Topical rosiglitazone is an effective anti-scarring agent in the cornea

Corneal scarring remains a major cause of blindness world-wide, with limited treatment options, all of which have side-effects. Here, we tested the hypothesis that topical application of Rosiglitazone, a Thiazolidinedione and ligand of peroxisome proliferator activated receptor gamma (PPARγ), can effectively block scar formation in a cat model of corneal damage. Adult cats underwent bilateral epithelial debridement followed by excimer laser ablation of the central corneal stroma to a depth of ~160 µm as a means of experimentally inducing a reproducible wound. Eyes were then left untreated, or received 50 µl of either 10 µM Rosiglitazone in DMSO/Celluvisc, DMSO/Celluvisc vehicle or Celluvisc vehicle twice daily for 2 weeks. Cellular aspects of corneal wound healing were evaluated with in vivo confocal imaging and post-mortem immunohistochemistry for alpha smooth muscle actin (αSMA). Impacts of the wound and treatments on optical quality were assessed using wavefront sensing and optical coherence tomography at 2, 4, 8 and 12 weeks post-operatively. In parallel, cat corneal fibroblasts were cultured to assess the effects of Rosiglitazone on TGFβ-induced αSMA expression. Topical application of Rosiglitazone to cat eyes after injury decreased αSMA expression and haze, as well as the induction of lower-order and residual, higher-order wavefront aberrations compared to vehicle-treated eyes. Rosiglitazone also inhibited TGFβ-induced αSMA expression in cultured corneal fibroblasts. In conclusion, Rosiglitazone effectively controlled corneal fibrosis in vivo and in vitro, while restoring corneal thickness and optics. Its topical application may represent an effective, new avenue for the prevention of corneal scarring with distinct advantages for pathologically thin corneas

Noninvasive Intratissue Refractive Index Shaping (IRIS) of the Cornea with Blue Femtosecond Laser Light

The present study investigated the feasibility and effectiveness of blue-IRIS in living corneal tissue by using 400-nm femtosecond laser pulses with 1-nJ pulse energy, 80-MHz repetition rate and 100-fs pulse duration. Blue-IRIS induced refractive changes up to 0.037 in the corneal stroma at fast scanning speeds, providing a possible alternative method of noninvasively altering corneal power

Keratocyte apoptosis and not myofibroblast differentiation mark the graft/host interface at early time-points post-DSAEK in a cat model.

To evaluate myofibroblast differentiation as an etiology of haze at the graft-host interface in a cat model of Descemet's Stripping Automated Endothelial Keratoplasty (DSAEK).DSAEK was performed on 10 eyes of 5 adult domestic short-hair cats. In vivo corneal imaging with slit lamp, confocal, and optical coherence tomography (OCT) were performed twice weekly. Cats were sacrificed and corneas harvested 4 hours, and 2, 4, 6, and 9 days post-DSAEK. Corneal sections were stained with the TUNEL method and immunohistochemistry was performed for α-smooth muscle actin (α-SMA) and fibronectin with DAPI counterstain.At all in vivo imaging time-points, corneal OCT revealed an increase in backscatter of light and confocal imaging revealed an acellular zone at the graft-host interface. At all post-mortem time-points, immunohistochemistry revealed a complete absence of α-SMA staining at the graft-host interface. At 4 hours, extracellular fibronectin staining was identified along the graft-host interface and both fibronectin and TUNEL assay were positive within adjacent cells extending into the host stroma. By day 2, fibronectin and TUNEL staining diminished and a distinct acellular zone was present in the region of previously TUNEL-positive cells.OCT imaging consistently showed increased reflectivity at the graft-host interface in cat corneas in the days post-DSAEK. This was not associated with myofibroblast differentiation at the graft-host interface, but rather with apoptosis and the development of a subsequent acellular zone. The roles of extracellular matrix changes and keratocyte cell death and repopulation should be investigated further as potential contributors to the interface optical changes

Immunohistochemistry for detection of alpha-smooth muscle actin (α-SMA) and fibronectin post-Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK).

<p>Corneal sections were stained with antibodies against α-SMA (red) to label myofibroblasts, antibodies against fibronectin (green), and 4′6′-diamidino-2-phenylondole dihydrochloride (DAPI) (blue) was used to label cell nuclei. (<b>A–C</b>) Photomicrographs of <i>ex vivo</i> cat corneal sections of the graft-host interface (arrows) on post-operative days 0 (<b>A</b>), 4 (<b>B</b>), and 9 (<b>C</b>)(scale bar for A – C = 0.2 mm). (<b>D and E</b>) The graft-host interface on days 0 (<b>D</b>) and 9 (<b>E</b>) (scale bar for D & E = 0.2 mm). (<b>F</b>) The central stroma of an unoperated cat cornea demonstrated an absence of α-SMA and mild diffuse fibronectin staining. (<b>G and H</b>) Incisional paracenteses wounds on day 0 (<b>G</b>) and day 9 (<b>H</b>) (scale bar for F – H = 0.4 mm). (<b>I</b>) An incisional paracentesis wound on day 9 at high magnification (scale bar for I = 0.2 mm). Note the lack of α-SMA staining at the graft-host interface (<b>C and E</b>), but positive α-SMA staining at the incisional wound (<b>H and I</b>) on day 9. On day 0, 4 hours after DSAEK, fibronectin staining was present extracellularly along the interface and also appeared to co-localize with DAPI in the cells of the adjacent host stroma (<b>A and D</b>). On day 9 post-DSAEK, there was faint fibronectin staining near the host stromal cells, but the interface fibronectin staining is much fainter and more consistent with the unoperated control.</p

Methods of Corneal Optical Coherence Tomography (OCT) Analysis.

<p>(<b>A</b>) OCT image of an <i>in vivo</i> cat cornea 4 hours after Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK). Note the greater intensity of backscatter at the graft-host interface (solid arrows) relative to the adjacent stromata. The perpendicular lines superimposed over the OCT image indicate the location of the areas analyzed for backscatter intensity (white and black) and thickness (red, orange, and maroon). Four perpendicular lines (white and black), 20 pixels wide and located +/−100 and +/−200 pixels from the central specular reflection were used to generate a pixel brightness profile. As depicted along the black line, intensity line graphs generated from each of the four perpendicular lines were then used to identify and measure intensity over three defined corneal regions; a 10 pixel thick region of interface (yellow), a 40 pixel thick region of the adjacent host stroma (green), and a 40 pixel thick region of the adjacent donor stroma (blue). Total corneal (red), host stromal (orange), and donor stromal (maroon) thickness measurements were taken in the central cornea as depicted by the central line. (<b>B</b>) A representative plot of normalized backscattered light intensity through a cat cornea post-DSAEK at a single time-point. This profile was generated as described above. These graphs were constructed for each of the line locations. Note the intensity peak at the interface (yellow) in comparison to that of the adjacent host (green) and donor stroma (blue).</p

Interface Intensity and Corneal Thickness Post-DSAEK.

<p>(<b>A</b>) Corneal optical coherence tomography (OCT) images of an <i>in vivo</i> cat cornea pre-operatively and at early post-Descemet’s Stripping Automated Endothelial Keratoplasty (DSAEK) time-points. Note the easily discernible post-operative interface between the host and donor stromata appears brighter than the adjacent tissue. The host stroma also has increased thickness immediately post-operatively, which improves with time. (<b>B</b>) Corneal OCT image-derived backscatter intensity at the graft-host interface (yellow) in comparison to the adjacent host stroma (green) and donor stroma (blue) at early post-DSAEK time points. Note that the graft-host interface was consistently brighter than the adjacent host stroma and adjacent donor stroma. This result was statistically significant at the majority of time-points; between interface and adjacent host stroma at days 0 (p = 0.0078), 2/3 (p = 0.0053), 4/5 (p = 0.0019) and 6/7 (p = 0.0003), and between interface and adjacent donor stroma at days 0 (p = 0.0078), 2/3 (p = 0.0005), and 6/7 (p = 0.0155). This difference was no longer observed at the 8/9 day time point. * = significant difference between mean interface and mean host stromal intensity. † = significant difference between mean interface and mean donor stromal intensity. (<b>C</b>) OCT image-derived corneal thickness measurements across early post-DSAEK time-points. Note the brisk increase in total thickness from pre-operative levels (Pre-Op) to immediate post-operative (0) levels associated with the addition of the donor tissue, and the subsequent gradual decline in total thickness to day 6/7.</p

First Demonstration of Ocular Refractive Change Using Blue-IRIS in Live Cats

PURPOSE. To determine the efficacy of intratissue refractive index shaping (IRIS) using 400-nm femtosecond laser pulses (blue light) for writing refractive structures directly into live cat corneas in vivo, and to assess the longevity of these structures in the eyes of living cats. METHODS. Four eyes from two adult cats underwent Blue-IRIS. Light at 400 nm with 100-femtosecond (fs) pulses were tightly focused into the corneal stroma of each eye at an 80-MHz repetition rate. These pulses locally increased the refractive index of the corneal stroma via an endogenous, two-photon absorption process and were used to inscribe three-layered, gradient index patterns into the cat corneas. The optical effects of the patterns were then tracked using optical coherence tomography (OCT) and Shack-Hartmann wavefront sensing. RESULTS. Blue-IRIS patterns locally changed ocular cylinder by À1.4 6 0.3 diopters (D), defocus by À2.0 6 0.5 D, and higher-order root mean square (HORMS) by 0.31 6 0.04 lm at 1 month post-IRIS, without significant changes in corneal thickness or curvature. Refractive changes were maintained for the duration they were tracked, 12 months post-IRIS in one eye, and just more than 3 months in the remaining three eyes. CONCLUSIONS. Blue-IRIS can be used to inscribe refractive structures into live cat cornea in vivo that are stable for at least 12 months, and are not associated with significant alterations in corneal thicknesses or radii of curvature. This result is a critical step toward establishing Blue-IRIS as a promising technique for noninvasive vision correction

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