Development of Selective Axonopathy in Adult Sensory Neurons Isolated From Diabetic Rats: Role of Glucose-Induced Oxidative Stress

OBJECTIVE

Reactive oxygen species (ROS) are pro-oxidant factors in distal neurodegeneration in diabetes. We tested the hypothesis that sensory neurons exposed to type 1 diabetes would exhibit enhanced ROS and oxidative stress and determined whether this stress was associated with abnormal axon outgrowth.

RESEARCH DESIGN AND METHODS

Lumbar dorsal root ganglia sensory neurons from normal or 3- to 5-month streptozotocin (STZ)-diabetic rats were cultured with 10 or 25–50 mmol/l glucose. Cell survival and axon outgrowth were assessed. ROS were analyzed using confocal microscopy. Immunofluorescent staining detected expression of manganese superoxide dismutase (MnSOD) and adducts of 4-hydroxy-2-nonenal (4-HNE), and MitoFluor Green dye detected mitochondria.

RESULTS

Dorsal root ganglion neurons from normal rats exposed to 25–50 mmol/l glucose did not exhibit oxidative stress or cell death. Cultures from diabetic rats exhibited a twofold (P < 0.001) elevation of ROS in axons after 24 h in 25 mmol/l glucose compared with 10 mmol/l glucose or mannitol. Perikarya exhibited no change in ROS levels. Axonal outgrowth was reduced by approximately twofold (P < 0.001) in diabetic cultures compared with control, as was expression of MnSOD. The antioxidant N-acetyl-cysteine (1 mmol/l) lowered axonal ROS levels, normalized aberrant axonal structure, and prevented deficits in axonal outgrowth in diabetic neurons (P < 0.05).

CONCLUSIONS

Dorsal root ganglia neurons with a history of diabetes expressed low MnSOD and high ROS in axons. Oxidative stress was initiated by high glucose concentration in neurons with an STZ-induced diabetic phenotype. Induction of ROS was associated with impaired axonal outgrowth and aberrant dystrophic structures that may precede or predispose the axon to degeneration and dissolution in human diabetic neuropathy.Diabetic sensory polyneuropathy in humans and animal models is associated with a spectrum of structural changes in peripheral nerves that includes microangiopathy, axonal degeneration, segmental demyelination, and ultimately loss of both myelinated and unmyelinated fibers (1,2). It has been proposed that high glucose concentrations induce toxicity and cell death in sensory neurons, and this triggers diabetic neuropathy through loss of nerve fibers (3). Cultured embryonic dorsal root ganglion sensory neurons were exposed to high nonphysiological concentrations of glucose that induced oxidative stress by increasing production of reactive oxygen species (ROS), and this was associated with mitochondrial dysfunction, which resulted in programmed cell death (4–6).Morphologic studies have provided a variety of results in relation to sensory neuron survival in animal models of diabetes. Long-term studies of 9 months in streptozotocin (STZ)-diabetic mice revealed a significant loss of sensory neurons (7). In STZ-diabetic rats of up to 12 months'' duration, no significant loss of adult lumbar dorsal root ganglion neurons was observed (8,9). Additionally, in 4-month diabetic BB rats, there was no dorsal root ganglion sensory neuron cell death (10); however, by 10 months there was progressive neuronal loss, but prominent only in the small dorsal root ganglion neuron population and not involving apoptosis (11). At the same time there was a significant decrease in the numbers of myelinated and unmyelinated fibers, but no evidence of structural changes in mitochondria in dorsal root ganglion sensory neurons (11). In STZ-diabetic mice, where loss of small neurons was also occurring, there was no sign of activation of the pro-apoptotic markers p38, caspase-3, and phosphorylated c-jun (12).Sural nerves from humans with diabetic neuropathy assessed using quantitative morphometry have significant endoneurial microangiopathy, early structural abnormalities in Schwann cells in myelinated fibers, and degeneration and loss of unmyelinated and myelinated fibers; however, in intact axons, mitochondria appeared structurally normal (1,13). In addition, studies performed on postmortem samples from type 2 diabetic patients have shown the occurrence of dystrophic changes in axon terminals and within the dorsal root ganglion and autonomic ganglia, but no evidence for significant neuronal cell loss (14,15).These results show that in vivo in animals and humans, the impact of diabetes on sensory neuron survival are discordant with the in vitro studies demonstrating toxic effects of high glucose concentration leading to apoptosis. We hypothesized that the underlying reason for this discrepancy was the use of embryonic sensory neurons for in vitro glucose toxicity studies (3). Cultured embryonic sensory neurons have phenotypic differences with adult sensory neurons and are dependent on neurotrophic factor–derived support for survival (16). Therefore, the aim of this study was to compare responses of adult dorsal root ganglion sensory neurons from age-matched control and 3- to 5-month STZ-diabetic rats exposed to high glucose concentration. To this end, the effect of high glucose concentration on oxidative stress and neuronal survival and axonal morphology was assessed.