Increased Expression of Osteopontin in Retinal Degeneration Induced by Blue Light-Emitting Diode Exposure in Mice

Abstract

Osteopontin (OPN) is a multifunctional adhesive glycoprotein that is implicated in a variety of pro-inflammatory as well as neuroprotective and repair-promoting effects in the brain. As a first step towards understanding the role of OPN in retinal degeneration (RD), we examined changes in OPN expression in a mouse model of RD induced by exposure to a blue light-emitting diode (LED). RD was induced in BALB/c mice by exposure to a blue LED (460 nm) for 2 h. Apoptotic cell death was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. In order to investigate changes in OPN in RD, western blotting and immunohistochemistry were performed. Anti-OPN labeling was compared to that of anti-glial fibrillary acidic protein (GFAP), which is a commonly used marker for retinal injury or stress including inflammation. OPN expression in RD retinas markedly increased at 24 h after exposure, was sustained through 72 h, and subsided at 120 h. Increased OPN expression was observed co-localized with microglial cells in the outer nuclear layer (ONL), outer plexiform layer (OPL), and subretinal space. Expression was restricted to the central retina in which photoreceptor cell death occurred. Interestingly, OPN expression in the ONL/OPL was closely associated with microglia, whereas most of the OPN plaques observed in the subretinal space were not. Immunogold electron microscopy demonstrated that OPN was distributed throughout the cytoplasm of microglia and in nearby fragments of degenerating photoreceptors. In addition, we found that OPN was induced more acutely and with greater region specificity than GFAP. These results indicate that OPN may be a more useful marker for retinal injury or stress, and furthermore act as a microglial pro-inflammatory mediator and a phagocytosis-inducing opsonin in the subretinal space. Taken together, our data suggest that OPN plays an important role in the pathogenesis of RD.

Keywords: inflammation; microglia; osteopontin; phagocytosis; retinal degeneration.

Figure 1

Characterization of blue light-emitting diode (LED)-induced retinal degeneration (RD) in mice. (A) Representative scotopic electroretinography (ERG) responses in unexposed control (black) and blue LED-induced retinas at 0 (orange), 24 (blue), 72 (red), and 120 h (green). All ERG components showed progressive reductions in a time-dependent manner. (B) Hematoxylin and Eosin (H&E) staining of representative retinal tissue sections. Consistent with ERG findings, retinal thickness decreased in a time-dependent manner. Prominent decreases were observed in the outer nuclear layer (ONL). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment and outer segment; OPL, outer plexiform layer; RPE, retinal pigment epithelium. (C) Terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) staining on a vertical section of eyecup taken from an RD mouse at 72 h after blue LED exposure. Numerous TUNEL-positive photoreceptors were observed in the ONL of the central retina (arrows) near the optic disc (od), while few TUNEL-positive cells were observed in the peripheral retina (arrowheads) near the ora serrata (os). 4′,6-diamidino-2′-phenylindole (DAPI) staining was used to label the nuclei of retinal cells.

Figure 2

Osteopontin (OPN) expression in blue LED-induced RD retinas. (A) Representative western blot. A representative 66-kDa band was identified with anti-OPN labeling and thus recognized as OPN. (B) Densitometric analysis of the 66 kDa OPN band. Results are expressed as percentages (%) relative to normal control retina values. Data represent the mean ± standard deviation (SD) for four mice in each group. *P < 0.05.

Figure 3

Temporal and spatial profiles of OPN expression in blue LED-induced RD retinas. Confocal micrographs taken from vertical sections of blue LED-induced RD eyecups processed for OPN immunoreactivity. (A) A representative normal control retina. Several ganglion cells (arrows) in the GCL were weakly labeled with OPN. INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment and outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. (B) A representative RD retina immediately after blue LED exposure (0 h). OPN expression was similar to that in the unexposed RD control. (C) A representative RD retina at 24 h after blue LED exposure. The central and mid-peripheral retina are magnified. Strong OPN immunoreactivity was observed in cells of the ONL (arrows) and in the subretinal space (bracket) in the central retina, but not in the peripheral retina. (D) A representative RD retina at 72 h after blue LED exposure. OPN expression was similar to that in the RD retina at 24 h after blue LED exposure; however, a relatively larger number of OPN-labeled cells were observed in the ONL of the central retina at this time point. OPN immunoreactivity in the subretinal space of the peripheral retina was negligible. (E) A representative RD retina at 120 h after blue LED exposure. OPN immunoreactivities in the ONL and subretinal space of the central retina were markedly decreased.

Figure 4

Cellular localization of OPN in blue LED-induced RD retinas. Confocal micrographs taken from vertical sections of blue LED-induced RD retinas processed for OPN (green) and Iba1 (red) immunoreactivity. (A–F) A representative RD retina at 72 h after blue LED exposure. OPN was observed in Iba1-labeled microglia (arrows) in the ONL and OPL. INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment and outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. A region including two OPN-labeled microglia in (A–C) are magnified in (D–F), respectively. OPN was localized in the processes of Iba1-labeled microglial cells. (G–J) A representative RD retina at 72 h after blue LED exposure. Iba1-labeled cells were observed in the INL, IPL, and subretinal space. OPN was observed in Iba1-labeled microglia of the ONL and microglia/macrophages of the subretinal space (arrow), while OPN expression was not observed in Iba1-labeled microglia of the INL or IPL. Two microglia/macrophages (arrows) showing Iba1 and OPN co-labeling are magnified in (J). (K) A representative RD retina at 120 h after blue LED exposure. Several Iba1-labeled microglia/macrophages were still observed in the ONL and subretinal space, while minimal OPN-labeling was observed in the ONL and subretinal space.

Figure 5

Immunogold electron microscopy of OPN in blue LED-induced RD retinas. Electron micrographs taken from vertical sections of an RD retina at 72 h after blue LED exposure that was processed for OPN immunoreactivity. (A) OPN localization in the ONL. OPN labeled with immunogold was observed in the cytoplasm of microglia (M) in the ONL. Photoreceptors (Ph) ongoing apoptosis (asterisks) or with degenerative changes (dPh) were frequently observed nearby. A portion of microglial cytoplasm (rectangle) is magnified in the inset. In the inset, immunogold is observed associated with vesicular structures proximal to Golgi complexes (g) but not with mitochondria (mt). (B,C) OPN localization in the subretinal space. Large numbers of immunogold particles were present in subretinal space (bracket). In a higher magnification view (C) of the rectangular region (B), immunogold is observed associated with the membranes of fragmented or degenerating outer segments (OS) packed with membranous discs, and inner segments (IS) containing numerous mitochondria. RPE, retinal pigment epithelium.

Figure 6

Glial fibrillary acidic protein (GFAP) immunoreactivity in blue LED-induced RD retinas. Confocal micrographs taken from vertical sections of the central (left panel) and peripheral (right panel) retina processed for GFAP (red) immunoreactivity in normal (A) and blue LED-induced RD mice (B–E). (A) A representative normal control retina. GFAP was expressed in astrocytes and Müller cell endfeet in the GCL. (B) A representative RD retina immediately after blue LED exposure (0 h). Expression of GFAP was similar to that in the normal control. (C,D) A representative RD retina at 24 h after blue LED exposure. GFAP immunoreactivity was slightly increased relative to 0 h. Thin GFAP-labeled Müller cell processes were found in the INL and the IPL. There were no differences in GFAP expression between the central (C) and peripheral (D) retina at this time point. (E,F) A representative RD retina at 72 h after blue LED exposure. GFAP immunoreactivity was increased in both the central (E) and peripheral (F) retina, particularly in the ONL of the central retina, where GFAP-labeled Müller cell processes (arrowheads) extended to the outer limiting membrane. (G,H) A representative RD retina at 120 h after blue LED exposure. GFAP immunoreactivity was decreased in both the central (G) and peripheral (H) retina, but GFAP-labeling (arrowheads) persisted in the outer limiting membrane.