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A Review of Audition, Aging, and

Caloric Restriction in Rhesus Monkeys

Ryan M. Anderson

Mentor: Joseph W. Kemnitz

Professor, Dept. of Cell & Regenerative Biology

University of Wisconsin-Madison

CRB 699-013 Independent Study

December 27, 2015

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Table of Contents

Chapter 1: Fundamentals of the Primate Auditory System 4

I. Introduction 4

II. Peripheral Auditory Nervous System 5

A. Outer Ear 5

B. Middle Ear 5

C. Inner Ear 6

D. Organ of Corti 7

E. Hair Cell 9

F. Inner Hair Cell 11

G. Outer Hair Cell 11

III. Central Nervous System 13

A. Cochlear Nucleus 13

B. Superior Olivary Complex 16

C. Inferior Colliculus 18

D. Medial Geniculate Body 19

E. Primary Auditory Cortex 20

Chapter 2: Research Review of the Rhesus Monkey Auditory System in the

Context of Aging and Caloric Restriction 22 I. The Wisconsin Study 22

A. Background of the Wisconsin Aging and Caloric Restriction Study 22

B. Methods and Materials of the Wisconsin Aging and

Caloric Restriction Study 22

C. General Methods of Auditory Testing in the Wisconsin Study Rhesus Monkeys 24

II. 2000 Article Review: Aging and middle ear function in rhesus monkeys.

Peter Torre III et al. 26

A. Introduction 26

B. Methods and Materials 27

C. Results 30

D. Discussion 31

III. 2002 Article Review: Effects of caloric restriction and aging on the auditory function

of rhesus monkeys: The University of Wisconsin Study.

Cynthia G. Fowler et al. 33

A. Introduction 33

B. Methods and Materials 33

C. Results for First Purpose: Does CR Preserve Monaural Auditory Function? 35

D. Results for Second Purpose: Does CR Preserve Binaural Auditory Function? 38

E. Results for Third Purpose: Does CR Preserve Auditory Thresholds? 38

F. Discussion 39

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IV. 2004 Article Review: Assessment of auditory function in rhesus monkeys: Effects of

age and calorie restriction. Peter Torre III et al. 41

A. Introduction 41

B. Methods and Materials 43

C. Results 45

D. Discussion 48

V. 2008 Article Review: Tympanometry in rhesus monkeys: Effects of aging and caloric

restriction. Cynthia G. Fowler et al. 53

A. Introduction 53

B. Methods and Materials 54

C. Results 55

D. Discussion 56

VI. 2010 Article Review: Auditory function in rhesus monkeys: Effects of aging and

caloric restriction in the Wisconsin monkeys five years later.

Cynthia G. Fowler et al. 58

A. Introduction 58

B. Methods and Materials 59

C. Results 60

D. Discussion 63

Chapter 3: Research Analysis and Suggested Areas of Further Study 67 I. Introduction 67

II. Analysis and Suggestions for Future Research 67

References 70

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Chapter 1 Fundamentals of the Primate Auditory System

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I. Introduction

Sound is a mechanical disturbance in the elastic medium through which it travels. As

defined in physics, the speed of sound is the product of its wavelength and frequency. In its most

simple form, sound is a single sinusoidal wave and is heard as a pure tone. The industrialized

world is full of sounds, most often being complex and is comprised of several sine waves of

differing amplitude and wavelength.

In the primate and other mammals, the dynamic range of the auditory system is about 12

orders of intensity. These intensities are expressed on the logarithmic scale of decibels. Volume,

however, is not synonymous with intensity and is on a liner scale. For every doubling of volume

there is an increased intensity by a factor of ten. 17th

century French mathematician and physicist

Joseph Fourier showed that a function, such as a sound wave, could be decomposed into its

substituent sinusoidal components. Primate audition computes sound information in a very

similar fashion with astounding sensitivity and accuracy on the scale of a hydrogen atom’s

width.

This paper covers the primate auditory system with a particular focus on the rhesus

macaque (Macaca mulatta). In this chapter, the basics of audition will be detailed sequentially

from the point of sound emission to the final integration of acoustic information at the primary

auditory cortex. In summary, sound first enters the outer ear and vibrates tiny ossicles at the

middle ear. These bone vibrations are transmitted into fluid movement in the cochlea which in

turn transduces the waves into electrical impulses. As the impulses course their way from the

1 The majority of the material within this chapter was mainly derived from the NTP 524 neuroscience course

headed by Prof. Tom Yin and Asst. Prof. Xin Huang at the University of Wisconsin-Madison.

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lower auditory centers in the brainstem and ascend to the cortex, sound information is

increasingly broken down, analyzed, and organized. Ultimately at the highest centers and

cortices, other inputs aid in more complex neuromechanisms such as sound meaning,

identification, and localization.

II. Peripheral Auditory Nervous System

A. Outer Ear

When sound is emitted from its source, it

first encounters the outer ear of the peripheral

auditory system. The pinna’s unique folds and

overall shape helps to collect and direct the sound

into the ear canal, which then channels sound to

the tympanic membrane. Localization of sound on the horizontal and vertical planes is also

enhanced by the pinna and over 20 muscles surrounding it. Mobility and filtering properties of

the pinna aid in the fine tuning of the localization of sound, the latter of which will be detailed

later.

B. Middle Ear

Sound waves coming in contact with the

tympanic membrane transduce vibrational energy

from the gas molecules in the air to the membrane.

Vibrations of the tympanic membrane are then

transmitted to a trio of tiny bones called the incus,

malleus, and stapes (Fig. 2). This ossicular chain

enables the movement of vibration through the middle

Figure 1. Overview of the primate ear 4

Figure 2. Ossicular chain of the middle 4

ear.

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ear to the inner ear. The stapes is the third bone in the chain and abuts a small opening, the oval

window, at the base of the cochlea. The stapes footplate covers the oval window and allows

vibrations to enter the cochlea of the inner ear via in-and-out movements in phase with the

frequency of the sound stimulus. Surface area from the tympanic membrane to the oval window

is a ratio of about 21:1 which provides an amplification of the pressure wave caused by sound. A

great change in pressure due to this difference in surface area allows the magnitude of the sound

stimulus to be more concentrated in the smaller passageways of the cochlea. Also in the middle

ear are the primate’s smallest muscles attaching to the ossicular chain, the tensor tympani and

stapedius. These muscles contract when a certain threshold of sound exceeds a safe range for

hearing and transmit less vibration to the inner ear, thus dampening the intensity of the actual

sound and protecting delicate hair cells in the cochlea.

C. Inner Ear

Transduction of the mechanical forces

into electrical signals occurs at the last third of

the peripheral auditory nervous system: the

cochlea of the inner ear. The cochlea is a small,

compartmentalized coil of bone deep inside the

ear. Attached to the cochlea are semicircular

canals via an intermediate bony space called the

vestibule, all three components making up the

“labyrinth of the inner ear”. Aside from the semicircular canals and vestibule, the cochlea

consists of three fluid-filled channels: the scala tympani, scala media, and scala vestibuli (Fig. 3).

The scala tympani and scala vestibuli are connected via the helicotrema, a small opening at the

Figure 3. Cross section of the cochlea.4

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apex of the cochlea that allows mixture of their perilymph fluid. It is here the pressure waves

reach the end of the cochlea and turn back towards the base via the perilymph of the scala

vestibuli. The round window resides at the vestibular end of the cochlea’s base. A membranous

covering of the round window allows the sound pressure waves to take their effect on the

perilymph that were induced by the stapes movement at the oval window. For every time the

stapes moves into the oval window, the round window bulges out, and vice versa. Along the

length of the cochlea, the scala media is bordered by the adjacent scala tympani and scala

vestibuli. Endolypmh fluid in the scala media vibrates from the pressure changes transmitted by

the perilymph via movement of neighboring membrane.

D. Organ of Corti

The organ of Corti is

located at the inner ear in the

scala media channel and

consists chiefly of specialized

epithelial cells: the inner and

outer hair cells. These cells are

responsible for transducing the

mechanical vibrations from

sound in the cochlear lymph into electrical impulses while maintaining frequency of the

stimulus. Immediately inferior to the organ of Corti is the basilar membrane and superior is the

tectorial membrane. The tectorial membrane comes in close contact with the hair cells of the

organ of Corti and vibrates tangentially with respect to the hair cells. Basilar membrane vibration

is in a vertical fashion, moving the hair cells up and down. If tangential and vertical movements

Figure 4. Cross section of the organ of Corti.4

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are too great (from intense sound stimuli), the shearing forces can damage the delicate stereocilia

that sit atop each hair cell and cause subsequent hearing loss.

The basilar membrane varies in width and stiffness from the base of the cochlea to the

apex. At the base, the basilar membrane is at its thinnest and most stiff, thus being responsible

for encoding high frequency sounds. At the apex of the cochlea, the basilar membrane is wide

and floppy, encoding for low frequency sounds. Four rows of hair cells stud the basilar

membrane; three rows of outer hair cells and one row of inner hair cells. These hair cells have

stereocilia that also vary in thickness and length, accompanying those characteristics of the

basilar membrane and thereby enhancing the quality of expressed frequencies along the cochlea.

Relatively flexible stereocilia are thinner and longer at the apex of the cochlea, whereas the

stiffer stereocilia are thicker and shorter at the base. This gradient of flexibility of the basilar

membrane and hair cells’ stereocilia underlies the tonotopic organization of the cochlea. Multiple

pitches of a complex sound cause multiple vibrations of the basilar membrane. The cochlea acts

as a frequency analyzer by decomposing the complex waveform and expressing the greatest

amplitudes of membrane vibration along the distance of the cochlea. The consequence of which

yields the greatest activation of the innervating neurons at that point of maximum amplitude,

reducing the complex sound into its substituent sinusoidal signals.2

This characteristic frequency of the hair cell necessarily means that there are threshold

tuning curves for auditory nerve fibers that match the frequency of basilar vibration. In humans

at lower frequencies of 2-3 kHz or less, the auditory nerve fibers exhibit phase locking to the

tone they’re receiving. Phase locking is a synchronized firing of the neurons with the phase of a

sound stimulus. Nerves can only do so to such an extent, hence the limit of phase locking up to

2-3 kHz. This is an important aspect of collecting sound information because phase-locked

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neurons provide a very high resolution of the stimulus and aid in the localization of low

frequency sound. For higher frequencies, the required rate of depolarization is too high for the

neurons to physiologically maintain and so phase locking is lost for frequencies generally greater

than 4 kHz.

E. Hair Cell

On top of each hair cell is a bundle of stereocilia, or a hair bundle, all protruding into the

scala media’s endolypmh. The stereocilia are arranged in a V-like fashion with graded hair

length. Taller “hairs” are on the outside of the “V” and shorter hairs are on the inside of the “V”.

Stereocilia are linked together through proteinaceous

connections called tip links. Whenever the endolymph current

rushes over the hair bundle, all of the hairs move in unison.

Unified movement of stereocilia direction via these tip link

connections allows the greatest depolarization of the

individual hair cell for the endolymph current present. When

the hair bundle moves toward the tallest stereocilium,

depolarization occurs. Hyperpolarization results from the hair

bundle moving towards the shortest stereocilium,

compressing the tip link and closing the transduction channel.

Concentration gradients of potassium and sodium are

reversed in inner ear hair cells compared to typical neurons.

Endolymph has a higher potassium concentration and the hair

cell’s intracellular environment is richer with sodium ions.

Stereocilia projecting into the endolymph are sensitive to the cytotoxic perilymph and die if

Figure 5: Hair cell mechanotransduction 4

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exposed to the fluid because of differing ion concentrations. The movement of each stereocilium

opens a transduction channel at the base which is quickly accompanied by a flood of the

potassium ions into the hair cell causing depolarization. Calcium ions also enter the cell with the

influx of potassium ions by way of cotransport. Calcium binds to the transduction channels and

influences them to open to an even greater extent. This increased opening of the channel

consequently is matched with the closing speed of the channel. A greater depolarization will

cause a greater repolarization, thus shortening the whole duration of the action potential. The

closing forces of the transduction channel concurrently take place with the incoming sound

wave. It is this event that causes the increase in distance individual stereocilia move in response

to that sound wave and therefore increases their sensitivity. Calcium current also flows into the

hair cell due to the cell’s overall depolarization. This calcium current is responsible for the

exocytotic vesicular release of neurotransmitter into the synaptic cleft to be received by the

auditory nerve fiber’s terminal bud. L-glutamate is the proposed neurotransmitter for this

excitation.3 For the majority of the central auditory system, most of the excitation is due to

glutamatergic inputs and inhibition from either GABAergic or glycinergic inputs. The main

exception to this is in the dorsal cochlear nucleus where many different kinds of cells use a

variety of neurotransmitters.10

It’s worthwhile mentioning the remarkable sensitivity to the primate auditory system.

Until the point of cell polarization, the entire transduction pathway is mechanical, not through a

second messenger system. The frequency of the stimulus sound is maintained from the air to

bone to the concentration changes within the hair cell. Response is so fast that the time it takes

for the hair bundle’s movement to translate into an electrical potential can happen as fast as 10

µsec. Such speed is needed for processes like accurate sound localization and would not be

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possible with second messenger systems. Amazing sensitivity is also exhibited in the lateral

movement of the hair cells. If the tallest hair were scaled up to the height of the Eiffel Tower, the

tip would only move a thumb’s width at the lowest threshold of depolarization. However,

sensitivity of this magnitude can be hazardous in our modern society of loud sounds and

contribute to the hearing loss and deafness.

F. Inner Hair Cell

Inner hair cells are the primary functional unit to the auditory nervous system. They

detect and transduce sound into neural signals according to the general pathway laid out above.

Each inner hair cell is innervated by approximately 20 never fibers. Of these 20 auditory nerve

fibers, 95% of them are afferent and the remaining 5% are efferent. In the grouping of afferent

fibers, 95% of those innervate inner hair cells, the remainder innervating outer hair cells. This

high allocation of nerve fibers and favor to afferent communication of the inner hair cells

indicates the greater degree of specialization and regulation.

G. Outer Hair Cell

Overall function of outer hair cells is thought to amplify sounds for low sound-pressure

levels at the minimum. A loss of outer hair cells results in hearing deficits, indicating that they

do serve some important role in audition. Auditory nerve fibers begin to lose their high

sensitivity and acute tuning for frequency, eventually atrophying from the lack of stimulation.2

It has been found that outer hair cells exhibit changes in shape in response to nerve

impulses. This behavior, termed electromotility, is the contraction or elongation of outer hair

cells that cause a mechanical force to be exerted onto the tectorial membrane and amplify the

ear’s sensitivity to soft sounds at specific frequencies. Changes in outer hair cell length occurs

very rapidly to keep in phase with the signal. Outer hair cells have this ability because they

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contain the contractile protein prestin. Prestin is highly concentrated along the lateral side of the

membrane in outer hair cells. The strength of hair cell stiffening due to the change in prestin

configuration is strong enough to move the whole organ of Corti up and down, which includes

all of the surrounding hair cells and basilar membrane. Outer hair cells contributing greater

vibration to the entire organ enables the detection of sounds on a larger spectrum. Specifically,

sounds can be amplified by up to 40dB by this mechanism, making quiet noises much easier to

hear.4

Prestin can also undergo these conformational changes in the absence of sound as well.

Electromotility is thought to usually enhance sounds, but in silence, can create “sounds” in the

inner ear. Since the basilar membrane is moving, hair cells still polarize to some extent and are

responsible for these spontaneous otoacoustic emissions, or SOAEs. Emissions are commonly

measured as a method to determine good inner ear health.

Outer hair cells are inhibited efferently via the superior olivary complex in the brainstem

projecting to the ipsilateral and contralateral cochleae.11

This being a cholinergic synapse,

acetylcholine is released onto the cochleae and reduce outer hair cell sensitivity. Reduced

sensitivity causes a broadening of the characteristic frequencies expressed along the basilar

membrane. Inhibition thus can be a protection mechanism for loud sounds while also enabling

the analysis of those loud sounds. A broadened characteristic frequency allows the central

auditory system to more accurately detect differences in the levels of the loud sounds at the

sacrifice for accurate frequency information. If there were no inhibition for complex loud

sounds, the characteristic frequency range would be tighter on the basilar membrane and those

hair cells would be overloaded with vibrational stimulation. Too much volume would greatly

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distort whatever frequency the stimulus was at, let alone put the outer hair cells at risk for

irreparable damage.

III. Central Nervous System2

A. Cochlear Nucleus

Depolarization of the hair cells

results in the vesicular release of the

excitatory neurotransmitter glutamate.

Once the hair cells transduce the

mechanical sound wave into a nerve

impulse, the auditory nerve fiber relays the

stimulus information to the central nervous

system. Immediately outside of the

cochlea the departing auditory nerve, or

cochlear nerve, merges with the vestibular

nerve. The vestibulocochlear nerve

diverges at the brainstem and

glutamatergically inputs to the cochlear

and vestibular nuclei to process hearing

and balance information, respectively. The

cochlear nucleus consists of three centers:

the anteroventral nucleus (AVCN), posteroventral nucleus (PVCN), and dorsal nucleus (DCN).

The PVCN will not be covered in this paper. Tonotopic representation from the cochlea is

2 Any mention of neurotransmitters without citation by a superscript comes from The Oxford Handbook of

Auditory Science: The Auditory Brain, reference number 10 in the reference list.

Figure 6: Overview of the central auditory system 4

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retained in the nuclei and throughout the higher centers of the central auditory nervous system.

Information processing occurs very differently, both at the dorsal and anteroventral cochlear

nuclei and at higher centers later in the pathways.

Phase locking and receiving a large, specialized synapse from the auditory nerve fiber

facilitates accurate passage of the impulse to higher systems from the AVCN. In R. Gomez-

Nieto’s and M.E. Rubio’s study on rhesus monkey cochlear nuclei, they found glycine to be the

main inhibitory neurotransmitter in the cochlear nucleus. AMPA, which mimics glutamate, was

suggested to be the main excitatory neurotransmitter, but the labeling for AMPA channels was

weak.9

Sound localization information of the horizontal plane is partially processed and passed

on through the AVCN via specialized cells called bushy cells. Phase locking exhibited by bushy

cells is to preserve temporal information and allow fine calculation for locating a sound source

below a given frequency. A trait that enables this maintenance of phase locking is the fast time

constant seen in bushy cell membranes.5 Depolarization of the potassium channels is achieved at

rather low thresholds and a fast time constant increases the rate of decay, thus decreasing the

duration charge is held in across the membrane. A shortened period of holding charge prevents

summation of signals from incoming depolarizations, allowing all wavelengths of the stimulus

frequency to be expressed. Maintaining the definition of frequency by keeping the stimulus in

phase is crucial to accurately compute localization of low frequency sounds at the superior

olivary complex where AVCN bushy cell project to.

Currently, the DCN of primates is much less understood than the AVCN. Along with

bushy cells, the DCN has several other types of cells each manipulating and transforming the

stimulus information in different and dramatic ways. There have been experiments done on cats

that suggest the DCN plays a role in vertical sound localization by using spectral cues. Young et

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al. found DCN glutamatergic neurons in cats projecting to the inferior colliculus are sensitive to

spectral notches, a unique frequency that helps sound localization along the vertical plane.6

When a lesion is made to these projecting neurons behavior of the cat’s orientation to the

stimulus sound is perturbed from their control behavior for both learned and reflexive head

orientation.7, 8

These results suggest that the DCN can be considered a location of early

processing of sound localization for elevation before being fully processed into auditory space at

the inferior colliculus. Rhesus monkeys seem to have the same glutamatergic excitatory

neurotransmitters commonly seen in lower mammals, such as rats. However, M. E. Rubio et al.

were unable to find if the inhibitory

neurotransmitters at play were glycine,

gamma-aminobutryic acid (GABA), or

both.8

Affecting the inputs to both the

AVCN and DCN is how sound enters

the ear and contacts the tympanic

membrane. The physical presence of

the torso, shoulders, head, ears, ear canal and especially the pinnae all contribute to head-related

transfer functions (HRTFs). HRTFs are the alterations of the sound wave due to its translation

from moving the air to colliding with all of the physical obstacles and vibrating the eardrum. In

this sense, the ear never hears the true sound in the way it was emitted from the free-field, but

only hears the sound after it has been altered from HRTFs and other confounding factors (type of

medium, other objects in environment, etc.). As the sound changes location, spectral cues result

from changing HRTFs which can be used in the localization of sound.

Figure 7: Detailed view of the central ascending auditory pathways 1

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B. Superior Olivary Complex

After the division of pathways at the cochlear nucleus, the AVCN’s projecting neurons

divide to synapse at contralateral and ipsilateral centers at the level of the mid-pons with the

release of glutamate to inspire excitation. The superior olivary complex (SOC) is among these

structures and is the first point at which sound from both ears are integrated via the decussation

of nerve fibers at the trapezoid body (Fig. 6). Integration here gives rise to the localization of

sound by comparing the differences between what each ear hears. Two different circuits are

responsible for the integration at the SOC, one at the medial superior olive (MSO) and the other

at the lateral superior olive (LSO). These different centers of integration are responsible for

different types of auditory information: interaural time differences (ITDs) and interaural level

differences (ILDs).

Interaural time difference (ITD) is the perception of sound at slightly different moments

in time by the MSO cells. MSO cells act as a coincidence detector to integrate the information of

different sound arrival times. Sound information reaches the ipsilateral and contralateral MSO

respective to the location of the sound, where depolarization occurs on the ipsilateral side

slightly before the contralateral

side. In the MSO, auditory nerve

fibers split to make several

parallel circuits. As the

depolarization wave from

auditory nerve fibers in the

cochlea takes its time to spread throughout these divisions in the MSO, MSO cells are

approaching threshold but never succeed in fully doing so. These cells need a coincident

Figure 8: Diagrammatic look of how the MSO integrates

sound information into location 4

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depolarization from the ipsilateral and contralateral side to attain a cumulative depolarization

great enough to reach threshold and fire an action potential. When the action potential is fired,

that MSO neuron is associated with a motor neuron that communicates head movement to the

direction the sound originated. As a sound source moves along the azimuth away from the

midline, the time of arrival between the ipsilateral and contralateral depolarizations is increased

and thus the head has to move a longer distance to face the sound. When the ears perceive no

difference in arrival of low frequency sound waves, the MSO localizes the sound to originate

from the midline of the observer.

Bushy cells from the AVCN project to the MSO and cause excitation via glutamate

neurotransmitter. Due to the projection’s decussation at the trapezoid body, MSO cells respond

to sounds in the contralateral sound field. The ears are separated by the distance of head width

and can detect the different arrival times for sounds that are below a certain frequency. For

humans, this frequency is anything less than 2 kHz. This is because the average human head

width is about 15cm and the wavelength of a 2 kHz frequency is also about 18cm. Auditory

nerve fibers’ electrophysiological ability to phase lock is also consequently lost for higher

frequencies. ITDs that can be perceived are very small, being on the order of 10 microseconds in

humans. Phase locking is absolutely necessary for detecting time differences in lower

frequencies. Perception of delayed sound arrival as small as 10 microseconds would not be

possible if the fine temporal resolution from phase locking was absent.

Interaural level differences (ILDs) are a compliment to ITDs for sound localization of

stimuli frequencies greater than 2 kHz. At higher frequencies, sound wavelength decreases and

the ears can no longer perceive the different times the wave hits one ear and not the other.

Instead the head acts as an acoustic obstacle and “shadows” the ear opposite to the sound.

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Intensity, or level, of the sound is perceived differently between the two ears from shadowing.

ILD cues are integrated by the LSO cells, being activated by ipsilateral ear stimulation and

inhibited by contralateral ear stimulation via glycine and GABA neurotransmitters. Inhibition of

the LSO arises from the medial nucleus of the inferior colliculus, or MNTB.4 This translates to

ipsilateral LSO activation and contralateral inhibition in the presence of a sound, a trait helpful

for localization of differing sound intensities. Axons of the LSO cells cross the midline to the

contralateral inferior colliculus for further integration at the level of the caudal midbrain.

C. Inferior Colliculus

Ascending neurons from the lateral and medial superior olivary complex travel to the

inferior colliculus via the lateral lemniscus and the nucleus of the lateral lemniscus. Glutamate

neurotransmitter is active at this excitatory input. The inferior colliculus serves as the point of

integration of azimuthal and vertical sound cues to yield an idea of auditory space for the

observer. ITDs and ILDs are derived from the MSO and LSO, respectively, to give the location

of a sound on the horizon of the field of observation. Localization of vertical sounds arises from

distinctly different mechanisms and is less understood than ITDs and ILDs.1

Vertical sound localization is essential in the generation of accurate auditory space

information for the observer. Unlike visual perception where space is physically mapped onto

brain areas, auditory space has to be calculated at the ICC using the ILDs, ITDs, and spectral

cues. Spectral cues arise from head-related transfer functions and the pinna’s unique shape for

filtering out vertically oriented sounds. Deformation of the pinna results is a considerable

hindrance for the filtering process. Amongst these cues are spectral notches. A spectral notch

occurs when the gain of an auditory signal drops dramatically at a certain frequency, making a

deep crevice, or notch, appear on the response recording. Localization of the sound’s elevation

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comes from the association made between the loss in signal intensity at a given frequency.

Higher elevations of sound sources have higher frequency spectral notches and lower elevations

of sound sources have lower frequencies. The pinna has a uniquely specialized structure to assist

in this perception of high versus low frequencies. There are central neuromechanisms that

contribute to the detection and computation of spectral notches, but those will not be covered in

this paper.1

Directly above the inferior colliculus is the superior colliculus, a center for visual

processing. This proximity suggests a location of integration between hearing and seeing to help

the movement of the eyes to a sound.12

Matching the focus of the eyes to what the ears detect

could be important for finding, identifying, and ultimately assigning value to a sound. Locating

predators or prey could provide an evolutionarily explanation as to why visual and auditory

information could start integration at the brainstem, the “primitive” part of the brain.

D. Medial Geniculate Body

After the integration of the horizontal and vertical auditory cues, the inferior colliculus

ascends projections to the medial geniculate body (MGB) at the thalamus. The MGB, chiefly the

ventral division, acts as a thalamic relay for further ascending projections to the primary auditory

cortex. Excitatory projections are principally glutamatergic and inhibitory projections

GABAergic, although there are a few minor exceptions.10

The MGB is the most inferior structure

where there is mixing of multiple characteristic frequency signals and has shown selectivity for

time intervals between the arrival of two frequencies.4 Similarly, Wang et al. in their 2008 study

found supporting evidence that the MGB acts as an intermediate stage of processing between the

inferior colliculus and primary auditory cortex. They found the first mixed presence of responses

phase-locked and non-phase-locked to the stimulus.13

Understanding speech requires the

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presence of both temporal and frequency information and thus the MGB could be considered the

first ascending center for speech processing.

E. Primary Auditory Cortex

The primary auditory cortex, or A1, is the final major structure in the auditory system.

Contributing the most projections to A1 is the MGB, sending glutamatergic and GABAergic

projections for excitation and inhibition, respectively. Glutamatergic projections also descend

back down to the MGB, inhibiting its activity. Located on the superior temporal gyrus in the

temporal lobe, A1 consists of Brodmann areas 41 and 42. Neuronal connections from the MGB

to A1 are point-to-point, ensuring a high fidelity of the ascending signal’s tonotopic

organization. The conscious perception of sound, like recognition of speech, would be much less

conceivable if it were not for the maintenance of this organization. Akin to the visual and

somatosensory systems with their sensory epithelia, the auditory system also has a topographical

map of the cochlea on A1. Lower frequencies corresponding to the apex are expressed on the

ventral side of A1 and higher frequencies corresponding to the base are expressed on the dorsal

side of the cortex. Patches of neurons are perpendicular to the cochlear tonotopic map, some are

excited by both ears (EE) and others excited by one ear and inhibited by the other (EI). The EE

and EI stripes alternate, similar to that seen in the primary visual cortex with ocular dominance

columns.

A1 has also been found toe exhibit duality in its signal representations of sound.13

Temporal representation is used to encode sound signals that change slowly over time whereas a

firing rate-based representation is used to encode rapidly changing sound signals. Expression of

auditory information in this manner gives perspective to the changes the signals underwent

coming from the MGB at the thalamus. Identifying and understanding speech and other animal

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sounds, as well as music, are some of the abilities gained from these temporal and rate-based

representations. These findings suggest that the cortical representations of sound are completely

distinct from the original “acoustic structures” which, Wang et al. argues, is necessary for the

auditory brain to do more complex calculations, such as sound segmentation, object processing,

and integration of multiple sensory perceptions. The secondary auditory cortex, A2, is

immediately proximal to A1 and is comprised of the Wernicke’s area, a center critical for the

comprehension of speech.

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Chapter 2 Research Review of the Rhesus Monkey Auditory System in the Context

of Aging and Caloric Restriction 3

I. The Wisconsin Study16, 17

A. Background of the Wisconsin Aging and Caloric Restriction Study

The following studies concerning auditory function in the rhesus macaque monkey

(Macaca mulatta) were carried out using the data collected from an earlier project. This long-

term study began in 1989 by Joseph Kemnitz et al. with the focus to develop the rhesus monkey

as a model for human aging and age-related disease. Through close monitoring of biomarkers,

the chief aim of the research was to establish the rate of aging in the rhesus macaque and

secondarily determine the effect of caloric restriction (CR) on the rate of aging. Non-human

primates offer greater translatability of the research to humans as opposed to similar aging

studies using animals like rats, spiders, guppies, or protozoa. Rhesus monkeys are ideal research

animals in this context because of their relatively long lifespan and their phylogenetic similarity

to humans seen both anatomically and neurophysiologically. It is this level of similarity that

allowed the following study summaries to build upon the 1989 CR-aging study and contribute to

further understanding of rhesus monkey auditory function in the context of aging and caloric

restriction.

B. Methods and Materials of the Wisconsin Aging and Caloric Restriction Study

This study utilized 76 rhesus monkeys that were all born at the University of Wisconsin-

Madison inside one of two facilities: the Wisconsin National Primate Research Center (WNPRC)

or the Harlow Center for Biological Psychology. In 1989, 30 adult rhesus monkeys in the age

3 Nearly the entirety of the material written in each article summary comes from that same article. These

references are included at the end of the paper. Any other external material is referenced as usual.

23

range of 8-14 years old (mean age = 9.3 years) were admitted to the CR and aging study as the

first cohort. Later in 1994, an additional two cohorts were admitted to the study. The first group

consisted of 30 females and a second group consisted of 16 males. All of these newly admitted

animals were aged to be in early adulthood aging around 10 years old. In total, there were 30

females and 46 males in this study. Within each cohort, monkeys were randomly chosen with the

randomization goal to create balance between the two groups according to age, pre-study body

weight, and baseline food intake.

Baseline measurements were made to determine the correct amount of food and nutrients

to supply for the following years of study. All animals were first fed ad libitum for 6-8 hours per

day for a 3-6 month period to determine the quantity of food for the control group and the diet

restricted group. The diet consisted of a pelleted, semi-purified food (Teklad, Madison, WI)

which contains 15% lactalbumin, 10% corn oil, and approximately 65% carbohydrate in the form

of sucrose and corn starch. A daily treat of fresh fruit was provided at the end of the day to all

monkeys, both the control and CR groups. Animals in the CR group had a similar diet which

included a supplement of 30% additional vitamins and minerals to ensure the monkeys, on

average, consume a similar amount of micronutrients and were not malnourished. Diet was

reduced in the CR group of by 10% increments over a period of three months for a total of 30%

diet restriction. Reduction quantities varied in each animal according to their individual baseline

averages. In the 1989 cohort of only males, the monkeys acclimated well to the palatable diet

change and gained weight. However, as time passed food consumption voluntarily decreased by

about 28% from initial levels. Once this lower level of ad libitum feedings stabilized, the new

quantities of food for the control and CR groups were established accordingly over this 18-month

24

assessment and later applied to the 1994 second and third cohorts. This resulted in a further

reduction of food in the CR animal diet by about 20%.

The rhesus monkeys were kept individually caged in large rooms. Room temperature was

kept around 21oC with a humidity of 50-65%. This allowed the control and accurate monitoring

of water and food intake as well as providing visual and auditory contact with the other

monkeys. Individual housing was the most reliable method to implement 30% of caloric

restriction in the diet restricted group while also deterring aggressive encounters. Also provided

were tree branches and other non-injurious objects for play. Environmental factors of the housing

space that were controlled included temperature, humidity, and 12-hour lighting periods from

0600 to 1800 to simulate day and night.

C. General Methods of Auditory Testing in the Wisconsin Study Rhesus Monkeys

18

All animals that underwent auditory testing had previously been involved in the

Wisconsin study researching the effects of caloric restriction on aging in rhesus monkeys.

Exposure to this study occurred 102, 42, or 36 months prior to admission for audition studies.

Testing utilized 68 rhesus monkeys consisting of 41 males and 27 females. There were fewer

monkeys used for testing due to a few animal deaths from complications in anesthesia,

cardiomyopathy, gastric bloat, cerebral edema, septicemia, severe gastritis, or amyloidosis. The

monkeys were within the age range of 11-23 years old when the current study began, but entered

the broader longitudinal aging study of caloric restriction 102, 42, or 36 months prior. For the

following article summaries, animals were termed adult or elderly based on several maturation

benchmarks of the rhesus monkeys. Average age of sexual maturity in the rhesus monkey is

about 5 years old and reaches adult stature at 8 years. Females enter menopause in their early

20s. In a laboratory environment, rhesus monkeys have a median life expectancy of 26 years and

25

a maximum life expectancy of about 40 years. Considering these points of significance in rhesus

monkey maturation, an adult is considered to be in the age range of 8 to 14 years old, whereas

elderly monkeys are above this range.

The following studies evaluated the preservation of auditory function under caloric

restriction and aging. Preservation of auditory function was assessed using methods such as

auditory evoked potentials like auditory brainstem response (ABR) and middle latency response

(MLR), distortion product otoacoustic emissions (DPOAE), acoustic admittance (Ytm), ear canal

volume (Vea), and tympanometric width (TW). The monkeys were anesthetized with ketamine or

Telazol via intramuscular injections in conjunction with other biomarker assessments. Valium,

xylazine, or isofluorane were used for additional sedation if needed. In the audition testing room,

background noise was accounted for and was not detectable in either the ABR or MLR

responses. Middle ear function was considered before any data collection to assure there was no

conductive hearing loss. Ear canals were checked for occlusions and blockages. Once these

checks were passed, the rhesus monkeys underwent a screening tympanometry test to again

check for conductive hearing loss. If a monkey had a visible occlusion or flat tympanogram,

testing was postponed until the conductive pathology causing the issues was identified and

corrected. Tympanometry is tested as a function of changing air pressures in the ear canal to

measure the dynamic admittance of the middle ear. Methods slightly vary from study to study for

choosing auditory function measurements, types of anesthesia, location of testing, and general

methodology. Any methodology that differs from what has been described above will be detailed

in the subsequent article summaries. Methodology will be completely covered in article

summaries that were not done by exclusively by the University of Wisconsin-Madison.

26

II. 2000 Article Review: Aging and middle ear function in rhesus monkeys.

Peter Torre III et al.

A. Introduction

The purpose of this study came from the idea that in order to better understand the

sensorineural mechanisms and functioning of the auditory system, conductive hearing must be

understood first. Further research of the rhesus monkey conductive auditory system is needed to

evaluate what is considered normal and not normal, thereby enabling future studies to ask more

specific research questions for both conductive and sensorineural hearing. Incoming sound to the

peripheral and middle ear can be perturbed by the pinna, ear canal, tympanic membrane,

ossicular chain, and the tensor tympani and stapedius middle ear muscles. Disturbance of the

sound before transduction into electrical signals can alter how the sound is sensed and perceived.

Central auditory signals are much too prone to these disturbances, creating a gap in auditory

understanding. It is for this reason why the study was carried out. While it has already been

identified that rhesus monkeys are relatively close to humans in a phylogenetic perspective, an

important difference to recognize is the size and morphology of the conductive components to

the outer and middle ear. Ossicles are smaller and lighter, which can vibrate faster and are

responsible for a higher frequency sound range to be heard, upwards of 40,000 Hz. Humans on

the other hand have a max frequency range of about 20,000 Hz.

The study consisted of two experiments. The first experiment measured the peak acoustic

admittance (Ytm) and equivalent ear canal volume (Vea). The second measured middle ear

resonance frequency. Peak acoustic admittance is the smallest magnitude of sound reflected off

the tympanic membrane and middle ear when a tone is emitted from the microphone in the

tympanometer rubber tip. Equivalent ear canal volume is the approximate volume between the

air tight tympanometric probe tip and tympanic membrane. To understand the definition of

27

resonance frequency, an understanding of conductance, spring susceptance, and mass

susceptance is needed. For audition, conductance is simply the in-phase movement of air with

the stimulus sound. Susceptance may be understood as the measure of how vulnerable air and

middle ear components are to conducting vibrational change. Resonance frequency is comprised

of spring susceptance and mass susceptance. Spring susceptance is the degree of how vulnerable

spring-like components of the middle ear, such as ligaments, muscles, the tympanic membrane,

and air will conduct vibrational change. Mass susceptance is the tendency for components of the

middle ear, such as the pars flaccida of the tympanic membrane, the ossicles, and the perilymph

of the cochlea, to impede the conduction of vibrational change. Spring susceptance leads

conductance by 90o in its sinusoidal traveling sound wave and mass susceptance lags

conductance by 90o. When spring and mass susceptances in the middle ear are equal, they cancel

out and create the greatest dampening of sound being transmitted through the system15

. The

dampened product wave was defined as the resonance frequency. Size and shape of the outer ear

and ear canal can affect the resonance frequency and thus the input to the central auditory

system. Tympanometry was used in this study to account for the Ytm, Vea, and resonance

frequency measurements. Since the effects of age and sex were relatively well documented in

humans, they were evaluated comparatively to rhesus monkeys. This was done with the intention

to give an idea of the translatability of findings in the rhesus conductive auditory system to the

human’s conductive auditory system.

B. Methods and Materials

In both experiments, otoscopy was performed for each animal to affirm the ear canal was

clear of any occlusion that could disrupt the results. The first experiment measured peak acoustic

admittance and equivalent ear canal volume with admittance tympanograms using a 220-Hz tone

28

emitted from the probe. The sample size consisted of 33 total adult rhesus monkeys, all of which

were not included in the Wisconsin CR-aging study. This cohort had adult and elderly monkey

groups. There were 17 elderly rhesus monkeys, nine females and eight males (mean age=26

years, SD=10 months). The adult monkey group contained 16 monkeys consisting of eight

females and eight males (mean age=10.9 years, SD=7 months). Before any testing had been

done, the monkeys were anesthetized intramuscularly with ketamine (15 mg/kg) and

intravenously with valium (1 mg/kg) as needed for additional sedation. Middle ear function was

measured with a Grason-Stadler tympanometer. A rubber-tipped probe was wedged into the ear

canal to create a hermetic seal, preventing ambient noises from entering the ear and isolating

only the emitted tones to be heard by the subject’s ears. Acoustic admittance tympanograms

were measured with a 220-Hz probe frequency for all monkeys. This was done using a “positive-

to-negative” direction pressure change and at a pump speed of 50 daPa/sec. The acoustic

admittance value at +200 daPa was used to estimate Vea.

The second experiment sought to evaluate the changes in the middle and peripheral ear

resonance frequencies due to aging. Resonance frequency of the middle ear was determined by

using a range of 20 tones from 226 Hz to 2000 Hz. Sixteen rhesus monkeys were studied. Eight

of the monkeys were older adults (4 males and 4 females, mean age=15.5 years, SD=1 month)

born at the University of Wisconsin-Madison. The other eight of the monkeys were all female

elders (age range=28-32 years old). All sixteen of these monkeys were part of an earlier study

researching the effects of the soft metal lead on auditory functioning through behavior and

development. Control animals never had additional exposure to lead. The lead-exposed animals

had their exposure end in1984 and were tested for this study in 1996. It is unlikely that the lead-

exposed monkeys had any auditory pathology since monkeys of the same age (15 years old) had

29

no different hearing function as determined by the methods used in this study. Of course,

however, it must be recognized that the lead exposure could alter results.

Auditory testing was done in an Industrial Acoustics Corporation sound booth. Monkeys

were sedated with an initial intramuscular injection of 5-6 mg/kg Telazol (Fort Dodge

Laboratories, Fort Dodge, IA). If needed, a following intramuscular injection of 5-10 ketamine

(Ketastet, Fort Dodge Laboratories) was given. Assessing of the resonance frequencies was done

via a Virtual 310 system connected to Apple Macintosh personal computer. Tympanograms were

measured at 20 different frequencies from 226 Hz to 2000 Hz with a consistent pressure change

from -200 through +500 daPa at 75-100 daPa/s.

Resonance frequency was measured implicitly at the surface of the probe microphone by

measuring the uncompensated acoustic admittance. This uncompensated acoustic admittance

consists of two components: the admittance of the ear canal and the admittance of the tympanic

membrane. By subtracting the uncompensated acoustic admittance from the admittance of the

ear canal (at +200 daPa), the tympanic membrane’s admittance can be estimated. This magnitude

of admitting sound into the tympanic membrane gives rise to the general auditory admittance to

the middle ear. Recall that resonance frequency was defined as the point when mass and spring

susceptance, the tendency to conduct or impede vibrational change in middle ear components,

are equal in magnitude and out of phase. At this point, the admittance is zero. To determine the

resonance frequency, admittance of the occluded ear canal (+200 daPa) was subtracted from the

uncompensated admittance.

30

C. Results

General results found that peak mean acoustic admittance, ear canal volume, and middle

ear resonance frequency were similar between monkeys and humans. More specifically, peak

acoustic admittance and mean ear canal volume in the monkeys were most similar to human

children. A trend of elder monkeys (28 to 32 years old) having lowered peak acoustic admittance

and ear canal volume appeared evident, but the trend held no statistical significance. There was a

significant main effect of sex similar to that found in humans where males had larger acoustic

admittance amplitudes and ear canal volumes than females, despite the age of the monkey. No

significant effects regarding age or sex in middle ear resonance frequency were found.

In the first experiment, a statistically significant effect of sex for the tympanogram was found

(p=0.008). Males had larger acoustic admittance peaks than females. There was marginal statistical

significance in age (p=0.07) and no significance in an age by sex interaction (p=0.48). Although there was

a trend of smaller ear canal volume with age. Similarly, there was statistical significance in sex for peak

acoustic admittance values (p=0.04). There were significantly larger ear canal volumes that belonged to

the males. The main effect of age (p=0.54) and age by sex interaction (p=0.43) did not reach statistical

significance. Table 1 shows how peak acoustic admittance and ear canal volume compared according to

sex and age group.

Figure 9 displays the results from the second experiment using feather plots to show

mean compensated and uncompensated calculated admittances for both the adult (15-year-olds)

and elderly monkeys (28-to-32-year-olds). Each vector represents one of 20 frequencies used in

Table 1. Peak acoustic admittance (Ytm) and ear canal volume (Vea) organized by sex and age group. Standard

deviations are in parentheses.14

31

the 226 Hz to 2000 Hz range.

Angle and length of the vector

arrows signifies the behavior of

the phase as probe tone

frequency changed. It was found

that uncompensated resonance

frequencies for the adult and

elderly age groups were very

similar. Compensated resonance

frequencies from the elderly

monkeys were higher than the adult

monkeys. However, there were no significant differences of resonance frequencies between the

age groups in either uncompensated or compensated resonance frequencies as determined by the

0.05 level. Any differences in resonance frequency by sex at the age of 15 years old were either

small or non-significant. When controlling for sex, non-significant differences between adult and

elderly monkeys for compensated resonance frequency were not eliminated.

D. Discussion

An underlying goal of this study was to assess translatability of the rhesus monkey

peripheral auditory system to humans. The effects of sex and age have been significantly more

thoroughly documented in humans and served as reference for the similar findings to rhesus

monkeys. Peak acoustic admittance decreased with age in adult monkeys. This may be explained

by the stiffening of the middle ear system in the tympanic membrane or ossicular chain. Even

though there were differences in peak acoustic admittance between adult and elder monkeys, the

trend was similar to the magnitudes and direction reported in human studies. With a greater

Figure 9. Feather plots displaying mean compensated and

mean uncompensated admittances for both the adult and

elderly rhesus monkey. 14

32

sample size and statistical significance, those studies found younger human adults had peak

acoustic admittances of 0.72 acoustic mmhos, whereas older human adults had peak acoustic

admittances of 0.66 acoustic mmhos. The peak acoustic admittance found in rhesus monkeys

was similar to the value found in human children, being an average of 0.5 mmhos.

Size and shape of the outer and middle ear is similar between humans and rhesus

monkeys. This is supported by mean ear canal volume of the monkeys falling in the 90th

percentile of human children (0.3-0.9 cm3). Differences in ear canal volume between the adult

and elder monkeys were small and not statistically significant in this study. It has also been

reported that ear canal volume decreases with age. Rhesus monkey ear canal volume differences

found in this study as a function of age is similar to the magnitude of difference reported in

human adults. Resonance frequency had no change with age in the rhesus monkeys, which is

consistent with human findings as well. A trend of increasing resonance was apparent but did not

reach statistical significance. Larger sample sizes are needed to dismiss or affirm the trend in

following studies.

Findings in significance of sex were females had smaller acoustic admittance values and

ear canal volumes than males. Smaller acoustic admittance volumes are likely attributed to the

stiffening of the ossicular chain from aging. Smaller ear canal volumes in females parallel the

findings in adult human studies. This is expected because of the size difference in the ear canal

and ossicles in males and females, for both rhesus monkeys and humans. There has been limited

research regarding the general anatomy of rhesus macaque middle ears and none, at the time of

this study, concerning the sex differences in the outer and middle ear systems. This study begins

to contribute to this gap in understanding of sex-related anatomical differences in the rhesus

monkey outer and middle ears. Female monkeys had slightly higher resonance frequencies than

male monkeys, which are also seen in humans. This is consistent with the finding that females

had slightly lower peak acoustic admittance values found in both rhesus monkeys and humans.

33

III. 2002 Article Review: Effects of caloric restriction and aging on the

auditory function of rhesus monkeys: The University of Wisconsin Study.

Cynthia G. Fowler et al.

A. Introduction

The purpose of this study was to gather data on presbycusis in the rhesus macaque and to

determine if caloric restriction preserves hearing function in tandem with the expected

improvement of health in later life. Presbycusis is the common pathological name of

predominately high-frequency and progressive sensorineural hearing loss. Caloric restriction’s

effects on auditory function was assessed along three dimensions: preservation of neural function

tested by auditory brainstem response (ABR) and middle latency response (MLR), preservation

of binaural function tested by ABR and MLR, and the preservation of auditory thresholds

measured by ABR. Measurements of neural function to estimate the auditory threshold is

assessed by both ABR and MLR measures.

In humans, it has been found that presbycusis begins earlier and is worse in men than

women. This is often attributed to a difference in lifestyle factors, such as type of occupation and

recreational activities. However, a 2001 study by Wiley et al. found that when equating the risk

factors that may negatively affect auditory function, men still have poorer hearing than women.

This may be due to some underlying pathophysiology regarding the variables under scrutiny in

this study, i.e., sex, age, and dietary conditions. Some physical changes that can be attributed to

hearing loss include some stiffening of the tympanic membrane and middle ear ligaments, as

well as the enlargement of the external auditory canal.

B. Methods and Materials

Three different protocols were followed to test the three focuses of this study. Latencies

were measured from the onset of the stimulus to the positive peak of each wave. Amplitudes

34

were measured with respect to the averaged baseline for that condition.

The study’s first purpose was to evaluate the preservation of auditory neural function in

aging, caloric restricted rhesus monkeys via administering ABR and MLR tests to one ear at a

time. Monaural clicks were given first to the right ear at 110 dB pSPL at a rate of 25/s for ABR

and 9.3/s for MLR. The process was

completed again for the left ear.

Peak latencies and amplitudes were

measured for ABR waves I, II, and

IV and MLR waves Po and Pa

(Figure 10). Interwave intervals I-IV

and IV-Pa were also noted.

The second focus of this study was to evaluate if caloric restriction in aging rhesus

monkeys preserves binaural function by ABR and MLR measures. This was tested similarly with

the first study objective, using the same click intensities and rates for ABR and MLR testing,

however the clicks were given binaurally. The binaural interaction component, or BIC, acted as a

measurement of how well the two ears worked in concert. BIC was derived from ABR and MLR

by subtraction of the binaural response from the computer summation of the left and right

monaural responses. Latencies and amplitudes were measured for wave V in the summated

binaural response, for left and right ears individually, and for their major positive peak in the

different waveform. Amplitude ratios of the BIC to the summed monaural response were used to

normalize the amplitudes of summed monaural responses across subjects.

Lastly, the third focus to evaluate if caloric restriction in aging rhesus monkeys preserves

auditory thresholds was done so by ABR measurements. To begin, the sound stimuli was given

Figure 10.Representative rhesus monkey waves in ABR on the

left panel and MLR on the right panel.18

35

to the right ear and reduced at 20 dB increments from high levels and 10 dB from low levels

until the sound could no longer be heard. The sound was then increased by 5 dB steps until the

monkey could hear the tones. Once this was established, this sound level was determined to be

the threshold for the right ear. After, the signal was given to the left ear at 5 dB lower than the

newly established right ear’s threshold. This was done to affirm that the better threshold had

been obtained in the right ear. Auditory threshold was defined as the lowest level at which a

response in the better ear could be identified by visual detection on the auditory brainstem

response recording.

C. Results for First Purpose: Does CR Preserve Monaural Auditory Function?

Investigation of whether or not CR preserves monaural auditory function in the aging

rhesus monkey was tested by ABR and MLR. Both of these auditory evoked potentials were

evaluated by analyzing the mean latencies and mean amplitudes, as well as the absolute latencies

and absolute amplitudes. There was no significant mean latency difference found between the

left and right ears in the subjects except for wave II. Waves I, IV, Pa, and Po did have significant

mean latency similarities, (p=0.038). Table 2 shows the wave latencies in milliseconds for each

Table 3. Mean latencies (in ms), for ABR and MLR waves. The average was taken from the left and right ears of

subjects in groups with respect to their diet and sex. Standard deviations are given in parentheses.18

Table 2. Latencies (in ms), for ABR and MLR waves in the left and right ears and their average. Standard deviations

are given in parentheses.18

36

ear and their averaged value. Wave II had a mean latency difference of 0.01ms which fell under

the measurement error of 0.02ms. The difference was deemed likely due to a sampling error

suggested by statistical analysis. With this in mind, it was determined that there were no

significant mean latency differences between the two ears. All wave mean latencies, including

wave II, were significantly correlated (p<0.001), with r values ranging to a significant correlation

low of 0.605 for Pa and a high of 0.855 for wave II. These correlation significances were great

enough to reasonably allow the averaging of latencies for any analysis that came afterward.

Table 3 shows these averages sectioned by dietary conditions and sex.

Absolute latencies for all waves had no significant differences save for wave Pa. Sex was

shown to be significant for wave Pa latency with females having shorter absolute latencies than

males. Age was also a significant factor and was linked to sex. Older adult males of 18 to 23

years old had longer latencies than older adult female monkeys of 18 to 19 years old, while

younger adult monkeys of both sexes had similar absolute latencies.

Mean amplitude differences were not significant between the left and right ears for any of

the waves (t-test). Wave II, IV, and Pa absolute amplitudes had significant correlation between

the ears (P<0.001). Between the two ears, there was a correlation low of 0.509 for wave IV and

high of 0.596 for wave Po. Wave I and Pa did not significantly correlate. As with the latency

measurements, the similarity of these amplitude values allowed the use of averages for

subsequent analysis. Table 3 gives the mean amplitudes for the left and right ears grouped

37

according to their dietary condition and sex. The

amplitude values are separated by their respective

wave and standard deviations are given in

parentheses.

Linear regression analysis was used for

the comparison of two variables, such as age and

sex. Wave I amplitude had significance from age

(p=0.011) and sex (p=0.001). Wave I amplitudes

decreased with age and overall were smaller for males than females. Amplitudes decreased faster

in wave I for males than females with age (p=0.005). Wave II had no significant effects for any

of the variables. Wave IV amplitude had significant effect on sex (p=0.028) where male

monkeys had smaller amplitudes than female monkeys. The effect of CR was stronger in females

than in the males (p=0.021). Females had an increase of 633% for wave IV amplitude compared

to control females (Figure 11). Control males had 17% larger wave IV amplitudes than the CR

males. This is theoretically attributed to the decreased efferent inhibition of the outer hair cells

from the superior olivary complex. Wave Po of the MLR approached significance for age

(p=0.073) and sex (p=0.059). Wave Pa amplitudes had no significance effects observed.

Interwave intervals I-IV and IV-Pa wave gaps were analyzed to measure brainstem

transmission time and neural transmission, respectively. While the interwave intervals came

from direct measurements, IV-Pa interwave gaps were implied measurements. This is because

the wave IV latency came from the ABR measure and the Pa latency from the MLR measure. A

significant effect of sex was found (p=0.043). Females had shorter interwave intervals than the

males had. There was also a significant sex by age interaction (p=0.027) in that older adult males

Figure 11. Mean wave IV amplitudes showing

the relationship between diet and sex. Negative

values are seen due to the reference of

amplitude values to the averaged baseline.18

38

had longer intervals than older adult females, and younger adult male and females had similar

interwave intervals.

D. Results for Second Purpose: Does CR Preserve Binaural Auditory Function?

Investigation of whether or

not CR preserves binaural auditory

function in the aging rhesus monkey

was tested by ABR and MLR. There

were no significant differences for

latencies in the scope of dietary

condition of the monkey or the

animal’s sex. However, binaural wave IV did have a significant interaction for dietary condition

by age (p=0.045). Using linear regression, the binaural wave IV amplitudes decreased faster in

control animals than caloric restricted animals as their age increased. The left and right ear sum

(L+R) for wave Pa had amplitudes that decreased with increasing age (p=0.029). A developing

trend for the caloric restriction by age interaction was also observed. L+R wave amplitude

decreased faster for older adult monkeys than younger adult monkeys, but to a greater extent in

control monkeys.

E. Results for Third Purpose: Does CR Preserve Auditory Thresholds?

Investigation of whether or not CR preserves auditory thresholds in the aging rhesus

monkey was tested by ABR. The third purpose to this study was to observe if caloric restriction

in aging rhesus monkeys preserves auditory thresholds as measured by ABR. Researchers

defined the threshold to be the smallest wave IV amplitude that could be visually identified.

Dietary group and sex showed a linked interaction trend where caloric restriction affected

Figure 12. Wave IV amplitude comparison of CR and control

rhesus monkeys. The regression line shows amplitude

decreases more rapidly as the monkeys age.18

39

threshold more in males than in females

(p=0.058). The diet restricted females had

thresholds 2 dB higher than their respective

controls whereas male monkeys had

thresholds 7 dB lower than their controls.

F. Discussion

Rhesus macaques were chosen as the

model research animal for this large study of senescence because of their evolutionary proximity

to humans on the phylogenetic tree as well as their relatively long life. Biomarkers of aging seen

in the rhesus monkey could be indicators of very similar aging mechanisms seen in humans.

Auditory signal amplitudes observed in this study are in need of additional scrutiny with

a focus on the effects of anesthesia. Anesthesia may affect some of the response results for the

amplitudes of signals in the ABR and MLR. In other studies, amplitudes were generally larger.

This study used the method of measuring peak amplitude relative to the average baseline.

Measuring relative to the baseline could buffer the apparent magnitude of some responses,

making them seem smaller than they actually are, whereas the other studies used peak-to-peak

measurements and provided an absolute perspective of the amplitudes.

It is important to note when looking at these results that the males were older on average

than the females. All of these monkeys entered this audition study after first being in the primary

study researching CR’s effects on aging. The monkeys were also divided across three cohorts

that entered the CR study at two different times: the first male-only cohort began in 1989 (mean

age=9.3 years), the second and third cohorts began in 1994 with 30 females and 16 males,

respectively (mean age= ~10 years). Data of this 2002 study pools the older, first cohort of only

Figure 13. Female and male ABR thresholds under

caloric restriction or control diet conditions.14

40

males and the last two cohorts of females and males that entered the study five years later, thus

skewing average male ages to be older.

Older adult males were at the stage when presbycusis began to become evident and could

be seen in the ABR measurements. Since the females were younger than the males on average,

the lessened degree of their poorer hearing may simply be a result of age. The older average

population of males may have also significantly skewed the results of findings such as mean

wave IV amplitudes showing the relationship between diet and sex (Figure 12). If all monkeys

were the same age, or if there were comparisons done only with the last two cohorts that entered

the current study, perhaps the findings would be less dramatic. Females did not exhibit any signs

of presbycusis in their thresholds in the diet restricted group or the control group. In the male

group, it is possible that they may be exhibiting more of the loss in cochlear function. It was

observed that males had a greater wave I amplitude reduction than females. Humans have wave I

amplitude reduction seen with high-frequency hearing loss that was not severe enough to

increase latencies. Also, all females in this study were pre-menopausal, which usually have an

onset age of 25-27 years old. Menopause may have an effect on auditory function and should be

further investigated.

The duration of caloric restriction needs to be considered for additional study. Males had

been a part of the caloric restriction study for 102 months before being tested in this study of

focus whereas the females had only done 42 months at the most. These differing period lengths

of caloric restriction may have had an effect on the thresholds. Any questions about this matter

can be addressed with simply continued monitoring to observe any of the same effects on MLR

and ABR testing arise in the female group. This would help indicate if the threshold differences

are due to age, caloric restriction, or some combination of both.

41

Focus of continued study should also include further inquiry of the relationship of age to

auditory brain waves. Monkeys in this study had wave Pa and IV-Pa interwave intervals increase

which suggests deficits in the upper brainstem or primary auditory cortex. In general, a trend was

observed showing ABR thresholds get poorer with age, particularly in control monkeys. The

threshold distribution across age for the entire group of monkeys suggested ABR thresholds

don’t start increasing until the age of about 18 years old. The oldest control monkeys had the

worst thresholds out of all of the monkeys. This takes into consideration that the males have

been, on average, in the dietary restriction study considerably longer and are older than the

females.

Presbycusis in humans is associated with the prevalence of cardiovascular disease. People

with experimental diets of reduced fat had drops in their cholesterol, lowered numbers of new

coronary artery disease onset, and a significantly better auditory threshold compared to those

with higher fat diets. Those who have cardiovascular disease have been reported to have worse

auditory function. The temporal bones in patients with hearing loss commonly also had a

narrowing internal auditory artery which may lead to a reduction in blood supply to the cochlea

and spiral ganglion thereby causing atrophy.

IV. 2004 Article Review: Assessment of auditory function in rhesus monkeys:

effects of age and calorie restriction. Peter Torre III et al.

A. Introduction

Caloric restriction (CR) has been shown to reduce the incidence of age-related disease

and retard physiological effects of aging in many different animals, including mice and rats. The

Wisconsin National Primate Research Center (WNPRC) and the National Institute on Aging

(NIA) have conducted roughly parallel studies investigating the effects of CR in aging rhesus

monkeys, which is a relatively close human ancestor that offers translatability of research to

42

human health. A 2002 WNPRC study conducted by Fowler et al. researched the age-related

changes of auditory function in rhesus monkeys and has already been summarized in this

chapter.18

This study done by the NIA largely parallels the WNPRC’s study done a couple years

ago to further add to the literature concerning aging, CR, and auditory function. The CR

monkeys involved with the NIA study were also first exposed to the long-term study of CR on

aging before becoming a part of this study. Otoacoustic emissions (OAEs) from the outer hair

cells are among the similarities already identified in the previous paper summaries of this

chapter.

OAEs are used in this study to assess cochlear function and to so, distortion product

OAEs, or DPOAEs, were measured. Recall that OAEs are created by outer hair cell

electromotility and, in effect, move the basilar membrane in the cochlea. Basilar membrane

movement causes depolarizations and hyperpolarizations which eventually lead to the emission

of sound from the inside of the middle ear. When two frequencies enter the ear simultaneously,

such as from a probe, their respective waves have an overlap on the basilar membrane and evoke

a distortion tone. A DPOAE is the resulting distortion tone from the wave overlap.19

Rhesus

monkeys have been shown to have similar DPOAEs to humans and among the monkeys

themselves, DPOAE magnitude decrease with age.

Auditory evoked potentials (AEPs) were used in this study to assess the auditory

brainstem responses (ABR) and middle latency response (MLR) measured via an

electroencephalogram (EEG). ABR peaks II and IV were the easiest to identify with the weakest

stimuli and are most comparable to a human’s V ABR wave. Recall that the 2000 Torre et al.

study found peak I, II, and IV ABR amplitudes were significantly smaller in elder monkeys

compared to the adult monkeys.14

Binaural peak IV amplitudes decreased significantly faster

43

with age than control monkeys compared to the CR monkeys. Elder monkeys had longer peak II

and IV latencies as well as fewer peak IV responses for lower stimulus levels. MLRs had no

found significances in Pa amplitude or latency in relation to age. All of these findings suggest

cochlear or neural function decline with aging.

In regards to the caloric restriction dimension of this investigation, previous studies that

used rodents had somewhat conflicting results. Beneficial, neutral, or even deleterious effects

from CR have been seen in lab mice and rats. In diet restricted rats, some strains had neither

auditory function nor longevity improved. Other strains had responses that indicated increased

auditory function and lifespan when exposed to CR.

B. Methods and Materials

The study used 50 total male rhesus monkeys consisting of 26 controls (mean age = 18.6

years, range 13-32 years, SD = 6.1 years) and 24 monkeys that underwent dietary restriction

(mean age = 20.4 years, range = 13-36 years, SD = 7.6 years). These CR monkeys had already

been in the CR study for 12-13 years. Control group animals were fed a low-fat diet high in fiber

at approximately ad libitum levels twice a day. CR animals were fed this same diet but had a

reduction in calorie intake by 30% compared to controls matched by age and weight. Diet was

provided with approximately 40% more vitamins and nutrients than the control levels while

maintaining the composition to reduce the chances of malnutrition in the CR monkeys. These

food allotments were modified based upon the National Research Council requirements

according to the monkey’s age and weight. All animals received a daily low calorie treat and had

ad libitum access to water.

Environmental control of the rhesus monkeys was quite comparable to that described in

the Wisconsin study by Fowler et al. in 2002.18

From the start, all of the animals were housed

44

continuously at the National Institutes of Health (NIH) Animal Center in Poolsville, MD. All

rhesus monkeys were singly housed in standard weight-appropriate sized cages and exposed to

only fluorescent lighting which was controlled to have 12-hour period, simulating day and night.

Room temperature was maintained between 22oC and 28

oC and humidity in the range of 50-

60%. The animals were kept in the same room to allow auditory and visual contact with other

monkeys. Toys and other manipulanda were included in the monkeys’ housing environment.

Intramuscular delivery of anesthesia with Telazol (4—5 mg/kg) (Elkins-Sinn, Inc.,

Cherry Hill, NJ) was performed to sedate the animal before any screening or testing. Additional

anesthetic of inhaled isofluorane was used when more restraint was needed. An otoscopic exam

was done before any testing to remove occlusions and was confirmed with a screening

tympanometry. This better ensured quality data by ruling out middle ear pathology of conductive

hearing that could influence the DPOAEs or AEPs. A rubber-tipped probe was used to obtain a

hermetic seal of the monkey’s ear canal. Applied pressure varied from -200 daPa to +200 daPa

and acoustic admittance was measured with a 226 Hz probe tone. Measurements of ABR, MLR,

and DPOAEs were used to determine any age-related changes of auditory function in the

observed male rhesus monkeys.

Distortion product otoacoustic emissions (DPOAE) were first recorded with a screening

test. The probe assembly was placed into the ear canal and the cochlea was stimulated with two

simultaneous frequencies (f1 and f2, where f2 > f1). Distortion product of the OAE was measured

by subtracting f2 from f1. Emissions were measured by using a f2/f1 ratio = 1.22 as f2 varied from

2000, 3000, 4000, and 5000 Hz. Levels of these frequencies were also kept constant (L1 and L2)

at 65 and 55 dB SPL, respectively. Absolute DPOAE and noise floor level were also measured.

Noise floor level is the amount of sound being detected in addition to the sound of interest,

45

which is the DPOAE in this case.19

DPOAE/noise ratio was determined by subtracting the noise

floor level from the DPOAE level. DPOAE level and DPOAE/noise ratios were used to

determine cochlear function.

Auditory evoked potentials (AEP) were assessed by auditory brainstem responses (ABR)

and middle latency responses (MLR). Rarefaction clicks were played into insert headphones at

the rate of 25/sec and at a level of 110 dB SPL. Monaural responses were measured for each

monkey and averaged at 10.24ms. This was attained by performing 500 sweeps. Responses were

replicated to make sure the waveform was reliable. Latencies and amplitudes for ABR waves I,

II, and IV were measured at 110 dB SPL. Peak latencies were measured from the stimulus onset

to the maximum point of the corresponding peak. Peak amplitudes were measured from the

maximum point of the peak to the following negative trough. Low-level ABR responses were

determined using 20 dB and 10 dB steps for high and low level sounds, respectively, and 5 dB

level steps to refine the found threshold. The ear that could detect the lowest threshold was

labeled the better ear. MLR was measured in the same fashion as the Fowler et. al study in

2002.18

An average was taken after 500 sweeps and collected in a 99.84ms timeframe. Peak

latencies and peak amplitudes were measured similarly to ABR peak measurements, from peak

to the subsequent trough.

C. Results

All data from all 50 monkeys involved in the study was analyzed because no middle ear

pathology or ear canal occlusions was detected via baseline tympanometry and DPOAE

screening tests. One CR monkey had a missing DPOAE and ABR measure and one control

monkey had a missing ABR amplitude measure because of physiologic or extraneous noise

interfering with the data collection. Six CR and eight control monkeys had no MLR measures for

46

their amplitudes and latencies. Any missing information was not included in the subsequent

analyses.

Right and left ears both had statistical significance. To avoid confusion about individual

or averaged measurements between the two ears, left ear data was ignored and only the right ear

data was used for analysis. Table 5 shows the Spearman comparisons between the left and right

ears for each dependent variable accounted for. DPOAEs of the right ear in a representative

monkey are shown in Figure 14. The top line with filled circles signifies the absolute DPOAEs at

specific frequencies 2000, 3000, 4000, and 5000 Hz. The bottom line with filled squares signifies

the noise floor at the same frequencies.

Figure 15 shows the ABR (top portion) and MLR (bottom portion) waves of the right ear

in a representative rhesus monkey. Significance was found in an age effect at each frequency for

DPOAE measurements determined by a multivariable ANCOVA. Linear regression analysis was

used to determine the direction of the associations. Absolute DPOAE level decreased with age

significantly at frequencies 2000 Hz (p < 0.05), 3000 Hz (p < 0.05), 4000 Hz (p < 0.05), and

5000 Hz

Table 5. Spearman correlations between left and right ears for dependent variables of focus.20

47

(p < 0.05). DPOAE/noise ratios were

similarly significant and decreased

with age at 2000 Hz

(p < 0.05), 3000 Hz (p < 0.05), 4000

Hz (p < 0.05), and 5000 Hz (p <

0.05). Regression analysis showed

absolute DPOAE level decreased at

about 0.70 dB (∼−0.06 × 12 months)

per year at 3000, 4000, and 5000 Hz.

There was only a decrease of 0.43 dB per year at the 2000 Hz frequency. DPOAE/noise ratios

decreased in the range of 0.43 to 0.66 dB per year for every increase with age, at these measured

frequencies.

Regarding the ABR measures, wave II’s peak amplitude significantly decreased

(p < 0.05) and wave IV’s peak latency significantly increased (p < 0.05) with age. ABR

threshold also had a significant age-related increase (p < 0.05). For every year the animals aged,

amplitude of peak II decreased by an average of 0.01 µV and latency of peak IV increased 0.01

ms. ABR threshold increased 1.2 dB per year.

Dietary effect was found to have no significance for any of the ABR, MLR, or DPOAE

variables as well as no significant interaction with age. The study population was divided into

age groups of old adult monkeys (13-18 years old) and elderly monkeys (26-36 years old) for

further analysis of how aging affects audition. Figure 16 shows changing absolute DPOAE levels

with age in the old adult and elderly monkeys. The similarity and lack of significant difference

between the control and dietary restricted groups can be seen relatively clearly. This lack of

Figure 14. DPOAE measures in the right ear of a rhesus

monkey. The top line shows absolute DPOAEs and the

bottom line shows noise floor levels.20

48

significance is reinforced further by the similarity in ABR thresholds between the control and

dietary restricted groups across all monkeys as well as elderly monkeys divided into the control

and CR groups.

D. Discussion

The primary focus of this study was to evaluate the change in cochlear and auditory

neural function in rhesus

monkeys as they age.

Concerning cochlear function,

DPOAE levels between 5 and

8 db SPL for the tested

frequencies (2000, 3000, 4000,

and 5000 Hz) were similar to

previous findings in younger

rhesus monkeys by Lasky et.

al in 200019

. However, this

study collected data in a

typical testing room, whereas

the Lasky study used a

commercial sound booth

which could have made a

difference in background noise

levels. Absolute DPOAE level

ranges have been observed to Figure 15. Representative auditory evoked potential

(AEP) waves from a rhesus monkey. ABR waves are on the

top panel and MLR waves on the bottom panel. 20

Figure 2 shows the ABR (top portion) and MLR (bottom

portion) waves of the right ear in a representative

rhesus monkey

49

decrease over time as the monkeys grow older. Torre and Fowler found younger monkeys (mean

age: 10.75 years) had absolute DPOAE levels at 10-15 dB SPL for frequencies 2000 through

5000 Hz.14

This 2004 study found older monkeys (mean age: 19.5 years) had absolute DPOAE

levels at 5-8 dB SPL for the same frequencies. Finally, the oldest monkeys (mean age: 25.9

years), which were in the Torre and Fowler study, had absolute DPOAE levels -0.3-5 dB SPL for

these frequencies.14

Also across these four frequencies (2000, 3000, 4000, and 5000 Hz), this

study found an approximate drop of 0.4-0.7 dB SPL in absolute DPOAE levels each year where

the other study found a 0.67 dB SPL drop in DPOAE levels each year.14

All of these findings

clearly indicate that there is some sort of age-related loss of cochlear function in audition for

rhesus monkeys and supports previous reports of cochlear degeneration in morphology. It’s

important to recognize that the DPOAE range of frequencies from 2000 to 5000 Hz is only a

small portion of the rhesus monkey’s auditory range that extends to 32 kHz. The narrow

frequency range studies here warrants further investigation of larger frequency ranges toward the

rhesus monkey frequency perception limit of 32 kHz. In the context of human comparison, older

humans had lower absolute DPOAE levels than younger adult humans.

Concerning neural function, ABR measures seen in this study and the Torre and Fowler

2000 study were rather comparable and fit with the pattern of data.14

Here they found the mean

latency peak of wave I and II to be 1.24ms and 2.20ms, respectively, for younger monkeys.

These values are equal to those found by Torre and Fowler14

. Mean peak IV latency for the

younger monkeys (mean age: 10.8 years) and older monkeys (mean age: 25.9 years) in the Torre

and Fowler study were on lower and higher ends, respectively, compared to the mean peak IV

latency for monkeys found in this study (mean age: 19.5 years). The researchers for this 2004

study found a peak IV latency increase in 0.01ms per year whereas other studies have found an

50

average of peak IV latency to increase by 0.025ms per year or had similar values. The

discrepancy of the value in peak IV latency increase per year from this study compared to others

may be attributed to a variety of factors such as difference in species and thus anatomical size,

level of the administered stimulus, which could lead to longer ABR peak latency measurements,

and age distributions of the monkeys. In humans, older people had longer ABR latencies and

reduced ABR amplitudes than younger people.

Peak Pa from the MLR was 24.6ms, which is similar to earlier findings. Peak II

amplitude significantly decreased at 0.01 µV per year, which was also previously reported.

However, this study had shown greater decreases in peak ABR amplitudes and peak MLR

amplitudes than other peak amplitudes in past studies. This could be due to a difference in the

layout of the electrode montages or age distributions from those studies. In further comparison to

the Fowler et al. 2002 findings18

, latencies found in mean ABR peak and peak Po from MLR

were similar in the control and CR males of this study. Peak Pa latency, however, was smaller in

the Fowler et al. study18

. Overall, some of the auditory evoked potential amplitudes were similar

to those found in Fowler et al. but different methods of measuring those amplitudes were

different.18

This study measured from the peak amplitude to the following trough, whereas

Fowler et al. used an averaged baseline approach. Male control monkeys and diet restricted

monkeys in this study and the Fowler et al. study had similar ABR thresholds, but CR monkeys

in the Fowler et al. study had lower ABR thresholds compared to this study. This may be due to

the fact that this study had older CR monkeys on average than the Fowler et al. study’s CR

monkeys. Aside from these measurements regarding neural functioning, no other ABR or MLR

waves tested for significance.

The second focus of this whole study was to investigate the effects of caloric restriction

51

on auditory function in the context of aging. Although many studies have come before the

WNPRC and NIA nonhuman primate aging studies researching caloric restriction, they were

done with mice which had many different strains. This makes it hard and ill-suited for

comparison, especially when considering the translatability of findings to humans. Diet did not

test into significance in any measure implemented in this study (Fig. 16), including no significant

age by diet interactions. However, older CR monkeys had similar measures to the younger

control monkeys in regards to mean peak latencies, peak amplitudes, and ABR thresholds. There

was a trend of worsened auditory function in the CR animals than in the control animals. This

could be attributed to the relatively late introduction of CR in the monkeys’ lifespan and suggests

that the timing of CR introduction is critical.

It should be noted that all of the findings found in this study were with male subjects

only. No factors of sex can be analyzed and compared within this study alone. Gender

comparison is crucial to further understanding of the mechanisms at play with the observed

effects, or lack thereof, in cochlear function, neural function, and caloric restriction.

While the methodology used in the WNPRC and NIA studies were rather similar, such as

housing and prepping the animal for testing, a key difference was the method of determining the

assigned food allotments. The NIA in this study measured the meals based on National Research

Council requirements according to monkey age and weight. A 40% addition to the NRCs

requirements was applied before implementing 30% CR to the entire diet restricted group while

maintaining the composition. A more personalized approach was used by the WNPRC. Food

allotments were based on a 3-6 month period of ad libitum feeding and 30% CR was determined

from those quantities to individually restrict the diets for each unique animal16

. The difference

52

between a standardized and individualized feeding scheme may explain some of the differences

in results found between the two studies.

V. 2008 Article Review: Tympanometry in rhesus monkeys: Effects of aging

and caloric restriction. Cynthia G. Fowler et al.

A. Introduction

The specific focus of this study was to determine if middle ear function in the aging

rhesus monkey is preserved by caloric restriction, as measured by tympanometry. Editors of this

Figure 16. Changing DPOAE levels in juvenile and old rhesus monkeys separated by dietary condition. 20

53

study belonged to the University of Wisconsin-Madison as well as outside institutions Oregon

Institute of Technology, University of Minnesota Medical Center, and University of Alabama.

All methods and results that came from this study were implemented and recorded at the

Wisconsin National Primate Center (WNPRC) at the University of Wisconsin-Madison. The

measures used here to evaluate how middle ear function is affected by aging and caloric

restriction were acoustic admittance (Ytm), ear canal volume (Vea), and tympanometric width

(TW).

The researchers identified a few anatomical changes that occur in aging humans which

could provide prospective for the recent findings as reported by the 2002 WNPRC study and

2004 NIA study. Thickening of the tympanic membrane and development of arthritis in the

ossicular chain have been found to be most evident in people older than 70. Equivalent ear canal

volume can reduce with age, which is attributed to the fact of collagen breakdown in the

cartilage of the ear canal, leading to collapsing canals, and thus decreased ear canal volume. In

anatomical comparison to rhesus monkeys, the tympanic membrane is more circular with a

diameter of 5.8mm and angles medially from the top of the canal by about 130o. Ossicular shape,

size, and orientation all are slightly different in the rhesus macaque.

Findings, however, have not been unanimous and differ considerably across institutions.

The researchers of this study wisely attributed the variability of results may be due to the fact

that as monkeys age, the data gathered will also become increasingly variable. This could be due

to confounding variables including physiological, neurological, and pathological change over

time as the animals mature and age. It was in this context and the large absence of data the

researchers defined the purpose of their study to evaluate tympanometry in rhesus monkeys to

determine the effect of age and caloric restriction. The concentration was divided into three

54

constituents. The first objective was to investigate if middle ear function changes as rhesus

monkeys age. The second objective was to investigate if caloric restriction affects middle ear

function in rhesus monkeys as they age. Finally, the last objective was to research in role rhesus

monkey sex may play in middle ear changes with age.

B. Methods and Materials

This study utilized the data collected from monkeys first exposed to the CR-aging study

(1989 and 1994) at the WNPRC for the first audition study done in 2002. Therefore, all methods

regarding the conditions the tested monkeys came from can be seen in section I of this chapter

discussing the background and methods of the Wisconsin CR-aging study.16,17

At the time of this

study, 52 of the original 76 monkeys had survived to serve as the subject population. All of the

rhesus monkeys aged between 15 and 26 years of age (mean age=20.5 years, SD=2.9 years). All

methods regarding how acoustic admittance and ear canal volume were measured can be seen in

section II when they were first reported in the Fowler et al. 2002 study.18

The exceptions to this

are the pressure sweep range, pump speed, and tone used for tympanometry. In this 2008 study,

the pressure sweep ranged from +200 daPa to -400 daPa. Pump speed of 600 daPa/s was used

and slowed to 200 daPa/s near the peak of the tympanometric reading to achieve better peak

Table 6. Summary of admittance data for Vea, Ytm, and TW across all monkeys .

(CR = caloric restricted, Cont = control).21

55

definition. The tone played through rubber-tipped probe was at +226 Hz. The only measure used

in this study that has not been introduced thus far is tympanometric width (TW). TW is defined

as the width of the tympanometry peak at 50% of the peak’s height.22

C. Results

Table 6 provides a summary of admittance data for Vea, Ytm, and TW across all monkeys.

Table 7 shows the pooled data from both ears. Data analysis used the pooled data because of how

well the data paired from the two ears. For Vea, no two-way interactions tested significance for

the variables sex, diet condition, and age. The effect of sex was the only variable that tested

completely statistically significant (p=0.003). Male rhesus monkeys had slightly larger mean Vea

(0.32cm3) than females (0.27cm

3). The effect of diet tested to be only marginally significant

(p=0.054). Diet restricted monkeys had slightly lower mean Vea (0.31cm3) than control monkeys

(0.28cm3). The effect of age did not test into significance (p>0.05)

Ytm also had no significant two-way interactions with age, diet condition, or sex. Sex and

diet effects did not test significant as well. The effect of age was marginally significant

Table 7. Pooled data from both ears from all monkeys grouped here by diet condition and sex

(CR = caloric restricted). 21

56

(p=0.067). Peak Ytm plotted as a function of age is shown in Figure 17. Ytm is averaged across

both ears. A general trend of reduction is apparent in the amplitude of peak Ytm as age increases.

Effects of diet were not significant in TW. The effect of age was significant (p=0.001)

with an increase in TW with age. Sex was marginally significant (p=0.080). Males had a more

narrow TW (73 daPa) than females (82 daPa). The interaction of age by sex was significant

(p=0.039). This suggests TW stays relatively the same in aging males whereas female TW

increases with age. TW averaged across ears is shown in Figure 18 plotted against age where

females and males have separate slopes. Age and TW for females was statistically significant

(p=0.028), but was not for males.

D. Discussion

Recall the main purpose of this

study was to determine if CR had any

effect on the development of aging as

measured by middle ear function in the

rhesus monkey. Peak acoustic admittance

(Ytm), tympanometric width (TW), and ear

canal volume (Vea) were the measures used

to evaluate these effects. In regards to Ytm, most of the measures showed no significant changes

in aging control monkeys. This suggests that there was no effect that could be altered by dietary

condition. Absolute peak Ytm in this study were smaller than the Torre et al. 2000 study, which is

very likely to be attributed to the larger number of younger monkeys in that study. This is

because most monkeys in the study at hand (mean age=20.5 years, SD=2.9 years) are aged

relatively closely to the older monkeys in that study (26 years, SD=10 months). Data for aging

Figure 17. Individual amplitudes for Peak Ytm plotted as

a function of age. The regression line though the data

points is shown. Some data points are obscured by

others. 21

57

humans is relatively similar to these findings in that there were either significant decreases or

non-significant trends of decrease in acoustic admittance with age. Some studies found there to

be significance in decreases in acoustic admittance but not with age, whereas other studies found

no significance at all. With such variance in these findings, it strongly suggests that the decrease

in acoustic admittance amplitude with age is small.

As for TW, values increased as the

monkeys aged, which is consistent with the

stiffening of the middle ear system. This

would naturally co-occur with a decrease in

Ytm with age. TW increase has also been

associated with aging in humans. This study

shows that TW remains relatively

unchanged in aging males, but increased in

widening for aging females. Further

longitudinal study is necessary to affirm the

expectation of continued increase in TW for

both females and males.

Vea in this study group of rhesus

monkeys was not affected by age. In the

2000 Torre et al. study, they did not find significantly different Vea between the studied age

groups, but did identify a trend for decreasing ear canal volume with advancing age. Those

researchers also found that male rhesus monkeys had significantly larger Vea than females and

these findings were reflected in this study. CR monkeys had slightly smaller Vea than control

Figure 18. Individual tympanometric data for

tympanometric width averaged across ears plotted

against age. Females and males have separate slopes

indicating the interaction of age and sex. Males (thin

line) have solid data points. Females (thick line) have

hollow data points.21

58

monkeys, but the mechanism for this is unknown. The researchers suggest that perhaps CR

exacerbates breakdown of cartilage in the ear canal. This effect would compound over time as

the monkey aged and is suggested to result in smaller Vea with aging. Further long-term study

should continue to better investigate this possibility. Female humans have been consistently

found to have smaller Vea than male humans over a wide range of ages. Vea averages in rhesus

monkeys are also quite comparable to those found in human children.

VI. 2010 Article Review: Auditory function in rhesus monkeys: Effects of

aging and caloric restriction in the Wisconsin monkeys five years later.

Cynthia G. Fowler et al. A. Introduction

The University of Wisconsin began the study of caloric restriction’s (CR) effect on

longevity and health span in the rhesus macaque monkey in 1989 with a cohort of only males.

Later in 1994, two more cohorts were added, one of only females and the other of only males. At

the time this 2010 study was published, recent findings from the Wisconsin study on CR and

aging in rhesus monkeys included improved health span and reduced mortality compared to the

control monkeys. Specifically, the improved health in CR monkeys constituted of lower body fat,

improved insulin sensitivity, and better cardiovascular function than the age-matched controls

being fed ad libitum. These physiological improvements also decreases the known risk factors

associated with hearing loss in humans, such as cardiovascular disease and diabetes. This

suggests the possibility that hearing could be preserved in the CR monkeys as they age. There

were three purposes of this study: (1) to continue research of the rhesus monkey as a human

model for presbycusis via auditory function evaluation, (2) to continue investigating the effects

of CR on auditory function within the context of age-related hearing loss (ARHL), and (3) to

determine the length of time CR needs to be present in order for protective effects against ARHL

59

to become evident. The first two purposes were evaluated by DPOAE measures obtained from

the Wisconsin study CR-aging monkeys. The last purpose was conducted with only the oldest

monkeys that entered the Wisconsin study in the first cohort in 1989.

B. Methods and Materials

Recall that the monkeys apart of this study had come from the Wisconsin study pool of

rhesus monkeys, which at the time of this study were 55 animals consisting of 22 females and 33

males. They had been subjected to the CR and aging study for varying amounts of times based

on when their cohort was first admitted. By the time these measurements were taken, the

monkeys had already participated in the Wisconsin study for 162, 102, or 96 months (13.5, 8.5,

and 8 years, respectively). Monkeys were randomly assigned with the randomization objective to

have balance between the two groups according to age, pre-study body weight, and baseline food

intake. Basic methods of this study were previously covered in the summary of the Wisconsin

study at the beginning of this chapter. To evaluate auditory function in the aging monkeys, the

methods of measurement used were distortion product otoacoustic emissions (DPOAE) and

auditory brainstem responses (ABR). An auditory evoked potential system (Intelligent Hearing

Systems) was used to measure DPOAEs (SmartOAE 4.31 USBez) and ABRs (SmartEP 3.62

USBez).

The animals who participated in the DPOAE testing included 22 females and 31 males

aged from 15-28 years old. Controls had a mean age of 20.5 years (range 15-26 years) and CR

monkeys had mean age of 20.6 (range 15-28 years). Procedure and methods were largely the

same as the 2004 NIA study except that the varying f2 frequencies played were different, using

2211 Hz, 3125 Hz, 4416 Hz, 6250 Hz, and 8837 Hz tones. Also, sixty-four sweeps were

performed for each DPOAE and then averaged.

60

ABR thresholds were measured with clicks and 8, 16, and 32 kHz tone bursts. In total, 48

monkeys had reliable data for the three frequencies and 54 monkeys had reliable thresholds for

clicks. Binaural stimuli was administered to ensure the threshold of the better hearing ear was

obtained. To obtain the click and 8 kHz thresholds an insert earphone (Etymotic ER-3A) was

used. Thresholds of 16 and 32 kHz were obtained with a high frequency transducer (IHS-3432)

placed 15 cm away from the ear. The center of the diaphragm was placed level with the ear canal

opening. Two trials of 700 sweeps were conducted at supra-threshold levels twice and 1000

sweeps at near-threshold levels. Finding the lowest threshold for the superior ear was conducted

similarly to the 2004 NIA study. Signal levels began at 100 dB pSPL and decreased at

20 dB steps for high levels and decreased at 10 dB levels near threshold, including one or two

steps below threshold. Threshold was defined as the lowest level the observer could still report a

hearing response.

C. Results

Regarding DPOAEs, the data were significantly correlated (p < 0.001) but were not

significantly different for the right and left ears (p=0.8049). Data from both ears of each monkey

were averaged at the five DPOAE f2 frequencies 2211, 3125, 4416, 6250, and 8837 Hz. A scatter

plot of these individual DPOAE levels scatter plotted against age in years can be seen in Figure

19. Even though the data points were quite variable for all of the monkeys, the regression line

slopes shown in Figure 19 are all nearly parallel. They all also display a consistent decrease level

with age. These DPOAE slopes were -0.983 for 2211 Hz, -0.928 for 3125 Hz, -0.965 for 4416

Hz, -0.807 for 6250 Hz, and -0.881 for 8837 Hz. All of the data show DPAOE levels declined at

a rate of nearly 1 dB/year for every frequency. DPOAE levels were not different between ears in

either groups divided by diet conditions or by sex. Refer to Table 8 for the diet divisions and

61

Table 9 for the sex divisions. DPOAE levels did not relate to either sex or diet after removing the

non-statistically significant two-way interactions from the statistical analysis. Trends for age

were essentially found to be null across the DPOAE levels at the five f2 frequencies as indicated

by the scatterplots. DPOAE levels decreased with increasing age for some of the frequencies

tested when diet and sex were controlled: 2211 Hz (p=0.0055), 3125 Hz (p=0.0026), and 4416

Hz (p=0.0184). Marginal significance was found in the DPOAE levels at frequency 6250 Hz

(p=0.0554). There was no significance for the DPOAE levels at frequency 8837 Hz. Background

noise levels were ruled out as an influencing factor when no significant effects were found with

age, sex, diet, and their interactions analyzed with the linear mixed models.

The researchers also tested the hypothesis for the length of time CR has to be exerted in

order for beneficial health effects are observed using the first cohort of the study, which

consisted of only males who had been exposed to CR for 13.5 years. This was to investigate if

CR had been implemented for a long enough duration for there to be a significant effect on

DPOAEs. There was no found no significant differences between the CR and control groups.

Figure 19. individual DPOAE levels scatter plotted against age in years. Regression lines for all five

frequencies are given. Some data points are obscured by others.23

62

However, the older cohort had increased loss in cochlear function at the highest frequencies

compared to the two younger cohorts. Slopes of DPOAE level by age functions were -0.531 at

2211 Hz, -0.462 at 3125 Hz, -0.726 Hz at 4416 Hz, -2.362 at 6250 Hz, and -2.054 at 8837 Hz.

ABR thresholds increased with age at each stimulus given, as seen in figure 3. None of

the interaction effects tested to be statistically significant and so were excluded from all other

analyses. Click thresholds had

marginal significance with the effect

of age (p=0.051), but there was a

significant curvilinear effect

(p=0.0341) which suggests that the

effects of age start to become

significant around the age of 21

years. Diet’s effect had marginal

significance on click thresholds

(p=0.0543) and controls had the

higher thresholds. Responses at 16

kHz the effect of age was marginally

significant (p=0.068) but also had a

significant curvilinear effect

(p=0.0485) which also suggests the

effects of age start to become

significant after the age of 21 years.

In 32 kHz responses, the effect of

Table 9. DPOAE levels for male and female monkeys and

total monkeys for left and right ears for the five f2 frequencies

tested.23

Table 8. DPOAE levels for left and right ears for the five f2

frequencies tested, for total, CR, and control monkeys.23

63

age was significant (p=0.0012) and the responses increased with age. Sex was marginally

significant (p=0.0514) and males had the higher thresholds. Duration of time spent in the study

was not statistically significant after controlling for age.

D. Discussion

The focus of the research in

this study was to address the effects of

CR, age, and sex on DPOAE levels

and ABR thresholds in aging rhesus

monkeys. All results found are only in

the context of developing ARHL in

rhesus monkeys and how it may be

related to auditory function in aging

humans. The effect of age had the

strongest statistical significance on auditory function. DPOAE levels decreased in a linear

fashion with increasing age and ABR thresholds increased with age, the latter meaning of the

tested group, the eldest monkeys had the greatest ABR threshold measures. Diet’s effect was

marginally significant showing lower thresholds at 8000 Hz for CR monkeys. The effect of sex

was marginally significant finding slightly greater ABR thresholds in males at 32 kHz.

The finding of linear decrease in DPOAE with increasing age is supported by other

studies using rhesus monkeys for human models. Direct comparison is difficult because of

varying DPOAE frequencies used along with the types of measurement used in these studies. For

example, the Torre et al. NIA study (2004) used five DPOAE frequencies of 2000Hz to 5000 Hz.

They found DPOAE levels decreased significantly with age for all frequencies used. Slopes

Figure 20. Individual ABR thresholds for monkeys by age for

each signal (clicks and 8, 16, and 32 kHz. Some data points

are obscured by others. 23

64

indicated 0.7 dB/year loss in 3000-5000 Hz frequencies and 0.4 dB/year for the 2000 Hz

frequency. This study by Fowler et al. (2010) found a 0.95 dB/year decrease with age in DPOAE

levels for the frequency range of 2211-4416Hz. However, the rate doubled in the frequencies

6250 Hz and 8817 Hz in the cohort which had been in the study considerably longer than the

other two cohorts, participating in the Wisconsin CR and aging study for 13.5 years. ABR

thresholds for clicks and 16 kHz tone bursts increased after 21 years in a curvilinear fashion.

These increases in ABR thresholds support the findings for DPOAE level decreasing with age.

Human studies on the effects of aging in DPOAE levels provide supporting findings to

this study on rhesus monkeys in that they also found decreasing levels with age and at similar

frequency ranges. A study done in 1991 by Lonsbury-Martin et al. found humans aged between

31 and 60 years (a rough equivalent age range when considering the increased rate of aging in

rhesus monkeys) had DPOAE levels drop at a rate of 0.3 dB/year.24

In another human aging

study, Dorn et al. reported decreasing DPOAE levels as a function of age in people 5 to 79 years

old.25

The decreases in DPOAE level had

increased rates among the f2 frequencies 6000

Hz and 8000 Hz, which were nearly the same

frequencies used in this study.

Change in ABR thresholds also declined

with age, meaning the same group of tested

monkeys had relatively unchanged ABR

thresholds compared to their respective values in

the study five years earlier. This was evident when

comparing this study’s ABR thresholds with those

Figure 21. ABR thresholds from Fowler et al. (2000)

(abscissa) and Torre et al. (2004) (ordinate). The line

is the best fit regression for the data. Some data

points are obscured behind others. 23

65

in the Fowler et al. 2002 study and is shown in Figure 21. The average ABR thresholds in this

study were 5 dB lower than the 2002 study, but may be attributed to the method of obtaining the

thresholds. Fowler et al. (2002) used unilateral stimuli in 5 dB steps whereas this study used

binaural stimuli and 10 dB steps.18

The comparison of ABR thresholds between the two studies

was significantly correlated (p<0.0001).

At the time this research was sent for publishing, the only other study that had

investigated change in cochlear function from applied CR was the Forre et al. study done in 2004

with the NIA rhesus monkeys (50 males aged 13-36). In that study, they found no significant

change in DPOAE levels according to dietary conditions using the f2 frequency range 2000 Hz to

5000 Hz. When also considering the findings of this Fowler et al. in 2010, there have been 103

total rhesus monkeys that have exhibited no significant effects in dietary condition on DPOAE

levels within the frequency range of 2000 Hz to 8817 Hz. Both the Torre et al. (2004) and

Fowler et al. (2010) studies took ABR measures as well, and both did not observe any effects of

caloric restriction on click-elicited responses. However, this study administered 8000 Hz tone

bursts and found marginal significance in the CR monkeys having lower ABR thresholds than

controls. This is of importance because this could mean click-stimuli may not be sufficient to

produce subtle changes in response that the high-frequency stimulus seems to do, calling. More

sensitive methods of measurement may be required to better observe meaningful subtleties in the

recordings.

It’s important to recognize the age of onset CR differed between the Torre et al. NIA

study (2004) and the Fowler et al. (2010) study. CR was applied to monkeys in the NIA study at

various life stages while the monkeys at Wisconsin were in early adulthood when CR began.

However, the different age of CR onset yielded no apparent differences between the two studies

66

in the monkeys’ development of ARHL. While other animals, such as rodents, have the duration

of CR and the associated onset age relatively well documented, these studies are the first with

monkeys that have a maximum age of about 40 years. Continued study is necessary to discover

how long CR must be present to begin to see how ARHL development is affected by dietary

condition.

67

Chapter 3 Research Analysis and Suggested Areas of Further Study

I. Introduction

Overall, the articles reviewed in chapter 2 served as contributions to the knowledge base

of the rhesus monkey auditory system. The variables of focus were age, sex, and caloric

restriction as well as the interrelatedness of these factors. Measurements used to evaluate

conductive and sensorineural hearing function included ear canal volume, tympanometric width,

acoustic admittance, resonance frequency, auditory evoked potentials including middle latency

response and auditory brainstem response, and otoacoustic emissions. The rhesus monkey is an

ideal research animal and is of critical importance because of the relatively higher degree of

research translatability to humans as compared to other common research animals, such as mice

and rats. Biological markers unique to aging pathology found in rhesus monkeys could be

indicators of similar aging mechanisms found in humans. Age-related hearing loss, or

presbycusis, is just one of many pathologies associated with aging. Advancements in the

understanding of hearing loss can lead to improving the quality of life for many who may suffer

from common challenges that come with hearing loss such as struggling with independence,

lowered productivity, social isolation, and depression. The following sections are analyses of the

core variables in the reviewed articles, being caloric restriction (CR), age, and gender, while

suggesting areas of further study.

II. Analysis and Suggestions for Future Research

Females were found to have smaller acoustic admittance values and ear canal volumes

than male rhesus monkeys.14

A smaller acoustic admittance volume may likely be attributed to

stiffening, and possibly arthritis, of the ossicular chain from aging. The finding of smaller ear

68

canals in human women parallels this finding.14

This is expected because of the size difference in

the ear canal and ossicles in males and females, for both rhesus monkeys and humans. A later

study done by Fowler et al. (2002) also found females to have a lessened degree of hearing.18

However, this may likely be due to the fact that the male monkeys were older on average than

the females. These were rhesus monkeys from the Wisconsin aging and CR study and one of the

two male cohorts began CR much earlier than the second cohort consisting of females. At this

point of the study, the older group of males was at the stage when presbycusis began to become

apparent. An additional analysis including only the latter two cohorts consisting of 30 females

and 16 males at approximately the same age would perhaps provide less dramatic results.

Tympanometric width (TW) exhibited increases with increasing age in the Wisconsin

study rhesus monkeys.21

This is consistent with stiffening of the middle ear system as well, and

would naturally co-occur with a decrease in acoustic admittance with age. The same CR

monkeys were measured to have slightly smaller ear canal volume than control monkeys.

Researchers from the study postulated this was perhaps from caloric restriction exacerbating the

breakdown of cartilage in the ear canal, thus losing structural support and decreasing in size.21

Regarding anatomy of the rhesus macaque, there has been little research on the general anatomy

of the middle ear and even less concerning the sex differences of the inner and outer systems.

Findings from continued longitudinal study could elucidate the details of conductive hearing and

thus aid in further understanding of sensorineural hearing, since the neural pathways are

dependent on conduction to receive their stimuli.

Duration of caloric restriction needs to be seriously considered for additional study.

Auditory brainstem response (ABR) threshold differences found in the Fowler et al. (2002) study

are hard to assign to age or CR factors or some combination of both.18

Torre et al. (2004) found

69

no effects of CR on auditory function.20

This suggests the male monkeys were either resistant to

the beneficial effects of CR or they began CR too late in life for it to be beneficial for

preservation of hearing. CR began at varying life stages in the male NIA 2004 rhesus monkeys

whereas Fowler et al. (2010) began CR in early adulthood.20, 23

The different ages of CR onset

produced no apparent differences in the two sets of monkeys. All of these findings demonstrate

the need for continued study to determine how long CR must be exerted to observe when age-

related hearing loss development begins to be affected by dietary condition.

Lastly, Torre et al. (2004) and Fowler et al. (2010) both observed a DPOAE level

decrease as rhesus monkeys grew older.20, 23

In the NIA study (50 males aged 13-36), there was

an observed DPOAE decrease rate of 0.4-0.7 dB SPL per year. The Wisconsin study (22

females, 33 males, aged 15-28) observed DPOAEs decreases at 0.95 dB SPL per year for

frequencies 2211 Hz-4416 Hz. This rate doubled at frequencies 6250 Hz and 8817 Hz for the

male monkeys a part of the oldest cohort, participating in the Wisconsin study for approximately

13.5 years. These findings strongly indicate that there must be some sort of age-related loss of

cochlear function and supports previous reports of cochlear degeneration. The rhesus monkey

has a frequency perception limit of 32 kHz and considering the data gathered was only between

2000 Hz and 8817 Hz, additional research in expanded frequency ranges is important.

70

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