Müller glial cells from transgenic mice are more resistant to iron-mediated stress than Müller glial cells from wild-type mice

A: Culture of Müller glial (MG) cells from wild-type (WT) and transgenic (Tg) mice at the first passage were treated with 100 µM of FeCl-NTA (FN), during 96 h. The number of cells was evaluated by counting in comparison to control condition. Each column represents the mean ±SEM. The triple asterisk represent statistical significance of differences between treated and control, respectively, for WT and Tg, and between control and treated, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

Transferrin secretion in medium of Müller glial cells from 8 to 12-day-old wild-type and transgenic mice decreases with the subcultures

A: Mouse transferrin (mTf) secretion in medium of cultured Müller glial (MG) cells from wild-type ( WT) and transgenic (Tg) mice at confluency after primary culture (P0) and at the first (P1) and the second passages (P2) was measured by radioimmunoassay (RIA; ng/ml). Each column represents the mean ±SEM. The asterisk represents statistical significance of differences between WT and Tg at P0, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

Mouse transferrin and human transferrin proteins expression are modulated after iron stress

A: Mouse transferrin (mTf) secretion was measured by radioimmunoassay (RIA) in the culture medium of Müller glial (MG) cells from wild-type (WT) and transgenic (Tg) mice in control condition or after addition of 100 µM of FeCl-NTA (FN) during 96 h. Each column represents the mean ±SEM. The double asterisk represents statistical significance of differences between treated and control, respectively, for WT and Tg, p<p><b>Copyright information:</b></p><p>Taken from "The protective role of transferrin in Müller glial cells after iron-induced toxicity"</p><p></p><p>Molecular Vision 2008;14():928-941.</p><p>Published online 20 May 2008</p><p>PMCID:PMC2391081.</p><p></p

Morphological analysis of flat-mounted retina labeled with FITC-conjugated lectin from <i>Bandeira simplicifolia</i>.

<p><b>(a) Illustration of the procedure followed with “ImageJ” software.</b> (<b>a₁</b>) Selection of seven areas per group, in mid (700x500 px) and extreme (400x300 px) peripheries on mosaics (3007x2904 px) made with microscopic images at low magnification (x4). (<b>a₂</b>) Application of steerable filters for ridge detection in the selected image, with the plug-in “Feature Detector” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017462#pone.0017462-Jacob1" target="_blank">[57]</a>. (<b>a₃</b>) Transformation of the filtered image into a binary image allowing selection of the retinal vessels to calculate the vascularized area (area covered by vessels out of total area, <b>Av/At</b>). (<b>a₄</b>) Derivation of the vascular skeleton from the binary image, using the procedure “skeletonizes”. This second binary picture was used to calculate the tortuosity index corresponding to the ratio: real vessel length (<b>L2</b>, in red) out of the length of an imaginary straight line on the measured vessel (<b>L1</b>, in blue) as shown in d₁ and d₂. <b>(b–c) Quantitative analysis of the retinal vasculature from control (CTL) and PGF ET-treated eyes. (b)</b> Vascularized area calculated from the binary images which are illustrated in c₁ and c₂. <b>All data</b>: CTL, 18.8%±0.5%; PGF-ET, 23.7%±1.1%; ** p = 0.0034. <b>Mid periphery</b>: CTL, 19.4%±0.7%; PGF-ET, treated eyes 25.1%±0.8%; Bonferroni, ** p<0.01. <b>Extreme periphery</b>: CTL, 18.2%±0.7%; PGF-ET treated eyes 21.2%±1.7%; Bonferroni, ns. (<b>c</b>) Tortuosity index of capillaries calculated from the second binary images. CTL, controls 1.053±0.005; PGF-ET, treated eyes 1.180±0.014; *** p<0.0001; PGF ET/CTL = 1.12.</p

Immunostaining for PGF (a–e), GFAP (b, c) and von Willebrand factor (d, e) in membrane excised surgically from a 60-year-old woman with proliferative diabetic retinopathy.

<p>(<b>a</b>) PGF was immuno-detected on the epi-retinal membrane. It was localized in GFAP (<b>b–c</b>, arrows) and von Willebrand factor (vW) (<b>d–e</b>, arrows) immuno-positive cells. Red stars indicate cells expression only GFAP (c) or vW (d), and green stars show non-endothelial cells expressing PGF.</p

PGF expression in diabetic retina.

<p><b>(a–b) Comparison of PGF staining in non diabetic (a) and diabetic (b) retinas.</b> (<b>a</b>) Sections of eyes from adult control non diabetic rat showed co-expression of PGF and GFAP in glial Muller cells from the gcl (arrowheads) to the inl, and PGF expression in glial Müller cells which are not immuno-reactive for GFAP (white star). Scale bar = 50 µm. (<b>b</b>) A similar pattern was observed on retinal sections of eyes from three-month-old diabetic rats, with a strong immuno-reactivity for PGF at the gcl level. <b>(c) PGF detection by Western-blot in diabetic and non-diabetic retinas, from 1, 2, 5 and 12 month-old rats</b>. For each lane in which 40 µg of proteins were deposited, the blood sugar level of the represented rats is indicated between parentheses. <b>(d–e) Immunostaining for PGF and GFAP in sections from pVAX2-rPGF-1 ET- treated diabetic rat eyes, one month after ET.</b> Sections show PGF-expressing infiltrating cells in the sub retinal space (<b>d</b>, arrows) and confirmed PGF expression by RMG cells (<b>d</b>, arrowheads). GFAP staining showed gliosis induced by RMG cells (<b>d, e</b>). <b>ch</b>, choroid; <b>gcl</b>, ganglion cell layer; <b>inl,</b> inner nuclear layer; <b>ipl</b>, inner plexiform layer; <b>onl</b>, outer nuclear layer; <b>rpe</b>, retinal pigmented epithelium; Scale bar = 100 µm.</p

Fluorescein angiograms of Brown-Norway rat eyes.

<p><b>(a) Observations with a classic angiograph (Pro III Fundus camera, Kowa), 4 and 6 weeks after pVAX2-rPGF ET</b>. Angiograms were established with a scan angle of 30°. Vascular abnormalities were scored from 0 to 5 in accordance to the following grading: <b>Grade 0</b>, normal retinal vasculature, as observed in control fundus at week 6 (<b>ONH</b>, Optic Nerve Head); <b>+1 point</b> for each of the following changes - dilated (between white arrowheads) or tortuous (white arrows) vessels, microaneurysmal-like hyperfluorescent dots (black arrowheads) <10 or hyper-fluorescence around the ONH; <b>+2 points</b> for microaneurysmal-like hyper-fluorescent dots >10. <b>(b) Observations with a confocal scanning Laser Ophthalmoscope (cSLO, Heidelberg Retina Angiograph I), 2, 3 and 5 weeks after pVAX2-rPGF ET</b>. The same grading was used to score vascular abnormalities. The higher resolution of the cSLO allowed the observation of early fluorescein leakage (<b>Grade 1</b>), and the detection of strong vascular abnormalities at later stage (<b>+ 1 point</b>).</p

Analysis of flat-mounted diabetic retinas labeled with FITC-conjugated lectin from <i>Bandeira simplicifolia</i>, one month after ET.

<p><b>(a) Quantitative analysis of the retinal vasculature from control and PGF ET-treated diabetic rat eyes. (b) Microscopy of flat-mounted control (b₁, b₃) and PGF ET-treated (b₂, b₄) diabetic retinas.</b> (<b>b₁–b₂</b>) Optic microscopy on mosaics made with microscopic images at low magnification (x4) showing no evident difference of the retinal vascularization between control (b₁) and pVAX-2-rPGF-1 ET-treated eyes (b₂), except a weak retinal venous dilation in treated retinas (between arrowheads). (<b>b₃, b₄</b>) Confocal microscopy showing lectin-labeled cell infiltration (arrow) and micro-aneurysmal-like structures observed in both group (insets).</p

Effect of PGF over expression on retinal histology and on RPE cells.

<p><b>(a–b) Histological sections of retinas from eyes embedded in historesin and stained by Toluidine blue, three months after saline (a₁, b₁) and pVAX2-rPGF-1 ET (a₂–a₄, b₂–b₄).</b> (<b>a₁</b>) Normal histological section of the retina after pVAX2 ET. <b>ch</b>, choroid; <b>gcl</b>, ganglion cell layer; <b>inl</b>, inner nuclear layer; <b>onl</b>, outer nuclear layer; <b>rpe</b>, retinal pigmented epithelium; <b>Sc</b>, Sclera. Scale bar (a1–4)  = 100 µm. (<b>a₂</b>) Histological section of pVAX2-rPGF-1 ET-treated retinas, showing vascular retinal abnormalities in the inner part of the peripheral retina. (<b>a₃</b>) Sections showing retinal detachment (star) associated with pre-retinal proliferation (arrow) and edema at the ONL level (magnified in b3 inset). (<b>a₄</b>) Sections showing retinal detachment (star) associated with RPE barrier breakdown. (<b>b₁</b>) Section at high magnification showing normal RPE cells after PBS ET. Scale bar (a1–4)  = 10 µm. (<b>b₂–b₄</b>) Magnified RPE cells of the retinal sections (a₂–a₄) demonstrating morphological changes of RPE cells which appears swollen, and dilation of the chorio-capillaries (b₂, b₄ black arrowheads). Background has been subtracted in all pictures. <b>(c) Sustained blood-retinal barrier breakdown induced by rPGF-1 over expression.</b> Tight junctions were observed by occludin immuno-histochemistry on whole flat-mounted RPE cells. (<b>c1–3</b>) Normal tight junction-associated occludin was observed in PBS-ET treated eyes. Scale bar = 20 µm. (<b>c2–4</b>) Two months after pVAX2-rPGF-1 ET, junctions remained opened between some RPE cells (white arrows). At this opened junction level, infiltrating cells (stars) are also detected by the DAPI staining and recognized by the small size of their nuclei as compared to those of RPE cells.</p

Control of rPGF-1 expression.

<p><b>(a) PGF and VEGF expression analysis at the mRNA level, two months after pVAX2-rPGF ET.</b> Mean relative band intensity ±SEM determined after detection of rPGF-1, rVEGF and GAPDH mRNA expression in control eyes (in white, n = 5) or in pVAX2-rPGF-1 ET treated eyes (in black, n = 5); **, p = 0.0079. Representative RT-PCR products visualized by ethidium bromide staining are shown. <b>(b) Localization of rPGF-1 expression on sections of the ciliary muscle, one month after saline ET (Control) or pVAX2-rPGF ET (PGF ET).</b> Alpha-smooth muscle actin (αSMA) - Texas-Red labeling allows localization of the ciliary muscle (<b>cm</b>). Compared to control eye, white arrowheads indicate that after pVAX2-rPGF-1 ET, the protein of interest which is labeled in green, is localized in muscle fibers and in the epithelium of ciliary bodies (<b>cb</b>). No staining was observed when the primary antibody was omitted. Scale bars: 100 µm.</p