Figure 5: Histological examination of the effects of shikonin on streptozotocin (STZ)/hypoxia-induced diabetic retinopathy in C57BL/6 mice.

(A) Morphology of the retina from the control group on day 28 (upper part of the picture), the untreated DM/hypoxic group, and the 50 mg/kg shikonin-treated group is shown by H&E staining. The lower panel shows the light blue, TUNEL-positive, cells in the retina from a representative untreated DM/hypoxic mouse (red arrow). (B) Inner nuclear layer (INL) cell count. (C) Outer nuclear layer (ONL) cell count. Data are expressed as mean ± SD from 5 animals. ***p < 0.001 compared with the control group; ^###p < 0.001 compared with the untreated DM/hypoxic group. (D) The left eye of three randomly selected mice from each group (5 animals) was individually collected and subjected to tissue homogenization at high speed for 2 × 30 sec using a Minilys tissue homogenizer (Bertin Technologies, France). The homogenate was then centrifuged at 14,000 g for 10 min at 4 °C and the supernatant was collected. The protein concentration in the supernatant was determined with Bradford reagent. Absorbance at 595 nm was measured using a spectrophotometer (Optizen POP, Korea). Lysate proteins (80 μg) from each eye were analyzed by separation on 10% reducing SDS-PAGE gels and western blotting.