Figure 1.
BMD treatment and induction of transient retinal ischemia.
(A) Diagram for BMD administration before and after ischemic injury. BMD or vehicle were injected by IP for short terms or administrated systemically for 4 weeks using osmotic pumps. (B) IOP elevation in the rat eyes following transient ischemic injury. (B) Mean IOP was 76.1±4.2 mmHg during anterior chamber perfusion with saline. In contrast, mean IOP of contralateral control eyes was 11.2±1.7 mmHg (n = 10). BMD, brimonidine.
Figure 2.
BMD-mediated protection of RGC survival in ischemic injury.
BMD or vehicle were administrated systemically for 4 weeks using osmotic pumps. (A–C) Brn3a whole-mount immunohistochemistry. High magnification showed representative images from the middle area of retinas. In comparison with normal control retina (A), vehicle-treated ischemic retina showed greater RGC loss (B). In contrast, BMD significantly increased RGC survival in ischemic retina (C). (D) The quantitative analysis of RGC loss. Values are mean ± SD (n = 6 retinas/group). *Significant at p<0.05 compared with non-ischemic contralateral control retina or vehicle-treated ischemic retina. BMD, brimonidine. Scale bar = 100 µm.
Figure 3.
BMD-mediated blockade of apoptotic pathway.
BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ischemia. Vehicle-treated ischemic retina significantly increased Bax, Bcl-xL, and pBad protein expression. In contrast, BMD significantly decreased Bax protein expression, but increased Bcl-xL and pBad protein expression compared with vehicle-treated ischemic retina. Values are mean ± SD (n = 4 retinas/group). *Significant at p<0.05 compared with non-ischemic contralateral control retina or vehicle-treated ischemic retina.
Figure 4.
BMD-mediated blockade of the upregulations of GFAP, NMDA receptors and SOD2 expression in ischemic retina.
BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ischemia. (A) Vehicle-treated ischemic retina significantly increased GFAP protein expression compared with control retina. In contrast, BMD significantly decreased GFAP protein expression in ischemic retina. (B–E) GFAP immunohistochemisty. When the primary antibody for GFAP was omitted, there was no labeling of the secondary antibody (B). In comparison with control retina (C), vehicle-treated ischemic retina showed activation of müller cells (arrowheads) and astrocytes (D). In contrast, BMD significantly decreased activation of müller cells and astrocytes (E). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale = 20 µm. (F) Vehicle-treated ischemic retina significantly increased NR1, NR2A and SOD2 protein expression compared with control retina. In contrast, BMD significantly decreased NR1, NR2A and SOD2 protein expression in ischemic retina. Values are mean ± SD (n = 4 retinas/group). *Significant at p<0.05 compared with non-ischemic contralateral control retina or vehicle-treated ischemic retina.
Figure 5.
Alterations of Tfam and OXPHOS complex protein expression in ischemic retina.
BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation and administrated intraperitoneally for 72 hours after ischemia. Acute IOP elevation significantly increased GFAP, Tfam and OXPHOS complex (I, II, and IV), but did not change OXPHOS complex III and ATP synthase protein expression in ischemic retina. Values are mean ± SD (n = 4 retinas/group). *Significant at p<0.05 compared with non-ischemic contralateral control retina. Cx, Complex.
Figure 6.
BMD-mediated restoration of Tfam protein expression in ischemic retina.
BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ischemia. (A) BMD significantly decreased Tfam protein expression compared with vehicle-treated ischemic retina. Values are mean ± SD (n = 4 retinas/group). *Significant at p<0.05 compared with vehicle-treated ischemic retina. *Significant at p<0.05 compared with vehicle-treated ischemic retina. (B–E) Tfam immunohistochemisty. When the primary antibody for Tfam was omitted, there was no labeling of the secondary antibody (B). In comparison with control retina (C), vehicle-treated ischemic retina increased Tfam protein expression in the OPL, INL, IPL, and GCL. Note that Tfam immunoreactivity was increased in neurons in the GCL (arrowheads) (D). In contrast, BMD significantly decreased in neurons in the GCL (arrowhead) (E). (F and G) Tfam and Thy1.1 (red) double labeling. Neurons containing Tfam immunoreactivity were colabeled with Thy1.1, a marker for RGCs (arrows), indicating that RGCs contained Tfam protein. (H) There was no significant difference between vehicle- and BMD-treated non-ischemic contralateral control retinas (p = 0.5). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; BMD, brimonidine. Scale bar = 20 µm.
Figure 7.
BMD-mediated restoration of OXPHOS complex protein expression in ischemic retina.
BMD or vehicle were injected once by IP at 24 hours before and at the time of initial IOP elevation for 24 hours after ishcemia. Note that BMD significantly decreased OXPHOS complex (I, II, III and IV) but did not change ATP synthase protein expression in ischemic retina. Values are mean ± SD (n = 4 retinas/group). There was no significant difference between vehicle- and BMD-treated non-ischemic contralateral control retinas. *Significant at P<0.05 compared with vehicle-treated ischemic retina. Cx, Complex; BMD, brimonidine.