Uniaxial stretch-induced axonal injury thresholds for

Bulletin of the JSME

Journal of Biomechanical Science and EngineeringVol.12, No.1, 2017

© 2017 The Japan Society of Mechanical EngineersJ-STAGE Advance Publication date: 15 February, 2017Paper No.16-00598

[DOI: 10.1299/jbse.16-00598]

1. Introduction

Traumatic brain injury (TBI), major cause of morbidity and mortality (Fault et al., 2010) , is associated with the

progressive neurodegeneration followed by extended or permanent loss of sensory, motor and/or cognitive function (Smith

et al., 1997; Adelson et al., 2000; Bramlett and Dietrich, 2002; Povlishock and Katz, 2005) and has a wide spectrum of

mechanisms of injury and pathologies. Main differences between the types of brain trauma were characterized under two

major categories, focal and diffuse brain injury, where focal brain injury is usually related with impact forces that may

produce cerebral contusions and hematomas, on the other hand, diffuse brain injury is dependent on the inertial forces

from rapid head accelerations/decelerations as well as the propagation of force through the brain after impact, usually

caused by traffic accidents, assaults and falls, that deform the white matter and eventually lead to diffuse axonal injury

(DAI) (Adams et al., 1989; Browne et al., 2011; Gupta and Przekwas, 2013).

The two distinct types of axonal pathology arising from DAI are the swellings that induced by the neurofilament

accumulation as a result of the damaged neurofilament structure in the axonal cytoskeleton and focal compaction and/or

impaired transport due to the mechanical insult (Povlishock, 1992) along the axons followed by secondary axotomy, and

the axonal bulbs which likely represent complete axonal disconnection (Chen et al., 1999; Smith et al., 2003a; Johnson et

al., 2013). These cytoskeletal abnormalities which proceed to the formation of the swellings and secondary axotomy are

the morphological indication of DAI whereas primary axotomy is a relatively rare event (Stone et al., 2004; Chung et al.,

2005; Kelley et al., 2006).

Therefore, DAI can be detected histologically by the visualization of the above mentioned morphological indication using

immunohistochemical labeling of multiple proteins which accumulates in injured axons (Gultekin and Smith, 1994; Ng et

al., 1994). Amyloid precursor protein (APP) is one of the strongest candidates amongst those proteins since it accumulates

in axonal swellings, and bulbs 2-3 h after brain trauma (Blumbergs et al., 1995; McKenzie et al., 1996) due to its

transportation by fast axonal transport and used as the indicative of impaired transport (Smith et al., 2003a; Suehiro and

Povlishock, 2001). Increased levels of tau protein accumulation, particularly in nonmyelinated axons of cortical

interneurons, serves as yet another example of a consistent TBI marker.

While most common injury cascades of TBI are recognized and summarized as cytoskeletal damage, calcium influx,

neurotransmitter release, and mitochondrial dysfunction, a standard treatment protocol has not yet been established

Uniaxial stretch-induced axonal injury thresholds for axonal dysfunction and disruption and strain rate effects on thresholds

for mouse neuronal stem cells

Abstract In this paper, the proposed study aims to achieve a better understanding of neuronal tolerance and contribute to the prediction of the secondary degeneration of diffuse axonal injury (DAI). Therefore, a uniaxial stretching device which subjected cultured neurons to uniaxial stretch was employed to evaluate the effect of strain and strain rate along axon to realize the injury threshold. Neurons differentiated from mouse neuronal stem cells were injured and the morphology was observed before and after stretching with strains of 0.10, 0.12, 0.18, 0.23 at strain rates of 8, 11, 19, 26 s-1 respectively. Results suggest that the threshold for axonal dysfunction is around 0.18 strain whereas the threshold for axonal disruption is around 0.23 and the results of strain rate effect investigations on axonal dysfunction and disruption around these threshold values indicated that higher strain rate values such as 50 s-1 may have diminishing effects on threshold for axonal disruption.

Key words : Traumatic brain injury, Diffuse axonal injury, Mouse neuronal stem cells, Uniaxial stretch

Evrim KURTOGLU *, Hiromichi NAKADATE *, Kazuhiro KIKUTA *, Shigeru AOMURA *

and Akira KAKUTA ** * Graduate School of System Design, Tokyo Metropolitan University, Tokyo, Japan

E-mail: [email protected] ** Advanced Course of Mechanical and Computer Systems Engineering, Tokyo National College of Technology

1220-2, Kunugida-machi, Hachioji-shi, Tokyo 193-0997, Japan

1

Received: 1 November 2016; Revised: 29 December 2016; Accepted: 7 February 2017

2© 2017 The Japan Society of Mechanical Engineers

Kurtoglu, Nakadate, Kikuta, Aomura and Kakuta,Journal of Biomechanical Science and Engineering, Vol.12, No.1 (2017)

[DOI: 10.1299/jbse.16-00598]

(Coronado et al., 2011). Moreover, although being the most common type of pathology in TBI with an approximately 40-

50% of occurrence rate out of the reported cases (Iwata et al., 2004; Meythaler et al., 2001), the diagnosis of DAI without

through histopathological examination, especially early or exact recognition of the extent of axonal injury, still remains as

a major challenge since these injuries are not promptly detectable with standard techniques such as computed tomography

(CT) or magnetic resonance imaging (MRI) scans as a consequence of the microscopic and disperse nature of the axonal

pathology of DAI (Adams et al., 1985; Diaz-Marchan et al., 1996). Thus, investigation of injury neuromechanics is crucial

in understanding neural tolerance and developing relevant therapies and/or diagnostic procedures for DAI. Applying the

defined levels of physiological injury must be realized through reliable and accurate models in order to assess the extent of

injury.

To the present, numerous in vitro models have been developed varying from using dissociated cells (Galbraith et al., 1993;

Cullen et al., 2007) to organotypic tissue slices (Cater et al., 2006) and from applying uniaxial (Pfister et al., 2003;

Nakadate et al., 2014) to biaxial strains (Nakadate et al., 2012; Sahay et al., 2002), to understand the mechanical stimuli of

the impact and the following responses of tissue and cells (Cargill and Thibault, 1996; LaPlaca and Thibault, 1997), and to

investigate the different aspects of DAI such as post-injury rise in calcium level (LaPlaca and Thibault, 1998; Rzigalinski

et al., 1997; Rzigalinski et al., 1998), electrophysiological responses of neurons (Galbraith et al., 1993; Tavalin et al.,

1995), neurofilament structure and formations of axonal swellings (Smith et al., 1999).

Although said in vitro models have provided some insight on understanding the cellular mechanism of neuronal injury, and

suggested that the degree of electrophysiological impairment and morphological damage of neurons is directly

related to the magnitude and rate of axonal stretch, the exact mechanisms that initiate secondary degeneration in DAI are

yet to be fully characterized. The neuromechanics of underlying pathways determining axonal injury is very complex

hence it is difficult to discern the mechanical principles that govern physical and functional tolerance. It is considered that

prediction of secondary degeneration resulting in DAI might be possible by quantifying axonal injury. In this study, axonal

injury induced by uniaxial stretch on differentiated mouse neuronal stem cells in order to clarify the relation between the

impulsive strain, strain rate and axonal injury. Herein, evaluation is performed by immunohistochemical labeling, with β-

APP and tau protein accumulation as biochemical markers of choice.

2. Materials and Methods

2.1. Uniaxial Stretching Device

The uniaxial stretching device consists of a servo actuator (RCS3-SA8C, IAI, Shizuoka, Japan), a servo actuator

controller (SCON-C; IAI), a linear sensor for measuring tensile displacement (LP-20F, Midori Precisions, Tokyo, Japan), a

load cell for measuring tensile loading (TCLS, Toyo Sokki, Kanagawa, Japan), a load cell converter (LC14111,

Unipulse, Tokyo, Japan), a programmable logic controller, an A/D converter unit (KV-3000 CPU, Keyence, Osaka, Japan),

an AC power unit (KV-U7, Keyence) and a polydimethylsiloxane (PDMS) chamber which can be seen in Fig. 1.

Fig. 1 The uniaxial stretching device and its components. The device consists of a PDMS chamber on a microscope stage,

a linear sensor, a load cell, a servo actuator and a wire. The PDMS chamber is clamped to the microscope stage at one

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[DOI: 10.1299/jbse.16-00598]

edge, and the other edge of the chamber is connected to the stainless plate those displacement is stopped by hitting the

stopper. The stainless plate is connected to the slider of the actuator through a wire and other tip of the wire is connected to

an iron piece. The iron piece is attracted strongly with the magnet set on the slider.

A detailed description of the device configuration and loading mechanism has been recently published (Aomura et al.,

2016). The Green-Lagrange strains of the culture substrate in the PDMS chamber were calculated by microscope images

before and after the stretching. The strains on the central point of the culturing substrate in the stretching direction and

perpendicular direction were obtained for every 0.5 mm displacement to 4 mm in total and plotted in Fig. 2A. In the

experiment, the compressive strain perpendicular to the stretching direction was smaller than 0.05 for the tensile strain

0.23 in the stretching direction at the maximum displacement with 4 mm.

Fig. 2 Displacement measurement of PDMS chamber: Strain and displacement of the culturing substrate fixed on the PDM

S chamber for the uniaxial stretching were observed experimentally. Relation between the forced displacement and distorti

ons around the center where cells are cultured was obtained. The strain in the direction perpendicular to the stretching dire

ction remains within 10% compared with the longitudinal strain (A). The displacement corresponds to the impact (strain 0.

10, 0.12, 0.18, 0.23 strain rate 8, 11, 19, 26 s-1

) is expressed as the function of time (B).

Strain rate was obtained by dividing the maximum strain by the time to maximum strain. The curves stood up in the first

10 msec by stretching, and converged in the last 10 msec by releasing with the same ratio as the standing up. It is expected

that the neuron on the substrate is stretched/compressed with a similar deformation history through the substrate.

2.2. Fabrication of the PDMS Chamber

The basic structure of the PDMS chamber with a well and a thin substrate is shown in Fig 3.

Fig. 3 PDMS chamber.

The base, a PDMS-prepolymer and the curing agent (SYLGARD 184 Silicone Elastomer Kit, Dow Corning, Midland, MI,

USA) were mixed in 10:1 mass ratio and the mixture was deaerated in a round desiccator connected to a dry vacuum

pump. The deaerated mixture was poured into a polystyrene square case and stainless mold to be 0.3 and 10 mm in

thickness, respectively, and was cured on a hot plate at 65°C for 1h. After curing, the two parts were bonded by the

deaerated mixture and cured at 65°C for 1h.

2.3. Cell Culture

Homogeneous mouse neuronal stem cells (NSCs) were produced via unidirectional neuronal differentiation from mouse

embryonic stem cells (ECSs) by using the neural stem sphere (NSS) method (Nakayama et al., 2003; Nakayama and Inoue,

0

1

2

3

4

5

0 5 10 15 20 25

Dis

pla

cem

ent

[mm

]

Time [ms]

10%8s-1

12%11s-1

18%19s-1

23%26s-1

B

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[DOI: 10.1299/jbse.16-00598]

2006). NSCs were plated onto Matrigel (BD MatrigelTM

Basement Membrane Matrix Growth Factor Reduced; Invitrogen,

Carlsbad, CA) coated dishes and allowed to proliferate exponentially in proliferation medium (PM) consisting of

Neurobasal medium (Invitrogen) supplemented with 1% Glutamax-I (Gibco), 1% penicillin-streptomycin supplement

(Sigma-Aldrich), 2% B-27 (Invitrogen) and 20 ng/ml fibroblast growth factor-2 (FGF-2) (R&D Systems, Minneapolis,

MN). The medium was replaced every 2 days. Then the cells were seeded into Poly-D-lysine and Matrigel coated culture

substrate (10 mm × 30 mm) of PDMS chamber at 5 × 104 cells/cm

2 and to induce differentiation of NSCs into neurons and

glia, PM was changed to differentiation medium (DM) consisting of Neurobasal medium supplemented with 2% B-27 and

10% astrocyte conditioned medium (ACM). Differentiation to NSCs from ECSs was confirmed by observing the

exponential increase in cell number, proliferation and differentiation into neurons and glia throughout the culturing

procedures (Shibata et al., 2016; Omori et al., 2014). The cells were cultured for 6 days and the medium was changed on

the third day of differentiation where differentiated cells can be seen in Fig 4.

.

Fig. 4 Fluorescent image of cultured neurons β-APP (Red) and Tau (Green) Merged.

The neurons matched in age to those subjected to strains were cultured in the PDMS chamber as a sham control, after

which they were set in and removed from the uniaxial stretching device without receiving any mechanical load. The

medium in the PDMS chamber was not removed during the experiment which was completed within 5 min. The

temperature of the device and surroundings were kept at 37°C. The PDMS chamber was returned to the CO2 incubator

after the experiment.

2.4. Immunostaining Analysis

A primary effect of dynamic deformation of axons following stretching is the disruption of axonal transport,

resulting in accumulation of transported materials in axonal swellings within just hours (Smith et al., 1999). Swellings

appear in a periodic interval along the connected axons, like beads on a string, to form a pathological phenotype referred to

as “axonal varicosities” (Rand and Courville, 1946). Axonal pathology found shortly after traumatic brain injury (TBI) is a

single swelling at a disconnection point on an axon, described as a terminal “axonal bulb” (previously referred to as a

“retraction ball”) (Smith and Meaney, 2000; Smith et al., 2003b; Strich , 1956). In this study, β-APP and Tau were stained

at 3 h post loading and observed by using an inverted fluorescence microscopy (FSX100, Olympus, Japan) equipped

with a fluorescence mirror unit (U-MWIG3, Olympus). Cultures were rinsed with Dulbecco’s phosphate-buffered

saline (DPBS) with Ca2+

and Mg2+

and fixed with 4% paraformaldehyde for 20 minutes at room temperature.

After permeabilization with 0.3% Triton X-100 for 5 minutes at room temperature, cultures were blocked with 5% goat

serum for 60 minutes at room temperature and incubated in rabbit polyclonal anti-β-APP (51-2700, Invitrogen, Life

Technologies) and Tau Monoclonal Antibody (TAU-5) (MA5-12808, Invitrogen, Life Technologies) at a dilution of 1:50

as the primary antibody overnight at 4°C. Subsequently, 10 μg/mL Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L)

secondary antibody (A-11037, Molecular Probes, Life Technologies) and 10 μg/mL Alexa Fluor 488-conjugated goat

anti-mouse IgG (H+L) secondary antibody (A-11029, Molecular Probes, Life Technologies) were applied for 60

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[DOI: 10.1299/jbse.16-00598]

minutes at room temperature. β-APP and TAU accumulated axonal swellings and axonal bulbs formed after stretching

were observed and counted by fluorescent images as shown in Fig 5.

Fig. 5 β-APP & Tau immunostaining: Formation of bulb (left) and swellings (right). β-APP is shown in red (I), Tau in

green (II) and merged in yellow (III).

Dysfunction of the axonal transport was defined as the rate of neurons that have β-APP and Tau accumulated axonal

swellings. Disruption of the axonal transport was defined as the rate of neurons that have β-APPand Tau accumulated

axonal bulb. In addition, we evaluated the number of axonal swellings per 100 μm and the length of the axon in which

axonal bulb was observed, respectively. The axon length was measured manually by Image J (National Institutes of Health,

Bethesda, MD, USA). Results were expressed as the mean ± standard deviation (SD) of 4 independent experiments. 200–

300 neurons per a PDMS substrate were analyzed totally. Means were compared by Steel’s multiple comparison test. A p

value of less than 0.05 was considered significant.

3. Results Cultured NSCs in PDMS chamber (Fig. 4) were subjected to impulsive strain of 0.10, 0.12, 0.18, 0.23 and strain rate 8, 11,

19, 26 s-1

respectively and the displacement profile of PDMS chamber under these impacts is shown in Fig. 2. 3 hours after

stretching, β-APP and Tau were stained to analyze the 2 most common pathologies of DAI; axonal swellings and axonal

bulb. Dysfunction of the axonal transport was defined as the rate of neurons that have β-APP and Tau accumulated

axonal swellings. Disruption of the axonal transport was defined as the rate of neurons that have β-APP and Tau

accumulated axonal bulb. β-APP and Tau accumulated axonal swellings and axonal bulbs formed after stretching were

observed and counted by fluorescent images as shown in Fig 5.

In order to clarify the relation between the impulsive strain, the strain rate and the axonal injury, first we focused on the

effects of the applied strain on axonal dysfunction and disruption separately.

As shown in Fig. 7, results suggested that the threshold for axonal dysfunction is around 0.18 strain whereas the threshold

for axonal disruption is around 0.23.

I II III

Stretch

ing

directio

n

directio

n

I II III

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[DOI: 10.1299/jbse.16-00598]

Fig. 6 Rate of neurons that have β-APP and Tau-accumulated axonal swellings (upper) and axonal bulbs (lower). The *

symbol represents a statistically significant difference (p < 0.05) versus sham control at each condition using Steel’s

multiple comparison test. Results are expressed as the mean ± standard deviation (SD) of 4 independent experiments.

Subsequently, the effects of strain rate on axonal dysfunction and disruption were investigated around the threshold values

which the above mentioned results indicated.

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[DOI: 10.1299/jbse.16-00598]

Fig. 7 Rate of neurons that have β-APP and Tau-accumulated axonal swellings (upper) and axonal bulbs (lower). The *

symbol represents a statistically significant difference (p < 0.05) versus sham control at each condition using Steel’s

multiple comparison test. Results are expressed as the mean ± standard deviation (SD) of 4 independent experiments.

Strain rate variations showed no significant influence on axonal dysfunction, however significantly affected axonal

disruption as can be seen in Fig. 8. Results suggested that higher strain rate values such as 50 s-1

may have diminishing

effects on threshold for axonal disruption.

4. Discussion

In this study, a uniaxial stretching device which subjected cultured neurons to uniaxial stretch was employed to evaluate

the effect of strain and strain rate along axon to realize the injury threshold in order to achieve a better understanding of

neuronal tolerance and contribute to the prediction of the secondary degeneration of DAI. Neurons differentiated from

mouse neuronal stem cells were injured and the morphology was observed before and after stretching with strains of 0.10,

0.12, 0.18, 0.23 at strain rates of 8, 11, 19, 26 s-1

respectively. First, the effects of the applied strain on axonal dysfunction

and disruption separately was studied where results suggested that the threshold for axonal dysfunction is around 0.18

strain whereas the threshold for axonal disruption is around 0.23. Similar injury tresholds were also suggested in several

previous studies (Bain and Meaney, 2000; Margulies and Thibault, 1992; Morrison III et al., 2003; Shreiber et al., 1997),

but to our knowledge, this is the first study that approaches axonal injury treshold for dysfunction and disruption

seperately.

Furthermore, the effects of strain rate on axonal dysfunction and disruption were investigated around the threshold values

which the previously mentioned results indicated. Strain rate variations showed no significant influence on axonal

dysfunction, however significantly affected axonal disruption. Results suggested that higher strain rate values such as 50 s-

1 may have diminishing effects on threshold for axonal disruption. Although there has been a few previous studies on the

effects of the strain rate on axonal injury (LaPlaca et al., 1997; Cargill and Thibault, 1996; Carter et al. 2006; Lusardi et al.,

2004; Geddes et al., 2003; Geddes and Cargill, 2001), this subject still merits more research effort since the researchers

still have not reached an agreement.

It is also vital to study these conditions on oriented axons since previous computational and experimental studies (Sahoo et

al., 2016; Wright and Ramesh, 2012; Nakadate et al., 2014) suggested that axonal injury thresholds strongly dependent on

the direction of axons since oriented axons on stretching direction are more susceptible to injury. However, the

strain/strain rate combinations analyzed in above mentioned studies are limited to determine axonal injury thresholds

accurately.

Indeed, studying the axonal injury thresholds for dysfunction and disruption with a wider range of strain/strain rate

combinations and also on oriented axons to have a better understanding on neuronal tolerance will be the future work for

this proposed study.

5. Conclusion

Neuronal injury is a widely observed, but difficult to study phenomenon since the neuromechanics of underlying pathways

determining axonal injury is very complex hence it is difficult to discern the mechanical principles that govern physical

and functional tolerance. The exact mechanisms that initiate secondary degeneration in DAI are yet to be fully

characterized. In this study, axonal injury induced by uniaxial stretch on differentiated mouse neuronal stem cells in order

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[DOI: 10.1299/jbse.16-00598]

to clarify the relation between the impulsive strain, strain rate and axonal injury. Herein, evaluation is performed by

immunohistochemical labeling, with β-APP and tau protein accumulation as biochemical markers of choice. Results

suggest that the threshold for axonal dysfunction is around 0.18 strain whereas the threshold for axonal disruption is

around 0.23 and the results of strain rate effect investigations on axonal dysfunction and disruption around these threshold

values indicated that higher strain rate values such as 50 s-1

may have diminishing effects on threshold for axonal

disruption.

Acknowledgment The authors gratefully thank Dr. Hiroyuki Omori at the Laboratory of Regenerative Neurosciences, Graduate School of

Human Health Sciences,Tokyo Metropolitan University for the gift of NSCs and his technical advice regarding cell

culture.This work was supported by a Grant-in-Aid for Scientific Research (B) (25289064) and a Grant-in- Aid for

Scientific Research (C) (26350509) from the Japan Society for the Promotion of Science.

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