1. The Laryngoscope
Lippincott Williams & Wilkins, Inc., Philadelphia
© 2000 The American Laryngological,
Rhinological and Otological Society, Inc.
Effects of Dietary Restriction and
Antioxidants on Presbyacusis
Michael D. Seidman, MD
Objectives/Hypothesis: The premise of this study failed (range, 18 –28 mo; average, 25 mo). This testing
is that the membrane hypothesis of aging, also known included auditory sensitivity studies using auditory
as the mitochondrial clock theory of aging, is the ba- brainstem response (ABR) testing, as well as tissue
sis for presbyacusis. Furthermore, it is proposed that analysis for mtDNA deletions using molecular biolog-
treatment with antioxidants or dietary restriction ical techniques. At the conclusion of the study, ani-
can attenuate age-related hearing loss. Many studies mals underwent a final ABR test and were tested for
have demonstrated a reduction in blood flow to spe- mtDNA deletions in brain and inner ear tissues, and
cific tissues, including the cochlea, with aging. Hypo- the opposite ear was used for histological analysis.
perfusion leads to the formation of reactive oxygen Results: Results indicated that the 30%-caloric-
metabolites (ROM). ROM are highly toxic molecules restricted group maintained the most acute auditory
that directly affect tissues including inner ear struc- sensitivities, the lowest quantity of mtDNA deletions,
tures. In addition, ROM can damage mitochondrial and the least amount of outer hair cell loss. The
DNA (mtDNA), resulting in the production of specific antioxidant-treated subjects had improved auditory
mtDNA deletions (mtDNA del4977 [human] or mtDNA sensitivities, and a trend for fewer mtDNA deletions
del4834 [rat]; also known as the common aging dele- was observed compared with the placebo subjects.
tion]. Previous corroborating data suggest that the The placebo subjects had the poorest auditory sensi-
common aging deletion mtDNA4834 may be associated tivity, the most mtDNA deletions, and the greatest
not only with aging but also with presbyacusis, thus degree of outer hair cell loss. Conclusions: Interven-
further strengthening the basis of the current studies. tion designed to reduce reactive oxygen metabolite
In this study, experiments provide compelling evi- damage appears to protect against age-related hear-
dence that long-term treatment with compounds that ing loss specifically and aging in general. This is re-
block or scavenge reactive oxygen metabolites atten- flected by an overall reduction in mtDNA deletions.
uate age-related hearing loss and reduce the impact These data also suggest that the common aging dele-
of associated deleterious changes at the molecular tion appears to be associated with presbyacusis, as
level. Study Design: Prospective randomized study. demonstrated by an increased frequency of the
Methods: One hundred thirty rats were randomly as- mtDNA del4834 in the cochleae with the most signifi-
signed to one of six groups with appropriate controls. cant hearing loss. Nutritional and pharmacological
Animals were divided into the following treatment strategies may very well provide rational treatment
arms: group 1, 30% caloric restriction; group 2, vita- options that would limit the age-associated increase
min E oversupplementation; group 3, vitamin C over- in ROM generation, reduce mtDNA damage, and re-
supplementation; group 4, melatonin treatment; duce the degree of hearing loss as the organism ad-
group 5, lazaroid treatment; and group 6, placebo. In vances in age. Key Words: Antioxidants, reactive oxy-
addition, 10 animals were used to determine the ap- gen metabolites, anti-aging, presbyacusis.
propriate caloric restriction. All subjects underwent Laryngoscope, 110:727–738, 2000
baseline and every-3-month testing until their health
INTRODUCTION
The premise of this study is that the membrane hy-
Presented as a Candidate’s Thesis to the American Laryngological, pothesis of aging, also known as the mitochondrial clock
Rhinological and Otological Society, Inc. Recipient of the Fowler Award.
From the Department of Otolaryngology—Head and Neck Surgery,
theory of aging, is the basis for presbyacusis. This study
Henry Ford Health System, West Bloomfield, Michigan. expands on the fact that aged animals have a reduction in
Supported by NIDCD grant No. DC00101-01-05. blood supply to the cochlea. It is hypothesized that this
Editor’s Note: This Manuscript was accepted for publication January generalized hypoperfusion leads to the generation of reac-
5, 2000.
tive oxygen metabolites (ROM). The ROM damage mito-
Send Correspondence to Michael D. Seidman, MD, Department of
Otolaryngology—Head and Neck Surgery, Henry Ford Health System, chondria, manifesting as a reduction in mitochondrial
6777 West Maple Road, West Bloomfield, MI 48323, U.S.A. membrane potentials and an increase in mitochondrial
Laryngoscope 110: May 2000 Seidman: Presbyacusis
727

2. DNA (mtDNA) deletions with concomitant hearing loss. concepts he had outlined remain relevant today is an
This study also demonstrate that long-term treatment affirmation of his stature as a scientist and clinician.
with compounds that either block or scavenge reactive Aging research has fortified these precepts as an accurate
oxygen metabolites have a protective effect on age-related and meaningful perspective on senescence and its many
hearing loss and the associated alterations at the genomic manifestations, such as presbyacusis.
level. Presbyacusis is characterized by the progressive de-
This study investigates new hypotheses designed to terioration of hearing associated with aging and is the
enhance the understanding of the molecular and biological most common cause of adult auditory deficiency in the
mechanisms of presbyacusis. The primary purpose of United States, affecting approximately 23% of the popu-
these experiments was to determine whether the mem- lation between 65 and 75 years of age and 40% of the
brane hypothesis of aging has a fundamental role in age- population older than 75 years of age.9 Thus the socioeco-
associated hearing loss. Furthermore, these experiments nomic ramifications are staggering. While presbyacusis is
were designed to determine whether diet restriction and common in industrialized societies, age-related hearing
treatment with compounds known to reduce damage from loss is less pronounced in other societies. This discrepancy
ROM will affect the aging process in general, and presby- has been attributed to many factors, including genetics,
acusis in particular. diet, and socioeconomic and environmental factors.10,11
The process of aging is associated with many molec-
ular, biochemical, and physiological changes including in- Reactive Oxygen Metabolites or Free Oxygen
creases in DNA damage, reduction in mitochondrial func- Radicals
tion, decreases in cellular water concentration, ionic Reactive oxygen metabolites are species that contain
changes, vascular insufficiencies, and decreased elasticity an unpaired number of electrons making them chemically
of cellular membranes. One contributing factor to this reactive and extremely toxic to cellular and subcellular
process is altered vascular characteristics, such as re- structures. It has been speculated that ROM are involved
duced erythrocyte velocity and vascular plasticity, as well in more than 100 clinical conditions.12 They are produced
as increased vascular permeability.1,2 These age-related in vivo during mitochondrial respiration, as well as via
changes result in reductions in oxygen and nutrient deliv- auto-oxidation of chemical and biological molecules. ROM
ery, as well as waste elimination.3,4 Such functional alter- are also environmental contaminants, can be formed from
ations favor the production of ROM. In addition, there is ionizing and ultraviolet radiation, and occur in response to
support in the literature for an age-associated reduction in hypoperfusion or ischemia followed by reperfusion.
endogenous enzymes that protect from ROM damage, in- There is extensive support in the literature for the
cluding superoxide dismutase, catalase, and glutathione protective effects of antioxidants and reactive oxygen me-
peroxidase.5 Collectively, these changes enhance the gen- tabolite scavengers. For example, tocopherols (vitamin E)
eration of ROM. ROM are responsible for producing mito- decrease atherosclerosis and delay death from myocardial
chondrial DNA damage by causing mutations in the mi- infarction, presumably by inhibiting lipid peroxidation.13
tochondrial genome. Specific mutations are known to be Carotenoids, such as -carotene and other plant pigments,
directly proportional to aging, such as the common aging may also have preventive effects against cancer and car-
deletion in humans, which is 4,977 base pairs (bp) in diovascular disease.14,15 Available data also suggest that
length, and in rats (4,834 bp). When mtDNA deletions many of the pathological correlates of Alzheimer’s disease
reach a certain level, the mitochondria become bioener- are precipitated by free radical–induced and oxidative
getically inefficient. Investigators have shown that aged stress–induced mechanisms.16 Similarly, Parkinson’s dis-
rats, monkeys, and humans have reductions in mitochon- ease has also been associated with oxidative stress, in-
drial function and increases in mtDNA deletions in heart, creased lipid peroxidation, reduced levels of glutathione,
brain, liver, and skeletal muscle.6 – 8 To develop the basis high concentrations of iron, and free radical generation
for this study, the following topics are discussed: presby- via autocatalytic mechanisms within neuromelanin-
acusis, reactive oxygen metabolites, mitochondrial func- containing catecholaminergic neurons.17
tion and mitochondrial DNA deletions, aging, and antioxi- Experimentally and clinically, it is well known that a
dants. primary source of ROM generation is through oxidative
phosphorylation, ischemia/reperfusion, or prolonged hypo-
perfusion, such as is seen in myocardial infarction, cere-
HISTORICAL PERSPECTIVES AND REVIEW OF brovascular accidents, aging, and, possibly, sudden senso-
THE LITERATURE rineural hearing loss and presbyacusis. It is clear that in
the aging cochlea there is a significant reduction of blood
Presbyacusis
supply2 and the ongoing need for energy generation
“Biological aging appears to be a physiological
through oxidative phosphorylation. Thus these two pro-
process engendered by the genotype and the adap-
cesses allow for the generation of ROM within the cochlea.
tive norm of the species. Superimposed on the inher-
ited processes seems to be the gradual accumulation
Vascular Changes Associated With Aging
of errors in DNA, which is probably related to a
The aging process is clearly linked with alterations in
decline in normal mechanisms of repair.”
circulatory function. Studies have demonstrated markedly
Dr. Schuknecht displayed tremendous insight when decreased flow within the circulatory system in the elderly
he wrote these lines some two decades ago. That the population.3,4 Prolonged periods of reduced blood flow
Laryngoscope 110: May 2000 Seidman: Presbyacusis
728

3. (hypoperfusion) may lead to decreased oxygen tensions (cytochrome c oxidase), and 2 of the 12 subunits of respi-
within tissues culminating in the generation of damaging ratory complex V (ATP6 and ATP8) (ATP synthase). The
ROM. remaining subunits of these complexes are encoded by
Various lines of research have centered on the con- nuclear DNA.29
cept that altered blood flow and/or oxygen delivery results It has been proposed that mitochondrial genomic mu-
in inner ear hair cell damage and hearing loss. In a human tations may be a major cause of human diseases. The first
cadaveric study of cochlear vessels, an age-related, grad- patient reported with a mtDNA deletion as a cause of illness
ual loss of capillaries in the spiral ligament of the scala was reported in 1959.30 Luft is often credited with demon-
vestibuli was observed, with an increase in intravascular strating the importance of mitochondrial medicine.31 Mito-
strands and avascular channels. Similar but less pro- chondrial mutations followed by cytoplasmic segregation has
nounced findings were noted in the spiral ligament in the been shown to contribute to neuromuscular disorders such
scala tympani.18 Another human cadaver study, using a as Kearns-Sayre/chronic external ophthalmoplegia plus syn-
microsphere technique to quantify blood flow, found di- drome,32 mitochondrial encephalomyopathy, lactic acidosis,
minished flow in morphometrically normal–appearing and stroke-like episodes (MELAS),33 subacute necrotizing
basal turn capillaries.19 Changes in whole blood viscosity encephalomyopathies, SNE (Leigh syndrome) and progres-
and red blood cell rigidity have been correlated with high- sive neuronal degeneration of childhood (Alpers syn-
frequency hearing loss in elderly humans.20 Furthermore, drome),34 and myoclonic epilepsy associated with ragged-red
a series of in vivo experiments using intravital microscopy muscle fibers (MERRF).29 Interestingly, as many as 67% of
of the cochlear microvasculature demonstrated age- patients with mtDNA disorders are known to manifest sen-
dependent, statistically significant reductions in mean sorineural hearing loss.35
erythrocyte velocity and significant increases in capillary Experiments performed since 1989 have demon-
permeability.2 In contrast, Axelsson21 noted no significant strated that mtDNA deletion with gene induction is also
differences in cochlear blood vessels of young and old associated with hypoxia. Corral-Debrinski et al.36 have
guinea pigs. However, there were more failures in con- found a specific mtDNA deletion occurring at increased
trast filling of the older animals’ vessels, because of more frequency in ischemic hearts. They concluded that isch-
frequent ruptures of the vessels.21 It is possible that this emic hearts have increased mtDNA damage and oxidative
may in part be due to increased vascular fragility that is a phosphorylation gene expression that may be associated
common feature of aged vasculature, a speculation en- with oxidative phosphorylation deficiency. In addition,
dorsed by Dr. Axelsson. Furthermore, these findings may there is evidence suggesting 5- to 12-fold elevations in
also be secondary to increased vascular permeability, mtDNA deletions in the brain associated with chronic
which is a common sequela of ischemic injury. hypoxia.37
Deletions of mtDNA with subsequent segregation
Effects of Hypoxia on the Inner Ear and enrichment of the deletions throughout life are con-
The effects of cochlear hypoxia on auditory function tributing factors in the aging process.6 It has been shown
have been extensively studied. There is a direct correla- that mtDNA deletions in the human heart are age depen-
tion between hypoxia and decreased hearing sensitivity. dent.6 Mitochondrial mutations occur and accumulate
Studies inducing hypoxia by inspiration of 100% nitro- continuously until death. Cells that accumulate large
gen,22 tracheal clamping and cessation of respiration,23 numbers of mitochondrial mutations become bioenergeti-
and vessel occlusion24 have all shown significant alter- cally deficient, which explains their important role in
ations of auditory sensitivity as measured by compound many human diseases.38
action potentials, summating potentials, cochlear micro- Mitochondrial and nuclear DNA are exposed to a
phonics, and otoacoustic emissions.25,26 significant amount of oxidative damage. Studies have
Decreased blood flow from a variety of causes includ- shown that increasing amounts of ROM occur as a func-
ing hypertension, atherosclerosis, noise exposure, or aging tion of age, which leads to an increase in membrane per-
promotes hypoxia with formation of ROM and resultant oxide content and rapidly exceeds the capacity of homeo-
tissue/organ damage.27,28 static protection.39 Thus there is extensive support that
mtDNA deletions accumulate with age and disease.40,41
Mitochondrial DNA Deletions Deafness has also been shown to have an association
Mitochondria are unique organelles possessing their with mtDNA deletions. It has been suggested that mito-
own DNA as well as their own enzymatic constituents to chondrial diseases should be considered in cases of pro-
allow for transcription and translation of genetic informa- gressive sensorineural hearing loss, especially with the
tion into proteins. Each mtDNA codes for a complete set of coexistence of multisystem involvement.42 A 10.4-kb
ribosomal (rRNA) and transfer RNA (tRNA). In addition, mtDNA deletion has been identified in association with
mtDNA codes for 13 of the approximately 60 polypeptides maternally transmitted diabetes and deafness, without
necessary for oxidative phosphorylation. These include 7 ophthalmoplegia or mitochondrial myopathies, which was
of the 25 subunits of respiratory complex I (ND1–ND4, 4L, the hallmark of mtDNA deletion syndromes.43 Other stud-
5, and 6) (reduced nicotinamide adenine dinucleotide ies have identified mutations in the tRNA(Leu)(UUR)
[NADH]-ubiquinol-oxidoreductase), 1 of the approxi- gene in a large pedigree with maternally inherited dia-
mately 9 subunits (cyt b) of respiratory complex III betes mellitus type II and deafness.44 A 3,243-point mu-
(ubiquinol-cytochrome c oxidoreductase), 3 of the 13 sub- tation (A3 G) has been demonstrated in a patient
units of respiratory complex IV (COI, COII, and COIII) with sensorineural deafness without diabetes.45 Several
Laryngoscope 110: May 2000 Seidman: Presbyacusis
729

4. human studies have demonstrated an association of mito- ROM lead to lipid peroxidation, polysaccharide depoly-
chondrial DNA mutations and presbyacusis, including a merization, nucleic acid disruption, and oxidation of sulf-
study demonstrating that older patients with presbyacu- hydryl groups leading to enzyme inactivation.52 Therefore
sis had a higher frequency of the common aging deletion the membrane hypothesis of aging suggests that ROM-
(4,977 bp) compared with similar-aged patients without induced cell membrane structural damage is the primary
presbyacusis.46 A significant difference in mtDNA dele- mediator in cellular aging.53
tion was noted in human archival temporal bones. Specif- Careful analysis of the above mechanisms suggests
ically, 14 of 17 aged patients with presbyacusis had the that certain aspects of the three leading theories of aging
4,977-bp deletion compared with 8 of 17 control patients may be interrelated. That is, free radical species lead to
with normal hearing.47 genetic and cellular alterations resulting in cellular dys-
function and, consequently, senescence. It is even more
Aging intriguing to realize that a trigger for the Bax gene is
Aging is the progressive accumulation of metabolic ROM.54 Thus a critical analysis of the prominent hypoth-
and physiological changes associated with an increasing eses of aging suggests that aspects of all three theories are
susceptibility to disease. There are many hypotheses in likely to apply. Specifically, the generation of ROM dam-
the current literature providing explanations for senes- ages cellular integrity, which may lead to alterations in
cence. Three of the most convincing theories are the te- gene expression, including telomere shortening and acti-
lomerase theory of aging, the “dysdifferentiation” hypoth- vation of Bax genes resulting in aging, apoptosis, and,
esis of aging, and the membrane hypothesis of aging (also ultimately, death.
referred to as the mitochondrial clock theory of aging).
The telomerase theory of aging suggests that there is Primary Treatment Interventions
a reduction in telomere length over time. The end of a The primary treatment interventions used in these
chromosome is made up of a structure called the telosome. experiments were the following:
The tip of the telosome is a region of DNA repeat se- Diet restriction. It is well known that a 30% caloric
quences and associated proteins called the telomere. It is dietary restriction can enhance longevity by 30% to 50%.
hypothesized that DNA transcription and replication are The putative mechanism is a reduction in metabolism and
affected by position effects mediated by the telomere. Re- oxidation reactions and hence, a significant reduction in
duction in the length of the telomere and alterations in its the generation of ROM.
chromatin assembly may explain the instability that oc- Vitamin E. Vitamin E (tocopherol) is a potent fat-
curs during senescence, as well as the immortalization soluble antioxidant. The most active naturally occurring
process in vitro.48 Although many aspects of telomerase compound is RRR- -tocopherol. Polyunsaturated fatty ac-
activity remain undefined, it has been hypothesized that ids (PUFAs) are liable to auto-oxidation. Each fatty acid
the balance between telomere shortening and telomerase free radical that is oxidized damages about three other
activity underlies cellular aging processes. PUFA molecules, thus producing a chain reaction that
The dysdifferentiation theory suggests that aging is a expands geometrically. Vitamin E can readily donate hy-
continuum of programmed differentiation leading to ei- drogen atoms to terminate this chain reaction. The vita-
ther a cessation of normal gene activity or a systematic min also appears to be essential for the protection of
activation of genes whose effects are deleterious to cellular circulating lipoproteins and the correct functioning and
function. Support for this theory is provided by apoptosis stability of cell membranes.
(programmed cell death) studies in hermaphroditic Vitamin C. Vitamin C or ascorbic acid is a water-
worms. These experiments elegantly elaborated the ge- soluble vitamin. It cannot be endogenously synthesized in
netic mechanisms responsible for controlling cell death. certain species, including humans; therefore its require-
The maintenance of homeostasis for cellular metabolism ment must entirely be met by dietary intake. It is an
and function consumes a large fraction of total body en- effective scavenger for superoxide (O2.-), thiol (sulfur-
ergy expenditure. This is engineered by the delicate bal- centered), singlet O2, and hydroxyl radicals. It has been
ance between cellular proliferation and death. For exam- shown to recycle -tocopherol in lipid membranes, thus
ple, the Bcl-2 gene appears to prevent oxidative damage to delaying the onset of peroxidation in isolated human low-
cellular organelles and lipid membranes.49,50 Another pro- density lipoproteins (LDLs).
tein that appears to operate as an accessory to Bcl-2 is a Melatonin. Melatonin (N-acetyl-5-methoxytryptamine)
21-kD protein referred to as Bax. The ratio between Bcl-2 is an indole hormone primarily secreted by the pineal gland.
and Bax appears to determine survival or death following Although primarily known for its effect on entrainment
an apoptotic stimulus. Specifically, elevated expression of pathways of the biological clock and actions on photoperiodic
Bcl-2 appears to be preventive, while that of Bax favors time, melatonin is also an important neural antioxidant and
the apoptotic process.49 free radical scavenger. It is believed to work via electron
The membrane hypothesis of aging states that aging donation to directly detoxify free radicals such as the hy-
is related to decreasing effectiveness of cellular protective droxyl radical. In addition, melatonin has been found to
and reparative mechanisms. This yields biochemical and protect cells (in vivo as well as in vitro) against oxidative
metabolic errors, which progressively accumulate, result- damage induced by a variety of free radical– generating
ing in cell death.51 The hypothesis further postulates that agents and processes. It is also effective in protecting nuclear
cellular senescence is attributable to the cross-linking ac- DNA, membrane lipids, and, possibly, cytosolic proteins
tion of ROM within the cellular membrane. In addition, from oxidative damage. In addition, evidence suggests that it
Laryngoscope 110: May 2000 Seidman: Presbyacusis
730

5. enhances the defense capacity of many antioxidative en- vitamin C, two pellets implanted with 200 mg vitamin C, each
zymes such as superoxide dismutase, glutathione peroxi- delivering 4.44 mg/d; melatonin, one pellet implanted with 9 mg
dase, and nitric oxide synthase. melatonin, each delivering 0.1 mg/d; and lazaroid, one pellet
Lazaroids. Lazaroids are 21-aminosteroids with no implanted with 112.5 mg drug, each delivering 1.25 mg/d.
In addition, random assays were performed on animals to
glucocorticoid, mineralocorticoid, or other hormonal prop-
determine blood levels of vitamin E and melatonin groups at
erties. They are multimechanistic inhibitors of lipid per- three separate time points during the life of the pellet. The
oxidation. Lazaroids scavenge free radicals and stabilize technique to perform the assays used high-pressure liquid chro-
cell membranes by decreasing fluidity, preserving vitamin matography.
E content in these membranes, and increasing surface
viscosity. They have demonstrated efficacy in improving
Auditory Brainstem Response Measurements
neurological outcome after central nervous system Auditory brainstem response (ABR) measurements were
trauma, subarachnoid hemorrhage, and ischemia. collected every 3 months from 2 months of age until the death of
the animal. Each ear was studied for auditory sensitivity. Sub-
MATERIALS AND METHODS jects were anesthetized using ketamine and Rompun (100 mg/kg
Fischer 344 rats were obtained from Harlan Inc. (Indianap- and 15 mg/kg, respectively, intramuscularly) and supplemented
olis, IN). A total of 130 2-month-old rats were used for these with ketamine as required. The subject’s temperature was main-
experiments. All procedures and protocols were reviewed and tained at 38°C 1°C with a heating blanket and monitored with
approved by the institution’s Care for Experimental Animals a rectal probe.
Committee. All animal experiments were performed in a humane Signal generation. The test stimulus consisted of tone-
manner according to standards established by the National In- bursts with a rise/fall time of 1 millisecond, a duration of 15
stitutes of Health (Bethesda, MD). milliseconds, and a period of 100 milliseconds. Intensity series
Subjects were maintained in individual cages at 21°C to were obtained at 3, 6, 9, 12, and 18 kHz, ranging from 0 dB sound
22°C with a 12:12-hour light-dark cycle initiated at 7:00 A.M. Food pressure level (SPL) to 100 dB SPL. These stimuli were generated
and water were available ad libitum to all groups with the excep- using a digital-to-analogue converter (model DA3–2, Tucker
tion of diet-restricted subjects. Davis Technologies, Gainesville, FL). The output of the digital-
To determine the appropriate dietary restriction, a group of to-analogue converter was connected to a programmable attenu-
10 animals was originally studied for a period of 1 month. These ator (model PA4,Tucker Davis Technologies), a weighted summer
10 animals were placed in metabolic cages where strict caloric (model SM3, Tucker Davis Technologies), a headphone buffer
intakes and urinary and stool outputs were recorded. The ani- (model HB6, Tucker Davis Technologies), and an earphone (mod-
mals ingested 16 g 3 g of a standard rodent diet formulation per el DT-48, Beyer Dynamic, Heilbronn, Germany) that was placed
day. Thus the appropriate caloric restriction was calculated to be in close approximation to the tympanic membrane of the animal.
11.2 g (30% restriction) of feed per day for the caloric-restricted A sampling frequency of 100 kHz was used to generate the signal.
group. The signals were calibrated at the tympanic membrane using a
The remaining 120 rats were randomly assigned to one of probe microphone (model ER-7C, Etymotic Research, Elk Grove
the following six groups: group 1: 30% caloric restriction (n 20); Village, IL). The output of the microphone was connected to an
group 2: vitamin E oversupplementation (2.475 mg/d) (n 20); analogue-to-digital converter (model AD2, Tucker Davis Technol-
group 3: vitamin C oversupplementation (4.44 mg/d) (n 20); group ogies) and a Pentium computer. An automatic calibration routine
4: melatonin treatment (0.1 mg/d) (n 20); group 5: lazaroid treat- was used for on-line calibration.
ment (1.25 mg/d) (n 20); and group 6: placebo (matrix pill) Recording. Sterile platinum needle electrodes (model E2,
(n 20). Grass Instruments, Quincy, MA) were placed under each pinna
The subjects were initially anesthetized using ketamine and and at the vertex of the subjects (under anesthesia). The elec-
xylazine (100 mg/kg and 15 mg/kg, respectively, intramuscularly) trodes were connected to a biological amplifier (model P5 series,
and supplemented with ketamine as required. Grass Instruments) with the gain setting at 100,000. The
To simplify identification of each individual rat and to elim- response was filtered between 0.3 and 3 kHz. The output of the
inate the possibility of inadvertently misidentifying the subject, amplifier was connected to an analogue-to-digital converter (mod-
an electronic identification (ID) microchip (Avid, Norco, CA) was el AD2, Tucker Davis) and a Pentium computer. The responses
inserted into each animal and read with a scanner before any were digitized with a sampling frequency of 50 kHz. For each
testing or treatment. In brief, the posterior nuchal region was recording, a 20-millisecond neural response was averaged 1,024
prepared and draped in a sterile manner. A 3-mm incision was times. Thresholds were identified when an N1 response 1 V
made posteriorly, and the ID microchip inserted. The wound was above baseline was observed, using a standard protocol.
closed with an interrupted 4-0 Vicryl suture.
After the microchip insertion, a separate incision was made
immediately cephalad to the microchip incision and the appropri- Isolation of Intact Mitochondria From Muscle
ate pellet (per previous randomization) was inserted into the Samples
subcutaneous tissues. This wound was closed with one 4-0 Vicryl Isolation of mitochondria from muscle was performed as
suture. The pellets were purchased from Innovative Research previously described.55 In brief, the muscle specimen, weighing
(Sarasota, FL). The pellets have been tested extensively to verify approximately 1 g, was freed of any fat or connective tissue and
stated timed-release parameters, appropriate dosing, and appro- rinsed in cold, nonionic isolation buffer containing 210 mmol/L
priate blood levels. The pellets selected for these experiments mannitol, 70 mmol/L sucrose, 1 mmol/L ethyleneglycol-bis-(beta-
were 3-month pellets and were guaranteed to provide basal levels aminoethylether)-N,N,N ,N -tetra-acetic acid (EGTA), 0.5% BSA,
of the particular drug or vitamin throughout the entire time and 5 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic
course. acid (HEPES), pH 7.2. The specimen was processed in approxi-
mately 0.5-g batches with a Thomas (Thomas Scientific, Swedes-
Drug Dosages boro, NJ) tissue slicer. The thinly sliced muscle was collected into
Dosages of drugs used were as follows: vitamin E, one pellet a 50-mL tube, suspended in 10 mL isolation buffer/g muscle
implanted with 222.75 mg vitamin E, each delivering 2.475 mg/d; tissue. The muscle tissue was homogenized using 10 passes in a
Laryngoscope 110: May 2000 Seidman: Presbyacusis
731

6. Teflon-glass homogenizer with moderate rotation speed. The re- cycles of denaturation at 94°C (for 30 s), annealing at 56°C (for
sulting sample was centrifuged at 1500 g for 5 minutes at 4°C. 30 s), and extension at 72°C (for 1 min).
The supernatant was decanted into a fresh tube, the pellet was
discarded, and the sample underwent repeat centrifugation. The
supernatant was decanted and centrifuged at 8000 g for 15
Gel Electrophoresis
The amplified PCR products were separated by electro-
minutes. The mitochondrial pellet was suspended in 30 mL iso-
phoresis on 1.5% agarose gel containing ethidium bromide. The
lation buffer and centrifuged again at 8000 g for 15 minutes.
gels were read under ultraviolet light and imaged. Agarose gel
The washed mitochondrial pellet was resuspended in 0.1 mL of
concentrations vary depending on fragment length. Thus for
the isolation buffer for each gram of muscle used. This sample
larger fragments a 1% agarose gel was used.
could then be stored at 70°C. For protein estimation, a 30- L
aliquot was removed and centrifuged at 8000 g for 15 minutes,
and the pellet was resuspended in isolation buffer without BSA. DNA Sequencing
Details of sequencing have been described previously.57 The
Tissue Sources age and, possibly, presbyacusis-dependent mtDNA region was
The tissues harvested from the animal subjects included selectively amplified, and the resulting fragment was gel purified.
brain, auditory nerve, and stria vascularis. All tissues were used Asymmetric PCR was performed with primers at a 1:100 ratio. A
immediately or stored at 70°C for later use. direct sequencing of the single-stranded mtDNA was made by
asymmetric PCR using the “fmol” DNA Sequencing System (Pro-
mega Co., Madison, WI).
DNA Extraction
Animal tissues were harvested and stored at 70°C, until
the time of DNA extraction. The samples were homogenized in 10 Histology
mmol/L Tris (pH 8) containing 1 mmol/L EDTA buffer and incu- Subjects were deeply anesthetized. Cardiac puncture was
bated overnight at 56°C with 15 L Proteinase-K (10 mg/mL) in performed, and the animal was perfused with 2.5% glutaralde-
0.5 mL digestion buffer consisting of 10 mmol/L Tris (pH 8), 10 hyde solution in a veronal acetate buffer freshly normalized to a
mmol/L EDTA, 50 mmol/L NaCl, and 2% sodium-dodecyl sulfate. pH of 7.4. The bullae were rapidly removed, and the cochleae
Standard extraction protocols for DNA were used with phenol, were reperfused through the oval and round windows. The co-
chloroform, and isoamyl alcohol. The proteins were removed from chleae were stored at 4°C for 48 hours, then perfused with 1%
the sample solution with phenol and chloroform, (25:24) both of osmium tetroxide in veronal acetate buffer normalized to a pH of
which served as separate organic solvents and hence deprotein- 7.4 for 30 minutes. After perfusion, the organs were dehydrated
ized more efficiently. The tissue extracts were centrifuged at using ethanol of ascending strength from 30% to 70%. The co-
10,000 g at room temperature to separate mtDNA from cellular chleae were decalcified overnight in 0.35 mol/L of EDTA in vero-
debris and protein. The supernatant was drawn off, and the nal acetate buffer normalized to a pH of 7.4 at room temperature.
residual phenol removed with equal volumes of chloroform and After decalcification, the organ of Corti was dissected in 70%
isoamyl alcohol (24:1). This subsequent extraction with chloro- ethanol. Each turn was mounted with glycerol and examined
form removed the remaining traces of phenol from the prepara- under an optical microscope. The hair cells were counted at orig-
tion. A 1/10 volume of 3 mol/L NaOAc and 1/100 volume of 1 mol/L inal magnification 630, using a Carl Zeiss GFL (Carl Zeiss Inc.,
MgCl2 were added, and mtDNA was recovered by precipitation Goettingen, Germany) differential interference contrast micro-
with 2 volumes of cold ethanol. This preparation was stored at scope. The data were used to generate cytocochleograms as a
70°C for 60 minutes, and the precipitate was recovered by frequency position map based on the following mathematical
centrifugation at 12,000 rpm for 30 minutes (4°C). The superna- derivation:
tant was removed, and the pellet washed with 70% ethanol,
air-dried, and redissolved in Tris-Edta buffer at the desired con- F A(10ax k),
centration. DNA concentrations were determined spectrophoto-
metrically using optical densities of 260 and 280 nm, and aliquots where F stands for frequency, A 0.64, a 2.1, x is expressed as
were used for polymerase chain reaction (PCR). a proportion of basilar length, and k 0.85.
There are technical differences in isolating DNA from skel-
etal muscle compared with other tissues. The methodology neces- Statistical Methodology
sitates special attention for the isolation of tightly coupled mito- Outcome variables. The hearing levels for five frequencies
chondria. Our laboratory has used the modifications as (3, 6, 9, 12, and 18 kHz) were tested separately.
previously described.56 Multiple comparisons. A Bonferroni adjusted P value of
.01 (P .05/5) was used to determine statistical significance for
Polymerase Chain Reaction each overall test for a treatment group effect for the five frequen-
Oligonucleotide primers were designed in our laboratory cies. If the overall test was not significant, tests between individ-
and synthesized by Fisher Biotech (Pittsburgh, PA) to amplify ual treatment groups were not performed. If the overall test was
several distinct regions of the rat, mouse, and human mtDNA significant, comparisons between treatment groups, two at a
genome. Of particular interest is the cytochrome b gene, the ND1 time, were performed and a P value of .05 or less was considered
segment of the 16S rRNA region and a 4,834-bp deletion in rats. statistically significant.
PCR is used to amplify the base-pair products that correspond to Analysis methods. To compare the baseline, pretreatment
these regions. Appropriate positive and negative controls were values, one-way ANOVA was performed. To make use of serial
run and then sequenced to authenticate the PCR products. The observations, repeated-measures analysis was performed using
PCR reaction contained 100 to 200 g of test sample, 200 mmol/L the SAS PROC MIXED58 procedure. The model assumed a corre-
of each deoxynucleotide triphosphate, 50 mmol/L KCl, 10 mmol/L lation between observations that decreased with the time (i.e., a
Tris-HCl (pH 8.3), 1.5 mmol/L MgCl2, 0.01% (wt/vol) gelatin, 1 lower correlation between the observations at months 15 and 25
mmol/L of each primer, and 5 U of Taq polymerase in a final compared with those between months 15 and 20 or between
volume of 100 L. The thermal cycling parameters were as fol- months 20 and 25). The PROC MIXED procedure allows subjects
lows: initial denaturation at 94°C for 3 minutes, followed by 30 to be included even if observations are not available for all times.
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732

7. The model included an adjustment for the average levels ob- better than placebo results at all frequencies except 12
served at each month. kHz. Lazaroid was better than placebo only at 12 kHz.
Four analyses were performed. These were restricted to all There was a progressive reduction of auditory sensi-
data available at 25 months, all data from 20 months onward, all tivity which began at approximately 6 to 9 months of age
data from 15 months onward, and all data from 12 months on-
in all groups. The greatest amount of hearing loss oc-
ward. The data from the oldest animals were of greatest interest,
but earlier data were also included.
curred over the last 12 months of life. The least amount of
presbyacusis was observed in the diet-restricted subjects.
The other treated animals experienced better hearing sen-
RESULTS sitivities than the control group, and this was statistically
This randomized prospective study was completed significant for all groups (P .01) with the exception of
over a 3-year period. A total of 120 subjects were initially the lazaroid group, and the overall effect varied with fre-
tested, and all subjects were studied throughout their life quency. Figure 1 is a graph depicting cumulative audio-
span. At the conclusion of the experiments, there were five logical data of all groups.
animals available for analysis in the diet-restricted group Table I summarizes the tests for overall treatment
(mean age, 27 mo), four in the vitamin E group (mean age, effects for each of the four analyses. The first four frequen-
26 mo), four in the vitamin C group (mean age, 24.5 mo), cies showed significant treatment effects in all of the anal-
three in the melatonin group (mean age, 25 mo), three in yses. A treatment effect could not be confirmed for a fre-
the lazaroid group (mean age, 26 mo), and three in the quency of 18 kHz for the two analyses restricted to
placebo group (mean age, 26). animals observed for 20 months or more.
The means for the various groups and the results of
the comparisons between groups within the repeated-
Blood Levels of Vitamin E and Melatonin
measures analysis are available in the detailed thesis
To verify the appropriate functioning of the matrix
(unpublished data).
drug delivery system, serum for vitamin E and melatonin
testing was obtained. Other assays are available for the
Mitochondrial DNA Deletions
remaining treatment options, but they are considered un-
To study changes in mitochondrial DNA, we evalu-
reliable and extremely expensive. Testing was performed
ated brain, auditory nerve, and stria vascularis for cyto-
at four separate time points: at baseline, 1 week after
chrome b, the ND-1 16S rRNA region of the mitochondrial
implantation of the 90-day pellet, 6 weeks after implan-
genome and the common aging deletion (mtDNA4834). Cy-
tation, and just before the pellet was due to be changed
tochrome b and ND-1 16S rRNA are used as controls to
(85– 89 d). Levels of the supplemented vitamin or hormone
verify the presence of mitochondrial DNA while the
were in the appropriate range.
mtDNA4834 is the test of interest that provides a molecu-
The vitamin E group baseline levels were 9 0.5
lar marker of aging. All specimens were positive for cyto-
nmol/mL. The levels at 1 week, 6 weeks, and just before
chrome b and ND-1 16S rRNA, verifying the presence of
pellet change were 16.4 nmol/mL 1.7 nmol/mL. Melato-
mtDNA. A small piece of muscle was harvested at every
nin levels are subject to diurnal variation. Testing was
3-month testing interval, thus allowing the opportunity to
performed at approximately 10:00 A.M. and revealed a
determine when the deletion was first apparent. The com-
baseline level of 59.5 pg/mL 5 pg/mL. The levels at 1
mon aging deletion gradually appeared at 9 months of age
week, 6 weeks, and just before pellet change were 1,446
in all groups except the restricted-diet group. This group
pg/mL 145 pg/mL.
did not show evidence of the common aging deletion until
16 months of age. Quantification studies revealed an age-
Auditory Sensitivity Results appropriate increase in the mtDNA4834, and DNA se-
Baseline auditory thresholds for all subjects at 2 to 4 quencing verified the authenticity of the PCR products.
months of age were in the range of previous studies in our The most rapid accumulation of the mtDNA4834 was evi-
laboratory as well as other laboratories, and were between dent in the ad libidum diet (placebo); the other treated
15 and 35 dB in the frequencies tested. Over the span of 24 groups had a slower accumulation of the deletion (Figs.
to 27 months the thresholds shifted the least in the diet- 2A–C).
restricted groups and the treated subjects. The threshold
shifted the most in the placebo group as tested at 26 Histology
months. The difference between the restricted-diet group The histological results demonstrated loss of outer
and the placebo group was statistically significant (P hair cells that is typically observed with aging. The differ-
.01). The antioxidant-treated groups had a statistically ences between the groups showed less hair cell loss in the
significant protective effect when compared with the pla- restricted-diet group and a slight reduction in hair cell
cebo group, but this effect was frequency specific (P .05). loss in the treated animals as compared with the control
In other words, not all frequencies showed a statistically subjects. Fig. 3A and B shows cochlear histograms from
significant response. Specifically, the vitamin E group ex- the diet-restricted and placebo groups, respectively.
perienced statistically better auditory thresholds at 6, 9,
and 12 kHz (P .05), but not at 3 or 18 kHz. Vitamin DISCUSSION
C–treated animals showed a statistically significant lower The results of this prospective study demonstrate
threshold compared with the placebo group at all frequen- that with the normal aging process (the placebo group)
cies except 3 kHz. Melatonin results were statistically there is a progressive reduction in auditory sensitivity
Laryngoscope 110: May 2000 Seidman: Presbyacusis
733

8. Fig. 1. Auditory thresholds of the tested groups at the last auditory brainstem response (ABR) recording across all five frequencies. The X-axis
plots frequency; the Y-axis plots decibels.
similar to presbyacusis observed in humans. This reduc- cant burden not only for the sufferers, but also for those
tion in hearing accelerates during the last 12 months of who communicate with them. The medical and socioeco-
life. Furthermore, there is an age-appropriate accumula- nomic costs are staggering, and with the expansion of the
tion of the common mtDNA deletion. This progression of world population and the numbers of elderly individuals
age-related hearing loss and accumulation of mtDNA de- expecting to more than double by the year 2030, this
letions can be attenuated by long-term treatment with problem is escalating.
antioxidants and scavengers of ROM. The findings ob- There is significant evidence supporting the concept
served in the histological results are a bit more difficult to that altered blood flow and/or oxygen delivery results in
interpret, but overall, they support these conclusions. hair cell damage. Atrophy of striae has been reported in
Presbyacusis or age-related hearing loss is the lead- elderly human cadavers, even in the absence of classic
ing cause of hearing loss in the world, creating a signifi- atherosclerotic changes.18 Similar changes have been
identified in the stria vascularis of gerbils.59 Prazma et
al.19 have observed diminished flow in capillaries of the
TABLE I. basal turn of the cochlea, and blood viscosity and red blood
Overall Treatment Effect for Adjusted Averages Over Various cell rigidity appear to correlate with high-frequency hear-
Observation Intervals. ing loss in elderly human subjects.20 A relationship be-
Month Month Month Month tween vascular atrophy and presbyacusis has been pro-
Frequency 25 Only 20–25 15–25 12–25 posed.60 In addition, studies have demonstrated a
(Hz) (n 25) (n 59–66) (n 108–116) (n 180–198) significant reduction in erythrocyte velocity, increased
3,000 .005 .009 .001 .001 vascular permeability, a trend for reduced capillary diam-
6,000 .007 .001 .001 .001 eters, and a decrease in auditory sensitivity with aging.2
9,000 .001 .001 .001 .001 Thus genetics, dietary factors, and environment have been
12,000 .007 .001 .001 .001 suspected to play a pivotal, yet poorly defined, role. The
18,000 .208 .114 .005 .001
current experiments support previous literature and ex-
pand on earlier findings by observing the progression of
Laryngoscope 110: May 2000 Seidman: Presbyacusis
734

9. Fig. 2. A. Electrophoretic gel depicting the mitochondrial DNA data from stria vascularis. Similar data (not shown) exist for brain and auditory
nerve. The gel provides the data for identification of cytochrome b from stria vascularis. This represents a 365-base-pair (bp) product and
verifies the integrity of the polymerase chain reaction (PCR) and the presence of mitochondrial DNA. B. Data for identification of ND1–16S rRNA
from stria vascularis. This represents a 601-bp product and also verifies the integrity of the PCR and the presence of mitochondrial DNA. C.
Data identifying the 4,834-bp deletion (common aging deletion) from stria vascularis. This represents a 597-bp product. Lane 1 negative
control; lane 2 placebo; lane 3 diet restriction; lane 4 vitamin C; lane 5 vitamin E; lane 6 melatonin; lane 7 lazaroid; lane 8
molecular weight standard curve. DNA concentrations were normalized to 200 ng/50 L of PCR reactant.
age-related hearing loss in a prospective randomized trial. parison with the placebo group support the hypothesis of
Furthermore, the identification of significant protection of this proposal; specifically, diet restriction, which reduces
auditory thresholds in the diet-restricted groups and the the formation of ROM, and antioxidants, which attenuate
partial protection observed in the treated subjects in com- age-associated hearing loss.
Fig. 3. A. Representative animal cochlea from a diet-restricted subject (original magnification 400) . Note the three outer hair cell rows (1,
2, and 3). There are some hair cells missing, which correlates well with the moderate degree of hearing loss noted on ABR testing. B.
Representative animal cochlea from a placebo subject (original magnification 400). Note the three outer hair cell rows (1, 2, 3). There are
many hair cells missing, which correlates well with the severe-profound hearing loss noted on ABR testing.
Laryngoscope 110: May 2000 Seidman: Presbyacusis
735

10. This report represents the first prospective random- a trend showing fewer deletions in the treated groups
ized study designed to investigate the effect of antioxi- compared with placebo. Given the long-term effect of dam-
dants, nutritional supplementation, and dietary restric- age to cells and tissues exposed to the normal amounts of
tion on hearing loss specifically and on aging in general. ROM, the findings of protection in auditory sensitivity and
Furthermore, these studies propose that the mechanism mtDNA deletions are encouraging.
involving the generation of ROM and resultant damage to The results of this study clearly demonstrate a pro-
mitochondria may be responsible in part for presbyacusis. tective effect of dietary restriction and nutritional over-
The observations from this study that dietary restric- supplementation on age-related hearing loss and mtDNA
tion and other nutrient supplementation reduce the pro- deletions. This beneficial effect most likely represents a
gression of age-related hearing loss is an important initial slowing of the mitochondrial clock theory of aging. Specif-
step toward outlining strategies that may slow the pro- ically, dietary restriction and other mechanisms aimed at
gression of presbyacusis. All of the interventions used reducing the overall burden of ROM provide an internal
affect the generation of ROM. milieu that is more favorable to the organism. The find-
There is an increasing body of evidence implicating ings support that the other treatment arms were protec-
ROM in the damage associated with cochlear ischemia, tive overall. Vitamins E and C, melatonin, and lazaroid
noise trauma, aging, presbyacusis, and ototoxicity. Specif- appear to offer at least a partial protective effect. This
ically, localized inner ear ischemia and hypoxia induced effect is not as evident as with dietary restriction. This
via selectively clamping the anterior inferior cerebellar limited benefit may be explained in part by the fact that
artery normally abolishes the compound action potential each animal was oversupplemented with individual com-
within seconds and the effect becomes permanent after 8 pounds. There is compelling evidence that supplementing
minutes of ischemia. When subjects were pretreated with with only one antioxidant may not be as effective as sup-
allopurinol or superoxide dismutase followed by ischemia, plementing with multiple antioxidants. This makes tele-
compound action potential thresholds were maintained.61 ological sense when considering that nutritional antioxi-
In addition, these experiments were extended to study dants work by a variety of mechanisms. Some stabilize cell
noise- induced hearing loss, which, like aging, has also membranes, others scavenge ROM molecules, yet others
been shown to cause vascular perturbations. enhance the role of other antioxidants. However, in exper-
Despite the potential for prevention of some of these imental design, it is crucial to test supplements individu-
disorders using antioxidants, there are other studies ally, first, to elucidate specific properties and effects.
which have not demonstrated a benefit of treatment using Based on such experimentation, studies subsequently can
antioxidant compounds such as superoxide dismutase be designed to observe the effect of supplements in com-
(SOD)62,63 and -tocopherol.64 Critical analysis of these bination. Indeed, experiments to investigate such a com-
negative studies reveals several flaws ranging from insuf- bined effect have clearly shown a synergistic positive ef-
ficient statistical power to lack of appropriate controls, use fect on generalized system function.70 Furthermore,
of the wrong conjugates of SOD (i.e., those with a short ongoing studies in our laboratory are addressing this spe-
half-life of 1– 4 s), and design difficulties. For example, one cific issue using a combination of vitamins and compounds
study gave SOD 8 hours after closed-head injury, when that can enhance mitochondrial function.
the majority of the damage had already occurred.62
There is a significant body of literature providing CONCLUSION
evidence that oxidative damage is important in the aging This study presents empirical evidence in support of
process. It has been demonstrated that life span differ- an integrated hypothesis of aging and presbyacusis. In
ences in certain rodent and primate species depend in part addition, presented data provide a framework for the con-
on free radical scavenging systems. Investigators ob- tinuing identification and evolution of pharmacological
served that animals with longer life spans had higher and nutritional strategies designed to attenuate age-
levels of SOD activity.65 Many studies have investigated associated hearing loss. Areas of scientific inquiry have
dietary increases in antioxidants and have demonstrated also been identified that require further investigation
life span increases of 13% to 30%.66,67 with the ultimate goal of enhancing our understanding of
Metabolic rate is also closely linked to the aging the mechanisms that surround senescence, both as a phe-
process. Many investigators have shown an inverse rela- nomenon and as an intrinsic biological property.
tionship between metabolic rate and life span.68,69 The
relationship between aging and metabolic rate is clear
because the production of toxic oxygen radicals is propor- ACKNOWLEDGMENT
tional to the rate of oxygen utilization (i.e., the metabolic My gratitude is extended to Drs. Wayne Quirk, Uma
rate), and this has a close direct relationship with dietary Bai, Mumtaz Khan, Wen Xue Tang, and Najeeb Shirwany,
intake. Collectively, these studies provide both indirect who assisted over the years, and Mr. Joseph Henig, and
and direct evidence that ROM are responsible for many Mrs. Cristy McAuley. Additional gratitude is expressed
clinical diseases including cochlear damage from isch- for the clinical insight and support of Drs. Richard Ni-
emia, noise, and, possibly, aging. chols, Malcolm Graham, and Herbert Silverstein.
The results of the mitochondrial experiments clearly
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