Acute changes in inner ears of laboratory animals caused by simulated sonic booms

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ACtJrE CHANGES IN INNER EARS OF LABORATORY ANIMALS

September, 1976

CAUSED BY SIMULATED SONIC BOOMS

by

Stanis1av Reinis

ti

st?

\971

UTIAS Report No. 211 CN ISSN 0082-5255

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ACUTE CHANGES IN INNER EARS OF LABORATORY ANIMALS CAUSED BY SIMULATED SONIC BOOMS

by

Stanislav Reinis

University of Toronto Institute for Aerospace Studies

and

Department of Psychology University of Waterloo

Submitted September, 1976

ACKNOWLEDGEMEN'r

I wish to thank D;r. I. I. Glass for his encouragement to initiate

my

research on the effect of sonic boom on anima~s, for his interest throughout the course.ofthis work, and for critical reading of several manuscripts including th~s one.

To Dr. H. S. Ripner I would like to offer my thanks for most helpfUl discussions.

I gratefully acknowledge the time and consideration that Dr. J~ J. Gottlieb has shown and would like to thank him for reading my manuscr~pts and many helpfuI ideas.

Valuable assistance was.supplied by Dr. N. D, ~lis and R. Gnoyke in operating the UTIAS loudspeaker-driven booth and travelling-wave horn sonie-boom simulators,

The financial assistance provided by the Canadian Ministry of Transport and the Transportation Development Agency is acknowledged with thanks.

The typing of the manuscript, with its numerous changes and additions, by Mrs. Mavis Hutter is appreciated with thanks.

..

' 1. Contents Acknow1edgement Tab1e of Contents Preface Sunnnary REVIEW OF LITERATURE 1.1 Introduction

1.2 Structure of the ear 1.2.1 Outer ear 1.2.2 Middleear 1.2.3 Inner ear

1.2.4 Nerve fibers innervating the 1.2.5 Blood supp1y of inner ear 1.2.6 Chemistry of inner ear

1.2.7 Production and importance of 1.3 Physical characteristics of sound 1.4 Physio1ogical mechanism of hearing 1.5 Acoustic reflex

1.6 Effect of noise on ear 1.7 Effects of impulse noise 1.8 Effects of sonic boom 2. R.ATIONALE OF STUDY

3 • MATERlALS AND METHODS 3.1 Apparatus

3.2 Test procedure with the inbred mice 3.3 Dissection of inner ear

3.4 Test procedures with chinchi11as 4. RESULTS

cochlea

f1uids of inner ear

4.1 Structure of organ of Corti in several inbred strains of mice

4.2 Effect of sonic boom on inner ear of inbred mice 4.3 Experiments with chinchi11as

5. DISCUSSION OF RESULTS AND CONCLUSIONS FURTHER PERSPECTIVES REFERENCES T.A;BLES ii iii iv v 1 1 1 1 1 2 2

3

4

5

6

7

8

8

12 14 16 17 17 18

19

21 22 22 22 24 24 28

29

PREFACE

Although many studies of the.effeets of sonie boom on humans have been made, some of them.deseribing even the behavior of entire populations ofeities and.regions, detailed studies with animals have not been done. It is therefore an unusual situation that.this report. dea1.s.with the effects of sonie boom on small animals af ter many human beings have already been subjeeted to it without fully.knowin~the possible effects. For example, any toxie .. agent.or .. drug.before being administ:ered to people is first tested on thousands of experimental animals. Similar tests were 'not performed on animals before humans were subjected to sonie booms. The.major.reasons for this situation are that both.peopleand.animals.have been subjeeted to thunder sinee primeval times that.was far.more intense than sonie booms from supersonietransports. (SST~s), and finally the diffieulty in sealing the effects produeed by sonie booms in small a~imals

to humans.

Nevertheless, an. experimentaL'study describing some changes in the inner ears .of labQra$@ry miee and ehinehillas subjeeted to sonie boom. is -long. over.due-. Both species have a substantially higherhearingsensitivity than humans, and· the observed changes may not beo applicabie to humans. On the other hand, if supersonie transport-overfli-ghts.are.to be permitted over populated areas humanexposure~~l be prolonged, and the pathologiealehanges.determined.herein -might show up eventually. However, if seeond-generation.SST's.'W'i.ll be designed sueh that the sonie-boom rise-time andoverpressure will be substantially

redueed, then pathologieal changes might be eliminated.

(

1. 1. Glass

Institute for Aerospaee Studies

SUMMARY

C57BL/6 inbred mice and_chinchillas were subjected to simulated sonic booms of different rise time, intensity, and number of sonic booms. The most important pathological change was found to be bleeding in the basal.turn of the scala tympani of the inner ear. Even a single boom having a rise time of

0.1 msec, peak overpressure of 3.3 psf, and duration of 120 msec was found to cause bleeding in at. least one inner ear of each exposed mouse. Similar results occurred for a single "superboom" having a 10-psf peak overpressure but a longer rise time of 5 msec. the frequency of the appearance of blood clots in the scala

tympani was found to increase with the number of booms administered. This cumulative effect was observed even when the booms were

administered at the rate of.one every

24

hours. The traces of bleeding usually disappeared.within.a period of 8 weekso The extent of the bleeding in.the scala tympani did not increase when mice were.exposed to a rapid.succession of simulated sonic booms (18,000 booms of 100 msec, 6 psf overpressure and 5 msec rise time in one hour). However, thre'e out of twenty mice suffered a rupture of the basilar membrane and destruction of the Corti organ in the basal turn of the cochlea.

The consequencesof the sonic boom in chinchillas are similar, but careful dissection of the inner ears, perfusion of the circulation and careful perfusion of the inner ear is needed in order to obtain unequivocal results.

Animal models of pathologicalstates are widely utilized in experimental medicine. Although some of these models are imperfect, they may indicate the character and quality of possible pathological changes taking place in humans. The structure of the inner ear of the mouse and the human is very

similar~ the main difference.being the size and (maybe) resistance to damage. Since we found a cumulation of the pathological changes due to the sonic bOOIT, one may postulate that even in humans, larger numbers and intensities of sonic booms may cause similar pathological changes. Thus, exposure to intense sonic boom, and in particular superboom, should be avoided before more data are accumulated.

1. R!WIEW OF LITERATURE 1.1. Introduction

A sonic boom may affect"several physiological functions of human and animal organisms. It may:

1. cause startle and annoyance. 2. interfére with sleep.

3. have an effect on cardiovascular and other somatic systems.

4.

cause hatching failure in birds, disturbed reproduction in mammals, along with other behavioral changes.

5.

induce damage to the inner ear.

The present study is concerned exclusively with the possibility of inner-ear damage caused by sonic boom. In order to adequately explain the pathological changes, it.is helpful to concentrate first on the normal structure and function of the ear. Some

attention is then paid to the effeets of noise in general, followed by the particular effects of impulse noise and sonie boom on the inner ear.

1.2. Strueture of the earo

1.2.1. Outer ear.

The outer ear consists of the irregularly-shaped shell of skin and cartilage ealled the pinna, or auricle, and a short funnel-shaped canal.ealled.the external auditory meatus (see Fi~. 1.1.). The inner end of the.eanal .is blind; closed by a thin tympanie, or drum, membrane .. This membrane forms a flexible partition between the outer and middle ears.

1.2.2. Middle ear

The middle ear lies.onthe inner side of the tympanic membrane, and is a small chamber hollowed out of the temporal bone. A chain of three small bones, or ossieles, is slung from the drum membrane to the inner (medial) walLof the middle ear. The outer-most ossiele is ealled themalleus and is attached firmly to the drum membrane. The middle bone is the incus, and the innermost has been named the stapes.

The medial wallof the middle ear is also the outer wall of the inner ear. A small ovaLopening in this wall is called the oval window, and on i t lodges _ the .. foot-plate of the stapes which is held in place by an annular ligament. Lower down on the inner wall is the round window, which is closed by a thin membrane. The ossicles of the middle earare.held by two tiny muscles, the tensor tympani and the musculus stapedius.

1.2.6. Inner ear

The inner ear contains-the.essential organ of hearing, the cochlea, and along with it the vestibule and the semicircular canals which act as the organ of bodily equilibrium (Fig.

1.2.).

The cochlea is a spiral passage wound around the modiolus, a short conical structure with .. its .. peak directed forward and out-warde In different animal speeies, there are two to five cochlear turns. The canal forming this structure is longitudinally divided into three passages called.scalae (lat. staircase, due to their resemblance to a spiral staircase).. The upper is known as the scala vestibuli, the middle the seala media, and the lower one the

sca~a tympani. The scala vestibuli and scala tympani communicate

at the apex of the cochlea by an aperture called the helicotrema. A membranous partition, the. vestibular (or Reissner's) membrane, divides scala vestibuli from scala media, while the basilar membrane divides-the scala media from the scala tympani. The basilar membrane carries the organ of Corti which contains the auditory receptors, the hair cells. On their surface, these cells bear fine hair-like.structures which are arranged in rows passing along the long axis of the cochlear canal.

The organ of Corti iscovered completely by a tectorial membrane, an amorphous mass of collagen and mucopolysaccharides. lts surface attached to the hair cells is covered with Hardesty's membrane. The outer margin of the Corti organ is limited by Low

Claudius cells and higher Hensen's cells. Three or four rows of hair celIs, called the outer hair cells, are supported by Deiters' cells and localized on the outer side of the canal of Corti, which is formed by outer and inner pillars. Inner hair cells on the inner side of the organ of Corti are supported by inner phalangeal cells and inner border cells (Fig. 1.3.). The basilar membrane on which these structures lie is formed by parallel thin fibers. The

shortest fibers (contributing to the perception of high-pitched tones) are in the basal turn of the cochlea; the longest fibers, resonant to low-pitched tones, are in the apical turn of the cochlea

(Fig. 1.

4. ) .

1.2.4.

Nerve fibers innervating the cochlea.

The afferent fibers conducting the nerve impulses from the inner ear into the brain are assembIed in the cochlear nerve which is a part of the eighth cranial nerve, the statoacoustic nerve. Each auditory nerve has about 28,000 fibers, and each hair cell receives a nerve filament. About

95%

of the nerve fi~ers, however, innervate the single row of inner hair celIE;. All nerve fibers in the cochlea belong to the nerve cells in the so-called spiral ganglion. These cells send their axons into the cochlear nerve and via the statoacoustic (auditory) nerve into the medulla

oblongata, the axons terminating here in the so-called cochlear nuclei.

The inner ear also receives nerve fibers conducting impulses from the.central nervous system to the cochlea. These fibers are probably responsible for the adaptation of the inner ear to changing functional conditions.

The efferent fibers conducting impulses from the central nervous system to the.cochleahave been studied intensively over the past decade. The histochemical determination of acetyl-cholinesterase, the enzyme inactivating the neurotransmitter acetylcholine, enabled the detection and identification of these efferent fibers. The fibers are concerned with the transmission of neural impulses.from the.olivocochlear bundle to the hair cells

(Spoendlin and Gacek, 1963;.Kimura and Wersall, 1962). Their activity influences the action potentials of the acoustic nerve, cochlear microphonics and summating potentials (Iurato, 1962) in an inhibitory manner (Dayal, .1968). These fibers may represent an important mechanism protecting the hair cells from unfavorable effects of excessive acoustic stimulation.

1.2.5. Blood supply of inner ear.

The blood supply to the inner ear probably plays some role in the pathogenic mechanisms of inner ear damage, and so requires a description. The cochlear blood supply stems from the blood vessels inside the skull and the main blood vessels accompany the stato-acoustic nerve, while the interior of the cochlea is sealed off from the blood supply of tne middle ear (Anson et al., 1966). The most important.vascular systems are the capillaries in the basilar membrane, which may be observed directly in a living ear (Lawrence, 1971), and the capillaries in the stria vascularis •.

This stria vascularis is.a network.of blood vessels accompanied by several types of cells, e.g., chromaffine cells (Hilding, 1965) and others, responsible for the control of the blood flow through this area, this control being by both adrenergic and cholinergic systems

(Suga and Snow, 1969, 1969a).

The capillariesof.the.inner ear may easily be demonstra-ted by a histochemical reactionor by injection with a contrast material •. They contain the enzyme alkaline phosphatase which

reacts with several substitutedtnaphthyl phosphates and diazo stains. The precipitate of the reactioniproducts indicates the presence of . the enzyme and, by implication,jthe capillaries (Nomura and Hiraide, 1969). The.blood.supply to.the!cochlea is relatively high in young children and experimental animat s, and decreases with age (Johnsson and Hawkins, 1972). This maybe the reason for the hair cell de-generation in the aging ear. This dede-generation begins in the basal turn of the cochlea and proceeds towards the apical turn (Johnsson

and Hawkins, 1972a). In elderly people, the hearing of high frequencies is affected firsto This phenomanon is called presbya-cusiso

1.2.6. Chemistrl of inner ear.

A knowledge of the chemical processes occurring within the inner ear is a necessary precondition for the understanding of normal and pathological cochlear functioning.

The inner ear, formed during the early periods of embryonic development,.has manycells which do not later divide, so that their destruction probably represents a terminal loss. All hair cells and cells of the canal of Corti belong to this category. The reason forsuch a condition is that the cells of the organ of Corti do notform any new deoxyribonucleic acid (DNA) and only very small amounts of ribonucleic acid (RNA) which are necessary for the maintenance of' the vital activity of the cells. In fact, in many hair cells, no labeling has been found af ter an injection of a radioactive precursor of RNA synthesis (Lobe, 1974). This could very well be the reason why the hair cells may be

relatively easily damaged by excessive noise (Rupen, 1969). Sound, stimulation of moderate intensities, on the other hand, is able to induce RNA synthesis in most hair cells (Kraus et al., 1975).

The energy metabolism of the inner ear is not very intensive if compared with other tissues. The cells contain some amount of glycogen, the storage form of glucose in the body. This glycogen content is high in the outer hair cells and low in the ::. inner hair cells and Deiters' cells (Falbe-Hansen and Thomsen, 1963). The energy reserves in the organ of Corti are thus

rela-tively high, higher than in stria vascularis and spiral ganglion. In spite of this reserve, themetabolic rate of the organ of Corti is much lower than the metabolic rate of stria vascularis. Stria vascularis has a permanent oxygen and energy supply from the blood circulation, whereas the organ of Corti receives its oxygen and nutrients by slow diffusion through the endolymph. The energy stores and metabolic rate are the~fnr~ geared to the availability of the substances from the circulation (Thalmann et al. , 1972). Thus, the organ of Corti has sufficient amount of energy fin the form of high-energy phosphate (ATP)] even af ter three minutes of circulatory blockage (Matschinsky and Thalmann, 1967).

The activity of different enzymes necessary for the metabolism of glucose is also distributed according to the oxygen and glucose availability. The enzymes of oxidative phosphorylation

(requiring oxygen and finally producing high energy phosphates) are high in the stria vascularis and low in the organ of Corti

(Thalmann et al., 1970). The organ of Corti contains high activity of enzymes.necessary for anaerobic glycolysis, providing energy without the consumption of oxygen (Vosteen, 1961; Webster and Staek,

The energy produced in the stria vascularis is primarily used for the active transport of ions into the endolymph. The two enzymes necessary for thistransport are: carbonic anhydrase and Na, K-dependent adenosine triphosphatase. Both of these are in very high concentrations in strja (Erulkar and Maren, 1961; Iinuma. and Daly, 1968; J ohnson and

Spo~dlin, 1966; etc.), while their concentration is not very remarkable

in the organ of Corti.

The chemistry of other components of the inner ear (lipids, mucopolysaccharides, etc.) has also been studied by several authors. These substances are mainly on the surface of the cells, in the basilar and tectorial membranes, and, although they are probably important for the function of the inner ear, more detailed knowledge of this

relation-ship is lacking (Belanger, 1953; Webster and Stack, 1967; Lim, 1970; Saito and Daly, 1971; Kuttner and Geyer, 1972).

1.2.7 Production and imwortance of fluids of inner ear.

As was mentioned previously, the cochlea is a membranous tube formed by three parallel spaces, scala vestibuli, scala tympani and scala media. Both scala vestibuli and scala tympaJ:li are filled with perilymph, the scala media with endolymph. The organ of Corti is localized between

scala media and scala tympani and the canal of Corti is filled wi th Cortilymph.

The perilymph probably originates from the cerebrospinal fluid filling the spaces around the brain and spinal cord. This fluid finds its way into the inner ear through the spaces around nerve sheaths of the acoustic nerve, and gets into the scala tympani and vestibuli. The

composition of the perilymph resembles that of cerebrospinal fluid, but there are a few differences probably caused by the secretory activity of blood vessels in the wallof the membranous cochlea (Silverstein et al., 1969). There is a controversy in the literature concerning the origin

of different protein components and enzymes in the perilymph. This controversy is at least partly due to the small amounts of fluids which may be collected and the fast postmortem changes of the proteins

(Sil verstein and Schuknecht, 1966; Sil verstein and Griffin , 1970; Silverstein, 1971; Palva and Forsen, 1970; Palva et al., 1972; etc.). The perilymph contains many enzymes involved in the metabolism of glucose (Palva and Raunio, 1967; Palva et al., 1970; etc.).

The pressure of the cerebrospinal fluid is also reflected in the perilymph pressure, and indirectly, through Reissner' s membrane, in the endolymph pressure (Beentjes, 1972).

The endolymph differs from the perilymph in many respects. It contains less protein,. much more potassium ions and much less sodium ions and glucose. Endolymph is probably acti vely produced in stria vascularis, the band of blood capillaries in the wallof scala media (Silverstein, 1966; 1966a). The thin Reissner' s membrane dividing scala vestibuli and scala media probably does not have its own secretory activity and does not contribute to the production of endolymph (Prazma, 1969). The cells.

of the stria vascularis contain a high concentration of an enzynJe

.necessary for the active transport of sodium and potassium ions, Na, K-activated adenosine triphosphatase (Kuijpers et al.,

1967;

1969).

The presence of bcth perilYIrlPh and endolymph is necessary

for normal hearing (Robertson,

197

4

)

.

The Cortilymph, fluid insidethe Corti canal, is probably also deri ved from the cerebrospi~al fluid (Ruderrt and Schreiner,

1968).

The difference in the concentration of ions in the endolymph

and perilymph depends also on the oxygen supply to the cochlea and the permanent availability of energy supplied by a high-energy phosphate, ATP (adenosine triphosphoric acid).

The difference in ionic concentrations determines the electrical poterrtials in different spaces within the cochlea. The tectorial membrane (covering the hair eells) has zero electrical potential, scala meAia is electrically positive and Cortilymph and

scala tympani are electronegati ve. The electroposi ti ve potential of scala media is produced by the secretory activity of stria vascularis (Misrahy et al.,

1958)

and depends on energy and the cochlear oxygen supply. Several substances interfering with oxidation and ion transport, such as cyanide or ouabain, reduce the potentials (Tanaka and Brown,

1970).

The endocochlear DC potential is equal from the basal to the apical turn of the cochlea (Suga et al.,

1964)

.

1.3 Physical characteristics of sound.

Acoustics, the science of sound, is interdisciplinary by nature. From physiciststo sociologists and psychologists, researchers from several disciplines are concerned with sound. Sound is caused by sensations produced in the ear as a result of fluctuations in air pressure . The size and rate of these vibrations of the air determine the loudness

and pitch of sound.

The intensi ty of sound may be determined in several different ways. These include the description of sound as a flow of energy (sound power), or as an energy flow per unit area (sound intensity), or as a fluctuation in air pressure (sound pressure) • The range of magnitudes betwe.en the faintest sound and the values for painfully loud sound is of the order of one millionfold.

It is usual to express the magnitude of sounds in terms of ratios relative to some reference value. The ratios are specified in logarithmic ratios called bels with one bel equal to ten decibels. The reference level for this scale is the quietest audible sound, considered to be

0.00002

Newtons/m2 (Pascals). This intensity may be perceived only at frequencies of sound around

2000

Hz. At lower or higher frequencies, the sensitivity of the ear to sound sharply decreases, and physical value

of

0.00002

N/m2 becomes unrealistic. Several scales were constructed which

take this finding into consideration. Of the three usual arbi trary scales, called A, B and C, the A-scale has becomethe most popular weighting

network because its frequency response corresponds to the way the human ear perceives sound. Where reference is made to the A-weighted network it is indicated as tldBA".

It is believed that the human ear is able to perceive sound frequencies from about 20 to about 16,000 Hz .. Precise limits have not been established partly due to an extensive ind~vidual variability, partly because even higher or lower intensities may be perceived when the intensity of such sound is sufficiently high.

1.4 Physiological mechanism of hearing.

Sound causes the vibration of the eardrum which is transferred through the ossicles into the oval window. The mechanical vibratións transferred to the cochlea produce travelling waves in the fluid. This travelling wave causes a maximum displacement in a given region of the basilar membrane, and the location of this maximum displacement on the membrane is related to the frequency of the tone. The high-frequency tones selectively distort regions of the basilar membrane close to the base of the cochlea, intermediate tones distort a portion of the membrane

trom the base to an intermediate region, and low frequency tones tend to distort the entire membrane. The cochlea therefore acts simply as a complex mechanical analyzer of the auditory stimulus. The region of greatest distortion of the basilar membrane produces the greatest amount of bending of hair cells and consequently the greatest differential activation of the auditory nerve fibers.

During the passage of the sound wave through the scala vesti-buli and tympani, the vibrations are believed to be transformed first

into the changes of the endocochlear DC potential and then int.o the pattern of nervous impulses in the cochlear nerve. The changes of the en.docochlear DC potential are called the cochlear microphonic potential and its waves relatively precisely follow the shape and frequency of the sound waves. It is clear that the relative movements of the tector-ial membrane to the hair cells causes the deformation of the hairs. The deformation of the hairs is somehow transformed into the cochlear micro-phonic potential. The mechanics of hair-cell stimulation differ substan-tially in different vertebrate species (Wever, 1970).

The most common theory of the generation of cochlear microphon-ics is the electromechanical theory (Davis, 1958). The endolymph potent-ial produces a current flow from endolymph to perilymph in the scala tympani which may. excite the nerve fibers in the surroundings of the hair cells. The sensory hairs are variabIe resistors which change their

resistance when they are bent. These variations in resistance may reach up to 120 ohms. The other theories proposing that there are enzymatic changes in sensory hairs, that hair cells act as generators producing the electrical current, etc., do not engender any widespread support.

The inner hair cells are probably more important for the genera-tion of nervous acgenera-tion potentials, because about 95% of afferent cochlear neurons are associated with inner hair cells and only 5% with outer hair cells (Spoendlin, 1972).

The impulse sound generates some nonlinearity and vorticity in the endocochlear fluids which causes the asymmetrical response of the hair cells. It is possible that this phenomenon contributes to the damage of the hair cells due to impulse sound (Tonndorf,

1969).

The tectorial membrane according to Davis

(1968)

mayalso be involved in the nonlinear mechanisms of the cochlea and possibly in the temporary threshold

shift •

1.5 Acoustic reflex.

The difference between the weakest perceived sound and that sound causing a pain sensation is very wide .. The ear however adapts to high intensity sounds, and the fine structures of the cochlea are protected against damage. At high intensities, over 130 dB SPL (sound pressure level), the response of the middle ear becomes nonlinear (Guiman and Peake,

1967)

due to the physical characteristics of the transmission. However, in an awake experimental animal, the middle ear system would not operate linearly above 60-70 dB SPL, which is .the threshold of the acoustic reflex.

The acoustic reflex is actually a contract ion of the stapedius and tensor tympani muscles which causes the malleus to pull away from the ear-drum (which therefore relaxes) and the stapes to recede from the oval window. The result of this movement is a decrease of amplitude of vibrations transferred into the inner ear and, thus, the attenuation .of hearing. This may be objectively measured by the decrease of the cochlear microphonic potential.

The acoustic reflex is effective only at low frequencies, between 200-600 Hz. When elicited by high-frequency sounds, the muscle reflex cannot produce its regulatory action, becoming very small above approx-imately 1500 Hz. The latent period of the acoustic reflex is, in human ears, between

40

and 160 milliseconds. Such variability exists not only among subjects, but for the same subject from trial to trial as well. The existence of the latent period has important practical and theoret-ical consequences. For example, due to the latency, the reflex is unable to perform its protective role when elicited by a very brief stimulus, no matter how intense this might beo

The relaxation of the middle ear muscles is much slower than the contraction and takes about two seconds. The relaxation does not depend on the intensity of the stimulus. Contraction of the muscles in the middle ear constitutes an essential part of the startle reaction (Greisen

and Neergaard ,

1975).

1.6. Effect of noise on ear.

As a result of exposure to noise of sufficient intensity for a long enough time, a person experiences a decrease in hearing sensitivity. This decrease in sensitivity is a complex phenomenon and consists of a

temporary componeIlt, temporary threshold shift (TTS), that recovers in time and a permanent component, permanent threshold shift (PrS). The proportion of decreased hearing sensitivity that is temporary or permanent depends on the history of the noise exposure. The decrease in hearing sensitivity may extend from a few decibels to almost complete hearing loss and is dependent on increasing noise intensity and duration of exposure.

For lower sound intensities, hearing sensitivity will not continue to decrease indefinitely to produce total deafness with continued exposure. Under these conditions, the decrease appears to be asymptotic (Melnick, 1964). This may have important implications for estimating the hazards of environment al and industrial noise.

Some basic data on the hearing loss due tO noise exposure have been known for over one hundred years. "Boilermaker' s" and "artillery-men' s" ears have been known to be defective with the cause of the

deficit well-known. In recent years, the effects of noise on human life have become a matter of serious concern for several reasons. Our rap~dly grow~ng population and advancing technology result in ever increasing noise levels. Noise is an undesirable by-product of virtually every aspect of present-day life. It not only affects humans, but also the whole ecological system in which life exists.

Man may even be exposed to some subjectively pleasant, but unexpeetedly damaging, sources of noise. Ear damage from exposure to rock and roll music has been found, not only in teenagers (Lebo and Oliphant, 1968), but also experimentally with guinea pigs exposed to 88 hours of 122 dB music as played in a well-known discotheque in Knotville (Lipseomb, 1969). Aeoustie trauma in sport hunters due to gun-firing is another example of this "leisure-indueed" disease (Coles and Riee, 1966; CHABA criteria, 1968; Odess, 1972). Also snowmobile engine noise may first cause a temporary threshold shift and, with further exposure, a permanent shift (Bess and poynor, 1972).

The threshold shifts are aceompanied by a temporary or permanent decrease of the eochlear microphonic potential, whieh, as mentioned above, exactly reproduees the sound pattern reeeived by the ear. With inereasing intensity of the sound, the eoehlear mierophonie inereases up to a certain level. However, with an additional inerease of the sound intensity, it levels off to show that there is a eertain limit to the eleetrieal response of the inner ear to astrong stimula-tion • Sound stronger than that sound causing a maximum eoehlear micro-phonic eauses a temporary or permanent threshold shift (Priee, 1967, 1968, 1968a, 1972, 1972a).

The impairment in hearing is eaused by damage to the hair eells of the Corti organ, althaugh the meehanism of this damage is

larg~ly unknown. Immediately following exposure to acoustic stimula-tion af more than 120 dB over one hour, the hair eells have a highly

distorted appearance. The cell nuclei are of ten swollen and have moved from their normal position at the base to lie somewhere in the cytoplasm of the cello In addition, the cell membrane can be ruptured. At first, the support-ing cells, the pillars and Deiters' cells remain intact, although the smooth endoplasmic reticulum is shifted and now found in the infranuclear zone.

A few days af ter acoustic overstimulation, the severely damaged sensory cells disintegrate completely and are expelled into the endolymphatic space. Outside the principal damage area the sensory cells are only partially affected and only a few deteriorate completely. This process is of ten initi-ated by cytoplasm leaking through the cuticula-free gap in the cell surface of the hair cello

The mild and probably reversible alteration one of ten sees is the proliferation of the smooth endoplasmic reticulum in the form of several so-called Rensen bodies in the outer hair cello Another reaction of the outer hair cells to acoustic overstimulation is observed in the form of fibroua condensations of their cytoplasm. Following acoustic trauma, selective alterations at the receptor pole, especially of the inner hair cells, are frequent. As a typical change, one sees luxation of the stereocilia, in which several cilia may join to form a thick club.

At the nerve endings of the outer hair cells acoustic overstimula-tion causes alteraoverstimula-tions which occur at the efferent endings. Af ter a latency of several days the mitochondria accumulate into clumps, and myelin figures are formed. The nerve ending continues to contain normal synaptic vesicles and some normal mitochondria. The degenerative changes in the Corti organ usually develop during several weeks af ter the damaging exposure.

Chronic acoustic trauma due to repeated stimulation with less

intense noise leads to alterations of a different nature. There is a relative-ly localized reduction of the sensory cells within the area of the tonotopic representation of the stimulation tone in the cochlea. Rowever, a great

number of sensory cells survive. They usually have an absolute increase in the number of lysosomes in the outer hair cells and dense inclusions in the apical lysosomes. Damage to the mitochondria has not been seen. The inclusions can fill up the lysosome to such an extent that the outer limiting membrane

disappears and the inclusions appear to lie free in the cytoplasma At scattered points between the otherwise intact cells, some hair cells are entirely missing. This illustrates the all-or-none pattern of cell damage. Either a cell

degenerates completely in a relatively early stage or it survives and maintains its structural integrity.

The standard pattern of degeneration therefore follows a well-defined order. First, the hair cells of the Corti organ degenerate, then the neural elements, and finally the supporting elements of the organ itself

(Duvall and QUick,

1969).

Isolated damage to the hair cells is uaually not seen (Johnsson,

1974).

events underlying the degeneration, the first phases of this process are extremely important. The very first event is the formation of blobs on the surface of the sensory hairs (Lim and Melnick, 1971, 1971a). This is followed immediately by changes in the smooth endoplasmic reticulum. Since the endoplasmic reticulum is concerned with proteosynthesis, it is possible that the degeneration of the Corti cells is associated with the failure of proteosynthesis. Failure of proteosynthesfus has been described by several authors. RNA metabolism, necessary for the synthesis of proteins in the cell, promptly reacts to loud noise (Nakamura, 1967; Wustenfeld and Halbfas,1965). Af ter intense stimulation the synthesis of RNA in the Corti organ decreases (Kraus et al., 1975). This is then followed by other changes such as accumulation of lysosomes, the organelles necessary for destruction and degradation of proteins and other macromolecular substances, deformation of cuticular plates of sensory celIs, and finally cell rupture and lysis. The space occupied by destroyed cells is then sealed off by supporting cells (Deiters' celIs).

The ~hreshold shifts may appear without large structural changes

of the sensory celIs. Beagley (1965) found structural changes only af ter the appearance of a threshold shift of 80dB. Even a permanent threshold shift need not be accompanied by hair cell damage (Hunter-Duvar and Elliott, 1972). This indicates that functional and biochemical changes appear before the cell is obviously damaged. The damage is uBually more intensive in young experimental animals (Falk et al., 1974), although it is also

depend-ent on the animal species. A 12-minute pure tone exposure of chinchillas produced greater losses than seen in squirrel monkeys exposed 12 hours to the same stimulus (Hunter-Duvar and Bredberg, 1974).

The biochemical changes accompanying hair cell damage have been mostly studied from two viewpoints: one is the energy metabolisE of the celIs; another is the sodium-potassium exchange accompanying the overloading.

The Corti organ has a relatively large amount of glycogen, which is higher in Deiters' cells than in hair celIs. This glycogen represents an energy store which the cells use during a period of increased activity (Ishii et al., 1969). Short-term, high-intensity stimulation may quickly exhaust the glycogen (Stack and Webster, 1961, 1971a), but this is not accompanied by any prominent increase of the enzymes of glucose aerobic metabolisme On the contrary, succinic dehydrogenase, an enzyme necessary for the energy metabolism of the cell (af ter exposure to the 110dB sound), decreases (Quade and Geyer, 1973).

Af ter acoustic stimulation, the partial pressure of oxygen in the organ of Corti decreases, which may mean that this structure consumes more energy (Misrahy et al., 1958). The oxygen in the cochlea may be ex-hausted within seconds (Mayahara and Perlman, 1972).

It is possible that vasoconstriction of the blood vessels of the inner ear may contribute to the pathogenesis of the cochlear damage

(Hawkins et al., 1972). This vasoconstriction means that the cells of the organ of Corti obtain less oxygen and quickly enter into a state of hypoxia and exhaustion. Thus, attempts have been made to improve the blood flow

through the cochlea by different vasodilating agents or agents supplying energy. Low molecular weight dextran supposedly improves the capillary blood flow and causes a much smaller numerical hair cell loss due to impulse sound (Kellerhah1~ 1972).

The finding presented by Nakashima et al. (1970) reveals another possible mechanism. They found that an intensive acoustic stimulation

(2000 Hz~ 100-140 dB SPL for two minutes) causes an increase of sodium ions and decrease of potassium ions in the endolymph. Af ter this intense stimulation the level of sodium did not return to the resting levels. The objection that the permanent change of ionic levels in endolymph may be caused by the rupture of Reissner's membrane and mixing of endolymph and perilymph Corti organ must be preserved for the cationic change to occur. When the Corti organ was destroyed (e.g., by previous strong noise) there was no increase of sodium following strong stimulation (Snow et al., 1970; Nakashima et al.~ 1973). The changes in the level of sodium and potassium in the endolymph af ter intense stimulation are identical with those changes following hypoxia (Suga et al., 1970). This comparison may indicate that the primary change in the inner ear during intense acoustic stimulation is the vasoconstriction of cochlear blood vessels with the subsequent damage to the Corti organ and changes of ions in the endolymph (Leonard et al., 1971). There is also a slight increase in protein in the endolymph (Scheibe et al.,

1975)~ which mayalso indicate vascular damage.

On the other hand, the mechanical changes also contribute to hair cell damage. Intensive sound may even cause perforations of Reissner's and the basilar membrane (Voldrich, 1972). These mechanical causes of damage are probably more important at higher frequencies and during gradually accumulating effects (StockweIl et al., 1969). The nonlinearity patterns, formation of eddies and pressure waves, are important in the localization of these changes (Watanuki et al., 1969).

The response of the inner ear to the intense sound is rather

individual and there are large differences not only among different species, but also between individual animaIs. This individual sound damage suscepti-bility has been explained, for example, by a differential acoustic reflex which reduces the stapes movements due to intense sound (Simmons, 1963). The state of the organism mayalso affect the extent of the pathological changes. Anesthesia, for example, increases the damage (Rubinstein and Pluznik, 1976).

The preceding review serves to indicate that the mechanism of cochlear damage due to intense sound, and particularly, impulse sound, is rather incompletely known and research in this area will probably provide much new data:.

1.7. Effects of impulse noise.

One of the most dangerous sounds from the standpoint of ear safety and health is impulse sound. The absence of an evaluation and measurement

of impulse noise effects on the human ear has long been the largest gap in OUT knowledge of acoustical pathology and this absence contributes to the lack of proper guidance in matters of hearing conservation.

There are several types of impulse noise (Coles and Rice, 1968):

Type A. These are occasional widely-separated impulses, typified by gunfire and other very intermittent explosive noise sources.

Type B. These are repetitive but discrete impulses covering ratios of peak-to-background level in the wave-envelope pattern of not less than about 6dB and impact rates of about 0.5 to 10 per seconde

Type C. These are highly repetitive noises in which the rep$tition rate is greater than about 10 per second and the ratio of peak-to-minimum level in the wave-envelope pattern is less than about 6dB. This is the commonest impulse noise type found in industry.

The risk of damage due to impulse noise depends on the peak pressure level, the duration of the impulse, ri se time and the total number of impulses. The effect of impulse noise has been detected in, among others, hunters,

artillerymen, and in particular in persons working in military armour units. The recognition of the danger of impulse noise was followed by several sets of criteria, the first being formulated by the NAS-NRC Committee on Hearing and Bio-Acoustics in 1968 (CHABA DRC), another in the U.S. Occupational Safety and Health Act of 1970, and a third has been set by U.S. Environmental Protection Agency in 1974.

The hearing loss due to impulse noise cannot be distinguished from loss due to continuous noise, but there are several differences between the TTS found af ter exposure to each (Luz et al" 1971):

1. When impulses are repeated, the TTS seen at the most suscept-ible audiometric frequencies increase as a linear function of the number of repetitions. This contrasts with the logarithmic growth seen with continuous noise.

2. When equal amounts of TT3iiare induced by continuous and by

impulse~moise, the TTS fr om impulse noise takes longer to

recover.

3.

Impulse noise-caused TTS recovers in an irregular fashion. In addition to a logarithmic recovery known from continuous noise exposure, diphasic plateau or rebound recovery has also been found (Luz and Hodge, 1971).

4. Typically, one observes more within subjects variance with impulse noise than with continuous noise.

These differences have been observed both in human and in animal ears. There are, however, some inter-species differences, chinchilla being more sensitive than Rhesus monkey or man when exposed to the same impulse noise source (Luz and Lipscomb, 1973). On the other hand, the chinchilla's loss is small (10-20dB) but broad (ranging from 300 Hz to 14.5 kHz); the monkeys' loss was peaked at higher fre~uencies (above 4 kHz) and large (above 80 dB loss at some fre~uencies) (Guthrie and Luz, 1973). Some research

results in this area are paradoxieal, in that lower (155dB peak SPL) intens-ities are potentially more hazardous to the hair cell population than the higher intensities (Hamernik and Henderson, 1974). This is, according to Kryter and Garinther (1965), a reflection of "tough" and "tender" ears in the exposed group. The explanation for this result offered by these authors is probably that at high intensities the tympanie membrane ruptures, thereby reducing the effective sound transmission to the cochlea. The rupture

supposedly heals within a matter of days and the cochlea is left in better condition than af ter the lower exposure.

The superimposed combination of exposure to continuous and impulse noise also has some surprising conse~uences. Cohen et al. (1966) measured the TTS af ter exposure to continuous noise, impulse noise and a combination of the two classes of noise. The addition of the 90 to 100 dB background noise to the impulse actually reduced the level of TTS, probably due to the presence of the acoustic reflex which protected the inner ear. Although several other authors (Walker, 1972; Lutman, 1972) verified these results, Hamernik et al. (1974) who used really damaging levels of both types of noise, found traumatic effects that more than exceeded the additive effects of either component.

In the pathogeny of the effect of impulse noise, several other factors play some role. The absence of the acoustic reflex, whose latency is longer than the duration or rise time of the impulse noise, is one of them. Another factor is the transformation of the sound in the outer ear. There is actually an enhancement of the intensity of sound at the eardrum

(maximum is about 20dB for stimuli in 3.0~~Hz region), and an increase of the rise time (Price, 1974). Impulse noise distends and misshapes the organ of Corti. This wavy distortion disappears within a few days (Lipscomb, 1974). Other than those differences noted above, impulse noise has effects on the inner ear very similar to those of continuous noise. There is even a

decrease of succinodehydrogenase similar to the decrease caused by continuous noise. This, according to Guttmacher et al. (1973), is due to hypoxia to which the hair cells are exposed as a conse~uence of noise.

1.8. Effects of sonic boom.

The effect of sonic boom on hearing is still a controversial topic. The physiological and pathological effect of sonic booms very much resembles the effect of impulse noise. The sonic boom is actually only a special case of impulse noise, and many described effects, such as a startie effect, sleep disturbance and annoyance, are identical for both short impulse noise and sonic boom (Griefahn, 1975). This review will not deal with the effects of sonic boom on human behavior or that of wild and laboratory animais.

All aireraft flying supersonieally ereate a near-field pressure disturbanee whieh ultimately forms a far-field N-wave "signature", whieh manifests itself as a sonie boom. If the duration is greater than about 100 ms, the bow and tail shock waves are heard as two sharp "elaps". The eharaeteristie pressure signature of the Coneorde eru±sing at twice the

speed of sound at an altitude of 16 km has a duration of about 250 ms. The overpressure is about 2.5 psf (pounds per square feet). (In SI units this is equivalent to 120 Pascals, where 1 psf

=

47 .. 88 N/in.2

=

47.88 Pa). During aireraft manoeuvres sueh as turns, pushovers and aeeelerations it

is pos si bIe to generate superbooms where the N-wave overpressure ean exëeed the eharacteristie value.

There are few papers available whieh analyze the effects of this type of impulse noise on the inner ear and hearing. Until recently, the available data seemed to suggest that even sonic booms of maximum intensity do not produce direct medieal injury.

In a recent laboratory study (Von Gierke and Nixon, 1972), human volunteers were exposed to an acoustic stimulus "very similar in signature overpressure level to the near~ield sonic boom generated by fighter-type aireraft at very low altituäe". Otologie al and audiometrie examination of the subjects revealed no adverse effects on the tympanie membrane or on auditoryacuity. The sonie boom-type aeoustie impulses of the l69dB

(110-115 psf, over fifty-fold greater than the eruising Coneorde boom) were shown to be "safe" for the partieipating subjeets.

Rice and Coles (1968) measured TTS for subjeets exposed to the simulated sonie booms, and the magnitude of temporary threshold shift suggested that exposure to N-waves (superbooms) of 17 psf (152dB) would not constitute an acoustie hazard. Cottereau (1974) in his review paper also eoneludes that the sonie booms due to the flight of supersonie planes have no troublesome biologieal eonsequenees.

Majeau-Chargois (1969) and Majeau-Chargois et al. (1970) reported on a study of damage to the auditory mechanism of guinea pigs subjeeted to simulated sonic booms. The animals were exposed to 1000 booms at I-sec intervals. The sound-pressure level at the ear was measured to be l30dB. In this unusually severe laboratory test, hair eell damaged oceurred in the apical turn of the coehleae of the exposed guinea pigs. Other turns were unaffected and the damage to the hair cells was eonsidered to be permanento However, the studies did not deseribe any impairment of hearing, as tested by the Preyer reflex (movement of the pinna as a response to the sound).

Bobbin and Gondra (1975) exposed guinea pigs to intense low fre-queney tones whieh they eonsidered to be analogous to sonic booms, because they are present in sonie booms and eontain most of the energy of the

impulse. They found that these frequencies may damage the eoehlear function and structure. However, they eonclude that sonie booms do not eontain

suffieiently intensive low frequeneies whieh may damage the cochlea. This review of the literature immediately relevant for our own

research shows that, with the exception of two papers presented by a single group, there is practically no experimental literature which has investi-gated the structural and functional effects of sonic boom on the inner ear. The validity of the papers by Majeau-Chargois is moreover hampered by the severity of the treatment given the animals--about 1000 bangs were admin-istered at I-sec intervals.

2. RATIONALE OF STUDY

The main purpose of this study is to provide basic data on the response of the inner ear to the sonic boom. The parametric study of different intensities, rise times and numbers of sonic booms is the main component of this study. The pathological changes which may appear from the first minutes after the exposure are followed and investigated.

Most authors who have been interested in the problem of the effect of sonic boom on the inner ear followed just one indicator of the inner ear activity--namely audiometric curves. In two papers by Majeau-Chargois et al., the absence of hair cells was also noted.

However, many authors who studied the conse~uences of inner ear

damage by excessive sound have shown that these changes are of ten not parallel. Temporary or permanent threshold shifts were found when the structure of the Corti organ was normal, and vice versa. Thus several other changes have to be followed in parallel, such as vascular changes and histochemical reactions in the organ of Corti and nerve cells of the spiral ganglion.

An experimental study using laboratory animals should always precede human experimentation. This is not to say that the results obtained with animals can be directly applied to humans. Although the basic response of the inner ear to noise is probably almost identical in many mammalian species, there are well-known ~uantitative differences in the sensitivity of the inner ear to damage. Small rodents, such as the mouse or chinchilla, used in this study, are much more sensitive to noise damage than, for example, infrahuman primates. This was the reason for using small animaIs. In order to detect pathological changes, one must be sure that they will be present. On the other hand, one has to be aware of much greater resistance of the human ear to damage (see Section 1.7).

The use of mice has one additional advantage. The extent of the

fre~uencies which they are able to hear and respond behaviorally is enormous (from about 100 Hz to more than 80,000 Hz). Mouse pups with their mothers and males with females actively interact at the fre~uency of more than 50kHz. This ability is ~uite important because the changes due to impulse noise were mainly found in the basal turn of the cochlea, in the area where

high-fre~uency sounds are perceived.

One phenomenon, which was already noticed during the first pilot experiments, was bleeding into the scala tympani of the inner ear due to the simulated sonic booms. Bath in mice and chinchillas, the bleeding can

be observed when the inner ears are fixed and disseeted, but with more c.are than had been previously deseribed in the literature • The present study is d,evoted to the detailed investigation of this phenamenon.

3 .

MATERIALS AND METHODS

3.1

Apparatus

On the day of the experiment, the experimental animals were trans-ported from the University of Waterloo to the University of Toronto,

Institute for Aerospaee Studies (urIAS) • At urIAS, groups of animals were exposed to simulated sonie booms, whieh were generated by either the Loud-speaker-Driven Booth or the Travelling-Wave Horn. A detailed deseription of tb,e two sonie-boom simulators ean be found in the paper by Glass, Ribner and Gottlieb

(1973):

The Loudspeaker-Driven Simulator is in the form of a solidly-built booth, whieh is about 70 eubie fe et in volume. Owing to the flexibility of the eleetronie equipment and loudspeakers, features of the sonie-boom overpressure signature inside the booth ean be adjusted at will. Thus, response to the variation of sueh eharaeteristics of sonie boom as N-wave over-pressure, rise time and duration can be evaluated. The Travelling-Wave Horn is in the form of a concrete horizontal pyramid, which is 80 feet long with a 10 x 10 foot base. At the apex of the horn a

specially designed mass-flow valve is used to control the flow of high-pressure air into the horn, which

generates a travelling simulated sonie boom N-wave

of suitable amplitude, rise time and duration. The interior of the horn eontains a high-frequeney sound absorber to reduce undesirable jet noise, and the open end is covered by a large porous piston to eliminate the undesirable reflection and subsequent eehoes.

The sonie boom that can be simulated by the Loudspeaker-Driven Booth and Travelling-Wave Horn is illustrated in Fig.

3.1.

The most important parameters used to

describe the idealized overpressure signature include the peak overpressure, duration, rise time, and waveform

(which may vary somewhat from the N- shape) • The spectral contents of the N-wave cover the frequency range from a fraction of one hertz to several thousand hertz. More over , most of the energy of the N-wave is coneentrated in the low frequencies below 200 Hz. It is worth noting that values of peak overpressure, duration, and rise time for

Ang1o-French Concorde and Soviet TU-144 aircraft, are of the order of 2 psf (pounds force per square foot or about 100 Pa), 300 msec, and 1 msec,

respectively. In the case of military supersonic fighter aircraft the duration is correspondingly less at about 100 msec.

An exarnple of the waveform actual1y produced in the Loudspeaker-Driven Booth is presented in Fig. 3.2. There is some superimposed noise but the shape of the N-wave is well preserved.

3.2 Test procedure wi,th the inbred mice.

C57BL/6J mice, all males, were born and raised in the vivarium of the Department of Psycho1ogy of the University of Waterloo. The animals were three months old when they were exposed to the simulated sonic booms.

Several experiments were made to evaluate the effects of simulated sonic booms on the inner ears of mice. An attempt was made to correlate the presence of the acute pathological changes with a single or succession of sonic booms of different intensi ty and rise time. Since the main patho-logical change was the bleeding in the scala tympani of the basal and some-times middle turn of t.he cochlea, it was also of interest to determine the time period for reabsorption of the extravasate and normalization of the changes.

For the first. se-t of experiments an attempt was made to correlate

the basic patho1ogical finding, bleeding into the basal turn of cochlea, with the intensity of a single boom. Four different g,roups of mic.e were each subjected to a single simulated sonic boom having the same rise time of 5 msec and duration of 100 msec. Tbe peak overpressure was varied., having the values 1.3,

3

.

0,

4.0 and 10 psf. These vaJ.ues represent 62, 143, 191 and 479 Pa, respective1y. The Loudspeaker-Driven Booth was used for the experiments for wbich the peak overpressure was 1.3, 3.0 and 4.0 psf)

whereas the Travelling-Wave Hom having the capability of generating power-ful simulated sonic booms was used for -the one experiment for which the peak overpressure was 10 psf. The mice (6 to 10 in each group) we re sacri-ficed 72 hours af ter their exposure to the single boom.

In the second set of experiments the effeGts of various rise times were stuQied. Groups of;6 to 10 mice were exposed to N-waves having a peak overpressure of 3.3 psf, a duration of 121 msec, but rise times that were varied from 0.1 to 10 msec.

In the same group of experiments, the cumulative effect of repeated sonic booms were also studied. Different groups of mice were put in the UTIAS Loudspeak~T-Driven Booth and exposed to 1, 2, 3, 4 or 5 booms at a rate of one every 10 seconds. After the experiment the mice were transported back to the University of Waterloo, where they we re sacrificed 24 hours af ter their exposure. Control mice, not exposedto simulated sonic booms, were transported

and handled with the experimental mice.

Af ter it was found that five repeated sonic booms can invariably cause bleeding in the cochlea, it was of interest to compare these results with the effect of booms administered at 24-hour intervals. This time interval resembles more closely a possible real situation when the individ-uals are exposed to sonic booms irregularly, once or several times a day. For this set of experiments, groups of mice were each put in the 1oudspeake~­ Driven Booth exposed to simulated sonic booms having a rise time of

5 msec,

a duration of 100 msec, and a peak overpressure of 3.0 psf. One group was exposed to only one boom, while another group was subjected to the same boom on five consecutive days. These animals were sacrificed 72 hours af ter the last boom.

In this experiment, we also studied the rate of absorption of the blood clot from the scala tympani. Thus, groups of animals were also sacri-ficed one, two, four, six and eight weeks af ter the last exposure to the sonic booms.

For the last experiment, twenty mice were subjected to a series of identical simulated sonic booms in the Loudspeaker-Driven Booth. Here an attempt was made to find out what other changes may appear when large numbers of sonic booms are administered in a short time. The mice experienced 18,000 booms, at a frequency of

5

booms per second for one hour. In this experiment only, the N-wave was slightly imperfect in that the peak overpressure of the compression portion was 4 psf and differed from that of the expansion part (,:

which was

6

psf. The N-wave ri se time was

5

msec and the duration was 100 msec. Of the group of twenty mice subjected to this treatment, ten were sacrificed 72 hours af ter exposure to the booms, five one week af ter, and five two weeks af ter exposure.

3.3. Dissection of inner ear.

This study required the dissection of large numbers of inner ears of mice. Consequently, a suitable method had to be found for this purpose. The mefihod of Engstrom et al. (1963) is excellent for precise preservation of the Corti organ. However, the decapitation of an unanesthetized animal may in itself cause bleeding into the inner ear, and during the dissection of the inner ear, the bleeding from surrounding tissues may easily conceal the tiny blood clot. Further, the perfusion of the cochlea with osmium

tetroxide may wash out the extravasated blood celIs. Therefore, in the pilot experiment several methods were tested to dissect and fix the inner ear. The following methods were employed:

1. Decapitation of unanesthetized animal with fast dissection of the inner ear and perfusion with osmium tetroxide. 2. Decapitation of unanesthetized animal with fast dissection

of the inner ear, opening of the cochlea near apex and fixation in 10% formalin.

3.

Nembutal anesthesia with opening of both carotid arteries and jugular veins and subsequent fixation in 10% formalin.

4.

Ne.mbutal anesthesia followed by perfusion of the circulation with saline and 10% forroalin with

sub-sequent fixation of temporal bones in formalin.

There were ten mice in each group in this experiment. The decap-itation of unanesthetized aniroals with subsequent fixation was accompanied by bleeding into the cochlea in

6

out of 20 dissected inner ears fixed with osmium tetroxide and in

8

out of 20 dissected ears fixed with forroalin. On the other hand, the mice which were anesthetized and either bIed or perfused did not show any bleeding into cochlea. This finding was verified later with more than seventy control mice which accompanied all experimental groups. Thus, for this study, the following method was used:

All mice were sacrificed by first deeply anesthetizing them with ether and then opening both the carotid arteries and jugular veinë. Af ter the bleeding stopped the top of the skull was opened, the brain was removed and both temporal bones were extracted and fixed in a 10% formalin solution. The middle ear was opened widely and the stapes was pulled out from the oval window. Af ter at least

24

hours of fixation, all structures of each middle ear were removed under the preparation microscope. The cochlea was then clearly visible under the medial wallof the middle-ear cavity. Later on, the bone covering the cochlea was decalcified with a eanlab decalcifying solution. This took at least 15 minutes. Thereafter, this solution was carefully washed away with distilled water and the softened bone was cut away with opthalmological scissors. The cochlea was carefully dissected and the segroents transferred into a droplet of hematoxyline-eosine.

The course of the staining was observed with the preparation micro-scope. When the stain became sufficiently intense (about

5

to 10 minutes) the segroents were transferred to a drop of water, and the excess hematoxyline was washed away. The parts of the outer wallof the cochlea and of the mod-iolus, which may interfere with observation, were trimmed with an ophthalmo~ ..

logical scalpel, and the segroents of the cochlea were arranged and mounted without dehydration in Hydramount (Gurr) or glycerol (for simultaneous phase-contrast observation). This technique, although it requires some skill and patience, is not difficult and roay be used not only for hematoxyline eosin staining, but also for several histochemical determinations.

It was possible not only to see clearly the stained structures, the basilar membrane and the nerve cells in the modiolus, but if phase contrast was used, the hairs of the hair celIs, the borders of the hair cells and the nerve fibers crossing the canal Corti. During the preparation procedure, the spaces in the membraneous cochlea were checked for the presence of blood clots and other pathological inclusions.