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Middle-ear development VIStructural maturation of the rat conducting apparatus.

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THE ANATOMICAL RECORD 239:475-484 (1994)
Middle-Ear Development VI: Structural Maturation of the Rat
Conducting Apparatus
WAYNE M ZTMMER, DEBORAH F ROSIN, AND JAMES C. SAUNDERS
Department of Otorhinolarvngologv. Head and Neck Surgery 1Jni11~rsity
of Punnylrmncrr,
Philadelphia, Pennsylvania
ABSTRACT
Background: The contribution of middle-ear development
to the overall development of hearing has not been explored in great detail.
This presentation describes the maturation of conductive elements in the
rat middle ear, and provides the basis on which future studies of middle-ear
functional development will follow.
Methods: The middle-ear apparatus was examined at nine different ages
(between 1 and 80 days postpartum) in Long Evans rats. A t each age elements of the conducting apparatus were observed with either light or scanning electron microscopy (SEM), and quantitative measurements were
made from video enhanced photomicrographs. Tympanic membrane area
and cone depth, the length of the malleus and incus arms, ossicular weight,
stapes foot plate and oval window areas, and bulla volume were all measured. Development of the area and lever ratios were derived from these
measurements. The data were fitted to exponential equations and the time
in days required to reach 90% of the adult level determined.
ResuZts: The pars tensa achieved 90% of total area by 17 days. The oval
window achieved the 90% criterion by 13 days, while the area ratio was
within 10%of its adult size by 8 days. The ossicles took between 26 and 34
days, while bulla volume took 59 days to reach the 90% level.
Conclusions: Middle-ear growth was very orderly and systematic in the
data reported. When maturation of the area ratio was considered against
development of the endocochlear potential or the round window compound
action potential, it was clear that the growth of this important aspect of the
middle ear preceded the onset of cochlear function. o 1994 WiIey-Liss, Inc.
Key words: Middle ear, Auditory, Hearing development, Ossicles, Tympanic membrane, Rat (Long Evans strain)
A number of animal models are currently being used
to study auditory development and these include the
chick, mouse, gerbil, guinea pig, and kitten. These species are interesting because their auditory systems, at
birth, may be either highly developed (as in “precocial”
species like the chick or guinea pig) or poorly developed
(as in “altricial” species like the mouse or kitten). The
rat is another altricial laboratory animal for which
there is a growing literature on the development of
hearing. In this species the first indication of cochlear
function is seen in the endocochlear potential which
emerges between the 8th to 10th day after birth (Rybak
et al., 1992). This is followed shortly by the first soundevoked potentials in the cochlea between 11 and 12
days. Adult-like compound action potentials (CAP) in
the cochlea were observed between the 17th and 20th
days after birth (Rybak et al., 1992; Uziel et al., 1981).
The development of hearing in the peripheral region
of the auditory system represents a n interplay between
the maturation of structures that permit the onset and
development of functional capability (Burda, 1985).
The middle-ear system and its capacity to conduct vi0 1994 WILEY-LISS, INC.
brational energy to the cochlea is important in this
process. Indeed, the serial nature of information processing in the peripheral ear suggests that the conductive apparatus may play a n essential role in the development of more central measures of auditory ability. If
sound does not reach the cochlear analyzer, regardless
of the inner ear’s ability to process vibrational energy,
hearing capacity will be severely limited (Saunders et
al., 1993).
This investigation describes the morphological development of the conductive apparatus in the rat, and
is a precursor to other investigations that will consider
the functional development of sound transmission in
the middle ear and its relation to the onset and maturation of hearing capacity in this species (Igic et al.,
1994).
Received January 12, 1994; accepted March 16, 1994.
Address reprint requests to Dr. James C. Saunders, 5 SilversteinORL, 3400 Spruce St., Philadelphia, PA 19104.
476
W.M. ZIMMER ET AL.
METHODS
Animals and Groups
Pregnant female rats of the Long Evans strain were
obtained from commercial breeders and held in the animal quarters at the University of Pennsylvania School
of Medicine. The birth of pups was determined with a n
accuracy of 2 6 hours, and these were maintained and
nursed by their mothers until the age of sacrifice. Animals were divided into nine groups aged 1,3,6,12,16,
22, 34, 60, and 80 days after birth. For each of the
middle-ear variables examined (see below) a t least five
different specimens from separate animals were sampled in each age group.
Tissue Preparation
The animals in each group were sacrificed with a n
intraperitoneal injection of ethyl carbamate (Urethane). Following decapitation the skin, muscle, and
external auditory meatus were removed taking care to
avoid damage to the bulla and tympanic ring. The skull
was split sagitally in animals 12 days or older and the
temporal bones on both sides were isolated. A 1 mm
hole was made in the inferior lateral aspect of the bulla
and a solution of 10%phosphate buffered formalin (pH
7.0) was injected into the cavity. The bones were then
immersed in the 10% formalin for at least 48 hours.
After fixation the specimen was rinsed with water and
a small wick was placed into the bulla hole to remove
gross fluid. The specimen was then set aside and allowed to dry in air overnight.
Paraffin wax was used to seal the eustachian tube
orifice of the dry bones so that the bulla would be fluid
tight for volume measurements. The specimen was
then weighed on a mass balance to a n accuracy of 10
pg. Next a watedsurfactant (Photoflo, Kodak Co.,
Rochester, NY) mixture was gently injected into the
bulla hole. The surfacant facilitated filling the bulla
and assured that all the recesses of the cavity were
filled. Water was injected into the cavity until the meniscus surrounded the injection needle. The needle was
then removed and the specimen immediately reweighed. The weight of the fluid was obtained by subtracting the bulla empty from the bulla filled weight.
The volume was calculated by dividing the fluid
weight by fluid density (the waterlsurfacent had a density of 1.00035 glcc). This method was a n effective and
accurate means of determining middle-ear volume
without damaging the elements of the conductive system (Vrettakos et zl., 1988; Cohen et al., 1992a).
After the bulla volume was determined, the specimen was further dissected. The temporal bone was
viewed with a stereomicroscope at magnifications between 20 and 50 x . The bone forming the terminal zone
of the external auditory canal (DeMaio and Tonndorf,
1978) was removed with a high speed drill using a
small diamond burr (3.0 mm). This allowed us to
clearly expose the tympanic ring. The specimen was
placed on the stage of a Nikon Multiphot micro/macro
photography system equipped with a TV camera and
image enhancing electronics. A calibrated millimeter
rule was placed alongside the specimen and this was
used in the subsequent measurements of membrane
area. A 10.3 x 7.5 cm video micrograph was obtained
using a Mitsubishi (Model P-4Ou) video printer. Re-
flected light was used to photograph the tympanic
membrane and the magnification was adjusted until
the membrane just filled the area of the video micrograph. These same video microscopic procedures were
used to obtain video pictures of all the middle-ear components.
The tympanic membrane and most of the lateral extent of the bulla was then removed to reveal the ossicles contained in the bulla cavity. Pictures of the ossicles were obtained either in situ under reflected-light
illumination, or after removal using transmitted light.
The latter approach proved useful for pups 12 days old
or younger, where the ossicles were transparent and
there was little contrast between the developing bony
structure and the surrounding mesenchyma. These
video prints were again used to measure the lengths of
the ossicles and their lever arms. I n some specimens
the tissue was prepared for scanning electron microscopy (SEM) using procedures described elsewhere
(Huangfu and Saunders, 1983). Polaroid micrographs
taken with the SEM were used to make comparisons
with the video prints, and to examine middle-ear structures in greater detail when needed. Length or area
measurements from SEM or video photos were the
same.
Fleischer (1978) described the morphology of many
rodent middle ears as exhibiting the so-called "microtype" organization. This design has two characteristic
features: the malleus is fused to the tympanic ring a t
the gonial, and there is a large mass a t the head of the
malleus called the orbicular apophysis (Cocherell e t al.,
1914; Huangfu and Saunders, 1983; Saunders and
Garfinkle, 1983). Fleischer (1978) suggested t h a t the
ossicles in the microtype middle ear exhibit two modes
of vibration. Two modes were indeed identified in a n
examination of sound driven tympanic membrane and
ossicular motion in the mouse middle ear (Saunders
and Summers, 1982). The r a t also exhibits a microtype
organization, and for the purpose of examining the lever arms in this species, two axes of rotation were defined. The first axis is indicated by line AB in Figure 1
and extends from the gonial through the incudomalleal
joint to the posterior ligament of the incus. The corresponding malleus lever (Ml) extends from the tip of the
umbo to form a 90" angle with the AB intercept. The
incus lever (11)extends from the distal tip of the incus
at the lenticular process and also forms a 90" angle
with the AB axis. The second presumed axis, as described for the mouse (Saunders and Summers, 1982;
Fleischer, 1978), extends from the gonial to the center
of the orbicular apophysis (line CD in Fig. 1). The
malleus lever (M2) for this axis extends from the tip of
the umbo to form a 90" angle with CD. The incus lever
(12) extends from the distal tip and forms a 90" angle
with CD. The functional significance of these axes in
the rat remains to be determined, but both were traced
during development for this presentation.
Prior to removal of the incus and malleus, the incudostapedial joint was gently separated, and the tensor
tympani tendon carefully cut. This provided excellent
exposure to the stapes and the stapedial artery coursing between the crura. Fine microscissors were used to
cut the artery as i t passed in a n antero-inferior direction between the stapes crura. A fine needle was used
to mobilize and pull the artery through the crura pos-
477
MIDDLE-EAR DEVELOPMENT
B
+lrnrnd
/
C,
/
\
//
\
/
/
/
/
/
A'
Fig. I . This line drawing of the adult rat ossicular system is viewed
from above with the tympanic membrane removed. The two axes of
rotation (AB, CD) are indicated as are the respective lengths of the
lever arms for axis AB (Ml, 11) and for axis CD (M2, 12). We have
chosen to measure the lever arms as a line segment perpendicular to
the axes of rotation. The long arm of the malleus is defined by M2
while the long arm of the incus is defined by 11. Ma1 = malleus; In =
incus; St = stapes; OA = Orbicular apophysis; Gon = gonial. The
tympanic ring is seen as the outer perimeter of the drawing.
tero-superiorly. The stapedius tendon was cut, and a
pick was used to "rock" the stapes back and forth in
order to rupture the annular ligament. This greatly
facilitated removal of the intact stapes bone. Because of
the extremely soft, nearly gelatinous texture of the
stapes in 12-day-old and younger pups, this maneuver
was vitally important in removing the ossicle without
crushing it. Video micrographs of the stapes and oval
window were then obtained. The remaining ossicles
were disarticulated and allowed to dry for a t least 24
hours. The malleus and incus were then weighed on a
mass balance to a n accuracy of 10 pg.
Rat pups younger than 12 days were prepared differently to facilitate dissection of the tympanic structures
in these very immature, technically challenging age
groups. The animals were decapitated and the skulls
were maintained intact. The external auditory canal
and the epidermis in the immediate vicinity were removed leaving most of the head covered with skin. This
provided substance to the otherwise soft skull and prevented damage due to crushing during the dissection.
Minimal soft tissue was removed in exposing the tympanic ring and the bony structures that defined the
tympanic cavity. Dissection was performed with forceps and blunt instruments as all structures were soft
and cartilaginous. It was impossible to assess bulla volume in these pups because of the immaturity of this
cavity and the fact that it was filled with mesenchyme.
Photomicrographs of the ossicles were obtained immediately after their removal to avoid desiccation.
Structural Analysis
The purpose of the dissections described above was to
expose the important components of the middle-ear apparatus so that they could be accurately photographed
and measured. We wanted to determine the development of the tympanic membrane area, including that of
its two divisions, the pars tensa and pars flaccida. The
478
W.M. ZIMMER ET AL.
area of the stapes foot plate and the oval window was
also measured. In addition, the depth of the tympanic
membrane cone, the lengths of the ossicular lever arms
(the long arm of the malleus and incus), and the volume of the middle-ear cavity (the bulla) were also
quantified with increasing postnatal age.
The identified structure was visualized under the
Multiphot system and carefully oriented perpendicular
to the optical axis of the microscope. Video image analysis hardware and software (Model OC-200, Correco,
Inc., Ville St.-Laurent, Quebec, Canada) was used to
enhance contrast and capture the image, and a video
photo of the image was then produced as described earlier. The video picture was then placed on a digitizing
tablet and the area of interest circumscribed with a
sensing pointer. Computer software (Sigma Scan by
Jandel Scientific, Inc., Sausalito, CA) integrated with
the digitizing tablet was then used to calculate either
areas, lengths, or angles. The lengths or area measurements were corrected for their magnification with respect to the millimeter rule which accompanied each
video print and each of these measurements was expressed a s millimeters or square millimeters.
RESULTS
The mean and standard deviation for each middleear parameter within all age groups were calculated. A
regression line of the form:
y
=
A (1 - e( - t h ) ) + B
was calculated for each set of data. The estimated size
of the structure a t birth is given by B, t is the number
of days after birth, A is the asymptotic or adult size of
the structure, and T is the time constant of the regression line. This regression analysis permitted quantitative comparisons among the development of each middle-ear structure examined by determining the number
of days needed to achieve 90% of the adult size (the
approximate asymptote in each equation).
General Observations
The skull of the rat was not fused until about 12-16
days after birth. The tympanic cavity was virtually
non-existent through 6 days of age since the medial
wall (which consisted of the cochlea and vestibule) was
in contact with the tympanic ring and membrane. At
this stage, mesenchyme filled the tympanum and surrounded and adhered to the middle-ear elements. The
homogeneous appearance of all the conductive structures made visual identification extremely difficult in
these early age groups, and the ossicles could only be
identified after carving them out of the “mesenchymal
gravy.” Indeed, identification was only possible because the dissections were performed sequentially beginning with the oldest and then progressing to
younger groups. This allowed us to acquire a sense of
how the various middle-ear structures were oriented
and where they were located before tackling the more
challenging, young animals. The tympanic cavity underwent impressive qualitative and quantitative
changes during development, and these are not presented.
Tympanic Membrane
Morphologic development proceeded rapidly through
the first 20 days. Between 1 and 3 days of age the
tympanic membrane (TM) was transparent, thin, and
slightly convex in all places except where fused to the
malleus. The malleus itself was not clearly discernible
except for the influence it had on the morphology of the
membrane. This is seen in the top panel of Figure 2
where the long process of the malleus a t 1 day of age
produces a depression on a n otherwise flat surface.
There are no other discernible features of the ossicles
visible. The TM served to contain the gelatinous mesenchyme, which blanketed the floor of the cavity and
surrounded the ossicles. As time progressed, the TM
became more fibrous and concave, and by 12 days this
membrane formed the lateral wall of the air-filled
bulla. By 22 days the TM achieved its adult appearance
which is presented in the lower panel of Figure 2. The
transparency of the membrane is now apparent with
the ossicles below clearly visible. The average areas of
the total tympanic membrane, as well as the components of pars tensa and pars flaccida, were calculated
for each of the age groups and are presented in Figure
3. The vertical bars give a n indication of one standard
deviation above or below the mean. The total tympanic
membrane area, as well as that of the pars tensa were
between 48% and 49% of adult size at 1 day. These
expanded to 90% of adult size by 23 and 17 days of age,
respectively. As Figure 3 shows, the pars tensa matured earliest and was responsible for most of the expansion of the tympanic membrane. The pars flaccida
was 37% (0.94 mm2) of asymptotic size at 1 day and
exhibited a slower rate of maturation, reaching 90% of
adult size (2.25 mm2) by 49 days postpartum. The expansion of pars flaccida was responsible for most of the
overall tympanic membrane area expansion after 22
days of age.
The development of tympanic membrane cone depth
is shown in Figure 4 and the insert indicates how this
variable was measured. The cone depth was determined from photomicrographs of the umbo taken in a
plane parallel to the tympanic ring. Although the
umbo was clearly below the plane of the tympanic ring
a t 6 days of age, it did not form the apex of a cone since
the tympanic membrane was, for the most part, completely flat. The malleus was connected to the tympanic membrane by a thin fold which appeared to expand progressively starting a t approximately 6 days.
During the next 6-10 days this process resulted in formation of the conically shaped adult tympanic membrane. When the cone was first identified clearly, it
was 67% of adult depth and reached 90% when the pup
was 15 days old. The exponential time constants of
growth for pars tensa area (see Fig. 3) and cone depth
were very similar, suggesting the uniform maturation
of the pars tensa in three dimensions.
It is worth noting that previously we have corrected
for the conical shape of the chick tympanic membrane
in calculating its surface area (Cohen et al., 1992a). In
the case of the gerbil (Cohen et al., 1992b), and now
with the present data for the rat, this correction has
proven very frustrating. The complex shape of the pars
tensa cone, due to the long arm of the malleus being
attached to the membrane along its length, and the fact
that the umbo is offset from the exact center of pars
tensa, made a n accurate calculation of the conical area
very difficult. One should realize that the tympanic
membrane areas reported in Figure 3 modeled the
479
MIDDLE-EAR DEVELOPMENT
Total TM Area
Pars Flaccida
Pars Tensa
1
l.Omm
H
0
40
20
80
60
AGE IN DAYS
Fig. 3. The surface area of pars tensa (PT), pars flaccida (PF), and
the total tympanic membrane (TM) area, measured in square millimeters, is presented for animals between 1 and 80 days of age. These
areas are modeled as flat plates and the depth of the pars tensa cone
was not factored into these measures. Each data point is the average
of 5 animals and all three measures were obtained in the same animal. The solid line is the exponential best fit to the data. The vertical
bars show one standard deviation above, below, or about the mean.
Fig. 2. Two photomicrographs of the tympanic membrane taken in
reflected light are presented. The top panel illustrates a 1-day-old pup
while the bottom panel shows a n 80-day-old animal. The change in
size is apparent and in the older animal the transparent tympanic
membrane reveals the ossicular structures below.
2
-I
-I
0.4 -
I
z
0.2
I
l-
membrane a s a flat plate. The increasing depth of the
cone indicates t h a t the actual area of the membrane
would be slightly larger than that presented.
Oval Window, and Stapes Foot Plate
Figure 5 illustrates the changes in oval window and
stapes foot plate areas. The oval window area increased
by 50% from 0.24 mm2 a t 1 day to its adult size (0.36
mm2) at 60 days after birth. The stapes foot plate underwent a n expansion of 76% from 0.21 mm2 a t 1day to
0.37 mm2 at 60 days. The oval window and foot plate
areas reached 90% of their asymptotic sizes by 13 and
17 days, respectively.
The development of the oval window and stapes foot
plate area plotted in Figure 5 demonstrated a n interesting phenomenon. As might be expected, the surface
area of the oval window, prior to 15 days, exceeded that
of the stapes foot plate, with the space between them
occupied by the annular ligament. However, after 16
days a reversal occurred, with the foot plate area exceeding the oval window area by approximately 5%.An
explanation for this latter observation lies in the defi-
,
Tympanic Ring
c Ii
Uhbo
t
a
W
0.0
1
f
W
-
?
I
I
0
n = 5
J
I
20
I
I
40
I
I
60
I
I
1
I
80
AGE IN DAYS
Fig. 4. Changes in the conical depth of pars tensa is presented as a
function of age. The insert in the figure illustrates how the depth was
estimated. The solid line represents the exponential fit to the data.
nition we used for oval window and foot plate areas.
The oval window was defined by the opening into the
vestibule. The stapes foot plate area was defined by its
outer perimeter when viewed from below. In 1-day-old
specimens the sides of the foot plate were a t nearly
right angles to the under surface of the plate (Fig. 6A).
In the adult specimen the lateral walls of the foot plate
formed a n acute angle of nearly 30" to the lower surface, and this relationship is seen in the adult specimen
480
W.M. ZIMMER ET AL.
80 DAYS
I
w
5
1 DAY
0.35
1
A
w
E 0.30
U
0
3
Oval Window
v Foot Plate
a
v,
Z
- 0.25
a
I
TI
[ i
w
w
0.20
0
1.0 mm
n = 5
I
20
I
40
8
60
I
1
80
AGE IN DAYS
Fig. 5. The changing area of the stapes foot plate and the oval
window area are shown during an 80-day interval from birth. The
solid lines are the exponential fits to the data. The fact that the oval
window appears to be smaller than the foot plate beyond about 15
days of age is explained in the text. The vertical bars (one standard
deviation) are shown in only one direction and for only the oval window to keep from cluttering the figure. Foot plate variability was
nearly the same as that of the oval window.
of Figure 6A. Thus, the perimeter of the oval window
opening in adults is smaller than the outermost dimension of the foot plate. This aspect of foot plate development is clearly seen in the micrograph of Figure 6B,
where the right edge of the foot plate forms a n acute
angle with the actual oval window opening. This observation is consistent with earlier reports on annular
ligament development in the chick and gerbil. Cohen e t
al. (1992a,b) have suggested that the ligament progressively decreases in size to accommodate the foot plate,
which, due to its slower maturational rate, continues to
expand after the oval window reaches its adult size.
Bulla Volume
The changes in bulla volume were the most dramatic
of all the middle-ear aspects examined. At 3 days after
birth the middle-ear cavity consisted of a tiny mesenchyme filled space enveloping the immature ossicles.
Indeed, much of the medial wall was in contact with
the tympanic membrane and tympanic ring. Over the
next 9 days these structures separated rapidly and by
12 days all mesenchyme was absorbed allowing reproducible measurements of bulla volume. Our measures
showed that bulla volume expanded by 235% from
0.018 cc at 12 days of age to 0.061 cc in the adult specimens (Fig. 7). The exponential fit to these data showed
it to be the slowest maturing aspect of the middle-ear
and after 59 days bulla volume was 90% of its asymptotic level.
Malleus and incus
The ossicles underwent marked qualitative changes
from a soft and gelatinous composition with very high
water content a t 3 days to a firm and bony appearance
I
1.0mm
I
Fig. 6. A The two line drawings depict the shape of the adult and
1-day-old stapes in the rat. The 1-day-old specimen only retained its
shape in water since it was formed entirely of cartilage. The scanning
electron micrograph (B) shows the adult stapes in situ. ST = stapedius muscle; FP = foot plate of the stapes; SA = stapedial artery; OA
= orbicular apophysis; TT = tensor tympani; C = the incudostapedial joint has separated.
by 22 days. By 6 days after birth the first signs of
ossification were identified a s a diffuse speckling uniformly distributed throughout the ossicles. These
“speckles” represented the centers of ossification. The
ossification became concentrated in the cortical margins of the ossicles by 12 days, and by 22 days they
were fully mineralized. Prior to 12 days after birth the
ossicles had a n extremely high water content and were
observed to shrival into formless masses less than 50%
of their original size upon even modest dehydration.
The severe loss of mass after drying prevented us from
accurately measuring the weight of the malleus prior
to 6 days old and that of the incus prior to 12 days. The
upper panel of Figure 8 shows the length of the long
arm of the malleus and the long arm of the incus measured from video micrographs taken immediately after
removal of these structures. These measures extend
481
MIDDLE-EAR DEVELOPMENT
3.5
v,
CY
3.0 -
W
I-
I
-
W
I-
0.04
rn
3 0.02
0
w
w
E
-I
2.5
-
=Z-
2.0
-
I
1.5 -
2
0
0
-
Z
Q
T
v,
0.06
5
I
f
k
0
Malleus
Incus
Bulla Volume
I
I
0.00
a
n = 5
L
0
20
i
40
L
l
60
I
I
80
AGE IN DAYS
Fig. 7. The bulla volume averaged over 5 samples, is plotted against
age in days. The fitted exponential line shows that after 80 days the
volume was still increasing slightly. The vertical bars indicate one
standard deviation about the mean.
from the tip of the arms to the approximate center of
the malleus and incus a s defined by axes AB and CD in
Figure 1. These arms increased by nearly the same
magnitude, 67 and 65%, respectively, from 1 day to
their adult size. The arm of the incus, however,
achieved 90% of its adult length in 26 days while the
malleus took 34 days.
The lower panel of Figure 8 depicts the increase in
the malleus and incus dry weight. The malleus dry
mass grew by 800%from 0.1 pg, when it could first be
measured at 6 days, to its adult value of 0.9 pg at 35
days. Nearly half of that increase was realized between
6 and 12 days, when it more than tripled. The weight of
the incus could not be determined prior to 12 days,
because of its high water content. Interestingly, the
exponential functions revealed that the time constants
for increasing mass were nearly identical for the
malleus and incus, with both structures attaining 90%
of their asymptotic size by 36 and 34 days of age, respectively.
The relationship between mass and length changes,
for the malleus and incus, are shown in Figure 9. We
elected to plot these variables as a percentage of adult
size so that they could be compared against each other
more easily. The time constant describing the maturation of length exceeded that of weight, for each of these
ossicles, by a narrow margin, and the incus developed
slightly faster than the malleus with regard to both
parameters.
Maturation of the Elements of Sound Transfer
While the process of pressure amplification from the
tympanic membrane to the stapes foot plate is complex,
most of the amplification can be accounted for quantitatively by the ratio between the tympanic membrane
(pars tensa) and oval window areas, and the ratio between the length of the long arms of the malleus and
the incus along the AB axis. These are the so-called
0.0
‘
0
40
20
AGE
IN
60
80
DAYS
Fig. 8. The changing length (in millimeters) of the long arm of the
malleus and incus (top panel) are plotted against age. These are the
M1 and I1 lines in Figure 1.The lower panel shows the development
of dry weight for the malleus and incus (in micrograms) over the same
age interval. The solid lines show the best fit exponential functions.
“areal” and “lever” ratios. The development of the area
ratio is plotted in Figure 10, and shows a n increase
from 17.0 a t 1 day to 24.8 by 80 days. An area ratio of
22.3 was 90% of the adult value when the pup reached
8 days of age.
The lever ratios along the AB and CD axes were
calculated by MU11 and M2/12 (see Fig. l), respectively, and are presented a s a function of age in Figure
11. These ratios show a relatively constant value during maturation, and essentially fluctuate about the
adult values. This reflects the fact that the relative
lengths of the malleus and incus lever arms increased
simultaneously (see Fig. 8). The mean lever ratios for
the AB and CD axes were 2.25:l and 2.21:1, respectively. As can be appreciated from Figure 11,the variation in the mean values for different ages was quite
small. The maximum difference between the high and
low values for MU11 was only 0.36 while for M2iI2 it
was 0.86.
DISCUSSION
The middle-ear conductive apparatus is responsible
for the transfer of sound vibrations from the tympanic
W.M. ZIMMER ET AL.
0
0
0
Lever Ratio (A - B)
A
Lever Ratio ( C - D)
Weight of Malleus
Weight of Incus
n = 5
n=5
I
0
20
40
60
a0
0
AGE IN DAYS
4
I
0
c
Q
21
fx
1
li
n = 5
17t
0
40
60
a0
AGE IN DAYS
Fig. 9. The results in Figure 8 are replotted as a percent of the
maximum value. In this way the relative rate of development for
weight and arm length of the malleus and incus can be compared. As
seen, the ossicular arms gain length before their mass increases up to
about 20 days of age.
‘
20
Pars Tensa/Oval Window
Fig. 11. The changes in the lever ratio, for the levers in axis AB
(MU111and axis CD (MWIZ), are compared over age. Little systematic
change is seen over the 80-day interval.
the rate of hearing development measured from more
central locations in the auditory system (Saunders et
al., 1993). While these studies provided intriguing evidence for the hypothesis that middle-ear maturation is
a n important contributor to the rate of auditory functional development, contrary observations exist.
Studies in the cat (Thomas and Walsh, 1990) demonstrated that a mature area ratio between the tympanic
membrane and stapes foot plate was present at the end
of the first week, a t a time when action potential
thresholds recorded from central auditory nuclei were
very insensitive. This observation suggested that the
kitten cochlea was quite immature at a time of mature
middle-ear structural development. In addition, the development of umbo velocity in the chick, a n important
measure of middle-ear function, was found to be independent of the maturation of auditory thresholds measured from brainstem nuclei (Cohen et al., 1992a).
15
0
20
40
60
a0
AGE IN DAYS
Fig. 10. The area ratio (pars tensaioval window area) is averaged
over 5 specimens between 1 and 80 days of age. The solid line is the
exponential fit to the data. The area ratio reaches asymptotic conditions within 10 days. Thereafter the expansion of both structures is
proportionally the same.
membrane to the fluid-filled spaces of the cochlea via
the stapes foot plate. It stands to reason that maturation of the conductive system is intimately tied to
measurement of hearing development recorded in the
cochlea, the eighth nerve, or other higher auditory processing centers. In fact, studies on the mouse (Huangfu
and Saunders, 1983; Doan et al., 1993), the gerbil (Cohen et al., 1992a,b), and hamster (Relkin and Saunders, 1980) suggested that both the structural and
functional maturation of the middle ear sets a limit on
Comparison With Other Data
To our knowIedge this is the first report describing in
detail the structural changes in the rat middle-ear during early postnatal life. A summary of developmental
times for various parameters to reach 90% of adult size
in the rat and two other mammals (mouse and gerbil)
is given in Table 1. In general, the time required for
various rat middle-ear structures to reach 90% of their
adult size were significantly longer than those of the
mouse (Huangfu and Saunders, 1983) and gerbil (Cohen e t al., 199213). Perhaps the most interesting difference between these species was the early maturation of
the area ratio in the rat. This reflects the fact that pars
tensa and oval window growth combined in such a way
to achieve large area ratios very early.
Structural Development of the Pressure Amplifier
The long arms of the malleus and incus also expanded a t similar rates with both achieving adult
483
MIDDLE-EAR DEVELOPMENT
TABLE 1. Summary of developmental time to achieve
90%of adult value'
W
Y
Y
LL
I
T
r
I
Age in days
Structure
Pars tensaiOW ratio
TM cone depth
Pars tensa area
Oval window area
Stapes foot plate area
Tympanic membrane area
Incus length
Malleus length
Incus mass
Malleus mass
Pars flaccida area
Bulla volume
Rat
Mouse2
8
15
17
13
17
23
26
34
34
36
49
59
17.5
10
18.5
6
19.5
15
16
Gerbil3
12
11.5
10
4
7
11.5
4
6
-
20.5
18
1
1
i
~
LL
~
13
36
'The 90% values were determined from the corresponding exponential
regression line for each of the middle ear structures measured.
'From Huangfu and Saunders (1983).
'From Cohen et al. (1992a).
O
w
a
I
I
I
I f
I /I
2ot
0
W
20
CAP at 8.0 kHz
40
60
1
80
AGE IN DAYS
length prior to adult mass. This, in part, accounts for
the minimal variation in lever ratios seen during the
course of development (see Fig. 11).The amplification
effect of the M2/I2 lever as defined here, was very close
to that of the more traditional MU11 lever. However,
we wonder whether this would actually be the case in
the middle ear response to sound, since rotation on the
CD axis requires both pivoting around the gonial and
stretching at the posterior ligament of the incus.
Stretching at the ligament seems likely since the ligament and incus were mobile at that location during
the dissections. Pivoting may be less likely since the
malleus was solidly attached to the tympanic ring a t
the gonial. Indeed, separating the malleus from the
tympanic ring during dissection required that the tympanic ring be either broken or cut at its fusion point. In
light of these observations, i t is likely that considerable
stiffness will be associated with pivotal motion a t the
gonial.
It seems unreasonable to expect that sound transmission occurred through the middle ear earlier than 6
days postpartum. The ossicles were soft and completely
flaccid, and the middle-ear cavity was filled with gelatinous mesenchyme at that time. During the next 6
days the bulla gradually aerated, the ossicles became
much more rigid (although not yet fully ossified), and
the area ratio was near its adult value. On the basis of
structural maturity alone i t is possible that sophisticated sound transmission through the middle ear was
well established by 12 days of age. It has been shown
(Uziel et al., 1981) that the cochlear microphonic and
whole-nerve AP response, evoked by tone bursts or filtered clicks of various frequencies, were within the
adult range when 15 days old, and this, of course,
would indicate mature sound conduction by this time.
Sound Conduction and Hearing Development
The time course for the development of cochlear function has been well established in the rat (Uziel et al.,
1981; Rybak e t al., 1992). The endocochlear potential
(EP) was first reported a t 7 days, increased gradually
until 11 days, rapidly escalated through 13 days, and
then increased more slowly to achieve a n adult level 17
Fig. 12. The growth of the area ratio, the CAP threshold (at 8.0
kHz), and the endochochlear potential (EP) is plotted as a percentage
of its maximum value. The relative rate of growth can be compared
among the conditions. Rapid maturation of the areal ratio appears to
occur prior to the onset of electrophysiologic events in the cochlea. The
lines in this figure are smoothed between data points.
days after birth (Rybak et al., 1992). Eighth nerve compound action potentials (CAP) elicited by click stimuli
were first detected between 11 and 13 days after birth.
However, very loud clicks, up to 100 dB SPL, were
required to produce a response a t this time. Adult-like
waveforms in the CAP response were identified between 16 and 17 days, while the CAP thresholds
showed substantial improvement in sensitivity from 13
to 20 days of age. This was followed by continued gradual improvement until adult thresholds were reached
between 25 and 28 days.
The relationship between middle-ear structural maturation, given by the area ratio, and cochlear auditory
development represented by the E P and CAP threshold
(at 8.0 kHz) is summarized in Figure 12. Although the
EP is not a sound driven potential, its presence is necessary for the normal transduction of sound in the cochlea (Rybak et al., 1992). Thus, its inclusion as another indicator of cochlear functional development is
justified. We calculated the percent difference in the
measured unit from the youngest age a t which data
could be obtained to the adult level. While this distorts
the results somewhat by forcing upper and lower age
boundaries, it produces a plot in which the value a t the
youngest age was 0% while the adult value was loo%,
for each variable examined. By normalizing all curves
against a common percent axis, a direct comparison of
the relative rate of development across ages can be
made. An examination of Figure 12 reveals that the
area ratio matured earlier than the EP, and the CAP
thresholds. Indeed the 90% of the maximum response
occurred a t about 10 days for the area ratio, 16 days for
the EP and after 28 days for the CAP. Electron micrographic studies of the developing rat cochlea (Burda,
1985; Roth and Bruns, 1992) also revealed striking
484
W.M. ZIMMER ET AL.
changes between 4 and 12 days after birth. Thus, at
approximately the tirne of hearing onsetat 12 days (as
was found
defined by the CAP responses), the
to be structurally mature. The time course of area ratio
and CAP maturation suggests that the middle ear and
the cochlea develop independently of each other with
sound conduction preceeding cochlear function.
This study has provided information regarding the
static development of the middle ear. The results presented here provide a strong foundation for further examination of functional middle-ear development in the
rat (Igic et al., 1994).
ACKNOWLEDGMENTS
This research was supported by a n award from the
NIDCD (ROl-DC00510) to J.c.s. The authors greatly
appreciate the assistance of Daryl Doan, Petar kit,
and Ms. Linda Markoff. Portions of this paper were
presented at the 1994 Mid-Winter Meeting of the AsSt. peterssociation for ~~~~~~~hin Oto~aryngo~ogy,
berg Beach, FL.
LITERATURE CITED
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Cohen, Y.E., H.N. Hernandez, and J.C. Saunders 1992a Middle ear
development: 11. Morphometric changes in the conducting apparatus of the chick. J. Morphol., 212r257-267.
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DeMaio, F.H.P., and J . Tonndorf 1978 The terminal zone of the external auditory meatus in a variety of mammals. Arch. Otolaryngol., 104.5570-577.
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Igic, P.G., D.E. Doan, and J.C. Saunders 1994 The development of the
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Relkin, E.M., and J.C. Saunders 1980 The development of tympanic
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