Figure 4
Reduction of vascular leakage in the diabetic Ins2Akta mouse retina by PEDF derivatives. Retinas were evaluated 30 min after animals received tail-vein injections of BSA-FITC. (A) Left: Cross-section of a diabetic retina (NT) showing diffusion of fluorescently labeled BSA into the adjacent parenchyma of a blood vessel. The arrow points to a large vessel in the inner retina (OS, outer segment; ONL outer nuclear layer; INL, inner nuclear layer). Middle: Control: Retina flat mount of a diabetic retina treated with vehicle alone (AT). The arrows point to several areas of BSA-FITC accumulation in the retinal parenchyma indicative of hemorrhaging. The vascular bed contains vessels that are thin and discontinuous. Right: Retina flat mount from a diabetic animal treated with the peptide eye drops. Fewer focal leakage areas and a thicker more continuous vascular bed are seen after treatment. (B) Quantitative measurements confirm that the number of hemorrhaging areas in the retina is reduced by ~55% after treatment, with either peptide or a combination of the two when compared with controls (AT) (p < 0.05). Differences were statistically insignificant between P60, P78 and combination treatments (n = 15). (C) Fluorometric measurements confirm a reduction in total BSA-FITC content in retina homogenates by ~40% after treatment with the PEDF derivatives compared with controls (FU, fluorescence units; p = 0.004). (D) Reduced levels of leaked albumin in the diabetic retina after treatment with either peptide was verified by Western blots of retina lysates (M, molecular weight standard; +, positive control — purified albumin protein). (E) Densitometry measurements of Western blots confirm a significant decrease in leaked albumin content in the peptide-treated group compared with AT controls (p = 0.002). Difference between P60 and P78 was statistically insignificant (n = 5). Values are means ± standard deviation (SD).