| Abstract No.: | A-C1104 |
| Country: | Canada |
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| Title: | CELLULAR COMPARTMENT DEPENDENT ALTERATIONS IN REACTIVE OXYGEN SPECIES GENERATION IN ADULT SENSORY NEURONS FROM DIABETIC RATS WITH SPECIAL REFERENCE TO DYSTROPHIC AXONAL SWELLINGS. |
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| Authors/Affiliations: | 1 Elena Zherebitskaya*; 1 Zuocheng Wang; 2 Paul Fernyhough;
1 Division of Neurodegenerative Disorders, St. Boniface Hospital Research Centre ; 2 Division of Neurodegenerative Disorders, St. Boniface Hospital Research Centre and Dept. of Pharmacology & Therapeutics, Winnipeg, MB, Canada
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| Content: | Objectives. Sensory neuropathy, which is induced by diabetes in humans and in experimental animal models, can present such complications as sensory loss, foot ulceration and infection. Lower limb gangrene and amputation can follow. In diabetes high blood glucose concentration and lack of insulin signaling are key factors in induction of abnormalities in polyol pathway flux, protein glycation and neurotrophic support in the peripheral nervous system (PNS). Combination of those factors can enhance oxidative stress and trigger distal nerve damage in PNS. Elevated generation of reactive oxygen species (ROS) and mitochondrial dysfunction has been proposed as a one of the critical factors in nerve degeneration at diabetes. We tested the hypothesis that sensory neurons exposed to long term type 1 diabetes in vivo would exhibit enhanced ROS and oxidative stress in perikarya and axons and tested if this stress was associated with impaired regenerative axon outgrowth in cell culture.
Methods. Lumbar dorsal root ganglia (DRG, L1-L6) sensory neurons were isolated from age matched normal and 3-month streptozotocin (STZ)-diabetic rats and cultured in defined F-12 media supplemented with N2 additives under either normal (10mM) or high (25-50mM) glucose concentrations. Levels of cell survival and axon outgrowth were assessed. Also, level of ROS was analyzed in perikarya and axons using dihydrorhodamine 123 and 5-(and-6)-chloromethyl-2’, 7’-dichlorohydrofluorescein diacetate (CM-H2DCFDA) using real time video confocal microscopy. In addition, mitochondrial preparations from DRG were assessed for rates of electron transport by measuring rates of oxygen consumption (Clarke electrode). Additionally, immunofluorescent staining was used to detect expression of phosphorylated neurofilament H (antibody SMI31), neuron-specific beta-tubulin III and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE). Furthermore, MitoFluor green dye was used for colocalization of mitochondria in the axons in formalin-fixed cultures of DRG neurons.
Results. DRG sensory neurons isolated from diabetic rats exhibited a 2-fold (P<0.001) elevation of ROS levels in axons after 24 hrs of culture compared with control. However, the perikarya exhibited no change in ROS levels. At 4 days in vitro levels of neurotrophin-induced axonal growth were significantly reduced by 2.3-fold (P<0.001) in diabetic cultures compared with control. Acute and longer term (24 hr) treatment with 1mM N-acetyl-cysteine significantly lowered axonal ROS levels and prevented the deficit in axonal outgrowth in diabetic neurons. Mitochondrial preparations from DRG of diabetic rats demonstrated enhanced rates (P<0.05) of coupled and uncoupled electron transfer. We also discovered that axonal morphology of diabetic neurons was abnormal with appearance of dystrophic swellings that were filled with neurofilament, mitochondria and amino acid adducts of 4-hydroxy-2-nonenal.
Conclusions. We show for the first time that DRG sensory neurons with a history of diabetes express high levels of ROS in their axons but not in the perikarya. Oxidative stress is limited to the axonal compartment in diabetic neurons, is associated with elevated mitochondrial electron transport and results in impaired axon outgrowth
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