Morphometric analysis of vascular network remodeling after BRVO.

<p>(A and B) Representative micrographs of CollIV-labeled retinal flatmounts of a control retina (A) and of the occluded vein after BRVO at 3d (B). Positions of the measurements at 800, 1200 and 1600μm from optic nerve were indicated by arrowhead. (C) Measurements of the retinal vein diameter of control retinas or of non occluded side and in the occluded vein at 1, 3 and 7d and indicated distance from the optic nerve after BRVO. Values in histograms are mean ± SEM of diameter (μm) of control or occluded vein in the retina from 4–11 eyes per group. Mann-Whitney non parametric test, * p<0.05 compared to control (0), non occluded veins of the same eye and intact eyes. (D and E) Representative micrographs of CollIV-labeled retinal flatmounts of the superficial capillary network of a control retina (D) and upstream of the occluded vein at 3d (E). (F and G) Quantification of the surface covered by CollIV-positive capillaries (F) and length (capillary length per surface) at different time points. Values in histograms are mean ± SEM of surface or length of capillaries in the retina from 4–18 eyes per group. Mann-Whitney non parametric test, * p<0.001 compared to control (0), non occluded veins of the same eye and non-occluded eyes. Scale bar = 100μm.</p

BRVO induced vascular and inflammatory markers expression.

<p><i>TGFβ1</i>, <i>TSP-1</i>, <i>COX-2</i>, <i>VEGF</i>, and <i>FGF2</i> real-time RT-PCRs in the occluded retina at indicated time points. The results were normalized to S26 expression. Values in histograms are mean ± SEM of mRNA expression of occluded area from 4 eyes per group. Mann-Whitney non-parametric test, * p<0.05 compared to non occluded control.</p

NG2-staining and pericyte counts after BRVO.

<p>(A-D) Representative micrographs of NG2 (green)-CollIV (red) double labeled retinal flatmounts of control (A and B) and at 7d after BRVO (C and D). DAPI is represented in blue in the insets of panel B and D. (E) Quantification of NG2 positive pericytes of control capillaries and capillaries upstream of the occluded vein at indicated time points. Values in histograms are mean ± SEM of number of NG2+ nuclei/mm² of vascular area. n = 4–18 per group. Mann-Whitney non parametric test, * p<0.05 compared to control (0), intact retina or opposite part of the BRVO retina. Scale bar = 100μm, 25 μm for insets.</p

Retinal thickness and hemorrhages after experimental BRVO.

<p>(A and B) Representative SD-OCT image of the superior pole at ∼900μm of the optic nerve of a control retina (A) and after BRVO (B; ∼400μm upstream of the occlusion) at d1. (C) Quantification of the thickness of occluded and sham-lasered inner retina, measured from the OPL to the retina, at ∼900μm of the optic nerve at different time points. Values in histograms are mean ± SEM of thickness of inner retina from 3–16 eyes per group. Mann-Whitney non parametric test, * p<0.05 compared to control (0). Control are values of the retina before BRVO.D and E: Photographs of the appearance of inner retinal hemorrhages at d1 (* occlusion site). IPL: inner plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; Scale bar A and B = 20μm.</p

Vascular endothelial cell apoptosis and proliferation after BRVO.

<p>(A and B) Representative micrographs of TUNEL (green)—CollIV (red) double labeled retinal flatmounts at 1d after BRVO of the occluded vein (A) and the upstream capillary bed (B). (B- right panels) Detail of a DAPI(blue)-labelled upstream capillary depicting a normal nucleus (arrow) and an apoptotic TUNEL⁺nucleus (arrowhead). (C) Quantification of TUNEL⁺ECs of control retinas and in the occluded vein and upstream capillary bed at 1, 3 and 7d after BRVO. Values in histograms are mean ± SEM of number of TUNEL⁺ECs of retina from 5–10 eyes per group. Mann-Whitney non parametric test, * p<0.05 compared to control (0), intact retina or opposite part of the BRVO retina. (D and E) Representative micrographs of EdU (green)—CollIV (red) double labeled retinal flatmounts at 7d after BRVO of the occluded vein (D) and the upstream capillary bed (E). (F) Quantification of EdU positive ECs of control retinas and in the occluded vein and upstream capillary bed at 1, 3 and 7d after BRVO. Mice were daily injected intraperitoneally with EdU at d0 just after laser photocoagulation until sacrifice.Values in histograms are mean ± SEM of number of EdU+ cells of retina from 5–10 eyes per group. Mann-Whitney non parametric test, * p<0.05 compared to control (0), intact retina. Scale bars A, B, D, and E = 100μm, 20μm for insets, B right panels: 10μm.</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

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

STZ leads to transient hyperglycemia without growth retardation in neonates.

<p>P1 rat pups were injected with low doses of streptozotocin in citrate buffer (STZ) or with the control vehicle (citrate buffer, CTL). <b>A</b>. Measurements of glycemia from P1 to P21 in both groups. The STZ group displayed a moderate increase in glycemia from P3 to P6, averaging 214 to 241 mg/dl (11.9–13.4 mmol/l). <b>B</b>. Insulin concentration in serum at P6 in control (white) and STZ treated (black) animals. The STZ group demonstrated a decreased level of insulin. <b>C</b>. Survival curve in STZ- and CTL-groups. Mortality was similar in both groups. <b>D</b>. Weight from P1 to P21 in STZ- and CTL-groups. Weight gain was not affected in STZ-injected animals when compared to control animals. Data in A and D were analyzed by a two-way ANOVA followed by a Bonferroni post test. Data in B were analyzed by an unpaired t-test. Data in C were analyzed by a Log-rank test. * P<0.05.</p

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