1. CX3CR1+ cells in the PNS play a key role in development of neuropathic pain in mice
Jianguo Cheng, MD, PhD, LiPing Liu, MD, PhD, Yan Yin, MD, Fei Li, MD, Zhen Hua, MD, PhD
Departments of Pain Management and Neurosciences, Cleveland Clinic, Cleveland, Ohio
AUA Poster # 1349
Introduction
The mechanisms of neuropathic pain are complex and far from clear. Neuroinflammation in both the central nervous system (CNS) and peripheral nervous system (PNS) has been specifically implicated (1-2). Fractalkine receptor (CX3CR1) is expressed constitutively in microglia and has been used as a specific marker for microglia in the CNS (3). It is a unique chemokine receptor that binds only to the chemokine, fractalkine (CX3CL1). We for the first time identified a unique population of CX3CR1+ cells in the PNS and investigated the role of this population of cells in the development of neuropathic pain by utilization of CX3CR1GFP knock-in mice, CX3CR1 knock-out mice, and chimeric mice with CNS or PNS CX3CR1 deficiency.
Methods
With IACUC approval, CX3CR1GFP/+ and CX3CR1GFP/GFP transgenic mice were used to induce neuropathic pain by chronic constrictive injury (CCI) of the sciatic nerve. Paw withdrawal thresholds were evaluated on post-surgical days 0, 7, 14, 21 and 28. The animals were sacrificed and perfused at these time intervals to collect samples of the sciatic nerve, DRG, and spinal cord of both sides for immunohistochemistry and flow cytometry examination of CX3CR1+ cells (4). We reconstituted irradiated CCR2RFP/+ or CX3CR1GFP/GFP mice with CX3CR1GFP/GFP or CCR2RFP/+ bone marrow cells to produce PNS CX3CR1 deficiency mice (CX3CR1GFP/GFP → CCR2RFP/+) or CNS CX3CR1 deficiency mice (CCR2RFP/+ → CX3CR1GFP/GFP) and used these mice to determine the role of CX3CR1+ cells the development of neuropathic pain. Statistical analyses were made using two-way analysis of variance followed by paired comparisons with Bonferroni corrections.
Results
CX3CR1 was expressed not only in microglia in the CNS but also in cells residing in the sciatic nerve and DRG in mice (Fig 1). The morphology of CX3CR1+ cells in the sciatic nerve and DRG was different from that of microglia in the spinal cord. These cells were positive for IBA1, a macrophage/microglia marker, and positive for CD45, a hematopoietic marker, suggesting this population of cells is a subtype of macrophages residing in the PNS. We further demonstrate that these cells were negative for NF-H (neuronal marker), myelin basic protein (MBP, marker for myelin), glutamine synthetase and Kir4.2 (markers for satellite cells), suggesting they were neither neurons nor Schwann cells, nor satellite cells. Immuno-Electronic microscopy confirmed that CX3CR1 was exclusively expressed on these cells. Interestingly, the number and morphology of CX3CR1+ cells were dramatically increased in the sciatic nerve and DRG, started from post-surgical day 3 and peaked at the day 14, in sync with hyperalgesia (Fig 2). Mice with CX3CR1 deficiency in both the CNS and PNS were resistant to the development of neuropathic pain (Fig 3). Mice with PNS CX3CR1 deficiency only (CX3CR1GFP/GFP → CCR2RFP/+) or CNS CX3CR1 deficiency only (CCR2RFP/+ → CX3CR1GFP/GFP) were partially resistant to the development of neuropathic pain (Fig 4-6). c
Results
CCI d
Fig 1. Distinct populations of CX3CR1+ cells in the sciatic nerve, dorsal root ganglion, and spinal cord. Scale bars: 10 μm for sciatic nerve and DRG; 50 μm for spinal cord.
Fig 2. Dynamic changes of CX3CR1+ cells in the sciatic nerve, DRG, and spinal cord after CCI. Scale bar: 50 μM for sciatic nerve and DRG; 200 μM for spinal cord.
Fig 3. CX3CR1 KO mice were resistant to the development of neuropathic pain after CCI of the sciatic nerve.
Fig 4. CNS CX3CR1 KO mice were partially resistant to neuropathic pain compared to chimeric control mice
Fig 5. PNS CX3CR1 KO mice were partially resistant to neuropathic pain compared to chimeric control mice
Fig 6. PNS CX3CR1+ cells contributed equally to the development of neuropathic pain, compared to microglia in the CNS.
Conclusions
We discovered a specific population of resident CX3CR1+ cells in the PNS (the sciatic nerve and DRG) which were morphologically different from blood CX3CR1+ cells and CNS CX3CR1+ microglia. PNS CX3CR1+ cells played a role in the development of neuropathic pain that was as important as microglia in the CNS. Further experiments will further clarify how this population of CX3CR1+ cells contribute to the initiation and/or maintenance of neuropathic pain.
Key References
Clark AK, et al. Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain. PNAS. 2007;104:
Staniland AA, et al. Reduced inflammatory and neuropathic pain and decreased spinal microglial response in fractalkine receptor (CX3CR1) knockout mice. J. Neurochemistry. 2010;114:
Verge GM, et al. Fractalkine (CX3CL1) and fractalkine receptor (CX3CR1) distribution in spinal cord and dorsal root ganglia under basal and neuropathic pain conditions. European J. Neurosci. 2004;20:
Liu L, et al. Isolation and flow cytometry analysis of inflammatory cells in the sciatic nerve and DRG after chronic constriction injury. J Neurosci. Methods, 2017, in press.
Acknowledgements: Supported by grants from the Department of Defense (DoD) and Cleveland Clinic Anesthesiology Institute.