Fig. 8
From: Reactive astrocytes undergo M1 microglia/macrohpages-induced necroptosis in spinal cord injury
Involvement of TLR4/MyD88 signaling in M1 microglia/macrophages-induced necroptosis of astrocytes. a, b Double-staining of GFAP with TLR2 or TLR4. Notice that many GFAP-positive cells around the lesion center express TLR4 (arrows). Bars = 50 μm. c, d Double-staining of RIP3/TLR4 and MyD88/GFAP. Notice that there are many RIP3/TLR4 and MyD88/GFAP double-positive cells (showed by arrows) in the injured mouse spinal cord. Bars = 50 μm. e Western-blotting of TLR4 and MyD88 in cultured astrocytes after treatment with TLZ or vehicle control. TLZ treatment dramatically induced the expression of TLR4 and MyD88. f–h Western-blotting of RIP3 and PI-staining in astrocytes treated with vehicle control, TLZ, TLZ plus control peptide (MyD88 con pep), and TLZ plus a MyD88 inhibitory peptide (MyD88 inh pep). Notice that MyD88 inhibitory peptide significantly suppressed the effects of TLZ on the expression of RIP3 and MLKL and PI-labeling. **P <0.01, *P <0.05. n = 3. Bar = 50 μm. i Western-blotting of TLR4, MyD88 in normally cultured astrocytes, and astrocytes treated with M0 CM, M1 CM, and M2 CM from microglia/macrophages. Notice that M1 CM significantly increased the expression of TLR4 and MyD88. **P <0.01, *P <0.05. n = 3. j Western-blotting of RIP3 in astrocytes treated with DMEM control, M0 CM, M1 CM, and M2 CM. k, l Double-staining of GFAP with TLR4 and MyD88 at 5 dpi in DMEM injected, GdCl3 treated or M1 macrophages transplanted mice. Notice that GdCl3 treatment significantly decreased, while transplantation of M1 macrophages significantly increased the percent of astrocytes expressing TLR4 or MyD88 in adjacent regions which were 400 μm rostral and caudal to cavity border. *P <0.05. Bars = 50 μm. n = 3