During neuropathic pain, sensory neurons in the spinal cord communicate with surrounding glial cells, including astrocytes and microglia. A number of chemokines are well recognized to contribute to this process, with most studies focusing on the effects of these secreted proteins on microglia. Now, a new study pinpoints a role for another chemokine—this time acting directly on astrocytes—in nerve injury-induced pain. Researchers led by Yong-Jing Gao, Nantong University, China, report that spinal neurons release a chemokine, C-X-C motif chemokine 13 (CXCL13), which binds to its receptor, CXCR5, on astrocytes to cause mechanical allodynia and thermal hyperalgesia in the spinal nerve ligation (SNL) model of neuropathic pain in mice.
“The novelty of this study resides in the identification of CXCL13 expression in dorsal horn neurons and CXCR5 in astrocytes,” wrote Marzia Malcangio, King’s College London, UK, in an email to PRF. Still, Malcangio emphasized that CXCL13/CXCR5 signaling is but one of many ways by which spinal neurons and glia communicate in neuropathic pain. “Several cytokines and chemokines are involved in this type of mechanism,” she added, something that previous studies of microglia and pain have illustrated.
The findings were published January 11 in The Journal of Clinical Investigation.
Zeroing in on CXCL13
Communication between neurons and microglia, including a role for chemokines, has long been a focus of efforts to understand neuropathic pain (see PRF related news stories here and here). More and more, it has been recognized that astrocytes are important players in neuropathic pain, too, and can also serve in this role via chemokine signaling (see PRF related news story here).
In the new study, to further understand the role of chemokines and astrocytes in neuropathic pain, lead authors Bao-Chun Jiang, De-Li Cao, Xin Zhang, Zhi-Jun Zhang, and colleagues began by asking which chemokine genes were most upregulated in neuropathic pain. To this end, the authors used a gene expression microarray 10 days after mice underwent SNL or sham surgery. They found that among dozens of detectable chemokine genes in the spinal cord, the gene for CXCL13 was the most highly upregulated of all of them.
Further analysis of the time course of CXCL13 gene expression revealed an increase as early as one day after SNL, which persisted for at least 21 days. Therefore, “I felt CXCL13 would be an important chemokine in regulating neuropathic pain,” Gao wrote PRF.
To pinpoint which cell types in the spinal cord expressed CXCL13, the authors combined in situ hybridization and immunohistochemistry to visualize CXCL13 messenger RNA (mRNA) and cell type-specific markers. In naïve mice, CXCL13 colocalized with NeuN, a neuronal marker, but not with glial fibrillary acidic protein (GFAP) or ionized calcium-binding adapter molecule (IBA-1), markers of astrocytes or microglia, respectively. By using immunohistochemistry, they found that, in SNL animals, CXCL13 was expressed at much higher levels in the dorsal horn ipsilateral to the nerve injury compared to naïve animals. Thus, nerve injury induced CXCL13 expression specifically in spinal neurons.
Next, the authors tested whether the induction of CXCL13 contributed to pain-like behaviors that result from SNL. Mice were injected in the dorsal horn with either a lentivirus expressing short hairpin RNA (shRNA) to knock down CXCL13 expression or with a control virus. The animals were then assessed for mechanical allodynia using von Frey filaments and heat hyperalgesia using the Hargreaves assay. For both measures, mice that received the shRNA displayed attenuated pain sensitivity compared to controls. This suggested that CXCL13 played an important role in the development of allodynia and hyperalgesia, although the cell type to which CXCL13 signaled remained unclear.
Finally, in an effort to understand how nerve injury induced CXCL13 expression, the investigators found that expression of a microRNA, called miR-186-5p, was downregulated after SNL. Furthermore, overexpression of miR-186-5p in the dorsal horn using a lentivirus decreased expression of CXCL13 in spinal neurons and alleviated both mechanical allodynia and heat hyperalgesia in the SNL animals. This hinted that the low levels of miR-186-5p observed after nerve injury contributed to induction of CXCL13 in neurons, resulting in pain-like behaviors.
From neuron to astrocyte
Since CXCR5 is thought to be the only receptor for CXCL13, the authors next examined whether its expression was also upregulated after nerve injury. CXCR5 mRNA was, in fact, increased in the dorsal horn one day following SNL and remained elevated for at least 21 days. Moreover, immunostaining showed that CXCR5 expression was low in the dorsal horn of naïve animals but increased after SNL. In contrast to CXCL13, however, CXCR5 primarily colocalized with GFAP, suggesting that CXCL13 serves as a signal from neurons to astrocytes in neuropathic pain.
To further test that idea, Gao and colleagues compared astrocyte activation, using GFAP immunoreactivity, between wild-type and CXCR5 knockout mice after SNL. While nerve injury enhanced GFAP immunoreactivity in the dorsal horn of wild-type mice, it did so to a lesser degree in CXCR5-knockout animals, implying that CXCR5 played a role in recruitment of astrocytes after nerve injury. Similar results were observed when assessing microglia activation with IBA-1 staining. Gao found this surprising, as “many publications have demonstrated that microglia activation precedes astrocyte activation in neuropathic pain.” Therefore, “we had thought that microglial activation would not be affected in CXCR5-knockout mice after SNL,” she added.
Similar to what was observed with knockdown of CXCL13, CXCR5 knockout mice displayed decreased mechanical allodynia and heat hyperalgesia after SNL compared to wild-type animals. Consistent with this finding, pain sensitivity was also reduced by knocking down CXCR5 using virally expressed shRNA in the dorsal horn, compared to mice that received a control virus. Thus, spinal CXCR5 appeared important for pain sensitivity after nerve injury.
Lastly, to link CXCL13 to CXCR5 in neuropathic pain, the authors intrathecally injected CXCL13 or vehicle into either wild-type or CXCR5 knockout mice. Although CXCL13 was sufficient to trigger mechanical allodynia and heat hyperalgesia in wild-type mice for at least 24 hours, these effects were reduced in CXCR5 knockout animals, suggesting that CXCL13 can cause pain-like behavior, at least in part, via CXCR5.
Overall, “this study provides evidence for a role of CXCL13 and CXCR5 in mediating neuron-glia communication in the spinal cord during chronic pain,” wrote Malcangio.
Gao and colleagues are now pursuing whether such signaling could be a viable target to treat neuropathic pain and other types of chronic pain in patients. In the current study, the authors found that both CXCL13 and CXCR5 were present in spinal cord tissue from healthy human donors. Gao also told PRF that her group has preliminary findings showing that CXCL13 levels in cerebrospinal fluid are elevated in patients with post-herpetic neuralgia.
Because the neuron-to-astrocyte signaling identified by the current study is embedded in a larger network of pain signaling involving multiple pathways, targeting CXCL13-CXCR5 signaling alone will probably not be sufficient to fully treat neuropathic pain. “It is unlikely that neuropathic pain would be reversed completely by targeting a single pathway in neuron-glia communication,” Malcangio said.
Matthew Soleiman is a neuroscientist-turned-science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.
Image credit: Jiang et al., reproduced with permission of the American Society for Clinical Investigation