Abstract
Abstract: :
Purpose: To study the natural movement of corneal epithelial cells in the normal adult mouse with in vivo microscopy. Methods: A transgenic mouse line that was engineered to ubiquitously express green fluorescent protein (GFP) was used to visualize the corneal epithelial cells. For histology, the cornea of GFP mice at various ages was fixed in formaldehyde and the epithelial cells were observed under a fluorescence microscope. For in vivo time-lapse studies, male mice were used at age 6 to 10 weeks at the beginning of the observation. An anesthetized mouse was placed on a custom-made observation platform under a fluorescence microscope, and the epithelial fluorescence was imaged. Ten to twenty overlapping microscopic fields covering approximately 1 mm2 of the epithelium were digitally recorded 3 times a week up to 6 weeks, and the rate of cell movements was determined from the time-lapse sequences. Results: Histology of the GFP mice at various ages showed that the high GFP expression in the corneal epithelium was ubiquitous only up to about 2 weeks after birth. After this stage the proportion of epithelial cells with bright GFP expression decreased gradually, resulting in the appearance of patches of several bright epithelial cells surrounded by the cells with a low level of GFP. Examination of adult mice at 6-10 weeks showed that the brightest cells were in the middle and basal epithelium, but not in the superficial layer. In these corneas, GFP positive epithelial cells collectively formed narrow streaks, pointing toward the center of the cornea, frequently displaying a swirling, or vortex, pattern near the center that has been previously described in the human cornea. Due to the unique shape, a cluster of several cells with a high GFP expression could be relocated and tracked in a living mouse over 1-4 weeks. Although individual cells were not identified, the centripetal movement of these cell clusters was evident from the time-lapse recordings, with an average rate of movement ranging from 20 to 30 µm/day. Conclusion: The corneal epithelium of adult GFP mice exhibits a pattern of GFP expression that is suitable for studying their movement in the normal, and also injured, cornea. In vivo time-lapse observations suggest that at least some of the epithelial cells at the basal or mid layers move centripetally in these mice.
Keywords: 372 cornea: epithelium • 474 microscopy: light/fluorescence/immunohistochemistry • 370 cornea: basic science