Download 2013 Ultrasturcture of the Tympanic Membrane

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Investigating the Ultrastructure of the Tympanic Membrane in Rats
April 10th 2013
Gord Locke
Supervisors: Dr. Hanif Ladak & Dr. Jian Liu
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Introduction
The ear is a highly complex organ used to detect sounds and detect the
internal balance of the body. The ear can be subdivide into three different parts: the
inner ear, the middle ear and the outer ear. The outer ear is responsible for
gathering the sound waves and focusing them on the middle ear. The middle ear is
responsible for transmitting and magnifying the sound waves and the inner ear for
converting them into neurological signals. A disruption in any of these areas can
lead to hearing loss but the most frequent area of disruption is in the middle ear. 3
out of 4 children will experience a middle ear infection by the time they are 3 years
old [1]. These infections can become chronic and damage the middle ear.
The tympanic membrane, which is commonly referred to as the eardrum, is a
key component in the middle ear. Things such as trauma, disease and loud noises
can affect the integrity of the tympanic membrane, which can lead to temporary or
permanent hearing loss. If the membrane becomes ruptured, the membrane will
heal the wound but the underlying structure of collagen fibers may remain damaged
[2]. Using scanning electron microscopy, the structure of the collagen fibers in the
tympanic membrane can be observed which will allow for better artificial eardrums
to be produced.
The purpose of this research was to examine the arrangement of fibers in rat
tympanic membrane, measure the diameter and subunit frequency of individual
fibers and look at the how the fibers differ in various areas of the tympanic
membrane.
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Theory
The Middle Ear
In both rat and human ears, the middle ear is comprised of the tympanic
membrane, three small bones called the ossicles and several muscles. The middle
ear primary function is to act as an impedance matcher for sound waves [3]. If the
middle ear did not act as an impedance matcher, then most of the energy carried in
sound wave would be reflected and lost. The middle ear by acting as an impedance
matcher prevents this energy from being reflected and increases the sensitivity of
the ear greatly.
The middle ear also takes advantage mechanical properties when
transmitting sound. The natural position of the ossicles is arranged in a lever
system. As shown in figure 1, as the tympanic membrane is shifted from the sound
waves, the stapes is moved with far greater magnitude, which allows for the
vibrations to be magnified. The surface area of the tympanic membrane is far larger
than that of the oval window as shown in figure 2, which the sound waves are
transmitted through. The energy of the sound wave is distributed across the large
area of the tympanic membrane. Through the principle of conservation of energy,
the same amount of energy is applied on the oval window, which has a far smaller
surface area. This causes the sound waves to be magnified and improves the
performance of the ear.
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Figure 2
Figure 1
Figure 1: This figure is of the ossicles showing how the size and orientation of the
bones make the ear an effective lever. The lever arm attached to the membrane, Lm
is longer than the lever arm attached to the inner ear, Li. As a result of Lm having a
larger arm than Li, Li will have a greater displacement relative to the displacement
of Lm.
Figure 2: figure is of the ossicles showing how the surface areas of the tympanic
membrane, Ad and the oval window, Af relate to each other. Pressure is defined as
Force per area and as Pressure is conserved the force on the oval window is higher
relative to the force on the tympanic membrane. This is a way that the middle ear
amplifies the sound waves.
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The Tympanic Membrane
The tympanic membrane is not a uniform and flat surface. There are several
distinct regions of the membrane that contribute to the properties of the eardrum.
The pars tensa is the main structural component of the membrane. The manubrium
is the attachment site of the malleus; one of the ossicles. The pars flaccida is a
smaller and more compliant part of the membrane. These areas are shown in figure
3.
Figure 3: This figure is a diagram of a
human tympanic membrane
The tympanic membrane is composed of four different layers as shown in
figure 4; an epidermal layer, a mucosal layer and two layers that contain collagen
fibers. One of these layers contains collagen distributed in a radial manner,
extending from the center of the eardrum to the outer diameter. The other layer
contains fibers that are organized in a circumferential manner [4]. The organization
of these fibers is shown in figure 5.
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Figure 4: The layers of the tympanic membrane
Figure 5: The organization
of collagen fibers in the
tympanic membrane
These layers of collagen fibers are the primary structures, which determine
the shape, and the structure. It is thought that the radial collagen fibers are
primarily responsible for the conduction of sound across the membrane and the
circumferential fibers responsible for providing structural strength to the
membrane.
Scanning Electron Microscopy
Scanning electron microscopy or SEM is an imaging technique that allows for
both biological and non-biological objects to be imaged with a very high
magnification. With this magnification SEM is able to resolve points that are less
than 1 nanometer apart. In order for biological materials to be imaged, they need to
be coated with a heavy metal. This is necessary because the surface of the object to
be imaged needs to be electrically conductive in order to interact with the electrons
being focused on them. Biological samples must also contain no liquids as SEM
requires a vacuum to function, if liquid remained in the specimen it would
evaporate and disturbed the imaging.
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Methods
The tympanic membranes were obtained through surgically removal from
healthy deceased rats and then incubated in a 1 % tripsin in phosphate buffer
solution for 24 hours to remove the epidermal layer and the mucosal layer from the
membrane. The membranes were then rinsed with a phosphate buffer solution and
then fixed in 2.5% glutaraldehyde solution. The membranes were then fixed in
ascending concentration series of ethanol and critical point dried with liquid carbon
dioxide. The membranes were then coated with 3nm of osmium and imaged with a
scanning electron microscope.
The images were then exported into ImageJ, an open source imageprocessing tool. Using imageJ, the contrast of the fibers was adjusted using
histogram equalization. The diameter and subunit frequency of the fibers were then
measured using imageJ.
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Results
d
c
a
b
Figure 6
Figure 6: This figure is an overhead view of a rat tympanic membrane. a. The
manubrium of the eardrum b. a tear in the eardrum the occurred during the surgical
removal from the rat. c. The pars tensa of the eardrum d. The pars flaccida of the
eardrum.
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Figure 7
Figure 7: This figure is an image of collagen fibers located in the pars tensa. The
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lines on the figure indicate the trend of radial fibers having aligned orientation.
Figure 8
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Figure 8: This figure is an image of radial fibers in the pars tensa of the tympanic
membrane. The arrow in the figure is showing
Figure 9
Figure 9: This figure shows collagen with axial periodicity exhibited in the bands.
This image was taken at the upper manubrium of the ear. As these bands are not
exhibited in the radial fibers of the pars tensa.
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Figure 10
Figure 10: This figure shows collagen fibers taken at the manubrium. There are
distinct bundles of collagen fibers that share common orientation but overall the
fibers are randomly oriented compared to the fibers located in the pars tensa.
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Figure 9
Figure 12: This figure is a histogram of the measured diameters of collagen fibers in
the pars tensa. The mean diameter of the fibers was 36.1nm with a standard
deviation of 3.6nm from a sample n=30
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Figure 10
Figure 13: This figure is a histogram of the measured subunit periodicity of collagen
fibers in the manubrium. The mean subunit periodicity of the fibers was 52.2 nm
with a standard deviation of 5.7 nm from a sample n=26
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Discussion
While the structure of the tympanic membrane has been looked at
previously, the advances made in the imaging field allow for higher resolutions
views of the tympanic membrane and allow for individual fibers to be measured.
The mean diameter of the collagen fibers was measured and the histogram of the
collected data exhibited a low standard deviation and a very clear peak Images of
collagen that were taken at manubrium exhibited an axial banding periodicity that
was not seen in radial collagen fibers in the pars tensa. The presence of this banding
pattern indicated that these fibers are a different type of collagen than the collagen
fibers in the pars tensa.
The orientations of the fibers provide some insight into their function in the
membrane. While the radial fibers are highly organized and exhibit uniform
direction, which is consistent with the results found in literature [5], other collagen
structures were found that had not been reported in the literature. Collagen fibers at
the manubrium were organized in comparatively small bundles and did not exhibit
the collective orientation unlike the radial fibers. As the manubrium is the
attachment site for the malleus, it is likely that these fibers primary purpose is to
attach and secure the tympanic membrane and the malleus and that the random
orientation provides better attachment.
Incorporating these orientations and sizes into computer models of the
tympanic membrane are one avenue in which this research hold potential. Finite
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element models of the eardrum have existed for several decades but the models are
constantly being refined to become more accurate. Due to the anisotropy of the
tympanic membrane, it responds unequally in different areas of the membrane to
different frequencies. Human speech has a spectrum from 20 to 20,000 Hz [6] so it
is important to have an accurate model that responds accurately to different
frequencies. Geometry is the primary factor that determines how a finite element
model responds and by incorporating the structure of the collagen fibers into the
model design, the model can provide improved accuracy. The degree of attachment
of the manubrium to the malleus is an aspect of finite element modeling that has a
significant impact of the results of the model [7,8].
This research also has applications in improving existing medical technology.
Currently, a ruptured eardrum is left to heal without medical intervention. However,
there are some ruptures that are too large for the body to heal on its own or chronic
infection prevents the healing of the eardrum. Currently, eardrum grafts primary
goal are to close the hole in the eardrum. They accomplish this by providing
scaffolding for the endothelial and mucosal layers of the tympanic membrane to
grow over. Eventually this graft will disintegrate and the rupture will be sealed but
the collagen fibers will not be replaced. This has been shown to affect the detection
of high frequency sound waves [9]. The loss of these high frequencies can affect an
individual’s ability to distinguish phonetics sounds both in person and over
telecommunications. By learning more about the structure and arrangement of the
collagen fibers, grafts which properly mimic the structure of the collagen fibers can
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be developed and prevent the hearing loss that would otherwise be an acceptable
loss in the healing process.
When measuring the diameter and subunit periodicity sample sizes were
chosen based on previously published literature. The numbers of samples could be
increased to reduce potential error, especially with respect to the axial periodicity,
which as shown with the histogram which exhibited a skew, which could be due to
the number of fibers measured.
Conclusion
Using scanning electron microscopy, collagen fibers in the tympanic
membrane were imaged. The diameters of the collagen fibers and the axial
periodicity of the collagen fibers along the manubrium were measured. Qualitative
data about the orientation of the fibers was acquired and analyzed. Radial fibers
exhibited common uniform orientation and fibers found on the manubrium were
disorganized and composed of a different type of collagen.
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References
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http://www.nidcd.nih.gov/health/hearing/Pages/Default.aspx. Updated 2013.
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