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Marco Mura,1– 3 Wendy Martin,4 K Keven Williams,4 Dina Joy K Abulon5 1Department of Translational Medicine, University of Ferrara, Ferrara, Italy; 2Department of Ophthalmology, University of Illinois, Chicago, IL, USA; 3Vitreoretinal Division, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia; 4Preclinical Development, Alcon Vision LLC, Fort Worth, TX, USA; 5Global Medical Affairs, Alcon Vision LLC, Irvine, CA, USACorrespondence: Marco MuraDepartment of Translational Medicine, University of Ferrara, Via Luigi Borsari 46, Ferrara, 44121, ItalyTel +390532455845Fax +390532207351Email marco.mura@unife.itPurpose: Novel 3D digital display systems, such as NGENUITY 3D digitally assisted visualization system (DAVS), can provide enhanced illumination, depth of field, and digital filtering. This study compared vitreous removal using NGENUITY 3D DAVS with a standard surgical microscope.Methods: This was an in vivo, 2-arm laboratory study in 15 Yorkshire pig eyes. The LuxOR LX3 microscope with NGENUITY 3D DAVS (arm 1) and the LuxOR LX3 microscope alone (arm 2) were used with 5× optical magnification and Oculus BIOM 200 mm optics. Standard core and peripheral vitrectomy without scleral depression was performed using the CONSTELLATION Vision System. Residual vitreous weight was assessed in enucleated eyes by a masked observer. Axial length and vitreous weight of contralateral eyes were compared from an additional 14 Yorkshire pigs to confirm that eyes from a single animal were essentially identical.Results: After vitrectomy, mean ± SD residual vitreous was significantly smaller with NGENUITY versus standard microscope (0.143 ± 0.146 versus 0.580 ± 0.269 g, respectively; P < 0.0001). Based on a mean initial vitreous weight of 2.5 g, as determined by assessment in contralateral eyes from an additional 14 Yorkshire pigs, the mean percentage vitreous removal was 94% ± 6% versus 77% ± 11%, respectively. Further, vitreous weight and axial length for contralateral eyes from any single animal in these additional 14 animals were essentially identical, as mean differences were 0.046 ± 0.035 g and 0.11 ± 0.08 mm, respectively.Conclusion: Vitrectomy with NGENUITY 3D DAVS resulted in significantly less residual vitreous in pig eyes compared with standard microscopy. NGENUITY may improve vitreous removal during vitreoretinal surgery by enhancing visualization.Keywords: 3D digital display systems, NGENUITY, vitrectomy

Purpose These studies examined corneal healing rates, Type-IV collagen and zonula occludens membrane-associated protein (ZO-1) expression, as well as aqueous PGE(2) and IL-1 beta concentrations in pigmented rabbits treated with either moxifloxacin 0.5%, gatifloxacin 0.3% or BSS following anterior keratectomy. Methods Anterior keratectomy surgery was followed by topical administration with commercial ophthalmic formulations of either moxifloxacin or gatifloxacin or BSS (TID for 96 h). Images of the fluorescein-stained healing corneas were analyzed for wound area. At 48 or 96 h following surgery, aqueous humor samples were collected and analyzed for the inflammatory mediators PGE(2) and IL-1 beta using an ELISA. The corneas were subsequently evaluated using both scanning and transmission electron microscopy. In a second parallel study, corneas were evaluated at both 48 and 96 h for Type-IV collagen and ZO-1 expression using immunohistochemistry. Results Fluorescein-stained corneal images at 96 h postsurgery demonstrated that 90% +/- 8% re-epithelialization for moxifloxacin, 81% +/- 14% for gatifloxacin, and 88 +/- 6% for BSS((R)) (P > 0.05). PGE(2 )levels in the aqueous humor of fluoroquinolone treated eyes were reduced at 48 h compared to BSS treated eyes. IL-1 beta was undetectable in all samples. No differences in Type-IV collagen or ZO-1 expression were observed between any treatment groups. There were no differences between groups in histological appearance or in ultrastructural healing processes. Conclusions These studies demonstrated that the commercial ophthalmic formulations of moxifloxacin and gatifloxacin were similar to each other in their effects on the levels of aqueous humor PGE(2) and rates of corneal wound re-epithelialization.

Purpose: Comeal wound healing was compared with topical ocular applications of moxifloxacin ophthalmic solution 0.5%, gatifloxacin ophthalmic solution 0.3%, or BSS Sterile Irrigating Solution after a 6-mm penetrating comeal incision during a 1-week observation period or after creation of a comeal flap during a 3-week observation period. Methods: Fifteen pigmented rabbits were randomized into 3 test groups of 5 rabbits per group (group 1, moxifloxacin 0.5%; group 2, gatifloxacin 0.3%; group 3, BSS Sterile Irrigating Solution). All animals underwent surgery using a microkeratome to create a comeal flap approximately 8.5 mm in diameter and 160 μm deep in the right eye (OD). The animals were dosed 4 times a day with the appropriate test article for 7 days after surgery. Biomicroscopic examinations and postmortem histopathology were conducted to compare the different treatments. In a second study, 9 pigmented rabbits were divided into 3 test groups of 3 rabbits per group. Each animal underwent bilateral surgery to create a 6-mm linear, penetrating incision into the central cornea, using a diamond keratome. The wound was subsequently sutured, and the animals were treated bilaterally (topical ocular) for 7 days with moxifloxacin (3 times a day), gatifloxacin (4 times a day), or BSS Sterile Irrigating Solution (4 times a day). On day 7, the left eye (OS) of each animal was processed and analyzed using standard hematoxylin-eosin histopathology, whereas the right eye of each animal underwent processing and analysis using scanning and transmission electron microscopy. Corneas were examined to evaluate wound healing progress, including the epithelial plug, cellular infiltrates, and stromal precipitates. Results: For both the comeal flap and linear incision studies, no appreciable differences were noted between groups with regard to slit-lamp or histopathologic examinations. In the linear incision study, electron microscopy revealed normal remodeling processes in all treatment groups, with no evidence of abnormal electron densities of the basal epithelial cells, basement membrane, anchoring filaments, collagen fibrils, extracellular matrix, or stromal keratocytes. Conclusions: Results showed that moxifloxacin and gatifloxacin were not significantly different from BSS in their effects on comeal wound healing and comeal haze after creation of both superficial and penetrating corneal wounds.

Purpose: Comeal epithelial wound closure rates in rabbit eyes were compared after topical applications of moxifloxacin 0.5% ophthalmic solution, gatifloxacin 0.3% ophthalmic solution, and BSS Sterile Irrigating Solution. Methods: Thirty rabbits were divided into 3 randomization groups of 10 rabbits each. Animals in each randomization group were predosed 4 times a day with a different test article in each eye for 1 day before surgery. Group 1 animals received BSS in 1 eye and moxifloxacin in the other eye; group 2 animals received BSS in 1 eye and gatifloxacin in the other eye; and group 3 animals received moxifloxacin in 1 eye and gatifloxacin in the other eye, resulting in each test article's being administered to 20 eyes. Rabbits underwent a bilateral procedure to remove the full thickness of the central comeal epithelium within a 5-mm trephine mark. After wounding, the eyes were dosed 4 times a day with the same respective predose test articles, and epithelial wound closure was recorded using slit-lamp photography. The data were analyzed to determine the rate of wound closure. Results: The mean wound radius closure rate was 71.1 ± 11.7 μm/h for BSS-treated eyes, 67.6 ± 7.1 μm/h for moxifloxacin-treated eyes, and 65.9 ± 8.0 μm/h for gatifloxacin-treated eyes. Conclusions: The relative order of wound healing rates was BSS > moxifloxacin > gatifloxacin; however, differences between treatment groups were not considered significant.

Gap junctional communication in the epithelium and endothelium of human corneas was studied. The influence of corneal storage on endothelial gap junctions was also examined.Donor human corneal cells were injected with carboxyfluorescein while surrounding cells were monitored for traces of fluorescence. Dye-spread coefficients were measured in corneal endothelial cells. Western blot and immunohistochemical analysis of the endothelium and epithelium was employed to determine if connexin 43 was present.Dye coupling occurs in both the epithelium and endothelium of the human cornea. Epithelial dye coupling was extensive in the basal layers but less apparent in the superficial layers. Endothelial dye coupling was similar to that reported for rabbit corneas. Western blot and immunohistochemical analyses demonstrated the presence of connexin 43 in both cell types.Gap junctional communication occurs in the endothelium and epithelium of human corneas, and both cell types express connexin 43. These results are similar to previous rabbit studies, thereby strengthening the use of the rabbit cornea as a gap junction model.

In newborn pig pial arterioles and cocultures of cerebral microvascular endothelial and smooth muscle cells, hypercapnia increases cAMP. In the intact cerebral circulation, both the increase in cAMP and the accompanying vasodilation require the presence of PGI2. Using piglet cerebral microvascular smooth muscle in primary culture, we addressed the hypothesis that, in the presence of PGI2, hypercapnia-induced changes in extracellular pH cause increases in cAMP. The stable PGI2-receptor agonist iloprost did increase production of cAMP in response to combined extracellular pH and pHi (11 ± 6 vs. 32 ± 10% in the absence and presence of 10−10 M iloprost, respectively). However, there was no positive dose-response relationship between iloprost concentration and stimulation of cAMP production by acidosis (e.g., 58 ± 9 vs. 41 ± 5% in the presence of 10−12 and 10−9 M iloprost, respectively). Rapid decreases in pHi stimulate the cAMP production. Decreases in extracellular pH do not appear to contribute further. The G protein inhibitor pertussis toxin did not augment cAMP production in response to decreasing pHi. We conclude that PGI2 receptor activation permits another mechanism to enhance cAMP generation in response to intracellular, but not extracellular, acidosis and that the mechanism of the permissive effect of PGI2 does not involve inhibition of a pertussis toxin-sensitive G protein.

Purpose: To determine whether pupillary responses to dilute tropicamide could be used as a diagnostic test for Alzheimer disease (AD). The authors also investigated whether concurrent use of an oral acetylcholinesterase inhibitor (tacrine) alters the pupillary response to dilute tropicamide in patients with AD, and whether pupillary responses to dilute tropicamide differ in young versus older control subjects. Methods: Pupillary diameter and area of both eyes were measured in light and darkness, at 10-minute intervals for 40 minutes after random instillation of 0.01 % tropicamide to one eye. Four groups of subjects were studied: 9 patients with AD, 10 who were treated with tacrine, 11 older control subjects, and 10 young control subjects. Results: Mean change in anisocoria was not significantly different among groups at any of the measurement time points. Mean percent change in diameter of the treated eyes showed a trend toward faster maximum dilatation in the AD groups, but change in pupillary measurements did not identify individuals with AD. Conclusion: Pupillary response to dilute tropicamide did not effectively distinguish individual patients with AD from young or older control subjects.

These studies examined corneal reepithelialization rates and type IV collagen expression in rabbits treated with either moxifloxacin HCl ophthalmic solution 0.5% as base or gatifloxacin 0.3% ophthalmic solution following anterior keratectomy.Animals (n = 6 per group) underwent surgery to create an 8-mm anterior keratectomy in the right eye. Rabbits were subsequently dosed with 1 drop, 3 times per day for 4 days with either moxifloxacin, gatifloxacin, or a commercially available irrigating solution. Fluorescein images were collected daily for the duration of the study. Approximately 96 h following surgery, the eyes were processed and evaluated for the presence of type IV collagen using immunohistochemical techniques. In two similar parallel studies, epithelial tissues were collected after the 48-h slit-lamp examination for a quantitative comparison of type IV collagen using either Western blot or quantitative polymerase chain reaction (Q-RT-PCR) techniques.Analysis of fluorescein images demonstrated that there were no significant differences in reepithelialization rates between the groups at any time point. At 96 h, 87%+/- 8% reepithelialization for moxifloxacin-treated eyes was observed compared with 77%+/- 10% for gatifloxacin-treated eyes and 85%+/-14% for BSS-treated eyes. The wound healing rates for the parallel studies demonstrated similar levels of reepithelialization for all groups. No discernable differences in type IV collagen expression were observed between treatment groups in the animals. The Q-RT-PCR analysis yielded no significant quantifiable difference in type IV collagen expression between any of the treatment groups. Expression values for alpha1 type IV collagen relative to the 18 S ribosomal RNA control were 0.0306+/-0.005 for BSS, 0.0251+/-0.002 for moxifloxacin, and 0.0254+/-0.006 for gatifloxacin.These studies indicate that there are no significant differences in corneal reepithelialization rates and type IV collagen expression between moxifloxacin ophthalmic solution 0.5%, gatifloxacin ophthalmic solution 0.3%, and the commercially available irrigating solution in this anterior keratectomy model.

Examine the mechanism of bicarbonate maintenance of cell-to-cell coupling in rabbit corneal epithelium and endothelium.Carboxyfluorescein was microinjected into rabbit corneal epithelial and endothelial cells. Adjacent cells were observed for fluorescence. Bathing solutions were buffered with bicarbonate, HEPES, phosphate, or acetate-citrate. The influence of intracellular pH and transmembrane voltage (V m ) were examined.Bicarbonate was the only buffer to increase dye coupling. Substitution of bicarbonate structural analogs bisulfite and carbamate in a HEPES-buffered solution increased dye coupling in both cell types. Intracellular pH and V m alterations in corneal epithelial cells bathed in HEPES vs. bicarbonate buffered media had no significant effects on dye coupling.Bicarbonate increases intercellular communication in the corneal epithelium and endothelium. This effect appears to result from an interaction of the bicarbonate molecule (or one of its structural analogs) with either gap junction proteins or an intermediary. We also demonstrate the presence of Cx43 in the rabbit corneal endothelium.

Microelectrode dye injection of 5,6-carboxyfluorescein was used to investigate gap junctional communication in the corneal epithelium.Dye injection started in the superficial layer and proceeded stepwise into the underlying epithelial layers until spread was observed. Intracellular [Ca2+] was manipulated by exposing the cornea to the calcium ionophore A23187 (global increase) or by increasing the [Ca2+] in the injection electrode (source cell increase). Intracellular pH was manipulated by exposing the cornea to nigericin in a low-pH KCI Ringer's (global decrease) or by lowering the pH in the injection electrode (source cell decrease). Heptanol was tested for its ability to uncouple gap junctions. Gap junctional communication was based on the layer at which spread was first observed and on the apparent dye travel distance from the point of injection.Control dye spread occurred, on average, in the third layer from the surface. Increased [Ca2+] in the source cell resulted in an initial spread occurring in the second layer. Globally increasing [Ca2+] with A23187 resulted in no change in the average initial spread layer. Lowering intracellular pH of the source cell did not affect the initial dye spread layer. Globally lowering intracellular pH resulted in significant gap junctional inhibition in a time-dependent manner. Dye spread distance was not significantly affected by [Ca2+] or pH manipulations. Heptanol (2.5 mM) completely inhibited dye coupling.All cell layers of the corneal epithelium contain functional gap junctions, although it appears that intercellular communication in the superficial layers does not occur under our control conditions. Intercellular communication through these junctions can be altered by various manipulations of [Ca2+] and pH.

The purpose of this presentation is twofold: (1) to summarize our studies as to the direct effect of 12(R)HETE, 8(R)HHDTrE on the corneal endothelium, and (2) to illustrate the diffusion and metabolism of 12(R)HETE by the cornea. Our data has shown that 12(R)HETE causes corneal swelling in a dose-dependent manner when perfused directly to the corneal endothelium. As reported by Schwartzman et al, 12(R)HETE is one of the major cytochrome P450-dependent arachidonate metabolites of the corneal epithelium and is an endogenous inhibitor of Na+/K+ATPase. Contact lens-induced hypoxic stress has also been shown to stimulate the endogenous formation of 12(R)HETE by the corneal epithelium. This increased formation of 12(R)HETE was found to correlate to increased corneal thickness and to changes in the corneal endothelial structure.These results suggest that 12(R)HETE produced by the corneal epithelium might be capable of diffusing across the cornea and affecting endothelial function.

The central endothelia of 48 eye bank corneas from donors ranging in age from 5 weeks to 88 years were photographed using in vitro specular microscopy. Computer-assisted morphometric analysis of the size and shape of endothelial cells was performed, and cell density was calculated. Histologic examination of the corneas after specular microscopy determined endothelial cell counts using x40 objective magnification. The mean endothelial cell counts from five different high-power fields were calculated. Results showed that there is a direct correlation between cell number and specular microscopy cell density (r = .91 and Spearman rank correlation, 0.69; both significant at P less than .01). A nomogram was developed to estimate corneal endothelial cell density from high-power field cell counts of pathologic specimens.