Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin

Abstract

Purpose: To characterize the reactions of retinal glial cells (astrocytes and Müller cells) to retinal injury in mice that lack glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) and to determine the role of glial cells in retinal detachment (RD)-induced photoreceptor degeneration.

Methods: RD was induced by subretinal injection of sodium hyaluronate in adult wild-type (WT) and GFAP-/-Vim-/- mice. Astroglial reaction and subsequent monocyte recruitment were quantified by measuring extracellular signal-regulated kinase (Erk) and c-fos activation and the level of expression of chemokine monocyte chemoattractant protein (MCP)-1 and by counting monocytes/microglia in the detached retinas. Immunohistochemistry, immunoblotting, real-time quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used. RD-induced photoreceptor degeneration was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and measurement of outer nuclear layer (ONL) thickness.

Results: RD-induced reactive gliosis, characterized by GFAP and vimentin upregulation, Erk and c-fos activation, MCP-1 induction, and increased monocyte recruitment in WT mice. Absence of GFAP and vimentin effectively attenuated reactive responses of retinal glial cells and monocyte infiltration. As a result, detached retinas of GFAP-/-Vim-/- mice exhibited significantly reduced numbers of TUNEL-positive photoreceptor cells and increased ONL thickness compared with those of WT mice.

Conclusions: The absence of GFAP and vimentin attenuates RD-induced reactive gliosis and, subsequently, limits photoreceptor degeneration. Results of this study indicate that reactive retinal glial cells contribute critically to retinal damage induced by RD and provide a new avenue for limiting photoreceptor degeneration associated with RD and other retinal diseases or damage.

FIGURE 1

Upregulation of GFAP and vimentin in astroglial cells after RD. Representative photographs of immunodetection of GFAP and vimentin in retinal sections of the control (RD^-) and detached (RD⁺) retinas 3 days after surgery show increased expression of GFAP and vimentin in the retina with RD. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer.

FIGURE 2

Absence of GFAP and vimentin suppresses RD-induced Erk and c-fos activation in retinal glial cells. (A) Representative photographs of retinal sections that reveal immunoreactivity of pErk or c-fos in retinal sections of WT mice and GFAP^-/-Vim^-/- mice 3 days after sham operations (RD^-) or RD(RD⁺). Arrowheads: pErk⁺ or c-fos⁺ astrocytes in the GCL. Arrows: pErk⁺ or c-fos⁺ Müller cells in the INL. Quantification of pErk⁺ (B) and c-fos⁺ (C) cells in the INL. After RD, the number of pErk⁺ cells in retinal sections of WT mice was significantly higher than in GFAP^-/-Vim^-/- mice.

FIGURE 3

Western blot analysis of Erk phosphorylation after RD. (A) Representative photograph of immunoblots reacted with anti-pErk (pErk) and anti-total Erk (Erk) antibodies 3 days after mice underwent sham operation or RD. (B) Quantification of the relative ratio of pErk to total Erk in WT retinas (gray) and in GFAP^-/-Vim^-/- retinas (black, G^-/-V^-/-). pErk was significantly induced after RD in WT but not in GFAP^-/-Vim^-/- mice.

FIGURE 4

MCP-1 induction in WT but not GFAP^-/-Vim^-/- retinas after RD. (A) Representative photographs of anti-MCP-1 staining in WT and GFAP^-/-Vim^-/- mice, and triple labeling of a WT retina with anti-MCP-1, anti- glutamine synthetase (GS) and DAPI 3 days after sham operations (RD^-)or RD (RD⁺). Arrows: MCP-1⁺ cells in the INL. (B) Quantification of MCP-1 protein expression by ELISA in retinas with (RD⁺) or without RD (RD^-). (C) Quantification of MCP-1 mRNA levels by RT-PCR in retinal tissues with RD in WT (WT) or GFAP^-/- Vim^-/- mice (G^-/-V^-/-). Expression of MCP-1 was significantly increased in the retinas of WT but not of GFAP^-/-Vim^-/- mice 3 days after RD.

FIGURE 5

Monocyte recruitment into the detached retinas of WT but not of GFAP^-/-Vim^-/- mice. IHC of anti- CD11b, a monocyte marker, in retinal sections of WT (A) and GFAP^-/- Vim^-/- (B) mice 3 days after RD. CD11b⁺ cells were detected in the OPL (arrows) and SRS (arrowheads) of WT mice. Quantification of CD11b⁺ cells in the OPL (C) and SRS (D) with or without RD. The number of CD11b⁺ cells was significantly reduced in GFAP^-/-Vim^-/- retinas after RD.

FIGURE 6

RD-induced photoreceptor apoptosis in WT and GFAP^-/-Vim^-/- mice. TUNEL assay in the detached retinal areas of WT (A) and GFAP^-/- Vim^-/- (B) mice 3 days after RD. (C) Quantification of TUNEL-positive cells. Note that the number of TUNEL-positive cells is significantly lower in the retinas of GFAP^-/-Vim^-/- mice than in those of WT mice.

FIGURE 7

Absence of GFAP and vimentin genes prevents RD-induced photoreceptor degeneration. (A) Hematoxylin and eosin-stained retinal sections taken from WT and GFAP^-/-Vim^-/- mice 7 days after sham operation (RD^-) or RD (RD⁺). (B) Quantification of ONL thickness. Note that the decrease in ONL thickness after RD is significantly reduced in retinas of GFAP^-/-Vim^-/- mice than in those of WT mice.