The Role of Bax as a Regulator of Corneal Epithelial Homeostasis

Abstract

Abstract: : Purpose: The pro-apoptotic oncogene Bax may play an important role in the regulation of corneal epithelial homeostasis. To better understand the physiological importance of Bax expression in the normal cornea, we examined epithelial thickness, proliferation and surface cell exfoliation rates in the central cornea of mice nullizygous for the Bax gene. Methods: Bax null homozygotes (HO), Bax heterozygotes (HT), and control wild type (WT) C57BL/6-Bax mice 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 106.3±4.0µm (WT, N=9), 102.0±7.6µm (HT, N=10) and 89.1±8.3µm (HO, N=9) (P<0.01, One-way ANOVA); mean epithelial thickness measurements were 36.1±2.0 µm (WT), 36.4±2.5µm and 31.9±2.7 µm (HO) (P<0.01). BrdU-labeling showed 889±265 BrdU-positive cells/mm² (WT, N=7), 917±178 cells/mm² (HT, N=7) and 1077±182 cells/mm² (HO, N=7) (P=0.231, power 0.129); and pilot live/dead assays demonstrated an increase in total dead cells on the corneal surface for the HO versus WT. Conclusions: In the Bax knockout mice, basal cell proliferation rates demonstrated a hyper-proliferative response associated with concurrent increases in surface cell death and central epithelial thinning. This data suggests an increased epithelial cell layer turnover rate in the absence of Bax. Further immunocytochemical and molecular studies evaluating potential mediators in this pathway are needed.

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