Taurine prevents the RGC death in NMDA-treated retinal explants.

<p>A) Digitalized reconstruction of a whole flat-mounted retinal explant immunolabeled with the POU4F1 (Brn3a) antibody. B–E) Representative enlarged fields from flat-mounted retinal explants acquired with the automated platform, showing POU4F1-immunopositive RGCs in a control untreated condition (Control; B) after 100 µM NMDA application (NMDA; C), after co-application of NMDA with taurine (1 mM; NMDA+Taurine; D) or after co-application of NMDA with MK801 (100 µM; NMDA+MK801; E) for 4 days. F) Quantification of RGC densities from whole flat-mounted retinal explants using the automated counting platform in Control group (n = 33, white bar); NMDA group: (n = 31, hatched bar); NMDA+Taurine group (n = 23, black bar) and Taurine group (n = 6, grey bar). G) Quantification of POU4F1-immunopositive RGC densities from whole flat-mounted explants under the control group (white bar), or the NMDA group (hatched bar), the NMDA plus MK 801 group (black bar), or finally the MK-801 group (grey bar) (n = 6 for each group). Data are expressed as means ± s.e.m. from n independent experiments. ***p<0.001, *p<0.05; One-way ANOVA followed by a Bonferroni post-hoc test. The scale bar represents 100 µm in panels (B–E).</p

Implication of the taurine transporter in retinal ganglion cell (RGC) survival.

<p>A–C) Gene amplification (PCR) of the taurine transporter (Tau-T; A), rhodopsin (Rho; B) and POU4F1 (Brn-3a; C) performed on total cDNAs, previously obtained through a reverse transcription of total RNA extracted from freshly purified rat RGC (not cultured), rat full retina (Ret), rat liver (Liv), or rat kidney (Kid). Tau-T gene amplification revealed a high expression in pure RGCs (A). The purity of the RGC preparation was indicated by the high level of PCR products for POU4F1 (Brn-3a), a specific RGC transcription factor in the retina (Brn-3a in C) whereas no cDNA amplification was obtained for Rhodopsin (Rho), a major rod photoreceptor marker (Rho in B). D) Quantification by qPCR of mRNA expression for Brn3a, Rhodopsin (Rho) and the taurine transporter (Tau-T) in freshly purified RGCs (RGC, white bars) compared to the entire retina (Ret, black bars) from Long-Evans rats. The absence of rhodopsin cDNA amplification indicated that the taurine transporter expression in freshly purified RGCs (not cultured) cannot be attributed to a rod photoreceptor contamination. Results, normalized to GAPDH expression and presented as arbitrary units (A.U.), are means ± s.e.m. from 4 independent experiments for each gene tested. ***p<0.001 Ret groups compared to RGC groups, Two-tailed Student’s t-test. E–G) Representative confocal images showing Tau-T immunolabelling (green; F) in βIII-tubulin positive RGCs (red; E) after 6 days in culture. H–J) Representative confocal image of rat retinal sections immunolabelled with POU4F1 (red; H) and Tau-T (green; I) antibodies to illustrate localization of Tau-T in the ganglion cell layer (merge; J). K) Suppression of the taurine-elicited RGC survival by the taurine uptake inhibitor, GuanidinoEthane Sulfonate (GES). Quantification of RGC densities after 6 DIV either in control conditions (Cont, n = 11; white bar), in the presence of 1 mM taurine (Taur, n = 10; black bar), in the presence of 1 mM GES (GES, n = 9; oblique hatched bar) or in the presence of both taurine and GES (Taur+GES, n = 11; horizontal hatched bar). Illustrated data are means ± s.e.m. from independent experiments. ONL: outer nuclear layer; INL: Inner nuclear layer; GCL: Ganglion cell layer. ***p<0.001, **p<0.01 and *p<0.05, one-way ANOVA followed by a Dunns post-hoc test. Scale bars represent 10 µm in panels (E–G) and 20 µm in panels (H–J).</p

Taurine supplementation prevents RGC degeneration in glaucomatous Long-Evans rats following episcleral vein occlusion.

<p>A) Taurine plasmatic levels in Long-Evans rats drinking taurine-free water (Wat; green bar; mean ± s.e.m., n = 21) or taurine-supplemented water for 3 months (Taur; red bar; mean ± s.e.m., n = 24; **p<0.01, student’s t-test). B) Intraocular pressure (IOP) levels measured at regular time intervals after episcleral vein occlusions by cauterization on the operated right eyes (Caut) and unoperated left eyes (Cont) in rats drinking taurine-free water (Wat, green bars) or in taurine-treated rats (Taur; red bars). The difference between the IOP remained statically significant between the operated and unoperated eyes in both animal groups throughout the study (mean ± s.e.m., n>14 for all groups; ***p<0.001 as compared to water/control eyes, one-way ANOVA followed by a Bonferroni post-hoc test). C) Representative photopic electroretinograms (ERG) recorded from an operated right eye (Caut eye) and an unoperated left control eye (Cont eye) after 3 months of episcleral vein occlusion in taurine-treated rats (Taur, red traces) and in control rats drinking taurine-free water (Wat; green traces). D) Quantification of photopic ERG amplitudes measured from the left unoperated eyes (Cont eyes) and right operated eyes (Caut eyes) in taurine-supplemented rats (Taur; red bars; mean ± s.e.m., n = 24) or control rats drinking taurine-free water (Wat; green bars; mean ± s.e.m., n = 32 for control eyes and 40 for cauterized eyes) after 3 months of vein occlusion. E) Complete rat retinal section viewed with a digital fluorescence scanner (Nanozoomer) showing POU4F1 (Brn3a) immunolabeling (red) and nuclear staining with DAPI (blue). F-I) Representative confocal images of retinal cryo-sections showing the POU4F1 (Brn3a) positive RGC immunolabeling (red) and cell nuclei staining (DAPI; blue) performed in unoperated left eyes (Cont; F,H) and cauterized right eyes (Caut; G,I) in rats without (Wat; F, G) or with taurine supplementation (Taur; H, I) added to their drinking water. J) Quantification of POU4F1 (Brn3a) positive RCG densities in both unoperated left (Cont eye) and operated right (Caut eye) eyes from rats without (Wat; green bars; mean ± s.e.m., n = 11 and 10 for control and cauterized eyes, respectively) or with taurine supplementation (Taur; red bars; mean ± s.e.m., n = 11 and 9 for control and cauterized eyes, respectively). *p<0.05 and ***p<0.001 as compared to indicated group in (B, D, J), one-way ANOVA followed by a Bonferroni post-hoc test. Scale bars represent 4 mm in panel (E) and 50 µm in panels (F-I).</p

Taurine prevents RGC degeneration in P23H rats with blood vessel atrophy.

<p>A, B) Representative pictures showing the lectin staining of blood vessels at the periphery of flat-mounted retinae from a one-year old Sprague-Dawley rat (A) and a one-year old heterozygous P23H rat (B). C,D) Quantification of number of blood vessel branching points (C) and percentage of blood vessel coverage (D) obtained on the whole retina by an automated platform in Sprague-Dawley wild-type animals (WT; white bar), in untreated heterozygous P23H rats (P23H Wat; grey bar) and in taurine-supplemented P23H rats (P23H Taur; black bar). These quantifications demonstrated the absence of taurine effect on blood vessel atrophy (means ± s.e.m., n = 5 for each group). E) Quantification of POU4F1 (Brn3a) immunopositive RGCs in retinal cryosections from Sprague-Dawley wild-type animals (WT, white bar), from untreated heterozygous P23H rats (P23H Wat; grey bar) and from taurine supplemented P23H rats (P23H Taur; black bar). Data expressed as RGC per mm of retinal section, are means ± s.e.m. from n = 7 animals for each group. ***p<0.001 and *p<0.05 as compared to indicated groups, one-way ANOVA followed by a Bonferroni post-hoc test. Scale bar represents 100 µm in panels (A–B).</p

Taurine stimulates the survival of pure adult retinal ganglion cells (RGCs) in culture.

<p>A–F) Purity of RGC cultures. Confocal representative images of pure RGC cultures immunolabeled with specific RGC markers, NF-200 (red in A) or βIII-Tubulin (red in B), as well as with a specific marker for microglia/macrophages, Cd11b/c (red in C), while all isolated cells were stained by the nuclear dye DAPI (blue in D–F). Note that most cells were immunolabeled with the RGC markers (NF-200 or βIII-Tubulin) (A,B,D,E) whereas a very few cells were positive for the macrophage marker (C,F). G–H) Effect of taurine on pure RGCs. Representative images showing viable cultured RGC labeled with calceinAM, after 6 days <i>in vitro</i> (6 DIV), in the negative control condition (Cont; G) and following 1 mM taurine application (Taur; H). I) Quantification of RGC densities after 6 DIV either in the control condition (Cont; white bar), with 1 mM taurine application (Taur; black bar), or with the B27 supplement, providing a positive control condition (Pos; grey bar). In each experiment, the respective RGC densities were expressed as a percentage of the negative control condition at 6 DIV. Illustrated data are means ± s.e.m. from 21 independent experiments. ***p<0.001, one-way ANOVA followed by a Dunns post-hoc test. Scale bars represent 100 µm in panels (A–H).</p

Taurine supplementation prevents RGC degeneration in DBA/2J mice, a genetic animal model of pigmentary glaucoma.

<p>A–C) Iris dispersion in DBA/2J mice. Normal iris in C57BL/6J mouse (A) and iris with pigment dispersion in DBA/2J mice drinking either taurine-free water (B) or taurine-supplemented (0.2 M) water (C) for 4 months. D) Taurine plasmatic levels in 12-month old C57BL/6J mice (C57/wat; purple bar) and DBA/2J mice drinking taurine-free water (DBA/Wat; green bar) or taurine-supplemented water (DBA/Taur; red bar). Data are means ± s.e.m. obtained from 8–10 animals per group. E) Intraocular pressure (IOP) levels measured at regular time intervals (8 to 12 months) on right eyes either from C57BL/6J mice (C57/wat; purple bars), from control DBA/2J mice (DBA/Wat; green bars) or from taurine-treated DBA/2J mice (DBA/Taur; red bars). Note the increased IOP in DBA/2J mice when compared to C57BL/6J mice but the lack of effect for the taurine supplementation on this increased IOP. Data (mm of Hg) are means ± s.e.m., n>10 animals for each group. F) Complete mouse retinal section viewed with a digital fluorescence scanner (Nanozoomer) showing POU4F1 (Brn3a) immunolabeling (red) and nuclear staining with DAPI (blue). G–I) Representative confocal images of retinal cryosections showing POU4F1-positive RGC immunolabeling (Brn3a; red) and retinal cell nuclei staining (DAPI; blue) in taurine-treated DBA/2J mice (DBA/Taur; H) and control DBA/2J drinking taurine-free water (DBA/Wat; H, I) as compared to C57BL6/J mice (C57/Wat; G). J) Quantification of RGC density on right eye retinal sections in C57BL/6J mice drinking taurine-free water (C57/Wat; purple bar; mean ± s.e.m, n = 6), in DBA/2J mice drinking either taurine-free water (DBA/Wat; green bar; mean ± s.e.m., n = 7) or taurine-supplemented water for 4 months (DBA/Taur; red bar; mean ± s.e.m., n = 6). ***p<0.001, **p<0.01 and *p<0.05, as compared to C57/Wat group in (E) or as compared to indicated groups in (D, J), One-way ANOVA followed by a Bonferroni post-hoc test. Scale bars represent 2 mm in pannel (F) and 50 µm in pannels (G–I).</p