A biochemical basis for induction of retina regeneration by antioxidants

Abstract

The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. Embryonic chicks are able to regenerate their retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye only if a source of exogenous factors, such as FGF2, is present. This study shows that NAC modifies the redox status of the CM, initiates self-renewal of the stem/progenitor cells, and induces regeneration in the absence of FGF2. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with different biochemical structures and modes of action, and found that NAC induces regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway.

Keywords: NAC; Regeneration; Retina.

Conflict of interest statement

Competing interests

No competing interests declared.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Fig. 1

Redox status in the chick ciliary margin post-retinectomy (PR). (A) Histological section of an embryonic day 4 (E4) chick eye showing the structures of the eye. Anterior (Ant) and posterior (Post) regions. L: Lens; CM: Ciliary margin; NE: neuro epithelium; RPE: Retinal pigmented epithelium. (B–M) Immunohistochemistry using the immunospin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) antibody on sections of the CM of chick eyes at (B) E4, (C) E5, (D) 6h and (E) 24h post-retinectomy (PR) only, as well as with the following treatments: (F, G) NAC at 6h and 24h PR respectively, (H, I) Vitamin C at 6h and 24h PR respectively (J, K) XJB 5–131 at 6h and 24h PR respectively, and (L, M) FGF2 at 6h and 24h PR respectively. Scale bar in M is 125 μm and applies to all. (N, O) Graphical representations of the ratio (intensity/area) of the signals detected in B–E and F–M respectively. Statistical analysis was performed using Dunnett multiple comparisons. The first analysis compared developmental samples and retinectomy samples and are shown in (N). Retinectomy significantly increased the level of oxidized proteins at both 6h and 24h PR (Dunnett 6h * = 0.05 and 24h * = 0.04). The second analysis compared NAC, Vitamin C, XJB 5–131, and FGF2 to Ret and results are shown in (O). NAC significantly reduced the level of oxidized proteins at both 6h and 24h PR (For NAC compared to Ret, Dunnett 6h * = 0.04 and 24h ** = 0.005; for XJB 5–131 compared to Ret, Dunnet 6h *= 0.05 and 24h *= 0.05). As an aside test, FGF2 was compared to Ret and showed marginal significant differences (Dunnett 6h = 0.07 and 24h = 0.06).

Fig. 2

The antioxidant NAC induces chick retina regeneration. (A–C, E, F) Histological analysis of chick eyes collected 7d PR treated with (A) NAC, (B) FGF2, (C) retinectomy + vector (PBS), (E) Vitamin C, or (F) XJB 5–131. Scale bar in F is 125 μm and applies to all. L: Lens; RPE: Retinal pigmented epithelium; ciliary regeneration (CR) and RPE transdifferentiation (TD). (D) Quantitative analysis of the mean level of regeneration for each treatment: NAC (n = 10), FGF2 (n = 10). Statistical analysis was performed using nonparametric Mann-Whitney-Wilcoxon tests for both CR and TD. There was no significant difference in the regeneration induced from the CM by NAC and FGF2 treatments (Wilcoxon S = 115.0, P = 0.4813), however, FGF2 treated eyes showed a significant increase in RPE TD when compared to NAC (Wilcoxon S = 138.0, *P = 0.0107).

Fig. 3

NAC-treatment enhances cell proliferation in the CM after retina removal. (A) Graphical representation of the mean level of EdU+ or (B) PH3+ cells after treatment with NAC for indicated times. Statistical analysis was performed using Dunnett multiple comparisons. Treatment with NAC resulted in a significant number of EdU+ cells compared to eyes receiving retinectomy only at 6h, 24h, and 3d PR (Dunnett for EdU+ cells: *P6hPR = 0.0170, **P24hPR = 0.0061 and **P3dPR = 0.0065; and for PH3 it was only significant at 3d PR. Dunnett for PH3+ cells: P6hPR = 0.3694, MP24hPR = 0.0587 and **P3dPR = 0.0024). M = marginal significance.

Fig. 4

Cell differentiation during retinal regeneration. (A–X) Immunofluorescence for cell makers for the different cell types of the retina at 7d PR and 11d PR: (A–D) Visinin labeled photoreceptors, (E–H) Vimentin labeled Müller glia, (I–L) Brn3a labeled ganglion cells and NAPA 73 labeled ganglion axons, (M–P) Ap2 labeled amacrine cells, (Q–T) Lim 1/2 labeled horizontal cells, (U–X) Pax6 labeled amacrine, horizontal and ganglion cells and Vsx2 labeled bipolar cells. Scale bar in X is 125 μm and it applies to all panels. (Y) Graphical representation of the number of positive cells for each marker. After 7d PR the number of immunofluorescent+ cells present in NAC treated eyes was significantly lower compared to eyes treated with FGF2 for Visinin (*P = 0.0118), Vimentin (***P = 0.0006), Brn3a (***P = 0.0008), Ap2 (*P = 0.0123), Lim1/2 (*P = 0.0119), Pax6 (**P = 0.0026) and Vsx2 (**P = 0.0051) and marginal for Napa 73 (MP = 0.0672). After 11d PR the number of immunofluorescent+ cells present in NAC treated eyes was significantly lower compared to eyes treated with FGF2 for Vimentin (***P = 0.0009), Ap2 (*P=0.0415) and Lim1/2 (*P = 0.0115), marginal for Visinin (MP = 0.0641), but was not significant for Brn3a (P = 0.9721), Napa 73 (P = 0.1300), Pax6 (P = 0.7596) and Vsx2 (P = 0.2537). The number of immunofluorescent+ cells was also compared between 7d PR with 11d PR in the NAC treated eyes, and there was significance difference for Vimentin (*P = 0.0120), Pax6 (***P = 0.0002) and Vsx2 (***P = 0.0001), marginal for Ap2 (MP = 0.0529), but not for Visinin (P = 0.1141), Brn3a (P = 0.1360), Napa 73 (P = 0.3078), and Lim1/2 (P = 1.00). The significance is not shown in the graph for the comparison of 7d PR and 11d PR NAC treated eyes. Note that the Pax6+ cells used for the comparisons were only the ones present in the INL excluding horizontal cells, so it is representative of amacrine cells. Mixed model ANOVA was used for the statistical analysis.

Fig. 5

The presence of stem/progenitor markers are enhanced during NAC-induced retina regeneration. (A–D) Immunofluorescence for the cell surface antigen SSEA1 indicates the presence of progenitor cells in the (A, B) anterior (ANT) and (C, D) posterior retina (POST) at 7d PR. Eyes were treated with (A, C) NAC or (B, D) FGF2. (E–H) Double immunofluorescence for the transcription factors, Vsx2 (green) and Pax6 (red) indicates the presence of progenitor cells in the (E, F) anterior and (G, H) posterior retina at 7d PR in eyes treated with (E, G) NAC or (F, H) FGF2. Scale bar in D is 125 μm and it applies to A–D, and in H, it is 60 μm and it applies to E–H. CM: ciliary margin; CR: ciliary regeneration; L: lens. (I) Quantitative analysis of the mean presence of SSEA-1 and Vsx2/Pax6+ cells in NAC and FGF2 treated eyes at 7d PR. There is a significant difference in the ANT, between eyes treated with NAC and FGF2 for both markers SSEA1: (F(1,4) = 212.64, ***P = 0.0001); Vsx2/Pax6: (F(1,4) = 14.77, *P = 0.0184). In the POST, there was a significant difference in eyes treated with NAC for Vsx2/Pax6 (F(1,4) = 8.82, *P = 0.0412) but not for SSEA1 (F(1,4) = 4.62, P = 0.0981). Mixed model ANOVA was used for the statistical analysis.

Fig. 6

Increased levels of glutathione (GSH) are not required during NAC-induced retina regeneration. (A) Schematic representation of the GHS pathway indicating the inhibitory mechanism of L-Buthionine sulfoximine (BSO) on γ-glutamyl-cysteine synthase, and diethyl maleate (DEM) on GSH. (B) Graphical representation of the GSH/GSSH ratio in the CM after 6h and 24h PR in eyes treated with DMSO (n = 10), NAC (n = 10), NAC + BSO (n = 10), and NAC + DEM (n = 10). Values were: at 6h PR with NAC: GSH/GSSH = 29:1, after 24h PR: NAC: GSH/GSSH = 9:1, compared to untreated controls after 6h PR: GSH/GSSH = 1:4, after 24h PR: GSH/GSSH = 0.2:8. The use of the inhibitors decreased the levels of GHS in presence of NAC. At 6h PR BSO+NAC: GSH/GSSH = 1:2.33, DEM+NAC: GSH/GSSH= 1:8, at 24h PR BSO+NAC: GSH/GSSH = 2.8:2, DEM+NAC: GSH/GSSH = 1:4. (C–E) Histological analysis of eyes collected 3d PR and treated with NAC and (C) BSO, (D) DEM, or (E) DMSO. Scale bar in E is 250 μm and it applies to all panels. L: lens; RPE: retinal pigmented epithelium; CR: ciliary regeneration. (F) Dunnett multiple comparisons was used as statistical analysis. Quantitative analysis of the regenerated area showing no statistical significance in regeneration between DMSO+NAC and BSO+NAC (P = 0.949) nor with DMSO+NAC and DEM+NAC (P = 0.4639).

Fig. 7

pErk activation is necessary for NAC-induced regeneration. (A) Quantitative analysis of pErk+ cells at 6h, 24h, 3d and 7d PR (intensity of the signal/area). Statistical analysis was done using Dunnett multiple comparisons. After 6h PR, 24h PR, 3d PR and 7d PR no significance in the intensity of pErk signal was observed when compared with FGF2 (Dunnett P6h = 0.40, P24h = 0.81, P3d = 0.38, P7d = 0.25). (B–G) Histological analysis 3d PR in eyes treated with (B) NAC + PD98059, (C) NAC + PD 173074, (D) NAC + DMSO, (E) FGF2 + PD98059, (F) FGF2 + PD173074, (G) FGF2 + DMSO. Scale bar in G is 250 μm and applies to all panels. L: lens; RPE: retinal pigmented epithelium; CR: ciliary regeneration. (H) Quantitative analysis of eyes that regenerated in presence of the different inhibitors at 3d PR. The area of the CM was included in the measurement of the amount of regeneration. The statistical analysis for the inhibitors was performed using Dunnett multiple comparisons. The amount of regeneration is significantly higher in the DMSO+NAC treated eyes compared to the eyes treated with PD98059 (MEK inhibitor) + NAC (***P = 0.0008). There is no significant difference between the DMSO+NAC and PD173074 (FGFR inhibitor) + NAC (P = 0.4973) treated eyes. However, there is a significant difference in the DMSO+FGF2 treated eyes compared to eyes treated with PD173074+FGF2 (***P = 0.0001) and PD98059+FGF (**P = 0.0013) that were used as controls for the treatments.

Fig. 8

The thiol group is critical for induction of retina regeneration. (A–C) Histological analysis of eyes collected 3d PR and treated with (A) N-acetylglycine (NAG), n = 12, (A′) NAG structure; (B) NAC, n = 10, (B′) NAC structure; or (C) N-acetylserine (NAS), n = 10, (C′) NAS structure. Scale bar in C is 180 μm and it applies to A and B. Histology after 3d PR of embryos treated with different antioxidants. L: lens; RPE: retinal pigmented epithelium; CR: ciliary regeneration.

Fig. 9

A model for the induction of chick retina regeneration by NAC. Arrows in yellow represent the different roles of the antioxidant NAC. Arrows in red represent the different molecules that feed into the specific roles of NAC. When the GSH synthesis role of NAC was tested through the use of specific inhibitors, regeneration still occurred. The use of another N-acetyl amino acid, N-acetylglycine (NAG), which can feed into the GSH synthesis pathway, did not result in regeneration. When different molecules that can scavenge ROS were used, no regeneration occurred. In addition, N-acetylserine (NAS) which is similar in structure to NAC but lacks the thiol group in carbon 3, and instead has a hydroxyl group, was unable to induce regeneration. Our data suggests that NAC’s thiol group is essential for the activation of Erk and subsequent induction of retina regeneration.