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P01.53. Spheroid formation and axonal severing in adult neurons during oxidative stress: role of calcium


Axonal severing is critical to the irreversible disability that occurs over the course of multiple sclerosis (MS). Reactive oxygen species (ROS) are implicated in neurodegenerative aspects of MS: axonal spheroid formation, severing, and axoplasmic Ca2+ elevation. However, the exact role of Ca2+ in spheroid formation remains unclear. The mechanism of action of natural anti-oxidants such as lipoic acid, which provide neuroprotection during oxidative stress in MS model, also remains unclear.


Primary cortical neurons from adult mice were subjected to physiologically-relevant levels of H2O2. Ca2+ dynamics and its sources were examined during spheroids formation using real time imaging, ratiometric Ca2+ indicators and immunocytochemistry.


Exposure to ROS led to a 3.5 fold increase in axoplasmic Ca2+ by 30 min. Onset of axonal spheroid formation began at 15 min when Ca2+ increase was 2.2 fold. Axonal severing occurred at sites of spheroids around 90-120 min. Analysis of small axonal segments revealed an uneven distribution of Ca2+ during exposure to H2O2. Micrometers apart, focal Ca2+ increases in small axonal domains ranged from 2.8 to 4.4 fold. Domains with a 3.8 to 4.4-fold increase correlated with the sites of spheroids, suggesting high focal extracellular Ca2+ influx at these sites. Several treatments significantly attenuated Ca2+ increase and completely abolished spheroid formation under ROS: removal of extracellular Ca2+; N-type Ca2+ channel blocker omega-conotoxin GVIA; L-type Ca2+ channel blocker amlodipine; and reverse Na+/ Ca2+ exchanger (NCX1) blocker KB-R7943. Aggregation of reverse NCX1 and N-type voltage-gated Ca2+ channel was detected at spheroids.


Our results reveal a correlation between focal axoplasmic Ca2+ and spheroid formation and suggest that focal aggregation of the reverse NCX1 and N-type Ca2+ channel plays central role in high focal Ca2+ increase during oxidative stress. These findings provide a basis for investigating the neuroprotective mechanism of the natural anti-oxidant lipoic acid during oxidative stress.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Barsukova-Bell, A., Forte, M. & Bourdette, D. P01.53. Spheroid formation and axonal severing in adult neurons during oxidative stress: role of calcium. BMC Complement Altern Med 12 (Suppl 1), P53 (2012).

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