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

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

Retinal angiogenesis is inhibited in hyperglycemic animals.

<p>P1 rat pups were injected with low doses of streptozotocin (STZ) or with the citrate buffer vehicle (CTL). <b>A–B</b>. Retinal flatmounts of P6 CTL (A) or STZ (B) animals were stained using anti-collagen IV antibody. Vascularized area circumferences are highlighted in red. <b>C</b>. Vascular area measurement in hyperglycemic (black bars) or control (white bars) animals. Values in histograms are mean ± SEM of vessel area of retinas from 4–8 animals per group from 3 different experiments. * <i>P</i><0.05 compared to CTL, two-way ANOVA, Bonferroni post-test. <b>D</b>. Mean tube length, branch point density and total tube length density were determined at P6 in hyperglycemic (black bars) or control (white bars) animals. Values in histograms are mean ± SEM of retinas from 4–8 animals per group from 3 different experiments. <b>E</b>. Higher magnification of P21 retinal flatmounts of CTL and STZ animals stained with collagen IV antibody. <b>F</b>. Mean tube length, branch point density and total tube length density were determined at P21 in hyperglycemic (black bars) or control (white bars) animals. Values in histograms are mean ± SEM of retinas from 4 animals per group from 2 different experiments. Scale bar 1 mm in A–B; 200 µm in E–F. No statistical differences in vessel parameters were found in D and F between STZ and control groups using unpaired t-tests.</p

Astrocytes and pericytes phenotype in NHIR.

<p>P1 rat pups were injected with low doses of streptozotocin (STZ) or with the citrate buffer vehicle (CTL). <b>A–D</b>. Retinal flatmounts and sections of P6 CTL (A–B) and STZ (C–D) were co-stained using anti-collagen IV antibody and anti-GFAP antibody. <b>E–F</b> Retinal flatmounts of P6 CTL (E) and STZ (F) were co-stained using anti-collagen IV antibody and or anti-Ng2 antibody. <b>G–H</b>. STZ animals with hyperglycemia >400 mg/DL were co-stained using anti-collagen IV antibody and anti-Ng2 antibody. Nuclei were counterstained with DAPI in A and C. Scale bar 50 µm in A–G, g and g′; 5 µm in E and F insets and H.</p

Effect of neonatal hyperglycemia on macrophage/microglial cells (MP/MC) recruitment.

<p><b>A–F</b>. Retinal flatmounts and sections were stained with Iba-1 antibody. Representative flatmounts and sections of control (A, B) and STZ (D, E) P6 animals or P21 animals (C, F). <b>G</b>. Quantification of Iba-1 positive cells at different time points in CTL (white bars) and STZ (black bars) animals. MC recruitment peaked at 5–6 days postnatal. Values are mean +/− SEM. *p<0.05 Two-way ANOVA, Bonferroni post-test. <b>H–K</b>. Retinal sections and flatmounts of CTL (H–I) and STZ animals (J–K) were stained with Iba-1 antibody. MC/MP were located deeper in the retina of in hyperglycemic animals, reaching the inner nuclear layer (J) and displayed a change in their morphology, with round, bloated bodies and short ramifications indicative of an activated state (K). <b>L</b>. Cell body size, number of processes, length of the processes and perimeter length of Iba1 positive cells were determined for control (white bars) and hyperglycemic (black bars) animal at P6. Values in histograms are mean ± SEM of at least 50 cells selected in 3 different experiments. *p<0.05, Unpaired t-tests. <b>M</b>. Real-time PCR of <i>Ccl2</i>, <i>Tnfα</i> and <i>Il-1β</i> in P6 rat pups retinas exposed to hyperglycemia (STZ, black bars; n = 5) compared to controls (CTL, white bars, n = 5). *p<0.05, Unpaired t-tests compared to controls. Nuclei were counterstained with DAPI in H and J. Scale bars: 1 mm in A and D; 50 µm in B–C, E–F and H–K. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer.</p

Intravitreal injection of STZ does not modify retinal vasculature.

<p><b>A</b>. Glucose transporter GLUT-2 expression (RT-PCR) in newborn and adult retinas, as compared to adult pancreas. GLUT-2 expression was similar in P6 and adult retina but 22.6 fold lower than in adult pancreas. <b>B–F</b>. P1 rat pups were injected intravitreously with streptozotocin (STZ) or with the citrate buffer vehicle (CTL). <b>B</b>. Retinal flatmounts of P6 CTL or STZ animals were stained using anti-collagen IV antibody. <b>C</b> Representative automated analysis of vascular density using Metamorph software. Branch points are represented by green points and tubes by solid gray lines (lower panel). <b>D–G</b>. Vascular area, mean tube length, branch point density and total tube length density were determined at P6 in hyperglycemic (black bars) or control (white bars) animals. Values in histograms D–G are mean ± SEM of retinas from 8 animals per group from at least 2 different experiments. Data were analyzed by unpaired t-tests. No significant differences were found in these parameters.</p