The microglial activation profile and associated factors after experimental spinal cord injury in rats

Figures & data

Figure 1 Compressive SCI model.

Notes: (A) Under an operating microscope, compressive injury was induced by a 2-minutes compression SCI using a 10 g aneurysm clip. (B) After removing the aneurysm clip, a clear imprint (white arrow) indicating the injury position can be seen.
Abbreviation: SCI, spinal cord injury.

Figure 2 Line chart of the BBB scores of rats in the SCI and sham operation groups.

Note: The BBB scores of both sides were significantly lower in the SCI group than the ones in the sham operation group at days 1, 3, 7, 14, and 28 post-operative ***(P<0.001, n=108).
Abbreviations: SCI, spinal cord injury; BBB, Basso, Beattie, and Bresnahan Locomotor Rating Scale.

Figure 3 Myelination of the spinal cord after SCI investigated through staining of LFB.

Notes: (A) Representative images of LFB staining in the sham group and 1, 3, 7, 14, and 28 days after SCI. The left side of the figure was low-power field (100×) and the right one was high-power field (400×). Scale bar =100 µm (left column) and scale bar =25 µm (right column). (B) Bar graphs of myelination in different groups. It was progressively aggravated during the acute period, reached the trough at day 7 post injury *(P<0.05, n=36), and then gradually recovered to close to the level of the sham operation group at day 28 post-operative. *P<0.05 compared with sham group.
Abbreviations: d, days; SCI, spinal cord injury; LFB, Luxol fast blue.

Figure 4 Immunohistochemistry of MBP in the SCI and sham operation groups.

Notes: (A) Representative images of MBP staining in the sham group and SCI group 1, 3, 7, 14, and 28 days post injury. The images on the left are a low field of vision (100×), while a high-power view (400×) is on the right. Scale bar =100 µm (left column) and scale bar =25 µm (right column). (B) Bar graphs of the degree of injury of oligodendroglia in different groups. The mean IODs of MBP declined during the acute period, reached the lowest level at day seven after SCI ***(P<0.001, n=36), and then gradually rose during 28 days post-operative. The mean IODs of MBP at day 3 and 14 post-operative were lower than ones in the sham operation group *(P<0.05, n=36). *P<0.05, and ***P<0.001 compared with the sham group.
Abbreviations: d, days; MBP, Myelin basic protein; SCI, spinal cord injury; IOD, integrated optical density.

Figure 5 Immunohistochemistry of LPL in tissue from different time points after SCI and sham operation groups.

Notes: (A) Representative images of LPL staining in the sham group and SCI group 1, 3, 7, 14, and 28 days post injury. On the left are the figures under a low field of vision (100×), while on the right are the ones under a high-power view (400×). Scale bar =100 µm (left column) and scale bar =25 µm (right column). (B) Bar graphs of the degree of injury of oligodendroglia in different groups. The mean IODs of LPL gradually declined during the acute period, reached the lowest level at day 7 after SCI ***(P<0.001, n=36), and then began to rise afterward during 28 days postoperative. ***P<0.001 compared with sham group.
Abbreviations: d, days; LPL, rabbit anti-Plastin L; IOD, integrated optical density; SCI, spinal cord injury.

Figure 6 The protein expression of MBP was detected using Western blotting analyses.

Notes: (A) Representative images of Western blot in the sham group and SCI group 1, 3, 7, 14, and 28 days post injury. (B) Bar graphs of the level of MBP protein in different groups. The ratio of MBP and β-actin declined firstly, reached the lowest level at day 7 after SCI ***(P<0.001, n=36), and then rose during 28 days post-operative ***(P<0.001, n=36). ***P<0.001 compared with the sham group.
Abbreviations: d, days; MBP, Myelin basic protein; SCI, spinal cord injury.

Figure 7 Double staining of ED-1 and MHC-II were used to investigate the microglial activation.

Notes: (A) Representative ED-1/MHC-II/DAPI photomicrographs of the spinal cord tissue in the different groups. In addition to pictures of the right-hand column which are a high-power field (400×), the other columns are low-power field (200×). Fluorescence colors: MHC-II: red, ED-1: green, and DAPI: blue. Scale bar =50 µm (left three columns) and scale bar =25 µm (right-hand column). (B) Bar graphs of the mean number of the activated microglial involved in immune-mediated responses in the different groups. ED-1⁺MHC-II⁺ cells at day 7 after SCI were significantly more than ones of the sham operation group ***(P<0.001, n=36). The number of double positive cells decreased at 4 weeks after injury, but it was still higher than that of sham operation group *(P<0.05, n=36). *P<0.05, **P<0.01, and ***P<0.001 compared with sham group.
Abbreviations: d, days; DAPI, 4′,6-diamidino-2-phenylindole; SCI, spinal cord injury.

Figure 8 Bar graphs of iron, glutamate, ATP and potassium concentrations in the tissues around the injury site were compared in the different groups.

Notes: (A, B) Compared with the sham operation group, both iron and glutamate concentration began to rise significantly at 1 day (**P<0.01, and ***P<0.001, n=36), peaked at 7 days (**P<0.01, and ***P<0.001, n=36), and then declined until day 28 post-operative (**P<0.01, and ***P<0.001, n=36). (C, D) Conversely, both ATP and potassium concentration began to decline significantly at 1 day (***P<0.001, and ***P<0.001, n=36), reached a low valley at 7 days (***P<0.001, and ***P<0.001, n=36), and then gradually recovered. But at day 28 post-operative, they still were below that of the sham operation group (***P<0.001, and ***P<0.001, n=36). **P<0.01, and ***P<0.001 compared with sham group.
Abbreviations: d, days; SCI, spinal cord injury.

Figure 9 Bar graphs of the protein levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, were compared in the SCI and sham operation groups.

Notes: (A–C) Level of above pro-inflammatory cytokines increased significantly at 1 day ***(P<0.001, n=36), peaked at 7 days ***(P<0.001, n=36), and gradually decreased. After 4 weeks, the three cytokine concentrations remained nearly 3-fold higher than ones of the sham operation group ***(P<0.001, n=36). ***P<0.001 compared with sham group.
Abbreviations: d, days; SCI, spinal cord injury.