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F. Luengo Gimeno, V. Lavigne, L. Correa, S. Gatto, J. O. Croxatto, J. E. Gallo; Gender Analysis of Limbal Samples During the Follow-Up of Rabbits Implanted With Allogeneic Epithelial Corneal Cell Sheets. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1794.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the presence of Y chromosome in cultured limbal cells of female rabbits implanted with male epithelial corneal sheets after severe mechanical injury of the anterior cornea.
Limbal biopsies were taken from male rabbits; cells were expanded for 15 days, seeded on a fibroblast feeder layer grown on clotted autologous platelet poor plasma (PPP) and after six days corneal epithelial sheets were ready to be grafted. Limbal Stem Cell Deficiency (LSCD) was induced in 13 female rabbits by mechanical keratectomy and immediately after, grafts were sutured over the exposed stroma. Limbal biopsies were obtained at 4 and 7 months and cells were cultured to obtain samples for G-band karyotyping. A minimum of 40 metaphase cells was examined for each individual. Rabbits were euthanized at 12 months for clinical, histological and immunofluorescence (k3/12, k19) results. Control groups consisted of no implant or an implant of clotted PPP
No signs of graft rejection were observed. Different proportions of Y chromosomes were detected at 4 and 7 months postoperatively. One rabbit had endophthalmitis and another died of no apparent cause. The rest of the animals treated had no inflammation, showed a stratified epithelium, keratin 3/12 expression and no expression of keratin 19. At one year, 7 of 8 rabbits showed no LSCD or corneal rejection signs. All controls showed LSCD signs, erratic epithelium and minimal cell differentiation. They revealed a slight expression of K3/12 and an expression of K19 in patchy patterns.
Implanted cells are present for at least 7 months after corneal allograft transplantation. The implanted epithelial sheets favored re-epithelialization and allogeneic cells survived for a long period of time without the use of systemic chronic immune suppression. G-banding of cultured cells has inherent limitations in sensitivity but not in accuracy; this means that irrespective of the percentage of male cells found, the presence of them undoubtedly indicates that there are donor cells still present in the cornea, but no conclusion can be made based on the absence of the male cells. Demonstrating the persistence and acceptance of an XY genotype graft over an XX receptor is of paramount importance in the pursuit of a successful therapeutic strategy.
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