Abstract
Purpose: :
To investigate the effect of neurotrophin-4 (NT-4) on the endoplasmic reticulum (ER) stress-related neuronal apoptosis in cultured rat retinas exposed to high-glucose (HG).
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 were used. After the rats were euthanized, the retinas were isolated and cultured in serum-free medium. One group of explants was cultured in 7.5 mM normal glucose (NG) and the other groups in 45 mM HG medium with and without 100 ng/ml NT-4. 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 PERK and CHOP/GADD153.
Results: :
The numbers of TUNEL-positive, and PERK- and CHOP-immunopositive cells in the ganglion cell layer (GCL) were significantly higher in retinas culture in HG medium than in the NG medium and in the NG+M medium (32.5±11.6% vs. 19.9±7.2%, P=0.0007: 32.5±11.6% vs. 20.3±9.4%, P=0.0052; 30.5±17.4% vs. 12.0±4.0%, P=0.003; 30.5±17.4% vs. 12.5±7.1%, P=0.007; 21.2±7.4% vs. 11.6±8.0%, P=0.0183; 21.2±7.4% vs. 11.5±6.2%, P=0.0072). In retinas incubated in HG medium supplemented with NT-4, the numbers of TUNEL-positive, and PERK- and CHOP-immunopositive cells were significantly lower than in the HG medium without the NT-4 (14.9±5.3% vs. 32.5±11.6%, P<0.0001; 7.0±3.8% vs. 30.5±17.4%, P=0.0004; 6.9±2.9% vs. 21.2±6.5%, P=0.0004).
Conclusions: :
The neuronal cell death in retinas exposed to HG is, in part, caused by ER stress. The decrease in the number of apoptotic cells in the HG medium containing NT-4 is correlated with the suppression of the expression of PERK and CHOP, both ER stress-related factors.
Keywords: diabetic retinopathy • neuroprotection • apoptosis/cell death