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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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Axonal swellings in neurons from STZ-diabetic rats exposed to high glucose represent accumulations of mitochondria and phosphorylated NFH, which are eliminated by antioxidant treatment. In the left column of immunofluorescent images is MitoFluor Green staining; the right column exhibits phosphorylated NFH staining (orange). Double-staining is yellow. Dorsal root ganglion sensory neuron cultures were derived from age-matched control or 3- to 4-month STZ-diabetic rats and maintained in defined media for 4 days. STZ-diabetic rat cultures were grown in 25 mmol/l glucose without insulin. During the final 24 h of culture time, selected control or diabetic cultures were treated with 1 mmol/l NAC. A– F: Control (A and B), STZ-diabetic (C and D), and STZ-diabetic culture treated for 24 h with 1 mmol/l NAC (E and F). White arrows indicate areas of colocalization of mitochondria and phosphorylated NFH. G: Quantification of the number of axonal swellings in each treatment group. Values are the means ± SE, n = 3 replicate cultures. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Axonal swellings in neurons from STZ-diabetic rats exposed to high glucose represent accumulations of mitochondria and phosphorylated NFH, which are eliminated by antioxidant treatment. In the left column of immunofluorescent images is MitoFluor Green staining; the right column exhibits phosphorylated NFH staining (orange). Double-staining is yellow. Dorsal root ganglion sensory neuron cultures were derived from age-matched control or 3- to 4-month STZ-diabetic rats and maintained in defined media for 4 days. STZ-diabetic rat cultures were grown in 25 mmol/l glucose without insulin. During the final 24 h of culture time, selected control or diabetic cultures were treated with 1 mmol/l NAC. A– F: Control (A and B), STZ-diabetic (C and D), and STZ-diabetic culture treated for 24 h with 1 mmol/l NAC (E and F). White arrows indicate areas of colocalization of mitochondria and phosphorylated NFH. G: Quantification of the number of axonal swellings in each treatment group. Values are the means ± SE, n = 3 replicate cultures. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: Experiments now determined whether oxidative stress was causally linked to aberrant axonal structure in neurons derived from STZ-diabetic rats. The antioxidant N-acetyl cysteine (NAC) was tested for its ability to lower ROS. Cultures from control rats were assessed for ROS using DHR123 imaging and treated acutely with 1 mmol/l NAC (Fig. 6). Figure 6A–D shows that NAC rapidly neutralized ROS levels. In a separate experiment, STZ-diabetic cultures were plated for 1 day in the presence or absence of 1 mmol/l NAC, and effect on axonal outgrowth was determined; NAC was clearly able to enhance levels of total axonal outgrowth (Fig. 6E). Cultures of sensory neurons from control or STZ-diabetic rats were maintained for 3 days (at this time point, diabetic neurons exhibited 4-HNE staining) (Fig. 5C and D). During the last 24 h of culture, control and STZ-diabetic neurons were treated with 1 mmol/l NAC for a further 24 h. After 4 days all groups of cultures were treated with MitoFluor Green dye to specifically stain mitochondria and then fixed and immunostained for phosphorylated NFH.Figure 7A and B shows control neurons with normal axonal structure and uniform axonal localization of mitochondria. Cultures from STZ-diabetic rats demonstrated abnormal accumulations of mitochondria in axonsthat colocalized with phosphorylated NFH staining (Fig. 7C and D). Treatment with 1 mmol/l NAC significantly lowered the number of axonal swellings in normal and STZ-diabetic cultures (Fig. 7E–G).


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)

Axonal swellings in neurons from STZ-diabetic rats exposed to high glucose represent accumulations of mitochondria and phosphorylated NFH, which are eliminated by antioxidant treatment. In the left column of immunofluorescent images is MitoFluor Green staining; the right column exhibits phosphorylated NFH staining (orange). Double-staining is yellow. Dorsal root ganglion sensory neuron cultures were derived from age-matched control or 3- to 4-month STZ-diabetic rats and maintained in defined media for 4 days. STZ-diabetic rat cultures were grown in 25 mmol/l glucose without insulin. During the final 24 h of culture time, selected control or diabetic cultures were treated with 1 mmol/l NAC. A– F: Control (A and B), STZ-diabetic (C and D), and STZ-diabetic culture treated for 24 h with 1 mmol/l NAC (E and F). White arrows indicate areas of colocalization of mitochondria and phosphorylated NFH. G: Quantification of the number of axonal swellings in each treatment group. Values are the means ± SE, n = 3 replicate cultures. *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 7: Axonal swellings in neurons from STZ-diabetic rats exposed to high glucose represent accumulations of mitochondria and phosphorylated NFH, which are eliminated by antioxidant treatment. In the left column of immunofluorescent images is MitoFluor Green staining; the right column exhibits phosphorylated NFH staining (orange). Double-staining is yellow. Dorsal root ganglion sensory neuron cultures were derived from age-matched control or 3- to 4-month STZ-diabetic rats and maintained in defined media for 4 days. STZ-diabetic rat cultures were grown in 25 mmol/l glucose without insulin. During the final 24 h of culture time, selected control or diabetic cultures were treated with 1 mmol/l NAC. A– F: Control (A and B), STZ-diabetic (C and D), and STZ-diabetic culture treated for 24 h with 1 mmol/l NAC (E and F). White arrows indicate areas of colocalization of mitochondria and phosphorylated NFH. G: Quantification of the number of axonal swellings in each treatment group. Values are the means ± SE, n = 3 replicate cultures. *P < 0.05. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: Experiments now determined whether oxidative stress was causally linked to aberrant axonal structure in neurons derived from STZ-diabetic rats. The antioxidant N-acetyl cysteine (NAC) was tested for its ability to lower ROS. Cultures from control rats were assessed for ROS using DHR123 imaging and treated acutely with 1 mmol/l NAC (Fig. 6). Figure 6A–D shows that NAC rapidly neutralized ROS levels. In a separate experiment, STZ-diabetic cultures were plated for 1 day in the presence or absence of 1 mmol/l NAC, and effect on axonal outgrowth was determined; NAC was clearly able to enhance levels of total axonal outgrowth (Fig. 6E). Cultures of sensory neurons from control or STZ-diabetic rats were maintained for 3 days (at this time point, diabetic neurons exhibited 4-HNE staining) (Fig. 5C and D). During the last 24 h of culture, control and STZ-diabetic neurons were treated with 1 mmol/l NAC for a further 24 h. After 4 days all groups of cultures were treated with MitoFluor Green dye to specifically stain mitochondria and then fixed and immunostained for phosphorylated NFH.Figure 7A and B shows control neurons with normal axonal structure and uniform axonal localization of mitochondria. Cultures from STZ-diabetic rats demonstrated abnormal accumulations of mitochondria in axonsthat colocalized with phosphorylated NFH staining (Fig. 7C and D). Treatment with 1 mmol/l NAC significantly lowered the number of axonal swellings in normal and STZ-diabetic cultures (Fig. 7E–G).

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