2006. Cancer pain and its impact on diagnosis, survival and quality of life. Nat. Rev. Neurosci. 7, 797–809. Menetski, J., Mistry, S., Lu, M., Mudgett, J.S., Ransohoff, R.M., Demartino, J.A., Macintyre, D.E., Abbadie, C., 2007. Mice overexpressing chemokine ligand 2 (CCL2) in astrocytes display enhanced nociceptive responses. Neuroscience. 149, 706–714. O'Callaghan, J.P., Miller, D.B., 2010. Spinal glia and chronic pain. Metabolism 59 (Suppl. 1), S21–S26. Old, E.A., Malcangio, M., 2012. Chemokine mediated neuron-glia communication and aberrant signalling in neuropathic pain states. Curr. Opin. Pharmacol. 12, 67–73. Serrano, A., Pare, M., McIntosh, F., Elmes, S.J., Martino, G., Jomphe, C., Lessard, E., Lembo, P.M., Vaillancourt, F., Perkins, M.N., Cao, C.Q., 2010. Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain. Mol. Pain 6, 90. Thacker, M.A., Clark, A.K., Bishop, T., Grist, J., Yip, P.K., Moon, L.D., Thompson, S.W., Marchand, F., McMahon, S.B., 2009. CCL2 is a key
7
mediator of microglia activation in neuropathic pain states. Eur. J. Pain 13, 263–272. Van Steenwinckel, J., Reaux-Le Goazigo, A., Pommier, B., Mauborgne, A., Dansereau, M.A., Kitabgi, P., Sarret, P., Pohl, M., Melik Parsadaniantz, S., 2011. CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury. J. Neurosci. 31, 5865–5875. Vit, J.P., Ohara, P.T., Tien, D.A., Fike, J.R., Eikmeier, L., Beitz, A., Wilcox, G.L., Jasmin, L., 2006. The analgesic effect of low dose focal irradiation in a mouse model of bone cancer is associated with spinal changes in neuro-mediators of nociception. Pain 120, 188–201. Wang, L.N., Yang, J.P., Zhan, Y., Ji, F.H., Wang, X.Y., Zuo, J.L., Xu, Q.N., 2011a. Minocycline-induced reduction of brain-derived neurotrophic factor expression in relation to cancer-induced bone pain in rats. J. Neurosci. Res. Wang, L.N., Yao, M., Yang, J.P., Peng, J., Peng, Y., Li, C.F., Zhang, Y.B., Ji, F. H., Cheng, H., Xu, Q.N., Wang, X.Y., Zuo, J.L., 2011b. Cancer-induced bone pain sequentially activates the ERK/MAPK pathway in different cell types in the rat spinal cord. Mol. Pain 7, 48. Watkins, L.R., Wieseler-Frank, J., Milligan, E.D., Johnston, I., Maier, S.F., 2006. Chapter 22 Contribution of glia to pain processing in health and disease. Handb. Clin. Neurol. 81, 309–323. White, F.A., Sun, J., Waters, S.M., Ma, C., Ren, D., Ripsch, M., Steflik, J., Cortright, D.N., Lamotte, R.H., Miller, R.J., 2005. Excitatory monocyte chemoattractant protein-1 signaling is up-regulated in sensory neurons after chronic compression of the dorsal root ganglion. Proc. Natl. Acad. Sci USA 102, 14092–14097. White, F.A., Wilson, N.M., 2008. Chemokines as pain mediators and modulators. Curr. Opin. Anaesthesiol. 21, 580–585. White, F.A., Miller, R.J., 2010. Insights into the regulation of chemokine receptors by molecular signaling pathways: functional roles in neuropathic pain. Brain Behav. Immun. 24, 859–865. Yao, M., Yang, J.P., Wang, L.N., Cheng, H., Zhang, Y.B., Xu, Q.N., Wu, Y.W., 2008. Feasibility of establishment of rat model of bone cancer pain by using Walker 256 cells cultured in vitro or in vivo. Zhonghua Yi Xue Za Zhi. 88, 880–884. Zhang, J., De Koninck, Y., 2006. Spatial and temporal relationship between monocyte chemoattractant protein-1 expression and spinal glial activation following peripheral nerve injury. J. Neurochem. 97, 772–783. Zhang, J., Shi, X.Q., Echeverry, S., Mogil, J.S., De Koninck, Y., Rivest, S., 2007. Expression of CCR2 in both resident and bone marrowderived microglia plays a critical role in neuropathic pain. J. Neurosci. 27, 12396–12406. Zhuang, Z.Y., Gerner, P., Woolf, C.J., Ji, R.R., 2005. ERK is sequentially activated in neurons, microglia, and astrocytes by spinal nerve ligation and contributes to mechanical allodynia in this neuropathic pain model. Pain 114, 149–159. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110.