Effect of minocycline on polarization of types M1/M2 microglia in spinal cord in rats after spinal nerve ligation

Abstract

Objective To investigate the effect of minocycline (Mino) on the polarization of types M1/M2 microglia (pro- and anti-inflammatory type) in the spinal dorsal horn of rats with neuropathic pain (NP) induced by spinal nerve ligation (SNL) and its underlying mechanism. Methods A total of 36 adult male SD rats were randomly stratified into Sham-operation (Sham) group, SNL group and Mino+SNL group by stratified random sampling based on body weight. Mechanical pain threshold and cold nociceptive thresholds of rat hind paw were measured in 1 d before and 14 d after modelling. Spinal cord tissue at the lumbar 5 (L5) segment was taken at 14 d after modelling, and the total number of microglia as well as the numbers of M1 and M2 microglia in the spinal dorsal horn were measured with immunohistochemistry and stereology. With aid of bioinformatics techniques, the core target in the spinal cord, Cst7, was selected. Then, the protein levels of microglia marker Iba-1, M1 microglia marker iNOS, M2 microglia marker CD206, Cst7 encoded protein cystatin F (CF) and pathway CatS/CX3CL1/CX3CR1 were detected with Western blotting. The expression levels of TNF-α, IL-6 and IL-10 in the spinal cord tissues were measured with ELISA. Results The mechanical pain and cold nociceptive thresholds were both significantly higher in the M+SNL group than the SNL group at 7~14 d after modelling (P < 0.01). The total number of microglia and the numbers of M1/M2 microglia in the spinal dorsal horn as well as the expression levels of CatS, CX3CL1, CX3CR1, TNF-α, IL-6, and IL-10 in the spinal cord tissues were obviously increased, and the expression level of CF was notably decreased in the SNL model group than the Sham group (P < 0.01). While, Mino treatment remarkably reversed above phenomena, with decreased total number of microglia and number of M1 microglia as well as expression levels of CatS, CX3CL1, CX3CR1, TNF-α and IL-6, and increased number of M2 microglia as well as CF and IL-10 levels when compared with the SNL group (P < 0.05). Conclusion Mino alleviates SNL induced neuropathic pain, probably through up-regulating CF in the microglia, and thus inhibiting the CatS/CX3CL1/CX3CR1 signaling pathway, promoting the conversion of microglia from type M1 to M2 to balance the imbalance in the M1/M2 polarization, and thus reducing neuroinflammation.

Keywords

• minocycline
• cystatin f
• microglia
• spinal nerve ligation
• neuropathic pain
• stereology
• rats