The Sheep Cornea: Structural and Clinical Characteristics.

PURPOSE: The aim of this study was to perform qualitative and quantitative analyses to characterize the corneas of young, healthy sheep. MATERIALS AND METHODS: Eight healthy male sheep, 10 months to 1 year of age, were included as experimental subjects. Central corneal thickness was measured using a handheld pachymeter, and an Easygraph corneal topographer provided topographic maps. Microstructural imaging of corneal layers was achieved by using the Heidelberg Retina Tomograph III Rostock Corneal Module in vivo corneal microscope (IVCM). An Ocular Response Analyzer (ORA) provided quantitative measurements of intraocular pressure (IOP), corneal hysteresis (CH), and corneal resistance factor. Tissue histology and immunohistochemistry were carried out to obtain detail on the corneal layers. RESULTS: Light microscopy and immunohistochemical labeling revealed a stratified epithelium, a limbus with numerous limbal crypts, a thick basement membrane, a thin Bowman's layer, a thick corneal stroma with a dense population of keratocytes, and a thick, hyper-reflective Descemet's membrane. Using IVCM, the cell density of the basal layer was noted to be significantly higher than that of other epithelial cell types. The density of keratocytes was significantly higher (P value = 0.0223) in the anterior compared to the posterior stroma. The endothelial cells were organized in a characteristic honeycomb pattern. The mean and standard deviation values for central corneal pachymetry were 623.14 ± 19.5 µm and 616.37 ± 34.87 µm for the left and right eyes, respectively. ORA-derived mean values for IOPcc and CH for the left and right eyes were 14.93 ± 1.73 mm Hg and 15.16 ± 2.02 mm Hg and 3.56 ± 0.72 mm Hg and 3.73 ± 0.49 mm Hg, respectively. CONCLUSIONS: The anatomical and clinical characteristics of the sheep cornea, as outlined in this study, make the sheep a suitable and relevant model for corneal research. This study provides researchers with important data on the suitability of sheep as a model for ophthalmic experiments.

Comparison of stem cell properties in cell populations isolated from human central and limbal corneal epithelium.

PURPOSE: The limbus of the cornea is said to be the niche for limbal stem cells (LSCs) and the primary source of corneal epithelial maintenance. Previously, we aimed to have shown that central human epithelial cells are capable of corneal regeneration after wounding. In this study, we aimed to investigate whether central epithelial cells in human corneas have LSC properties. METHODS: Human corneal epithelial cells were separated from the central cornea and the limbus. Isolated cells were collected for sphere-forming assay, and spheres formed subsequently were analyzed using immunohistochemistry. Fluorescence-activated cell sorting (FACS) was also used to analyze epithelial cells from central cornea, limbal rim, older donors, younger donors, and dissociated spheres. These analyses were based on cell size and Hoechst 33342 dye efflux ability, and side populations and non-side populations were isolated for colony growth measurement and sphere-forming assay. RESULTS: Human central and limbal epithelial cells were capable of forming spheres, in a 1:2 ratio, that were positive for p63 immunolabeling. In FACS, central and limbal epithelial cells showed no significant difference in cell size and dye efflux ability. There were almost 10 times more large cells with good dye efflux ability from younger donors than from older donors, and the gated side population showed more than 4 times faster rate of colony growth than the non-side population. Dissociated sphere cells, however, did not follow a similar pattern to tissue-derived cells using FACS analysis. In these, there were more than twice as many large cells than small cells with good dye efflux ability. CONCLUSIONS: Both limbal and central epithelial cells are capable of forming spheres in cultures that have stem cell properties. Central and limbal epithelial cells cannot be differentiated using FACS, but younger donor tissues give rise to greater numbers of large cells with high dye efflux. Therefore, results indicate that human central corneal epithelium contains cells with stem/progenitor properties, and these stem properties decline with age.

A novel method of organotypic brain slice culture using connexin-specific antisense oligodeoxynucleotides to improve neuronal survival.

Organotypic slice cultures obtained from immature brain tissue represent a well-established model system for neuroscience research. Current culture methods, however, do not allow long-term culture of mature brain slices. Slice cultures from mature animals would provide an in vitro experimental environment suitable for investigation of neuropathologies, which in human, predominate in aged individuals. We hypothesized that damage, incurred by slicing of the brain, is propagated through intercellular connexin43 (Cx43) gap junction channels and that this damage is not easily repaired in mature central nervous system (CNS) tissue that lacks the pluripotency of immature tissue. We investigated the role of Cx43 gap junctions in long-term survival of mature brain tissue using antisense oligodeoxynucleotide (AsODN) technology. The application of Cx43 AsODN immediately after slicing of the mature brain led to a significant but transient knockdown of Cx43 protein. This treatment was associated with the long-term survival of hippocampal neurons with normal morphology within whole brain slices taken from 14 and 40-day-old adult rats.