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

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          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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          Most cited references42

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          High glucose-induced oxidative stress and mitochondrial dysfunction in neurons.

          The current study examines the association between glucose induction of reactive oxygen species (ROS), mitochondrial (Mt) depolarization, and programmed cell death in primary neurons. In primary dorsal root ganglion (DRG) neurons, 45 mM glucose rapidly induces a peak rise in ROS corresponding to a 50% increase in mean Mt size at 6 h (P<0.001). This is coupled with loss of regulation of the Mt membrane potential (Mt membrane hyperpolarization, followed by depolarization, MMD), partial depletion of ATP, and activation of caspase-3 and -9. Glucose-induced activation of ROS, MMD, and caspase-3 and -9 activation is inhibited by myxothiazole and thenoyltrifluoroacetone (P<0.001), which inhibit specific components of the Mt electron transfer chain. Similarly, MMD and caspase-3 activation are inhibited by 100 microM bongkrekic acid (an inhibitor of the adenosine nucleotide translocase ANT). These results indicate that mild increases in glucose induce ROS and Mt swelling that precedes neuronal apoptosis. Glucotoxicity is blocked by inhibiting ROS induction, MMD, or caspase cleavage by specific inhibitors of electron transfer, or by stabilizing the ANT.
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            Actin-ATP hydrolysis is a major energy drain for neurons.

            In cultured chick ciliary neurons, when ATP synthesis is inhibited, ATP depletion is reduced approximately 50% by slowing actin filament turnover with jasplakinolide or latrunculin A. Jasplakinolide inhibits actin disassembly, and latrunculin A prevents actin assembly by sequestering actin monomers. Cytochalasin D, which allows assembly-disassembly, but only at pointed ends, is less effective in conserving ATP. Ouabain, an Na(+)-K(+)-ATPase inhibitor, and jasplakinolide both prevent approximately 50% of the ATP loss. When applied together, they completely prevent ATP loss over a period of 20 min, suggesting that filament stabilization reduces ATP consumption by decreasing actin-ATP hydrolysis directly rather than indirectly by modulating the activity of Na(+)-K(+)-ATPase, a major energy consumer.
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              Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons.

              RM Lindsay (1988)
              Largely on the basis of studies with nerve growth factor (NGF), it is now widely accepted that development of the peripheral nervous system of vertebrates is dependent in part on the interaction of immature sensory and autonomic neurons with specific survival factors that are derived from peripheral target fields. I have found, in marked contrast to an absolute requirement for NGF during development, that adult rat dorsal root ganglion sensory neurons are not dependent on NGF or other survival factors for long-term (3-4 weeks) maintenance in vitro. When dissociated and enriched, at least 70-80% of adult DRG neurons survived and extended long processes either in the absence of exogenously added NGF or upon the removal of any possible source of endogenous NGF or other neurotrophic activity (i.e., nonneuronal cells, in chemically defined culture medium, in the presence of an excess of anti-NGF antibodies, or when cultured as single neurons in microwells). Although not required for survival or expression of a range of complex morphologies, both NGF and brain-derived neurotrophic factor (BDNF) were found to stimulate the regeneration of axons from adult DRG neurons.
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                Author and article information

                Journal
                Diabetes
                diabetes
                diabetes
                Diabetes
                Diabetes
                American Diabetes Association
                0012-1797
                1939-327X
                June 2009
                27 February 2009
                : 58
                : 6
                : 1356-1364
                Affiliations
                [1] 1Division of Neurodegenerative Disorders, St Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada;
                [2] 2Department of Pharmacology & Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada.
                Author notes
                Corresponding author: Paul Fernyhough, paulfernyhough@ 123456yahoo.com .
                Article
                0034
                10.2337/db09-0034
                2682687
                19252136
                bd55f63c-f72f-48d4-8aa0-155d9ad3510e
                © 2009 by the American Diabetes Association.

                Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

                History
                : 8 January 2009
                : 19 February 2009
                Categories
                Original Article
                Complications

                Endocrinology & Diabetes
                Endocrinology & Diabetes

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