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. 2016 Jul;55:82-92.
doi: 10.1016/j.bbi.2015.11.007. Epub 2015 Nov 11.

Microglial P2Y12 Receptors Regulate Microglial Activation and Surveillance During Neuropathic Pain

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Free PMC article

Microglial P2Y12 Receptors Regulate Microglial Activation and Surveillance During Neuropathic Pain

Nan Gu et al. Brain Behav Immun. .
Free PMC article

Abstract

Microglial cells are critical in the pathogenesis of neuropathic pain and several microglial receptors have been proposed to mediate this process. Of these receptors, the P2Y12 receptor is a unique purinergic receptor that is exclusively expressed by microglia in the central nervous system (CNS). In this study, we set forth to investigate the role of P2Y12 receptors in microglial electrophysiological and morphological (static and dynamic) activation during spinal nerve transection (SNT)-induced neuropathic pain in mice. First, we found that a genetic deficiency of the P2Y12 receptor (P2Y12(-/-) mice) ameliorated pain hypersensitivities during the initiation phase of neuropathic pain. Next, we characterised both the electrophysiological and morphological properties of microglia in the superficial spinal cord dorsal horn following SNT injury. We show dramatic alterations including a peak at 3days post injury in microglial electrophysiology while high resolution two-photon imaging revealed significant changes of both static and dynamic microglial morphological properties by 7days post injury. Finally, in P2Y12(-/-) mice, these electrophysiological and morphological changes were ameliorated suggesting roles for P2Y12 receptors in SNT-induced microglial activation. Our results therefore indicate that P2Y12 receptors regulate microglial electrophysiological as well as static and dynamic microglial properties after peripheral nerve injury, suggesting that the microglial P2Y12 receptor could be a potential therapeutic target for the treatment of neuropathic pain.

Keywords: 2-Photon imaging; Electrophysiology; Microglia; Neuropathic pain; P2Y12 receptor; Surveillance.

Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Microglial P2Y12 Receptors Are Upregulated to Promote Neuropathic Pain
A–B, Representative low and high magnification confocal images of both the ipsilateral and contralateral dorsal horns of the spinal cord at post-operative day (POD) 7 following spinal nerve transection (SNT). Microglia are shown in green in CX3CR1GFP/+ mice in both WT and P2Y12−/− tissues and P2Y12 immunoreactivity is shown in red. Scale bar is 200 µm and 20 µm for lower and higher magnification images, respectively. C, Quantified P2Y12 immunoreactivity is higher in the ipsilateral compared to the contralateral dorsal horn in WT mice but remains absent in the dorsal horn of P2Y12−/− mice at POD 7. n=4 mice per group. Data are shown as mean ± SEM. *** P < 0.001. D–E, Pain hypersensitivities including thermal hyperalgesia (D) and mechanical allodynia (E) are reduced in P2Y12−/− mice compared to WT mice. n=7–8 per group. Data are shown as mean ± SEM. *** P < 0.001.
Figure 2
Figure 2. P2Y12 Receptors Limit Microglial Electrophysiological Activation during Neuropathic Pain
A–B, Representative tracings showing (A) and quantified summaries of (B) outward currents in response to ATP (1mM) puff application to microglia from sham and SNT-surgery WT and P2Y12−/− mice. n=3–5 cells per group. Data are shown as mean ± SEM. * P < 0.05; *** P < 0.001. C–D, Representative tracings showing (C) and quantified summaries of (D) depolarization steps from −100mV to 80mV in microglia from sham and SNT-surgery WT and P2Y12−/− mice. n=10 cells for WT Sham group, n=17 cells for WT POD3 group, n=3 cells for P2Y12−/− Sham group, n=11 cells for P2Y12−/− POD3 group. Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; *** P < 0.001 compared with WT POD 3; ###P < 0.001 compared with WT Sham.
Figure 3
Figure 3. P2Y12 Receptors Limit Microglial Morphological Activation during Neuropathic Pain
A, Representative two-photon z-stack images of GFP-expressing microglia in the spinal cord dorsal horn of sham (left) ipsilateral (left and right center) and contralateral (right) dorsal horn from WT (top) and P2Y12−/− (bottom) tissue slices. Scale bar is 200 µm. B, Representative raw (green) and transformed skeletal (gray) images from sham (left) ipsilateral (left and right center) and contralateral (right) dorsal horn from WT (top) and P2Y12−/− (bottom) tissues. Scale bar is 20 µm. C, Quantification of microglial process length in WT sham as well POD 3 and 7 following SNT surgery showing progressive shortening of process lengths. n=6–9 slices per group (from 3 mice). Data are shown as mean ± SEM. *** P < 0.001. D, Quantification of microglial process length in WT and P2Y12−/− microglial at POD 7 following SNT surgery in WT and P2Y12−/− tissues. n=8–11 slices per group (form 4 mice). Data are shown as mean ± SEM. *** P < 0.001. E–F, Sholl (E) and endpoint voxel (F) analysis of microglia from WT and P2Y12−/− tissues in the sham and at POD 7 following SNT surgery. n=14–25 cells per group from 5–7 mice for Sholl analysis and n=8–11 slices per group from 4 mice for endpoint voxel analysis. Data are shown as mean ± SEM. n.s., no significance. * P < 0.05; *** P < 0.001
Figure 4
Figure 4. P2Y12 Receptors Limit Microglial Dynamics during Neuropathic Pain
A–F, Representative difference images used to quantify microglial motility indices of microglia from WT mice in vivo (A–B) WT slices (C–D) and P2Y12−/− slices (E–F) under sham conditions (left), and POD 7 following SNT surgery in the ipsilateral dorsal horn. Scale bar is 25 µm. G–H, Quantification of microglial motility indices from WT and P2Y12−/− slices during 15 minute long imaging sessions represented as a summary through time (G) as well as an average for each condition (H). n=4–6 mice per group for in vivo images and n=6–11 slices from 3–6 mice per group for slices images. Data are shown as mean ± SEM. n.s., no significance. *** P < 0.001.

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