Corneal cellular proliferation and wound healing

Abstract

Background: Cellular proliferation plays an important role in both physiological and pathological processes. Epithelial hyperplasia in the epithelium, excessive scar formation in retrocorneal membrane formation and neovascularization are examples of excessive proliferation of cornea cells. Lack of proliferative ability causes corneal degeneration. The degree of proliferative and metabolic activity will directly influence corneal transparency and very evidently refractive result after photorefractive keratectomy (PRK). The mechanisms behind regulation of the proliferative activity is not fully understood. In this work aspects of corneal cell proliferation in normal and wounded corneas was studied. Techniques to detect the substances produced naturally by proliferating cells to replace the methods that identify artificial DNA precursor labeling have recently been developed and was evaluated in this study. The location and behaviour of corneal epithelial stem-cells were studied both experimentally and clinically. The influence of leukocytes on cellular proliferation, neovascularization and hyaluronan production in the wounded corneas were studied with a novel technique that prevent the leukocytes from entering the corneal injury. The proliferating potential of corneal cells were compared for two corneal wound models.

Methods: Cellular proliferation was studied in the normal rabbit cornea and after different wound models including excimer-laser induced wounds, alkali wounds and manual epithelial debridement. Epithelial stem cell grafting was evaluated in 8 human subjects. In order to prevent leukocytes from leaving the blood stream and enter the wound, fucoidin was administered i.v in rabbits that received alkali injuries to one cornea. The technique was evaluated and the influence on cell proliferation and hyaluronan production was assessed. Hyaluronan was detected by a receptor-binding technique and evaluated semiquantitatively. Proliferative Cell Nuclear Antigen (PCNA) antibodies and Ki 67 was used to study cell proliferation, and the two techniques were compared.

Results: Autologous stem cell grafting in 8 humans where one eye had been injured with a chemical burn and subsequently conjunctivalized, showed good effect on visual outcome and the chronic irritation diminished. In specimens from corneas that later underwent a corneal graft the epithelial cells were of corneal origin. In the normal rabbit cornea, basal epithelial cells in the limbus presented PCNA whereas very few cells in the epithelium, stroma or endothelium were positive. Central injury to the epithelium by PRK induced proliferative activity in the limbal area first. The bare central stroma was covered first by sliding cells that later became proliferative in the leading edge and then over the entire wound surface. In the stroma, keratocytes started proliferative activity later than the epithelium and finished later. Leucocytes enter the wounded cornea via the tear film after PRK and via the limbus and tear film after alkali injuries. Intravenously administered fucoidin efficiently prevented leucocytes from entering corneal alkali injuries. Leukocytes entered the wound as early as 12 hours after injury. The absence of leucocytes in the wound retarded the proliferative activity during early wound healing in all cell layers. Fucoidin prevented also the invasion of leukocytes in the limbal area after alkali injury. Without leukocytes there was no proliferative activity found in the vascular endothelial cells and there was no new synthesis of hyaluronan.

Conclusion: PCNA is expressed in the limbal basal cells and colocalize with the stem-cells that renew the epithelial cell surface and constitute the barrier to the conjunctival epithelium and vessels. Injury to the central epithelium, by an unknown signal initiate more proliferative activity in the limbal epithelial cells. Human eyes that suffer damage to the limbus where the stem cells are proposedly located and the barrier is maintained can be successfully restored by autologous stem cell grafts. Leukocytes influence proliferative activity during wound healing. Two early signs of neovascularization disappear when leukocytes are excluded from the limbus following alkali injury. There is no proliferative activity in the vascular endothelial cells and hyaluronan is not synthesized.