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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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Cultured sensory neurons from STZ-diabetic rats exhibit abnormal morphology and reduced levels of axon outgrowth. A: Upper and lower panels show phase contrast images of neurons derived from normal rats at 4 days in culture in defined F12 + modified N2 medium with 10 nmol/l insulin. B: Upper and lower panels show images of cultures derived from 3- to 4-month STZ-diabetic rats and grown in defined media with 25 mmol/l glucose and no insulin. White arrows show areas of abnormal axonal structure highlighted by swelling and beading. The insert shows a ×3 image exhibiting the phase-dark axonal swellings. White arrowheads are rare swellings in normal neurons. Black arrowheads are nonneurons. C: Quantification of axonal outgrowth, longest axon, after 1 and 4 days of culture from control or STZ-diabetic rats. Values are the means ± SE, n = 58–97 axons. *P < 0.001.
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Figure 2: Cultured sensory neurons from STZ-diabetic rats exhibit abnormal morphology and reduced levels of axon outgrowth. A: Upper and lower panels show phase contrast images of neurons derived from normal rats at 4 days in culture in defined F12 + modified N2 medium with 10 nmol/l insulin. B: Upper and lower panels show images of cultures derived from 3- to 4-month STZ-diabetic rats and grown in defined media with 25 mmol/l glucose and no insulin. White arrows show areas of abnormal axonal structure highlighted by swelling and beading. The insert shows a ×3 image exhibiting the phase-dark axonal swellings. White arrowheads are rare swellings in normal neurons. Black arrowheads are nonneurons. C: Quantification of axonal outgrowth, longest axon, after 1 and 4 days of culture from control or STZ-diabetic rats. Values are the means ± SE, n = 58–97 axons. *P < 0.001.

Mentions: We next determined whether cultured neurons from STZ-diabetic rats were also refractory to high glucose concentration. Neurons from age-matched control rats were grown in defined F12 + modified N2medium under low (10 mmol/l) glucose with 10 nmol/l insulin, whereas neurons from 3- to 4-month STZ-diabetic rats were grown in F12 + modified N2medium with 25 mmol/l glucose with no insulin support (to attempt to mimic diabetes in vivo). Cell survival of neurons from normal and diabetic rats over a 4-day period and under defined conditions were identical. Figure 2 shows the phase contrast images of sensory neurons from control (Fig. 2A, upper and lower panels) and STZ-diabetic (Fig. 2B) rats after 4 days in culture. Diabetic dorsal root ganglion neurons exhibited abnormal morphologic changes in neurite outgrowth, which were characterized by less axonal outgrowth (Fig. 2C) and appearance of phase-dark axonal swellings and beading along neurites (Fig. 2B, lower panel, inset). Separate cultures from control or STZ-diabetic rats were grown for 1 day and assessed for level of ROS in cell bodies and axons using CM-H2 DCFDA (Fig. 3A, B, and E) or DHR123 (Fig. 3C, D, and F) fluorescence imaging. ROS levels in cell bodies did not differ between control and diabetic neurons (not shown). Axonal ROS levels in neurons from STZ-diabetic rats showed at least a twofold elevation compared with control using both dyes (Fig. 3E and F). Cultures were then grown for 4 days and immunostained for 4-HNE adducts and β-tubulin III. Control cultures exhibited normal axons with very rare instances of axonal swellings (or varicosities); such swellings were negative for 4-HNE adduct staining (Fig. 4A, C, and E). In comparison, cultures from STZ-diabetic rats demonstrated large numbers of axonal swellings that were characterized with positive staining for 4-HNE adducts (Fig. 4B, D, and F). The cell bodies of control and diabetic cultures revealed similar levels of highly positive staining for 4-HNE adducts.


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)

Cultured sensory neurons from STZ-diabetic rats exhibit abnormal morphology and reduced levels of axon outgrowth. A: Upper and lower panels show phase contrast images of neurons derived from normal rats at 4 days in culture in defined F12 + modified N2 medium with 10 nmol/l insulin. B: Upper and lower panels show images of cultures derived from 3- to 4-month STZ-diabetic rats and grown in defined media with 25 mmol/l glucose and no insulin. White arrows show areas of abnormal axonal structure highlighted by swelling and beading. The insert shows a ×3 image exhibiting the phase-dark axonal swellings. White arrowheads are rare swellings in normal neurons. Black arrowheads are nonneurons. C: Quantification of axonal outgrowth, longest axon, after 1 and 4 days of culture from control or STZ-diabetic rats. Values are the means ± SE, n = 58–97 axons. *P < 0.001.
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Related In: Results  -  Collection

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Figure 2: Cultured sensory neurons from STZ-diabetic rats exhibit abnormal morphology and reduced levels of axon outgrowth. A: Upper and lower panels show phase contrast images of neurons derived from normal rats at 4 days in culture in defined F12 + modified N2 medium with 10 nmol/l insulin. B: Upper and lower panels show images of cultures derived from 3- to 4-month STZ-diabetic rats and grown in defined media with 25 mmol/l glucose and no insulin. White arrows show areas of abnormal axonal structure highlighted by swelling and beading. The insert shows a ×3 image exhibiting the phase-dark axonal swellings. White arrowheads are rare swellings in normal neurons. Black arrowheads are nonneurons. C: Quantification of axonal outgrowth, longest axon, after 1 and 4 days of culture from control or STZ-diabetic rats. Values are the means ± SE, n = 58–97 axons. *P < 0.001.
Mentions: We next determined whether cultured neurons from STZ-diabetic rats were also refractory to high glucose concentration. Neurons from age-matched control rats were grown in defined F12 + modified N2medium under low (10 mmol/l) glucose with 10 nmol/l insulin, whereas neurons from 3- to 4-month STZ-diabetic rats were grown in F12 + modified N2medium with 25 mmol/l glucose with no insulin support (to attempt to mimic diabetes in vivo). Cell survival of neurons from normal and diabetic rats over a 4-day period and under defined conditions were identical. Figure 2 shows the phase contrast images of sensory neurons from control (Fig. 2A, upper and lower panels) and STZ-diabetic (Fig. 2B) rats after 4 days in culture. Diabetic dorsal root ganglion neurons exhibited abnormal morphologic changes in neurite outgrowth, which were characterized by less axonal outgrowth (Fig. 2C) and appearance of phase-dark axonal swellings and beading along neurites (Fig. 2B, lower panel, inset). Separate cultures from control or STZ-diabetic rats were grown for 1 day and assessed for level of ROS in cell bodies and axons using CM-H2 DCFDA (Fig. 3A, B, and E) or DHR123 (Fig. 3C, D, and F) fluorescence imaging. ROS levels in cell bodies did not differ between control and diabetic neurons (not shown). Axonal ROS levels in neurons from STZ-diabetic rats showed at least a twofold elevation compared with control using both dyes (Fig. 3E and F). Cultures were then grown for 4 days and immunostained for 4-HNE adducts and β-tubulin III. Control cultures exhibited normal axons with very rare instances of axonal swellings (or varicosities); such swellings were negative for 4-HNE adduct staining (Fig. 4A, C, and E). In comparison, cultures from STZ-diabetic rats demonstrated large numbers of axonal swellings that were characterized with positive staining for 4-HNE adducts (Fig. 4B, D, and F). The cell bodies of control and diabetic cultures revealed similar levels of highly positive staining for 4-HNE adducts.

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