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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

Corneal epithelial cell cultures on stromal carriers.

Exposure of denuded rabbit corneal stromal carriers for 24 hr at 37 degrees C to suspensions of rabbit corneal epithelial cells grown in tissue culture resulted in the establishment of a cell layer on the carriers. The cell layers persisted for at least 1 week of incubation and were one to three cells thick. They consisted of healthy-appearing cells with normal intracellular organelles and intercellular desmosomal connections. After 2 to 7 days of incubation the cells were still capable of DNA replication and produced hemidesmosomes and basement membrane. This system is useful for in vitro studies of substrate requirements for hemidesmosome and basement-membrane formation by corneal epithelial cells.

Limbal epithelium in ocular surface wound healing.

The regenerated epithelium derived from limbal epithelium was histologically and biochemically compared with epithelia regenerated from corneal and bulbar conjunctival epithelia. The histologic results indicated that regenerated epithelium of limbal origin increased in thickness with time after healing and showed no goblet cell appearance on the cornea. This suggests that regenerated epithelium from the limbus is more like regenerated epithelium of corneal origin than that of bulbar conjunctival origin. However, the glycogen content and protein pattern profile showed that regenerated epithelium of limbal origin had characteristics intermediate between those of corneal and bulbar conjunctival origin. Thus it is proposed that there are three distinct types of ocular surface epithelia--corneal, bulbar conjunctival, and limbal--and that limbal epithelium behaves differently from corneal and conjunctival epithelia in ocular surface wound healing.

Basement membrane synthesis by human corneal epithelial cells in vitro.

PURPOSE: Collagen gels may prove to be potential carriers for transplantation of cultured corneal epithelial cells. The purpose of this study was to evaluate the suitability of collagen gels in comparison with corneal stromal blocks as the substrate to support the growth of human corneal epithelial cells in culture and the synthesis and deposition of the basement membrane components by these cells. METHODS: Corneal epithelial sheets, freed from the culture dishes using Dispase II (Boehringer Mannheim, Indianapolis, IN), were cultured on corneal stromal blocks. Deposition of laminin, type IV collagen, type VII collagen, and perlecan (heparan sulfate proteoglycan) were evaluated immunohistochemically after 4 days, 7 days, 2 weeks, and 3 weeks. Human limbal explant cultures were established on collagen gels prepared from bovine type I collagen with or without addition of cultured human corneal fibroblasts. After 1, 2, 3, and 4 weeks, the deposition of the basement membrane components was evaluated immunohistochemically. RESULTS: Corneal epithelial cells, cultured on corneal stromal blocks as well as on collagen gels with or without fibroblasts, deposited laminin, type IV collagen, perlecan, and type VII collagen at the interface of the cells and the substrates. However, different substrates differentially influenced the temporal pattern of the deposition of various basement membrane components. On the stromal blocks, deposition of laminin, type IV collagen, and perlecan by the epithelial cells was evident at 1 week. Type VII collagen was detected at 2 weeks. On the collagen gels with fibroblasts, deposition of laminin, type IV collagen and perlecan was detectable at 1 week. In the epithelial cultures on the collagen gels without fibroblasts, only perlecan was detectable at 1 week. At 2 weeks, all of the basement membrane components, including type VII collagen were detectable on the collagen gels, either with or without fibroblasts. CONCLUSION: Human corneal epithelium cultured on collagen gels or on corneal stromal blocks can synthesize and deposit basement membrane components, including laminin, type IV collagen, type VII collagen, and perlecan within 2 weeks in culture. Therefore, collagen gels may serve as potential carriers for human corneal epithelial transplantation.

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.