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Bcl-2 and Bax regulation of corneal homeostasis in genetically altered mice
Danielle M Robertson1, Patrick M Ladage, Nobutaka Yamamoto
1Department of Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA. Danielle.Robertson@utsouthwestern.edu
Purpose:
The antiapoptotic protein Bcl-2 and its proapoptotic counterpart Bax may play an important role in the regulation of corneal epithelial renewal. To understand the physiologic importance of Bcl-2 and Bax in the healthy cornea, epithelial and stromal thickness, proliferation, and surface cell exfoliation rates were examined in the central cornea of genetically altered mice overexpressing Bcl-2 and lacking Bax.
Methods:
Adult Bcl-2 transgenic (n = 55) and Bax knockout (n = 82) mice and wild-type controls were clinically prescreened at no less than 2 months of age for any ocular developmental abnormalities. Polymerase chain reaction was used to confirm genotype. Corneal epithelial renewal was examined by in vivo tandem scanning confocal microscopy to measure total corneal and sublayer thickness and by immunohistochemistry on whole mount corneal tissues to determine basal epithelial cell proliferation using 5-bromo-2-deoxyuridine (BrdU) and surface cell exfoliation with a Calcein AM-Ethidium homodimer assay (live/dead). Stained corneas were scanned with a laser scanning confocal microscope, and images were digitized, and cell counts were obtained. Levels of Bcl-2 protein were assessed by Western blots.
Results:
No significant changes in proliferation or cell death were found in either group compared to wild-type littermate controls; however, epithelial and stromal thicknesses were greater in the Bcl-2 transgenic group compared to wild-type and decreased in the Bax knockout. Western blotting confirmed that there was no change in the level of Bcl-2 expression in the corneal epithelium in the Bcl-2 transgenic or Bax knockout strains.
Conclusions:
The significant difference in epithelial and stromal thickness suggests a functional role for Bcl-2 and Bax in the maintenance of corneal homeostasis; however, the lack of significant alterations in proliferation, cell exfoliation, and levels of Bcl-2 protein in the adult corneal epithelium suggest that these phenotypic changes are a result of a new stable homeostatic equilibrium (proliferation + migration = shedding). Additional experiments to delineate the role of Bcl-2 and Bax during development and in the adult cornea are necessary.
