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Diffuse axonal injury (DAI) is an evolving axonopathy commonly characterized clinically as widespread damage to the white matter tracts. In recent electrophysiological studies, researchers have proposed that myelinated and unmyelinated axons differ in their vulnerability and functional recovery following DAI. In this study we present for the first time an in vitro stretch-injury approach that utilizes a novel myelinating co-culture system to determine the differential response between myelinated and non-myelinated axon bundles to injury. In implementing this technique we demonstrate that myelinated axon bundles are less vulnerable to stretch injury compared to caliber-matched non-myelinated bundles. Interestingly, moderate axonal strain did not induce demyelination, but instead caused an increase in the proportion of degenerated myelin basic protein over time. Additionally, there were no significant differences in the expression of axonal swellings, which is indicative of disrupted axonal transport. In summary, we present an ideal in vitro model that permits further mechanistic investigations into the role of myelin and oligodendrocyte-neuron interactions in response to DAI.

The ubiquitin proteasome system (UPS) plays a vital role in the regulation of protein degradation. Ubiquitination of proteins has been implicated in the pathological cascade associated with neuronal degeneration in both neurodegenerative disease and following acquired central nervous system (CNS) injury. In the present study, we have investigated the role of the UPS following mild to moderate in vitro axonal stretch injury to mature primary cortical neurons, a model of the evolving axonal pathology characteristic of diffuse axonal injury following brain trauma. Transient axonal stretch injury in this model does not involve primary axotomy. However, delayed accumulation of ubiquitin in neuritic swellings at 48 h post-injury (PI) was present in axonal bundles, followed by approximately 60% of axonal bundles progressing to secondary axotomy at 72 h PI. This delayed accumulation of ubiquitin was temporally and spatially associated with cytoskeletal damage. Pharmacological inhibition of the UPS with both MG132 and lactacystin prior to axonal injury resulted in a significant (p < 0.05) increase in the number of axonal bundles progressing to secondary axotomy at 48 and 72 h PI. These results demonstrate that, following mild to moderate transient axonal stretch injury, UPS activity may assist structural reorganization within axons, potentially impeding secondary axotomy. Protein ubiquitination in the axon may therefore have a protective role relative to the diffuse axonal changes that follow traumatic brain injury.

We report a new model of transient axonal stretch injury involving pressurized fluid deflection of bundles of axons, resulting in a transient 1-6% increase in original axon length to investigate the slow progression of axonal alterations that are characteristic of diffuse axonal injury (DAI). We found no discernable difference in axon bundle morphology or cytoskeletal neurofilament protein arrangement between unstretched and stretched axonal bundles at 24 h post-injury. However, by 48 h post-injury, there was a stereotypical response of stretched axons involving characteristic neurofilament alterations that bear similarities to in vivo neuronal responses associated with DAI that have been reported previously. For instance, neurofilament protein immunoreactivity (SMI-312) was increased in axons contained within 51% of all injured axon bundles at 48 h compared to surrounding unstretched axon bundles, suggestive of neurofilament compaction. Furthermore, axonal bundle derangement occurred in 25% of injured axon bundles, with individual fibres segregating from each other and becoming undulating and wavy. By 72 h post-stretch, 70% of injured axon bundles underwent secondary axotomy, becoming completely severed at the site of initial stretch injury. While these results suggest a temporal series of stereotypical responses of axons to injury, we were able to distinguish very clear differences between mildly (100-103% increase in original axonal length) injured and strongly injured (106%+) axons. For instance, mildly injured axons developed increased neurofilament immunoreactivtity (SMI-312) within 48 h, and the marked development of ring-like neurofilament immunoreactive structures within axonal bundles, which were rarely axotomized. Conversely, at more severe strain levels increased neurofilament immunoreactivity was less apparent, while axons often became distorted and disorganised within axonal bundles and eventually became completely disconnected. Almost no ring-like neurofilament structures were observed in these severely injured axonal bundles. This suggests that axons do not respond in a stereotypical manner to a transient stretch insult, and indeed that variable degrees of stretch injury activate different responses within axons, with dramatically different outcomes. Hence, it is possible that the cytoskeletal characteristics that we have used in this study may be useful parameters for discriminating between mildly and severely injured axons following TBI.