Morphological and Autoradiographic Studies on the Corneal and Limbal Epithelium of Rabbits

Abstract

The investigation was centered on the morphological features of the conjunctiva-cornea transition (limbus) of the rabbit eye and the proliferative behavior of its epithelium. The eyes were processed for examination with light and electron microscopy, as well as for autoradiography after intravitreal injection of [ 3 H]thymidine ([ 3 H]TdR). At the sites of extraocular muscle insertion, the vascularization of the stroma extended to the peripheral cornea, and the limbal epithelium was thin with its basal stratum made up by clear cuboidal cells. In between the muscle insertions, the cuboidal clear cells, as well as the stroma blood vessels, were scarce. At the light microscope level, the basement membrane was distinct in the cornea but not in the limbus or the conjunctiva. Autoradiographs demonstrated that, at the limbus, the basal cells migrated very quickly to the suprabasal region and remained there up to the 28-day interval. Labeled cells were identified in all epithelial layers of the cornea, including the basal one, at 21 and 28 days but not in the limbal basal clear cells. The rate of renewal of conjunctival epithelium was similar to that observed for the transition with scarce clear cells. The high-resolution autoradiographs demonstrated that the basal cuboidal clear limbal cells exhibit a quick renewal and that they are not label-retaining cells. These latter ones were detected all over the corneal epithelium and in the suprabasal layers of the limbus up to 28 days, in physiological conditions, without the need of stimulation by damage to the corneal epithelium. Anat Rec, 291:191-203, 2008. 2008 Wiley-Liss, Inc.Key words: cornea; conjunctiva; epithelium; stem cell; limbus; renewal; autoradiographyThe human limbus is the transitional zone between the cornea, conjunctiva, and sclera, being 1.5 mm wide and 2 mm thick in the vertical plane. Several features have been used to get a more precise delimitation of the limbus at the microscope level. Among them the following are outstanding: (a) the termination of the Bowman's layer and the Descemet's membrane; (b) vascularization of the stroma; (c) differences in the occurrence of proteoglycans (keratan predominance in the cornea and presence of dermatan sulfate and hyaluronic acid in the limbus and sclera, for example); (d) loss of the orderly packing of collagen fibrils occurring in the cornea; (e) a

A Rabbit Model of Acanthamoeba Keratitis That Better Reflects the Natural Human Infection

“…At this injection site, it is possible to form a space to hold a certain amount of fluid containing the parasites. Our model allows slow infiltration of the protozoa with a certain degree of self-containment, which is similar to the natural condition under which Acanthamoeba infection of the cornea occurs, when the corneal surface is slightly injured (Goes et al, 2008). …”

Autoradiographic study on the regenerative capability of the epithelium lining the center of the cornea after multiple debridements of its peripheral region

“…All the eyes injected with 3 H-TdR, belonging to both groups above mentioned, had their corneas processed for autoradiography using paraffin as well as 0.7-0.8 µm-thick resin semithin sections [23,24,34,43,44]. The labeling index (number of labeled nuclei per 100 cells) was used to evaluate the results.…”

“…The rabbits were killed in groups of two at 12, 24 and 40-44 hr, and 2, 7 and 17 days afterwards. The processing of the corneas for histology and autoradiography as well as the technique of isotope injection have already been fully described before [34]. Pieces of the same cornea were used for paraffin as well as for resin embedding.…”

“…These proposals were based on surgical procedures such as elimination of the corneal epithelium plus limbus [38,39] or epithelial transplantation [40,41] or in vitro biochemical studies [42]. These reports were not supported by recent studies [13,33,34], indicating that germinative (or oligopotent or stem) cells may be found throughout the corneal epithelium.…”

“…Autoradiographic studies [28] intended to characterize stem cells by locating slowcycling and label-retaining cells exclusively in the basal layer of the limbal epithelium [29][30][31][32] have equally been found inadequate for this purpose, since these reports had not been confirmed by other investigators [13,[33][34][35][36][37].…”