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. Author manuscript; available in PMC: 2009 Nov 1.

Published in final edited form as: J Cereb Blood Flow Metab. 2008 Jul 9;28(11):1845–1859. doi: 10.1038/jcbfm.2008.75

Fig. 6 — Roscovitine treatment decreases activation of microglial and astroglia at 7 days after injury, as indicated by immunostaining for p22^PHOX, ED1, Galectin-3 and GFAP. All images are cropped from the indicated areas of larger multi-tile images (data not shown); multi-tile images are used for the cell counting data presented. (A) Representative composite confocal images after double-immunostaining for GFAP (red) and p22^PHOX (green) of the core and periphery of the injury show that injury-dependent increase in the number of p22^PHOX-positive microglia is concentrated in the core, whereas the increase in GFAP-positive astroglia, occurs especially in the periphery of the lesion. Roscovitine attenuated both changes. There is no colocalization between markers for microglia and astroglia. Images showing the separate p22^PHOX and GFAP channels are included. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in both p22^PHOX (n=3 sections, threshold=50, unpaired t test, ** P=0.0029) and GFAP-positive cells (n=3 sections, threshold=150, unpaired t test, * P=0.0298) per brain section. (B) Representative composite confocal images after double-immunostaining for ED1 (red) and cyclin G1 (green) of the injury core show the increased number of ED1-positive microglia after TBI. These cells do not have increased cyclin G1 staining. Roscovitine treatment attenuated the ED1 staining increase. There is no colocalization between ED1 and cyclin G1 immunostaining. Images with separate ED1 and cyclin G1 channels are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in ED1-positive cells (n=3 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section. (C) Representative composite confocal images after immunostaining for Galectin-3 (red) and staining with TO-PRO-3 (blue, DNA stain) of the area of the injury core illustates the increased number of Galectin-3-positive microglia. Roscovitine treatment attenuated the increased Galectin-3 staining. The separate Galectin-3 and TO-PRO-3 images are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in Galectin-3-positive cells (n=4 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section. (D) Representative composite confocal images after immunostaining for Iba-1 (green) and staining with TO-PRO-3 (blue, DNA stain) of the area of the injury core illustates the increased number of Iba-positive microglia. Roscovitine treatment attenuated the increased Iba-1 staining. The separate Iba-1 and TO-PRO-3 images are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in Iba-1-positive cells (n=4 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section.

Fig. 6 — Roscovitine treatment decreases activation of microglial and astroglia at 7 days after injury, as indicated by immunostaining for p22^PHOX, ED1, Galectin-3 and GFAP. All images are cropped from the indicated areas of larger multi-tile images (data not shown); multi-tile images are used for the cell counting data presented. (A) Representative composite confocal images after double-immunostaining for GFAP (red) and p22^PHOX (green) of the core and periphery of the injury show that injury-dependent increase in the number of p22^PHOX-positive microglia is concentrated in the core, whereas the increase in GFAP-positive astroglia, occurs especially in the periphery of the lesion. Roscovitine attenuated both changes. There is no colocalization between markers for microglia and astroglia. Images showing the separate p22^PHOX and GFAP channels are included. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in both p22^PHOX (n=3 sections, threshold=50, unpaired t test, ** P=0.0029) and GFAP-positive cells (n=3 sections, threshold=150, unpaired t test, * P=0.0298) per brain section. (B) Representative composite confocal images after double-immunostaining for ED1 (red) and cyclin G1 (green) of the injury core show the increased number of ED1-positive microglia after TBI. These cells do not have increased cyclin G1 staining. Roscovitine treatment attenuated the ED1 staining increase. There is no colocalization between ED1 and cyclin G1 immunostaining. Images with separate ED1 and cyclin G1 channels are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in ED1-positive cells (n=3 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section. (C) Representative composite confocal images after immunostaining for Galectin-3 (red) and staining with TO-PRO-3 (blue, DNA stain) of the area of the injury core illustates the increased number of Galectin-3-positive microglia. Roscovitine treatment attenuated the increased Galectin-3 staining. The separate Galectin-3 and TO-PRO-3 images are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in Galectin-3-positive cells (n=4 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section. (D) Representative composite confocal images after immunostaining for Iba-1 (green) and staining with TO-PRO-3 (blue, DNA stain) of the area of the injury core illustates the increased number of Iba-positive microglia. Roscovitine treatment attenuated the increased Iba-1 staining. The separate Iba-1 and TO-PRO-3 images are also shown for both vehicle- and roscovitine-treated animals, respectively. Cell counting using ImageJ indicates that roscovitine treatment results in a significant decrease in Iba-1-positive cells (n=4 sections, threshold=50, unpaired t test, **** P<0.0001) per brain section.