Effect of post-operative treatments on stromal and epithelial thickness.

<p><b>A.</b> Plot of change in central epithelial thickness in the 4 experimental groups showing the average amount of epithelial thickness scraped off prior to laser ablation (time-point 0) and the hyperplasia in this layer that was already evident 2 weeks later in all but the untreated eyes, remaining relatively stable thereafter. By 8 weeks post-PRK, there is no longer a significant difference between experimental groups. <b>B.</b> Plot of change in central stromal thickness averaged across eyes in the 4 experimental groups, showing the amount of stromal thickness removed by laser ablation (at time-point 0) and the significant stromal thickening observed as soon as 2 weeks later in all groups (albeit to different extents). No significant differences were noted between groups after 4 weeks post-PRK. Error bars = SEM. N = number of eyes, which differs at different time-points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070785#pone-0070785-t001" target="_blank">Table 1</a>). DMSO/Cell = DMSO/Celluvisc treated eyes. Cell = Celluvisc treated eyes. <b>C.</b> In vivo confocal imaging of the endothelial layer pre-operatively and 2 weeks post-laser ablation (at the end of topical treatment) in one DMSO/Celluvisc-treated cat eye and one eye that had received 10 µM Rosiglitazone post-operatively. Note tightly packed, mostly hexagonal cells, with no gaps between them, which are evident in both treatment groups.</p

Optical imaging and the impact of Rosiglitazone treatment on wound healing in the cat cornea.

<p>Illustrative confocal images were taken 15 µm below the basal epithelial layer of the central cornea in one eye treated with DMSO/Celluvisc vehicle solution (<b>A, C, E</b>), and one eye treated with 10 µM Rosiglitazone (<b>B, D, F</b>) daily for 2 weeks after laser ablation. Pre-operative images (<b>A, B</b>) show a quiescent stroma that appears quite similar across the two eyes. Two weeks after laser ablation, just after the end of topical treatments, both corneas showed increased reflectivity, which was highest in the vehicle-treated eye (<b>C</b>) and least in the Rosiglitazone-treated eye (<b>D</b>). By 12 weeks post-operatively, the Rosiglitazone-treated eye’s reflectivity was back down to basal levels (<b>F</b>), while the vehicle-treated eye remained hazier than pre-operatively (<b>E</b>). OCT images of a Rosiglitazone-treated eye collected pre-operatively (<b>G</b>), 4 weeks (<b>H</b>) and 12 weeks (<b>I</b>) post-operatively illustrate a bright zone of reflectivity at 4 weeks post-op, which was not present pre-operatively and is lost by 12 weeks post-op. <b>J</b>. Plot of normalized intensity change in the anterior 30% of the stroma relative to pre-operative values (dotted line at zero), measured from OCT images such as those in G–I. <b>K</b>. Similar plot of normalized pixel intensity change against post-operative time for the posterior 30% of the stroma in either untreated, vehicle-treated or Rosiglitazone-treated cat corneas post-excimer laser ablation. Error bars = SEM. N = number of eyes, which differs at different time-points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070785#pone-0070785-t001" target="_blank">Table 1</a>). DMSO/Cell = DMSO/Celluvisc treated eyes. Cell = Celluvisc treated eyes.</p

Number of eyes that underwent laser ablation, followed by OCT imaging, confocal imaging and/or wavefront sensing (WFS) at each post-operative time-point.

<p>Note that the number of eyes used for wavefront analysis (in brackets, where applicable) is identical to the number of eyes imaged at the 12wk post-operative timepoint.</p

Change in higher order wavefront aberration root mean square (RMS) 2 and 12 weeks after laser ablation relative to pre-operative levels.

<p><b>A.</b> Plot of change in higher order RMS (HORMS) at 2 and 12 weeks post-laser ablation in Rosiglitazone-, DMSO/Celluvisc and Celluvisc -treated cat eyes. HORMS increased significantly relative to pre-operative levels in all groups of eyes, and this elevation was maintained all the way out to 12 weeks post-PRK, but there were no significant inter-group differences in the magnitude of the change, whose significance was likely driven predominantly by spherical aberration (SA). <b>B.</b> Plot of change in residual HORMS illustrating significantly smaller increases in Rosiglitazone-treated eyes relative to both vehicle-treated eyes. These differences are maintained out to 12 weeks post-operatively. <b>C.</b> In contrast, the magnitude of increase in coma RMS is not significantly different between treatment groups, at either 2 or 12 weeks post-PRK. <b>D.</b> Finally, spherical aberration (SA) RMS does not appear significantly increased 2 weeks post-laser ablation in Rosiglitazone or DMSO/Celluvisc-treated eyes, but all 3 groups show a similar, positive change in SA RMS relative to pre-operative values by 12 weeks post-PRK. Error bars = SEM, N = number of eyes, * p<0.05, Student’s t-test.</p

Anti-fibrotic effects of Rosiglitazone on cultured feline corneal fibroblast.

<p><b>A</b>. Representative western blots showing protein levels for αSMA. Tubulin levels were assayed as a loading control. For this experiment, cells were pretreated with 25 µM, 50 µM, and 75 µM Rosiglitazone for 30 min, before adding 1 ng/ml of TGFβ in DMEM/F12 containing 1% HS. The cells were cultured in this treated medium for 1, 2 or 3 days and then harvested for western blotting. While some effect could be observed at lower doses, Rosiglitazone clearly inhibited αSMA expression at 75 µM, while tubulin levels remained stable. <b>B</b>. Plots of relative expression of αSMA normalized to densitometric values obtained in cells stimulated with 1 ng/ml TGFβ for each culture day sampled. Data shown are means±SD, averaged over 3 experiments, and they confirm a statistically significant inhibitory effect of Rosiglitazone on αSMA. * P<0.05, Student’s t-test <i>relative to TGFβ-only condition</i>.</p