Up-regulation of different angiogenic factors in vitreous samples.

<p><b>A.</b> Total VEGF was significantly up-regulated in vitreous samples of DR patients (p = 0.002). B. VEGF-A was also significantly up-regulated in DR patients (p<0.001). <b>C.</b> Additionally, PGF was upregulated in DR samples compared to controls (p<0.001). Each symbol depicts an individual patient. The horizontal bar indicates the mean cytokine concentration per group. **: p<0.01, ***: p<0.001.</p

Contribution of pleiotropic immunomodulatory cytokine levels in vitreous samples.

<p><b>A.</b> A difference in IL-2 (p = 0.62) level was not measurable between control and DR patients. <b>B.</b> There was no difference in IL-4 level between control and DR patients (p = 0.78). <b>C.</b> A trend for the down-regulation of IL-13 level (p = 0.36) was noted in DR patients. Each symbol depicts an individual patient. The horizontal bar indicates the mean cytokine concentration per group.</p

Clinical patient data for both groups: Controls and diabetic retinopathy (DR) patients.

<p>Y = Year; M = male, F = female; OD = right eye; OS = left eye. DR stages are: 1 = mild to moderate NPDR, 2 = severe NPDR, and 3 = PDR.</p

Cytokine concentration (pg/ml) in vitreous samples of control and diabetic retinopathy (DR) patients measured via ELISA.

<p>Significant p-values are in bold.</p

Retinal Ganglion Cell Loss Is Accompanied by Antibody Depositions and Increased Levels of Microglia after Immunization with Retinal Antigens

<div><h3>Background</h3><p>Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats.</p> <h3>Methodology/Principal Findings</h3><p>Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3⁺retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP⁺ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1⁺ microglia cells (p = 0.003) was observed in retinas of RGA animals.</p> <h3>Conclusions/Significance</h3><p>Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.</p> </div

Detection of autoreactive antibodies against ocular tissues.

<p>Mean scores of autoreactive antibodies during the study. Levels of antibodies against retina (A) and optic nerve (B) continuously increased in sera of RGA animals after immunization in comparison to controls (CO). At four and six weeks significantly higher antibody reactivity was detected in the RGA group. Values are mean±SE; p<0.05 in bold.</p

IgG deposits in the ganglion cell layer.

<p>(A) Deposits were observed in retina cross-sections (n = 10 eyes/group) of RGA animals (circled) and (B) counts revealed a significantly higher number of these deposits in the RGA group in relation to controls (p = 0.02). (C) DAPI, IgG, and IgG₁ staining in a representative retina cross-section of a RGA immunized animal. Co-localization of IgG and IgG₁ is evident in the merged rightmost picture. The majority of the detected deposits were of the IgG₁ subtype. (D) Confocal image of the retina of an RGA animal. A colocalisation of the IgG deposit and an Iba1⁺ microglia can be seen. Values are mean±SE. Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, PRL = photoreceptor layer. (*, p<0.05; scale bars in A and C: 10 µm and in D: 7.5 µm).</p

Distribution of iba1⁺ microglia in retina.

<p>Number of Iba1⁺ somas counted in each retinal layer. Significantly higher numbers Iba1⁺ microglia were present in the nerve fiber, ganglion cell and inner plexiform of retinas from RGA immunized animals. Values are mean±SE; p<0.05 in bold.</p

Examination of optic nerves.

<p>Exemplary LBF/Nissl (A) and H&E (B) stained longitudinal optic nerve sections of a control (CO) and an RGA animal six weeks after immunization. (C) Immunized animals showed significantly more signs of demyelination. Values are mean±SE. (**, p<0.01; scale bar: 50 µm).</p

GFAP stain in retina sections.

<p>(A) GFAP expression (red) in representative retina cross-sections of a control (left) and a RGA animal (right) at six weeks. A significantly higher GFAP staining intensity given as integrated density (B) as well as an enhanced %Area of GFAP⁺ staining (C) was detected for the RGA group in comparison to controls Values are mean±SE. Abbreviations: GCL = ganglion cell layer, IPL = inner plexiform layer, INL = inner nuclear layer. (**, p<0.01; ***, p<0.001; scale bar: 10 µm).</p