Corneal epithelium regeneration in rabbit eyes

Author(s)

Flávia Leão Barbosa

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2003-09-04

Examining board members

Eduardo Miguel Laicine; Antonio Haddad; Sidney Julio de Faria e Sousa

• Advisor: Eduardo Miguel Laicine

Abstract

Up to now, it has been accepted that the stem cells for the corneal epithelium are localized exclusively at its peripheral portions or more precisely in the basal stratum of the epithelium on the esclerocorneal zone or limbal region. This investigation was undertaken to check the potentiality of the central portion of the corneal epithelium as a source of stem cells for the epithelial regeneration of any region of the cornea. For this purpose, in a first experimental group, the peripheral epithelium of the cornea of albino rabbits (Orictolagus cuniculus), was removed by scraping; this procedure was associated to limbal extirpation in a second experimental group and, in a third group, the central portion of corneal epithelium was removed. At end of the surgical procedures, the eyes were intravitreally injected or not with 3hthymidine (3H-TdR). The animais were killed at different times afterwards and their corneas were processed to obtain histological sections. These were processed for autoradiography, immunohistochemistry and, eventually, for special staining techniques. With the immunohistochemical study, using the AE5 antibody, which is known as corneal epithelium cell marker, the cells of newly formed epithelium were immunostained. Using the 4G10.3 antibody, regarded as a stem cell marker, unequivocal reaction occurred with the cells of the basal stratum, distributed in the transitional and newly formed epithelium or in spots in the central region. The observations of the autoradiographic preparations showed that the scraped surface of the cornea was covered with cells derived of the original central epithelium and that, differently from control corneas, there was a large number of labeled cells in the endothelium and stroma. Regarding the epithelium, it was detected a great increase in the labeling index compared to the control corneas, that is, practically 100% in experimental corneas versus about 40% in the control ones. Besides, it was found that the first labeled cells in the superficial stratum appeared 7 days after the surgical procedures and 3H-TdR injection instead of the 14 days observed in physiological conditions. The cells of the newly formed epithelium, which were significantly labeled in the days after removing the epithelium were just lightly labeled or no labeled at all 2 to 3 months after 3H-TdR injection. These results allowed the following conclusions: 1. The newly formed epithelium was reconstituted with typical corneal epithelium cells; 2. There were cells with characteristics of stem cells in all basal stratum of the cornea epithelium, not only in the limbal zone one; 3. The surgical procedures stimulated the cells of the cornea to divide, effect that was a striking one for endothelial and stroma cells, which rarely divide; 4. The newly formed epithelium was derived by replication of almost all the cells of the basal stratum, mainly from the remaining central epithelium; 5. The rate of renewal of the original epithelial cells was practically two times that observed in physiological conditions; 6. The labeling pattern of the newly formed epithelium found at longer time intervals after the surgical procedures and the 3H-TdR injections indicated that its renewal rate is higher than in the original one. (AU)