Review and analysis of the biomedical and kinematic properites of tensile strength within the spine during elongation

1. Probing the Influence of Myelin and Glia
on the
Tensile Properties of the Spinal Cord
DeAndria Hardy
December 2, 2008

2. Traditional medical data, until this point, concerning
the glia matrix has primarily focused on classifying it as
merely a structural component. The article reviewed sought
to expand on traditional knowledge. Researchers David
Shreiber, Hailing Hao, and Ragi Elias proposed that the
glia matrix in connection with the myelin sheath
contributes to the overall mechanical properties present
within the spinal cord. Shreiber et al hypothesized that a
disrupted glia matrix and a demyelinated spinal cord
decreased the spinal cord’s stiffness and ultimate tensile
stress. To evaluate this conjecture the researchers
performed an experiment involving uniaxial tensile testing
of chick embryos on day 18 of development.
The experiment was borne out of a desire of the
researchers to expand on the accepted classification of
glia. The first step in this process was to compile
characteristics of glia from known data and establish the
proper niche for the results of the experiment’s
hypothesis. The glia matrix is composed of non-neuronal
cells. These cells along with being the binding force among
neurons provide many regulatory functions for the central
nervous system. The glia matrix provides nutrition, it
maintains homeostasis, and forms myelin. These cells are
broken down into divisions of the central nervous system

3. (CNS): astrocytes, oligodendrocytes, radial glia, and
ependymal cells. The researchers focused exclusively on
astrocytes and oligodendrocytes. These particular cells
contribute the most to structural “cellular scaffolds” of
the glia matrix (Schultze 1866).
Mechanical properties of
the spinal cord are greatly impacted by the scaffolds. They
increase the tissues of the CNS’s ability to withstand
loading and inherent stresses. In general load bearing
tissues do not include those of the CNS. These tissues are
commonly accepted as bones, tendons, and blood vessels.
That is because these tissues experience continual loading
during everyday activities and they accept and disperse the
load to reduce damage to the human body. Observing this as
the case for load bearing tissues, Shreiber et al. relied
on the previous research of Qing Yuan to draw the
following: glia could be considered as a load bearing
tissue because during everyday activities where the flexion
occurs in the spinal cord it endures a 6-10% strain and
certain glia cells protect against this flexion to reduce
pressures and abnormal forces from causing the spinal cord
injury. The particular glia cells responsible for the
dispersion of stresses are the oligodendrocytes and
astrocytes. These cells accomplish this by creating a
“cellular crosslink”. The crosslink stems from the

4. interconnections present between oligodendrocytes and axons
and astrocytes and blood vessels.
Once it was established that the mechanical properties
of the spinal cord were a result of the “cellular
crosslink” the researchers had to choose the proper method
to disrupt this network. The experiment’s hypothesis
required a method that would target the three components in
question: astrocytes, oligodendrocytes, and myelin. The
researchers were forced to rely on two methods to
accomplish the desired disruptions (Graca and Blakemore
1986). The first method involved a chemical interference
using ethidium bromide (EB), an agent that is cytotoxic to
oligodendrocytes and astrocytes. Ethidium bromide
selectively targets dividing cells and leaves cells like
neurons intact. The other method was immunological. This
method used galactocerebroside (GalC), an antibody that
specifically targeted myelin producing oligodendrocytes but
did not cause astrocyte damage.
The first step of the experiment was to introduce the
agents for myelin suppression. There were four reagents
involved, two for suppression and two as controls. To
inject the reagent into the chick eggs, small windows were
created in the shells. After the injections the windows
were sealed with cellophane tape until stage E18 of

5. development. Chick eggs at stage E14 were injected with
either 0.01% EB in 0.1% saline or its control 0.1% saline.
An Immunoglobulin G (IgG) rabbit-αGalC antibody at a 1:25
dilution with a 20% serum complement in 0.1M Phosphate
Buffered Saline (PBS) was injected into chick eggs at stage
E12. Its control was a pure rabbit IgG at a 1:25 dilution
with a serum complement solution. Each chick egg received
two 3µl injections, one in the cervical spinal cord and one
into the thoracic spinal cord. At stage E18 the spinal
cords from each of the chick eggs were extracted for
testing.
Upon excising the spinal cords at stage E18, they were
quantified for myelination. The researchers used myelin
basic protein (MBP) immunohistochemistry and osmium
tetroxide treatment for the quantifications. Three spinal
cords from each of the four experimental conditions were
examined. From each spinal cord five sections were taken
and five areas within the white matter were selected at
random for testing. The immunohistochemistry
quantifications were performed by first harvesting the
stage E18 spinal cords and immersion fixed in 4%
paraformaldehyde. The spinal cords were then incubated in a
20% sucrose-saline solution at 4oC overnight. The next day
longitudinal sections were cut from the frozen spinal cords

6. with a cryostat. The sections were labeled with a 1:400
dilution of rabbit anti-MBP and a 1:100 dilution of mouse
α-NeuroFilament-200 (Sigma). Then the sections were
incubated in a goat anti-rabbit Alexa 488 dye and goat
anti-mouse Alexa 546 dye. The Alexa 488 was used to
visualize the MBP and the Alexa 546 was used to visualize
the neurofilaments. The osmium tetroxide quantifications
were performed by placing 5µm frozen transverse sections on
microslides that were pre-treated with a 2% osmium
tetroxide solution for 30 minutes and then dehydrated by an
alcohol wash. The slides were coverslipped and the myelin
sheaths were counted at high magnification under a
brighfield microscope. The number of myelin sheaths were
averaged for each slide, spinal cord, and experiment
condition. The data was then normalized to the control
condition.
To test the role of glia in the mechanical properties
of the spinal cord, each of the chick spinal cords were
stretched uniaxially at a low strain rate until failure.
The excised stage E18 spinal cords were exposed ventrally
and an 11 segment section that extended from the first
nerve root was measured. The measurement was performed
three times for accuracy. The dorsal half of the vertebrae
was then removed and the spinal cord re-measured. This too

7. was performed three times. The resulting section was
visually checked for any damages that could have occurred
during excising. The spinal cords were then marked off by
reflective plastic dots into a 12mm section. Three
additional dots were added as a means of monitoring
uniformity during testing. Afterward the spinal cords were
placed in a Bose/Enduratec ELF 3200 with a 0.5N cantilever
load cell for uniaxial testing. The ends of the spinal
cords were placed on polyethylene plates that were 10mm
apart of the load cell crossheads, with the plastic dots
marking the 12mm section exactly at the edge as seen in
Figure 1.
Each spinal cord was stretched once at 0.012mm/s
with a .001 s-1 strain rate. Images were taken every .5mm to
assess the uniformity of the strain. The load and
displacement of each spinal cord were recorded at 1.67 Hz
and then converted to a nominal stress. The stress-stretch
curves were plotted and the ultimate tensile stress, σUTS,
and the stretch at the ultimate tensile stress, λUTS, were
identified.
The results showed that after injection the spinal
cords treated with EB and αGalC were significantly shorter
than the controls (Table 1). In general the spinal cords
injected with the IgG or saline controls exhibited similar

8. MBP immunoreactivity to embryos without any treatment.
While the other spinal cords injected with EB and αGalC
exhibited a decrease in immunoreactivity or a decrease in
the number of detectable myelinated axons (Figure 2).
Immunohistochemistry was use to asses the demyelinated
axons. Alpha-Glial Fibrillary Acidic Protein (α-GFAP) was
used to stain and test for astrocytes. GalC was used to
stain and test for oligodendrocytes. The green
immunofluorescence in Figure 2e, 2b, and 2d shows the
experiment control. In Figure 2a and 2c there is a red
immunofluorescence. This exhibits the myelin decrease.
The results of the uniaxial testing illustrated nonlinear, strain stiffening behavior (Graph 1). This behavior
was made apparent when each condition was fit to the Ogden
strain energy potential function: W = 2G/α2 (λα1+ λα2 + λα3−
3). The intermittent peaking on the graphs depicts
microfractures and recovery within the spinal cords. Even
after the ultimate tensile stretch is achieved these
intermittent peaks can be seen, showing that the spinal
cord was still attempting to accept and redistribute the
loading on it. The graph also depicts significantly lower
ultimate stress for the spinal cords treated with EB and

9. αGalC. The two treatment conditions of EB and αGalC express
a significantly lower shear modulus (Table 2).
All of the results from the uniaxial tensile testing
and subsequent calculations verified the researchers’s
hypothesis that glia is more than just a binding element of
the CNS. The assumption that if the glia matrix indeed
contributed to the mechanical properties of the spinal cord
a disruption would decrease the overall ultimate tensile
stress of the spinal cord was quantified. In experimental
conditions where the primary components of the glia matrix,
astrocytes and oligodendrocytes, were interrupted a
substantial decrease presented itself.
When critiquing this experiment that researcher’s
success is an obvious positive note. More impressive than
the experiment’s success is the niche in which the
researcher’s chose. Amidst all of the current research that
exist concerning demyelination and the spinal, as it
relates to diseases like Multiple Sclerosis, the
researchers explored it mechanical properties. It is
commendable to go against the norm of signal transduction
and myelin. The researchers also chose a different form of
deformation than any previous experiment. Other experiments
done on the spinal cord have been in reference to force
present on the spinal cord with compressive or shear

10. forces. Shreiber et al. chose to test in tension versus the
other accepted methods. This gave the experiment and its
result validity separate from conclusion drawn from other
experiments.
The experiment’s methodology was also impressive. The
experiment did not rely on just one method to demyelinate
the spinal cord. By using both a chemical and immunological
method of disruption, it eliminated skewed results. For
instance, if the researchers only used one method of
disruption the results could be called into question
because the results were all inclusive. Also the method was
very precise and easily reproduced. In review of other
experiments, the methods are so tedious and materials are
so difficult to acquire it become almost impossible to
recreate the experiment. Finally, the most notable aspect
of the experiment is that is has the ability for other
avenues of research. This research provides a “leaping
point” for other research to explore remyelination. Current
research has focused on remyelination solely to assist in
signal transduction. This research opens the opportunity to
explore how remyelination affects the stability and other
mechanical properties of the spinal cord. It could lead to
other discoveries on how to improve the quality of life for

11. those who suffer from demyelinating diseases like Multiple
Sclerosis.
Figure 1- Schematic of uniaxial testing setup
Figure 2- Immunoreactivity of
experiment conditions

12. Table 1- Length and area measurements of stage E18 spinal
cords
EB
(n=6)
Length
(mm)
Area
(mm2)
Saline
(n=5)
αGalC
(n=5)
IgG
(n=5)
Control
(n=6)
22.35±0.2
22.83±0.25
21.99±0.35
22.89±0.36
23.03±0.49
1.47±0.05
1.54±0.04
1.28±0.04
1.58±0.11
1.51±0.02
Graph 1- Stress-stretch curves for various experiment
conditions
Table 2- Results of uniaxial testing
σUTS
λUTS
G(kPa)
α
EB
28.4 ± 9.38
1.38 ± 0.09
17.4 ± 5.70
8.32 ± 2.55
Saline
77.8 ± 19.6
1.43 ± 0.09
29.2 ± 7.38
8.49 ± 1.34
αGalC
55.9 ± 28.9
1.45 ± 0.10
17.7 ± 6.80
8.74 ± 0.84
IgG
93.5± 37.3
1.45 ± 0.10
30.0 ± 7.26
9.00 ± 1.62
Graph 2- Stress-stretch comparison curves
Control
85.2 ± 17.7
1.42 ± 0.03
32.8 ± 9.53
8.22 ± 1.27

14. References
Bain AC, Shreiber DI, Meaney DF (2003) Modeling of
microstructural kinematics during simple elongation of
central nervous system tissue. J Biomechanical Engineering
125(6): 798–804.
Elias Ragi, Hao Hailing, David Shreiber (2008) Probing the
influence of myelin and glia on the tensile properties of
the spinal cord. Biomedical Model Mechanobiology
Graca DL, Blakemore WF (1986) Delayed remyelination in rat
spinal cord following ethidium bromide injection.
Neuropathology Applied Neurobiology 12(6):593–605.
Schultze M (1866).Zur Anatomie und Physiologe der Retina.
Max Cohen & Sons, Bonn