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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 Atlas6
https://t.me/med1917
Step 10: Mounting
The stained sections are mounted on 1 × 3 inch glass
slides, coverslips are placed, and lead weights are used
to compress the coverslips onto the slides. These are
arranged in sequential order in cardboard folders.
The slides are now ready for microscopic
examination.
REFERENCES
1. Schuknecht H. Temporal bone removal at autopsy. Prepa-
ration and uses. Arch Otolaryngol.
2. O’Malley JT, Merchant SN, Burgess BJ, Jones DD, Adams
JC. Effects of fixative and embedding medium on morphology and immunostaining of the cochlea. Audiol Neu-
rootol. 2009;14(2):78–87. Epub 2008 Oct 1.PMID: 18827478
1968 Feb;87(2):129–137.
CHAPTER 2
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Special Temporal Bone Histology Techniques
for Both Preparation and Analysis
Alicia M. Quesnel, Reuven H. Ishai, and Michael J. McKenna
INTRODUCTION
Donated human temporal bone specimens from patients who had otologic diseases are extremely valuable because the human inner ear is inaccessible during life for pathologic analysis. These temporal bone
specimens provide the material from which the pathologic basis of otologic disorders can be determined.
Traditional light microscopy of temporal bone sections
stained with hematoxylin and eosin provides excellent
cellular level detail, as detailed in Chapter 1. In this
chapter, we review special temporal bone histology
techniques for both preparation and analysis, including preparation of specimens with otologic implants,
two-dimensional reconstruction of the cochlea, auditory, and vestibular hair cell and neuron quantification,
as well as radiologic imaging, immunostaining, electron microscopy, and DNA extraction from human temporal bone specimens. The utility of these techniques
is illustrated by selected studies that have contributed
to further understanding of the pathophysiology of multiple otologic disorders.
SPECIAL CONSIDERATIONS IN
HISTOLOGIC PROCESSING
Once the temporal bone has been removed and prepared
as detailed in Chapter 1, histologic processing of these
valuable specimens is laborious and time-consuming.
Each specimen may take up to 2 years to process, in
part due to a lengthy period of decalcification required
for the dense otic capsule bone. Production of highquality sections with minimal artifact is dependent
on the skill of the technician, but readily achieved by
experienced technicians with a process that has been
perfected over many years. This process is described in
detail in Chapter 1, but summarized briefly here.
Histologic processing begins with fixing the tissue, either by embalming or fixation of the temporal
bone specimen immediately on removal. The postmortem time, that is, the duration of time from death to
fixation, is the biggest determinant of postmortem artifact in an experienced laboratory.
Next, the specimen undergoes decalcification for
months. A chemical test is performed to determine the
endpoint. Radiographs may be used to confirm that
the specimen has been adequately decalcified. This is
followed by dehydration via immersion in alcohol solutions of escalating concentrations. The specimen is
then embedded in a material to allow for sectioning
without compression artifact or shearing of the tissue.
Celloidin is the most commonly used embedment material, as it offers excellent histologic preservation as
well as the ability to remove the celloidin for immunostaining at a later point.
The specimen is then blocked or oriented for sectioning on the microtome. Horizontal or vertical sections are obtained (Figure 2–1). Sectioning of temporal
bone specimens is most commonly done in the horizontal plane. Horizontal sections are easy for the otologist to interpret given the familiarity of anatomical
structures in this plane on axial radiologic imaging
(such as axial computed tomography images). In addition, horizontal sections are optimal for identification of
pathology within the tympanic membrane and pathology affecting the ossicular joints, given the sections fall
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Temporal Bone Histology and Radiology Atlas8
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Figure 2–1. A. Mid-modiolar section through the cochlea in a horizontally sectioned temporal bone. B. Near mid-modiolar
section through the cochlea in a vertically sectioned temporal bone.
roughly perpendicular to these structures. Pathologic
changes at the anterior and posterior aspect of the footplate, such as otosclerosis, can be readily identified on
horizontal sections (Figure 2–1a). The traditional method
of two-dimensional reconstruction of the cochlea, which
is used for quantification of hair cells and spiral ganglion neurons and for correlation of pathology to the
location along the cochlear duct, is based on horizontal
sections. Vertical sectioning in the plane of the superior
semicircular canal is optimal for the evaluation of superior semicircular canal dehiscence (Figure 2–2).
Vertical sectioning is also preferred for the evaluation of the Eustachian tube and related tubal muscle
pathology, and middle fossa dura pathology such as a
defect associated with a spinal fluid leak, arachnoid
granulations, or encephaloceles.
SPECIMENS WITH OTOLOGIC IMPLANTS
Temporal bone specimens from patients who received
otologic implants during life, including tympanostomy
tubes, ossicular prostheses, stapedectomy prostheses,
and cochlear implants, require special care in processing. A stellite-tipped steel microtome blade can section
through various plastic materials, including polytetrofluoroethylene, silastic, and polyethylene, preserving the architecture of delicate tissues surrounding the
implant. Experience at the Massachusetts Eye and Ear
Infirmary laboratory has demonstrated that the fibrous
capsule, which typically surrounds implanted foreign
materials, is routinely preserved when the sectioning
is completed with plastic implants in situ. Therefore,
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temporal bones with plastic tympanostomy tubes or
ossicular prostheses are sectioned with the implant in
situ. This also enables the histopathologist to assess
the position of the prosthesis within the middle ear or
tympanic membrane. Metal implants, which primarily include stapes prostheses and cochlear implants,
cannot be sectioned in situ, as the microtome blade becomes damaged, resulting in unusable and torn sections. Specimens with stapes prostheses are embedded
in celloidin and sectioned to the level of the implant. A
photograph of the section at the level of the implant is
Figure 2–1. (continued )
taken to capture the position of the implant within the
middle ear and vestibule (Figure 2–3).
The implant is then gently removed, and sectioning is completed. Most often, the majority of the fibrous
capsule remains intact after manual removal of the stapes prosthesis, so that the position of the implant can
also be assessed by examination of the fibrous capsule
on the histologic sections.
For temporal bone specimens from patients who
had cochlear implants placed during life, special
care is required during the removal, processing, and
Figure 2–2. Superior semicircular canal dehiscence demonstrated in a vertically sectioned temporal bone.
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Figure 2–3. Temporal bone specimen from a patient who had undergone stapedectomy during life, Toluidine blue stain. This
demonstrates the in situ appearance of a stapes prosthesis in good position, with the hook of the prosthesis crimped around the
incus and the piston in the vestibule. At this point, the prosthesis is then delicately removed, and sectioning of the specimen
continues. Photo credit: Barbara Burgess.
sectioning steps. The electrode array is transected in the
mastoid via a postauricular incision prior to removal
of the specimen with the oscillating saw. This avoids
intracochlear shearing artifact that may occur if the oscillating saw contacts the electrode. The major goal for
cochlear implant specimens is to preserve the ability to
assess the position and depth of electrode within the
cochlea, while minimizing artifact so that pathologic
changes related to cochlear implantation may be accurately assessed. The initial specimens with cochlear
implants that were received in the 1990s were embed-
ded in araldite, which enabled sectioning of the temporal bone specimen with the electrode maintained
in situ. The disadvantages of araldite as an embedding material include slightly reduced cellular level
structural detail (as compared to hematoxylin and eosin stained paraffin or celloidin embedded) and the inability to perform immunohistochemical studies. Given
these disadvantages with araldite, cochlear implant
specimens processed in our laboratory in recent years
have been embedded in celloidin and the electrode
is gently removed prior to sectioning. The electrode
Temporal Bone Histology and Radiology Atlas12
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position can be determined by the pattern of intracochlear inflammatory reaction and/or fibrous capsule.
In addition, a computed tomography (CT) scan of the
specimen prior to removal of the electrode and sectioning is taken, and the electrode position may be determined from the CT.1
COMPUTED TOMOGRAPHY OF SPECIMENS
Specimens may undergo imaging by CT after fixation
and prior to decalcification. The specimen is mounted in
a custom-designed non-metallic temporal bone hol der
in order to orient the specimen so that it approximates
the orientation of the temporal bone during clinical imaging of patients (Figure 2–4).
The plastic holder is needed to avoid metallic artifact.2 Specimens undergo CT imaging with the same
protocol used for high resolution temporal bone imaging in patients, with a 0.5 mm slice thickness. Reconstructions in any plane can be made after data acquisition using a reformatting program.
Direct comparison of CT imaging and pathology
in the same temporal bone specimen is a powerful tool
(Figure 2–5). The accuracy of radiologic diagnoses can
be assessed, and pathologic correlates to radiologic
findings can be determined. The evaluation of the role
of CT imaging in the diagnosis and management of
otosclerosis is an example of the utility of obtaining
imaging of the specimen prior to histologic processing. Traditionally, the accuracy of CT in the diagnosis
of otosclerosis has been based on correlation to audiologic and intraoperative findings, rather than to pathology. If a total stapedectomy is performed, such that
Figure 2–4. Custom-designed temporal bone specimen holder for computed tomography imaging of the speci men prior to
histologic processing and sectioning. Photo credit: Jennifer O’Malley.
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Figure 2–5. Computed tomography (CT) correlation with histology in a temporal bone specimen with otosclerosis. A. Note the
hypodensity of bone anterior and posterior to the stapes footplate, and surrounding the cochlea (called the halo sign) in the CT.
This corresponds to areas of otosclerosis in the matched histologic section shown in (B).
the entire stapes with its footplate is removed, then
pathology can be obtained from the stapes. However,
since otosclerosis typically begins in the otic capsule
anterior to the footplate, and may result in multiple
foci within the otic capsule, postmortem human temporal bone pathology remains the best comparison for
CT findings in otosclerosis. In a recent, blinded, randomized study comparing high resolution CT imaging
of human temporal bone specimens to the pathologic
findings of the specimens, the sensitivity for diagnosis
of otosclerosis by CT was 80% and the specificity was
92%. The foci of otosclerosis that were not visible on
CT tended to be small foci without any large pseudovascular spaces. In the few specimens that received an
errant diagnosis of otosclerosis, there was a hypodensity on CT that corresponded to an island of connective
tissue or slight expansion of a vascular space in the otic
capsule.
3
Multifocal cochlear endosteal margin involvement by otosclerosis has been associated with mixed
or sensorineural hearing loss in otosclerosis.
4,5
Bisphosphonates, which are medications that slow bone turnover and are commonly used for osteoporosis, may
also slow the progression of sensorineural hearing loss
Temporal Bone Histology and Radiology Atlas14
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Figure 2–5. (continued )
in otosclerosis for those patients who develop a sensorineural component of hearing loss related to otosclerosis.6 Interestingly, CT was found to underestimate
the incidence of cochlear endosteal margin involvement by otosclerosis when CT imaging was compared
to pathology in the same temporal bone specimens.3
Clinical data indicates that magnetic resonance (MR)
CLINICAL CAVEAT: These findings may guide the
clinician in how to interpret CT findings in both
oval window and cochlear otosclerosis, and in making a more informed decision about when to treat
with bisphosphonates.
imaging may be the more sensitive modality in which
to assess response of otospongiotic lesions to bisphosphonate therapy.
7
TWO-DIMENSIONAL RECONSTRUCTION OF
THE COCHLEA AND THE SPIRAL GANGLION
A graphical reconstruction of the cochlea in a twodimensional plane may be created from horizontal
histologic sections. This enables the otopathologist to
map pathologic findings from each histologic section
through the cochlea on a diagram that indicates the
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Figure 2–6. A. Two-dimensional reconstruction of the cochlear duct based on identication of key points (indicated by numbers 1 to 8) and measurement of the distance between points A, B, and C. The tick marks indicate approximate 1 mm increments
along the cochlear duct. B. Histologic section through the hook region of the cochlea demonstrating the location of points A,
B, and C, which are needed for measurements to create the two-dimensional reconstruction.
distance along the cochlear duct from base to apex. The
classic two-dimensional reconstruction technique was
described by Guild in 1921,
8
with additional technical
details presented by Schuknecht in 1953.9 Some distortion occurs in the translation of a three-dimensional
structure to a two-dimensional graphical reconstruction,
but this error on average at various points throughout
the cochlea is less than 5%.
This reconstruction method is based on 20 µm histologic sections through the temporal bone at a plane
horizontal to the lateral semicircular canal. Every
10th section has been stained with H&E is used for this
process. The inner and outer pillar cells are traditionally used as the basis for the two-dimensional cochlear
reconstruction. The calculated cochlear duct length rep resents the spiral distance from base to apex through the
point of the inner and outer pillar cells.
To reconstruct the cochlea, the most superior (first)
and most inferior (last) sections containing the cochlea
are identified (Figure 2–6). Next, eight key points or sections are identified and the slide numbers are recorded.
The first six points are the sections in which the inner
and outer pillar cells are tangential to the basal, middle,
and apical turns and will define the vertical midpoint
through the two-dimensional cochlear reconstruction.
Moving from superior to inferior through the sections
containing the cochlea, points 1, 2, and 3 are the tangential sections in which the basal, middle, and apical turns
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