The Mechanisms Of CXCL1in Spinal Cord Astrocytes Underlying Neuropathic Pain

In recent years, it has been increasingly recognized that besides neurons, the glialcells(astrocytes and microglia)in the CNS also play a critical role in the development andmaintenance of neuropathic pain. Reactive glial cells contribute to the immunoreaction andregulate the function of neurons through producing pro-inflammatory substances. Amongthese substances, the cytokines and chemokines have been implicated to play a role inchronic pain. In this study, we investigated the effect of CXCL1in the spinal cord onneuropathic pain and its mechanisms following spinal nerve ligation(SNL)in mice.Methods1. The neuropathic pain model was established by ligation of the left L5spinal nervein mice.2. The mechanical allodynia and heat hyperalgesia were evaluated by behavioral test.3. The expression of CXCL1and TNF-α mRNA was detected by Real-time PCR.4. Immunofluorescence staining was used to detect the expression of CXCL1, GFAP,OX-42, IBA1, CXCR2, pERK, and c-Fos in the spinal cord. The distribution of CXCL1-and CXCR2-IR cells was examined by immunofluorescence double-staining.5. The changes of CXCR2protein were detected by western blot.6. Combination of ISH with immunofluorescence labeling was used to identify thedistribution of CXCL1mRNA.7. To mimic the role of astrocytes in neuroinflammation in vivo, the culturedastrocytes were treated with TNF-α. The effect of MAPK on the CXCL1expression ofastrocytes induced by TNF-α was investigated by using JNK inhibitor SP600125, MEKinhibitor U0126, and p38inhibitor SB203580.8. Intrathecal injection of CXCL1neutralizing antibody to observe the effect onSNL-induced mechanical allodynia and heat hyperalgesia.Results:1. The surgery of SNL produced no effect on the growth of body weight and motor ability. Shortly after the ligation on L5spinal nerve, mice developed neuropathic pain inthe ipsilateral hindpaws. The mechanical allodynia and thermal hyperalgesia weredeveloped at day1and lasted for more than21days after SNL.2. Real-time PCR assay shows the expression of CXCL1mRNA in spinal cordincreased significantly at3d after SNL(P<0.05vs. na ve spinal cord).3. Immunofluorescence staining shows that SNL induced a marked increase ofCXCL1-IR cells in the ipsilateral side of L5spinal cord at3,10and21days after SNL(P<0.05vs. na ve spinal cord). The most significant increase of CXCL1-IR cells wasobserved in SNL animals at10d(P<0.001vs. na ve and sham10d spinal cord). ELISAassay shows the expression of CXCL1protein in L5spinal cord was lower in naive groupthan SNL3d and10d groups. The content of CXCL1protein is approximate7pg/ml,11pg/ml and15pg/ml in na ve, SNL3d and10d L5spinal cord, respectively.4. Immunofluorescence double-staining shows that CXCL1is colocalized with GFAP,a marker for astrocytes, but not with NeuN, a marker for neurons or OX-42, a marker formicroglia. Double staining with in situ hybridization and immunofluorescence also showsthe colocalization of CXCL1mRNA and GFAP in the ipsilateral spinal cord after SNL.5. Immunofluorescence staining for GFAP and IBA1show the astrocytes andmicroglia in L5spinal cord were activated after SNL. Pearson's correlation analysis showsthe increase of CXCL1was correlated with the activation of astrocytes after SNL.6. The increase expression of TNF-α mRNA was earlier than the change of CXCL1mRNA after SNL. The expression of TNF-α mRNA in SNL1d spinal cord had increasedabout8.8times as great as that in na ve spinal cord, but the marked increase of CXCL1mRNA was observed until at3day after SNL. Intraperitoneal injection of soluble TNFreceptor (Etanercept) prevented SNL-induced pain hypersensitivity and decreasedSNL-induced CXCL1mRNA upregulation. It suggests that TNF-α may be the motivationof the CXCL1mRNA expression afte SNL.7. Intrathecal application of TNF-α, induced upregulation of CXCL1expression inthe spinal cord at1and3h after injection(P<0.05vs. na ve spinal cord). Statistic analysisof CXCL1-IR shows that3h after intrathecal injection of TNF-α, the intensity ofCXCL1-IR was increased in the dorsal horn of spinal cord(P<0.01vs. na ve spinal cord).Immunofluorescence double-staining shows the CXCL1-IR was colocalized with the astrocytic marker GFAP in the dorsal horn of spinal cord after TNF-α intrathecal injection.8. TNF-α(10ng/ml)could induce upregulation of CXCL1expression in culturedastrocytes. The JNK-inhibitor SP600125inhibited TNF-α(10ng/ml,1h)-induced CXCL1upregulation at the doses of10,20,50μM. However, the MEK-inhibitor U0126(20μM)or p38MAPK-inhibitorsSB203580(10,20,50μM)could not effectively inhibit theupregulation of CXCL1expression that induced by TNF-α.9. Intrathecal injection CXCL1neutralizing antibody(1or4μg)could dose-dependently elevate the paw withdrawal threshold and prolong the paw withdrawal latencyat10d after SNL.10. Spinal injection of CXCL1peptide(10or100ng)induced a dose-dependent heathyperalgisa in na ve mice. It also induced ERK activation in the neurons of superficial(Ⅰ-Ⅱ), but not the deep(Ⅲ-Ⅴ)laminae of spinal cord and c-Fos upregulation in neuronsof spinal cord dorsal horn(Ⅰ-Ⅴlaminae).Immunofluorescence double staining shows thatthe major receptor of CXCL1, CXCR2was expressed in neurons of the spinal cord.11. The number of CXCR2-IR neurons was increased in the dorsal horn of spinal cordat3d after SNL. Western blot shows the expression of CXCR2protein in SNL3d wasincreased3.4fold as much as that in na ve (P<0.05). Intrathecal application of CXCR2antagonist (SB225002),3d after SNL could elevate the paw withdrawal threshold, andmaintain for at least1h. It also could prolong the paw withdrawal lantency, and maintainfor at least3h.Conclusion1. Tight ligation of spinal nerves(SNL)in mice can induce a long-lasting hyperalgesia(mechanical allodynia and heat hyperalgesia). The SNL model is a reliable and stableanimal model for peripheral neuropathic pain.2. The mRNA and protein of CXCL1in spinal cord can be increased after SNL. TheCXCL1was primarily expressed by astrocytes.3. TNF-α in spinal cord can be upregulated by SNL. TNF-α upregulates CXCL1expression via JNK pathway in astrocytes.4. Spinal injection of CXCL1can induce heat hyperalgesia, activate ERK andincrease expression of c-Fos in the spinal cord dorsal horn. 5. SNL can upregulate CXCR2, the primary receptor of CXCL1in spinal cord. TheCXCR2was primarily expressed by neurons.6. Inhibiting the action of CXCL1and CXCR2at the level of spinal cord canattenuate the neuropathic pain.7. Under neuropathic pain, CXCL1and CXCR2interact between astrocytes andneurons, and contribute to central sensitization and neuropathic pain.