Mild Axonal Stretch InjuryIn VitroInduces a Progressive Series of Neurofilament Alterations Ultimately Leading to Delayed Axotomy

Abstract

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.

Therapy Development for Diffuse Axonal Injury

“…Two general models have emerged that induce uniaxial (one direction) stretching of axons spanning two populations of neurons via either rapid extension of an elastic substrate in the longitudinal direction of the attached axons or using a pressurized fluid jet. 31,33,58,61,62,[95][96][97][98] Important findings from these models include demonstration of primary rupture of axonal microtubules, evolving proteolysis, and loss of ionic homeostasis, collectively revealing multiple potential therapeutic targets as discussed below.…”

Dorsal root compression produces myelinated axonal degeneration near the biomechanical thresholds for mechanical behavioral hypersensitivity

“…Following nerve root trauma, axonal changes indicative of dysfunction and degeneration develop, which depend on the specific mechanical inputs of tissue loading (Olmarker et al, 1989;Kobayashi et al, 1993Kobayashi et al, , 2004Colburn et al, 1997Colburn et al, , 1999Hashizume et al, 2000;Kobayashi and Yoshizawa, 2002;Sekiguchi et al, 2004;Chung et al, 2005;Singh et al, 2006). Nerve root compression produces endoneurial edema, membrane leakage, and Wallerian degeneration (Olmarker et al, 1989;Kobayashi et al, 1993Kobayashi et al, , 2004Colburn et al, 1997Colburn et al, , 1999Hashizume et al, 2000;Kobayashi and Yoshizawa, 2002;Sekiguchi et al, 2004).…”

“…Axonal degeneration and inflammation can lead to increased electrical activity in adjacent intact axons, and can result in persistent allodynia-like behavioral hypersensitivity (Li et al, 2000;Obata et al, 2003). Axonal injury can also produce a breakdown in fast axonal transport, producing accumulations of transported proteins such as neurofilament (NF) and β-amyloid precursor protein (βAPP) (Chen et al, 1999;Chung et al, 2005;Singh et al, 2006). In fact, βAPP accumulation increases as early as 3-4 hours after injury in a rat model of lumbar nerve root stretch; βAPP accumulation increases with both applied tissue strain and strain rate (Singh et al, 2006).…”

Clinical and Finite Element Analysis of Acute Whiplash

“…Axonal injury relates directly to magnitude and rate of strain increase [66]. Rapid occurrence of these strains can exceed the material properties of the tissue, leading to tissue disruption; however, even mild stretch can induce progressive neurofilament alteration and delayed axotomy [67]. The degree of injury appears to be related to the peak strain of the tissue and the loading rate.…”

“…Stretching of the axolemma may result in several levels of injury: a conduction block due to myelin damage, or membrane injury with irreversible changes, decreased amplitude and increased latency [66]. Deformative stress acting upon the Na + channel mechanoreceptors increases Na + influx, causing reversal of the cation exchange pumps and depolarization of voltage-gated Ca ++ channels, with subsequent pathological influx of Ca ++ [67,69].…”