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MicroRNA-124 as a Novel Treatment for Persistent Hyperalgesia

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MicroRNA-124 as a Novel Treatment for Persistent Hyperalgesia

Hanneke L D M Willemen et al. J Neuroinflammation.

Abstract

Background: Chronic pain is often associated with microglia activation in the spinal cord. We recently showed that microglial levels of the kinase G protein-coupled receptor kinase (GRK)2 are reduced in models of chronic pain. We also found that mice with a cell-specific reduction of around 50% in GRK2 level in microglia/macrophages (LysM-GRK2+/- mice) develop prolonged inflammatory hyperalgesia concomitantly with ongoing spinal microglia/macrophage activation. The microRNA miR-124 is thought to keep microglia/macrophages in brain and spinal cord in a quiescent state. In the present study, we investigated the contribution of miR-124 to regulation of hyperalgesia and microglia/macrophage activation in GRK2-deficient mice. In addition, we investigated the effect of miR-124 on chronic inflammatory and neuropathic pain in wild-type (WT) mice.

Methods: Hyperalgesia was induced by intraplantar IL-1β in WT and LysM-GRK2+/- mice. We determined spinal cord microglia/macrophage miR-124 expression and levels of pro-inflammatory M1 and anti-inflammatory M2 activation markers. The effect of intrathecal miR-124 treatment on IL-1β-induced hyperalgesia and spinal M1/M2 phenotype, and on carrageenan-induced and spared nerve injury-induced chronic hyperalgesia in WT mice was analyzed.

Results: Transition from acute to persistent hyperalgesia in LysM-GRK2+/- mice is associated with reduced spinal cord microglia miR-124 levels. In our LysM-GRK2+/- mice, there was a switch towards a pro-inflammatory M1 phenotype together with increased pro-inflammatory cytokine production. Intrathecal administration of miR-124 completely prevented the transition to persistent pain in response to IL-1β in LysM-GRK2+/- mice. The miR-124 treatment also normalized expression of spinal M1/M2 markers of LysM-GRK2+/- mice. Moreover, intrathecal miR-124 treatment reversed the persistent hyperalgesia induced by carrageenan in WT mice and prevented development of mechanical allodynia in the spared nerve injury model of chronic neuropathic pain in WT mice.

Conclusions: We present the first evidence that intrathecal miR-124 treatment can be used to prevent and treat persistent inflammatory and neuropathic pain. In addition, we show for the first time that persistent hyperalgesia in GRK2-deficient mice is associated with an increased ratio of M1/M2 type markers in spinal cord microglia/macrophages, which is restored by miR-124 treatment. We propose that intrathecal miR-124 treatment might be a powerful novel treatment for pathological chronic pain with persistent microglia activation.

Figures

Figure 1
Figure 1
MicroRNA-124 and CCAAT-enhancer-binding protein (C/EBP)-α expression after intraplantar interleukin (IL)-1β in wild-type (WT) and LysM-G protein–coupled receptor kinase (GRK)2+/−mice. Control WT and LysM-GRK2+/− mice received an intraplantar injection of 1 ng IL-1β. (A) Percentage decrease in heat withdrawal latency after the injection in control WT and LysM-GRK2+/− mice (n = 12). At baseline (n = 9) and 24 hours (n = 6) after IL-1β injection, lumbar (L2 to L5) spinal cord microglia were isolated to determine (B) miR-124 expression (normalized for small nucleolar (sno)RNA55) and (C) the lumbar and thoracic mRNA expression of C/EBP-α normalized for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin) in control WT (n = 6) and LysM-GRK2+/− (n = 6) mice. n = One sample contains isolated microglia from four mice. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2
M1 and M2 phenotype in spinal cord after intraplantar IL-1β. Wild-type (WT) and LysM-G protein–coupled receptor kinase (GRK)2+/− mice received an intraplantar injection of 1 ng IL-1β. At 15 hours after injection, spinal cord was collected, and frozen sections of (A) lumbar spinal cord (L2 to L5) and as control (B) thoracic spinal cord (T6 to T10) were stained for M1 (CD16/32) and M2 (CD206 and arginase-I) phenotypic markers. A representative example of M1 and M2 staining in the dorsal horn of one of the four mice per group is displayed. Scale bar indicates 20 μm. (C) Quantification of microglia/macrophages expressing M1 and M2 phenotypic markers in spinal cord from WT and LysM-GRK2+/− mice. Expression was quantified in approximately 10 to 15 dorsal horns of spinal cords per group (4 mice per group). The level of expression in the lumbar or thoracic area from control WT mice was set at 100%. Data are expressed as means ± SEM. **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
Gene expression of M1-associated and M2-associated genes in spinal microglia after intraplantar injection of interleukin (IL)-1β. Wild-type (WT) and LysM-G protein–coupled receptor kinase (GRK)2+/− mice received an intraplantar injection of 1 ng IL-1β into the hind paws. At 24 hours after injection lumbar (L2 to L5) spinal cord and (as a control) thoracic (T6 to T10) spinal cord was collected from WT and LysM-GRK2+/− mice, and microglia were isolated for analysis of mRNA expression by quantitative RT-PCR of (A) IL-1β, (B) inducible nitrous oxide synthase (iNOS) and (C) transforming growth factor (TGF)-β (n = 6 quantiatitve PCR samples per group; one sample contains isolated microglia from 4 mice). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01.
Figure 4
Figure 4
Effect of microRNA (miR)-124 treatment on interleukin (IL)-1β-induced hyperalgesia. (A) LysM-G protein–coupled receptor kinase (GRK)2+/− and (B) wild-type (WT) mice received an intrathecal injection of 20, 50 or 100 ng miR-124 per mouse or 100 ng negative control miRNA 1 day before intraplantar injection of 1 ng IL-1β and the percentage change in heat-withdrawal latency was determined (n = 4 to 10 per group). Data are expressed as means ± SEM. **P < 0.01, ***P < 0.001 for 50 ng miR-124 versus control miRNA; #P < 0.05, ###P < 0.001 for 100 ng miR-124 versus control miRNA.
Figure 5
Figure 5
MicroRNA-124 treatment influences the M1/M2 balance and abolishes the genotype differences after interleukin (IL)-1βinjection. Mice received an intrathecal injection of 100 ng miR-124 the day before intraplantar injection of 1 ng IL-1β. At 15 hours after IL-1β injection, the spinal cord was collected, and frozen sections of lumbar spinal cord (L2 to L5) were stained for M1 and M2 phenotypic markers. M1 and M2 marker expressions were compared in wild-type (WT) versus LysM-G protein–coupled receptor kinase (GRK)2+/− mice, and expression was quantified in 18 dorsal and 18 lumbar spinal-cord sections per group from 4 mice per group. The level of expression in IL-1β-treated WT mice was set at 100%. Data are expressed as means ± SEM.
Figure 6
Figure 6
Effect of microRNA(miR)-124 treatment on carrageenan-induced persistent hyperalgesia in wild-type (WT) mice. Wild-type (WT) mice received an intraplantar injection of 20 μl 2% λ-carrageenan. At day 6, mice were still hyperalgesic. At this time point, mice received an intrathecal injection of 100 ng miR-124 or control miRNA, and the percentage change in heat withdrawal latency was determined (miR-124 n = 8; control miRNA n = 4). Data are expressed as means ± SEM. ***P < 0.001.
Figure 7
Figure 7
Effect of miR-124 treatment on SNI-induced mechanical allodynia in WT mice. Spared nerve injury was performed on WT mice and mice were treated daily with 100 ng miR-124 or control miRNA intrathecally (n = 7 per group). Mechanical allodynia was monitored in (A) ipsi- and (B) contralateral paw and is depicted as force needed for 50% withdrawal determined in accordance with the up and down method. Arrows indicate miRNA injection. (C) Spontaneous locomotor activity in an open field was monitored at day three after SNI to control for a potential effect of miR-124 treatment on motor function. Data are expressed as mean ± SEM. *** P < 0.01.

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