Abstract
Purpose: :
To determine whether the expressions of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Jun N-terminal kinase (p-JNK) are significantly associated with neuronal cell death in diabetic (DM) and high-glucose (HG) exposed rat retinal explants.
Methods: :
All of the procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The retinas of six adult male Sprague-Dawley rats and six streptzotocin (STZ)-induced diabetic rats were used. The rats were euthanized, and the retinas were isolated and cultured in serum-free medium. The explants from non-diabetic controls were cultured in 7.5 mM normal-glucose (NG) or 45 mM HG medium. For osmotic control, a 7.5 mM glucose with 37.5 mM D-mannitol (NG+M) solution was used. After 7 days in culture, the explants were fixed, cryosectioned, and stained by TdT-dUTP terminal nick-end labeling (TUNEL), and also immunostained for p-c-Jun and p-JNK. Sections were co-stained with DAPI and Fluoro-Jade B (FJB), a marker of degenerated neurons. Statistical analysis was performed with ANOVA (Fisher’s PLSD) and P<0.05 was considered significant.
Results: :
The numbers of TUNEL-positive cells in the ganglion cell layer (GCL) were significantly higher in the HG (32.7 ± 11.7%) and in the DM groups (31.0 ± 14.5%) than in the NG (18.8 ± 7.3%) and in the NG+M groups (19.7 ± 9.0%) (NG vs. HG, P=0.0005; NG+M vs. HG, P=0.0407; NG+DM P=0.0002). The numbers of p-c-Jun- and p-JNK-immunopositive cells were significantly higher in the HG (25.2±12.0%, 18.5±5.6%) and the DM groups (18.7±8.0%, 20.2±7.0%) than in the NG (11.2±5.1%, 9.3±4.4%) and the NG+M groups (12.2±9.1%, 12.7±10.0%)(NG vs. HG, P=0.0003; NG+M vs. HG, P=0.0014; NG vs. DM, P=0.00304; NG vs. HG, P=0.0005; ; NG+M vs. HG, P=0.0407, NG+DM, P=0.0002). The FJB signals in the GCL were mainly colocalized with p-c-Jun and p-JNK expression.
Conclusions: :
Increased expressions of p-c-Jun and p-JNK are associated with neuronal apoptosis in diabetic retinas and retinas incubated in HG medium.
Keywords: diabetic retinopathy • apoptosis/cell death