Probing the Influence of Myelin and Glia on the

Tensile Properties of the Spinal Cord

DeAndria HardyDecember 2, 2008

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

(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

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

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

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

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

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 non-

linear, 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+ λα

2 + 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

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

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

those who suffer from demyelinating diseases like Multiple

Sclerosis.

Figure 1- Schematic of uniaxial testing setup

Figure 2- Immunoreactivity of experiment conditions

Table 1- Length and area measurements of stage E18 spinal cords

EB

(n=6)Saline (n=5)

αGalC (n=5)

IgG (n=5)

Control (n=6)

Length (mm) 22.35±0.2 22.83±0.25 21.99±0.35 22.89±0.36 23.03±0.49Area (mm2) 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

EB Saline αGalC IgG ControlσUTS 28.4 ± 9.38 77.8 ± 19.6 55.9 ± 28.9 93.5± 37.3 85.2 ± 17.7λUTS 1.38 ± 0.09 1.43 ± 0.09 1.45 ± 0.10 1.45 ± 0.10 1.42 ± 0.03G(kPa) 17.4 ± 5.70 29.2 ± 7.38 17.7 ± 6.80 30.0 ± 7.26 32.8 ± 9.53α 8.32 ± 2.55 8.49 ± 1.34 8.74 ± 0.84 9.00 ± 1.62 8.22 ± 1.27

Graph 2- Stress-stretch comparison curves

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