Figure 1.
Clinical evaluation of acute effect of BAC on the ocular surface.
(A–D) Representative images of corneal fluorescein staining showing no substantial fluorescein staining was apparent in both control (A) and BAC treated eyes (C–D). (E–H) Representative images of corneal rose bengal staining showing the patches and diffuse corneal staining in BAC-treated eyes compared with no obvious staining in the untreated control eye. (I)Schirmer test. (J) BUT. (K) Rose bengal staining score. There were no statistically significant differences between the BAC-treated and control groups in aqueous tear production and BUT. However, rose bengal staining showing statistically significant differences between the BAC-treated and control groups. ** P<0.01 (Dunnett test).
Figure 2.
Acute effect of BAC on the morphology of rabbit corneal epithelial superficial and endothelial cells.
Note that surface cells in the corneal epithelium of eyes treated with BAC was smaller, with blurry boundaries and abnormal reflectivity nuclei, compared with those of control eye. In contrast, the morphology of corneal endothelial cells did not appear to differ between BAC treated and control eyes. EP: epithelium, EN: endothelium.
Figure 3.
Acute effect of BAC on the size of surface cells in the corneal epithelium, central corneal thickness, corneal TER and CF uptake.
Note that the size of surface cells in the corneal epithelium of eyes treated with high concentrations of BAC was significantly smaller than that of control (A), corneal TER was significantly decreased (C) and corneal CF uptake was significantly increased (D) in all BAC treated cornea. In contrast, there was no significant difference between BAC treated and control eyes in central corneal thickness (B). Data show mean ± SD of values from six eyes per group. * P<0.05, ** P<0.01 (Dunnett test).
Figure 4.
Acute effects of BAC on distribution of ZO-1, ZO-2 occludin and claudin-1 in the rabbit corneal superficial epithelium.
Corneal tissue blocks prepared from a control eye or from eyes treated with 0.005%, 0.01% or 0.02% BAC. ZO-1, ZO-2, occludin and claudin-1 staining was observed as a continuous linear pattern along with superficial cell-cell borders in normal rabbit corneal epithelial cells. ZO-1and ZO-2 staining was patchy and discontinuous in the eyes treated with BAC. In contrast, the pattern of occludin and claudin-1distribution in the eyes treated with BAC is similar to that of untreated cells. Data are representative of three independent experiments.
Figure 5.
Acute effect of BAC on expression of Ki67 in the rabbit corneal epithelial basal layer.
Corneal tissue blocks prepared from a control eye (A) or from eyes treated with 0.005% (B), 0.01% (C) or 0.02% BAC (D). Mean basal Ki 67+ cells were shown in (E). Data show mean ± SD of values from three eyes per group.
Figure 6.
Acute effects of BAC on rabbit corneal epithelial cell apoptosis.
Representative images for TUNEL assay (A–D). Upper panel shows representative images for TUNEL assay in the superficial epithelia. The lower panel shows an orthogonal view generated from cross-sectional studies. Mean superficial apoptotic cells were shown in (E). Data show mean ± SD of values from three eyes per group.
Figure 7.
Acute effect of BAC on the expression of ZO-1 mRNA and protein in the rabbit corneal epithelium.
(A) Corneal epithelial cells isolated from a control eye or from eyes treated with 0.005%, 0.01% or 0.02% BAC were subjected to western blot analysis with antibodies to ZO-1 or β actin (loading control). (B) Qutantitative analysis of ZO-1 band intensity in bolts similar to those shown in (A). Data were normalized by the corresponding β actin band intensity and mean ± SE of values from three eyes per group. (C) Corneal epithelial cells isolated from a control eye or from eyes treated with 0.005%, 0.01% or 0.02% BAC were subjected to RT-PCR analysis of ZO-1 mRNA. (D) Quantitative analysis of ZO-1 band intensity in gel similar to that shown in (A). Data were normalized by the corresponding G3PDH band intensity and are mean±SE of values from three eyes per group. Topical application of BAC had no significant effect on the amount of ZO-1 mRNA and protein (Dunnett test).