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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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Effect of STZ-diabetes on expression of MnSOD in axons of cultured sensory neurons. Lumbar dorsal root ganglion sensory neurons were isolated from age-matched normal or 5-month STZ-diabetic rats and cultured with 10 mmol/l (white or black bars) or 25 mmol/l (gray or red bars) glucose for 1 or 3 days. A– F: Cells were fixed and immunofluorescently costained for β-tubulin III (red; neuron specific) (panels A, C, and E show double stain) and MnSOD (green) (panels B, D, and F show single stain). Charts show 1 day (G) and 3 days (H) of MnSOD expression in axons assessed by fluorescence intensity at ×100 on a confocal microscope. Values are the means ± SE, n = 65–94 axons. *P < 0.05 vs. other groups; **P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 9: Effect of STZ-diabetes on expression of MnSOD in axons of cultured sensory neurons. Lumbar dorsal root ganglion sensory neurons were isolated from age-matched normal or 5-month STZ-diabetic rats and cultured with 10 mmol/l (white or black bars) or 25 mmol/l (gray or red bars) glucose for 1 or 3 days. A– F: Cells were fixed and immunofluorescently costained for β-tubulin III (red; neuron specific) (panels A, C, and E show double stain) and MnSOD (green) (panels B, D, and F show single stain). Charts show 1 day (G) and 3 days (H) of MnSOD expression in axons assessed by fluorescence intensity at ×100 on a confocal microscope. Values are the means ± SE, n = 65–94 axons. *P < 0.05 vs. other groups; **P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Increased ROS under high glucose concentration in diabetic neurons may reflect an impaired capacity to scavenge free radicals. Therefore, the effect of diabetes and high glucose on the expression of MnSOD was assessed in isolated mitochondrial preparations from dorsal root ganglia and in cultured sensory neurons. In Fig. 8, we present Western blot data demonstrating that MnSOD expression was reduced by 32% (P < 0.005) in mitochondria isolated from dorsal root ganglia of STZ-diabetic rats, and this was prevented by insulin therapy.Figure 9A and B shows axonal staining of MnSOD in sensory neurons from age-matched control rats cultured for 1 day under 10 mmol/l glucose. Results were similar under 25 mmol/l glucose (images not shown). Neurons from STZ-diabetic rats cultured under 10 mmol/l glucose for 1 day exhibited diminished immunostaining for MnSOD (Fig. 9C and D); however, if cultured in the presence of 25 mmol/l glucose, then no alteration in MnSOD expression was observed (Fig. 9E and F). When cultures were maintained for 3 days in vitro, the ability of 25 mmol/l glucose to maintain or raise MnSOD expression was significantly impaired (Fig. 9G and H).


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)

Effect of STZ-diabetes on expression of MnSOD in axons of cultured sensory neurons. Lumbar dorsal root ganglion sensory neurons were isolated from age-matched normal or 5-month STZ-diabetic rats and cultured with 10 mmol/l (white or black bars) or 25 mmol/l (gray or red bars) glucose for 1 or 3 days. A– F: Cells were fixed and immunofluorescently costained for β-tubulin III (red; neuron specific) (panels A, C, and E show double stain) and MnSOD (green) (panels B, D, and F show single stain). Charts show 1 day (G) and 3 days (H) of MnSOD expression in axons assessed by fluorescence intensity at ×100 on a confocal microscope. Values are the means ± SE, n = 65–94 axons. *P < 0.05 vs. other groups; **P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 9: Effect of STZ-diabetes on expression of MnSOD in axons of cultured sensory neurons. Lumbar dorsal root ganglion sensory neurons were isolated from age-matched normal or 5-month STZ-diabetic rats and cultured with 10 mmol/l (white or black bars) or 25 mmol/l (gray or red bars) glucose for 1 or 3 days. A– F: Cells were fixed and immunofluorescently costained for β-tubulin III (red; neuron specific) (panels A, C, and E show double stain) and MnSOD (green) (panels B, D, and F show single stain). Charts show 1 day (G) and 3 days (H) of MnSOD expression in axons assessed by fluorescence intensity at ×100 on a confocal microscope. Values are the means ± SE, n = 65–94 axons. *P < 0.05 vs. other groups; **P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Increased ROS under high glucose concentration in diabetic neurons may reflect an impaired capacity to scavenge free radicals. Therefore, the effect of diabetes and high glucose on the expression of MnSOD was assessed in isolated mitochondrial preparations from dorsal root ganglia and in cultured sensory neurons. In Fig. 8, we present Western blot data demonstrating that MnSOD expression was reduced by 32% (P < 0.005) in mitochondria isolated from dorsal root ganglia of STZ-diabetic rats, and this was prevented by insulin therapy.Figure 9A and B shows axonal staining of MnSOD in sensory neurons from age-matched control rats cultured for 1 day under 10 mmol/l glucose. Results were similar under 25 mmol/l glucose (images not shown). Neurons from STZ-diabetic rats cultured under 10 mmol/l glucose for 1 day exhibited diminished immunostaining for MnSOD (Fig. 9C and D); however, if cultured in the presence of 25 mmol/l glucose, then no alteration in MnSOD expression was observed (Fig. 9E and F). When cultures were maintained for 3 days in vitro, the ability of 25 mmol/l glucose to maintain or raise MnSOD expression was significantly impaired (Fig. 9G and H).

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