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Abstract Background Though it is well-known that a high-salt diet (HSD) is associated with many chronic diseases, the effects of long-term high-salt intake on physiological functions and homeostasis remain elusive. In this study, we investigated whether and how an HSD affects mouse nociceptive thresholds, and myeloid cell trafficking and activation. Methods Healthy C57BL/6 male and female mice were fed an HSD (containing 4% NaCl in chow and 1% NaCl in water) from the time of weaning for 3 to 4 months. Circulating monocytes, nerve macrophages, spinal microglia, and associated inflammatory responses were scrutinized using flow cytometry, immunohistochemistry, and quantitative real-time polymerase chain reaction (qPCR) approaches. Mouse pain sensitivity to mechanical stimuli was monitored with von Frey tests along the experimental duration. Results Mice on an HSD have reduced mechanical thresholds. They feel more pain than those on a normal diet (ND), e.g., regular laboratory chow (0.3% NaCl in chow). An HSD induced not only a remarkable expansion of circulating monocytes, CCR2+Ly6Chi inflammatory monocytes in particular, but also an accumulation of CD11b+F4/80+ macrophages in the peripheral nerves and an activation of Iba-1+ spinal microglia. Replacing an HSD with a ND was unable to reverse the HSD-induced mechanical hypersensitivity or rescue the altered immune responses. However, treating HSD-fed mice with a chemokine receptor CCR2 antagonist effectively normalized the pain thresholds and immune cell profile in the periphery and spinal cord. An HSD failed to alter pain thresholds and myeloid cell activation in CCR2-deficient mice. Spinal microglial activation is required for HSD-induced mechanical hypersensitivity in male, but not in female mice. Conclusion Overall, this study provides evidence that an HSD has a long-term impact on physiological function. CCR2-mediated cellular response, including myeloid cell trafficking and associated inflammation, plays pivotal roles in salt-dietary modulation of pain sensitivity.

Introduction/Aim: Though several studies have shown that high salt diet (HSD) is related to the development of chronic diseases, the effect of long-term high-salt intake on the immune system and pain behavior remains elusive. Here, we aimed to investigate whether and how HSD affected mouse pain thresholds and innate immunity, especially myeloid cells. Methods: Healthy C57BL/6 male and female mice were fed with HSD (containing 4% NaCl in chow and 1% NaCl in water) right from weaning period (3-week after birth) for 1–3 months. Results: Behavioral analysis demonstrated that HSD-fed mice were hypersensitive to mechanical stimuli. Moreover, HSD induced an expansion of circulating monocytes, especially the proinflammatory monocyte (Ly6Chigh, CCR2+) subsets. HSD also increased the density of Iba-1+ microglia and the number of FcγII/III receptor (CD16/32) expressing cells in the lumbar spinal cord. Prolonged HSD feeding, meanwhile, resulted in increased infiltration of monocyte to the peripheral nerves, contributing to the monocyte-derived macrophage (CD11b+, F4/80+) population. Finally, shifting the HSD back to the regular laboratory chow (with 0.3% NaCl) cannot reverse the HSD-induced mechanical hypersensitivity myeloid cell expansion and polarization towards the inflammatory subsets in the periphery. However, treating HSD mice with CCR2 antagonist effectively normalized the pain threshold and myeloid cell profile in the peripheral blood and nerves. Discussion/Conclusions: Long term HSD feeding decreased mouse mechanical thresholds, in parallel with an expansion and polarization of myeloid cells towards inflammatory status. Such changes were not reversible when HSD-fed mice back to normal diet. However, blocking CCR2 signaling successfully attenuated HSD-induced hypersensitivity.

Introduction: Neuropathic pain is a debilitating condition. The importance of neuroimmune interactions in neuropathic pain has been evidenced by the involvement of different immune cells in peripheral and central sensitization of pathological pain. Macrophages and microglia are the most abundant immune cells activated in injured nerves and spinal cord, respectively. Several lines of evidence showed that macrophage/microglia survival, activation, proliferation, and differentiation require the involvement of macrophage-colony stimulating factor. In this study, we investigated whether blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling can be effective in relieving neuropathic pain. Materials and methods: Partial sciatic nerve ligation was performed in mice to induce neuropathic pain behavior. Mice were orally treated with a selective colony stimulating factor 1 receptor inhibitor, PLX5622, daily in both preventive (two days prior to surgery until D14 post-partial sciatic nerve ligation) and reversal paradigms (D28-D33 post-partial sciatic nerve ligation). Animal neuropathic pain behavior was monitored using von Frey hairs and acetone application. Phenotype of macrophages in injured nerves was analyzed at D3 and D33 post-injury using flow cytometry analysis. The effect of PLX5622 on microglia activation in lumbar spinal cord was further examined by immunohistochemistry using Iba-1 antibody. Results: Significant alleviation of both mechanical and cold allodynia was observed in PLX5622-treated animals, both in preventive and reversal paradigms. PLX5622 treatment reduced the total number of macrophages in injured nerves, it appears colony stimulating factor 1 receptor inhibition affected more specifically CD86+ (M1 like) macrophages. Consequently, the expression of various pro-inflammatory cytokines (TNF-α, IL-1β) was reduced. Microglia activation in dorsal horn of lumbar spinal cord following partial sciatic nerve ligation was significantly inhibited with PLX5622 treatment in both preventive and reversal paradigms. Conclusion: Macrophages in peripheral nerve and microglia in the spinal cord are required in the generation and maintenance of injury-associated neuropathic pain. Blocking macrophage-colony stimulating factor/colony stimulating factor 1 receptor signaling on these myeloid cells along the pain transmission pathway is an effective strategy to alleviate neuropathic pain. [ABSTRACT FROM AUTHOR]