Schematic Representation of NFκB Molecular Signaling in Ischemia-Independent Microenvironment and the Effects of YC-1.

<p>NFκB/p65: <b>Nuclear Factor Kappa B/P65</b>; α5β1: <b>Integrin Alpha-5 Beta-1</b>; ET-1: <b>Endothelin-1</b>; MMP-9: <b>Matrix Metalloproteinase-9</b>; FAK: <b>Focal Adhesion Kinase</b>; EPO: Erythropoietin.</p

The Expression of NFκB and Downstream Angiogenic Proteins in rho/VEGF Mouse Model.

<p>Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.</p

The Expression of NFκB and Downstream Angiogenic Proteins in rho/VEGF Mouse Model.

<p>Immunohistochemical analysis of NFκB/p65, FAK, α5β1, EPO, ET-1, and MMP-9, has indicated the expression levels of these proteins were significantly elevated in the rho/VEGF retinas that were left untreated. YC-1-treated retinas exhibited a significant decrease in the protein expression levels as compared with DMSO-treated retinas. Despite the sustained expression of VEGF, there were no detectable differences in the levels of CXCR4 and SDF-1 protein expression amongst the animals of all groups. Retinas were examined at 100× objective. Scale bar, 100 µm.</p

A–C. Quantification of Subretinal NV in Various Control and Experimental Groups.

<p>Metamorph image-analysis software was used to compute the number and area of neovascular lesions and the total area of NV on the outer surface of each retina. The figure displays; A) the number of NV lesions per retina; B) the total neovascular area per retina; and C) the average neovascular lesion per retina. Mice that were treated with YC-1 and SN50 had; 1) significantly fewer neovascular lesions, 2) significantly smaller NV area per retina, and 3) smaller area of NV lesion per retina, than did mice that were treated with DMSO and SN50M, respectively. Image analysis confirmed that there was no difference between DMSO- and SN50M-treated mice and mice that were left untreated.</p

Immunohistochemical Profile of Retinal Layers among the Various Mouse Groups.

<p>The retinal layers stained vividly. However, the grain intensity varied significantly from one layer to another. The intensity of immunoreactivity was graded as follows: strong (+++), moderate (++), weak (+), negative (−) (A). Retinal tissue specimens of YC-1 treated groups were compared to normoxic, non-treated rho/VEGF retinas, DMSO-treated-rho/VEGF mice and YC-1-treated rho/VEGF mice.</p

YC-1 Curtails Ischemia-Induced Downregulation of Basal ZnT8 Expression in the Injured Retina.

<p>Quantitative Real-Rime RT-PCR analysis was conducted by utilizing specific primer sets (A). The levels of <i>ZnT8</i> mRNA in the non-treated ischemic retinas were significantly downregulated by approximately 3.05 folds, as compared to nontreated normoxic retinas. Dual injections treatment with YC-1 has resulted in a significant upregulation of <i>ZnT8</i> (**<i>P</i><0.01) gene expression when compared with DMSO-treated hypoxic cells. ANOVA; Mean ± SEM of mRNA level normalized to β-actin were calculated, (**<i>P</i><0.01, as compared to DMSO-treated retinas). Data are representative of 3 independent experiments (B). Immunohistochemical localization of ZnT8 in the Mouse Retina has indicated that in the non-treated normoxic retina; the ONL, OPL, GCL, and NFL tissue layers of the retina exhibited the strongest ZnT8 expression, whereas the PRL, INL and IPL exhibited moderate ZnT8 immunoreactivity. An image with high level of magnification (marked with circle) shows staining of the cell bodies in the GCL. Scale bar: 200 µm (C).</p

Immunohistochemical Analysis of PDGF-B, <i>in vivo</i>.

<p>Photomicrographs of retinas from various OIR groups that were immunostained for PDGF-B. The expression of PDGF-B was upregulated in the non-treated ischemic and DMSO-treated groups, compared with non-treated normoxic group. While all protein immunoreactivities were downregulated in the YC-1-treated group, compared with DMSO-treated groups. Data are representative of 3 independent experiments. Scale bar: 140 µm.</p

A–I. The Activation of NFκB and Downstream Angiogenic Genes in the rho/VEGF Mouse Model.

<p>The mRNA levels for the genes; <i>NFκB/p65</i>, <i>α5</i>, <i>β1</i>, <i>ET-1</i>, <i>MMP-9</i>, <i>FAK and EPO</i>, were quantified by Real time RT-PCR. Selected experiments, which measured the mRNA levels of <i>NFκB/p65</i>, indicated that YC-1 and SN50 downregulated the mRNA levels of <i>NFκB/p65</i> as compared to DMSO or SN50M-treated retinas, respectively. For the other genes listed above, the mRNA levels were upregulated in the DMSO-treated retinas and the rho/VEGF group that was left untreated. In contrast, retinas from YC-1-treated retinas exhibited a significant downregulation of the mRNA expression as compared to retinas that were treated with DMSO. Despite the sustained expression of VEGF, there were no detectable differences in the levels of <i>CXCR4</i> and <i>SDF-1</i> mRNA expression in the animals of all groups. ANOVA was used for statistical analyses. Mean ± SEM of mRNA level normalized to β-actin were calculated, [***<i>P</i><0.001 and **<i>P</i><0.01, as compared to respective controls]. Data are representative of 3 independent experiments. <b>J. Sequence for the Primer Sets Used for the Quantitative Real-Time PCR Analysis</b>.</p

Characterization of ZnT8-immunoreactivity in Normal, Pathological and YC-1-Treated Retinas.

<p>Photomicrographs of retinas from various OIR groups that were immunostained for ZnT8 exhibit a downregulation of ZnT8 expression in the non-treated ischemic (B) and DMSO-treated groups (C), as compared with non-treated normoxic group (A). While ZnT8 immunoreactivities were upregulated in the YC-1-treated group (D), as compared with DMSO-treated groups. Data are representative of 3 independent experiments. Scale bar: 150 µm.</p

Quantitative Assessments of Retinal Immunohistochemical Staining Intensity of ZnT8 in Normal, Pathological and YC-1-Treated Retinas.

<p>The retinal layers stained vividly. However, the grain intensity varied significantly from one layer to another. The intensity of immunoreactivity was graded as follows: strong (+++), moderate (++), weak (+), negative (−) (A). Retinal tissue specimens of YC-1 treated groups were compared to normoxic, non-treated ischemic and DMSO-treated retinas. The collected images of the retinas were imported to the image analysis system Metamorph 7.1. All image analyses were conducted in a masked fashion. Values obtained from at least 5 retinal fields were used to calculate the average pixel intensity value per retina. Bar graphs exhibit the intensity of staining of ZnT8 in all four groups. The area of staining was measured in (um²/um²) in all four groups. Values (mean ± SEM), from 3 separate experiments from at least 10 images from 4 different eyes/group. (***<i>P</i><0.001 and **<i>P</i><0.01). Data are representative of 3 independent experiments (B).</p