The conjunctiva in corneal epithelial wound healing

BACKGROUND/AIMS—During the healing of corneal epithelial wounds with limbal involvement, conjunctival epithelium often migrates across the denuded limbus to cover the corneal surface. It is believed that, over a period of time, conjunctival epithelium covering the cornea assumes characteristics of corneal epithelium by a process referred to as conjunctival transdifferentiation. The purpose of this study was to examine, clinically, the fate of conjunctival epithelial cells covering the cornea and to assess the healing of corneal epithelial wounds when the conjunctival epithelium was removed or actively prevented from crossing the limbus and extending onto the cornea.
METHODS—10 patients with conjunctivalisation of the cornea were followed for an average of 7.5 months. Five patients in this group had their conjunctival epithelium removed from the corneal surface and allowed to heal from the remaining intact corneal epithelium. In another four patients with corneal epithelial defects, the conjunctival epithelium was actively prevented from crossing the limbus by mechanically scraping it off.
RESULTS—The area of cornea covered by conjunctival epithelium appeared thin, irregular, attracted new vessels and was prone to recurrent erosions. Conjunctivalisation of the visual axis affected vision. Removal of conjunctival epithelium from the cornea allowed cells of corneal epithelial phenotype to cover the denuded area with alleviation of symptoms and improvement of vision. It was also established that migration of conjunctival epithelium onto corneal surface could be anticipated by close monitoring of the healing of corneal epithelial wounds, and prevented by scraping off conjunctival epithelium before it reached the limbus.
CONCLUSION—This study shows that there is little clinical evidence to support the concept that conjunctival transdifferentiation per se, occurs in humans. "Replacement" of conjunctival epithelium by corneal epithelial cells may be an important mechanism by which conjunctival "transdifferentiation" may occur. In patients with partial stem cell deficiency this approach can be a useful and effective alternative to partial limbal transplantation, as is currently practised.

Keywords: corneal epithelium; conjunctiva; stem cells; transdifferentiation     

A new in vitro corneal preparation to study epithelial wound healing.

Corneal epithelial wound healing is an important process necessary for maintenance of visual integrity. Corneal epithelial wound healing occurs by cellular migration and proliferation. However, the molecular basis of reepithelialization is not known. To investigate individual molecular contributions to the wound healing process, an in vitro corneal preparation comparable to the in vivo condition is needed. This investigation developed a new whole mount in vitro rabbit cornea preparation and studied epithelial wound healing rates for epithelial and subepithelial wounds. The wound closure rates obtained in this study for epithelial and subepithelial wound healing (52 +/- 14 microns/hr and 38 +/- 7 microns/hr, respectively) are comparable to in vivo rates of wound healing determined by other laboratories for rabbits. This preparation, achieved by functionally separating the epithelial and endothelial sides of the cornea, allows application of agents to the cornea in a manner that approximates the in vivo condition. This in vitro system is promising for future studies designed to investigate corneal wound healing while reducing potential ocular discomfort associated with in vivo corneal wounding.     

Clinical patterns of corneal epithelial wound healing

We studied the reepithelialization of corneal abrasions in 21 patients. All abrasions, irrespective of the nature of injury, followed a consistent pattern during reepithelialization. Three to six convex leading fronts of migrating epithelial sheets developed along the circumference of the defect and progressed toward the center. Neighboring fronts met along their sides, resulting in the formation of various geometric shapes. In the final stage of the healing process a contact line shaped like a "Y" or two "Y's" placed with their long axes end to end was seen where the advancing fronts of migrating epithelial sheets met. The rate of healing of the abrasions was determined by measuring the area of the abrasions at daily intervals from serial photographs. The area of the epithelial defects decreased exponentially with time, indicating a constant rate of epithelial cell migration.     

Protein synthesis during corneal epithelial wound healing

Previous investigations have shown that corneal epithelium, migrating to cover a wound, synthesizes protein and glycoprotein at a faster rate than does normal stratified epithelium. The authors have found that the maximal rate of synthesis, as indicated by the incorporation of leucine and glucosamine, occurs 16 hr after wounding, 6 hr before wound closure. A comparison of total protein and protein synthesized during migration indicates that the increased synthesis is the result of the enhanced synthesis of many of the proteins present in unwounded epithelia. However, one protein band with a molecular weight of 110 K daltons was present to a much greater extent in migrating tissue than in normal epithelium. A time course analysis indicates that this band is apparent during migration and is not present either before wounding or 24 hr after wound closure.     

小鼠角膜上皮创伤再上皮化过程基本特征的动力学研究

目的 研究小鼠角膜上皮机械性创伤再上皮化过程炎性细胞迁移、血小板聚集的动力学特征.方法 计算小鼠角膜2 mm上皮缺损创面改变;免疫荧光染色观察角膜基底细胞分裂和中性粒细胞、血小板、γδT细胞及巨噬细胞迁移特征;电子显微镜观察角膜上皮细胞形态学、基质PMNs和角膜缘血小板的变化.结果 创伤后24 h基本再上皮化,96 h基底细胞恢复正常;伤后18 h和30 h分裂细胞达峰值;PMNs迁移峰为12 h和30 h,血小板聚集峰于12 h;γδT细胞的两峰为6 h和24 h,MΦ两峰滞后为24 h和42 h.结论 角膜上皮创伤修复过程分3期.基底细胞分裂增生和PMNs、MΦ以及γδT细胞迁移呈双相式;早期PMNs迁移与血小板聚集同步.     

The corneoscleral limbus in human corneal epithelial wound healing

We studied re-epithelialization of the ocular surface in 17 human eyes (14 patients) with large corneal and conjunctival abrasions. We focused on the healing of the limbal region. During re-epithelialization, cell movement was found to occur circumferentially along the corneoscleral limbus and centripetally from the corneoscleral limbus. In no patient did the central corneal defect close before the corneoscleral limbus had first re-epithelialized completely. Normal limbal healing was observed to occur by circumferentially migrating tongue-shaped corneal limbal epithelium. These tongue-shaped projections developed from either side of the remaining intact epithelium and advanced along the corneoscleral limbus until they met. A centripetal movement of cells from the corneoscleral limbus then completed the healing process. In three patients, however, the advancing conjunctival epithelium extended across the corneoscleral limbus before the tongue-shaped projections of corneal limbal epithelium had met. The surface of the cornea covered by conjunctival epithelium was thin and irregular, and later showed peripheral scarring, vascularization, and recurrent erosions.     

表皮生长因子对碱烧伤角膜伤口愈合的实验研究

目的　观察鼠表皮生长因子 (m ouse epiderm al grow th factor,m EGF)对角膜上皮创伤修复的作用 ,并探讨其量效关系。方法　采用兔角膜碱烧伤后上皮缺损模型 ,分别给予 0 .5、1、2 g· L- 1m EGF滴眼液治疗 ,生理盐水作对照组 ,每日荧光染色裂隙灯观察、记录 ,疗程 1周。结果　 2 g· L - 1m EGF治疗组与其他 3组有显著性差异 ,0 .5及 1g· L- 1m EG F与对照组之间无显著性差异。结论　尽管量效关系复杂 ,2 g· L- 1m EGF对角膜碱烧伤后上皮缺损的修复起到一定的促进作用。     

整合素在角膜上皮创伤愈合中的研究进展

整合素作为一类重要的细胞黏附分子,通过影响细胞的形态,介导细胞的黏附、迁移和增生,在角膜上皮创伤愈合中发挥了重要的作用。讨论α2β1、α3β1、α5β1、αvβ3、α6β4、α9β1和αvβ6这7种整合素在角膜上皮创伤愈合中的研究进展及其临床意义。α6β4整合素为半桥粒的主要组成部分,介导角膜上皮细胞在细胞外基质上的静态黏附,损伤后该黏附就转变为α2β1、α3β1整合素介导的动态黏附,细胞在黏附-去黏附的过程中实现迁移,从而修复创面。α6β4、α3β1整合素相互协调作用,实现上皮细胞的板层状运动。研究还发现α6β4、α3β1整合素的活化还能促进细胞的增生。损伤后上皮细胞表面α5β1、αvβ3整合素的表达上调,二者与黏着斑的形成密切相关。α9β1和αvβ6为近年来新发现的与角膜上皮创伤愈合有关的整合素,其具体作用尚有待进一步的研究。     

Epidermal growth factor in alkali-burned corneal epithelial wound healing

We conducted a double-masked study to evaluate the effect of epidermal growth factor on epithelial wound healing and recurrent erosions in alkali-burned rabbit corneas. Epithelial wounds 10 mm in diameter healed completely under the influence of topical epidermal growth factor, whereas the control corneas did not resurface in the center. On reversal of treatment, the previously nonhealing epithelial defects healed when treated with topical epidermal growth factor eyedrops. Conversely, the epidermal growth factor-treated and resurfaced corneas developed epithelial defects when treatment was discontinued. Histopathologic examination disclosed hyperplastic epithelium growing over the damaged stroma laden with polymorphonuclear leukocytes when treated with epidermal growth factor eyedrops, but it did not adhere to the underlying tissue. Hydropic changes were seen intracellularly as well as between the epithelial cells and the stroma.     

Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5

PURPOSE: To investigate the effect of annexin A5, a calcium-dependent phospholipid-binding protein, on corneal epithelial wound healing. METHODS: The effect of annexin A5 on migration of rabbit corneal epithelial (RCE) cells in vitro was examined in scrape-wounded cell monolayers. The effect of annexin A5 on the release of urokinase-type plasminogen activator (uPA) from cultured RCE cells was determined by zymography, fluorogenic assay of PA activity, and enzyme-linked immunosorbent assay. The proliferation of RCE cells was assessed by measurement of [3H]thymidine incorporation. The effect of annexin A5 on corneal wound closure in rabbits was investigated after removal of the corneal epithelium, either by exposure to iodine vapor or surgically. Eye drops containing annexin A5 were instilled into one eye and vehicle into the other. The area of the epithelial defect was measured at various times after wounding, and the healing rate was calculated by linear regression analysis. RESULTS: Annexin A5 significantly promoted the migration of RCE cells in a wounded monolayer. However, annexin A5 had no effect on RCE cell proliferation. Annexin A5 also increased the release of uPA both from wounded RCE cell monolayers and from nonwounded semiconfluent RCE cells. In both models of corneal wound closure, the healing rate was significantly increased by instillation of eye drops containing annexin A5 compared with that apparent in the eyes that received vehicle. CONCLUSIONS: Annexin A5 promoted corneal epithelial wound healing both in vitro and in vivo. Upregulation of uPA release from corneal epithelial cells may contribute to this effect of annexin A5.     

Alteration of epithelial paracellular permeability during corneal epithelial wound healing.

We studied the paracellular permeability to mannitol of corneas with epithelium of corneal, limbal, or conjunctival origin. Corneas with epithelial defects reepithelialized by corneal or limbal epithelium were nonvascularized; the corneal permeability was initially increased and returned to normal 3 days later. When epithelial defects extended beyond the limbus, they were healed by conjunctival epithelium. If corneas remained avascular or minimally vascularized, the conjunctiva-derived epithelium underwent a transdifferentiation process into a cornealike morphology in which the corneal permeability was initially increased upon complete reepithelialization, and gradually decreased to a level similar to that of normal cornea, 4 weeks after healing. However, when corneas became vascularized, the conjunctiva-derived epithelium retained its original phenotype, and corneal permeability remained increased throughout the 8-month period of study. The deranged barrier functions noted in the above vascularized cornea were demonstrated further by horseradish peroxidase tracer, which was found in the intercellular spaces of conjunctiva-derived epithelium of vascularized corneas but not in the avascular corneas with epithelia of corneal or limbal origin, or transdifferentiated conjunctival epithelium. To study further the effect of subsequent ocular surface trauma, conjunctival biopsy was performed on transdifferentiated avascular corneas 3 months after initial wounding. The biopsy resulted in extensive vascularization in three of eight previously nonvascularized corneas. Two weeks later, the corneal permeability was increased to a level similar to that of conjunctiva. These results indicate that corneal epithelial paracellular permeability correlates well with the status of the epithelial phenotype.     

Influences of methylcellulose on corneal epithelial wound healing.

We performed a study to evaluate the influence of methylcellulose (MC) on the corneal epithelial wound healing. A precise epithelial wound 2.0 mm in diameter and 70 microm in depth was created by an excimer laser on the cornea of a pig's eyeball. After treatment, the eyeballs were cultured in the incubator and perfused with TC-199 medium into the vitreous cavity. Topical MC with eight different combinations of concentrations and viscosities for experimental groups and BSS for the control group were applied on the epithelial defects three times. Wounds were evaluated 24 hours later with fluorescein stain and recorded with a scoring system. All of the experimental groups except one showed a more statistically significant superior wound-healing rate than the control group. MC possesses the effect to accelerate corneal epithelial wound healing.     

Cell-matrix and cell-cell interactions during corneal epithelial wound healing

The corneal epithelium serves as a barrier and contributes to the maintenance of corneal transparency and rigidity. In most instances, corneal epithelial defects caused by simple injury are resurfaced promptly. However, in individuals with certain clinical conditions, such as herpes simplex virus infection, neurotrophic keratopathy or diabetic keratopathy, corneal epithelial defects persist and do not respond to conventional treatment regimens because of delayed epithelial wound healing. After the corneal epithelium is removed by injury, the remaining epithelial cells migrate over the denuded surface of the cornea in a manner that is dependent both on the interaction of the cells with the underlying substrate and on cell-cell adhesion. In this review, we describe the specific roles of cell-matrix and cell-cell interactions during the course of corneal epithelial wound healing. The clinical implications of the basic research findings are also discussed.     

Macroscopic observations on corneal epithelial wound healing in the rabbit

A newly-developed macroscope was applied to observe the healing process of corneal epithelial wound in vivo. After removing epithelium of the central cornea, the changes of the corneal surface were observed with the macroscope and the findings were compared with histological examinations. At 12 hours after abrasion, areas unstained with Richardson's staining (R staining) appeared. In the histological section, a single layer of regenerating epithelial cells covered the same area. At 24 and 36 hours after abrasion, the epithelial defects became smaller but surrounding epithelium was rough and showed dot-like staining with R solution. By 2 days, the epithelial defects disappeared. On macroscopic observation, the central corneal surface showed a pavement-like appearance. Histology revealed that the regenerating epithelium still consisted of one or two layers. At 3 days, dot-like stainings were present only in the center and the corneal surface appeared considerably smooth. Histology also showed that regenerating epithelium became columnar and multilayered, thereby suggesting stratification. By 7 days, the abraded corneal surface had recovered its smooth appearance. Histologic sections also demonstrated that the epithelium had regained its normal structure. Thus, using this macroscope, findings suggesting the process of epithelial migration and proliferation could be observed.