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

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

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

Neuronal and Muller cell genesis in hyperglycemic animals.

<p><b>A–C</b>. Retinal sections of CTL and STZ-treated P6 rat pups were stained with various antibodies specific to neurons of the INL (A), photoreceptors cells (B) and glial cells (C) of the retina. <b>A</b>: INL neuron generation was not affected by hyperglycemia and similar patterns of staining (red) were observed in CTL and STZ animals for PKCα (bipolar cells), Ap2α (amacrine cells) and calretinin (CALR, horizontal cells). <b>B</b>. Photoreceptor generation was not affected by hyperglycemia and similar patterns of staining (red) were observed for peanut agglutinin (PNA, cones), Rho4D2 (R4D2, rods) in STZ P6 animals compared to CTL. <b>C</b>. Muller cells cell genesis was not affected by hyperglycemia when compared to CTL. Nuclei were counterstained with DAPI. Scale bars 50 µm. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer.</p

Hyperglycemia induces apoptosis and retinal degeneration.

<p><b>A–D</b>. Retinal sections of STZ (C, D) and CTL (A, B) P6 animals were stained with the TUNEL labeling kit (red) and counterstained with DAPI. TUNEL labeling revealed the presence of apoptotic and fragmented nuclei in the INL of STZ retina (E). <b>F–G</b>. Representative histological toluidin-blue stained retinal sections in CTL (F) and STZ (<b>G</b>) P14 rat pups. <b>H–I</b>. Quantification of the number of nuclei per 100 µm in the central part (C) and the periphery (P) of the ganglion cell layer (H) or the INL (I), in hyperglycemic (black bar) and normoglycemic P14 animals (white bar). <b>J</b>. Quantification of the number of row of photoreceptor nuclei in the central part (C) and the periphery (P) of the retina, in hyperglycemic (black bar) and normoglycemic P14 animals (white bars). Values are mean +/− SEM of 9–10 retinas from 2 different experiments (H; I; J). * p<0.05; one-way ANOVA followed by Bonferroni post-tests. The number of nuclei in the INL and ONL was significantly reduced in STZ animals compared to CTL; this effect was predominant at the periphery of the retina. <b>K–L</b>. Representative histological toluidin-blue stained retinal sections of P14 STZ rat pups showing advanced structural disorganization with rosettes (K) and folds in the outer nuclear layers (L). Scale bars: 100 µm in A and C; 50 µm in B and D; 5 µm in E; 20 µm in F, G, K and L. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer.</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