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, 21 (5), 642-51

Role of the CX3CR1/p38 MAPK Pathway in Spinal Microglia for the Development of Neuropathic Pain Following Nerve Injury-Induced Cleavage of Fractalkine

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Role of the CX3CR1/p38 MAPK Pathway in Spinal Microglia for the Development of Neuropathic Pain Following Nerve Injury-Induced Cleavage of Fractalkine

Zhi-Ye Zhuang et al. Brain Behav Immun.

Abstract

Accumulating evidence suggests that microglial cells in the spinal cord play an important role in the development of neuropathic pain. However, it remains largely unknown how glia interact with neurons in the spinal cord after peripheral nerve injury. Recent studies suggest that the chemokine fractalkine may mediate neural/microglial interaction via its sole receptor CX3CR1. We have examined how fractalkine activates microglia in a neuropathic pain condition produced by spinal nerve ligation (SNL). SNL induced an upregulation of CX3CR1 in spinal microglia that began on day 1, peaked on day 3, and maintained on day 10. Intrathecal injection of a neutralizing antibody against CX3CR1 suppressed not only mechanical allodynia but also the activation of p38 MAPK in spinal microglia following SNL. Conversely, intrathecal infusion of fractalkine produced a marked p38 activation and mechanical allodynia. SNL also induced a dramatic reduction of the membrane-bound fractalkine in the dorsal root ganglion, suggesting a cleavage and release of this chemokine after nerve injury. Finally, application of fractalkine to spinal slices did not produce acute facilitation of excitatory synaptic transmission in lamina II dorsal horn neurons, arguing against a direct action of fractalkine on spinal neurons. Collectively, our data suggest that (a) fractalkine cleavage (release) after nerve injury may play an important role in neural-glial interaction, and (b) microglial CX3CR1/p38 MAPK pathway is critical for the development of neuropathic pain.

Figures

Figure 1
Figure 1
Spinal nerve ligation (SNL) induces CX3CR1 expression in the spinal cord. (a) Western blot analysis shows a sustained increase in the levels of CX3CR1 in the L5 spinal dorsal horn, compared with the control without injury. ERK2 was used as the loading control. (b) Quantification of CX3CR1 levels in the L5-spinal cord from Western blotting. CX3CR1 levels were normalized against corresponding loading control. *, P<0.05, **, P<0.01; compared with control, ANOVA followed by Fisher’s PLSD post-hoc comparison. (c) Immunofluorescence indicates that SNL increases CX3CR1 levels in the ipsilateral spinal cord (L5) 2 days after the injury. Scale, 100 μm.
Figure 2
Figure 2
SNL induces CX3CR1 expression in microglia of the spinal cord. (a–c) Double immunofluorescence shows that CX3CR1 (a, red) is colocalized with OX-42 (b, green), a microglial marker, in the medial superficial dorsal horn (laminae II–III), 2 days after SNL. c is the merge of a and b. Yellow fluorescence indicates a double labeling. Scale, 50 μm. (d, e) Immunofluorescence indicates an increase of CX3CR1 expression in the dorsal horn on the ipsilateral (injured) side 10 days after SNL. Scale, 50 μm. (f, g) Double staining indicates that CX3CR1 (red) is not colocalized with astrocytic marker GFAP (f, green), but colocalized with OX-42 (g, green) in the medial ipsilateral dorsal horn (laminae II–IV) on post-SNL day 10. Two singly stained images were merged. Scale, 50 μm.
Figure 3
Figure 3
Suppression of SNL-induced neuropathic pain by a neutralizing antibody against CX3CR1. A single injection of a neutralizing CX3CR1 antibody (10 μg, 20 μl) into lumbar CSF space, 30 min before surgery, decreases SNL-induced mechanical allodynia on post-surgery day 1 and day 2. Control serum (10 μg) has no effect. **, P<0.01, compared to corresponding serum control, unpaired t-test, n=6.
Figure 4
Figure 4
Suppression of SNL-induced p38 activation by a neutralizing antibody against CX3CR1. (a, b) Compared to sham control (a), SNL induces robust p38 activation in the dorsal horn on day 2, as indicated by an increase in the number of p-p38-IR cells (b). (c) A single intrathecal injection of a neutralizing CX3CR1 antibody (10 μg, 20 μl), 30 min before surgery, inhibits SNL-induced p38 activation 2 days after nerve injury. Images in a-c were taken from the medial superficial dorsal horn (laminae I–III). Scale, 50 μm. (d) Number of p-p38-IR cells in the medial superficial dorsal horn (laminae I–III) of SNL rats receiving CX3CR1 antibody or control serum. Animals were perfused for p-p38 staining 2 days after SNL. *, P<0.05, compared to corresponding control serum, n=4, unpaired t-test.
Figure 5
Figure 5
Activation of p38 in the spinal cord following intrathecal infusion of fractalkine. (a, b) Immunofluorescence indicates an increase in p-p38-IR cells in the dorsal horn 2 days after fractalkine infusion via an osmotic pump (3 ng/h). Scale, 50 μm. (c) High magnification image shows p-p38-IR cells in the dorsal horn (lamina III), with morphology of microglia. Scale, 25 μm. (d) Number of p-p38-IR cells in the medial laminae I–III of each spinal section following infusion of saline (1μl/h for 48 h) and fractalkine (3ng/μl/h for 48 h). **, P<0.01, compared to saline control, n=3, unpaired t-test.
Figure 6
Figure 6
Spinal infusion of fractalkines induces mechanical allodynia. Fractalkine or saline was infused for 2 days as in Fig 5. **, P<0.01, compared to saline control, n=5, unpaired t-test.
Figure 7
Figure 7
SNL produces cleavage of fractalkine in the injured dorsal root ganglion. (a) Western blot with a fractalkine antibody reveals two bands of fractalkine. The top band (band 1, ≈100 kDa, a membrane-bound form) almost disappears one day after nerve ligation. Band 2 (≈ 80 kDa) is equivalent to the positive control (20 ng of recombinant full-length fractalkine). Molecular weight is indicated on the left edge. (b) Quantification of the large fractalkine band (100 kDa) in the L5 DRG of SNL-injured rats after 1 day and time-matched sham control rats. Fractalkine levels were normalized against corresponding loading control.
Figure 8
Figure 8
Fractalkine does not produce acute effect on synaptic transmission in the spinal cord. (a) Patch clamp recording in lamina II neurons of the isolated spinal cord slices shows spontaneous excitatory synaptic current (sEPSC) before, during, and after bath application of fractalkine (20 ng/ml, 5 min). (b, c) High magnification shows sEPSC before (a) and after (b) fractalkine (20 ng/ml) application. (d, e) Effects of fractalkine on the frequency (d) and amplitude (e) of mEPSC. Data are presented as the ratio of pre-treatment value. P>0.05, paired t-test, n=3.

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