Biochemical transformation of regenerating ocular surface epithelium.

Several biochemical parameters, including glycogen levels, and the activities of hexokinase, phosphorylase, and lactate dehydrogenase have been compared in regenerating epithelium of conjunctival and corneal origin in rabbits. The study was designed to determine the extent of biochemical transformation of conjunctival into corneal epithelium completed within 6 weeks. Although histological transformation, especially in the case of the chemically damaged eyes, is not. Glycogen and lactate dehydrogenase levels remained well below normal corneal epithelial levels for the period of observation.

Functional competence of regenerating ocular surface epithelium.

Several aspects of the ocular surface epithelium, including its clinical appearance, its effect on the strength of underlying stromal wounds, and its healing rates, have been compared between regenerating epithelium of conjunctival and that of corneal origin in rabbits. The results showed that conjunctival epithelium could not transform completely into functionally competent corneal epithelium within 6 weeks.

Ocular surface epithelium and corneal vascularization in rabbits. I. The role of wounding.

A new model for rabbit corneal vascularization, created by making a penetrating wound in corneas with epithelium of conjunctival origin, is described. Obligate resurfacing of the cornea from conjunctival epithelium usually leads to a small, but consistent peripheral superficial corneal vascularization. Subsequent penetrating wounds elicit, in 75% of cases, a marked vascular ingrowth. Normal eyes and eyes resurfaced by peripheral corneal epithelial cells do not vascularize after such wounds. The vessels are located in the anterior corneal stroma, and the regenerated epithelium has a conjunctival appearance. Although increased hydration plays a role in this vascularization, the extent of vascularization was much greater in the presence of regenerated epithelium of conjunctival origin than in the presence of regenerated epithelium of corneal origin.

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.

Comparison of limbal and peripheral human corneal epithelium in tissue culture.

Peripheral human corneal epithelium grows better in tissue culture than central epithelium, but it is not known whether ocular limbal epithelium grows even better than does the peripheral corneal epithelium. In this work we compared the growth kinetics of limbal and peripheral human corneal epithelial cells in tissue culture. Four 1-2 mm2 explants, removed from the limbus or from peripheral cornea (1-2 mm inside the limbus) of eye bank eyes, were grown to confluence in primary culture. Cells were then passaged at 2 X 10(5) cells per dish. At intervals thereafter, the cells were counted in a hemocytometer to determine plating efficiency and growth curves. Mitotic activity was determined 4 days after passaging by labeling cultures with 3H-thymidine and counting aliquots using the hemocytometer and scintillation counter. In the primary cultures, limbal epithelium grew as small, uniformly polygonal cells. Peripheral corneal cells grew to a variety sizes. The 24 hr plating efficiency and doubling time of limbal epithelial cells were 47 +/- 8% and 80 +/- 14 hr, respectively, while those of peripheral corneal cells were 41 +/- 10% (P less than 0.1) and 131 +/- 25 hr (P less than 0.001). The mitotic activity of limbal cells was significantly higher than that of peripheral (2.9 +/- 1.2 vs. 0.8 +/- 0.6) (P less than 0.01). These results indicate that human ocular limbal epithelium grows better in culture than does peripheral human corneal epithelium.

Collagen gel for ocular surface.

A replacement ocular surface requires a substrate that is easily manipulated surgically, does not cause an inflammatory reaction, and is nontoxic to epithelial cells. This work evaluates the usefulness of a collagen gel as a substrate for corneal epithelial cells by determining the ocular toxicity of the gel and the ability of the gel to support and maintain corneal epithelial cells in vitro. Collagen gels, made from Vitrogen, were easily manipulated and were well-tolerated in rabbit eyes for up to 6 wk (n = 3). Epithelial sheets placed on collagen gels and incubated at 37 degrees C for up to 13 days remained well-apposed to the gels and appeared normal, but thinned, from five to three layers. The basal cells extended cytoplasmic blebs into the gels, but only one sheet of five sheets showed basement membrane deposition by 6-13 days. Thus, the collagen gels appear to meet the criteria defined above and may be a suitable substrate in biofabricated ocular surfaces.

Conjunctival epithelium in healing of corneal epithelial wounds.

The mitotic rate and goblet cell content of conjunctival epithelium following total or central corneal epithelial removal using n-heptanol was measured to determine how the conjunctival epithelium responds to injury and whether conjuctiva responds to central corneal epithelial loss. One day following a wound that removed corneal, limbal, and 1-2 mm of bulbar conjunctival epithelium, the mitotic rate of the remaining conjunctival epithelium was ten times normal (P less than 0.001), proving that the conjunctiva responds to injury by cellular proliferation. At 1 and 2 days following a limited 10 mm diameter central corneal wound, the mitotic rate of peri-limbal conjunctival epithelium was three to four times normal (P less than 0.01), and even following a 5 mm diameter central wound, it was three to four times normal on day 1 (P less than 0.05). Goblet cell frequency was a less reliable indicator of conjunctival response to corneal injury: it was decreased following the largest and smallest wounds but not affected by the 10 mm diameter wound. These studies demonstrate that conjunctival epithelium peripheral to the cornea is affected by small central corneal wounds, and may therefore play a role in corneal epithelium healing.

Comparison of central and peripheral human corneal epithelium in tissue culture.

Past attempts to grow human corneal epithelium in culture had limited success, with confluence rarely attained. This work is to determine whether different areas of human corneal epithelium grow better in tissue culture. We compared the extent, the mitotic rates, and morphology of outgrowths and histology of explants from central and peripheral human corneas in culture. Explants, 2 mm in diameter, removed from eye bank eyes, were placed epithelial side up on a culture dish with modified SHEM tissue culture medium (Jumblatt et al, 1983). After 7 days, the tissues were fixed, stained and the area of outgrowths from explants measured using an image processor. For eight eyes from donors averaging 66 yr old, the average area of central outgrowths was 7.8 +/- 1.1 mm2, while that of peripheral outgrowths was 52.8 +/- 5.2 mm2 (P less than 0.001). The mitotic rate of outgrowths of central epithelium was significantly less than that of peripheral epithelium (1.1 +/- 0.5% vs 18.8 +/- 0.8%) (P less than 0.001). After 14 days, central outgrowths had not attained confluence and consisted of large cells. Peripheral outgrowths had attained confluence and consisted of small polygonal cells. Histology of explants showed that only one layer of epithelium remained on the stroma in central explants, but several layers were present on the peripheral explants. Thus, peripheral human corneal epithelium grows better in culture than does central human corneal epithelium.

Conjunctival epithelial wound healing.

In vivo conjunctival epithelial healing in albino rabbits was investigated by light microscopy following both n-heptanol and trephined conjunctival wounding. Reepithelialization occurred faster following n-heptanol treatment (3 days) versus trephination (6-7 days). No goblet cells were present in the migrating epithelium during reepithelialization. After 1 day of wounding, goblet cells disappeared several millimeters peripheral to the wound margin in both types of wounds. Goblet cells first reappeared peripherally 1 week after wounding before they appeared in the central wound area. These observations indicate that a large area of conjunctival epithelium surrounding a wound is involved with repair of that wound. Since the goblet cell content of conjunctival epithelium appears to change as a result of the stresses of epithelial repair, the goblet cell population may reflect the presence of reparative or proliferative processes in the ocular surface.

Conjunctival goblet cell frequency after alkali injury is not accurately reflected by aqueous tear mucin content.

Goblet cell counts have been used to evaluate the suitability of conjunctiva as a source of ocular surface epithelial cells. However, since tear mucin content can be determined without tissue excision, it seemed that the concentration of those compounds might be a useful indicator of conjunctival vitality. To test the extent to which aqueous tear composition reflects conjunctival goblet cell frequency, goblet cell frequency and aqueous tear mucin content were measured after alkali injury in rabbits. Mild alkali injury (0.1 N NaOH for 30 sec) caused a transient but substantial decrease in goblet cells (to 25% of normal at day 7) with a return to normal by six weeks. Tear mucin content was decreased to a lesser degree, from a normal value of 6.4 +/- 0.47 nmol oligosaccharide per microliter (n = 10) to a minimal value of 4.7 +/- 0.64 (n = 7) (73% of normal) at day 7, returning to normal 4 weeks after injury. Thus, the direction of the change was the same, but the magnitudes were quite different. These results suggest that conjunctival goblet cell frequency is not accurately reflected by aqueous tear mucin content, and therefore, that tear mucin content cannot be used directly as an indicator of conjunctival health.

Sex chromatin of donor corneal epithelium in rabbits.

The survival of donor corneal epithelium was investigated in rabbits after they received unilateral 8 mm diameter lamellar keratoplasties with living donor tissue of a different sex from the host. Three weeks, 6 weeks, and 12 weeks postoperatively, cultured corneal epithelia, grown from 5 mm diameter central buttons, the adjacent 0.75 mm wide donor rings, the peripheral 10 to 13 mm diameter rings, and 5 mm diameter central host buttons were used for sex-chromatin analysis. The results indicated that some of the donor corneal epithelium survived up to 12 weeks postoperatively. Even in the absence of overt epithelial rejection, however, a time-dependent decrease of donor corneal epithelium and simultaneous invasion of host corneal epithelium were demonstrated.

Effects of ultraviolet radiation on corneal epithelial metabolism.

Acute exposure to ultraviolet light to 257 nm wavelength produced a photokeratitis associated with characteristic metabolic alterations of corneal epithelial metabolism in rabbits. Significant increases in corneal hydration occurred simultaneously with decreased corneal epithelial glycogen content, but adenosine triphosphate content and enzyme activity of epithelial extracts were not affected despite clinical and histological damage to the corneal epithelium. The pattern and time course of ultraviolet damage to the cornea are distinctly different from those of other forms of trauma.

Corneal re-epithelialization from the conjunctiva.

After debridement of the entire corneal epithelium, epithelial cells of conjunctival origin cover the exposed corneal surface. Four to five weeks later, these cells undergo a morphologic transformation to normal-appearing corneal epithelium. To study this transformation the entire corneal epithelium was removed from rabbits with the use of n-heptanol, after which the histologic appearance of and the number of goblet cells in the regenerated epithelium were noted. Five stages of transformation were seen. Immediately after healing, the epithelium consisted of one to two squamous cell layers with no goblet cells apparent at the light microscope level (stage 1). In the following weeks goblet cells appeared at the limbal edge of the cornea (stage 2), reached a uniform distribution across the cornea (stage 3), and subsequently receded toward the limbus (stage 4), leaving an epithelium with normal corneal morphologic appearance (stage 5). To see if there was an ongoing centripetal cell migration from the conjunctiva across the cornea after initial healing, the central corneal epithelium was isolated from the periphery by a ring of glue. Such isolation resulted in a thinning of the central epithelium and a thickening of the peripheral epithelium. These studies suggest that (1) the transformation into corneal epithelium lags behind defect closure by 4 to 5 weeks, (2) goblet cells do not initially migrate as recognizable cells, and (3) there is a continuous centripetal cell motion even after the initial defect closure is accomplished.

Diabetes mellitus and the rabbit corneal epithelium.

Diabetes mellitus has been shown to be a factor in the development of corneal epithelial abnormalities in stressed human eyes, but the biochemical basis for this is not known. To see if sorbitol pathway activation might be involved, ocular surface epithelial healing rates and metabolites of the glycolytic and sorbitol pathways were measured in alloxan-diabetic rabbits. As in humans, corneal epithelial healing rates were not decreased in the diabetic rabbits, suggesting that the rabbit may be an appropriate model for human disease. Increased levels of glucose, glycogen, and sorbitol were found in the diabetic corneal epithelium compared with normal. However, the sorbitol accumulation only mounted to 1.0 mOsm/L of tissue water, which implies that osmotic damage secondary to corneal epithelial cell sorbitol accumulation might not be a significant factor in corneal epithelial abnormalities of diabetes.

Insulin sensitivity and sorbitol production of the normal rabbit corneal epithelium in vitro.

Insulin-depleted, normal rabbit corneal epithelium was incubated in vitro in tissue culture medium containing high concentrations of glucose (35 mM). These short-term incubations showed that the epithelium took up glucose equally whether or not insulin had been added to the medium, indicating that corneal epithelium is an insulin-insensitive tissue. Sorbitol accumulation showed that the normal corneal epithelium has a sorbitol pathway which can be activated in the presence of high intracellular glucose. The low level of sorbitol accumulation in these normal epithelia is probably not osmotically significant.

Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation.

The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing.

An ultrastructural study of rabbit ocular surface transdifferentiation.

When debridement of the rabbit cornea is followed by re-epithelialization from the conjunctiva, a process of transdifferentiation of the endothelium occurs. Goblet cells appear peripherally 1 week after healing of the epithelial defect, are widespread at 2 weeks, and disappear centrally at 3 to 4 weeks. Six weeks after closure of the defect, the epithelium has reverted to the customary corneal appearance. The morphology of the regenerating epithelium was studied by light, transmission and scanning electron microscopy. The precursor cells for the goblet cells were identified in stage 1, before PAS-positive cells were present, as pairs of cells with dark cytoplasm and prominent Golgi. Subsequently, goblet cells were present in pairs, indicating that goblet cells are derived from non-goblet epithelial cells, and that they do not simply migrate onto the cornea. At the time of transdifferentiation, loss of goblet cells was shown to occur both by desquamation from the surface and by in situ cell death.

Inhibition of vascularization in rabbit corneas by heparin: cortisone pellets.

The purpose of this work was to study the effectiveness of heparin plus cortisone, and of cortisone alone in control of corneal vascularization in rabbit eyes. Corneal vascularization was induced by de-epithelialization of the cornea and limbus and part of the bulbar conjunctiva with concurrent trephination and excision of a central 2-mm diameter corneal button. Inhibition of vascularization by polymer pellets impregnated with heparin (Panheprin, Abbott Laboratories; Chicago, IL) and cortisone, or neither drug was studied by implanting the pellets into the eyes at the time of injury and following the eye clinically and histologically. Wounded corneas with empty pellets developed vascularization extending from the limbus to the central cornea within 3 wk (n = 10). In other wounded eyes, heparin:cortisone pellets prevented vascularization (n = 10) while cortisone pellets slowed, but did not totally inhibit vascularization (n = 6). In other eyes, clear autografts were transplanted into vascularized eyes; and the ability of the drug-impregnated pellets to inhibit grafts vascularization was evaluated. In eyes with heparin:cortisone pellets inserted into the donor button at the time of keratoplasty, the autografts remained clear for at least 6 wk (n = 10) but subsequently vascularized if the sutures were not removed, while cortisone pellets slowed but did not block vascularization (n = 6). If heparin:cortisone pellets were inserted into the vascularized host tissue, rather than into the donor button, vascularization of the graft occurred (n = 6). Thus, heparin (Panheprin, Abbott Laboratories; Chicago IL) plus cortisone inhibited vascularization in rabbit cornea in the models studied: The effect of other commercially available heparins remains to be studied.

Corneal epithelial cell attachment with endogenous laminin and fibronectin.

PURPOSE: To evaluate the role of endogenously produced laminin and fibronectin as well as the effect of exogenous laminin and fibronectin in the attachment of human corneal epithelial cells in vitro. METHODS: Primary cultured human corneal epithelial cells labeled with 3H-thymidine were seeded onto plates coated with laminin or fibronectin, or onto uncoated bacteriologic plates. Attachment of cells was measured in the presence or absence of antisera against laminin or fibronectin, by counting radioactivity. RESULTS: Human corneal epithelial cells attached to plates coated with human laminin or human fibronectin in a dose-dependent manner, with 69% and 50% of cells attached to the wells coated with 40 micrograms/ml of laminin and fibronectin, respectively (P < 0.001). The percentage of attachment to uncoated bacteriologic plates increased from 1.2% at 45 min of incubation to 6.7% at 90 min, 22.2% at 3 hr, and 40.1% at 6 hr of incubation. Cycloheximide, a protein synthesis inhibitor, completely inhibited cell attachment. Rabbit antiserum against human fibronectin reduced cell attachment to the uncoated plates to 67% of the control value (P < 0.01), whereas rabbit antiserum against human laminin decreased the attachment to 52% of the control (P < 0.01). A combination of these two antisera reduced cell attachment to 46% of the control (P < 0.01). CONCLUSIONS: Endogenous laminin and fibronectin as well as exogenous laminin and fibronectin play significant roles in the attachment of human corneal epithelial cells in culture.

In vivo effects of 5-FU on ocular surface epithelium following corneal wounding.

We evaluated the dose relationship between the antiproliferative and toxic effects of 5-fluorouracil (5-FU) on the ocular surface epithelium following experimental corneal epithelial wounding in rabbits. Central corneal epithelial defects 8 mm in diameter were made using n-heptanol. 5-FU (0.05 mg, 0.5 mg, or 5.0 mg per day in divided doses) or saline was applied topically for up to 18 days beginning on the day of wounding. The animals were sacrificed at 1, 7 or 14 days after wound closure. The effect on the ocular surface epithelium was assessed by observation of the clinical and histological appearance, and determination of the rate of corneal epithelial defect closure, corneal epithelial mitotic rate and conjunctival goblet cell frequency. A daily dose of 0.05 mg of topical 5-FU for 18 days had no discernable clinical or histopathological effect compared to wounded, saline treated controls. Treatment with 0.5 mg daily prevented the high mitotic rate typically noted 1 day immediately following defect closure, yet had no significant effect on clinical appearance, histological appearance, or healing rate. Daily topical application of 5.0 mg of 5-FU reduced the corneal epithelial mitotic rate to approximately 1% of the wounded controls, with persistent epithelial defects occurring in 22% of the eyes in this group. In those eyes which did heal, the corneal epithelium was markedly thinner than controls 1 day after defect closure. Fourteen days after healing, epithelial thickness in this group varied from 2 to 13 cells across each cornea, with the thickest area occurring centrally and tapering gradually to the limbus.(ABSTRACT TRUNCATED AT 250 WORDS)