Fig 2: Blockage of the protective effects of melatonin on Sev-induced synaptic impairment and dysmyelination by MT1-shRNA (shMT1). (A) Illustration of injection of lentivirus expressing shMT1. (B, C) Western blotting and quantification of MT1 in hippocampus injected with different shMT1. ShMT1-#2 was most effective in silencing the expression of MT1 (D) Western blotting and quantification of GluR1, GluR2, Homer, and PSD95 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV (EV, empty vector), Sev + mel + shMT1. Notice that shMT1 blocked the up-regulation of GluR1, GluR2, Homer, and PSD95 by melatonin. (E) Immunostaining of vglut1. Notice the weak immunoreactivity of vglut1 in mice treated with shMT1 as compared with that in mice treated with EV. (F) Western blotting and quantification of PDGFRa, NG2, Sox10, and Olig2 in mice of the following groups: control + vehicle, Sev + vehicle, Sev + mel, Sev + mel + EV, Sev + mel + shMT1. Notice that shMT1 blocked the up-regulation of PDGFRa, NG2, Sox10, and Olig2 induced by melatonin. (G) Immunostaining of Olig2. N = 6 mice per group. Con, control. Sev, Sevoflurane. mel, melatonin. *p < .05. **p < .01. ***p < .001. One-way ANOVA

Fig 3: Flow cytometric analysis of primary microvessels: (a and c) the primary microvessels were cultured in the TGM2D medium for 3 (a and c) and 7 (c) days and analyzed by flow cytometry. Primary antibodies included CD31, NG2, and CD45. (b and d) the primary microvessels were cultured in the TGM2D medium for 7 days and immunostained by antibodies against CD31, NG2, CD45, and Ki67. DAPI stained nuclei. Scale bars, 100 µm.

Fig 4: Interaction between macrophages and pericytes(A)Multiplex immunofluorescence staining of macrophage markers CD68, CD163, and pericyte markers MCAM, NG2 in GBM samples.(B) StrataQuest software recognizes pericytes by NG2/MCAM. Quantify the number of cells at different distances from pericytes: 0–25µm (green area), 25–50µm (brown-yellow area).(C) The scatterplot shows the number of DAPI? cells, CD68? cells, CD163? cells, CD68?CD163? cells, NG2? cells, MCAM? cells, NG2?MCAM? cells.(D) The scatterplot shows the number of CD68?CD163? cells at different locations from NG2?MCAM? cells: 0–25µm, 25–50µm.(E) StrataQuest software recognizes pericytes by NG2/MCAM. Quantify the number of cells at different distances from pericytes: 0–25µm (green area), 25–50µm (brown-yellow area).(F) The scatterplot shows the number of DAPI? cells, CD68? cells, CD163? cells, CD68?CD163? cells, NG2? cells, MCAM? cells, and NG2?MCAM? cells.(G) The scatterplot shows the number of CD68?CD163? cells at different locations from NG2?MCAM? cells: 0–25µm, 25–50µm.

Fig 5: Fascin-1 is expressed partly in Iba1+ cells around the lesion but not in infiltrating Mac2+ macrophages, NeuN+ neurons or NG2+ cells in the injured spinal cord at 14 days. (A) Representative immunofluorescence images of Fascin-1 (Red) and Iba1 (Green). Arrowheads indicate the colocalization observed with a ×40 objective lens. The blue dotted lines delineate the boundary around the lesion core. (B–D) Representative immunofluorescence images of Fascin-1 (Red) staining with Mac2, NeuN, and NG2 (Green) staining, respectively, showing no apparent colocalization of staining. The asterisks indicate the lesion epicenter. Nuclear staining with DAPI is shown (Blue). Scale bars: low magnification, 200 µm; higher magnification, 20 µm. (E) Percentage of Fascin-1+Iba1+ cells relative to the total number of Fasicn-1+ or Iba1+ cells outside the blue dotted lines in the injured spinal cord. The data are presented as the mean ± SEM (n = 5 independent experiments).