Γ-Synuclein and Brn-3a localization were examined in the human retina by immunofluorescence

γ-Synuclein (red) was present in the cytoplasm of retinal ganglion cells (RGCs; triangles) and in the nerve fiber layer (NFL), while Brn-3a (green, arrowheads) was observed in RGC nuclei. Arrows mark cells in the retinal ganglion cell layer (GCL) that were not stained by either Brn-3a or γ-synuclein. Nuclei throughout the retina are counterstained with DAPI (blue), and little immunofluorescence is noted through the inner or outer plexiform layers (IPL and OPL, respectively), or inner and outer nuclear layers (INL and ONL, respectively). Thus γ-synuclein is localized both in the body of RGC and in their axons.<p><b>Copyright information:</b></p><p>Taken from "γ-Synuclein as a marker of retinal ganglion cells"</p><p></p><p>Molecular Vision 2008;14():1540-1548.</p><p>Published online 22 Aug 2008</p><p>PMCID:PMC2518532.</p><p></p

Experimental groups.

<p>3 animals were studied per group, but IOP and histology data for PBS-injected right eyes were pooled into groups irrespective of left eye treatment.</p><p>*1 month survival groups were only subjected to histology and iron staining, not IOP or immunofluorescence measurements.</p><p>**A set of uninjected animals was used exclusively for ERG measurements. IVT intravitreal; AC anterior chamber; PBS phosphate buffered saline.</p

Microglial activation in response to magnetic particles.

<p>Example CD11b/c immunofluorescence images for the PBS- (control), 50 nm- and 4 µm-injected animals at 1 hour, 1 week and 5 months as marked. There was no difference in microglial activation among PBS- and magnetic particle-injected animals at any time point surveyed. Scale bar, 50 µm.</p

Hematoxylin- and eosin-stained sections.

<p>Hematoxylin- and eosin-stained representative sections for the PBS- (control), 50 nm- and 4 µm- injected animals at 1 week and 5 months, as marked. Accumulation of 4 µm particles were noted layered against the retina (IVT injection, green arrows) and along the iris and in the angle (AC injection, blue arrows) out to 5 months. Yellow arrows highlight example dotted cell nuclei in the GCL (yellow dots), INL (green dots) and ONL (red dots) at higher magnification used for cytotoxicity counting. Black arrows point to corneal endothelial cells along the endothelial cell layer, used for cytotoxicity counting. Scale bar, 50 µm in all pictures.</p

Localization of magnetic nanoparticles at 1 week.

<p>At 1 week after IVT injection, immunofluorescence was used to localize 50 nm particles (green in a) and 4 µm particles (red in b) as shown by white arrows. In both cases, retinas were counterstained with GFAP and DAPI (nuclei, blue). At 1 week, both sizes of magnetic particles were detectable in the ganglion cell layer (arrows). Scale bar, 50 µm.</p

Iron deposits in the ocular tissues after particle injections.

<p>Representative images from Prussian blue histochemical staining for iron from control and magnetic particle-injected eyes at 1 week. Iron staining was observed only in the positive control, and not in any of the injected eyes, as marked. The 4 µm particles were visible in the iris and retinal tissues (arrows).</p

Measure of electroretinograms changes in response to magnetic particles.

<p>Electroretinograms taken at 9–14 weeks for 50 nm- and 4 µm-, AC- and IVT-injected animals, as well as control, uninjected animals, as marked. The solid line represents the average of the particle-injected left eyes for each group, and the dashed line represents the average of the PBS-injected right eyes for each group, except in the control animals (1^st column) in which neither eye was injected. There was no significant difference in the a- and b-waves for the control (uninjected) and nano- and microparticle injected animals with either IVT or AC injections.</p

Iron deposits in the ocular tissues due to injected magnetic particles at 5 months.

<p>Prussian blue histochemical iron staining images for control and magnetic particle-injected eyes at 5 months. Black arrow shows an example of background choroidal staining in an IVT PBS- (control) injected eye, frequently observed as a background choroidal staining in many of the sections. Neither IVT nor AC 50 nm-injected eyes showed iron staining, but positive iron stains were noted around 4 µm particles after IVT injection (white arrow), in the cornea (blue arrow), and in the ciliary body (green arrows) seen after AC injection. Scale bar, 50 µm.</p

Iron staining after particle injection – fraction of eyes staining with Perl's Prussian Blue.

<p>*Staining seen around clumps of beads.</p><p>**Staining seen in cornea and ciliary body (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017452#pone-0017452-g008" target="_blank">Figure 8</a>).</p

Measure of astrocyte activation using GFAP staining.

<p>Example GFAP immunofluorescence images for the PBS- (control), 50 nm- and 4 µm-intravitreally injected animals at 1 hour, 1 week and 5 months as marked. AC-injected animals are also shown for the 5 month time point. Astrocyte activation is seen along the ganglion cell layer (red) with nuclear counterstaining with DAPI (blue) highlighting the retinal layers. Astrocyte activation was similar among PBS- and magnetic particle-injected animals at all three time points for both types of injections. For IVT-injected animals, GFAP staining was seen primarily at the site of injection along the ganglion cell layer. Scale bar, 50 µm.</p