In vivo evaluation of microglia activation by intracranial iron overload in central pain after spinal cord injury

Abstract

Background: Central pain (CP) is a common clinical problem in patients with spinal cord injury (SCI). Recent studies found the pathogenesis of CP was related to the remodeling of the brain. We investigate the roles of iron overload and subsequent microglia activate in the remodeling of the brain after SCI.

Methods: An SCI-induced CP model was established in Sprague-Dawley rats that were randomly assigned to SCI, sham operation, deferoxamine (DFX), minocycline, and nitric oxide synthase inhibitor treatment groups. At 12 weeks, pain behavior and thermal pain threshold were evaluated in each group, and iron transferrin receptor (TfR)1 and ferritin (Fn) mRNA, as well as iron-regulatory protein (IRP)1, FN, lactoferrin, and nuclear factor (NF)-κB protein levels in the rat brains were measured. Microglia proliferation and differentiation and IRP1 expression were evaluated by immunohistochemistry.

Results: Autophagy was observed in rats after SCI, accompanied by reduced latency of thermal pain, increased iron content and IRP1 and NF-κB levels in the hindlimb sensory area, hippocampus, and thalamus, and decreased Fn levels in the hindlimb sensory area. TfR1 mRNA expression was upregulated in activated microglia. Treatment with an iron-chelating agent, or inhibitors of nitric oxide synthase or microglia suppressed microglia proliferation.

Conclusions: SCI may induce intracranial iron overload, which activates microglia via NF-κB signaling. Microglia secrete inflammatory factors that induce neuronal damage and lead to CP. Treatment with an iron-chelating agent or NF-κB or microglia inhibitors can relieve CP resulting from SCI.

Keywords: Central pain; Iron; Microglia; NF-κB; Spinal cord injury.

Fig. 1

a Thermal pain latency in rats. *P < 0.05 vs. SCI group, △P < 0.05 vs. sham operation group. b Thermal pain latency in rats over a 90-day period

Fig. 2

Brain iron content in rats. a Comparison of total brain iron content between groups. b Iron content in rat hippocampus, hindlimb sensory cortex, and thalamus. *P < 0.05 vs. SCI group, △P < 0.05 vs. sham operation group

Fig. 3

a Western blot analysis of IRP1, Fn, NF-κB, and LF expression in the hindlimb sensory cortex of rats. b The gray ratio of IRP1 and the loading control β-actin were plotted. Data represent the average of three experiments. c–e Expression levels of Fn (c), NF-κB (d), and LF (e). *P < 0.05 vs. SCI group; ΔP < 0.05 vs. sham operation group

Fig. 4

a TfR1 and b Fn expression in rat hindlimb sensory cortex, thalamus, and hippocampus, as determined by ELISA. *P < 0.05 vs. SCI group; ΔP < 0.05 vs. sham operation group

Fig. 5

a–d TfR1 gene expression in rat hindlimb sensory cortex, thalamus, and hippocampus. Melting curve of TfR1 (a) and expression levels in rat hindlimb sensory cortex (b), hippocampus (c), and thalamus (d). *P < 0.05 vs. SCI group; ΔP < 0.05 vs. sham operation group

Fig. 6

a–g Immunohistochemical analysis of microglia distribution in rat hindlimb sensory cortex

Fig. 7

a–e Immunohistochemical analysis of IRP1 expression in rat hindlimb sensory cortex