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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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Figure 10–1C
Figure 10–2A. Otosclerosis. Histologic (non-clinical) otosclerosis, which can be found during histologic examination of the
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temporal bone in around 10% of the population. ME – middle ear; V - vestibule; SF – stapes footplate; FN – facial nerve. Arrows point to the normal (uninvolved) stapedovestibular joints. A sizeable focus of otosclerosis (O) is seen just anterior to the
anterior stapedovestibular joint, and a smaller focus posterior to the posterior joint. Because there is no xation of the stapes
footplate, there was no clinically apparent conductive hearing loss.
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Figure 10–2B. Otosclerosis. Eighty-two-year-old male with history of prostate carcinoma and otosclerosis. Histopathologic exam
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of the left temporal bone shows clinical xation of the stapedial footplate due to active otosclerosis located anterior to the oval
window. ME – middle ear; M – malleus; I – incus; FN – facial nerve.
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Figure 10–2C. Otosclerosis. Eighty-nine-year-old female with over 30-year history of sensorineural (not conductive) hearing
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loss. Histopathologic exam reveals lateral cochlear wall otosclerotic involvement with the lesion beginning at the ssula ante
fenestram and extending anteriorly toward the cochlea, but sparing the stapedovestibular joint.
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Temporal Bone Histology and Radiology Atlas220
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(Figure 10–3). However, significant gaps remain in our
understanding of their molecular mechanisms. Recognizing that all of the many otological diseases require
sequential human temporal bone studies with good corollary clinical records, we must also be aware that there
are clinically diagnosed diseases that currently have no
or minimal histopathological studies. Examples include
auditory neuropathy, sudden sensorineural hearing loss,
and vestibular Meniere’s disease, among others.
Adaptation of new molecular and imaging techniques can provide researchers with the tools necessary to study both 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 preservation of proteins, DNA,
RNA, and other molecules. Breakthrough human otologic studies will require high-quality human ear tissues, advanced techniques, and highly trained researchers. Human studies should be performed within
short postmortem periods on human specimens that
contain all of 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.
CLINICAL CAVEAT: Clinical otosclerosis affects
about 0.3% of the population while histologic otosclerosis is found in around 10% of the population.
Foci of otosclerosis that do not affect the movement
of the stapes footplate or obstruct the round window niche do not cause conductive hearing loss
and will remain clinically inapparent—as seen in
Figure 10–2A. Cochlear involvement with otosclerosis—as seen in Figure 10–2C and D—will manifest as sensorineural hearing loss.
Figure 10–2D. Otosclerosis. Magnication of the otosclerotic focus from
Figure 10-2C reveals lateral cochlear wall involvement with spiral ligament and stria vascularis atrophy in addition to hair cell damage.
Figure 10–2E. Otosclerosis. Complete obliteration of round window with otosclerosis as well as pericochlear otosclerosis caus-
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ing permanent hearing loss.
Figure 10–2F. Otosclerosis. Round window otosclerosis that does not obliterate the window is observed as an incidental nding during histopathologic
exam. TM – tympanic membrane; ST – sinus tympani; P – posterior semicircular canal; O – otosclerosis in the round window niche.
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Figure 10–3A. Vestibular schwannoma. Seventy-two-year-old male patient with a history of sensorineural hearing loss and
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vertigo. Otopathological examination shows vestibular schwannoma located in the internal auditory canal.
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Figure 10–3B. Vestibular schwannoma. Magnied portion of Figure 10-3A
shows loss of cochlear hair cells, associated with serous labyrinthitis, in this
patient.
10. Future of Temporal Bone Studies 223
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IMMUNOHISTOCHEMISTRY
Immunohistochemistry of decelloidinized human temporal bones using the avidin-biotin-horseradish peroxidase method or immunofluorescent labeling is routinely
being performed (Figure 10–4). We have applied several
antigen retrieval methods including heat, chemical, and
proteolytic-induced epitope retrieval to existing specimens. We observed mucin upregulation by immunohistochemistry (Figure 10–5A–D) and mRNA by in situ
hybridization (Figure 10–5E–H) in mucoid otitis media
using human temporal bones with postmortem times
<6 hours.
4,5
Double Labeling
Using immunohistochemistry to analyze temporal
bones with cholesteatoma, we have found that Id1
(Figure 10–6A) and NF-kB p65 (Figure 10–6B) proteins
were co-expressed in the middle ear mucosa (Figure 10–6C). We demonstrated NF-kB p65 (stained with
MAB3026 antibody against “activated” p65) in the nuclei of cholesteatoma epithelium cells. Figure 10–6D
shows double-labeling in situ hybridization of the Eustachian tube. Two genes, MUC5AC (TRITC) and MUC5B
(FITC), were targeted on a specimen autopsied 3 hours
after death.
Expression of Toll-Like Receptors
in Chronic Otitis Media
Toll-like receptors (TLRs) are membrane proteins that
play a crucial role in induction and activation of innate immunity in the course of infection.6 Activation
of TLRs leads to the mobilization of cytokines, chemokines, interferons, defensins, lysozyme, and other molecules in otitis media.
and TLR4 were expressed in the middle ear mucosa
and granulation tissue in donors with chronic otitis
media (Figure 10–7).
7,8
We have found that both TLR2
THREE-DIMENSIONAL RECONSTRUCTION
Knowledge of many structures in human temporal
bones is better achieved using three-dimensional (3D)
reconstruction (Figure 10–8). We have applied 3D models generated from sections with 3D reconstruction
software (Amira, Visualization Sciences Group, Bordeaux, France; and Zuse Institute, Berlin, Germany).
To cite several examples, we used 3D models to study
the diameters of the facial nerve and canal in temporal
bones with Bell’s palsy
in temporal bones with bilateral and unilateral Meniere’s disease10; and epitympanic bony volume and
tympanic isthmus area in temporal bones with chronic
otitis media.
11
9
; the membranous labyrinth
PROTEOMIC ANALYSIS
We are also continuing proteomic analysis of celloidinembedded archived bones, and plan to start studies using our newly acquired temporal bones. 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-fixed, celloidin-embedded chinchilla temporal bones yielded more than 50 proteins including
collagen (alpha 1, 2, and 3), cytokeratins (CK-1, CK-10,
CK-2e), cochlin, vitronectin (isoform X1), and fibronectin (isoform X6). We believe those studies will provide
intact protein identification combined with a protein
database for diseased and nondiseased human ears. As
a result, protemic profile 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. These analyses will provide insight into the
underlying mechanisms of ear problems and propose
potential treatment approaches.
VALIDATION OF ANIMAL MODELS AND
POTENTIAL TO 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 (Figure 10–9), Meniere’s
disease, otosclerosis, and studying changes after cochlear implant surgeries (Figure 10–10), among others,
Figure 10-4. Seventy-two-year-old male with Meniere’s disease. He had a history of bilateral uctuating hearing loss, tin-
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nitus, and vertigo episodes. Histopathologic exam of the left ear showed profound cochlear hydrops (arrow) in all turns of the
cochlea. U - utricle.
Figure 10–5. A. Immunolabeling of Na+,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.
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Figure 10–6. A-D. Immunohistochemical analysis. Noninamed middle ear (ME) epithelia did not react with MUC4 (A) or
MUC5B (B) 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.
Figure 10–7. Double immunolabeling of proteins co-expressed in cholesteatoma. A. Id1 (green). B. NF-kB p65 (red). C. Yellow-
orange dots = overlap of both Id1 and NF-kB p65. D. In situ hybridization showing MUC5AC (arrowheads) and MUC5B (arrows).
so studying human specimens is critical. In addition,
animal models have serious limitations in terms of
relevance to human ear disorders. For example, only
75% of mouse genes have equivalents in humans.12
Furthermore, experimental animal models do not always mimic the human condition; experimental otitis
media has been induced by inoculation of bacteria or
obstruction of the Eustachian tube, whereas in humans,
acute otitis media is often preceded by viral upper respiratory tract infection.
spontaneous otitis media involving bacterial pathogens that are not common to human ear infections.15
13,14
Knockout mice can develop
Finally, animal and human ears have significant anatomic differences. We have had many years of experience validating our experimental animal models to our
human temporal bones, including their potential for
translational applications. For example, in our animal
model, we demonstrated passage of bacterial toxins
and inflammatory 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 to 5 times thicker), despite
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