Lipoxin A4 inhibits microglial activation and reduces neuroinflammation and neuropathic pain after spinal cord hemisection

Abstract

Background: Spinal cord injury (SCI) is a severe neurological disorder with many disabling consequences, including persistent neuropathic pain, which develops in about 40 % of SCI patients and is induced and sustained by excessive and uncontrolled spinal neuroinflammation. Here, we have evaluated the effects of lipoxin A4 (LXA4), a member of a unique class of endogenous lipid mediators with both anti-inflammatory and analgesic properties, on spinal neuroinflammation and chronic pain in an experimental model of SCI.

Methods: Spinal hemisection at T10 was carried out in adult male CD1 mice and Wistar rats. To test if LXA4 can reduce neuroinflammation and neuropathic pain, each animal received two intrathecal injections of LXA4 (300 pmol) or vehicle at 4 and 24 h after SCI. Sensitivity to mechanical stimulation of the hind paws was evaluated using von Frey monofilaments, and neuroinflammation was tested by measuring the mRNA and/or protein expression levels of glial markers and cytokines in the spinal cord samples after SCI. Also, microglia cultures prepared from murine cortical tissue were used to assess the direct effects of LXA4 on microglial activation and release of pro-inflammatory TNF-α.

Results: LXA4 treatment caused significant reductions in the intensity of mechanical pain hypersensitivity and spinal expression levels of microglial markers and pro-inflammatory cytokines induced by SCI, when compared to rodents receiving control vehicle injections. Notably, the increased expressions of the microglial marker IBA-1 and of the pro-inflammatory cytokine TNF-α were the most affected by the LXA4 treatment. Furthermore, cortical microglial cultures expressed ALX/FPR2 receptors for LXA4 and displayed potentially anti-inflammatory responses upon challenge with LXA4.

Conclusions: Collectively, our results suggest that LXA4 can effectively modulate microglial activation and TNF-α release through ALX/FPR2 receptors, ultimately reducing neuropathic pain in rodents after spinal cord hemisection. The dual anti-inflammatory and analgesic properties of LXA4, allied to its endogenous nature and safety profile, may render this lipid mediator as new therapeutic approach for treating various neuroinflammatory disorders and chronic pain with only limited side effects.

Keywords: Chronic pain; Lipoxin; Microglia; Neuroinflammation; Spinal cord injury.

Fig. 1

Lipoxin A4 attenuates mechanical allodynia induced by spinal cord hemisection in mice. a Treatment scheme for intrathecal injections of lipoxin A4 or vehicle and behavioral analyses. b, e Following SCI, adult mice develop mechanical allodynia in both ipsilateral and contralateral hindpaws. Fifty percent paw withdrawal thresholds are increased with LXA4 administration at 4 and 24 h, compared to vehicle-treated group. An additional intrathecal bolus injection of 300 pmol of LXA4 at 35 days still attenuated mechanical allodynia in both ipsilateral and contralateral paws from 3 h up to 24 h. c, f–d, g The area under the curve depicting variations of 50 % paw withdrawal thresholds in time are presented for treatment evaluation up to 35 days and after the bolus injection. Results are presented as mean ± SEM. Asterisk denotes p < 0.05 when comparing to sham-operated group and number sign denotes p < 0.05 when comparing to vehicle-treated group (two-way ANOVA followed by Bonferroni; n = 6 mice/group)

Fig. 2

ALX/FPR2 receptor gene knockdown with intrathecal siRNA treatment impairs the analgesic effect of LXA4 after SCI in mice. a No differences were observed on the 7th day post-surgery with the siRNA administration in the ipsilateral paw. b Gene knockdown suppresses the antihyperalgesic effects of LXA4 observed in the contralateral paw. c On the 7th day after surgery, there is a significant decrease on ALX/FPR2 receptor mRNA levels in the spinal cord of animals injected with the specific siRNA. Results are presented as mean ± SEM. Asterisk denotes p < 0.05 when comparing to control-siRNA-treated group (two-way ANOVA followed by Bonferroni, n = 6 mice/group)

Fig. 3

LXA4 attenuates the SCI-induced microglial reactivity in the spinal cord. Lipoxin A4 reduces the SCI-induced increase in Iba-1 expression in the spinal cord at 7 and 36 days after surgery. Staining for Iba-1 (red, a), GFAP (green, b), and DAPI (blue) in thoracic spinal cord from sham, vehicle-treated SCI, or LXA4-treated SCI mice. Scale 200 μm. Iba-1 staining was enhanced at 7 (a, b) and 36 days (d, e) after SCI and was reduced by LXA4 treatment. GFAP expression was significantly upregulated by SCI at 7 (a–c) and 36 days (d–f) but was not changed following LXA4 administration. Results shown in graphs are presented as mean ± SEM. Asterisk denotes p < 0.05 when comparing to sham-operated group. Number sign denotes p < 0.05 when comparing with vehicle-treated group (Student’s t test, n = 6 mice/group)

Fig. 4

Lipoxin A4 reduces the expression of microglial markers and TNF-α. a–h Spinal cord injury promotes a significant upregulation of ALX/FPR2 receptors, GFAP, IBA-1, P2Y₁₂, TNF-α, IL-6, iNOS, and TGF-β mRNA levels 7 days post-surgery, comparing to sham-operated mice. c–e LXA4 significantly reduced IBA-1, P2Y₁₂, and TNF-α mRNA levels. Data is expressed as mean ± SEM. Asterisk denotes p < 0.05 when comparing to sham-operated group. Number sign denotes p < 0.05 when comparing with vehicle-treated group (one-way ANOVA followed by Bonferroni; n = 4 mice/group)

Fig. 5

Lipoxin A4 attenuates microglial activation in primary cultures. a Microglia cells stained for DAPI (blue) and ALX/FPR2 receptor (red). Scale 50 μm. b Representative image of ALX/FPR2 mRNA expression on the spinal cord and microglia and astrocytes cultures. c LXA4 reduced p-p38 increase induced by IFN-γ. d IFN-γ induces a significant increase in TNF-α release, which is reduced by both concentrations of LXA4. Data is expressed as mean ± SEM. Asterisk denotes p < 0.05 when comparing to control. Number sign denotes p < 0.05 when comparing with vehicle (one-way ANOVA followed by Bonferroni; n = 4 cultures)