Superficial epithelial cells are spontaneously exfoliated from the corneal surface during blinking, 1,2 and recent studies have further demonstrated that apoptosis plays an important role in controlling this vital homeostatic process. 3–5 In general, the loss of surface cells is thought to be compensated by centripetal migration of transient amplifying cells from the limbus, additional proliferation of the basal epithelial cells, and the subsequent upward movement and terminal differentiation of cells leaving the basal lamina. 2,6 Surprisingly, no studies have examined the effects of eyelid closure on this normal process or determined the effects exerted by contact lens wear.
It has been reported in human subjects that overnight contact lens wear causes consistent pathophysiologic changes in the corneal epithelium such as microcysts and central epithelial layer thinning. 7–9 Paradoxically, however, clinical studies also demonstrated that the exfoliation rate of human corneal epithelial cells decreases during all forms of contact lens wear, independent of lens material rigidity, 9,10 and the number of exfoliating surface cells as measured with the irrigation chamber is consistently reduced in both daily and extended contact lens wear. 10–12 Taken together, these findings demonstrate a down-regulation of homeostatic surface cell death and exfoliation during all human contact lens wear.
Recently, we observed in the rabbit model that overnight wear of rigid gas permeable (RGP) contact lenses reduces the number of TUNEL-labeled corneal surface epithelial cells, demonstrating a direct lens-related apoptotic effect. 13 Additionally, these studies suggested that persistence of expression of Bcl-2 protein in surface cells may play a central role in modulating apoptotic surface cell exfoliation in both human and rabbit corneal epithelium as well as mediating the effects of contact lens wear. 5,13 These experiments, however, were limited in the total areas and number of cells surveyed by histologic sections, and, additionally, such studies could not entirely exclude other nonapoptotic pathways potentially mediating cell exfoliation. 5,13
The purpose of this study was, therefore, to examine the effects of eyelid closure and contact lens wear on spontaneous surface cell death and exfoliation over the entire rabbit cornea using a standard calcein-acetoxymethyl ester–ethidium homodimer (calcein AM–EthD-1) viability assay (Molecular Probes, Inc., Eugene, OR).
MATERIALS AND METHODS
Thirty-six New Zealand white rabbits, 10 to 16 weeks old and weighing 2.5 to 3.5 kg, were used for this study and humanely treated according to the ARVO statement for the use of animals in ophthalmic and vision research. All rabbits were healthy and free of clinically observable ocular surface disease. The nictitating membranes from both eyes of 32 rabbits were partially excised under anesthesia 2 mg/kg Rompun (Bayer, Shawnee Mission, KA) and 30 mg/kg Ketaset (Fort Dodge Animal Health, Inc., Fort Dodge, IA). Twenty-four rabbits with bilateral nictitating membranectomy were randomly assigned to four test groups for calcein AM–EthD-1assay [(1) low Dk/t RGP lens, (2) hyper Dk/t RGP lens, (3) hyper Dk/t soft lens, and (4) eyelid closure, six rabbits in each group]. Eight remaining rabbits with bilateral nictitating membranectomy were used for eye irrigation to collect exfoliating epithelial cells. In the remaining four rabbits, the nictitating membrane from only one eye was excised to control for the effects of nictitating membranectomy on the surface viability assay, and the other eye served as a control with an intact nictitating membrane. After overnight lens wear, rabbits were humanely killed with an overdose of 100 mg/kg Sleepaway (Fort Dodge Animal Health, Inc.). All experiments were done at 10 A.M. to standardize circadian effects.
Test Contact Lenses and Eyelid Closure
Table 1 shows the material and physical characteristics of the RGP and soft contact lenses. A trial RGP lens set with base curves 7.60, 7.80, and 8.00 was used to determine the best possible fit for each rabbit. All RGP lenses were exclusively designed for rabbit eyes with uniform thickness and plano power. The hyper Dk/t silicone hydrogel soft test lens had a base curve of 8.6 mm and a power of −3.00 D.
Complete eyelid closure was obtained by carefully suturing the upper and lower lids together under anesthesia using 7-0 black braided silk (Ethicon, Inc., Somerville, NJ). The oxygen tension at the ocular surface under closed eyelids has previously been reported to be 7.4 to 7.7%, compared with 21% under open-eye conditions at sea level. 14
All lens fitting and eyelid closure were done on one eye of each rabbit, randomly chosen by coin toss, at 10 A.M., and the contralateral eye served as a control. Before lens removal, 300 μL of warmed saline (0.9% sodium chloride, 35°C) was added to the ocular surface over the contact lens. The floating contact lens was then carefully taken out with a standard rubber suction device. The same volume of warmed saline (300 μL) was also applied to the paired control eye.
The calcein AM–EthD-1 assay provides the simultaneous determination of viable and nonviable cells with two probes that measure two parameters of cell viability. Calcein AM determines viable cells by the presence of intracellular esterase activity, converting enzymatically to intensely fluorescent calcein (excitation/emission 495 nm/515 nm). EthD-1 indicates nonviable cells by entering cells with damaged/compromised membranes, undergoing 40-fold enhancement of fluorescence on binding to nucleic acids (∼495 nm/∼635 nm).
Viability of Surface Epithelial Cells
The working solution was prepared in the sterile MEM culture media (pH = 7.20), Life Technology, Inc., Rockville, MD) containing sodium bicarbonate (44 mmol/L), RPMI vitamins and glutathione, and nonessential amino acids and ascorbic acid (0.63 mmol/L). Calcein AM and EthD-1 were added to the media (2 and 4 μM, respectively), just before use.
After excising the cornea carefully with an attached 2-mm scleral rim, the corneal surface was immediately immersed in 1.5 mL of calcein AM/EthD-1 working solution in a 12-well cell culture plate and incubated at 37°C for 20 minutes. A vertical strip of cornea (4 mm width) was then cut from the superior to inferior cornea, mounted epithelial side up on a glass slide and then immediately observed using a 10× objective and Leica Diaplan fluorescent microscope (Leica, Deerfield, IL) equipped with a Cohu high-performance monochrome charge-coupled device camera (Model 4912, Cohu, Inc., San Diego, CA). Sequential digital images (2.21 mm 2 ) were captured as sequential fields from superior to inferior cornea, along the corneal strip (total 15 fields = 33.15 mm 2 per cornea). The number of nonviable cells on the corneal surface was counted and recorded as cells/mm 2 per field. For statistical analysis, the data were organized in three compartments: superior peripheral (fields 1–5), central (fields 6–10), and inferior peripheral corneal epithelium (fields 11–15).
Viability of Exfoliated Corneal Epithelial Cells
Exfoliated corneal epithelial cells were collected from a precorneal tear film of anesthetized non–lens-exposed rabbits (12 eyes), using a corneal irrigation chamber method as previously reported. 12 Immediately after collection (9 mL/min), exfoliated cells in irrigation buffer 15 [116.3 mmol/L NaCl, 18.8 mmol/L KCl, 26.0 mmol/L NaHCO 3 , 0.08 mmol/L NaH 2 PO 4 , 0.6 mmol/L MgCl 2 · 6H 2 O, and 0.4 mmol/L CaCl 2 · 2H 2 O (pH = 7.40, 302 ± 2 mOsm/kg)] were incubated with 2 μM calcein AM and 4 μM EthD-1 for 10 minutes at 37°C. The suspension was rapidly filtered through a polycarbonate membrane with 0.5 μm pore size (Osmonics Inc., Livermore, CA, U.S.A.). Exfoliated cells were observed with a 63× objective and Leica Diaplan fluorescent microscope. As previously reported, it was possible to identify corneal epithelial cells from conjunctival or inflammatory cells with specimens. 12,15
SigmaStat for Windows Version 1.0 (Jandel Scientific, San Rafael, CA, U.S.A.) was used to perform two-way analysis of variance (ANOVA) (factors group and location) and Student-Newman-Keuls test to analyze the differences in each test group versus its control and between experimental test groups. One-way ANOVA (Kruskal-Wallis rank method) was also performed to test the distribution of nonviable cells in the normal cornea. A p value less than 0.05 was considered statistically significant.
Viability of Surface Epithelial Cells
Most epithelial surface cells of the normal cornea were viable and stained with calcein AM assay (Fig. 1A). Some surface cells however, showed a red nucleus (EthD-1 assay) (Fig. 1B), indicating that these cells were dead or in the process of dying.
Figure 2 shows the data plotted by location for all normal corneas (with membranectomy) examined in this study (n = 28 corneas). Nonviable cells had the highest density in the central cornea, gradually decreasing in density to the corneal periphery (approximate, center to periphery ratio = 2:1). There was a significant difference between the central region and the periphery (p < 0.001, Kruskal-Wallis one-way ANOVA on ranked data).
Figure 3A shows results of the viability assay after overnight wear of the low Dk/t RGP test lens wear (n = 6 rabbits). Overall, two-way ANOVA analysis showed a significant difference in the number of nonviable cells between the corneal epithelium of test group and control (p = 0.0013, two-way ANOVA); however, a significant decrease was only observed when the central region was compared with control eyes (p < 0.05, two-way ANOVA multiple-comparison Student-Newman-Keuls test), but no significant difference was noted in the inferior and superior peripheral regions (p > 0.05). A few isolated areas with patches of ethidium-positive surface cells were noted in two of the six specimens. These areas may represent massive necrotic cell death not associated with normal physiologic surface cell loss and therefore were excluded from the data analysis.
Figure 3B shows the effect of wear of the hyper Dk/t test RGP lens (n = 6 rabbits). Similar to the results obtained with low Dk/t RGP lens wear, there was a significant difference in the number of nonviable cells between the test group and control (p = 0.00428, two-way ANOVA). A significant decrease was also observed in the central region compared with control eyes (p < 0.05) but not in both peripheral regions (p > 0.05). No isolated areas of surface cell loss or nonviability were seen with hyper Dk/t RGP lens wear.
Both overnight hyper Dk/t soft lens wear (Fig. 3C, n = 6 rabbits) and eyelid closure (Fig. 3D, n = 6 rabbits) revealed results similar to RGP lens wear. There was a significant difference in the number of nonviable cells between the test group and the control (p = 0.0139 and p = 0.00758, respectively, two-way ANOVA) with a significant decrease in the central corneal epithelium compared with control eyes (p < 0.05) but not in both peripheral regions (p > 0.05). No surface areas with clusters of lost cells were seen.
Two-way ANOVA did not indicate a statistically significant difference between the experimental groups (p = 0.633); however, it should be noted that the power of the test performed was less than 0.8, and thus a larger sample size is required for a conclusive interpretation.
As a control experiment, the effects of nictitating membranectomy on the surface cell viability assay were also evaluated (n = 4 rabbits). There was no significant difference between the control eyes and membranectomized eyes in the number of nonviable cells (p < 0.05, data not shown).
Viability of Exfoliated Epithelial Cells
Exfoliated epithelial cells collected with the irrigation chamber were uniformly dead cells that contained fluorescent red nuclei (Fig. 1C, calcein AM; 1D, EthD-1), similar to the results of nonviable cells observed on the corneal surface (Fig. 1E, calcein AM; 1F, EthD-1). 3,15 Some of these cells also retained an intense punctate fluorescent green cytoplasm suggesting that calcein AM activity had not completely terminated in the subcellular structures (see Fig. 1C). The average number of dead exfoliated epithelial cells from the normal rabbit cornea collected by eye irrigation was 7.1 ± 2.6 cells/9.0 mL/min (mean ± SD , n = 12 eyes), which agrees well with previous studies. (3,15)
Given its central role in visual function, the paucity of studies of the homeostatic regulation of normal corneal surface cell exfoliation is indeed surprising. Using the calcium AM–EthD-1 assay, Ren and Wilson 3 previously reported that most surface cells on the normal corneal epithelium are viable, whereas a small subpopulation is nonviable. All exfoliated cells, however, appeared to be nonviable. 3,15 Our results confirm and significantly extend these findings, demonstrating for the first time a quantitative gradient of nonviable cells increasing from the corneal periphery to the center, with an approximate center-to-periphery ratio of 2:1. As expected, all exfoliated cells were dead or dying, in excellent agreement with the past studies. 3,15 Of considerable interest is the demonstration that the number of nonviable cells on the corneal surface decreases centrally with eyelid closure despite known closed eye hypoxia. Taken together, these observations provide strong support of the eyelid shearing force hypothesis, which postulates that the mechanical shearing forces induced by eyelid blinking reach the highest degree of stress at the apex (central region) of the corneal surface, predicting increased exfoliation of dead cells preferentially from the center. 15 To place the results obtained in this study with the rabbit model into proper perspective, it is important to note that several recent clinical studies in human patients have reported large, consistent decreases in spontaneous surface cell exfoliation from the corneal surface during all contact lens wear. 9,10 In another human trial, however, the parallel effect of non-lens-related hypoxia on surface cell exfoliation was also studied under open-eye conditions with normal blinking. Continuous wear of 100% nitrogen or 95% nitrogen:5% CO 2 goggles for 6 hours (hypoxia without eyelid closure) also led to a significant reduction in corneal surface cell exfoliation. 16
Consistent with this finding from human studies, the result of the present investigation also demonstrated that both overnight wear of any test contact lens (independent of lens type or oxygen transmissibility) or eyelid closure with hypoxia (no lens and no eyelid blinking) significantly decreased the number of nonviable cells on the central surface of the rabbit cornea.
Our most recent experimental studies further revealed that the Bcl-2 protein appears to play a critical central role in regulating normal surface epithelial cell exfoliation in both rabbit and human corneas. 5,13 Importantly, the data presented here are also in excellent agreement with those parallel studies demonstrating Bcl-2–mediated decreases in corneal surface cell death produced by overnight contact lens wear using the same test lenses in the rabbit model. 13,17
Using all currently available results from past and parallel reports, the findings in the current study suggest, therefore, a new overall hypothesis balancing hypoxia and lid shear forces in the regulation of normal and contact lens–effected surface corneal epithelial cell exfoliation. (1) Under open eye conditions (no contact lens), there is no hypoxia and therefore lid shearing forces presumably drive exfoliation of nonviable surface cells in the central cornea. If, however, hypoxia is introduced by 100% nitrogen gas exposure with goggle wear, surface cell exfoliation is significantly reduced despite eyelid blinking forces. (2) Under closed-eye conditions (no lens), corneal surface cell exfoliation is maximally suppressed (no eyelid shear forces + hypoxia). (3) Contact lens wear, however, decreases surface corneal epithelial cell death and exfoliation with the lens acting as an apparent shield or barrier to the effects of eyelid shearing forces. Thus, the control of corneal surface cell exfoliation seems to be regulated by oxygen (possibly Bcl-2 mediated) and/or lid shearing forces, with the contact lens behaving as a protective shield against the latter. Additional studies are clearly indicated to further validate this new hypothesis.
The central gradient of increasing surface cell death reported here also has an additional potential physiologic effect of great importance. If cells are exfoliated centrally after terminal differentiation and upward movement from the basal epithelial layer, a central driving force will exist that can explain the long-observed migration pattern of newly generated transient amplifying cells and basal epithelial cells from the limbus to the center of the cornea. Of great interest in this regard is the parallel finding from another recent study that demonstrates that basal epithelial cell proliferation is also suppressed by either eyelid closure or contact lens wear in the rabbit model. 18 Taken together with results in the current study, it appears that both surface cell exfoliation and basal epithelial proliferation are simultaneously suppressed by eyelid closure and contact lens wear. The central question of possible regulatory linkage between these two processes is an important area for future investigation.
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