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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_4399_Библиотеки_им_академика_М_И_Перельмана
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Temporal Bone Histology and Radiology Atlas226
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Figure 10–8. 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;
P = posterior compartment of human temporal bone.
structural similarity—possibly resulting, in humans, in
a slower rate of diffusion and decreased permeability.
In addition to our archived collection of human temporal bones, our collection contains 19 different species
of animals, including four different species of experimentally induced otitis media. The following are some
examples of human temporal bone correlations with
animal models. We studied human temporal bones
from donors with mucopolysaccharidoses I (MPS I).
An autosomal recessive disorder, MPS I is caused by a
mutation in the gene encoding α-L-idurodinase and affects many organs, including the ear. Otologic findings
include otitis media and a progressive hearing loss.
Our histologic analysis of human temporal bones with
MPS I showed residual mesenchyme and basophilic
concretions in the middle ear, as well as degeneration
of the organs of Corti16 (Figure 10–11A) and significant
loss of hair cells in all cochlear turns,17 explaining the
combined conductive and sensorineural deafness that
human patients experience. We saw similar pathologic
changes in our MPS I knockout mouse (Figure 10–11B),
validating it as an effective animal model.18 We then investigated gene therapy in this model, with an eye on
its potential for translation to humans with MPS I.19
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 various animal spe-
20,21
cies
(Figure 10–12) and evaluated its permeability
to different substances. Similar changes of inflammatory cell infiltration of the round window membrane
and scala tympani due to otitis media in our chinchilla
22–26
model
and our human temporal bones with otitis
media27 have been shown. We also demonstrated sensorineural hearing loss secondary to otitis media in our
chinchilla model,28 a sequela described in humans.
Figure 10–9A. Sixty-four-year-old male with history of otitic meningitis. He had bilateral chronic otitis media and presence
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of chlolesteatoma in the left ear. Note that the cholesteatoma has destroyed the ossicles and is located between the tympanic
membrane and the stapes footplate, creating a columellar effect on the sound conduction.
Figure 10–9B. Higher magnication of Figure 10-9A shows small cholesteatoma pearls under the mucosal surface of the stapedial footplate.
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Figure 10–10. This patient had a history of cochlear implant surgery. Histopathologic examination of the surgery site shows new
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bone formation and brotic changes in all turns of the cochlea indicating a more generalized postoperative reaction than expected.
Figure 10–11. Mucopolysaccharidosis. A. A human temporal bone with MPS I shows loss of outer and inner hair cells. B. In an
MPS I mouse at 1 year of age, organ of Corti (arrow) and neurolaments (N) were missing. SL - spiral ligament.
228
10. Future of Temporal Bone Studies 229
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Figure 10–12. In a chinchilla model, we have seen similar changes and mechanisms we see in human studies in terms of
interaction in between middle and inner ear. MEC - middle ear cavity; RWM - round window membrane; ST - scala tympani.
Inammatory cells such as polymorphonuclear cells, mononuclear cells, bacteria in the membrane, and scala tympani. Note
vacuolization and edema of the RWM. All specimens were processed in a similar manner to test the validity of the animal model
in human patients.
Our laboratory made significant progress in otitis
media-related translational research using our collection
of human temporal bones and experimental animal
models. We initiated the concept of the otitis media
continuum
lent otitis media” (Figure 10–14) or intractable tissue
pathology behind an intact tympanic membrane
and middle ear and inner ear interaction in acute and
chronic otitis media (Figure 10–15).33 This research transformed treatment of patients.
29,30
(Figure 10–13); described “chronic si-
34–36
31,32
CLINICAL CAVEAT: The concept of chronic silent
(masked) otitis media depends on the basis of clinical and otopathologic evidence of chronic infection
in the middle ear cleft in the absence of obvious
pathologic findings in the tympanic membrane. 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, as is seen in Figure 10–13.
Figure 10–13A. Five-year-old male with history of immunodeciency and bone marrow transplant. Histopathological examina-
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tion shows otitis media with effusion.
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Figure 10–13B. Otitis media with effusion. Higher magnication of the image in Figure 10-13A shows the continuation of
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effusion from serous to seromucoid and mucoid character in the middle ear.
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Figure 10–14. Eighteen-month-old patient with bilateral chronic otitis media and meningitis. Fibrocystic granuloma, choles-
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terol granuloma, and residual mesenchyme can be seen behind the intact tympanic membrane. Purulent labyrinthitis showed
biolm structures in the inner ear.
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Figure 10–15A. One-year-old with a history of otogenic meningitis. Horizontal sections of the right temporal bone show
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irreversible pathologies consistent with severe otitis media, such as granulation tissue and brous structures in the middle ear.
Note that the tympanic membrane is intact.
Figure 10–15B. Otogenic meningitis. Higher magnication of the round
window from Figure 10-15A indicates purulent secretion around the niche
in the middle ear that has crossed the membrane into the scala tympani.
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Temporal Bone Histology and Radiology Atlas234
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FUTURE DIRECTIONS IN HUMAN
TEMPORAL BONE RESEARCH
Future studies should correlate otopathologic analysis
with genotypic and clinical data in order to better understand the disease progression and pathogenesis. To
date, around 50 genes have been identified as a cause of
nonsyndromic deafness. Only a few otopathologic case
reports associated with genetic information have been
described. We desperately need studies comparing genetic information from tissues such as blood or cheek
swab with the identified gene mutations responsible for
the specific ear diseases and their protein expression in
human temporal bones by immunohistochemistry in
order to create targets for future treatment plans and
more importantly genetic counseling. For example, previous studies have shown that endolymphatic hydrops
can be the only histologic marker for Meniere’s disease; however, hydrops is not directly responsible for
its symptoms (hearing loss, tinnitus, and vertigo).
Paparella and co-workers reported various developmental anomalies involving and surrounding the endolymphatic drainage system, such as a reduced Trautmann’s triangle and hypopneumatization of mastoid air
cells, which could be an early manifestation of genetic
abnormalities.39 It is believed that these anomalies can
gradually lead to dysfunction of the endolymphatic
duct, primarily including malabsorption of endolymph,
but also possibly to dysfunctional interference with secretion and function of the local immune system—ultimately ending up with Meniere’s disease. These types
of studies will help researchers and clinicians determine
the gene(s) associated with this disease, facilitating potential therapeutic strategies.
The laboratory should have a multidisciplinary
team in temporal bone procurement, clinical data collection, and methodologic analysis, such as pathology, histology, immunohistochemistry, proteomics, polymerase
chain reaction, cDNA microarray, in situ hybridization,
light and electron microscopy, and 3-D reconstruction
from serial temporal bone sections to understand the
pathogenesis, pathophysiology, and etiology of the
hearing and balance disorders 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 PCR, in situ hybridiza-
40
tion, and immunohistochemistry.
Current standard
37,38
procedures have proven useful for many imaging and
molecular studies of human temporal bones; however,
because of long postmortem, fixation, and decalcification times, such procedures can fail to conserve the
structure of specimens or to preserve their nucleic acids and proteins. The limitations of DNA retrieval from
archived temporal bones are as follows: (1) the amount
of DNA extracted from a single section (~500 ng) is low;
(2) extracted DNA does not allow PCR amplification of
fragments exceeding ~300 to 400 bp; (3) extracted DNA
is labile and degrades rapidly; and (4) formalin fixation can induce alterations in template sequence that
can inhibit alignment.
41
In PCR amplifications, minor
alterations in DNA sequences due to formalin fixation
can be misinterpreted as a possible mutation (up to 1
artifact per ~500 bp).42 We found rapid degradation
of RNA in bones when postmortem times exceeded
6 hours. Long preparation and storage times can also
result in poor preservation of ear structures, in degradation of DNA/RNA, and in decreased antigenicity of
proteins. Contamination of DNA/RNA is a significant
problem.43 Some reasons for contamination of archived
specimens included: (1) before the development of molecular methods there was no reason to follow 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 specific requests regarding
disease, fixative, or embedding media, as well as microdissected structures, modified fixative, avoiding the
pitfalls of reusing embedding material and of storing
multiple specimens in the same solution, studies will
use ear tissues with less contamination and with better
preservation of morphology and molecular footprints,
enhancing immunohistochemical, electron microscopic, and proteomic analysis. However, transmission
electron microscopy can be applied to unstained celloidin sections; if those studies are performed in human specimens with minimal postmortem times, morphologic analysis will demonstrate excellent structural
detail.
SUMMARY
Because of the diversity of ear diseases, one main requirement of human temporal bone laboratories is the
ability to acquire and archive a large number of bones
from deceased donors with good clinical histories for
auditory and vestibular research. Because of human oto-
10. Future of Temporal Bone Studies 235
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pathologic studies, we will have a better understanding of the structural and molecular mechanism of various ear pathologies and otopathologic conditions that
could have a major impact, facilitating the development of new strategies for preventing and treating ear
diseases and their complications.
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