Fax +41 61 306 12 34

E-Mail [email protected]
www.karger.com
Audiol Neurotol 2009;14:350360
DOI: 10.1159/000241892
Principles of Local Drug Delivery to the
Inner Ear
Alec N. Salt
a
Stefan K. Plontke
b


a
Department of Otolaryngology, Washington University School of Medicine, St. Louis, Mo. , USA;

b
Department of Otorhinolaryngology, Head and Neck Surgery and Tbingen Hearing Research Center,
University of Tbingen, Tbingen , Germany
are more effective. However, if the applied substance does
not easily pass through the round window membrane, or if
a more widespread distribution of drug in the ear is required,
then intralabyrinthine injections of the substance may be
required. Intralabyrinthine injection procedures, which are
currently in development in animals, have not yet been
proven safe enough for human use.
Copyright 2009 S. Karger AG, Basel
Introduction
Local drug delivery to the inner ears of humans was
first used more than half a century ago for the treatment
of Mnires disease with local anesthetics [1, 2] and an-
tibiotics [3] . It was popularized in the 1990s as it became
accepted that locally applied gentamicin provided an ef-
fective treatment for the vestibular symptoms of patients
with Mnires disease with limited risk to hearing [46] .
In addition to aminoglycosides and anesthetics, a variety
of drugs have been applied extracochlearly to the round
window niche in humans, including neurotransmitters
Key Words
Animal Cochlea Perilymph Controlled release
Inner ear Human Local drug delivery Pharmacokinetics
Abstract
As more and more substances have been shown in preclini-
cal studies to be capable of preventing damage to the inner
ear from exposure to noise, ototoxic drugs, ischemia, infec-
tion, inflammation, mechanical trauma and other insults, it
is becoming very important to develop feasible and safe
methods for the targeted delivery of drugs to specific re-
gions in the inner ear. Recently developed methods for sam-
pling perilymph from the cochlea have overcome major
technical problems that have distorted previous pharmaco-
kinetic studies of the ear. These measurements show that
drug distribution in perilymph is dominated by passive dif-
fusion, resulting in large gradients along the cochlea when
drugs are applied intratympanically. Therefore, in order to
direct drugs to specific regions of the ear, a variety of de-
livery strategies are required. To target drugs to the basal
cochlear turn and vestibular system while minimizing expo-
sure of the apical cochlear turns, single one-shot intratym-
panic applications are effective. To increase the amount of
drug reaching the apical cochlear turns, repeated intratym-
panic injections or controlled-release drug delivery systems,
such as biodegradable biopolymers or catheters and pumps,
Received: April 1, 2009
Accepted after revision: July 17, 2009
Published online: November 16, 2009
Neurotology
Audiology
Dr. Alec N. Salt
Department of Otolaryngology, Box 8115
Washington University School of Medicine
660 South Euclid Avenue, St. Louis, MO 63110 (USA)
Tel. +1 314 362 7560, Fax +1 314 362 1618, E-Mail salta @ ent.wustl.edu
2009 S. Karger AG, Basel

Accessible online at:
www.karger.com/aud
A.N.S. and S.K.P. contributed equally to this paper. A.N.S. is a mem-
ber of the scientific advisory board of Otonomy; however, this work
was not supported by Otonomy.
Local Drug Delivery to the Ear Audiol Neurotol 2009;14:350360 351
and neurotransmitter antagonists for tinnitus [7] , mono-
clonal antibodies for autoimmune inner ear disease [8] or
apoptosis inhibitors (AM-111) for noise-induced hearing
loss [9] . However, glucocorticoids have become the most
widely used drugs for local application to the inner ear,
and have been given to treat Mnires disease [10] , idio-
pathic sudden sensorineural hearing loss [1113] , auto-
immune inner ear disease [14] and tinnitus [15] , even
though the evidence supporting their use is rather lim-
ited [16, 17] . Nevertheless, at present, dosing protocols
and the selection of drug delivery systems are almost to-
tally empirically based, and there is still only a limited
understanding of the pharmacokinetics of drugs in the
ear.
Pharmacokinetics of the Inner Ear
Although the LADME scheme was developed to de-
scribe the pharmacokinetic processes in the human body
following a given dosage regimen, it is helpful to adopt
this concept for understanding and investigating the
principles of drug movements in the inner ear after local
or systemic application. The LADME concept involves
liberation, absorption, distribution, metabolism and
elimination of drugs ( fig. 1 ). While for whole body phar-
macokinetics, the LADME processes are centered on
blood circulation, in the ear they are centered on the in-
ner ear fluids.
Liberation describes the release of the drug from its
dosage form.
Absorption refers to the movement of the drug from
the site of administration to the inner ear fluids (e.g. from
the middle ear to the perilymph of the scala tympani (ST)
through the round window membrane, RWM).
Distribution involves the processes by which the
drug diffuses, flows or is transferred within and be-
tween the different fluid-filled compartments (peri-
lymph and endolymph), and how it spreads from the
fluid spaces into the various tissue compartments of
the inner ear.
Metabolism is the chemical conversion or transforma-
tion of drugs into active moieties or compounds which
are easier to eliminate.
Elimination describes the removal of the unchanged
drug or metabolite from the inner ear (e.g. to blood, ce-
rebrospinal fluid or the middle ear).
Although these processes generally follow the above
sequence, they may occur simultaneously. While the
drug is still being liberated from a controlled release for-
mulation, previously absorbed drug may already have
been eliminated.
Liberation
Current efforts in the area of drug delivery in general,
and also specifically in inner ear therapies, include the
development of drugs which are liberated from a formu-
lation over a period of time in a controlled manner. Types
of sustained release formulations include liposomes,
drug-loaded biodegradable microspheres and drug poly-
mer conjugates, including gels [1822] . Drug release from
carrier systems may be driven only by the concentration
gradient (such as for a drug in a resorbable gelatin sponge
soaked with drug solution) or maintained by a gradual
breakdown of the carrier, either spontaneously or in-
duced by physical and chemical triggers (e.g. temperature
or pH), with subsequent release of drug.
Absorption
Absorption of a drug from the middle ear to peri-
lymph of the inner ear can occur through a number of
structures, including:
Fig. 1. Pharmacokinetic processes of the inner ear according to
the LADME concept, as described for an intratympanic applica-
tion of a formulated drug. Absorption occurs primarily through
the round window membrane. The drug, upon entering the peri-
lymph, distributes both within the scala tympani and into adja-
cent fluid and tissue-filled spaces. The drug is also subjected to
metabolism and elimination to blood or CSF.
Salt /Plontke

Audiol Neurotol 2009;14:350360 352
Round Window Membrane . The RWM in humans,
monkeys, felines and rodents consists of 3 main layers:
(1) an outer epithelial layer facing the middle ear cavity;
(2) a middle connective tissue layer; (3) an inner cellular
layer facing the ST perilymph [23, 24] . Tight junctions are
present between cells of the outer layer, while in the mid-
dle layer fibroblasts, fibrocytes, collagen, elastin, capil-
laries, and myelinated and unmyelinated nerves have
been described [25] . Many studies have demonstrated in
qualitative terms that substances applied to the middle
ear enter the basal turn of the ST, and may influence
structure and function of the ear [24, 2628] . In contrast,
few have performed quantitative measurements of drug
levels in the perilymph or measured RWM permeability.
Of the pharmacokinetic studies in the literature, a sub-
stantial proportion cannot be interpreted quantitatively
due to sampling methods that caused the fluid samples to
be highly contaminated with CSF [18, 29, 30, 31] . In these
studies, large volumes (10 l) relative to the volume of the
ST in the guinea pig (4.6 l) [32] were taken from the
basal turn of ST. As the cochlear aqueduct enters ST at
this location, samples taken nearby become severely con-
taminated with CSF that is drawn into the scala as the
sample is aspirated. Based on measurements with marker
ions, it was estimated that 10- l samples taken from the
basal turn of guinea pigs contained as little as 15% peri-
lymph and 85% CSF [33] . Sample measurements are more
readily interpreted when the samples are taken from a
location further from the cochlear aqueduct. A better
technique, in which multiple samples are taken sequen-
tially from the cochlear apex within a period of a few
minutes, allows both the concentration and the gradient
of drug along the ST to be quantified [34] . Results ob-
tained with this technique show that, following 2- to 3-
hour application of a drug or marker to the RWM, there
are substantial gradients along the ST. The gradients de-
termined for 3 substances are shown in figure 2 . For
TMPA (trimethylphenylammonium: an ionic marker),
gentamicin and dexamethasone, basal-apical concentra-
tion differences of over 1000-fold were found in most an-
imals. The presence of basal-apical gradients following
drug applications to the RWM is supported by a number
of histological studies that suggested markers were at
higher concentration or cellular damage was greater in
the basal turn than in apical regions [28, 35, 36] . The con-
centration measurements in figure 2 also show that the
basal turn concentration of drugs is variable, with over
10-fold differences between animals being common.
Measurements of entry rates using microdialysis have
confirmed that the variability between animals arises
from differences in RWM permeability [37] . RWM per-
meability has also been shown to be sensitive to experi-
mental manipulations. Permeability is increased by local
anesthetics [38] , endotoxins and exotoxins [39, 40] , hista-
mine [41] , drying through the use of suction near the
round window niche [42] , by osmotic disturbances and
by the presence of benzyl alcohol (a commonly used pre-
servative) in the applied solution [42] .
1000
100
10
1
10000
1000
100
10
1000
100
10
1
C
o
n
c
e
n
t
r
a
t
i
o
n

(

M
)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
m
l
)
C
o
n
c
e
n
t
r
a
t
i
o
n

(
m
g
/
m
l
)
TMPA Gentamicin Dexamethasone
0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 14 16
Distance along ST (mm)
Fig. 2. Concentration gradients along ST of the guinea pig follow-
ing 2- to 3-hour applications to the RWM. Distances are measured
along the ST from the basal end. Results are shown for the mark-
er ion TMPA [34] , gentamicin [53] and dexamethasone [54] . In
addition to the steep concentration gradients, it is also apparent
that the basal turn (02 mm) concentrations of each substance
vary by more than a factor of 10, due to inter-animal variations in
RWM permeability and elimination of the drug. Recently, it has
been shown that these concentration gradients are stable with
time [83] .
Local Drug Delivery to the Ear Audiol Neurotol 2009;14:350360 353
In addition, there is only limited knowledge about the
individual processes of transmembrane transport con-
tributing to substance absorption through the RWM, in-
cluding passive diffusion, facilitated diffusion through
carriers, active transport or phagocytosis.
Oval Window (Including the Stapes Footplate and An-
nular Ligament). Although a number of investigators
have suggested substances may enter perilymph of the
vestibule by this route [36, 43] , it is technically difficult to
measure the amount. Substances cross readily between
the ST and scala vestibuli (SV) [44, 45] , presumably pass-
ing through the spiral ligament (discussed further in
Distribution). Thus, the observation of drug or marker
in the vestibule or saccule does not confirm that it entered
through the oval window. In addition, attempts to oc-
clude the RWM (such as with dental cement [36] ) were
found to be only partially effective. It is undoubtedly pos-
sible for drugs to pass through the thin bone of the stapes
footplate or through the walls of the oval window niche
in amounts which may be significant in the human. The
amount of drug entering by this route, however, remains
uncertain and likely depends on drug size and charge, but
is thought to be small relative to that entering through the
RWM.
The Bony Otic Capsule . It has recently been shown that
when a drug is applied by filling the middle ear with so-
lution in guinea pigs, the highest drug levels are produced
in the apical regions of the cochlea [46] . This results from
the drug entering perilymph through the bony otic cap-
sule, which is very thin in the apical turns of animals such
as guinea pigs and chinchillas. This presents a consider-
able problem when studies in rodents are used as a mod-
el for drug delivery in the human, as it can be assumed
that the thicker bone of the human otic capsule will rep-
resent a more effective boundary. The resulting drug dis-
tribution patterns along the length of the cochlea are
therefore likely to differ markedly between animals and
humans following intratympanic applications.
Distribution
The fluids of the inner ear show little evidence of stir-
ring, i.e. no pronounced movement comparable to that
of the systemic circulation. In the intact state, rates of vol-
ume flow of perilymph and endolymph are both exceed-
ingly slow and the distribution of drugs within the fluid
spaces is dominated by passive diffusion. Diffusion is a
highly predictable process and its effects can be calcu-
lated with accuracy. The rate of substance movement by
diffusion is nonlinear with distance, allowing drugs to
spread rapidly over short distances (such as across a co-
chlear scala), but slowly over distances of more than a few
millimeters. This results in large gradients along the co-
chlea when substances are applied to the basal turn, as
shown earlier in figure 2 . Also contributing to the gradi-
Fig. 3. Reconstructed 3D anatomy of the fluid spaces of the guin-
ea pig inner ear derived by segmentation of an OPFOS (orthogo-
nal-plane fluorescence optical sectioning) image set [84] using
Amira software. The enlargement shows the basal turn with the
stapes removed (leaving an imprint of the footplate). V = Vesti-
bule; SL = spiral ligament; RW = round window. The SL follows
the periphery of the RW almost half way around, providing a ma-
jor route for drugs in the ST near the RW to diffuse across into the
vestibule. This anatomic pathway accounts for how drugs applied
intratympanically to the RW can gain access to vestibular struc-
tures. Blue = Endolymph; orange = perilymph of SV and V; yel-
low = perilymph of ST; green = spiral ligament; red = sensory
structures; purple = RW; magenta = cochlear aqueduct; brown =
stapes.
Salt /Plontke

Audiol Neurotol 2009;14:350360 354
ents along the length of the cochlea is the loss of drug
from the scala as it diffuses. Losses result from the distri-
bution into fluid spaces and tissue compartments adja-
cent to the scala, and through elimination from the ear
to other much larger compartments, such as blood. For
ST, substances readily distribute through the fluid spaces
of the spiral ligament into the SV and the vestibule. Fig-
ure 3 shows the anatomy of the spiral ligament in the
basal turn, which is seen to wrap around the periphery of
approximately half the RWM. Drugs present in the peri-
lymph at the base of ST close to the RWM would therefore
be expected to have ready access to perilymph of the ves-
tibule. In addition, drugs in the ST can pass through pas-
sages in the bone (canaliculi perforantes) into Rosenthals
canal and from there to the modiolus [47, 48] , and into
the fluid spaces of the organ of Corti [49] . Although the
canaliculi perforantes in the medial wall of the ST appear
rather large, data from experiments with neurotrophins
suggest that only a small proportion of a drug infused
into the ST reaches the vicinity of the spiral ganglion neu-
ron cell bodies, suggesting that other barriers to diffusion
may exist [50] .
In addition, some substances may also enter endo-
lymph, although this is likely to depend on the electrical
charge of the molecule, which subjects it to the influence
of cellular potentials and the endocochlear potential.
Cationic markers are excluded from endolymph [44] ,
while anionic markers are accumulated and retained
there [51] .
Apart from a drugs chemical characteristics, especial-
ly size and its hydrophobicity, distribution processes are
primarily dependent on concentration gradients, so that
the distribution of substances into different compart-
ments of the inner ear can be reversed if the concentra-
tion gradient changes. This is apparent during sequential
apical sampling, when CSF entering the basal turn of the
ST gains drug as it passes through the scala towards the
sampling site, and can be referred to as redistribution.
Indeed, the rate at which sequential sample concentra-
tions decline during rapidly repeated sampling from the
apex provides a valuable index of the rate and amount of
drug available to redistribute into the scala during sam-
pling.
It has also been suggested that protein levels in peri-
lymph may be sufficient to bind or buffer some drugs
[52] . Uptake of a drug into cellular structures of the ear
may also occur. By causing a loss of drug from the scala,
each of these processes acts to reduce the rate at which the
drug spreads along the scala by diffusion. However, in
contrast to the expected slowing of the rate of distribu-
tion caused by these processes, the measured gradients
along the ST in figure 2 could only be explained by the
substances spreading towards the apex at a slightly faster
rate than can be accounted for by diffusion. To account
for the data, it was necessary to incorporate a low rate of
apically directed perilymph flow into the simulations of
the experiments. The perilymph flow rates averaged
0.019, 0.021 and 0.009 l/min for TMPA [34] , gentamicin
[53] and dexamethasone [54] , respectively. While these
rates are extremely low, they suggest that perilymph flow
may contribute significantly to drug distribution when
its effects are accumulated over a period of hours or
days.
Metabolism
Metabolism or biotransformation considers the trans-
formation of the drug from one form to another with al-
tered (lower or higher) effectiveness by tissues and en-
zymes within the ear or in adjacent compartments, such
as the middle ear. There is, however, very limited quanti-
tative data about the metabolism of specific groups of
substances following their application to the inner ear. It
has been shown that dexamethasone-phosphate is me-
tabolized within the ear to the active moiety dexametha-
sone [54, 55] . There is also accumulating evidence that
extracellular purines in the cochlear fluids, important
modulators of cochlear function and potential targets for
manipulation [56] , are regulated by ectonucleotidases
[57] .
Elimination
The rate of elimination of a drug from perilymph is
one of the major factors influencing both the concentra-
tion achieved in perilymph and how far towards the apex
a drug is distributed following its application to the RWM.
For faster elimination rates, perilymph concentration
saturates at a lower value and reaches a steady state more
quickly than with slower elimination rates. In addition,
when the amount of drug eliminated from a specific re-
gion becomes equal to the rate of diffusion along the sca-
la at that point, then a steady state will be established and
the drug will never reach a higher concentration at more
apical locations. In order to determine how far a specific
drug will spread along the cochlea, knowledge of the
elimination rate is required. Determination of the elimi-
nation rate for a specific substance is, however, techni-
cally difficult. If drug is applied locally to the RWM, then
the decline in concentration at a location near the appli-
cation site (e.g. in the basal part of the ST) following the
application results from the combined effects of both dis-
Local Drug Delivery to the Ear Audiol Neurotol 2009;14:350360 355
tribution and elimination, which would distort quantifi-
cation of the elimination rate. If the time course of drug
decline is monitored by taking samples repeatedly over a
long period, such as samples taken hourly in the study by
Parnes et al. [29] , then the act of sampling, accompanied
by drawing CSF into the cochlea, also contributes to the
decline of drug concentration and prevents an accurate
estimation of the elimination rate. Similarly, the use of
microdialysis to follow perilymph concentration as a
function of time [37, 58] does not provide a valid indica-
tion of elimination rate as the microdialysis procedure
itself provides a major source of elimination [37] . As a re-
sult of these technical difficulties, there are relatively few
studies from which drug elimination rates can be reliably
derived. In a few studies, where all or most of the peri-
lymph in the cochlea was taken as a single sample, the
effects of redistribution were minimized. One series of
studies investigated the kinetics of gentamicin in the
chinchilla [59] , in which large samples of perilymph (15
l) were taken from the vestibule. These studies were an-
alyzed quantitatively [60] from which a perilymph elimi-
nation half-time of 500 min for gentamicin was deter-
mined. In another study, prednisolone kinetics were in-
vestigated in guinea pigs [61] , obtaining perilymph by
aspirating the entire contents of the ST and SV after the
temporal bone was removed from the animal. Quantita-
tive analysis of these data allowed an elimination half-life
of 130 min for prednisolone to be derived [62] . These ap-
pear to be the only studies in which the experimental de-
sign allowed a meaningful interpretation of the elimina-
tion rate for drugs.
While pharmacokinetic analyses generally consider
elimination of solutes from the scala fluids to blood, we
know from anatomic data that this is an oversimplifica-
tion. The major capillary beds in the ear are not located
in, or directly associated with, the scalae. Although arte-
rioles and venules run around the bony walls of SV and
ST, respectively, they run within bony canals separated
from the scala fluid spaces. Instead, the major capillary
beds are associated with the spiral ganglion, the lateral
wall and limited regions of the organ of Corti. Fluid path-
ways through the bone between the ST and the spiral
ganglion, and between the SV and the modiolar spaces,
have been demonstrated [47, 48] . Elimination of drugs
from perilymph is therefore likely to occur indirectly,
mediated by the vascularized tissues in communication
with perilymph. Whether spiral ligament or modiolar
sites dominate perilymph kinetics remains to be estab-
lished. It is also likely that distribution of many substanc-
es will be influenced by active transport processes at
physiological boundaries in the ear, comparable to those
established for the eye [63] , but given the complexities of
pharmacokinetic studies of the ear, influences of specific
transport processes on cochlear fluid pharmacokinetics
have yet to be demonstrated.
Delivery Systems and Protocols
Based on knowledge of how drugs enter and are dis-
persed in the ear, it is possible to build a framework for
how drugs can be delivered to the ear for specific pur-
poses, as shown in figure 4 . At present, the strategy for
local drug delivery in clinical situations is dominated by
intratympanic applications. For situations where the
drug does not readily pass the RWM, or where better con-
trol of drug level is necessary, then intralabyrinthine ap-
plications are necessary. However, none of the intralaby-
Fig. 4. Idealized flowchart for drug deliv-
ery to the ear, taking into account the
known distribution properties and limita-
tions of different delivery systems. IT = In-
tratympanic; SCC = semi-circular canal.
Salt /Plontke

Audiol Neurotol 2009;14:350360 356
rinthine application techniques used in animals has yet
been proven safe enough for use in humans, although this
field is advancing rapidly.
Intratympanic (Middle Ear) Applications
Delivery protocols used in conjunction with intratym-
panic applications have included single injections (a one-
shot), repeated injections (3 times a day over a period of
time, or longer intervals such as weekly), or continuous
delivery over a period of days using a microcatheter. The
influence of delivery protocol on the amount and time
course of drug in perilymph has not yet been document-
ed, but calculations show that a key variable influencing
the perilymph concentration achieved by each application
is the time the drug remains in contact with the RWM
[37] . As most of the decline in drug level near the applica-
tion site results from distribution of the drug into other
parts of the ear, longer application times help maintain
the drug level while nearby regions become loaded with
the drug. In clinical terms, this means that to reduce vari-
ability of perilymph drug levels to a minimum, the time
the drug remains in the round window niche must be con-
trolled as closely as possible. Calculations also show that
multiple injection or continuous application protocols not
only produce higher drug concentrations near the appli-
cation site, but also allow proportionately greater concen-
trations to reach higher turns of the cochlea [64] . The ap-
plication protocol can therefore be modified according to
the goals of the local drug application. For treatment of
Mnires patients with gentamicin, where the goal is to
suppress the balance system while minimizing gentami-
cin-induced hearing loss, a one-shot application is most
appropriate, as this results in the steepest drug gradient
along the cochlea, minimizing the gentamicin concentra-
tion reaching cochlear locations responsible for speech
frequencies. In contrast, when the goal is to distribute the
drug throughout the cochlea, then multiple applications
per day or continuous delivery protocols would minimize
longitudinal gradients to the greatest degree possible.
There are numerous other factors that influence the peri-
lymph drug level resulting from a specific application pro-
tocol, including the RWM permeability to the drug and
how fast the drug is eliminated from the round window
niche by the middle ear mucosa, through drainage to the
spaces of the temporal bone, and by the injected solution
leaving the middle ear through the eustachian tube.
Intralabyrinthine Applications
In the literature, the ambiguous terms infusion, per-
fusion and injection have been used to describe the de-
livery of drugs to the inner ear. The term perfusion of
the ear was originally used to describe the passage of so-
lution through the perilymphatic spaces from a site of
entry to an open outlet. Perfusion was, however, also used
to describe intratympanic application without perfora-
tion of the otic capsule, i.e. the solution passing solution
across the ear [14, 65] . Infusion (derived from the Latin
verb to pour into) also suggests a passive slow delivery
of solution to the ear. In contrast, the terms intralabyrin-
thine injection and intracochlear injection are preferred
as they more accurately represent a more rapidly occur-
ring and forceful introduction of solution directly into
the fluid spaces of the inner ear.
In general pharmacokinetics, the term bioavailability
is commonly used to describe the rate and extent of drug
input. For extracochlear applications, such as intratym-
panic injections to the round window niche, the percent-
age of drug entering the inner ear (bioavailability) is rel-
atively low. It was estimated that the basal turn con-
centration only reached a mean of 2.5% of the applied
gentamicin concentration and 1.4% of the applied dexa-
methasone-phosphate concentration with a round win-
dow irrigation protocol and 0.17% of the applied genta-
micin concentration when the bulla was filled with solu-
tion [46, 53] . Drugs injected directly into the perilymph
would theoretically be expected to exhibit 100% bioavail-
ability, and in this respect are comparable to an intrave-
nous application when considering the entire human
body. In reality, 100% bioavailability might not be reached
in all intralabyrinthine application techniques due to
leaks either at the application site or at sites of fluid efflux,
such as the cochlear aqueduct. Nevertheless, bioavailabil-
ity of drug within the inner ear can be substantially in-
creased by direct application of the drug through the
RWM, through the stapes footplate, into the lateral semi-
circular canals or into the endolymphatic sac.
Under conditions when the injection pipette is sealed
into the otic capsule, intralabyrinthine injections pro-
duced more consistent perilymph concentrations (of the
marker ion) than applications to the round window niche
[66] . Difficulties arise, however, when there is any form
of leak at the injection site or when an outlet for the in-
jected solution is provided. For the same reasons that per-
forations of the otic capsule cause contamination of fluid
samples with CSF, perforations to insert an injection pi-
pette cause a release of intracochlear pressure that results
in an artifactual volume flow from the cochlear aqueduct
entering the ST in the basal turn to the site of the perfora-
tion. In guinea pigs, CSF entry rates of 0.51 l/min oc-
cur, which is fast enough to displace drug solution from
Local Drug Delivery to the Ear Audiol Neurotol 2009;14:350360 357
the ear (especially from the basal turn of ST, where CSF
enters) within minutes. Attempts to seal injection sites
through the bone with bone dust, adipose tissue or fascia
may be effective after a number of hours or days as the
site scars, but are unlikely to provide an immediate fluid-
tight seal. Thus, the procedures used to seal the injection
site may critically influence the perilymph drug level. In
humans, the situation may be considerably different from
animal models as the cochlear aqueduct is narrower and
CSF pressure at the level of the aqueduct may be negative
when the patient is sitting or standing.
The spread of drug from the site of injection depends
on the rate of injection and the site (if any) where the
displaced perilymph exits the ear. For low injection
rates, the induced rate of perilymph flow may be low,
and spread from the injection site will be dominated by
diffusion. This has been demonstrated both by real-
time measurements of markers [67] and by functional
measures during pulsed injections into the basal turn of
the ST [68] . For higher injection rates, the spread of drug
can only be predicted if the fluid outlet site is known. As
a result of these factors, the region(s) of the inner ear af-
fected by intra labyrinthine injections depends to a large
extent on the site of injection. Possible injection sites (as
shown in fig. 4 ) include the RWM (through fenestra-
tions in the bone in the basal turn of ST), the stapes
footplate or the semi-circular canals, and the endolym-
phatic sac.
Many studies have reported the injection of agents
through the RWM using hand-held narrow-gauge hypo-
dermic syringes. However, when perilymph concentra-
tion was measured following injections through the
RWM with fine (20- M OD) bevelled pipettes held in a
micromanipulator, small leaks around the pipettes caused
significant washout of the drug [67] . It was calculated that
the leakage rate under these conditions averaged just 90
nl/min, which was sufficient to wash out the drug but was
not visible with an operating microscope. The leakage
around the pipette was markedly reduced when the round
window niche was filled with Healon gel before the injec-
tion pipette was inserted. These results suggest that injec-
tions through the RWM with handheld hypodermic sy-
ringes are likely to result in even higher rates of peri-
lymph leakage, and cannot be regarded as a quantitative
delivery method.
Cochlear Implants
There is accumulating evidence from animal experi-
ments that locally applied glucocorticoids can help pre-
serve acoustic hearing thresholds following cochlear
implantation [6971] . As there is increasing interest in
the possibility of implanting patients with high-fre-
quency hearing loss but good low-frequency hearing, it
has become an important goal to perform the implanta-
tion with minimal damage to residual hearing [7274] .
Other drug candidates include the use of neurotrophins
Fig. 5. Schematic of the balance between
the control of perilymph drug level by dif-
ferent application approaches and the risk
to the patients hearing and/or balance by
the drug application alone. Intratympanic
drug applications are low risk, but produce
variable perilymph levels. Improved con-
trol of drug levels may be achieved by ma-
nipulations of RWM permeability (ar-
rows). Intracochlear drug injections give
better control of perilymph drug concen-
tration, but the safety of such procedures
has not been demonstrated. Delivering
drugs from a cochlear implant may carry
little additional risk to the patient. As ex-
perience is gained, the goal remains to pro-
vide quantitative control of the drug level
in perilymph while minimizing risk to
hearing and balance function.
Salt /Plontke

Audiol Neurotol 2009;14:350360 358
to preserve spiral ganglion cells [75] and apoptosis in-
hibitors to minimize insertion trauma [76] . There are
several possible strategies of intracochlear drug delivery
in combination with cochlear implants, including ap-
plications to the RWM prior to surgery [77] , one-shot
injections into the ST at the time of implantation [78,
79] , bathing the electrode in drug solution or a gel
preparation prior to insertion into the cochlea, drug re-
lease from the electrode carrier itself which also func-
tions as a scaffold, drug release from a reservoir in the
electrode carrier, drug injection through an incorporat-
ed channel attached to a pump [80, 81] , or by surface
coating of the electrode carrier with a controlled-release
formulation [82] .
Clinical Considerations
At present, the most widely used approach to deliver
drugs locally to the inner ear is through the use of intra-
tympanic applications. This is in large part because of the
requirement for a safe and feasible way of drug delivery
to the inner ear, especially to minimize damage to hear-
ing, which became a major component of local gentami-
cin applications in the treatment of Mnires disease.
Animal studies, however, showed that perilymph drug
levels achieved with RWM applications are highly vari-
able [29, 37, 54, 61] and that more consistent perilymph
concentrations can be achieved by intracochlear injec-
tions of substances [67] . However, based on experience
with surgical procedures that involve perforation of the
inner ear such as stapedectomy or the cochlear implanta-
tion of patients with residual hearing, it is well known
that perforation and/or surgical manipulation of the ear
carries a significant risk of deafness. Appropriate tech-
niques for the intralabyrinthine injections of drugs have
not yet been developed and proven safe. This results in a
balance or trade-off between the ability of the delivery
method to control the perilymph drug level and the risk
of the drug delivery procedure to the hearing and balance
function of the patient as schematized in figure 5 . In or-
der to have good control of perilymph drug levels and ef-
fective drug delivery without damage to the ear, we either
need to reduce the variability and increase the bioavail-
ability associated with intratympanic applications (such
as by better control of the drug level in the round window
niche or by permeabilizing the RWM) or we need to de-
velop safe methods of intracochlear drug delivery that do
not damage the ear.
Acknowledgements
This study was supported by research grant RO1 DC01368 (to
A.N.S.) from the National Institute on Deafness and other Com-
munication Disorders (NIDCD), National Institutes of Health
(NIH), and by grants 0313844B and 0314103 (to S.K.P.) from the
Federal Ministry of Education and Research (BMBF), Germany.

References
1 Ersner MS, Spiegel EA, Alexander MH:
Transtympanic injection of anesthetics for
the treatment of Mnires syndrome. AMA
Arch Otolaryngol 1951; 54: 4352.
2 Vlger G: Beseitigung von Labyrinthliquor-
druckstrungen bei dem Menirschen
Symptomkomplex durch das Hyaluronida-
seprparat Kinetin (in German). HNO 1952;
3: 142147.
3 Schuknecht HF: Ablation therapy for the re-
lief of Mnires disease. Laryngoscope 1956;
66: 859870.
4 Lange G: Gentamicin and other ototoxic an-
tibiotics for the transtympanic treatment of
Menires disease. Arch Otorhinolaryngol
1989; 246: 269270.
5 Blakley BW: Clinical forum: a review of in-
tratympanic therapy. Am J Otol 1997; 18:
520526.
6 Carey J: Intratympanic gentamicin for the
treatment of Menieres disease and other
forms of peripheral vertigo. Otolaryngol
Clin North Am 2004; 37: 10751090.
7 Schwab B, Lenarz T, Heermann R: Use of the
round window micro cath for inner ear ther-
apy results of a placebo-controlled, pro-
spective study on chronic tinnitus (in Ger-
man). Laryngorhinootologie 2004; 83: 164
172.
8 Van Wijk F, Staecker H, Keithley E, Lefebvre
PP: Local perfusion of the tumor necrosis
factor alpha blocker infliximab to the inner
ear improves autoimmune neurosensory
hearing loss. Audiol Neurootol 2006; 11: 357
365.
9 Suckfuell M, Canis M, Strieth S, Scherer H,
Haisch A: Intratympanic treatment of acute
acoustic trauma with a cell-permeable JNK
ligand: a prospective randomized phase I/II
study. Acta Otolaryngol 2007; 127: 938942.
10 Barrs DM: Intratympanic corticosteroids for
Menieres disease and vertigo. Otolaryngol
Clin North Am 2004; 37: 955972.
11 Rauch SD: Intratympanic steroids for sen-
sorineural hearing loss. Otolaryngol Clin
North Am 2004; 37: 10611074.
12 Haynes DS, OMalley M, Cohen S, Watford
K, Labadie RF: Intratympanic dexametha-
sone for sudden sensorineural hearing loss
after failure of systemic therapy. Laryngo-
scope 2007; 117: 315.
13 Plontke SK, Lwenheim H, Mertens J, Engel
C, Meisner C, Weidner A, Zimmermann R,
Preyer S, Koitschev A, Zenner HP: Random-
ized, double blind, placebo controlled trial
on the safety and efficacy of continuous in-
tratympanic dexamethasone delivered via a
round window catheter for severe to pro-
found sudden idiopathic sensorineural hear-
ing loss after failure of systemic therapy. La-
ryngoscope 2009; 119: 359369.
Local Drug Delivery to the Ear Audiol Neurotol 2009;14:350360 359
14 Light JP, Silverstein H: Transtympanic per-
fusion: indications and limitations. Curr
Opin Otolaryngol Head Neck Surg 2004; 12:
378383.
15 Sakata E, Ito Y, Itoh A: Clinical experiences
of steroid targeting therapy to inner ear for
control of tinnitus. Int Tinnitus J 1997; 3: 117
121.
16 Harris JP: Inner ear steroid perfusion. Curr
Opin Otolaryngol Head Neck Surg 2007; 15:
303.
17 Hamid M, Trune D: Issues, indications, and
controversies regarding intratympanic ste-
roid perfusion. Curr Opin Otolaryngol Head
Neck Surg 2008; 16: 434440.
18 Arnold W, Senn P, Hennig M, Michaelis C,
Deingruber K, Scheler R, Steinhoff HJ,
Riphagen F, Lamm K: Novel slow- and fast-
type drug release round-window microim-
plants for local drug application to the co-
chlea: an experimental study in guinea pigs.
Audiol Neurootol 2005; 10: 5363.
19 Endo T, Nakagawa T, Kita T, Iguchi F, Kim
TS, Tamura T, Iwai K, Tabata Y, Ito J: Novel
strategy for treatment of inner ears using a
biodegradable gel. Laryngoscope 2005; 115:
20162020.
20 Tamura T, Kita T, Nakagawa T, Endo T, Kim
TS, Ishihara T, Mizushima Y, Higaki M, Ito
J: Drug delivery to the cochlea using PLGA
nanoparticles. Laryngoscope 2005; 115:
20002005.
21 Ge X, Jackson RL, Liu J, Harper EA, Hoffer
ME, Wassel RA, Dormer KJ, Kopke RD, Ba-
lough BJ: Distribution of PLGA nanoparti-
cles in chinchilla cochleae. Otolaryngol
Head Neck Surg 2007; 137: 619623.
22 Paulson DP, Abuzeid W, Jiang H, Oe T,
OMalley BW, Li D: A novel controlled local
drug delivery system for inner ear disease.
Laryngoscope 2008; 118: 706711.
23 Goycoolea MV, Lundman L: Round window
membrane. Structure function and perme-
ability: a review. Microsc Res Tech 1997; 36:
201211.
24 Goycoolea MV: Clinical aspects of round
window membrane permeability under nor-
mal and pathological conditions. Acta Oto-
laryngol 2001; 121: 437447.
25 Schachern PA, Paparella MM, Duvall AJ 3rd:
The normal chinchilla round window mem-
brane. Arch Otolaryngol 1982; 108: 550554.
26 Rahm WE, Strother WF, Gulick WL, Crump
JF: The effects of topical anesthetics upon the
ear. Ann Otol Rhinol Laryngol 1959; 68:
10371046.
27 Imamura S, Adams JC: Distribution of gen-
tamicin in the guinea pig inner ear after local
or systemic application. J Assoc Res Otolar-
yngol 2003; 2: 176195.
28 Wagner N, Cay-Thomasen P, Laurell G,
Bagger-Sjbck D, Thomsen J: Cochlear hair
cell loss in single-dose versus continuous
round window administration of gentami-
cin. Acta Otolaryngol 2005; 125: 340345.
29 Parnes LS, Sun AH, Freeman DJ: Corticoste-
roid pharmacokinetics in the inner ear flu-
ids: an animal study followed by clinical ap-
plication. Laryngoscope 1999; 109: 117.
30 Liu HJ, Dong MM, Chi FL: Dexamethasone
pharmacokinetics in guinea pig inner ear
perilymph. ORL J Otorhinolaryngol Relat
Spec 2006; 68: 9398.
31 Yang J, Wu H, Zhang P, Hou DM, Chen J,
Zhang SG: The pharmacokinetic profiles of
dexamethasone and methylprednisolone
concentration in perilymph and plasma fol-
lowing systemic and local administration.
Acta Otolaryngol 2008; 128: 496504.
32 Thorne M, Salt AN, DeMott JE, Henson
MM, Henson OW, Gewalt SL: Cochlear fluid
space dimensions for six species derived
from reconstructions of three-dimensional
magnetic resonance images. Laryngoscope
1999; 109: 16611668.
33 Salt AN, Kellner C, Hale S: Contamination
of perilymph sampled from the basal cochle-
ar turn with cerebrospinal fluid. Hear Res
2003; 182: 2433.
34 Mynatt R, Hale SA, Gill RM, Plontke SKR,
Salt AN: Demonstration of a longitudinal
concentration gradient along scala tympani
by sequential sampling of perilymph from
the cochlear apex. J Assoc Res Otolaryngol
2006; 7: 182193.
35 Stver T, Yagi M, Raphael Y: Cochlear gene
transfer: round window versus cochleosto-
my inoculation. Hear Res 1999; 136: 124
130.
36 Saijo S, Kimura RS: Distribution of HRP in
the inner ear after injection into the middle
ear cavity. Acta Otolaryngol 1984; 97: 593
610.
37 Hahn H, Kammerer B, DiMauro A, Salt AN,
Plontke S: Cochlear microdialysis for quan-
tification of dexamethasone and fluorescein
entry into scala tympani during round win-
dow administration. Hear Res 2006; 212:
236244.
38 Hft J: The permeability of the round win-
dow membrane and its changes by panto-
caine (tetracaine). Arch Klin Exp Ohren
Nasen Kehlkopfheilkd 1969; 193: 128137.
39 Ikeda K, Morizono T: Changes of the perme-
ability of round window membrane in otitis
media. Arch Otolaryngol Head Neck Surg
1988; 114: 895897.
40 Selivanova O, Maurer J, Ecke U, Mann WJ:
The effects of Streptolysin-O and sodium
hyaluronate on the permeability of the round
window membrane in guinea pigs an elec-
trophysiologic study (in German). Laryngo-
rhinootologie 2003; 82: 235239.
41 Chandrasekhar SS, Rubinstein RY, Kwartler
JA, Gatz M, Connelly PE, Huang E, Baredes
S: Dexamethasone pharmacokinetics in the
inner ear: comparison of route of adminis-
tration and use of facilitating agents. Otolar-
yngol Head Neck Surg 2000; 122: 521528.
42 Mikulec AA, Hartsock JJ, Salt AN: Perme-
ability of the round window membrane is
influenced by the composition of applied
drug solutions and by common surgical pro-
cedures. Otol Neurotol 2008; 29: 10201026.
43 Tanaka K, Motomura S: Permeability of the
labyrinthine windows in guinea pigs. Arch
Otorhinolaryngol 1981; 233: 6773.
44 Salt AN, Ohyama K, Thalmann R: Radial
communication between the perilymphatic
scalae of the cochlea. I. Estimation by tracer
perfusion. Hear Res 1991; 56: 2936.
45 Salt AN, Ohyama K, Thalmann R: Radial
communication between the perilymphatic
scalae of the cochlea. II. Estimation by bolus
injection of tracer into the sealed cochlea.
Hear Res 1991; 56: 3743.
46 Mikulec AA, Plontke SK, Hartsock JJ, Salt
AN: Entry of substances into perilymph
through the bone of the otic capsule after in-
tratympanic applications in guinea pigs: im-
plications for local drug delivery in humans.
Otol Neurotol 2009; 30: 131138.
47 Shepherd RK, Colreavy MP: Surface micro-
structure of the perilymphatic space: impli-
cations for cochlear implants and cell- or
drug-based therapies. Arch Otolaryngol
Head Neck Surg 2004; 130: 518523.
48 Rask-Andersen H, Schrott-Fischer A, Pfaller
K, Glueckert R: Perilymph/modiolar com-
munication routes in the human cochlea.
Ear Hear 2006; 27: 457465.
49 Ilberg C, Vosteen KH: Permeability of the in-
ner ear membranes. Acta Otolaryngol 1969;
67: 165170.
50 Richardson RT, OLeary S, Wise A, Hard-
man J, Clark G: A single dose of neurotro-
phin-3 to the cochlea surrounds spiral gan-
glion neurons and provides trophic support.
Hear Res 2005; 204: 3747.
51 Salt AN, DeMott JE: Endolymph volume
changes during osmotic dehydration mea-
sured by two marker techniques. Hear Res
1995; 90: 1223.
52 Swan EE, Mescher MJ, Sewell WF, Tao SL,
Borenstein JT: Inner ear drug delivery for au-
ditory applications. Adv Drug Deliv Rev
2008; 60: 15831599.
53 Plontke SK, Mynatt R, Gill RM, Salt AN:
Concentration gradient along scala tympani
following the local application of gentamicin
to the round window membrane. Laryngo-
scope 2007; 117: 11911198.
54 Plontke SK, Biegner T, Kammerer B, Delabar
U, Salt AN: Dexamethasone concentration
gradients along scala tympani after applica-
tion to the round window membrane. Otol-
ogy Neurotol 2008; 29: 401406.
55 Hargunani CA, Kempton JB, DeGagne JM,
Trune DR: Intratympanic injection of dexa-
methasone: time course of inner ear distri-
bution and conversion to its active form.
Otol Neurotol 2006; 27: 564569.
56 Housley GD: Extracellular nucleotide sig-
naling in the inner ear. Mol Neurobiol 1998;
16: 2148.
Salt /Plontke

Audiol Neurotol 2009;14:350360 360
57 Vlajkovic SM, Housley GD, Muoz DJ, Rob-
son SC, Svigny J, Wang CJ, Thorne PR:
Noise exposure induces up-regulation of
ecto-nucleoside triphosphate diphosphohy-
drolases 1 and 2 in rat cochlea. Neuroscience
2004; 126: 763773.
58 Hibi T, Suzuki T, Nakashima T: Perilym-
phatic concentration of gentamicin admin-
istered intratympanically in guinea pigs.
Acta Otolaryngol 2001; 121: 336341.
59 Hoffer ME, Balough B, Kopke RD, et al: Mor-
phologic changes in the inner ear of Chin-
chilla laniger after middle ear administra-
tion of gentamicin in a sustained-release
vehicle. Otolaryngol Head Neck Surg 1999;
120: 643648.
60 Plontke SK, Wood AW, Salt AN: Analysis of
gentamicin kinetics in fluids of the inner ear
with round window administration. Otol
Neurotol 2002; 23: 967974.
61 Bachmann G, Su J, Zumegen C, Wittekindt
C, Michel O: Permeabilitt der runden Fen-
stermembran fr Prednisolon-21-Hydro-
gensuccinat. HNO 2001; 49: 538542.
62 Plontke SK, Salt AN: Quantitative interpre-
tation of corticosteroid pharmacokinetics in
inner ear fluids using computer simulations.
Hear Res 2003; 182: 3442.
63 Hughes PM, Olejnik O, Chang-Lin JE, Wil-
son CG: Topical and systemic drug delivery
to the posterior segments. Adv Drug Deliv
Rev 2005; 57: 20102032.
64 Salt AN, Gill RM, Plontke SK: Dependence
of hearing changes on the dose of intratym-
panically-applied gentamicin: a metaanaly-
sis using mathematical simulations of clini-
cal drug delivery protocols. Laryngoscope
2008; 118: 17931800.
65 Shea JJ Jr: The role of dexamethasone or
streptomycin perfusion in the treatment of
Menieres disease. Otolaryngol Clin North
Am 1997; 30: 10511059.
66 Salt AN, Hale SA, Plonkte SK: Perilymph
sampling from the cochlear apex: a reliable
method to obtain higher purity perilymph
samples from scala tympani. J Neurosci
Methods 2006; 153: 121129.
67 Salt AN, Sirjani DB, Hartsock JJ, Gill RM,
Plontke SK: Marker retention in the cochlea
following injections through the round win-
dow membrane. Hear Res 2007; 232: 7886.
68 Chen Z, Kujawa SG, McKenna MJ, Fiering
JO, Mescher MJ, Borenstein JT, Swan EE,
Sewell WF: Inner ear drug delivery via a re-
ciprocating perfusion system in the guinea
pig. J Control Release 2005; 110: 119.
69 Ye Q, Tillein J, Hartmann R, Gstoettner W,
Kiefer J: Application of a corticosteroid (Tri-
amcinolon) protects inner ear function after
surgical intervention. Ear Hear 2007; 28: 361
369.
70 James DP, Eastwood H, Richardson RT,
OLeary SJ: Effects of round window dexa-
methasone on residual hearing in a guinea
pig model of cochlear implantation. Audiol
Neurootol 2008; 13: 8696.
71 Vivero RJ, Joseph DE, Angeli S, He J, Chen S,
Eshraghi AA, Balkany TJ, Van de Water TR:
Dexamethasone base conserves hearing
from electrode trauma-induced hearing loss.
Laryngoscope 2008; 118: 20282035.
72 Lehnhardt E: Intracochlear placement of co-
chlear implant electrodes in soft surgery
technique HNO (in German). 1993; 41: 356
359.
73 Kiefer J, Gstoettner W, Baumgartner W, Pok
SM, Tillein J, Ye Q, von Ilberg C: Conserva-
tion of low-frequency hearing in cochlear
implantation. Acta Otolaryngol 2004; 124:
272280.
74 Gantz BJ, Turner C, Gfeller KE, Lowder MW:
Preservation of hearing in cochlear implant
surgery: advantages of combined electrical
and acoustical speech processing. Laryngo-
scope 2005; 115: 796802.
75 Richardson RT, Thompson B, Moulton S,
Newbold C, Lum MG, Cameron A, Wallace
G, Kapsa R, Clark G, OLeary S: The effect
of polypyrrole with incorporated neuro-
trophin-3 on the promotion of neurite out-
growth from auditory neurons. Biomaterials
2007; 28: 513523.
76 Eshraghi AA, Wang J, Adil E, He J, Zine A,
Bublik M, Bonny C, Puel JL, Balkany TJ, Van
De Water TR: Blocking c-Jun-N-terminal ki-
nase signaling can prevent hearing loss in-
duced by both electrode insertion trauma
and neomycin ototoxicity. Hear Res 2007;
226: 168177.
77 Bird PA, Begg EJ, Zhang M, Keast AT, Mur-
ray DP, Balkany TJ: Intratympanic versus in-
travenous delivery of methylprednisolone to
cochlear perilymph. Otol Neurotol 2007; 28:
11241130.
78 Kiefer J, Gstoettner W, Baumgartner W, Pok
SM, Tillein J, Ye Q, von Ilberg C: Conserva-
tion of low-frequency hearing in cochlear
implantation. Acta Otolaryngol 2004; 124:
272280.
79 Paasche G, Tasche C, Stver T, Lesinski-
Schiedat A, Lenarz T: The long-term effects
of modified electrode surfaces and intraco-
chlear corticosteroids on postoperative im-
pedances in cochlear implant patients. Otol
Neurotol 2009; 30: 592598.
80 Garnham C, Reetz G, Jolly C, Miller J, Salt A,
Beal F: Drug delivery to the cochlea after im-
plantation: consideration of the risk factors.
Cochlear Implants Int 2005; 6(suppl 1):12
14.
81 Hochmair I, Nopp P, Jolly C, Schmidt M,
Schsser H, Garnham C, Anderson I: MED-
EL cochlear implants: state of the art and a
glimpse into the future. Trends Amplif 2006;
10: 201219.
82 Richardson RT, Wise AK, Thompson BC,
Flynn BO, Atkinson PJ, Fretwell NJ, Fallon
JB, Wallace GG, Shepherd RK, Clark GM,
OLeary SJ: Polypyrrole-coated electrodes
for the delivery of charge and neurotrophins
to cochlear neurons. Biomaterials 2009; 30:
26142624.
83 Hahn H, Biegner T, Kammerer B, Delabar U,
Salt AN, Plontke SK: Persistence of dexa-
methasone drug levels and base to apex gra-
dients in the perilymph of the scala tympani
after single shot injections through the
round window membrane (abstract). J Assoc
Res Otolaryngol 2009; 32: 115.
84 Voie AH: Imaging the intact guinea pig tym-
panic bulla by orthogonal-plane fluores-
cence optical sectioning microscopy. Hear
Res 2002; 171: 119128.