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Development of selective axonopathy in adult sensory neurons isolated from diabetic rats: role of glucose-induced oxidative stress.

Zherebitskaya E, Akude E, Smith DR, Fernyhough P - Diabetes (2009)

Bottom Line: Axonal outgrowth was reduced by approximately twofold (P < 0.001) in diabetic cultures compared with control, as was expression of MnSOD.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT

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.

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High glucose concentration does not impair adult sensory neuron survival or induce oxidative stress. A and B: Phase contrast images of 2-week cultures of adult dorsal root ganglion sensory neurons grown in defined F12 + 10% FBS medium with and without 50 mmol/l d-glucose. Note the phase-bright neuronal perikarya and phase-dark nonneuronal cells (mostly fibroblasts and Schwann cells). C: Levels of survival of dorsal root ganglion neurons for control (10 mmol/l d-glucose) or 50 mmol/l d-glucose over a 4-week period. Cell numbers were assessed by morphology under a phase contrast microscope. Values are the means ± SE, n = 4 replicate cultures. The insert graph shows no difference in total axonal outgrowth at 24 h. Values are the means ± SE, n = 3 replicate cultures. D and E: Levels of 4-HNE adduct expression or caspase 3 activation in neuronal perikarya in arbitrary units of fluorescence intensity at 2 and 4 weeks of culture. Values are the means ± SE, n = 85–108 neurons. *P < 0.05. glu, glucose; wks, weeks.
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Figure 1: High glucose concentration does not impair adult sensory neuron survival or induce oxidative stress. A and B: Phase contrast images of 2-week cultures of adult dorsal root ganglion sensory neurons grown in defined F12 + 10% FBS medium with and without 50 mmol/l d-glucose. Note the phase-bright neuronal perikarya and phase-dark nonneuronal cells (mostly fibroblasts and Schwann cells). C: Levels of survival of dorsal root ganglion neurons for control (10 mmol/l d-glucose) or 50 mmol/l d-glucose over a 4-week period. Cell numbers were assessed by morphology under a phase contrast microscope. Values are the means ± SE, n = 4 replicate cultures. The insert graph shows no difference in total axonal outgrowth at 24 h. Values are the means ± SE, n = 3 replicate cultures. D and E: Levels of 4-HNE adduct expression or caspase 3 activation in neuronal perikarya in arbitrary units of fluorescence intensity at 2 and 4 weeks of culture. Values are the means ± SE, n = 85–108 neurons. *P < 0.05. glu, glucose; wks, weeks.

Mentions: Initial experiments were designed to test the influence of high concentrations of glucose (50 mmol/l) on survival of sensory neurons in culture, the induction of apoptosis, and markers of oxidative stress. Sensory neurons from adult control rats were grown up to 4 weeks in F12 medium with 10% FBS. Healthy neurons were phase bright, and by 1 week, cultures exhibited high numbers of nonneuronal cells, including Schwann cells, satellite cells, and fibroblasts (Fig. 1A and B). High glucose concentration had no effect on survival of neurons at any time and did not affect axon outgrowth on day 1 (Fig. 1C and insert). Surviving dorsal root ganglion neurons did decline over time and had diminished by 50% by the 28th day; however, there was no effect of 50 mmol/l glucose on this process. Oxidative stress and/or induction of apoptosis was assessed by staining for adducts of 4-HNE and caspase 3 activation. There was no effect of 50 mmol/l glucose on caspase 3 or 4-HNE adduct expression in neuronal perikarya at 2 or 4 weeks (Fig. 1D and E). However, there was a small (10–20%), but statistically significant, increase of activation of caspase 3 and 4-HNE adduct expression in the 4- vs. 2-week group of dorsal root ganglion neurons; this effect was not perturbed by 50 mmol/l glucose.


Development of selective axonopathy in adult sensory neurons isolated from diabetic rats: role of glucose-induced oxidative stress.

Zherebitskaya E, Akude E, Smith DR, Fernyhough P - Diabetes (2009)

High glucose concentration does not impair adult sensory neuron survival or induce oxidative stress. A and B: Phase contrast images of 2-week cultures of adult dorsal root ganglion sensory neurons grown in defined F12 + 10% FBS medium with and without 50 mmol/l d-glucose. Note the phase-bright neuronal perikarya and phase-dark nonneuronal cells (mostly fibroblasts and Schwann cells). C: Levels of survival of dorsal root ganglion neurons for control (10 mmol/l d-glucose) or 50 mmol/l d-glucose over a 4-week period. Cell numbers were assessed by morphology under a phase contrast microscope. Values are the means ± SE, n = 4 replicate cultures. The insert graph shows no difference in total axonal outgrowth at 24 h. Values are the means ± SE, n = 3 replicate cultures. D and E: Levels of 4-HNE adduct expression or caspase 3 activation in neuronal perikarya in arbitrary units of fluorescence intensity at 2 and 4 weeks of culture. Values are the means ± SE, n = 85–108 neurons. *P < 0.05. glu, glucose; wks, weeks.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682687&req=5

Figure 1: High glucose concentration does not impair adult sensory neuron survival or induce oxidative stress. A and B: Phase contrast images of 2-week cultures of adult dorsal root ganglion sensory neurons grown in defined F12 + 10% FBS medium with and without 50 mmol/l d-glucose. Note the phase-bright neuronal perikarya and phase-dark nonneuronal cells (mostly fibroblasts and Schwann cells). C: Levels of survival of dorsal root ganglion neurons for control (10 mmol/l d-glucose) or 50 mmol/l d-glucose over a 4-week period. Cell numbers were assessed by morphology under a phase contrast microscope. Values are the means ± SE, n = 4 replicate cultures. The insert graph shows no difference in total axonal outgrowth at 24 h. Values are the means ± SE, n = 3 replicate cultures. D and E: Levels of 4-HNE adduct expression or caspase 3 activation in neuronal perikarya in arbitrary units of fluorescence intensity at 2 and 4 weeks of culture. Values are the means ± SE, n = 85–108 neurons. *P < 0.05. glu, glucose; wks, weeks.
Mentions: Initial experiments were designed to test the influence of high concentrations of glucose (50 mmol/l) on survival of sensory neurons in culture, the induction of apoptosis, and markers of oxidative stress. Sensory neurons from adult control rats were grown up to 4 weeks in F12 medium with 10% FBS. Healthy neurons were phase bright, and by 1 week, cultures exhibited high numbers of nonneuronal cells, including Schwann cells, satellite cells, and fibroblasts (Fig. 1A and B). High glucose concentration had no effect on survival of neurons at any time and did not affect axon outgrowth on day 1 (Fig. 1C and insert). Surviving dorsal root ganglion neurons did decline over time and had diminished by 50% by the 28th day; however, there was no effect of 50 mmol/l glucose on this process. Oxidative stress and/or induction of apoptosis was assessed by staining for adducts of 4-HNE and caspase 3 activation. There was no effect of 50 mmol/l glucose on caspase 3 or 4-HNE adduct expression in neuronal perikarya at 2 or 4 weeks (Fig. 1D and E). However, there was a small (10–20%), but statistically significant, increase of activation of caspase 3 and 4-HNE adduct expression in the 4- vs. 2-week group of dorsal root ganglion neurons; this effect was not perturbed by 50 mmol/l glucose.

Bottom Line: Axonal outgrowth was reduced by approximately twofold (P < 0.001) in diabetic cultures compared with control, as was expression of MnSOD.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus