Real time imaging of intra-axonal calcium flux in an explant mouse model of axonal Guillain-Barré syndrome

The acute motor axonal variant of Guillain-Barré syndrome is associated with the attack of motor axons by anti-ganglioside antibodies which activate complement on the axonal plasma membrane. Animal models have indirectly implicated complement pore-mediated calcium influx as a trigger of axonal damage, through the activation of the protease calpain. However, this calcium influx has never been imaged directly. Herein we describe a method to detect changes in intra-axonal calcium in an ex vivo mouse model of axonal Guillain-Barré syndrome and describe the influence of calcium on axonal injury and the effects of calpain inhibition on axonal outcome. Using ex vivo nerve-muscle explants from Thy1-TNXXL mice which axonally express a genetically encoded calcium indicator, we studied the effect of the binding and activation of complement by an anti-GD1b ganglioside antibody which targets the motor axon. Using live multiphoton imaging, we found that a wave of calcium influx extends retrogradely from the motor nerve terminal as far back as the large bundles within the muscle explant. Despite terminal complement pores being detectable only at the motor nerve terminal and, to a lesser degree, the most distal node of Ranvier, disruption of axonal proteins occurred at more proximal sites implicating the intra- axonal calcium wave. Morphological analysis indicated two different types of calcium-induced changes: acutely, distal axons showed swelling and breakdown at sites where complement pores were present. Distally, in areas of raised calcium which lacked detectable complement pores, axons developed a spindly, vacuolated appearance suggestive of early signs of degeneration. All morphological changes were prevented with treatment with a calpain inhibitor. This is the first investigation of axonal calcium dynamics in a mouse model of Guillain-Barré syndrome and demonstrates the proximal reach of calcium influx following an injury which is confined to the most distal parts of the motor axon. We also demonstrate that calpain inhibition remains a promising candidate for both acute and sub-acute consequences of calcium-induced calpain activation.