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Purpose. To determine recovery time needed for the corneal epithelium to return to a normal proliferation rate following 24-hour rigid gas-permeable (RGP) lens wear. Methods. An RGP lens (Dk/t = 10) was fitted at 9:00 AM on one randomly chosen eye of each rabbit (N = 16) and removed 24 hours later while the other eye served as a control. Following contact lens removal, the rabbits were injected at four different time intervals (1.2, 4, and 7 days) with 5-bromo-deoxyuridine (BrdU) at 9:00 AM to label dividing corneal epithelial cells. All animals were sacrificed 24 hours after BrdU injection, and corneas were stained for BrdU. A series of continuously adjacent digital images of the whole-mount epithelium was collected from superior limbus to central cornea, and all BrdU-labeled epithelial cell pairs on each digitized image were counted. Results. On day I following lens removal, the number of BrdU-labeled corneal epithelium cells was reduced by 50% centrally (C) and 36% peripherally (P) (P 0.001) compared with control eyes. At day 2, there was no statistically significant difference between control and experimental corneas (P = 0.1175). At day 4, a marked significant increase in BrdU-labeled cells was noted, averaging 83% (C) and 96% (P) (P < (1.001). At day 7. BrdU-labeling returned to control values, although it was still slightly elevated (14% Cl and 5% [P], P = 0. 045). Conclusions. The corneal epithelium in the rabbit required at least 1 full week to recover to its stable baseline proliferation rate following only 24 hours of RGP lens wear. Interestingly, hyperproliferation was noted within the limbal, peripherals and mid-peripheral corneal epithelium 4 days after lens removal.

To determine the effects of lens type and oxygen transmissibility on human corneal epithelium during daily lens wear (DW).Prospective, randomized, double-masked, single-center, parallel treatment groups clinical trial.Two hundred forty-six patients fitted with: (1) high oxygen-transmissible soft lenses (n = 36), (2) hyper oxygen-transmissible soft lenses (n = 135), and (3) hyper oxygen-transmissible rigid gas-permeable (RGP) lenses (n = 75).Irrigation chamber to collect exfoliated epithelial surface cells, confocal microscopy, and tear collection at baseline, 2 weeks, and 4 weeks of DW.(1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal epithelial surface cells, (2) central epithelial thickness, (3) superficial epithelial cell area, (4) epithelial surface cell exfoliation, and (5) tear lactate dehydrogenase (LDH).Four weeks of DW with the high oxygen-transmissible soft lens significantly increased PA binding from baseline 6.55 +/- 3.01 to 8.75 +/- 3.05 bacteria per epithelial cell (P0.01). By contrast, hyper oxygen-transmissible soft lens wear increased binding significantly less (6.13 +/- 2.45 to 7.62 +/- 3.06; P0.01), whereas hyper oxygen-transmissible RGP lens wear demonstrated no significant changes (5.91 +/- 2.40 to 6.13 +/- 2.17; P = 0.533). No significant change in central epithelial thickness was found after 4 weeks of DW in either soft lens; however, the epithelial thickness decreased by 9.8% (P0.001) with RGP lens wear. Epithelial cell surface area increased 3.3% and 4.1% with the high and hyper oxygen-transmissible soft lenses, respectively, and 10.5% with the hyper oxygen-transmissible RGP lens (P0.001). Epithelial desquamation significantly decreased in all groups (P0.001). Tear LDH levels increased for all test lenses (P0.001).Increased PA binding induced by wear of a conventional soft lens material is significantly greater than that induced by the new hyper oxygen-transmissible soft silicone hydrogel lens during DW. However, both soft materials showed significant increases in PA binding as compared with baseline controls. By contrast, hyper oxygen-transmissible RGP lens DW did not increase PA binding significantly. Taken together, these findings suggest for the first time both an oxygen effect as well as a difference between soft and rigid lens types on PA binding in DW.

To test prospectively a new biologic rationale for an advanced hyper oxygen-transmissible lens (HOTL) providing prospects for safer daily (DW) or extended (EW) contact lens wear.Prospective, randomized, double-masked, single-center, 13-month clinical trial.One hundred sixty-eight patients completed the DW study (1 month): control lens (n = 70); HOTL (n = 98). One hundred thirty-six patients finished 1 year of EW: controls (n = 56), HOTL (n = 25, 6 nights; n = 55, 30 nights).Irrigation chamber to collect corneal surface cells, confocal microscopy, tear collection at baseline, 2, and 4, weeks of DW, and 24 hours, 1, 3, 6, 9, and 12 months of EW.(1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness (CET); (3) superficial cell area (SCA); (4) epithelial surface cell exfoliation (DESQ); and (5) tear lactate dehydrogenase (LDH).Daily wear with control lens increased PA binding from 5.90 +/- 2.60 to 7.81 +/- 3.04 bacteria per cell (P0.01); HOTL wear increased PA binding significantly less (5.31 +/- 1.87-5.98 +/- 2.26; P0.01). Daily wear produced no significant changes in CET or SCA. Significant decreases in DESQ were seen with both lenses with no significant intergroup differences. Tear LDH increased significantly in DW with HOTL wear versus control (P = 0.0017), but not after 1 month of subsequent EW (P = 0.533). One to 3 months of EW with control lens showed significantly higher PA binding than HOTL wear (P0.01); binding adaptively decreased thereafter, returning to baseline at 9 to 12 months. Lens EW produced significantly enlarged SCA, thinning of CET (except 6-night HOTL wear), and decreased DESQ (P0.01). Some adaptive recovery was seen with CET and DESQ, but not SCA; importantly, the data indicated no significant difference between 6- or 30-night EW for all outcomes.Hyper oxygen-transmissible lens wear (DW or EW) produced significantly decreased PA binding compared with control lens wear, with no significant difference in wearing schedule (6 nights vs. 30 nights); additionally, there was a remarkable and unexpected adaptive recovery in the first 6 months of all soft lens wear, with a return to baseline PA binding levels and partial recovery for the other outcomes except SCA at 1 year. These results suggest that HOTL use should result in a decrease in the incidence of and risk(s) for lens-related microbial keratitis and that further epidemiologic studies should consider time in adapted EW in future risk and incidence analyses.

To determine the effects of open or closed eye and overnight contact lens wear on rabbit corneal epithelial surface cell death, detected by annexin V binding to cell surface phosphatidylserine and propidium iodide (PI) double-labeling.New Zealand white rabbits (n = 42) weighing 2.5 to 3.5 kg were divided into 7 study groups: hyper Dk/t rigid gas-permeable (RGP) lens; high Dk/t RGP lens; low Dk/t RGP lens; hyper Dk/t soft lens; high Dk/t soft lens; eyelid closure; and nictitating membranectomy study group (n = 6 rabbits for each group). Each rabbit was randomly chosen to have either one eye fitted with a test contact lens or both eyelids sutured closed; the contralateral eye served as a control. Rabbits were humanely sacrificed after 24 hours. Corneal buttons including the limbus were excised and stained with annexin V-FITC and PI to identify the number of nonviable epithelial surface cells. A series of sequential microscopic adjacent fields (200 microm X560 microm) from the inferior limbus to the central cornea were evaluated using epifluorescence microscopy, and the total number of cells stained with annexin V or PI, or both, was counted in each field.Nonviable cells (cells with positive staining) were lowest in density at the limbus and gradually increased in numbers towards the central cornea under normal open eye conditions (P0.05). Eyelid closure (no lens) caused a significant decrease in the numbers of nonviable cells in the peripheral and central cornea (P0.05) but not at the limbus (P0.05). Overnight wear of hyper Dk/t or high Dk/t RGP soft contact test lenses caused significant decreases in the numbers of nonviable cells on the central corneal surface (P0.05). Hyper Dk/t and high Dk/t soft lenses had similar suppressive effects on the number of surface nonviable cells, independent of lens oxygen transmissibility. By contrast, the number of nonviable cells was dependent upon RGP lens oxygen transmissibility; hyper Dk/t RGP lens wear produced significantly less suppression of the number of nonviable cells in the central cornea than high Dk/t RGP lens wear (P0.05). Wear of a nonphysiologic low oxygen transmissible RGP test lens however, produced a significant increase in nonviable cells in the central cornea. The number of nonviable epithelial surface cells was not affected by nictitating membranectomy under open eye conditions (P0.05).This study revealed in the rabbit model a gradient of nonviable surface epithelial cells increasing towards the central cornea under open eye conditions and with central suppression of surface cell death following closed eye (no lens) or physiologic contact lens wear. Taken together, the results suggest that eyelid closure or contact lens wear may protect the corneal epithelial cells against the shear stress forces exerted by eyelid blinking, which are believed to drive central epithelial surface cell death and exfoliation. However, under very low oxygen tensions combined with lens effect, such as in low Dk/t RGP lens wear, surface cell death may be accelerated.

To determine effects of lens type and oxygen transmissibility on human corneal epithelium during extended wear (EW).Prospective, randomized, double-masked, single-center, parallel treatment groups, 1-year clinical trial.One hundred seventy-eight patients completed the study: (1) high-O(2) soft lens (6-night [N] EW) (n = 27); (2) hyper-O(2) soft lens (6N-EW, n = 33) or (30N-EW, n = 66); and (3) hyper-O(2) rigid gas-permeable lens (RGP) (30N-EW, n = 52).Irrigation chamber to collect exfoliated corneal surface cells, confocal microscopy, and tear collection at baseline, 1, 3, 6, 9, 12 months of EW.(1) Pseudomonas aeruginosa (PA) binding to exfoliated corneal surface cells; (2) central epithelial thickness; (3) superficial epithelial cell area; (4) epithelial surface cell exfoliation; and (5) tear lactate dehydrogenase.Quantitative evidence demonstrated increased binding of PA to human exfoliated corneal epithelial cells during the first 3 months of soft lens EW; the control high-O(2) test lens showed significantly higher bacterial binding (P0.05). Binding activity gradually decreased thereafter and returned to baseline after 9 and 12 months. The corneal epithelium demonstrated enlargement of surface cell size, thinning of central epithelium, and a significant decrease in surface cell shedding (P0.05). Remarkably, there was subsequent partial adaptive recovery in cell shedding and epithelial thickness but not surface cell size. There was no significant difference between 6N and 30N continuous wear of the hyper-O(2) soft lens for all outcome measures. Importantly, hyper-O(2) RGP lens wear did not show significantly increased PA binding during 1 year.This study establishes three important new findings: (1) hyper-O(2) soft lens EW produces significantly less PA binding than the lower O(2) soft lens with no significant difference in PA binding with 6N versus 30N EW of the hyper-O(2) soft lens; (2) there is a remarkable adaptive recovery after 6 months with all soft lens wear with gradual return to prelens PA binding levels and partial recovery of other outcome measures for all test lenses EW except surface cell size; (3) 30N EW of the hyper-O(2) RGP lens produced no significant increases in PA binding over 1 year. Taken together, these results suggest that introduction of new hyper-O(2) transmissible lens materials into clinical use may offer safer EW, and future epidemiologic studies of ulcerative infectious keratitis should consider both lens type and time in lens EW in any incidence/risk analysis.

The purpose of this study was to determine the localization of Bcl-2 protein in the human cornea. Anti-human Bcl-2 monoclonal antibodies (MAbs) against selective Bcl-2 peptide sequences were used to localize Bcl-2 protein immunocytochemically in fresh eye bank donor human corneas (n = 4). Specificity of each MAb was determined by Western blot analysis of pooled protein extracted from human corneal epithelium (n = 3). Expression of Bcl-2 protein in apoptotic surface epithelial cells was detected by co-labeling with TUNEL assay and anti-Bcl-2 antibody staining. Two MAbs specific for amino acids residues (aa) 41-54 within the loop domain of Bcl-2 protein stained nuclei of all corneal epithelial cell layers. MAb specific for aa 61-76, also within the loop domain, produced faint nuclei and nuclear envelope staining. Occasional corneal surface epithelial cells however, consistently lacked anti-Bcl-2 nuclear staining with these three MAbs; concomitant TUNEL assay revealed that all TUNEL positive-surface cells were Bcl-2 negative. In the stroma, keratocytes showed similar but weak anti-Bcl-2 staining. All corneal endothelial cells showed intense nuclear staining with MAbs, with no gradient or absence of staining. In summary, Bcl-2 protein can be localized to the nuclei and nuclear envelope of corneal epithelial cells, keratocytes and endothelial cells with the use of MAbs specific for the loop domain of Bcl-2. TUNEL-labeled surface epithelial cells did not stain with MAbs to Bcl-2, suggesting degradation or epitope masking perhaps by specific phosphorylation of the loop domain during apoptosis. Taken together, these findings suggest that Bcl-2 protein may play a critical role in modulating apoptotic cell desquamation in the human corneal epithelium.

PURPOSE To examine the rabbit corneal epithelial cell proliferation rate after extended wear of disposable or silicone hydrogel contact lenses or prolonged eyelid closure. METHODS One randomly chosen eye of 40 New Zealand White rabbits was assigned to silicone hydrogel contact lens wear (n = 15, SH), disposable hydrogel contact lens wear (n = 6, DH), eyelid suturing (n = 15, SUT), or no intervention (n = 4). Contralateral eyes served as the control. After 24 hours or 1 week of lens wear, 5-bromo-2-deoxyuridine (BrdU) was injected intravenously to label dividing corneal epithelial cells, and animals were killed 24 hours after injection. Corneas were stained with monoclonal anti-BrdU antibody and FITC-conjugated secondary antibody. A series of continuous digital images of the wholemounted epithelium were collected from the superior to inferior limbus, and the number of BrdU-labeled cell pairs was counted. RESULTS SH, DH, and SUT caused a significant decrease in BrdU-labeled pairs of cells over the entire corneal epithelium at day 2 compared with the number in contralateral control eyes (P < 0.001). One week of SUT or SH caused a significant increase centrally in BrdU-labeled cells (P < 0.01). BrdU labeling at the limbus in all groups was not significantly different from the control. Unexpectedly, the proliferation rate of the control corneas was also significantly affected by contralateral lens wear and suturing. CONCLUSIONS Short-term overnight SH, DH, and SUT all significantly suppressed the cell proliferation rate in the rabbit corneal epithelium. However, adaptation, with central hyperproliferation of cells, appeared to occur at 8 days. The effects of lens wear and eyelid suturing on the cell proliferation rate in contralateral control eyes suggests a central mechanism that regulates corneal epithelial proliferation.