Atomic Force Microscopy Reveals Important Differences in Axonal Resistance to Injury

Abstract

Axonal degeneration after traumatic brain injury and nerve compression is considered a common underlying cause of temporary as well as permanent disability. Because a proper functioning of neural network requires phase coherence of all components, even subtle changes in circuitry may lead to network failure. However, it is still not possible to determine which axons will recover or degenerate after injury. Several groups have studied the pressure threshold for axonal injury within a nerve, but difficulty accessing the injured region; insufficient imaging methods and the extremely small dimensions involved have prevented the evaluation of the response of individual axons to injury. We combined microfluidics with atomic force microscopy and in vivo imaging to estimate the threshold force required to 1), uncouple axonal transport without impairing axonal survival, and 2), compromise axonal survival in both individual and bundled axons. We found that rat hippocampal axons completely recover axonal transport with no detectable axonal loss when compressed with pressures up to 65 ± 30 Pa for 10 min, while dorsal root ganglia axons can resist to pressures up to 540 ± 220 Pa. We investigated the reasons for the differential susceptibility of hippocampal and DRG axons to mechanical injury and estimated the elasticity of live axons. We found that dorsal root ganglia axons have a 20% lower elastic modulus than hippocampal axons. Our results emphasize the importance of the integrity of the axonal cytoskeleton in deciding the axonal fate after damage and open up new avenues to improve injury diagnosis and to identify ways to protect axons.

A microfluidic neuronal platform for neuron axotomy and controlled regenerative studies

“…These values are in the threshold range of previously reported values to create axonal injury (300 pN-4 nN). 28 However, evaluating the shear force due to the rapid velocity change in the channels crossing region, axon experience forces larger than 24 mN, when being crossed by air. The breaking of axons in the small region dened by the two uids interface is almost instantaneous.…”

Diagnostic tools for evaluating the impact of Focal Axonal Swellings arising in neurodegenerative diseases and/or traumatic brain injury

“…Using this technique, changes in axonal shape have been observed in response to the application of mechanical force (Magdesian et al, 2012;Hemphill et al, 2011). In order to accurately measure relative changes in axonal morphology, it is necessary to account for potential image distortions that may arise due to changes in lighting, cell marker fluorescence levels, or microscope drift.…”

“…Cellular membrane blebbing has been documented as a precursor to cell death occurring as the cell cytoskeleton degrades (Smith et al, 1999;Maxwell et al, 1997). Many studies report cognitive, behavioral, perceptual and sensorymotor impairments coinciding with the presence of FAS, yet no framework exists to explain the link between these macro-scale behavioral symptoms and the underlying micro-scale pathology (Magdesian et al, 2012;Hemphill et al, 2011).…”

Acute and Chronic Neural Stimulation via Mechano-Sensitive Ion Channels

“…Lusardi et al also made use of neurons plated on flexible substrates followed by a pneumatic pressure pulse to investigate the effects of mechanical stretching on a fraction of cells on the un-stretched cells with concurrent monitoring of acute calcium influx propagation. Pa. 137 The authors attributed the higher resistance of DRG neurons to compressions to the lower elasticity (20%) of their axons which allowed the axons of the DRG neurons to accommodate greater stresses. The finding is also aligned to a previous finding on the mechanical heterogeneity of the brain which may be connected to their susceptibility to mechanical damage and recovery.…”