Arachidonic acid containing phosphatidylcholine increases due to microglial activation in ipsilateral spinal dorsal horn following spared sciatic nerve injury

Abstract

Peripheral nerve injury induces substantial molecular changes in the somatosensory system that leads to maladaptive plasticity and cause neuropathic pain. Understanding the molecular pathways responsible for the development of neuropathic pain is essential to the development of novel rationally designed therapeutics. Although lipids make up to half of the dry weight of the spinal cord, their relation with the development of neuropathic pain is poorly understood. We aimed to elucidate the regulation of spinal lipids in response to neuropathic peripheral nerve injury in mice by utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry, which allows visualization of lipid distribution within the cord. We found that arachidonic acid (AA) containing [PC(diacyl-16:0/20:4)+K]+ was increased temporarily at superficial ipsilateral dorsal horn seven days after spared nerve injury (SNI). The spatiotemporal changes in lipid concentration resembled microglia activation as defined by ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry. Suppression of microglial function through minocycline administration resulted in attenuation of hypersensitivity and reduces [PC(diacyl-16:0/20:4)+K]+ elevation in the spinal dorsal horn. These data suggested that AA containing [PC(diacyl-16:0/20:4)+K]+ is related to hypersensitivity evoked by SNI and implicate microglial cell activation in this lipid production.

Fig 1

(A) Differential distribution of PCs in the spinal cord section obtained from 7 days after SNI mice. PCs were visualized as ion image of spinal cord section. PCs with identical FA compositions at the sn-1 position are placed horizontally, while those with identical FA compositions at the sn-2 position are arranged vertically. [PC(diacyl-16:0/20:4)+K]⁺ showed specific increase in the ipsilateral dorsal horn compared with the contralateral side (arrowheads). The other PCs showed no significant elevation in the ipsilateral side. Scale bar: 1mm. (B~E) Statistical analysis for [PC(diacyl-16:0/20:4)+K]⁺ distribution in the spinal dorsal horn section at 7 days after the initial operation. No changes were observed in sham group (B, C). After SNI, ion signal intensities significantly increased in the ipsilateral dorsal horn compared with the contralateral side (D, E). This increase was present in every animal of both groups. *p<0.05 Welch’s t test. (n = 4 for both SNI and sham). All error bars represent SEM. Scale bar: 500μm

Fig 2. The changes of microglia and astrocyte in the spinal cord sections after SNI in comparison with [PC(diacyl-16:0/20:4)+K]⁺.

The intensity of Iba-1 in the ipsilateral dorsal horn increased from day 3 (maximum at day 7) (A~C), while the GFAP did not show any difference at each time period (J~L). Magnified images of Iba1 (D~F) and [PC(diacyl-16:0/20:4)+K]⁺ (G~I) in the ipsilateral dorsal horn. Scale bar: 300μm. Significant increase of [PC(diacyl-16:0/20:4)+K]⁺ was observed in the ipsilateral dorsal horn at day 7, which resembled the change of microglia. Scale bar: 1mm

Fig 3. Statistical analysis for [PC(diacyl-16:0/20:4)+K]⁺ distribution with minocycline treatment.

The minocycline attenuated hypersensitivity to innocuous mechanical stimulation of the left hind-paw after SNI compared with control group (A). This was present in every animal and persisted until 7 days post SNI. Two-way ANOVA and Tukey. *p<0.01, minocycline versus control. All error bars represent SEM. Representative images of PC distribution in the spinal dorsal horn and quantification of the averaged ion signal intensities in each ROI in control group (B, C) and mino group (D, E). Control group showed significant ion signal elevation in the ipsilateral dorsal horn compared with the contralateral side (B, C). In mice receiving minocycline, ion signal elevation in the dorsal horn was significantly decreased (D, E). This was present in every animal *p<0.05 Welch’s t test. (n = 4 for both minocycline and control). All error bars represent SEM. Scale bar: 500μm.