M1-type microglia can induce astrocytes to deposit chondroitin sulfate proteoglycan after spinal cord injury

After spinal cord injury (SCI), astrocytes gradually migrate to and surround the lesion, depositing chondroitin sulfate proteoglycan-rich extracellular matrix and forming astrocytic scar, which limits the spread of inflammation but hinders axon regeneration. Meanwhile, microglia gradually accumulate at the lesion border to form microglial scar and can polarize to generate a pro-inflammatory M1 phenotype or an anti-inflammatory M2 phenotype. However, the effect of microglia polarization on astrocytes is unclear. Here, we found that both microglia (CX3CR1(+)) and astrocytes (GFAP(+)) gathered at the lesion border at 14 days post-injury (dpi). The microglia accumulated along the inner border of and in direct contact with the astrocytes. M1-type microglia (iNOS(+)CX3CR1(+)) were primarily observed at 3 and 7 dpi, while M2-type microglia (Arg1(+)CX3CR1(+)) were present at larger numbers at 7 and 14 dpi. Transforming growth factor-β1 (TGFβ1) was highly expressed in M1 microglia in vitro, consistent with strong expression of TGFβ1 by microglia in vivo at 3 and 7 dpi, when they primarily exhibited an M1 phenotype. Furthermore, conditioned media from M1-type microglia induced astrocytes to secrete chondroitin sulfate proteoglycan in vitro. This effect was eliminated by knocking down sex-determining region Y-box 9 (SOX9) in astrocytes and could not be reversed by treatment with TGFβ1. Taken together, our results suggest that microglia undergo M1 polarization and express high levels of TGFβ1 at 3 and 7 dpi, and that M1-type microglia induce astrocytes to deposit chondroitin sulfate proteoglycan via the TGFβ1/SOX9 pathway. The study was approved by the Institutional Animal Care and Use Committee of Anhui Medical University, China (approval No. LLSC20160052) on March 1, 2016.