18 research outputs found

    Light microscope and fluorescence micrographs of LE (A–B) and ZD-LE (C–D) rats.

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    <p><b>A</b>) Bright-field image of the retina - RPE – choroid complex in LE rat. Black arrowheads show the regularity of the Bruch's membrane. <b>B</b>) Fluorescence micrograph of ED1 positive cells in the same area as in (A). ED1 immunoreactivity (white arrows) is visible in the choroid. <b>C</b>) Bright-field image of the RPE – choroid complex in ZD-LE rat. Black arrowheads show the irregularity of the Bruch's membrane, white arrowheads indicate RPE cells. <b>D</b>) Fluorescence micrograph of ED1 positive cells in the same area as in (C). Small (<3 µm) (white arrow) and large (>3 µm) (white asterisk) ED1 positive cells are detected in the choroid. ED1 immunoreactivity is also shown around choroidal blood vessels and along the choriocapillaris just below the Bruch's membrane (white arrowheads). A thin ED1 positive cell simultaneously in contact with the Bruch's membrane and a RPE cell (black arrow) is shown. In B and D), note the strong autofluorescence of the photoreceptor outer segments frequently observed in paraffin sections due to the fluorescence emitted by the retinoids reflecting the clearance of toxic byproducts and the accumulation of lipofuscin-like fluorophores associated with the increase of lipid peroxidation with age in the retina.</p

    Light microscope semi-thin sections showing the retina-choroid complex of A) LE rat and of B) ZD-LE rat.

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    <p>In B, the choroid shows many large round cells (black arrows) which are absent in the control rats (A). <b>C</b>) Quantification of infiltrated pigmented cells with a diameter larger than 3 µm in the choroid of LE (1.3±1.9) <i>vs</i>. ZD-LE (19±1.6) rats. The increase observed in ZD-LE rats is statistically significant (p<0.0001). Abbreviations: ONL, Outer Nuclear Layer; POS, Photoreceptor Outer Segments; RPE, Retinal Pigment Epithelium; Ch, Choroid.</p

    Zinc mole fraction (in at%) of melanosomes in the RPE of control LE and ZD-LE animals as determined by quantitative EDX spectroscopy in the TEM.

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    <p>In the ZD-LE group (0.004±0.01 at% zinc) which is below the detection limit of 0.02 at%, the zinc mole fractions were significantly lower compared to the control group (0.04±0.02 at% zinc) (p = 0.02, t test).</p

    Effects of a Single Intravitreal Injection of Aflibercept and Ranibizumab on Glomeruli of Monkeys

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    <div><p>Purpose</p><p>It is known that endothelial cells in the kidney are also strongly VEGF-dependent. Whether intravitreal drugs can be detected within the glomeruli or affect VEGF in glomerular podocytes is not known. Therefore, the aim of this pilot study was to investigate the effects of a single intravitreal injection of aflibercept and ranibizumab on glomeruli of monkeys.</p><p>Methods</p><p>The kidneys of eight cynomolgus monkeys, which were intravitreally injected either with 2 mg of aflibercept or with 0.5 mg of ranibizumab, were investigated one and seven days after injection. Two animals served as controls. The distribution of aflibercept, ranibizumab and VEGF was evaluated using anti-Fc- or anti-F(ab)-fragment and anti-VEGF antibodies respectively. The ratio of stained area/nuclei was calculated using a semi-quantitative computer assisted method. Glomerular endothelial cell fenestration was quantified in electron microscopy using a systematic uniform random sampling protocol and estimating the ratio of fenestrae per µm.</p><p>Results</p><p>Compared to the controls, the anti-VEGF stained area/nuclei ratio of the ranibizumab-treated animals showed no significant changes whereas the stained areas of the aflibercept-treated monkeys showed a significant decrease post-treatment. Immune reactivity (IR) against aflibercept or ranibizumab was detected in aflibercept- or ranibizumab treated animals respectively. The number of fenestrations of the glomerular endothelial cells has shown no significant differences except one day after aflibercept injection in which the number was increased.</p><p>Conclusion</p><p>Surprisingly, both drugs could be detected within the capillaries of the glomeruli. After a single intravitreal injection of aflibercept, VEGF IR in the podocytes was significantly reduced compared to controls. Ranibizumab injection had no significant effect on the glomeruli's VEGF level. Whether this is caused by aflibercept's higher affinity to VEGF or because it is used in a higher stoichiometric concentration compared to ranibizumab remains to be investigated.</p></div

    Semi-quantitative computer assisted method used for the quantification and normalisation of the VEGF staining.

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    <p>Glomeruli were defined as the area of interest (AOI), and then the AOI were isolated using the image j software. The nuclei in the AOI were then counted and finally the stained area of each AOI was determined.</p

    Examples of representative transmission electron micrographs of (A) a fenestrated glomerular endothelium and of (B) peripheral <i>versus</i> mesangial portions of the glomerular endothelium (both one day after aflibercept injection).

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    <p>(<b>A</b>) Blood lumen on the upper part, urinary space on the lower part of the image. The healthy glomerular filtration barrier consists of three layers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113701#pone.0113701-Eremina2" target="_blank">[6]</a>: the fenestrated glomerular endothelial cells, the intervening glomerular basement membrane and the podocyte processes and slit diaphragm. GBM =  glomerular basement membrane, CL =  capillary lumen, POD =  podocyte. Arrows mark glomerular endothelial cells fenestrae (note the absence of diaphragm), asterisks mark podocyte foot processes, arrowheads mark podocyte slit diaphragm. (<b>B</b>) At this magnification, podocyte foot processes (asterisks) allow the clear identification of the capillary lumen (CL). In accordance with our definition, the peripheral portion begins where the endothelium and the glomerular endothelial basement membrane (GBM) run approximately parallel (marked by arrows). Arrows mark direction into which peripheral endothelium begins. In between the arrows the mesangium (Mes) and the mesangial portion of the capillary endothelium (MesE) is located. Note that in the mesangial portion there is no GBM adjacent to the fenestrated endothelium so that the described counting method is not applicable and the endothelium does not show the typical single- layered configuration. Magnification ×20000.</p

    Quantification and normalisation of the VEGF staining.

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    <p>Results of the analysis of aoi of glomeruli from kidneys of monkeys one and seven days after ranibizumab and aflibercept treatment and the corresponding controls after anti-VEGF staining; t-test against control: * for p<0.05, *** for p<0.0001; t-test ranibizumab day 1 <i>versus</i> aflibercept day 1 and ranibizumab day 7 <i>versus</i> aflibercept day 7: # for p<0.05, ### for p<0,0001; t-test aflibercept day 1 <i>versus</i> aflibercept day7: +++ for p<0.0001.</p

    Examples of transmission electron micrographs used for the quantification of the glomerular endothelial cells fenestrations.

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    <p>The red line drawn a long lamina rara interna, the length of line is in µm, the red crosses point out fenestrations. (<b>A</b>) after injection of the vehicle; (<b>B</b>) in the untreated control; (<b>C</b>) one day after injection of ranibizumab; (<b>D</b>) seven days after injection of ranibizumab; (<b>E</b>) one day after injection of aflibercept; (<b>F</b>) seven days after injection of aflibercept. Magnification ×20000.</p

    Towards a Quantitative OCT Image Analysis

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    <div><p>Background</p><p>Optical coherence tomography (OCT) is an invaluable diagnostic tool for the detection and follow-up of retinal pathology in patients and experimental disease models. However, as morphological structures and layering in health as well as their alterations in disease are complex, segmentation procedures have not yet reached a satisfactory level of performance. Therefore, raw images and qualitative data are commonly used in clinical and scientific reports. Here, we assess the value of OCT reflectivity profiles as a basis for a quantitative characterization of the retinal status in a cross-species comparative study.</p><p>Methods</p><p>Spectral-Domain Optical Coherence Tomography (OCT), confocal Scanning-La­ser Ophthalmoscopy (SLO), and Fluorescein Angiography (FA) were performed in mice (<i>Mus musculus</i>), gerbils (<i>Gerbillus perpadillus</i>), and cynomolgus monkeys (<i>Macaca fascicularis</i>) using the Heidelberg Engineering Spectralis system, and additional SLOs and FAs were obtained with the HRA I (same manufacturer). Reflectivity profiles were extracted from 8-bit greyscale OCT images using the ImageJ software package (<a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>).</p><p>Results</p><p>Reflectivity profiles obtained from OCT scans of all three animal species correlated well with ex vivo histomorphometric data. Each of the retinal layers showed a typical pattern that varied in relative size and degree of reflectivity across species. In general, plexiform layers showed a higher level of reflectivity than nuclear layers. A comparison of reflectivity profiles from specialized retinal regions (e.g. visual streak in gerbils, fovea in non-human primates) with respective regions of human retina revealed multiple similarities. In a model of Retinitis Pigmentosa (RP), the value of reflectivity profiles for the follow-up of therapeutic interventions was demonstrated.</p><p>Conclusions</p><p>OCT reflectivity profiles provide a detailed, quantitative description of retinal layers and structures including specialized retinal regions. Our results highlight the potential of this approach in the long-term follow-up of therapeutic strategies.</p></div