Steroids do not prevent photoreceptor degeneration in the light-exposed T4R rhodopsin mutant dog retina irrespective of AP-1 inhibition

Abstract

Purpose: AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model.

Methods: The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed ( approximately 15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry.

Results: Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain.

Conclusions: Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.

FIGURE 1

EMSA of AP-1 DNA-binding activity in retinal nuclear proteins from T4R RHO mutant dogs 1 hour after light exposure. The dogs were untreated or treated with systemic DEX (A) or were treated locally with TRIAM in the exposed (E) eye (B), the shielded (S) eye, or both (B) eyes (C). The exposed retina of the untreated dog (EM60) showed a marked increase in AP-1 binding, but it was suppressed by DEX treatment (EM67). Retinas receiving TRIAM in the E, S, or B eyes showed increased AP-1 binding in the exposed eye. The description below the gels represent the genotype at the RHO locus, the postexposure (PE) interval, and the treatment. The dashed lines in the gels indicate that the images represent lanes from the same gel that were not adjoining.

FIGURE 2

The AP-1 binding activity ratio (E/S, exposed/shielded) in retinas after exposure to light. (A) Without treatment, AP-1 levels increased significantly 1 hour after light exposure, but DEX pretreatment inhibited AP-1 activation. (B) Pretreatment with TRIAM in the exposed (E), or (C) shielded (S) or both (B) eyes resulted in minimal to no AP-1 inhibition. (C) The exposed, normal retina (EM102) did not show any increase in AP-1-binding activity. Note that the AP-1 binding ratio for untreated affected dogs in (C) (EMB29, -30, -34) represents the average of the three ratios. The description below the histograms represent the genotype at the RHO locus, the postexposure (PE) interval, and the treatment.

FIGURE 3

c-Fos immunoreactive pattern in nuclear proteins of light-exposed (E) or shielded (S) retinas. The normal retina (N164) showed a distinct c-Fos band of ∼50 kDa, and there were no differences between E and S eyes. The untreated, affected mutant retina (EM60) also showed a pattern similar to normal in S eye, but exposure to light (E) resulted in the appearance of a higher molecular mass band at ∼56 kDa, which represents phosphorylated c-Fos. Direct ocular treatment with TRIAM did not prevent c-Fos phosphorylation (EMB18). Exposure to light after systemic DEX resulted in a smear in the p-c-Fos position, an indication of slight and partial c-Fos phosphorylation (EM59, EM67). The description below the gels represents the genotype at the RHO locus, the postexposure (PE) interval, and the treatment. The dashed lines in the gels indicate that the images represent lanes from the same gel that were not adjoining.

FIGURE 4

Phosphorylated ERK1/2 in retinal postnuclear Rernatant fractions from T4R RHO mutant dogs 1 hour after exposure to light. (A) Comparable levels of phosphorylated ERK1/2 were detected by immuno-blot analysis in both light-exposed (E) and shielded (S) retinas of a dog treated with systemic DEX (EM67), but a significantly increased level of p-ERK1/2 was found in the exposed retina of untreated mutant (EM66). (B, C) Increased p-ERK1/2 levels in the E retinas were found in animals in which the TRIAM was administered to the exposed (B) or shielded (C) eyes. There was some degree of variability in the level of p-ERK1/2 phosphorylation in the exposed retinas of TRIAM-treated eyes, but all showed increased p-ERK1/2 levels with exposure to light. Tubulin or β-actin immunoblots were used as a loading control for normalization. The lines below the p-ERK1/2 gels represent the genotype at the RHO locus, and the treatment. The dashed lines in the gels indicate that the images represent lanes from the same gel that were not adjoining.

FIGURE 5

Ratios of phosphorylated ERK1/2 (E/S, exposed/shielded) in T4R RHO mutant retinas 1 hour after light exposure. (A) Untreated, exposed mutant retinas showed a marked increase in p-ERK1/2, but it was inhibited by systemic DEX. The exposed retinas showed light-dependent increases in p-ERK1 and p-ERK2 after treatment with TRIAM, which was administered to the exposed (B) or shielded (C) eyes. In general, p-ERK1/2 levels were variable between animals, and increases were more marked in p-ERK1 than for p-ERK2. The lines below the histograms represent the genotype at the RHO locus, and the treatment.

FIGURE 6

Photomicrographs of plastic-embedded retinal sections from eyes collected 2 weeks after clinical exposure to light. Images were taken approximately 1250 μm from the optic disc along both the superior (A₁, A₃; B₁, B₃; C₁, C₃; D₁, D₃) and inferior (A₂, A₄; B₂, B₄; C₂, C₄; D₂, D₄) meridians from the exposed (A₁, A₂; B₁, B₂; C₁, C₂; D₁, D₂) and shielded (A₃, A₄; B₃, B₄; C₃, C₄; D₃, D₄) eyes. (A) The untreated mutant (EM109) showed extensive outer retinal degeneration and loss of RPE in the exposed retina, but the shielded retina was normal; (A₂, arrow) intraretinal pigmented cell. (B) Systemic DEX (EM110) the photoreceptors and outer retinal layers degenerated in the exposed eye, but the shielded retina was normal. An incomplete, and structurally compromised RPE layer remained (B₁, B₂, *), and the ONL consisted of one to two incomplete rows of nuclei. (C) Sconj and IVit TRIAM administered to both eyes (OU) of a mutant dog (EM103) before exposure to light did not prevent RPE, photoreceptor, and outer retinal degeneration. Note presence of hypertrophied cells external to the inner nuclear layer (INL) with cytoplasmic inclusions that have cytologic characteristics of macrophages (C₁, arrows). The TRIAM-treated shielded, mutant retina remained normal (C₃, C₄). (D) Intravitreal TRIAM administered to both eyes (OU) of a normal dog (EM100) before exposure to light caused no adverse effects in the exposed or shielded retinas. OS, outer segment layer; IS, inner segment layer; OPL, outer plexiform layer. Scale bar, 20 μm.

FIGURE 7

Morphologic and immunohistochemical characterization of the retina from light-exposed (EM108, OS; EM112, OS) and shielded (EM112, OD) T4R RHO mutant dogs; dog EM108 was treated with systemic DEX, and dog EM112 was not treated. All sections from an individual eye were from a series of serial sections taken from the same region. (A₁) Marked thinning of the ONL, and loss of photoreceptor inner and outer segments in the DEX-treated, light-exposed mutant retina. Note the preservation of the RPE (arrows). (B₁) Similar loss of photoreceptors in the untreated, light-exposed retina. Note, however, the absence of the RPE. (C₁) Normal retinal structure and lamination in the shielded eye. (A₂) Weak RPE65 labeling (arrows; green) and absence of GFAP labeling (red) in the DEX-treated light-exposed mutant retina. (B₂) Strong GFAP labeling in the untreated, light-exposed retina; the absence of RPE65 labeling resulted from RPE loss. (C₂) The shielded mutant retina was normal, and RPE labeling was intense and distinct. (A₃) The majority of the remaining cell somata in the ONL of the DEX-treated, light-exposed mutant retina were human cone arrestin (hCAR) positive (arrowheads; red); punctate labeling of some cells with the rod opsin antibody is also present (arrows; green). (B₃) Similar findings in the untreated, light-exposed retina. (C₃) Normal pattern of rod opsin (green) and hCAR (red) labeling in the shielded mutant retina. TL, tapetum lucidum; OS, outer segments; IS, inner segments; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar, 20 μm.

FIGURE 8

Double fluorescence immunohistochemistry with p-c-Fos (green) and CRALBP (red) antibodies in T4R RHO mutant retinas 1 hour after exposure to light. Images were taken from the midperipheral region of the superior retinal meridian in light-exposed (A₁, B₁, C₁) and shielded (A₂, B₂, C₂) eyes of untreated animals (No Rx), or after steroid administration (systemic DEX or ocular TRIAM). Colocalization of p-cFos and CRALBP was seen in the Müller cell somata of the light-exposed retinas (A₁: EM141 OS; B₁: EM227 OS; and C₁: EM226 OS) but not in the shielded eyes (A₂: EM142 OD; B₂: EM227 OD; and C₂: EM226 OD). Scale bar: 20 μm.

FIGURE 9

Double fluorescence immunohistochemistry with p-ERK1/2 (green) and CRALBP (red) antibodies in T4R RHO mutant retinas 1 hour after exposure to light. Images were taken from the midperipheral region of the superior retinal meridian in light-exposed (A₁, B₁, C₁) and shielded eyes (A₂, B₂, C₂) of untreated animals (No Rx) or after steroid administration (systemic DEX or ocular TRIAM). Colocalization of p-ERK1/2 and CRALBP was seen in the Müller cell somata of the light-exposed retinas (A₁: EM141 OS; B₁: EM227 OS; and C₁: EM226 OS) but not in the shielded eyes (A₂: EM142 OD; B₂: EM227 OD; and C₂: EM226 OD). p-ERK1/2 labeling was reduced in the exposed retinas treated with either systemic DEX (B₁: EM227 OS), or ocular TRIAM (C₁: EM226 OS) in comparison with that observed in the untreated exposed retina (A₁: EM141 OS). Scale bar, 20 μm.

FIGURE 10

The events that occur after clinical light exposure that cause photoreceptor degeneration in the T4R RHO mutant retina in the presence or absence of systemic DEX. In the untreated mutant retina, exposure to light resulted in phosphorylation of ERK1/2 and c-Fos and increased DNA binding by AP-1 (AP-1 activation). Our previous results suggest that AP-1 activation results in a cell survival response initiated in Müller cells. Treatment with systemic DEX inhibits phosphorylation of ERK1/2 and c-Fos, and AP-1 DNA binding activity is not increased. This may result in a decreased cell survival response mediated by Müller cells. However, regardless of systemic DEX treatment, clinical light exposure of the T4R RHO mutant retina leads to photoreceptor cell death.