Figure 3.

Calpain activity in nondiabetic and diabetic mice. A and B: Fluorogenic calpain substrate was injected intravitreally (A) or added on fresh-frozen cryosections (B) of diabetic and nondiabetic mice. In A, the left panels represent a wide view of the retina (nasal), and the middle panels represent enlarged view of the boxed regions. Calpain activity is shown with green, and nuclei were stained with DAPI (blue). Calpain activity was predominantly localized to photoreceptors of diabetic retina (magenta arrows) as compared with nondiabetic retina. Focal deposits of staining was present in the IPL and the GCL (yellow arrows). In B, sections were pretreated with or without calpain inhibitor (CI) before the addition of calpain substrate (CS). The pretreatment of the sections with CI abolished the staining in photoreceptors, but not of the focal staining. C: Calpain activity was determined by the level of cleavage of spectrin. Protein lysates were subjected to Western blotting using anti-cleaved spectrin antibody (approximately 160 kDa) and β-actin antibody (approximately 42 kDa) as loading control. Scale bars: 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; IS, photoreceptor outer segment; OPL, outer plexiform layer; OS, photoreceptor outer segment.