Preprint

DOI:10.1016/j.jchemneu.2016.05.006
The axon as a physical structure in health and acute trauma
Matthew T. K. Kirkcaldie1,2
Jessica M. Collins2
1. School of Medicine
2. Wicking Dementia Research and Education Centre
Faculty of Health, University of Tasmania

Abstract
The physical structure of neurons dendrites converging on the soma, with an axon
conveying activity to distant locations is uniquely tied to their function. To perform their
role, axons need to maintain structural precision in the soft, gelatinous environment of the
central nervous system and the dynamic, flexible paths of nerves in the periphery. This
requires close mechanical coupling between axons and the surrounding tissue, as well as an
elastic, robust axoplasm resistant to pinching and flattening, and capable of sustaining
transport despite physical distortion. These mechanical properties arise primarily from the
properties of the internal cytoskeleton, coupled to the axonal membrane and the
extracellular matrix. In particular, the two large constituents of the internal cytoskeleton,
microtubules and neurofilaments, are braced against each other and flexibly interlinked by
specialised proteins. Recent evidence suggests that the primary function of neurofilament
sidearms is to structure the axoplasm into a linearly organised, elastic gel. This provides
support and structure to the contents of axons in peripheral nerves subject to bending,
protecting the relatively brittle microtubule bundles and maintaining them as transport
conduits. Furthermore, a substantial proportion of axons are myelinated, and this thick
jacket of membrane wrappings alters the form, function and internal composition of the
axons to which it is applied. Together these structures determine the physical properties and
integrity of neural tissue, both under conditions of normal movement, and in response to
physical trauma. The effects of traumatic injury are directly dependent on the physical
properties of neural tissue, especially axons, and because of axons extreme structural
specialisation, post-traumatic effects are usually characterised by particular modes of axonal
damage. The physical realities of axons in neural tissue are integral to both normal function
and their response to injury, and require specific consideration in evaluating research models
of neurotrauma.
1 The axon
In the mature nervous system, the primary purpose of the axon is to propagate and
regenerate action potentials at a consistent speed, and secondarily to this, to provide
support for the energetic and signalling needs of its distal processes. Accordingly, its interior
is structured to maintain its shape and calibre, and permit rapid internal transport along its
length, while remaining compliant across the range of everyday movements. In large part this
is attributable to the physical qualities of the axoplasm, which resembles a structured gel or
liquid crystal (Weiss & Mayr, 1971; Mukhopadhyay et al., 2004; Jones & Safinya, 2008) due
to the packed array of loosely interacting cytoskeletal proteins aligned with the main axis of
the fibre (Berthold & Rydmark, 1995; Hirano & Llena, 1995). In addition to the passive
mechanical properties of the individual elements, many are coupled by motor proteins
capable of using ATP-derived energy to generate physical force, adding an active element to
the properties of the network (Goriely et al., 2015). The axon cytoskeleton consists of
microtubules, which stiffen the axon and provide transport scaffolding; packed arrays of
neurofilaments filling the axoplasm; actin microfilaments and spectrins, forming a cortex just
inside the axolemma, and proteins which link these elements (fig. 1; Schnapp & Reese, 1982;
Berthold & Rydmark, 1995; Lasiecka et al., 2009).

1.1 Cytoskeleton
1.1.1 Microtubules
Microtubules (MTs) are the largest cytoskeletal element, consisting of spiral tubular polymers
22-23m in diameter, made from - and -tubulin heterodimers (see reviews by Fukushima,
2011; Kapitein & Hoogenraad, 2015). The microtubule-associated proteins (MAPs) facilitate
microtubule assembly and transport mechanisms, and bind to microtubules to form sidearms which interact with the surrounding environment, promoting complexing and stability;
the principal types employed in axons are MAP1A, MAP1B, MAP3, MAP5 and tau, whereas
MAP2 is found in the soma and dendrites (Mandlekow & Mandlekow, 1995; Fukushima,
2011). Although MTs are vital for structure, growth, the transport of organelles and proteins
(Gardel et al., 2008; Kapitein & Hoogenraad, 2015) and extending and organising growing
axons during development (Haynes & Kinney, 2011), in mature axons they are primarily
transport conduits and bracing structural elements (Brangwynne et al., 2006; Gardel et al.,
2008; Ouyang et al., 2013).
Early in the initial polarisation of developing neurons, characteristic MT modifications (e.g.
detyrosination and acetylation at specific residues) are enriched in the nascent axon,
recruiting the transport proteins and MAPs appropriate for the structural and regulatory
challenges of this extraordinary structure (Fukushima, 2011; Janke & Bulinski, 2011). In
mature axons, further dynamic post-translational modifications such as acetylation,
tyrosination, glutamylation and glycylation regulate MT stability on the local scale, alter MAP
binding, and probably alter the movements of the microtubule motors kinesin and dynein
(Janke & Bulinski, 2011).
1.1.2

Neurofilaments

Neurofilaments (NFs) are the only major cytoskeletal element unique to neurons, and are
correspondingly unique among intermediate filaments (Leterrier et al., 1996; Parry, 2011).
Although not all axons contain NFs, the axons which do are largely filled by them (Schnapp
& Reese, 1982; Berthold & Rydmark, 1995). Mature NFs are heteropolymers consisting of a
10nm-wide core of protofilament dimers arranged in coiled coils, with their carboxy-terminal
tail domains extending as perpendicular side-arms in a bottle-brush configuration (Fuchs &
Cleveland, 1998; Leermakers & Zhulina, 2010; Parry, 2011).
NFs assemble from five structurally related subunits: neurofilament light (NFL), medium
(NFM) and heavy (NFH) (66, 95-100 and 110-115 kDa respectively; Lee & Cleveland, 1996;
Janmey et al., 2003) and -internexin (INT; 66 kDa; Yuan & Nixon, 2011); peripherin also
contributes to NFs in peripheral and some central axons (58kDa; Parry, 2011). The aminoterminal rod domain is highly conserved between all subunits, but the tail domain is
extended and structurally complex in NFM and NFH compared to NFL, INT and peripherin
(Pant & Veeranna, 1995; Leermakers & Zhulina, 2010; Parry, 2011).
A series of lysine-serine-proline repeats in NFH and NFM tail domains provides multiple sites
for phosphorylation (Elder et al., 1998a; Jones & Safinya, 2008), making NFs, particularly
NFH, the most phosphorylated proteins in the nervous system (Pant & Veeranna, 1995). This
phosphorylation alters inter-filament interaction (sometimes called bridging, although this
term is misleading; Leterrier et al., 1996; Guadano-Ferraz et al., 1990; Mukhopadhyay et al.,
2004; Leermakers & Zhulina, 2010), prevents premature assembly prior to entering the axon
(Nixon, 1993; Lee & Cleveland, 1996), and alters interactions between NFs and microtubules
(Hisanaga & Hirokawa, 1990). In the axon, phosphorylation of NFM and NFH creates linearly
aligned, spaced lattices of NFs (Schnapp & Reese, 1982; Eyer & Leterrier, 1988; Gotow et al.,
1994; Janmey et al., 2003) whereas dephosphorylation favours collapsed meshes and
fascicles in the soma and dendrites (Hirokawa et al., 1984; Pant & Veeranna, 1995). This
process is locally regulated, since individual NFs can have phosphorylated and
dephosphorylated domains at different points along their length (Brown, 1998).
Axonal NFs, which become phosphorylated near the axon initial segment (Nixon et al., 1994;
Schlaepfer & Bruce, 1990) form a tough, elastic network, capable of bearing considerably
more elastic strain than other cytoskeletal elements (Kreplak & Fudge, 2007). Within the
arrays, filaments have characteristic spacing (Elder et al., 1998a; Kumar et al., 2002; Jones &

Safinya, 2008) but are otherwise distributed randomly (Price et al., 1988) or semi-randomly
(Hsieh et al., 1994a; Mukhopadhyay et al., 2004), suggesting that the interactions between
them are not fixed.
1.1.3

Actin and the axon cortex

The subsurface structure of the axolemma, the axon cortex, is largely composed of a
meshwork of actin microfilaments linked by ankyrins and spectrins (Schnapp & Reese, 1982;
Thi et al., 2004; Grintsevich & Reisler, 2013). These filaments provide a substrate for motor
proteins such as myosin, and are pinned to the axolemma at adherens junctions, connected
by catenins to cadherins in the membrane (Salzer, 1995; Thi et al., 2004; 2013). Motor
proteins permit the actin cortex to locally modulate stiffness and compliance, in ways which
may be linked to signalling mediated by extracellular attachments (Gardel et al., 2008).
At the axon initial segment, spectrins and ankyrins, assisted by transmembrane neurofascin,
regulate the structured array of channels, and static actin forms a diffusion barrier to
compartmentalise the axonal environment from the soma (Jones et al., 2014; Ebel et al.,
2014). In myelinated axons, specialised juxta-nodal membrane domains also use spectrins
to organise ion channels and myelin linkages of the paranodal junction, again in
transmembrane partnership with neurofascin (Buttermore et al., 2013; Zhang et al., 2013;
Ebel et al., 2014). Likewise, periodic bands of actin tethered by spectrin IIII tetramers
have been identified in unmyelinated axons (Xu et al., 2013) and all neurons and
oligodendrocytes in vitro, arranging channels similarly to paranodal regions (dEste et al.,
2016). Spectrins are also vital to membrane integrity for example, a lack of spectrin
causes axons of C. elegans to break during normal movement (Hammarlund et al., 2007).
1.1.4

Cytoskeletal coupling

The elements of the axon cytoskeleton interact with each other, organelles, the membrane
and the extracellular environment by a variety of mechanisms. One of the primary scaffolds
is the axon cortex, a mesh of actin filaments supporting the axolemma, cross-linked and
attached to the membrane by spectrins, myosin and filamin A, and attached via talin, vinculin,
paxilin, -actinin, and zyxin to integrins and thence the extracellular matrix (Bennett & Baines,
2001; Tyler, 2012; Grintsevich & Reisler, 2013; Ingber et al., 2014).
The axon cortex is also coupled to the internal cytoskeleton by a range of proteins which
also link NFs, MTs and organelles (Frappier et al., 1991; Fuchs & Cleveland, 1998; Elder et al.,
1998a; Hirokawa & Takeda, 1998; Young & Kothary, 2011). Myosin Va couples NFs to actin
and organelles (Rao et al., 2002; 2011), providing both NF motility and active structural
properties. Another important cytoskeletal linker, BPAG1n (dystonin) organises NFs and MTs
by interlinking them, and ties NFs to actin (Yang et al., 1996; 1999; Fuchs & Cleveland 1998;
Suozzi et al., 2012). Although the outermost NFs are linked to the axon cortex, the bulk of
the axon volume is structured by interactions between NFs, and between NFs and MTs.
In large arrays of parallel neurofilaments, NFM rather than the larger NFH is the major
regulator of filament spacing and thus axon calibre (Cleveland et al., 1991; Elder et al.,
1998b; Jacomy et al., 1999; Rao et al., 2003; Jones & Safinya, 2008). Computational
modelling of neurofilaments suggests that phosphorylation of NFM is critical in establishing
interfilament spacing (Chang et al., 2009; Leermakers & Zhulina, 2010), but other studies
suggest that phosphorylation of NFM does not affect the volume its tail occupies around the
filament core, and therefore should not influence spacing (Stevenson et al., 2011). This
apparent contradiction, along with the observation that NFM interactions can be attractive
rather than the electrostatic repulsion expected between highly phosphorylated proteins
(Rammensee et al., 2007; Jones & Safinya, 2008), implies another mechanism at work.
Simulations of interacting tails under physiological conditions suggests that their interaction
is independent of phosphorylation state, and that filament spacing could be due to entropic
exclusion of tails when the volume they occupy encroaches on those from adjacent
filaments (Stevenson et al., 2011; Jayanthi et al., 2013), with phosphorylation regulating tail
extension (Chang et al., 2009). The extreme elasticity of neurofilament networks, requiring
filaments to slide past one another, argues against fixed or strong linkage between filaments;
instead it appears that the tails act as entropic springs to hold filaments close without

linking them (Perrot et al., 2008; Jones & Safinya, 2008); however, there is some evidence for
stronger linkage (Rammensee et al., 2007). Phosphorylated NFM and NFH tails thus coat
individual NFs with a skin of organised cytosol which packs together as a structured gel
filling the axon, maintaining collinearity of the filaments (and therefore transport pathways)
while permitting considerable sliding and elasticity.
In the axoplasm, NF and MT domains are intermingled: NFs typically occupy four times the
cross sectional area of microfilaments but are about ten times more flexible, probably
because the rod tetramers at the NF core are free to slide past one another (Janmey et al.,
2003). NFs are also far more elastic than MTs, which break when stretched (Peter & Mofrad,
2012; Shamloo et al., 2015). For these physical properties to coexist, NFs and MTs need to
be loosely coupled. Whereas NFs interact by means of their own sidearms, microtubuleassociated proteins (MAPs) perform this role for MTs (Heimann et al., 1985; Perrot et al.,
2008). Like the interactions between NFs, MT-NF couplings are most likely dominated by
entropy: the reduced freedom of NF tails when MAPs such as MAP2 and MAP6 impinge on
their domain, leads to steric repulsion (Mukhopadhyay et al., 2004; Young & Kothary, 2011).
Powered motor proteins such as kinesins and dyneins link NFs to MTs, holding them in
tension by pulling against MTs (Wagner et al., 2004; Gardel et al., 2008; Young & Kothary,
2011; Janke & Bulinski, 2011); as well as facilitating the transport of small NF polymers (Xia
et al., 2003; Uchida et al., 2009; Lee et al., 2011). Fixed protein bridges suggested by
some studies (e.g. Schnapp & Reese, 1982; Hirokawa et al., 1984; Miyasaka et al., 1993) are
most likely to be artefact rather than bonded links (Mukhopadhyay et al., 2004), although a
family of larger intermediate filament associated proteins (IFAPs) such as Macf1 and the
plakins plectin and BPAG1n (dystonin) are known to couple NFs independent of sidearm
interactions (Fuchs & Cleveland, 1998; Yang et al., 1999; Rao et al., 1998; Wiche et al., 2015;
review in Young & Kothary, 2011). The ability of NFs to organise the axoplasm into a
structured, loose gel could be viewed as a way of protecting and buffering the fragile
microtubule network within the dynamic environment of the axon, particularly in the
peripheral nervous system. Deletion of dystonin, which decouples NFs from the actin cortex,
fragments both axonal NFs and MTs (Yang et al., 1999).
1.2 Axoplasm
These interactions between cytoskeletal elements profoundly influence the bulk behaviour of
axoplasm. Minor properties such as the coupling strength of entropic interactions, or the
local availability of ATP to power active coupling elements, can greatly alter mechanical
properties when scaled to entire networks (Bernal et al., 2007; Goriely, 2015).
Rheology, in which materials are subjected to mechanical loads and their physical responses
are measured, is difficult to apply to individual axons due to the small forces and tiny
displacements involved (Elkin et al., 2007; Gardel et al., 2008), and challenging in bulk tissue
because of its structural complexity (Goriely et al., 2015). However, mechanical properties of
axons have been measured with increasing precision in recent years. Many of these
properties are non-linear, such as stress-stiffening and stress-weakening, and depend on
the type and degree of interaction between cytoskeletal proteins. Entropic and active
couplings produce hybrid stress responses which are part passive and part active (Tyler,
2012); for example, axons deformed by a microneedle respond with passive viscoelastic and
active (molecular motor based) deformations, and initial relaxation may be followed by slow
active contraction (Bernal et al., 2007).
Neurofilaments give the axoplasm gel-like properties: Gilbert (1975) described the NF-rich
axoplasm of the syncytial giant axon of a marine worm, a strong, elastic hydrogel containing
4% NFs whose structure breaks down in the presence of calcium ions. The unusual entropic
coupling of NF sidearms means that in bulk, these filaments respond to large strains in nonlinear ways, although this is insignificant under physiological conditions (Rammensee et al.,
2007; Ingber et al., 2014). Helical NFs structure the gel at larger scales (see also Jones &
Safinya, 2008), minimising stored elastic energy and enabling elastic reconfiguration during
stretching and contraction (Gilbert, 1975) as well as maintaining the linearity of NF-mediated
transport pathways across normal movement ranges.

Balancing this elasticity, cytoskeletal disruptions followed by axon rheology suggest that
MTs are the primary contributor to structural rigidity in axons (Ouyang et al., 2013). Despite
this intrinsic stiffness, MTs frequently bend in vivo (Ingber, 2003a; Gardel et al., 2008;
Brangwynne et al., 2006), and are therefore under compression; conversely, NFs are
intrinsically flexible, and so their observed straightness in axons means they are either under
elastic tension (Janmey et al. 2003), or motor proteins on the MTs may pull on NFs to
straighten them (Ingber, 2003a; Gardel et al., 2008; Young & Kothary, 2011). In this respect
the axoplasm has been described as a tensegrity composite (Ingber et al., 2014), in which
couplings between NFs and MTs facilitate compressive load bearing in MTs, and reduce the
length scale of MT buckling under such loads (Brangwynne et al., 2006), while coupling the
internal structural elements to the axon cortex.
Based on these observed properties, axoplasm may be described as a structured gel which
is compliant and elastic along the axis of the axon, integrated into the membrane by the
axon cortex, and supported by cytoskeletal elements braced by active tension from motor
proteins, and passive tension due to MT stiffness braced against NF elasticity. This internal
structure ensures linear alignment of the major transport-related proteins, and prevents
collapse or pinching of the axoplasm during movement.
2 Extracellular environment
2.1 Extracellular matrix and surface ligands
During development, axons are formed by growth cones navigating through tissue,
generating mechanical traction by means of adhesions with the extracellular matrix and
other cells (OToole et al., 2008; Haynes & Kinney, 2011; Dent et al., 2011). These
interactions determine axonal morphology, and the shape of the axon reflects a balance
between internal cytoskeletal tension and the external physical environment (Bauer &
Ffrench-Constant, 2009). Many extracellular molecules link with proteins bound to the axon
membrane, including the immunoglobulin superfamily members Thy-1, integrins, cadherins,
nectins and Ig-CAMs (Leyton & Hagood, 2014; Salzer, 1995; Takeichi, 2007; Tyler, 2012;
Mori et al., 2014), as well as the protocadherins (Morishita & Yagi, 2007; Weiner & Jontes,
2013) and neurofascin, a membrane anchor involved in the bundling of axons and myelin
paranodes (Ebel et al., 2014). In addition to their functional properties, these molecules
provide a diverse array of molecularly patterned surfaces throughout the nervous system,
permitting precise local organisation and connectivity, for which the coupling of extracellular
interactions to internal cytoskeletal regulation is essential (Haynes & Kinney, 2011; Tyler,
2012).
Even in the mature nervous system, many of these couplings are not just organisational: a
range of evidence suggests that tension and mechanical forces induced in axons are
significant regulators of signalling, growth and intracellular traffic (e.g. Tyler, 2012; Goriely et
al., 2015). Many of the molecules mediating axons extracellular attachments are coupled to
the internal cytoskeleton (Salzer, 1995; Thi et al., 2013; Mori et al., 2014), through which
force transduction provides a signalling mechanism to which many internal processes are
sensitive. Ingber (1997; 2003a;b) suggests the term mechanochemistry to describe the
linkage between mechanical stress at the tissue and organ level and alterations to the
internal biochemistry of the cells in that tissue.
Cadherins link membranes together by binding calcium, and are connected to the actin
cortex via catenins at desmosomes; these adherens junctions include a diverse array of
signalling molecules depending on the specific cadherins and catenins involved (Salzer,
1995; Fuchs & Cleveland, 1998; Thi et al., 2004; Takeichi, 2007); E-cadherin at adherens
junctions interacts with talin, vinculin and the actin cytoskeleton, acting as a force transducer
to trigger internal signalling (Dingyu et al., 2015). Protocadherins do not form these
intracellular complexes, and may merely provide greater variety in the molecular coding of
membranes; they are also less abundant on axons (Morishita & Yagi, 2007; Yagi, 2012;
Weiner & Jontes, 2013). The integrin family of receptors binds to a wide array of extracellular
molecules, notably laminin, influencing growth and intracellular events by a range of internal
signalling pathways, and linking directly to actin and NFs (Salzer, 1995; Denda & Reichardt,
2007); for example, tyrosine kinases cluster at integrin binding sites (Salzer, 1995).

Extracellular glycoproteins and glycosaminoglycans such as hyaluronan, tenascin-R and the

chondroitin sulphate proteoglycans aggrecan, versican, neurocan and brevican, couple to
membrane-spanning proteoglycans, particularly the syndecans, providing tissue integrity but
also acting as coreceptors for integrins and growth factor receptors (Zimmermann & DoursZimmermann, 2008; Thi et al., 2013; Dauth et al., 2016). Such influences may be mediated
by distortion of the cytoskeleton, either proximal to events such as receptor binding, or by
forces propagating deeper into the cell (Ingber, 2003b).
2.2 Myelin
For many axons, their most significant extracellular interaction is with myelinating processes
from oligodendrocytes and/or Schwann cells. The complex intercellular crosstalk which
regulates myelination and demyelination is emerging as a key mechanism for CNS plasticity
(Young et al., 2013; Pajevic et al., 2014; Seidl, 2014; Fields, 2015), and is beyond the scope
of this short review. However, mechanical factors influence the process, and the resultant
axon-myelin complex has physical properties which differ significantly from the unmyelinated
axon.
The physical properties of individual axon fibres, especially their calibre, have a strong
influence on the interactions leading to myelination (Hirano & Llena, 1995; Simons & Lyons,
2013). It seems likely that the mechanical environment around the axon, moreso than
membrane signalling, induces the differentiation of oligodendrocytes and the initiation of
myelination (Rosenberg et al., 2008; Bauer & ffrench-Constant, 2009; Mitew et al., 2013),
although neurofilaments themselves have been shown to enhance proliferation and
maturation of cultured oligodendrocytes (Fressinaud et al., 2012). Myelin itself is an extreme
membrane conformation which must be induced in oligodendrocytes and Schwann cells by
axon contact, cell surface ligands including nectins, and activity-dependent signalling (for
reviews of ligands and signalling, see Mitew et al., 2013; Mori et al., 2014; Tomassy et al.,
2015). Initially, calibre and local membrane curvature are significant: in vitro studies of
oligodendrocytes cultured with polystyrene fibres indicate that an inert cylindrical structure
with diameter > 0.4m is sufficient to initiate myelination, although the resulting myelin is
disorganised and rarely compact (Lee et al., 2012); this interaction takes place alongside
specific ligand-mediated membrane interactions which may enhance or suppress
oligodendrocyte differentiation (Mitew et al., 2013). The process of myelin compaction is
physical and biochemical, in which membrane layers overcome their normal glycoproteinmediated repulsion by means of intracellular myosin IIa (Kippert et al., 2009) and
downregulating cell surface molecules (Bakhti et al., 2013). Active constriction by these
compacting lamellae compresses the axon calibre at nodes (Perrot et al., 2007).
In turn, ongoing interaction between the axon and the oligodendrocyte or Schwann cell is
required for functional maturation of the axon (Simons & Lyons, 2013). Extracellular presence
of the cell surface molecule MAG (myelin associated glycoprotein) modulates
phosphorylation of NFs, affecting the internal cytoskeletal properties of the axon (de Waegh
et al., 1992; Hsieh et al., 1994b; Kumar et al., 2002; Garcia et al., 2003). Shiverer mutant
mice, lacking myelin basic protein, produce myelin which is thinner than normal, causing
disruption to the axonal cytoskeleton and alterations in the ratios of neurofilament
monomers; their NFL also degrades faster (Brady et al., 1999).
Primary sensory afferents and motor and autonomic efferents span between the CNS and
PNS environments, so their axons are myelinated by both Schwann cells and
oligodendrocytes. The difference in myelinating cell interactions causes a transition in axonal
properties as they cross from the peripheral nervous system into the CNS (e.g. myelinated
fine PNS fibres losing myelin in the CNS; Fraher, 1992, fig. 16; Hsieh et al., 1994b; Berthold
& Rydmark, 1995; Hirano & Llena, 1995), presumably reflecting the differing physical
requirements for peripheral nerves and white matter tracts. The transition zone is sharply
demarcated, characterised by structural and chemotactic boundaries (Fraher, 1992; Mauti et
al., 2007; Osen et al., 2011).
Beyond a simple ensheathing of the axon, the physical transformation caused by myelination
dynamically regulates axon structure in response to activity (Young et al., 2013; Fields, 2015).
Patchy myelination of CNS axons (Tomassy et al., 2014) suggests fine-grained local

regulation of axon calibre and other parameters (de Waegh et al., 1992; Hsieh et al., 1994b;
Garcia et al., 2003) in order to maintain temporal coherence on very short time scales,
evident in the physical axon modifications observed in time-critical parts of the CNS (e.g.
Seidl et al., 2010; 2014; Ford et al., 2015). Timing constraints for peripheral axons may be
less rigorous: stimulation at the neuromuscular junction has significant jitter and a long
integration time in terms of muscle response (Katz & Miledi, 1965), which is only
mechanically significant after volleys of action potentials sustained over time.
Myelination also minimises energy overheads for information processing, minimising the area
of exposed membrane on which leak channels are needed to maintain potential, for which
ATP must be burned to sustain concentration gradients. The energy costs of myelinated
axons have been estimated at one third of their unmyelinated counterparts (Neishabouri &
Faisal, 2011).
3

Mechanical properties of CNS tissue

The brain and spinal cord are extremely soft tissues, which in vivo are suspended by
arachnoid trabeculae, and float in cerebrospinal fluid. Accordingly, their mechanical
properties as bulk tissue in vivo have proven difficult to measure; Meaney et al. (2014)
observe that recent measurements using sensitive techniques suggest that neural tissue is
an order of magnitude softer than widely cited measurements made in the 1970s to 2000s.
In bulk, living neural tissue has unusual nonlinear stress-stretch hysteresis due to motor
protein active responses, as well as the viscoelastic properties of the tissue elements
themselves; new approaches based on dynamic MRI imaging are rapidly adding to
descriptions of the brain as a physical object (Goriely et al., 2015).
Unsurprisingly, myelination strongly influences the mechanical properties of CNS tissues.
Hippocampal layers, which vary in their ratios of neurons, glia, axons and dendrites, differ in
their mechanical properties as measured by atomic force microscopy (AFM; Elkin et al.,
2007), with grey matter layers stiffer than white matter layers. Similarly, AFM and tensile
measurements suggest that the grey matter of mouse spinal cord is about twice as stiff as
white matter, more so in the dorsal horn, and that white matter is more elastic in the
longitudinal direction than transversely (Koser et al., 2015). Since white matter in the spinal
cord consists of aligned axons whose cytoplasm is filled with elastic neurofilament networks,
it is unsurprising that the meshlike structure of grey matter is stiffer and less compliant.
Despite greater myelination, rheology of fresh postmortem human brain samples from adults
and children shows that the adult brain is far stiffer than that of the child, perhaps due to the
increased deposition of extracellular matrix elements such as CSPGs (Orlando & Raineteau,
2015) or more extensive interconnection in the adult brain. Furthermore, the adult brainstem
is far stiffer than rest of brain (Chatelin et al., 2012). Adult axons are stiffer than neonatal
axons, perhaps because of their increased NF content (Lamoureux et al., 2010) and the
moderate degree of tension they are normally under in the adult brain (Xu et al., 2009; 2010).
4 Movement and mechanical loads
4.1 Normal movement
The physical environment in which axons must operate differs significantly between the CNS
and the PNS. In the CNS, the surrounding tissue is more static and mechanically isolated
from physical movement by arachnoid trabeculae and CSF cushioning (Meaney et al., 2014).
The only major movements experienced inside the blood-brain barrier are flexions and
torsions of bulk tissue, such as bending of the spinal cord within the vertebral column,
rotation of the head (turning the brain with respect to the spinal cord) and relative movement
of large anatomical structures such as the cerebellum and pons during voluntary movements
and changes in head orientation (Ji et al., 2004; Ji & Margulies, 2007; Bayly et al., 2012).
Both pulse and respiration move the spinal cord several tenths a millimetre within the
vertebrae (Winklhofer et al. 2014), applying minor stress to axons in the roots of spinal
nerves passing through the vertebrae.
At microscopic scales, the CNS is in constant motion: human brain tissue moves tens of
microns in response to the pulse pressure (Kucewicz et al., 2007) and neural signalling itself
causes movements on very small scales: mechanical waves down the axon, caused by the

changing electrical fields across the axolemma, accompany action potentials (e.g. Tasaki &
Iwasa, 1982; El Hady & Machta, 2015). These tiny forces may also be a factor in myelination
(Fields, 2011;2015).
In the PNS, nerves bend and stretch with body movement, leading to unevenly distributed
strain along the course of axons, particularly where they pass around joints (Tassler et al.,
1994; Phillips et al., 2004; Abolfathi et al. 2009; Javid et al., 2014). Although peripheral
nerves are well supported by perineurium and epineurium (Ushiki & Ide, 1990), they can
stretch about 20% without rupture (Tassler et al., 1994), which requires axons to be elastic
and physically robust presumably the reason that NFs are ubiquitous in peripheral axons,
whether myelinated or not (Berthold & Rydmark, 1995). Mechanically, there are distinct
domains within individual axons (Schnapp & Reese, 1982): in the axoplasm, NF networks
differ from the MTs embedded in them, and from the actin-spectrin network of the axon
cortex (Berthold & Rydmark, 1995). Any applied force may cause focal disruptions at the
interface between these domains.
4.2 Mechanical loading in acute trauma
During traumatic brain injury (TBI), the variation in mechanical properties between structures
of the CNS adds to the intracellular cytoskeletal anisotropies, leading to uneven propagation
and focal concentrations of force. Generally, the bulk tissue properties determine force
distribution, but local concentrations depend on macro- and microanatomy (Cloots et al.,
2010).
In closed and rotational head injury, tissue is strained, sheared and compressed causing
injury throughout the brain, including the most common pathology, diffuse axonal injury (DAI;
Adams et al., 1989; 1991; Holbourn, 1943; 1945; Strich, 1956; Strich & Oxon, 1961). When
force is applied to brain tissue, structural anisotropies cause focal strains in individual axons
around twice as high as those experienced by the tissue as a whole. Existing static tension
in axons of the mature NS (Xu et al., 2009; 2010) may be released by or exacerbate
traumatic forces (Ayali, 2010). Strain is focused at structural boundaries near vessels, at
the ventricles, and the glia limitans where axons deviate and change in stiffness (Cloots et
al., 2011). Experimental models of TBI demonstrate that intra-axonal loci of impaired axonal
transport, and thus DAI, are found where axons change their anatomical course (Povlishock,
1993).
In DAI, damaged axons are not sheared but undergo focal disruption of cytoskeletal
elements, due to mechanical anisotropy, local exceeding of breaking strain, and by delayed
biochemical processes. The latter include perturbation of ionic balance, including elevated
intra-axonal sodium and calcium levels (Wolf et al., 2001; Kilinc et al., 2009). This activates
calcium-sensitive proteases such as calpains, which may degrade NFs, MTs, spectrins and
ion channels (Iwata et al., 2004; Hammarlund et al. 2007; Perrot et al., 2008; Kilinic et al.,
2009; McGinn et al., 2009; Siedler et al., 2014; Johnson et al., 2015; Hill et al., 2016). The
axonal cytoskeleton can also be degraded by TBI-induced oxidative stress and lipid
peroxidation (reviewed by Johnson et al., 2013).
The primary consequences of diffuse injuries are mediated by their effects on the
cytoskeleton, since DAI damages both MTs and NFs. NFs may accumulate due to injuryinduced primary mechanical failure of the NF network (Meythaler et al., 2001). Additionally,
TBI can cause reduction of interfilament spacing, termed NF compaction, as a result of
dephosphorylation or proteolysis of neurofilament sidearms (Povlishock et al., 1997; Pettus
et al., 1994; Kanungo et al., 2011). In addition to the role of calpains (Ma, 2013; Siedler et al.,
2014) it is possible that damage to cytoskeletal integrity may open diffusion barriers
separating the soma from the axon (Jones et al. 2014), in which case the delayed effects of
somatic kinases and phosphatases gaining access to the axon may cause further disruption.
These effects may also impede or alter the course of regenerative processes by which axons
negotiate the environment of post-traumatic glial scarring (Yuan & He, 2013; Cregg et al.,
2014).
TBI-induced stretching of axons can cause the breakage, disorganization and disassembly
of MTs, resulting in impaired axonal transport and the formation of axonal swellings (Tang-

Schomer et al., 2010; 2012). Axonal MTs are usually under compression, but in trauma they
are subjected to tensile forces. Computational modelling of cross-linked MT bundles
demonstrates that under uniaxial tension they stiffen, until tensile stresses become too high,
when they elongate and weaken due to failure of bundle crosslinks (Peter & Mofrad, 2012).
MT bundle rupture due to tensile forces has also been simulated, showing that both the
magnitude and rate of force application are critical in determining whether MTs fail.
Furthermore, this model demonstrated that under tension, microtubules can rupture due to
rapid force changes (Shamloo et al., 2015). In support of the computational modelling data,
an analysis of axons subjected to dynamic stretch injury has also demonstrated the rupture
of axonal MTs, and that faster rates of tensile stretching lead to more axonal pathology
(Tang-Schomer et al., 2010). However, loading and unloading curves for transient stresses
differ, and it is possible to cause permanent structural changes even below the damage
threshold (Goriely et al., 2015).
It was initially hypothesized that MT disassembly and NF compaction were different aspects
of the same progressive pathology, culminating in impaired axonal transport. However, more
recent evidence has indicated that in the majority of injured axons, NF compaction occurs in
the absence of impaired axonal transport and swelling, indicating that the two pathologies
are distinct (Stone et al., 2001; Marmarou et al., 2005), perhaps reflecting a dissociation
between MTs damaged mechanically and NFs damaged by proteolysis or
dephosphorylation.
Impaired transport can cause two types of axon swellings: isolated bulbs in continuation
with either the proximal or distal axonal segment, and varicosities which occur intermittently
along the length of the axon (Hanell et al., 2014). The differing forms are hypothesized to
indicate the force distribution on the axon at the time of injury. Axons that have undergone a
uniform strain along their length will form varicosities, where periodic breaks in MTs cause
partial interruption of axonal transport (Smith & Meaney, 2000; Tang-Schomer et al., 2010).
Conversely, axons that have experienced a highly localized strain causing progressive
cytoskeletal damage, will develop a bulb in conjunction with disrupted transport and
eventual disconnection, termed secondary axotomy (Smith & Meaney, 2000).
Although better human data is slowly emerging (Bayly et al., 2012; Chatelin et al., 2012;
Meaney et al., 2014; Goriely et al., 2015), in considering traumatic loads on the brain we still
lack sufficient data to link mechanical load of the tissue and cellular injury (Cloots et al.,
2010, p414). A further uncertainty is the correlation between structural damage and
functional impairment for example, what mild forms of TBI may be tolerated by the CNS
without lasting detriment (Meaney et al., 2014). These issues are of critical importance in
understanding neurotrauma.
5

Conclusion

Popular science illustrations show neurons floating in the void, touching only at synapses.
The reality is almost completely the opposite in the CNS and peripheral nerves, every
micron of the surface of the soma, dendrites and axons is physically coupled to other
neurons, glia, ependymal cells, extracellular matrix or basal lamina. This tight integration,
and its crosstalk with the internal cytoskeleton, is fundamental to the function and physical
properties of neurons. Nowhere is this more evident than in the most extremely specialised
of neural structures, the axon. The great majority of axons protein content is precisionselected and regulated on the basis of structural qualities in particular, NFs and their
extremely phosphorylated tails create a viscous, elastic and linearly structured axoplasm
which is resistant to pinching or flattening, and maintains axon structure under a range of
physiological loads, aided and modulated by myelin wrapping of the outer membrane.
Under traumatic loads, the sensitive coupling of cytoskeletal elements to one another and to
membrane and extracellular proteins becomes a vulnerability, with focal concentrations of
stress leading to mechanical failure, aberrant activation of intracellular signalling,
cytoskeletal disruption and ongoing dysregulation of internal structure. It is fundamental, but
not always appreciated, that these processes are mediated by the transmission of force from
loads applied to the tissue, by means of linkages between the internal cytoskeleton and the
extracellular matrix. Since these couplings are essential to normal function and regulation,

the points of integration between the extracellular physical environment and the internal
structure of the axon are the primary nexus of injury mechanisms in neurotrauma. This needs
to be a major consideration in designing or evaluating experimental models of trauma:
isolated or naked axons in culture are useful in some aspects, but cannot capture the
dynamics of an axon subjected to traumatic disruption in vivo as part of an integrated tissue.

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Figure 1 legend: Axon cytoskeletal elements

Symbol

Name

Function in mature axons

References

Plasma membrane

Phospholipid bilayer to contain

axoplasm, and anchor transmembrane
proteins

Lasiecka et al., 2008

Microfilaments integrating cytoskeletal

elements throughout the axoplasm
Actin

Substrate for motor proteins providing

active structural properties

Nixon & Yuan, 2011;

Schnapp & Reese, 1982.

Anchors membrane proteins

Structural rigidity and bracing
Microtubules

Transport corridors for proteins and

organelles, via kinesins and dyneins

Microtubule
associated proteins
(MAPs)

Mediate interactions between MTs

and other cytoskeletal proteins

Neurofilaments

Structures axoplasm into an elastic,

liquid-crystal gel

(NF heavy,
NF medium,
NF light,
internexin,
peripherin)

MT bundling

Active bracing of MTs (dyneins,

kinesins)
Active mechanical properties (myosin
Va coupling to actin)

Kapitein & Hoogenraad,

2015; Janke & Bulinski,
2011
Mandelkow & Mandelkow,
1995; Fukushima, 2011.

Parry, 2011; Uchida et al.,

2009; Hisanaga & Hirokawa,
1990; Jayanthi et al., 2013.

Plectins

Linkers between actin, organelles,

MTs and NFs (putative)

Fuchs & Cleveland, 1998;

Wiche et al., 2015

Spectrins

Link actin cortex to the membrane and

internal cytoskeleton; organise
channels and extracellular proteins on
the axolemma; organise paranodes

Frappier et al., 1991;

Bennett & Baines, 2001;
Buttermore et al., 2013

Integrins

Link the extracellular matrix to the

axon cortex and/or NFs

Salzer, 1995; Denda &

Reichardt, 2007

Cadherins

Transmembrane linkers for anchoring

the cytoskeletons of adjacent cells

Salzer, 1995; Takeichi, 2007

Ankyrins

Anchoring linkages for membrane

proteins bound to the actin cortex

Bennett & Baines, 2001;

Jones et al., 2014

Dyneins

Retrograde transport along MTs

Roberts et al., 2013

Kinesins

Anterograde transport along MTs

Hirokawa et al., 2009

Transported items

Vesicles, mitochondria, organelles

Myosin Va

Active tension of NFs against actin

Rao et al. 2002; 2011

Kv, NaV

Voltage gated ion channels for action

potential propagation

Gu & Barry, 2011

Based on figures from Schnapp & Reese, 1982; Hirokawa, 1984; Tyler, 2012; Parry, 2011; Leapman et
al., 1997; Fuchs & Cleveland, 1998; Wagner et al. 2004; Rao et al. 1998; 2003; Castan et al. 2013;
Long et al. 2005; Payandeh et al., 2011; Jones et al., 2014; Young & Kothary 2011.