Quantification of laser-induced choroidal neovascularization (CNV) in C57BL6/JRj.

<p>(A) Laser-induced CNV (Yag 532 Eyelite parameters: 100 µm, 50 ms, 400 mW) was visualized immediately after laser impact using SD-OCT imaging as described in the “materials and methods” section. Based on this image, a CNV volume is extrapolated using the following formula (4/3π*a*b²)/2, in which <i>a</i> is the polar radius and corresponds to the measure along the vertical axis and <i>b</i> is the equator radius and corresponds to the horizontal axis. (B) Linear regression showing that data obtained from extrapolation or Imaris 3D reconstruction (described step by step hereafter) are statistically equivalent (r² = 0,94, n = 8). (C) Imaris software allows a 3D rendering of SD-OCT imaging. Data shown here arise from the same SD-OCT sequence than shown in panel A. (D) The neovascularization volume, just above the RPE cell layer, was delimitating manually (representative white dotted line in one slice) in about 20 slices (over 100) along z-axis to create a 3D mask. Based on this manual delimitation the Imaris software computed a 3D mask shown in yellow (E). The final visualization, that allowed CNV volume quantification, was obtained after automated mask thresholding (F). OPL: Outer Plexiform Layer, RPE: Retinal Pigmented Epithelium, CHO: Choroid. Scale bar: 50 µm.</p

Retinal layer thickness measures in C57BL/6JRj wild-type mice by SD-OCT and histology.

<p>Retinal thickness in nasal and temporal sides in SD-OCT image (A) and in corresponding histological section (B). (C) Measures of retinal layers thickness by SD-OCT and histology in C57BL/6JRj mice, n = 11, Mann Whitney test. (D) Retinal thickness evaluated by SD-OCT and histology in C57BL/6JRj mice. Each pair of point represents the whole retina thickness of the same eye measured with SD-OCT (blue dots) and histology (orange dots). IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, OLM: outer limiting membrane, RPE: retinal pigmented epithelium. SD: Standard Deviation. Scale bars: 50 µm.</p

Characterization of a retinal degeneration mouse model by SD-OCT.

<p>SD-OCT images of control mice retina (A) and <i>rho−/−</i> mice retina (B) from post-natal day 21 (P21) to 180 (P180). Magnification (X2.4) of P21 and P180 control mice outer retina (C) and <i>rho−/−</i> mice (D). (E) Measures of INL thickness obtained from SD-OCT data in control and <i>rho−/−</i> mice (P21: <i>p</i> = 0.0123; P180: <i>p</i> = 0.7125). (F) Measures of ONL thickness obtained from SD-OCT data in control and <i>rho−/−</i> mice (P21 and P180: <i>p</i><0.0001). (G) Measures of ONL thickness obtained from morphometric measurements on cryostat sections in control and <i>rho</i>−/− mice (P15 and P180: p = 0.0022). Statistical significance of the difference between groups was analyzed at the initial time-point (P15 or P21) and the latest time-point (P180) studied by Student's <i>T</i>-test for E and F (n = 23 per group) and by Mann Whitney test for G (n = 6 per group). IPL: inner plexiform layer, INL: inner nuclear layer, ONL: outer nuclear layer, OLM: outer limiting membrane, RPE: retinal pigmented epithelium. SD: Standard Deviation. Scale bars: 50 µm.</p

SD-OCT imaging in other pathological models: rd8 mutation and light-challenge.

<p>(A) Typical ocular lesions of <i>rd8</i> mutation in <i>crb1</i> gene (C57BL/6NRj mice in which presence of the <i>rd8</i> mutation was confirmed by genotyping). (B–C) SD-OCT follow-up of the outer retina during a light-challenge in C57BL/6JRj mice. Control unexposed three month-old mouse has a normal appearance with 4 bands of different reflectance corresponding to the PR segments (B). Mice were then exposed to light during 4 days as described in the “methods” section and the retina was imaged by SD-OCT at day 3 (D3), 7, 14 and 21 after starting the illumination (C). The light-challenge leads to a temporary abolition of the distinction between the two bands forming the outer segment, with a peak at D7 (right panels: enlargement of the area enclosed by a white box on the left view). INL: Inner Nuclear Layer, ONL: Outer Nuclear Layer, IS: Inner Segments, OS: Outer Segments. Scale bars: 50 µm.</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

Image_1_Potential contributions of the intrinsic retinal oscillations recording using non-invasive electroretinogram to bioelectronics.tif

Targeted electric signal use for disease diagnostics and treatment is emerging as a healthcare game-changer. Besides arrhythmias, treatment-resistant epilepsy and chronic pain, blindness, and perhaps soon vision loss, could be among the pathologies that benefit from bioelectronic medicine. The electroretinogram (ERG) technique has long demonstrated its role in diagnosing eye diseases and early stages of neurodegenerative diseases. Conspicuously, ERG applications are all based on light-induced responses. However, spontaneous, intrinsic activity also originates in retinal cells. It is a hallmark of degenerated retinas and its alterations accompany obesity and diabetes. To the extent that variables extracted from the resting activity of the retina measured by ERG allow the predictive diagnosis of risk factors for type 2 diabetes. Here, we provided a comparison of the baseline characteristics of intrinsic oscillatory activity recorded by ERGs in mice, rats, and humans, as well as in several rat strains, and explore whether zebrafish exhibit comparable activity. Their pattern was altered in neurodegenerative models including the cuprizone-induced demyelination model in mice as well as in the Royal College of Surgeons (RCS–/–) rats. We also discuss how the study of their properties may pave the way for future research directions and treatment approaches for retinopathies, among others.</p

Scleral modifications in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant eyes.

<p>Nissl staining of retinal sections in control (<b>A</b>) and mutant (<b>B</b>) eyes shows reduced scleral thickness at P90. The choroid and occasionally the RPE appear thicker at the posterior pole of the mutant eyes. Transmission electron micrographs of the posterior sclera wall (<b>C, D</b>) shows that the collagen fibrils form well-organized lamellae in the control sclera (<b>C</b>); in the mutant fibril-poor areas and impaired packing are evident (<b>D</b>). Contrary to the control sclera the collagen fibril density is lower in all layers of the mutant sclera (<b>E</b>). Transmission electron micrographs showed fibril collagen organization within a lamella of the posterior sclera (<b>F, H</b>) and in localized areas (<b>G, I</b>) of control (<b>F, G</b>) and mutant (<b>H, I</b>). Fibrils were morphologically abnormal with irregular contours and heterogeneous diameters in the mutants. Measurements of cross-sectional diameters of fibrils from the inner, middle and outer posterior sclera in control (<b>J</b>) and mutant eyes (<b>K</b>). The mean fibril diameter distribution is modified in the mutants in all three layers; the frequency of very small as well large diameter fibrils is increased. (ch) choroid, (fb) fibroblast, (Lg) longitudinal and (Tr) transversal orientation of the cross-sectioned fibrils. Two-tailed unpaired <i>t</i> test was used. ***<i>P</i><0.001, Values are mean ± SEM of 3 animals per genotype; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129518#sec009" target="_blank">methods</a>. Scale bars: 50 μm in A, B; 3.5 μm in C, D; 1.2 μm in F, H; 300 nm in G, I.</p

MRI analysis of post-natal eye growth in control and <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants over the first year of life.

<p>High resolution sagittal slices through the optical axis of the right eye of each mouse were acquired (<b>A-H</b>). A variety of optical parameters were extracted from the MRI images collected from groups of control and mutant mice at the ages indicated (<b>I-N</b>). Growth rate of axial length and vitreous chamber depth (<b>O</b>, <b>P</b>). A two-way ANOVA post hoc Tukey test was used, <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 4 animals per age and genotype. Scale bars: 1500 μm in A-H.</p

Decreased retinal cell density is associated with increased cell death in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant eyes.

<p>Reduced retinal cell density in the ONL, INL and GCL between P5 and P90. Values are expressed in % of normal thickness of the corresponding layers. Comparisons were calculated between P3 as 100% cell density and the other time points in each layer. Two-way ANOVA post hoc Tukey was used, ***<i>P</i><0.001, ns: non statistically significant, n = 5 animals per age. (<b>A</b>). The PH3 + cells are similarly distributed in control (<b>B</b>) and mutant retinal layers at P3 (<b>C</b>). Similar proliferation indexes in control and mutant retinas between E13.5 and P1, a significant reduction is seen in the mutants at P3 and P5 (<b>D</b>). In normal retinas TUNEL + cells are essentially seen in the INL and to a lesser extent in the ONL and GCL layers (<b>E</b>, upper panel). In the mutants the TUNEL signal appears stronger in the INL, ONL and IPL (<b>E</b>, lower panel). Cell death is significantly increased in the mutants between P5 and P21 (<b>F</b>). Distribution of caspase 3+ apoptotic cells in control and mutant retinas at P7 and P21 (<b>G</b>). Apoptosis is significantly increased in the mutants (<b>H</b>). The expression of the marker of autophagy Hsp70 is particularly strong in the mutant INL (<b>I</b>). Western-blot analysis of the indicated autophagic markers; GAPDH is used as an internal loading control (<b>J</b>). Two-way ANOVA post hoc Tukey test was used, **<i>P</i><0.01, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 5 animals per age and genotype; ***p<0.01. Scale bars: 50 μm in B, C, I; 30 μm in E, G.</p

Lrp2-deficient eyes are abnormally enlarged.

<p>Sagittal cryosections through the developing eye (<b>A-D</b>). Lrp2 is expressed in the developing neuroretina (nr) at E12.5 (<b>A</b>). From E15.5 onward the signal is restricted in the lens (L) facing inner layer of the ciliary body (cb) epithelium, a low expression is also seen in the outer layer of the CB and in the retinal pigmented epithelium (rpe) (<b>B</b>). Loss of Lrp2 signal in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants at E12.5 and E15.5 (<b>C, D</b>). Sagittal cryosections of control and mutant retinas at P60 showing retinal thinning and the presence of a posterior staphyloma in the mutant (<b>E</b>, <b>F</b>). Reconstruction of the mouse face using MRI at P60 (<b>G</b>, <b>H</b>). The <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants display bilateral eye enlargement, through the anterior-posterior axis and the equatorial diameter (double headed arrows in <b>G, H</b>). Horizontal MRI images showed that the retrobulbar space, between the orbit and the eyeball, (double-headed arrows in <b>I, J</b>) is decreased in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants. The corpus callosum (arrow in <b>I</b>) is not formed in the mutants (asterisk in J). (vz) ventricular zone. Scale bars: 25 μm in A-D; 600 μm in E, F.</p