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J. Lavinsky et al.
(a) Title and abstract: It is recommended that the study title
identify it as a randomized clinical trial. The abstract
should cover the trial design, methods, results, and
conclusions.
(b) Introduction: This should include the scientic back-
ground and explanation of the rationale, specic objectives, and hypotheses.
(c) Study design: Description of trial design, such as paral-
lel, including allocation ratio. Additionally, important
changes to methods after the clinical study’s outset,
such as eligibility criteria, are explained.
(d) Participants: The methods section should clearly dene
the eligibility criteria for participants, with information
on the locations where the data were collected.
(e) Interventions: The interventions for each group, with
sufcient details to allow replication, including when
they took place.
(f) Outcomes: Primary and secondary outcomes were
dened in advance, including how they were assessed.
Any changes to outcomes after the clinical trial has
commenced should also be stated.
(g) Sample size: State how the sample size was deter-
mined and whether there were any trial-stopping
guidelines.
(h) Randomization sequence generation: Show the method
used to generate the allocation randomization sequence.
Types of randomization, such as simple or blocking.
(i) Allocation: Mechanism employed to implement the ran-
dom allocation sequence (such as sequentially numbered containers), describing the steps taken to conceal
the sequence until interventions were assigned.
(j) Implementation of allocation: State who generated the
random allocation sequence, who enrolled participants,
and who assigned participants to interventions.
(k) Blinding: If done, who was blinded after the assignment
to interventions, and how was this done?
(l) Statistical methods: Describe the statistical methods
used to compare groups for primary and secondary outcomes, as well as for subgroup analyses and adjusted
analyses.
(m) Participant ow: It is strongly recommended that a dia-
gram be used. Show the numbers of participants who
were randomly assigned, received the treatment
assigned, and were analyzed for the primary outcome.
For each group, show losses and exclusions after randomization with their respective reasons.
(n) Recruitment: Dene when participants were recruited
and follow-up times. State the reasons for ending or
stopping the trial.
(o) Baseline data: Include a table showing baseline demo-
graphic and clinical characteristics for each group.
(p) Numbers analyzed: For each group, the number of par-
ticipants in each analysis with the denominator.
(q) Outcomes and estimates: For each primary and second-
ary outcome, the results for each group and the estimated effect size and its respective precision (95%
condence interval). For binary outcomes, it is recommended that both absolute and relative effect sizes are
presented.
(r) Ancillary analyses: Subgroup analyses and adjusted
analyses, distinguishing prespecied from exploratory.
(s) Harms: All important harms or unintended effects in
each group.
(t) Limitations: Indicate trial limitations, including sources
of potential bias and imprecision.
(u) Generalization: Describe the external validity and appli-
cability of the clinical trial ndings.
(v) Interpretation: Interpretation consistent with the results,
with a balance of benets and harms.
(w) Registration: Registration number and name of the reg-
istered clinical trial.
(x) Protocol: State where the full clinical trial protocol can
be accessed.
(y) Funding: State the sources of funding and other support,
and the role of funders.
This CONSORT checklist enables the reader of a randomized clinical trial of otitis media to check that it meets all
of the criteria recommended to consider it an appropriate
publication to possibly inuence clinical practice.
Systematic Reviews andMeta-Analyses
Systematic reviews and meta-analyses have been crucial in
dening otitis media management practice. They are used as
the starting point for developing instructions for clinical
practices. Guidelines for the publication of systematic
reviews and meta-analyses have been constructed based on
the denitions used by the Cochrane Collaboration. A systematic review is a review of a clearly formulated question
that employs systematic and explicit methods to identify,
select, and critically evaluate relevant research and collect
and analyze data from these studies, which are included in
the review. Statistical methods (meta-analysis) may or may
not be used to analyze and summarize the results of the studies included. Meta-analysis is the use of statistical techniques
to integrate the results of the studies included in a systematic
review.
An executive committee evaluated which items were crucial for systematic reviews and meta-analyses to be considered satisfactory, developing the PRISMA statement [6],
which denes the following checklist:
(a) Title: The title should identify the report as a systematic
review, meta-analysis, or both.
13 Critical Appraisal ofPublished Research inOtitis Media or How toSort theWheat fromtheCha!
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131
(b) Abstract: Should include a structured summary cover-
ing theoretical background, data sources, eligibility criteria, participants, interventions, study appraisal and
synthesis methods, results, limitations, conclusions, and
registration number.
(c) Rationale: Describe the rationale for the review in the
context of what is already known.
(d) Objectives: Make an explicit statement of questions
such as participants, interventions, comparisons, outcomes, and study design.
(e) Protocol and registration: Indicate whether a review
protocol exists that can be accessed and provide information about the registration of the review.
(f) Eligibility criteria: Dene characteristics of the study
and the report, such as eligibility criteria with
rationale.
(g) Information sources: Describe all information sources
used in the search (database, contact with authors) and
the date of the most recent search.
(h) Search: State the complete electronic search strategy,
including limits, so it can be repeated.
(i) Study selection: State the process for selecting studies,
that is, the search for eligible studies.
(j) Data collection process: Methods for extracting data
from articles and processes for obtaining and conrming data from researchers.
(k) List of data items: List all variables collected, with ref-
erences or simplications.
(l) Risk of bias in each study: Describe the methods used to
assess the risk of bias in each study and how this information was used in the analysis of the data.
(m) Summary measures: Should dene the principal mea-
sures for summarizing results.
(n) Synthesis of results: Describe the methods for analyzing
data and combining the results of studies with measures
of consistency for each meta-analysis.
(o) Risk of bias across studies: It is recommended that any
assessment of the risk of bias that may affect the cumulative evidence, such as publication bias, be specied.
(p) Additional analyses: All additional methods of analysis
should be described, and those that were prespecied
should be identied.
(q) Study selection: The results should state the number of
studies screened, assessed for eligibility, and included
in the review.
(r) Study characteristics: For each study, present character-
istics for data extraction with their respective citations.
(s) Risk of bias for each study and between studies: It is
recommended that data on risk of bias for each study
and outcome be presented in the results.
(t) Results of individual studies: For each study and for all
outcomes considered, present a simple summary of data
for each intervention group and effect estimates and
condence intervals using forest plots.
(u) Synthesis of results: Present results for each meta-
analysis conducted, including condence intervals and
measures of consistency.
(v) Additional analyses: Present additional analyses such as
subgroup analyses and meta-regressions.
(w) Summary of evidence: It is recommended that the
main ndings be summarized, especially the strength
of evidence for each outcome and its relevance to key
groups.
(x) Limitations: Discuss limitations at the level of studies
and outcomes and at the review level.
(y) Conclusions: Give a general interpretation of the results
and their relationship to other evidence. Similarly, state
the implications for future research.
(z) Funding: Declare sources of funding and the role of
funders in the review.
Publications in the form of meta-analyses and systematic
reviews are increasingly common in the area of otitis media.
We therefore consider that it is important that readers are
familiar with the PRISMA guidelines to enable them to use
the checklist to critically appraise a meta-analysis or systematic review.
Observational Studies
The majority of studies of otitis media are observational,
especially those conducted in the past. Such studies are
essential to enable us to understand the many different clinical entities related to otitis media and enable developments
in the area to be achieved through intervention studies.
However, it is common for observational studies to be conducted inappropriately, making it less likely that correct
extrapolation will be possible. An initiative known as
Strengthening the Reporting of Observational Studies in
Epidemiology (STROBE) published a 22-item checklist
named the STROBE Statement, containing recommendations on what should be included in a more precise and complete description of such studies [7].
(a) Title and abstract: Indicate the study design in the title
or abstract. Provide an informative and balanced summary in the abstract.
(b) Rationale: Include in the introduction the theoretical
background and the reasons for the research.
(c) Objectives: State the specic objectives and
hypotheses.
(d) Study design: The methods should describe the key ele-
ments of the study design.
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J. Lavinsky et al.
(e) Setting: Describe the setting with locations and impor-
tant dates, with periods of recruitment, exposure, follow- up, and data collection.
(f) Participants: For cohort studies, present the eligibility
criteria, sources, and methods of selection of participants and follow-up. For case-control studies, present
the eligibility criteria and the sources and diagnostic criteria for the separation of cases from controls. For crosssectional studies, present the eligibility criteria and the
sources and methods of selection of participants. In
cohort studies and paired case-control studies, criteria
for pairing and the number of exposed and non-exposed
participants should be given.
(g) Variables: All outcomes should be clearly dened, with
exposures, predictors, confounders, and effect modiers. Diagnostic criteria likewise.
(h) Data sources and measurement: For each variable, the
source of data and details of assessment methods are
described.
(i) Bias: List all methods used to avoid potential sources of
bias.
(j) Study size: The sample size should be clearly dened.
(k) Quantitative variables: It is recommended that the way
that quantitative variables were treated and any catego-
rizations used should be explained.
(l) Statistical methods: It is recommended that all statisti-
cal methods and the control of confounding factors
be described. Likewise, describes subgroup and inter-
action analyses, how missing data were dealt with,
losses to follow-up in cohort studies, pairing of cases
and controls, sampling strategies, and sensitivity
analyses.
(m) Participants: Number of participants in each stage and
number who completed follow-up.
(n) Descriptive data: Demographic characteristics of par-
ticipants and information on exposures and confound-
ers. State the number of participants with missing data
for each variable. For cohort studies, it is essential to
state the follow-up time.
(o) Outcomes: Number of outcome events or summary
measures over time for cohort studies. For case-control
studies, the number of participants in each exposure cat-
egory. For cross-sectional studies, the number of out-
come events or summary measures.
(p) Main results: Estimates unadjusted and, if necessary,
adjusted for confounding variables. If continuous vari-
ables were categorized, state the cut-offs used. If possi-
ble, transform relative risk estimates into absolute risk.
(q) Other analyses: Report on any analyses of subgroups
that have been conducted.
(r) Key results: In the discussion, it is recommended that
the main ndings be related to the study objectives.
(s) Limitations: In the discussion, it is recommended that
limitations be discussed, especially potential biases or
imprecision.
(t) Interpretation: The interpretation of results should be
cautious, taking into account objectives, limitations,
multiplicity of analyses, and results of similar studies.
(u) Generalization: The external validity of the results
should be discussed.
(v) Funding: State sources of study funding and the role of
funders.
Conclusions
The objective of this chapter was to present an organized
strategy for conducting a critical analysis of a scientic publication, specically for the otitis media area, in which there
have been signicant increases in both the quantity and quality of publications over recent years. Researchers and clinicians thus have the tools to independently form their own
opinions on the scientic content they analyze and incorporate this knowledge into their practice when they consider it
truly relevant.
References
1. Gelbach SH. Interpreting the medical literature. 4th ed. Norwalk:
Appleton & Lange; 2002.
2. Consolidated Standards of Reporting Trials, Altman DG, etal. The
revised CONSORT statement for reporting randomized trials. Ann
Intern Med. 2001;134:663–94.
3. Kassirer JP, Campion EW.Peer review: crude and understudied, but
indispensable. JAMA. 1994;287:259–60.
4. Percie du Sert N, Ahluwalia A, Alam S, etal. Reporting animal
research: explanation and elaboration for the ARRIVE guidelines
2.0. PLoS Biol. 2020;18(7):e3000411.
5. Schulz KF, Altman DG, Moher D, CONSORT Group. CONSORT
2010 statement: updated guidelines for reporting parallel group randomised trials. BMJ. 2010;340:c332.
6. Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group.
Preferred reporting items for systematic reviews and meta-analyses:
the PRISMA statement. PLoS Med. 2009;6(7):e1000097.
7. von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC,
Vandenbroucke JP, STROBE Initiative. The strengthening the
reporting of observational studies in epidemiology (STROBE)
statement: guidelines for reporting observational studies. Prev Med.
2007;45(4):247–51.
Translational Histopathology inOtitis
https://t.me/medicina_free
Media: TheReal Evidence-Based
Medicine!
SuanurM.Kayaalp, SebahattinCureoglu,
andMichaelM.Paparella
Pathology, the gold standard for discerning diseases in
humans, is essential to every discipline in the medical eld.
Human ear structures are usually inaccessible during life,
with the exception of surgical biopsies. However, such biopsies include only minuscule specimens of ear tissue and are
often unrepresentative of the overall condition of the ear.
Organs of the body, other than the ear, are routinely studied
in hospital pathology laboratories. However, because of the
difculties and expense of ear tissue acquisition and preparation, human otopathology has evolved as a separate research
discipline. The study of otitis media in human ear sections
has been and continues to be the foundation for advancements in patient care (Fig.14.1).
Although light microscopy of human temporal bones has
profoundly inuenced our knowledge of otitis media
(Fig.14.2a–c) and irreversible pathologies in the middle ear
(Fig.14.3), signicant gaps remain in our understanding of
their molecular mechanisms. The adaptation of new molecular and imaging techniques can provide researchers with the
tools necessary to study normal function and pathology of
the ear. However, the studies of underlying ear pathologies
and disease mechanisms heavily depend on postmortem
times, which affect middle and inner ear structures as well as
the preservation of proteins, DNA/RNA, and other molecules
(Fig. 14.4). Breakthrough human otologic studies will
require high-quality human ear tissues, advanced techniques,
and highly trained researchers. Human studies should be performed on human specimens with short postmortem periods
that contain all the structures of the ear, including the entire
Eustachian tube and mastoid, which is not possible in samples obtained at routine autopsies at this time.
The laboratory should have a multidisciplinary team in
temporal bone procurement, clinical data collection, and
methodologic analysis, such as pathology, histology, immu-
S. M. Kayaalp · S. Cureoglu (*) · M. M. Paparella
Otopathology Laboratory, Department of Otolaryngology,
University of Minnesota Physicians (UMP), University of
Minnesota, Minneapolis, MN, USA
e-mail: cureo003@umn.edu
14
Fig. 14.1 An 86-year-old patient with diverticulitis and diabetes who
was born deaf. Her ears showed multiple otopathologic problems,
including cholesteatoma, cholesterol granuloma, tympanosclerosis,
vestibular schwannoma, serous labyrinthitis, cochlear hydropic
changes, and otosclerosis, all of which could be managed surgically
nohistochemistry, proteomics, polymerase chain reaction,
cDNA microarray, in situ hybridization, light and electron
microscopy, and three-dimensional (3D) reconstruction from
serial temporal bone sections, to understand the pathogenesis, pathophysiology, and etiology of middle ear infections
through analysis of human ear tissues. The combined
approach of ultrastructural and molecular analysis with temporal bone pathology will greatly enhance our understanding
of the mechanisms involved in ear diseases.
We have previously shown that fresh dissected human
ear tissues are suitable for studying mRNA and protein
expression using techniques such as polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry (Fig. 14.5) [1, 2]. Current standard procedures have
proven useful for many imaging and molecular studies of
human temporal bones; however, because of long postmortem, xation, and decalcication times, such procedures can
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. V. Goycoolea et al. (eds.), Textbook of Otitis Media, https://doi.org/10.1007/978-3-031-40949-3_14
133
134
a
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S. M. Kayaalp et al.
b
c
Fig. 14.2 This patient had a long-term history of ear pain, prolonged
antibiotic treatment, and an immunocompromised status due to chronic
myeloid leukemia. His histopathologic examination showed (a) an
fail to conserve the structure of specimens or preserve their
nucleic acids and proteins. The limitations of DNA retrieval
from archived temporal bones are as follows [3]: (1) the
amount of DNA extracted from a single section (~500ng) is
low; (2) extracted DNA does not allow PCR amplication of
fragments exceeding approximately 300–400 bp; (3)
extracted DNA is labile and degrades rapidly; and (4) for-
intact tympanic membrane, (b) otomycosis of the tympanic membrane,
(c) in addition to bacterial communities such as biolm like structures
in the middle ear
malin xation can induce alterations in template sequence
that can inhibit alignment [4]. We found rapid degradation
of RNA in bones when postmortem times exceeded 6h [1].
Long preparation and storage times can also result in poor
preservation of ear structures, degradation of DNA/RNA,
and decreased antigenicity of proteins. Contamination of
DNA/RNA is a signicant problem [3]. Some reasons for
cd
gh
14 Translational Histopathology inOtitis Media: TheReal Evidence-Based Medicine!
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the contamination of archived specimens include: (1) before
the development of molecular methods, there was no reason
to follow the sterile technique; (2) temporal bones were processed in various concentrations of celloidin that were
reused in multiple specimens; and (3) multiple specimens
were stored in the same solutions. With newly acquired
human temporal bones with specic requests regarding disease, xative, or embedding media, as well as microdissected structures and modied xative, avoiding the pitfalls
of reusing embedding material and storing multiple specimens in the same solution, studies will use ear tissues with
Fig. 14.3 An 88-year-old male with hx of chronic otitis media and
dizzy spells showed mixed typed hearing loss and a perforated ear
drum, Note that ear canal keratinized epithelium moves into the middle
ear, creating acquired cholesteatoma
less contamination and better preservation of morphology
and molecular footprints, enhancing immunohistochemical,
electron microscopic, and proteomic analysis. However,
135
a
Fig. 14.4 Double immunolabeling of proteins co-expressed in cholesteatoma. (a) Id1 (green). (b) NF-kB p65 (red). (c) Yellow-orange dots=over-
lap of both Id1 and NF-kB p65. (d) In situ hybridization showing MUC5AC (arrowheads) and MUC5B (arrows)
bcd
ab
ef
Fig. 14.5 (a–d) Immunohistochemical analysis. Noninamed middle
ear (ME) epithelia did not react with MUC4 (a) or MUC5B (b) antibodies but reacted positively with MUC4 (c, arrows) and MUC5B (d,
arrows) antibodies in specimens with mucoid otitis media (MOM). (e–
h) In situ hybridization. Control mucosa expressed no MUC4 (e) or
limited MUC5B (f), while ears with mucoid otitis media expressed
abundant MUC4 (g) and MUC5B (h) mRNA transcripts
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S. M. Kayaalp et al.
ba
Fig. 14.6 (a) Immunolabeling of Na+ and K+-ATPase in the stria vascularis and spiral ligament Spiral ganglion cells immunostained with NE-14
(b) and NeuN (c) antibodies. (d) The nuclei, stained with DAPI. (e) Merge of (c) and (d) images
c
d
e
transmission electron microscopy can be applied to
unstained celloidin sections; if those studies are performed
in human specimens with minimal postmortem times, the
morphologic analysis will demonstrate excellent structural
detail. Immunohistochemistry of decelloidinized human
temporal bones using the avidin–biotin–horseradish peroxidase method or immunouorescent labeling is routinely
performed (Fig. 14.6). We have applied several antigen
retrieval methods (heat, chemical, and proteolytic-induced
epitope retrieval). We observed mucin upregulation by
immunohistochemistry (Fig.14.5a–d) and mRNA by in situ
hybridization (Fig. 14.5e–h) in mucoid otitis media using
human temporal bones with postmortem times <6h [2, 5].
A novel processing technique with a laser microtomeand
automatic noncontact sectioning of human temporal bone
without decalcication will be possible in the near future. As
the laser microtome is automatic and requires minimal tissue
manipulation, it improves the quality of sectioning, which is
Fig. 14.7 Example of a 3D model generated for volume calculation for
epitympanic compartments of the human temporal bone. A=anterior
compartment; L = lateral compartment; M = medial compartment;
MI= malleus/incus compartment; and P = posterior compartment of
human temporal bone
frequently affected by decient decalcication and humanrelated issues. It minimizes processing artifacts related to the
presence of metallic implants (such as cochlear implants), as
contactless sectioning avoids the sheer stress artifacts that
result from standard sectioning with rotary/sliding microtomes [6, 7]. Because of its built-in optical coherence tomography, it provides a means to preview and section areas of
interest in 3D: This allows optimal quality of whole- mount
sectioning, which yields tissue of uneven quality through
standard manual dissection even for highly-skilled technicians. It allows the use of different embedding media such as
Spurr epoxy and methyl-methacrylate, which can be better
for the preservation of proteins and nucleic acids than celloidin but are impractical to use in standard processing
because they are too dense for whole-specimen sectioning
using the sliding microtome. This novel system can process
the whole temporal bone, including the mastoid and
Eustachian tubes.
Double labeling. Using immunohistochemistry to analyze temporal bones with cholesteatoma, we have found
that Id1 (Fig.14.4a) and nuclear factor-kappa B (NF-kB)
p65 (Fig.14.4b) proteins were coexpressed in the middle
ear mucosa (Fig. 14.4c). We demonstrated NF-kB p65
(stained with MAB3026 antibody against “activated” p65)
in the nuclei of cholesteatoma epithelium cells.
Figure14.4d shows double-labeled in situ hybridization of
the Eustachian tube. Two genes, MUC5AC (TRITC) and
MUC5B (FITC), were targeted in a specimen autopsied 3h
after death.
Expression of toll-like receptors (TLRs) in chronic otitis
media (COM). TLRs are membrane proteins that play a cru-
cial role in the induction and activation of innate immunity in
the course of infection [8]. Activation of TLRs leads to the
mobilization of cytokines, chemokines, interferons, defensins, lysozyme, and other molecules in otitis media [9, 10].
We have found that both TLR2 and TLR4 were expressed in
the middle ear mucosa and granulation tissue of donors with
COM.
3D Reconstruction. Knowledge of many structures in
human temporal bones is better achieved using 3D reconstruction (Fig.14.7). We have applied 3D models generated
from sections with 3D reconstruction software (Amira,
Visualization Sciences Group, Bordeaux, France; and Zuse
Institute, Berlin, Germany). The 3D reconstruction of the
temporal bone is a validated mechanism to study the anatomical relationships between the structures within the ear
[11–13]. We have studied the 3D model of epitympanic bony
14 Translational Histopathology inOtitis Media: TheReal Evidence-Based Medicine!
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137
volume and tympanic isthmus area in temporal bones with
COM [14]. It is also possible, with these reconstructions, to
evaluate potential pathophysiological mechanisms leading to
different diseases, such as otitis media, Meniere’s disease,
and hearing loss of all causes [11–13].
An air-containing middle ear is fundamental for normal
hearing. The major aeration pathway of the epitympanum is
the tympanic isthmus, which provides a major ventilation
route to the upper epitympanum. The aeration pathway from
the Eustachian tube leads directly to the mesotympanic and
hypotympanic spaces, whereas the epitympanum is aside
from the direct air stream and aerated [16]. The epitympanic
diaphragm, which consisted of the incus, malleus, and their
folds, was described as the oor of the epitympanum, dividing the structure from the mesotympanum. It is believed that
the epitympanum and mastoid aeration occur through a tympanic isthmus (Palva 1991) [11, 15, 16]. Obstruction of the
tympanic isthmus is a very common nding in various types
of middle ear diseases, and that causes a disturbance in air
diffusion within the temporal bone pneumatic system [15].
Dysventilation of the tympanic cavity due to blockage of or
a narrowed ventilation/drainage route predisposes the middle ear to COM [16]. Swollen mucosa and tympanosclerotic
deposits can obstruct the tympanic isthmus, resulting in the
accumulation of exudates above the level of the epitympanic
diaphragm [15]. A PE tube placement will only aerate the
mesotympanum but not the epitympanic compartments if the
tympanic isthmus is blocked.
On the other hand, it has been known that Eustachian tube
dysfunction is one of the initial factors for chronic ear disease
development and retraction pockets. The upper segment of
the Eustachian tube, close to the tympanic cavity, is commonly exposed to the majority of childhood middle ear
inammatory diseases, making it a logical candidate for
acquired inammatory stenosis and irreversible damage to
the temporo-mastoid system. Eustachian tube dysfunction,
caused by a blockage or a narrow protympanic space, may be
the main factor that triggers middle ear diseases [17]. A
reduced volume of its bony segment, the protympanum, was
observed in our temporal bones with middle ear diseases [13].
Proteomic analysis. Proteomic analysis in formalin-xed,
parafn-embedded, and celloidin-embedded tissues has been
described in the literature [18, 19]. However, the degradation
of proteins can occur in celloidin-embedded tissues due to
the methods used to embed and remove celloidin before performing the proteomic analysis. We continue to perform proteomic analysis of celloidin-embedded archived bones and
plan to perform studies using our newly acquired temporal
bones from patients with COM.We generally develop methodologic protocols using our archived chinchilla temporal
bones before applying them to our valuable human specimens. We used tandem mass spectrometry at the Center for
Mass Spectrometry and Proteomics at the University of
Minnesota. Analysis of formalin-xed, celloidin-embedded
chinchilla temporal bones yielded more than 50 proteins,
including collagen (alpha 1, 2, and 3), cytokeratins (CK-1,
CK-10, and CK-2e), cochlin, vitronectin (isoform X1), and
bronectin (isoform X6). We believe those studies will provide intact protein identication combined with a protein
database for human specimens from otitis media cases. As a
result, the proteomic prole of the pathologic tissue can be
compared to the normal protein distribution. In addition,
abnormal proteins detected during proteomic analysis can be
further evaluated by immunohistochemistry. Those analyses
will provide insight into the underlying mechanisms of ear
problems and propose potential treatment approaches.
Validation of Animal Models and Potential for Clinical
Application. Many researchers have relied on experimental
animal models; however, the application of such models to
the human ear needs to be validated, given the genetic and
anatomic differences between various animals and humans.
Furthermore, no animal models currently exist for many otologic syndromes, irreversible ear pathologies such as cholesteatoma (Fig. 14.8a, b). Furthermore, experimental animal
models do not always mimic the human condition; experimental otitis media has been induced by the inoculation of
bacteria or obstruction of the Eustachian tube, whereas in
humans, acute otitis media is often preceded by a viral upper
respiratory tract infection [20, 21]. Knockout mice can
develop spontaneous otitis media involving bacterial pathogens that are not common in human ear infections [22].
Finally, animal and human ears have signicant anatomic
differences. We have had many years of experience validating our experimental animal models against our human temporal bones, including their potential for translational
applications. For example, in our animal model, we demonstrated the passage of bacterial toxins and inammatory
mediators through the round window membrane, thereby
damaging the inner ear and causing hearing loss. However,
our comparative studies of rodent models and the human
round window membrane have shown differences in thickness (the human membrane is about 4–5 times thicker),
despite structural similarity—possibly resulting in a slower
rate of diffusion and decreased permeability in humans.
We have conducted many studies validating various
aspects of the chinchilla model of otitis media (pioneered at
the University of Minnesota) with respect to our human temporal bones from deceased donors with otitis media. We
compared the ultrastructure of the normal human round window to that of various animal species [23, 24] (Fig.14.9) and
evaluated its permeability to different substances. Similar
changes of inammatory cell inltration of the round window membrane and scala tympani due to otitis media in our
chinchilla model [25–29] and our human temporal bones
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S. M. Kayaalp et al.
a
b
Fig. 14.9 In a chinchilla model, we have seen similar changes and
mechanisms to those we sseen in human studies in terms of interaction
in between the middle and inner ears. MEC middle ear cavity, RWM
round window membrane; and ST scala tympani. Inammatory cells,
such as polymorphonuclear cells, mononuclear cells, bacteria in the
membrane, and scala tympani. Note vacuolization and edema of the
membrane (RWM). All specimens were processed in a similar manner
to test the validity of the animal model in human patients
Fig. 14.8 A 64-year-old male patient had a history of otogenic meningitis. He had bilateral chronic otitis media and presence of cholesteatoma in the left ear. (a) Note the cholesteatoma destroyed ossicles and
is located in between tympanic membrane and the stapedial footplate,
creating a columellar effect in the sound conduction. (b) Higher magnication showed small cholesteatoma pearls under the mucosal surface
of the stapedial footplate
with otitis media [30] have been shown. We also demonstrated sensorineural hearing loss secondary to otitis media
in our chinchilla model [31], a sequela described in humans.
Our laboratory has made signicant progress in otitis
media-related translational research using our collection of
human temporal bones and experimental animal models. The
inner ear is intimately associated with the middle ear, both
functionally and anatomically. Functional changes in the
middle ear mechanics affect the transmission of sound to the
inner ear, while areas of anatomical connections allow for
the passage of microorganisms, cytokines, toxins, or drugs
from the middle to the inner ear with potentially serious
sequelae. The round window membrane, which is the only
soft tissue barrier between the middle and inner ear and
which lies within a niche and adjacent to the sinus tympani,
facilitating the accumulation of pus, is vulnerable to toxic
substances within the middle ear (Fig.14.10a, b) [32]. This
research transformed the treatment of patients with otitis
media [33].
Dr. Paparella and his colleagues initiated the concept of
the otitis media continuum [34, 35] (Fig.14.11). It has been
suggested that all types of otitis media represent different
stages of a continuum of events. It can occur along a continuum, with the acute stage of otitis media leading to the
chronic stage. One form of otitis media may resolve or lead
to a more chronic stage of the disease. Although otitis media
with effusion is characterized by the presence of a clear uid
in the middle ear cleft, there is no evidence of secretory cell
proliferation at an earlier stage. Over time, subepithelial
space is widened by edema, vascular dilation, and the inltration of host defense cells. With the reabsorption of watery
components of the uid, the increased density of goblet cells,
and the formation of secretory glands, the middle ear effusion becomes mucoid, in addition to the chronic inammatory changes in the middle ear cleft.
COM has been clinically dened as a chronic discharge
from the middle ear in the presence of a perforation of the
tympanic membrane. It is dened when the inammatory
a
14 Translational Histopathology inOtitis Media: TheReal Evidence-Based Medicine!
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process has entered a chronic phase and permanent alteration
of middle ear structures is seen [36]. The pathologic changes
in the tympanic membrane due to COM include perforation,
retractions, myringosclerosis, pseudocystic spaces, and hemorrhage [37]. Paparella et al. introduced the concept of
chronic silent otitis media based on histopathologic evidence
of chronic infection in the middle ear cleft in the absence of
obvious ear-related symptoms and changes in the tympanic
membrane or intractable tissue pathology behind an intact
tympanic membrane [38, 39]. Because the chronic pathology
b
is undetected, there is a lack of clinical treatment for otitis
media, increasing the risk of complications due to the underlying problems [39]. As a result of the silent middle ear
infection process, intratemporal problems such as endolymphatic hydrops, labyrinthitis, facial palsy, sensorineural hearing loss, and/or intracranial complications such as meningitis
or brain abscess can be seen [39, 40]. The clinician should,
therefore, be aware that an intact tympanic membrane does
not necessarily preclude the presence of pathologic changes
in the middle ear cleft (Fig.14.12). In addition, ventilation
tube placement does not guarantee the resolution of middle
ear effusion if there are associated pathologies, such as granulation tissues in hidden areas, including the facial recess or
sinus tympani (Fig.14.13). As seen in the section, a dimeric
membrane is observed due to several tube placements and
residual effusion in addition to posterior brocystic granulation tissue around the sinus tympani and round window niche
(Fig.14.13).
As a result of human otopathologic studies, we will have
a better understanding of the structural and molecular mech-
Fig. 14.10 A 1-year-old patient had a history of otogenic meningitis.
(a) Horizontal sections of the right temporal bone showed irreversible
pathologies such as granulation tissue and brous structures in the middle ear. (b) Higher magnication of round window membrane indicated
purulent secretion around the niche in the middle ear has been crossing
the membrane into the scala tympani as an example of middle ear/inner
ear interaction
anisms, which will have a major impact on facilitating the
development of new strategies to prevent and treat different
types of otitis media and their complications.
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