Short‐term hyperglycemia produces oxidative damage and apoptosis in neurons

Abstract

Dorsal root ganglia neurons in culture die through programmed cell death when exposed to elevated glucose, providing an in vitro model system for the investigation of the mechanisms leading to diabetic neuropathy. This study examines the time course of programmed cell death induction, regulation of cellular antioxidant capacity, and the protective effects of antioxidants in neurons exposed to hyperglycemia. We demonstrate that the first 2 h of hyperglycemia are sufficient to induce oxidative stress and programmed cell death. Using fluorimetric analysis of reactive oxygen species (ROS) production, in vitro assays of antioxidant enzymes, and immunocytochemical assays of cell death, we demonstrate superoxide formation, inhibition of aconitase, and lipid peroxidation within 1 h of hyperglycemia. These are followed by caspase-3 activation and DNA fragmentation. Antioxidant potential increases by 3-6 h but is insufficient to protect these neurons. Application of the antioxidant alpha-lipoic acid potently prevents glucose-induced oxidative stress and cell death. This study identifies cellular therapeutic targets to prevent diabetic neuropathy. Since oxidative stress is a common feature of the micro- and macrovascular complications of diabetes, the present findings have broad application to the treatment of diabetic patients.

SOD2 protects neurons from injury in cell culture and animal models of diabetic neuropathy

“…To produce a hyperglycemic insult, 20 mM additional glucose (total 45 mM glucose) was added to the media for the period specified in individual experiments. This model of hyperglycemic insult has been described previously Russell et al, 2002;Vincent et al, 2005a). Media glucose concentrations did not significantly decrease during the treatment periods as determined by standard gas chromatography-mass spectrometry (data not shown).…”

“…We previously published the time-course of oxidative stress and markers of DRG neuron injury in our hyperglycemia model (Vincent et al, 2004b;Russell et al, 2002;Vincent et al, 2005a). The time-points used in the present studies are based on the previous experiments.…”

“…The time-points used in the present studies are based on the previous experiments. We previously demonstrated that DCFDA oxidation peaked at 3-5 h, caspase-3 activation at 5-6 h, and differences in neurite growth peaked at 12 h. After 3 d in culture, H 2 O 2 generation was assessed by loading DRG neurons with the H 2 O 2 probe CM-H 2 -DCFDA (Russell et al, 2002;Vincent et al, 2005a). Reduced CM-H 2 -DCFDA is nonfluorescent.…”

“…The cells were rinsed in HBSS and examined in a fluorescence plate reader. DCF fluorescence was assessed at 485 nm excitement and 520 emission with 1 sec integration (Vincent et al, 2005a;Vincent et al, 2005b). In experiments involving Ad.GFP infected DRG neurons, the GFP signal was assessed prior to glucose exposure and this value was subtracted from the final green fluorescence.…”

“…TRAP is determined by a luminometric method (Alho et al, 1998;Vincent et al, 2005a). DRG neurons are lysed in ultra-pure water, then sonicated to complete the breakdown of cellular structures.…”