8 research outputs found

    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 aflibercept/ranibizumab staining.

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    <p>Results of the analysis of aoi of glomeruli from kidneys of monkeys one and seven days after aflibercept (A) or ranibizumab (B) treatment and the corresponding controls, respectively after staining with the anti-Fc-fragment antibody for aflibercept and the anti-Fab fragment antibody for ranibizumab immune reactivity analysis; t-test against the corresponding control was performed: ** for p<0.001,*** for p<0.0001.</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

    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

    Box plot representation of the quantification of the fenestrations per µm depending on the treatment and its duration.

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    <p>Application of the Student's test showed no significant differences in the number of fenestrations per µm except one day after aflibercept's injection where the number was increased compared to all other groups (*, p<0.05).</p

    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