Figure 4.

Cell death analysis in corneal tissue by TUNEL assay and cleaved caspase 3 staining. (a) Cell nuclei were stained with DAPI (blue). Cell death was evaluated by TUNEL (green) staining. High numbers of TUNEL-positive nuclei were detected in the cornea of the UVB control mice. Contrary, few TUNEL-positive nuclei were observed in nano-AST-treated mice, and AST oil administration causes a minor reduction of TUNEL-positive cells. Lutein and bilberry extract did not result in a noticeable reduction in numbers of TUNEL-positive nuclei. The nonirradiated group (naïve) showed background levels of TUNEL-positive cells, potentially associated with tissue harvesting process. (b) Cleaved caspase 3 staining findings were consistent with TUNEL staining results, with marked increased c-caspase 3-positive cells through the whole corneal epithelial layer in the UVB control, AST oil-, lutein-, and bilberry-treated groups. Nano-AST-treated animals, however, demonstrate only a few c-caspase 3-positive cell signals on the surface of the epithelial layer. No specific c-caspase 3 signals were detected in naïve corneas. (c) Numbers of TUNEL-positive cells were evaluated and averaged. Quantitative analysis confirmed the significant reduction of apoptotic cells by nano-AST (p < 0.001) compared to the UVB control group. AST oil, lutein, and bilberry extract had similar numbers of TUNEL-positive nuclei as the UVB control group (p > 0.05). n = 8 (eyes) per group. n.s., p > 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.