1. Volume 15, Issue 5, Pages 516-526 (May 2014)
Contribution of Chemokine CCL2/CCR2 Signaling in the Dorsal Root Ganglion and Spinal Cord to the Maintenance of Neuropathic Pain in a Rat Model of Lumbar Disc Herniation 
Xiang Zhu, Su Cao, Ming-Di Zhu, Jin-Qian Liu, Jun-Jie Chen, Yong-Jing Gao 
The Journal of Pain 
Volume 15, Issue 5, Pages (May 2014)
DOI: /j.jpain
Copyright © 2014 American Pain Society Terms and Conditions

2. Figure 1
Time course of NP-induced mechanical allodynia and heat hyperalgesia in rats. NP application induced rapid (<1 day) and persistent (>28 days) mechanical allodynia (A). However, the heat hyperalgesia developed later (7 days) and recovered earlier (21 days) than mechanical allodynia (B). *P < .05, **P < .01, ***P < .001 versus sham. BL, baseline. Two-way ANOVA followed by Bonferroni test. n = 5 rats per group.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

3. Figure 2
NP application induces CCL2 upregulation in DRG neurons. CCL2 mRNA was increased in the DRG after NP application, but not in sham animals (A). CCL2 protein was also increased at 5 days and 10 days after NP (B–E). Double staining showed that CCL2 was colocalized with CGRP (F), IB4 (G), and NF200 (H). *P < .05,**P < .01, ***P < .001 versus naïve, #P < .05, ###P < .001 versus sham. Student's t-test. n = 5 for RT-PCR, n = 4 for immunohistochemistry.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

4. Figure 3
NP application induces CCR2 upregulation in DRG neurons. CCR2 mRNA was increased at 1 day, peaked at 10 days, and was maintained at 21 days (A). CCR2 was constitutively expressed in naïve DRG (B, C) and increased at 5 days (B) and 10 days after NP (D) or sham operation (E). At 10 days in NP group, CCR2 were colocalized with MAP2 (F) and ED-1 (G), but not with GFAP (H). *P < .05,**P < .01, ***P < .001 versus naïve, #P < .05, ##P < .01 versus sham. Student's t-test. n = 5 for RT-PCR, n = 4 for immunohistochemistry.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

5. Figure 4
NP application induces the increase of CCL2 mRNA and protein in activated astrocytes in the spinal cord. CCL2 mRNA was increased from 1 day to 21 days (A). CCL2-IR was also increased at 5 days, 10 days, and 21 days (B–F). Double staining showed that CCL2 was expressed in GFAP-positive astrocytes (G), but not in NeuN-positive neurons (H), or OX-42-positive microglia (I). Western blot showed that GFAP expression was increased from 1 day to 7 days (J). Immunostaining showed that GFAP-IR was mild in the sham-operated animal and increased in the NP animals at 10 days after surgery (K). *P < .05, **P < .01, ***P < .001 versus naïve, ###P < .001 versus sham. Student's t-test. n = 5 for RT-PCR, n = 4 for Western blot, and n = 5 for immunohistochemistry.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

6. Figure 5
NP application induces the increase of CCR2 mRNA and protein in spinal neurons. CCR2 mRNA was increased from 3 days to 10 days (A). CCR2-IR was also increased at 5 days and 10 days, but not at 21 days (B–F). Double staining showed that CCR2 was predominantly expressed in NeuN-positive neurons (G–I). *P < .05, ***P < .001 versus naïve, ###P < .001 versus sham. Student's t-test. n = 5 for PCR, n = 5 for immunohistochemistry.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

7. Figure 6
Intrathecal injection of CCR2 antagonist RS attenuates NP-induced pain hypersensitivity. Intrathecal injection of RS at the dose of 25 μg attenuated mechanical allodynia (A) and heat hyperalgesia (B) at 10 days after NP application. RS (10 μg) had no effect on either mechanical allodynia or heat hyperalgesia at 10 days (A, B). Intrathecal injection of RS (25 μg) did not affect mechanical allodynia when it was injected at 1 day, but attenuated mechanical allodynia when it was injected at 3 days (C). *P < .05, **P < .01, versus vehicle. BL, baseline. Two-way ANOVA followed by Bonferroni test. n = 6 rats per group.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions

8. Figure 7
Schematic shows how CCL2 and CCR2 in the DRG and spinal cord regulate NP-induced pain. In the DRG, NP application induces CCL2 increase in neurons and CCR2 increase in neurons and macrophages. CCL2 may act on CCR2 in neurons to increase neuronal excitability and in macrophages to induce their infiltration into the DRG. In the spinal cord, NP induces CCL2 and CCR2 increase in astrocytes and neurons, respectively. CCL2 can also be released from primary afferents. The released CCL2 acts on CCR2 in neurons and increases neuronal excitability or induces the expression of late-responses genes that further contribute to the maintenance of chronic pain.
The Journal of Pain  , DOI: ( /j.jpain )
Copyright © 2014 American Pain Society Terms and Conditions