The Effect of Bcl-2 Overexpression on Corneal Epithelial Homeostasis in Transgenic Mice

Abstract

Abstract: : Purpose: Bcl-2 is an anti-apoptotic oncogene that may play an important role in homeostatic control of the corneal epithelium. Using transgenic Bcl-2 mice, the aim of this study was to examine the effects of Bcl-2 overexpression on the corneal epithelium. Methods: Bcl-2 transgenic heterozygotes (TG), and control wild type (WT) BALB/c littermates were clinically pre-screened for any ocular developmental abnormalities. Corneal epithelial homeostasis was examined by (1) in vivo confocal microscopy to measure corneal and epithelial thickness, (2) immunocytochemistry on wholemount corneal tissues to determine (a) basal epithelial cell proliferation using 5-bromo-2-deoxyuridine (BrdU) and (b) surface cell exfoliation with a Calcein AM–Ethidium homodimer assay (live/dead). Stained corneas were scanned with a laser scanning confocal microscope, images were digitized and cell counts were obtained. Results: Mean total corneal thickness measurements were 100.88±6.97µm (WT, N=9)) and 113.67±3.20µm (TG, N=6) (P=0.004, Mann-Whitney Rank Sum Test); mean epithelial thickness measurements were 50.13±3.99 µm (WT) and 54.11±2.33 µm (TG) (P=0.004, T-test). Preliminary BrdU-labeling showed 1067±263 BrdU-positive cells/mm² (WT, N=6) versus 1272±340 cells/mm² (TG, N=6) (P=0.313, power 0.06); and pilot live/dead assays suggest an increase in total dead cells on the corneal surface for the TG versus WT. Conclusions: Overexpression of Bcl-2 in TG mice is associated with a thickened epithelial phenotype and with a trend towards increased proliferation and surface cell death. This study supports the hypothesis that Bcl-2 expression is important for the regulation of corneal epithelial homeostasis.

Keywords: cornea: epithelium • transgenics/knock-outs • cell death/apoptosis