Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_4399_Библиотеки_им_академика_М_И_Перельмана
.pdf
Temporal Bone Histology and Radiology Atlas16
https://t.me/med1917
Figure 2–6. (continued )
first appear, and points 4, 5, and 6 are the sections in
which the turns disappear. The slide containing the helicotrema is point 7. The slide containing the beginning
and end of the hook region (basal-most portion of the
cochlea, including up to the round window) is point 8.
Two measurements are needed from the slide identified
for point 8. Measurement AB represents the distance in
millimeters between the inner/outer pillar cells in the
basal turn to the inner/outer pillar cells in the proximal
hook. Measurement BC represents the distance in millimeters between the inner/outer pillar cells in the apical portion of the hook and the inner/outer pillar cells
in the basal portion of the hook region. Using the eight
points, and two measurements, a two-dimensional spiral diagram of the cochlea can be drawn on graph paper
or plotted using a computer program. If graph paper is
used, each box represents 0.2 mm.
A two-dimensional diagram of Rosenthal’s ca-
nal and the spiral ganglion may be constructed in an
analogous manner to that for the cochlear duct reconstruction. Rosenthal’s canal, which contains the spiral
ganglion neurons, forms a 1½ turn spiral, whereas the
cochlear reconstruction is a 2½ to 3 turn spiral. First,
the slides that contain spiral ganglion neurons are
identified, and, if using graph paper, these slide numbers should be used to label the y-axis. Rosenthal’s canal commonly begins at approximately section 200 and
ends at approximately section 390. Five key points are
needed. Points 1 and 2 are the sections in which spiral
ganglion neurons first appear in the modiolus adjacent
to the basal and middle turns, respectively. Points 3
and 4 are the sections in which the last spiral ganglion
neurons are seen in the basal and middle turns, as sections are viewed from superior to inferior. Point 5 is the
most basalward (or superior) section in which spiral
ganglion neurons are seen in the hook region. Rosenthal’s canal is typically divided into Segments I, II, III,
and IV based on a vertical midline (Figure 2–7).
Figure 2–7. Two-dimensional reconstruction of Rosenthal’s canal based on identication of slides that contain spiral gan-
https://t.me/med1917
glion neurons (indicated by numbers 1 to 5). The location and number of spiral ganglion neurons can be plotted on this twodimensional reconstruction.
17
Temporal Bone Histology and Radiology Atlas18
https://t.me/med1917
QUANTIFICATION OF AUDITORY HAIR CELLS
AND SPIRAL GANGLION NEURONS
After creating a two-dimensional reconstruction, inner and outer hair cells may be easily quantified by
marking their presence or absence on the graph in the
basal, middle, and/or apical turns for each slide section through the cochlea. Hair cells may be identified
by their location within the Organ of Corti, presence
of stereocilia which is frequently visible at the light
microscopic level, and oblong cell shape with round
nucleus. Both premortem degeneration of hair cells or
postmortem artifact can make identification challenging and there is some subjectivity to determining the
presence or absence of a cell in some specimens.
Spiral ganglion neurons are traditionally quantified by the Abercrombie method of utilizing a correction
factor to account for the “double counting” that may occur when a structure straddles two sections. Spiral ganglion neurons may be counted by the number of nuclei
or nucleoli. Theoretically, a count based on the number
of nucleoli may be more accurate as the nucleolus is
smaller than the nucleus. Counting a smaller object necessitates less correction of the actual count because the
structure is less likely to straddle two sections (i.e., be
double counted); the corrected count is therefore more
similar to the empirical count, which should minimize
error. Postmortem degeneration or premortem pathology, however, may result in a pyknotic nucleus, in
which identification of the nucleolus is not possible. In
addition, given the small size of the nucleolus, counting
the number of nucleoli must be done at a magnification
of at least 400×, which makes the quantification process
more time-consuming. Therefore, due to practical constraints, quantification of spiral ganglion neurons is often based on counts of the spiral ganglion nuclei.
As noted previously, typically every 10th histologic section is mounted on a slide, stained, and available for review in the histologic slide set. To quantify
the number of spiral ganglion neurons, every 10th section through the modiolus is reviewed, and the number of spiral ganglion neuron nuclei or nucleoli are
counted. The count is multiplied by 10 to account for
intervening sections. This number, which represents
the actual count, is then multiplied by a correction
factor. The Konigsmark correction factor
the diameter of the structure (nucleus or nucleolus)
counted and the section thickness, as follows:
correction factor = section thickness /
(section thickness + diameter
of the structure counted).
10
is based on
The average diameter of the spiral ganglion neuron
nucleolus is 2 µm, and the typical section thickness is
20 µm. The correction factor for counts based on the
nucleolus is 0.91.
11
Nadol verified the accuracy of the
Konigsmark correction factor in 1988 by comparing empirical counts of spiral ganglion cells to spiral ganglion
nucleoli in a human specimen sectioned at a thickness
of 2 µm.
nucleus is 0.68.
10,11
The correction factor for counts based on the
12
An audio-cytocochleogram provides a graphical
summary of remaining cellular structures within the
cochlea (Figure 2–8). The audiogram is combined with
a graph of the quantified cellular structures, which are
plotted along a linear representation of the cochlea that
describes both frequency and location. This frequencylocation map for the human is based on temporal bone
specimens from patients who had abrupt hearing losses
over a defined frequency range so that the location of
the correlative pathologic findings could be mapped to
the frequency.13
Alternatively, a three-dimensional reconstruction
of the modiolus provides a very accurate quantification of spiral ganglion neurons, and avoids estimations
and correction factors needed with two-dimensional
reconstructions and quantification. However, threedimensional reconstruction is extremely time-intensive
and requires photographing each section and a specialized computer program. It is therefore not practical
for most applications or scientific inquiries requiring
quantification of the spiral ganglion neurons in multiple specimens. Optical methods of spiral ganglion neuron quantification have been successfully performed
in guinea pig temporal bones.14 The application of stereology to human archival temporal bones for spiral
ganglion neuron quantification has suggested that the
traditional methods of two-dimensional counting with
applied corrections factors may underestimate counts.15
The method of spiral ganglion neuron quantification based on two-dimensional reconstruction (as described here) has been applied in many animal and
human temporal bone studies. One notable application
was the classic study by Schuknecht describing four
basic types of presbycusis in humans.16 He quantified
hair cells and spiral ganglion neurons by these methods, compared the pathology to audiometric findings
in these patients, and identified the typical audiologic
features associated with predominant degeneration of
the hair cell, spiral ganglion neuron, and stria vascularis. Another important use for these methods has been
in investigations of the relationship between cochlear
implant performance and spiral ganglion neuron cell
counts. A recent study demonstrated that, in patients
with bilateral cochlear implants who had donated
Figure 2–8. Audiocytocochleogram. This right temporal bone specimen came from a 96-year-old man who had progressive
https://t.me/med1917
sensorineural hearing loss. The audiogram demonstrates a sharply downsloping mild to profound sensorineural hearing loss. The
cytocochleogram demonstrates partial loss of inner and outer hair cells in the apical and middle turns, with complete loss of
hair cells in the basal turn. There is variable partial loss of stria vascularis. Signicant loss of spiral ganglion neurons throughout
the cochlea is shown. The profound high-frequency hearing loss corresponds on the frequency–location map to the complete
loss of hair cells in the basal turn. The reduced word recognition score may be explained by both hair cell and spiral ganglion
neuron loss.
19
Temporal Bone Histology and Radiology Atlas20
https://t.me/med1917
their temporal bones, the ear with the higher number
of spiral ganglion neurons was associated with better
cochlear implant performance.12 This affirms that preservation and maintenance of the spiral ganglion neuron
population in cochlear implant patients is important;
furthermore, it supports the merit of research directed
toward maximizing spiral ganglion neuron survival,
such as with electrical stimulation and/or neurotrophic
support.
QUANTIFICATION OF VESTIBULAR HAIR CELLS
AND SCARPA’S GANGLION NEURONS
Gross vestibular hair cell loss (i.e., more than about
25%) may be assessed using standard light microscopy,
but more specific quantification of hair cells requires
differential interference contrast (DIC) microscopy.17
Using DIC microscopy with magnification to 100×,
type I and type II vestibular hair cells can be reliably
distinguished from each other and the surrounding
sustentacular cells.18 Type I vestibular neurons have a
flask shape and a surrounding nerve chalice and type II
vestibular neurons have a cylindrical shape without the
surrounding chalice. The neurons can be distinguished
from the supporting cells by the stereocilia, which are
easily seen in well-preserved specimens using DIC microscopy. Similar to auditory hair cell quantification,
vestibular hair cells are counted on every 10th section
through the vestibular end organs (semicircular canals,
utricle, and saccule), and a correction factor is applied.
The vestibular hair cell count is reported as a density
(i.e., number of cells per 0.01 mm2), and losses relative
to normative data may be determined.19
When the vestibular nerve is intact and preserved
as part of the temporal bone specimen, vestibular neurons may be quantified by reviewing all stained sections from superior to inferior through the internal auditory canal. Vestibular neurons containing a nucleolus
are counted, and a correction factor of 0.88 is applied.17
The percent loss compared to normal vestibular neuron
population for age may be determined from normative
data.20
IMMUNOSTAINING
Immunostaining of human archival temporal bone
specimens has progressed significantly in the past decade, and has proven to be very successful for some
antigens while challenging for others. Immunohistochemistry can detect the presence and location of
proteins in tissue sections, and has the potential to significantly contribute to our understanding of pathophysiology in otologic disorders through its application to human temporal bone specimens. Some of the
challenges in immunostaining in archival temporal
bone specimens include postmortem degeneration, removal of the embedment material, and variability in
the concentration of antibody needed. Robust immunostains, such as anti-neurofilament (Figure 2–9), often result in good immunostaining of specimens with
more than 24 hours postmortem time prior to fixation,
while other immunostains yield more variable results
based on the postmortem time.
Since celloidin embedment is advantageous for
inner ear morphology preservation, temporal bone
specimens are routinely embedded in celloidin. A process for successful removal of celloidin in preparation
for immunostaining has been developed.
col for immunostaining for each new antibody must
be somewhat empirically derived, and so new immunostains are first tried on mouse temporal bone specimens that have been histologically processed and embedded by the same technique as the human archival
specimens. In addition, variations in the fixative and
decalcification process affect whether an immunostain
is ultimately successful in the human temporal bone
specimens. The addition of acetic acid to formaldehyde fixative, for example, has been found to improve
immunostaining results in the cochlea.22 New temporal
bone specimens processed in our laboratory are therefore initially fixed in formaldehyde with acetic acid to
optimize future immunostaining results.
Positive immunostaining for macrophages, B-cell
lymphocytes, and T-cell lymphocytes with anti-CD68,
anti-CD20, and anti-CD3 antibodies was demonstrated
surrounding the electrode within the cochlea, consistent with a cellular immune response.23 A new class of
resident macrophages/microglia was recently found
throughout the human temporal bone, including within
the spiral ganglion/modiolus, endochondral bone surrounding the cochlea, spiral limbus, Organ of Corti, and
spiral ligament. Immunostaining for known markers of
macrophages and microglia (anti-CD163, anti-IBa1, and
anti-CD68) led to the discovery of this cell type within
the human temporal bone.24 (Figure 2–10) The significance of these cells is not yet determined. Given the ubiquitous distribution throughout the inner ear and their
known role in innate immune defense elsewhere, they
may play a role in the development of multiple otologic diseases.
21
The proto-
2. Special Temporal Bone Histology Techniques for Both Preparation and Analysis 21
https://t.me/med1917
Figure 2–9. High power image of human Organ of Corti from a temporal bone specimen immunostained for neurolament, using anti-neurolament antibody. The nerve terminals at the base of the inner and outer hair cells (arrows) and dendrites (open
triangle) are highlighted with this stain (as shown by the dark brown color). Photo credit: Jennifer O’Malley.
CLINICAL CAVEAT: Immunostaining in human
temporal bone specimens from patients who underwent cochlear implantation during life has been
able to characterize the inflammatory cell infiltrate
frequently seen in the cochlea after implantation.
detail at the sub-cellular level, and detail on the morphologic structure of cells beyond light microscopy.
Rapid fixation of the temporal bone within several
hours of death is a requisite for electron microscopy.
Significant artifact at the ultra-structural level occurs
in specimens with a longer postmortem time prior to
fixation. For EM, the inner ear is perfused with glutaraldehyde via the oval and round windows in order to
and then the temporal bones
,
ELECTRON MICROSCOPY
rapidly initiate fixation
may be subsequently removed and placed in fixative.25
The cochlea is then sectioned into separate turns, post-
fixed in osmium tetroxide, dehydrated, embedded in
Electron microscopy (EM) of rapidly fixed human temporal bone specimens can provide exquisite structural
Epon, further sectioned, and stained with toluidine
blue.25
Figure 2–10. A. Immunostaining for ionized calcium binding protein 1 (anti-IBA1), which stains macrophages and microglia,
https://t.me/med1917
in human archival temporal bone tissue demonstrates an abundance of macrophages/microglia in the spiral ganglion of the
cochlea. Arrows indicate spiral ganglion neurons. Open arrow heads indicate macrophages/microglia. B. The multiple processes
and morphology of these macrophages/microglia can be seen with anti-IBA1 staining as well at high power. Photo credit: Jennifer O’Malley.
22
2. Special Temporal Bone Histology Techniques for Both Preparation and Analysis 23
https://t.me/med1917
Figure 2–11. Electron microscopy of the human spiral ganglion neuron demonstrating the difference in morphology between
the type I and type II spiral ganglion neurons. Photo credit: Barbara Burgess and Joseph Nadol, Jr, MD.
EM of the human temporal bone has led to the
discovery of new anatomic findings, provided insight into the pathophysiology of presbycusis, identified the pathologic correlates of several types of syndromic hearing loss, and confirmed the validity of an
animal model of hearing loss. With light microscopy,
the study of neural degeneration is generally limited
to the presence or absence of spiral ganglion cell bodies within the modiolus. Using EM, individual afferent nerve fi bers, nerve terminals, and synapses can be
studied, characterized, and the density per hair cell may
be determined (Figure 2–11). Reciprocal synapses, in
which both afferent and efferent synaptic specializations exist at the same nerve terminal, were first identi-
fied at the basal poles of human outer hair cells using
EM in 1981.26 These synapses may enable communication between outer hair cells through a local neural network interconnected by type II spiral ganglion nerve
fibers, and modulated by the olivocochlear system.27
EM studies of the human cochlea also identified a significant reduction in the number of afferent spiral ganglion neu ron fibers and synapses at the outer hair cells
in cases of presbycusis with normal hair cell populations, that is, an early primary neuropathy.25 More recent work in animal models of age-related hearing loss
using immunostains for nerve terminals, presynaptic
ribbons, and postsynaptic receptors demonstrated an
analogous pattern of degeneration of synapses, and
Temporal Bone Histology and Radiology Atlas24
https://t.me/med1917
furthermore, reported that this synaptic degeneration
precedes measurable age-related decrements in hearing.28 Although there are known differences in the sensorineural elements of the cochlea in humans versus
rodents and other mammals, the prior findings from
the human temporal bone EM study help confirm the
validity of animal models of age-related hearing loss.
In Alport’s syndrome, which results in progressive renal failure and hearing loss, EM revealed an abnormal
separation of the basilar membrane from the basement
membrane of cells of the Organ of Corti.29 This ultrastructural pathology is similar to the findings in the
kidney, in which the layers of the glomerular basement
membrane separate.
DNA EXTRACTION FROM SPECIMENS
There has been remarkable progress in the discovery of
genetic mutations causing hearing loss in recent decades,
but associated pathologic findings in the human cochlea
have been described in only a small number of genetic
hearing loss conditions. Furthermore, with the development of genome-wide screening tools, extraction of
genetic material from the temporal bone spec imens,
in which pathology can be determined and au diologic
findings can be correlated, would be particularly useful.
Unfortunately, efforts to extract DNA from human archival temporal bone specimens have been largely unsuccessful, in part due to cross-contamination of DNA
from the storage of specimens together. RNA extraction is not possible due to the postmortem time prior
to tissue procurement, which typically exceeds multiple
hours. In recent years, registered temporal bone donors
have been asked to donate a buccal swab, which has
been stored for later use in DNA analysis. Of note, there
are multiple temporal bone specimens from patients
in which genetic mutations causing hearing loss were
diagnosed during life. This does allow correlation of
pathologic findings with genetic findings.
CONCLUSION
Human temporal bone specimens remain a relevant
and important source for the continued discovery of
pathophysiologic mechanisms of hearing loss. The traditional method for two-dimensional reconstruction of
the cochlea and Rosenthal’s canal is used for mapping
pathology along the length of the cochlea, and assists
in quantification of hair cell or neuronal losses. Computed tomography imaging of specimens yields the
ability to definitively correlate radiologic and histologic findings in the same specimen, and has been particularly useful in otosclerosis and in determining intracochlear electrode position. Immunohistochemistry
on archival human temporal bones has been successful
with multiple immunostains thus far, and has enabled
characterization of the cellular immune response surrounding a cochlear implant electrode and discovery
of resident macrophages/microglia within the inner
ear. EM of rapidly fixed human temporal bone specimens can produce high-quality images with excellent
ultrastructural detail, and may be used to confirm the
validity of animal models or identify new anatomical
structures or pathology at the sub-cellular level.
ACKNOWLEDGMENTS
The authors acknowledge the contributions from members of the Massachusetts Eye and Ear Otopathology
Laboratory, particularly Dr. Joseph Nadol, Jr, Jennifer
O’Malley, Barbara Burgess, Diane Jones, Meng Yu Zhu,
Dr. Joseph Adams, and the late Dr. Saumil Merchant.
We also thank Garyfallia Pagonis for her assistance in
preparing the figures.
REFERENCES
1. O’Malley JT, Burgess BJ, Zhu MY, Curtin HD, Nadol JB Jr.
Correlation between histologic and radiographic reconstruction of intracochlear electrode position in human
temporal bones. Audiol Neurootol. 2014;19(3):184–192.
2. O’Malley J, Mitchell R, Jones D, Burgess B, Curtin HD,
Merchant SN. The utility of CT scanning prior to processing human temporal bones in celloidin. Abstracts of the 32nd
Midwinter Research Meeting of the Association for Research in
Otolaryngology. Baltimore, MD. February 14–19, 2009.
3. Quesnel AM, Moonis G, Appel J, O’Malley JT, McKenna
MJ, Curtin HD, Merchant SN. Correlation of computed tomography with histopathology in otosclerosis. Otol Neu-
rotol. 2013;34(1):22–28.
4. Kwok OT, Nadol JB Jr. Correlation of otosclerotic foci and
degenerative changes in the organ of Corti and spiral ganglion. J Otolaryngol. 1989;10(1):1–12.
5. Parahy C, Linthicum FH Jr. Otosclerosis and otospongiosis: clinical and histological comparisons. Laryngoscope.
1984;94(4):508–512.
2. Special Temporal Bone Histology Techniques for Both Preparation and Analysis 25
https://t.me/med1917
6. Quesnel AM, Seton M, Merchant SN, Halpin C, McKenna MJ. Third-generation bisphosphonates for treatment of sensorineural hearing loss in otosclerosis. Otol
Neurotol. 2012;33(8):1308–1314.
7. De Oliveira Vicente A, Chandrasekhar SS, Yamashita HK,
Cruz OL, Barros FA, Penido NO. Magnetic resonance imaging in the evaluation of clinical treatment of otospongiosis: a pilot study. Otolaryngol Head Neck Surg. 2015;
152(6):1119–1126.
8. Guild SR. A graphic reconstruction method for the study
of the Organ of Corti, Anat. Rec. 1921;22:141–157.
9. Schuknecht HF. Techniques for study of cochlear function and pathology in experimental animals: development of anatomical frequency scale of the cat. AMA Arch.
Otolaryngol., 1953:58;377–397.
10. Konigsmark BW. Methods for the counting of neurons.
In Nanta WJH, Ebbesson SOE, eds. Contemporary Research
Methods in Neuro-anatomy. Berlin: Springer-Verlag, 1970:
315–340.
11. Nadol JB Jr. Quantification of human spiral ganglion
cells by serial section reconstruction and segmental density estimates. Am J Otolaryngol. 1988;9(2):47–51.
12. Seyyedi M, Viana LM, Nadol JB Jr. Within-subject comparison of word recognition and spiral ganglion cell
count in bilateral cochlear implant recipients. Otol Neu-
rotol. 2014 Sep;35(8):1446–1450.
13. Schuknecht HF. Auditory and cytocochlear correlates of
inner ear disorders. Otolaryngol Head Neck Surg. 1994;
110(6):530–538.
14. Wrzeszcz A, Reuter G, Nolte I, Lenarz T, Scheper V. Spiral
ganglion neuron quantification in the guinea pig cochlea
using confocal laser scanning microscopy compared to
embedding methods. Hear Res. 2013;306:145–455.
15. Tang Y, Lopez I, Ishiyama A. Application of unbiased stereology on archival human temporal bone. Laryngoscope.
2002;112(3):526–533.
16. Schuknecht HF. Presbycusis. Laryngoscope. 1955;65(6):
402–419.
17. Degeneration of Auditory and Vestibular End Organs.
In: Merchant SN, Nadol JB Jr, eds. Schuknecht’s Pathology
of the Ear, 3rd ed., Shelton, CT: People’s Medical Publishing House, 2010.
18. Merchant SN. A method for quantitative assessment
of vestibular otopathology. Laryngoscope. 1999;109(10):
1560–1569.
19. Merchant SN, Velázquez-Villaseñor L, Tsuji K, Glynn RJ,
Wall C III, Rauch SD. Temporal bone studies of the human
peripheral vestibular system. Normative vestibular hair
cell data. Ann Otol Rhinol Laryngol Suppl. 2000;181:3–13.
20. Velázquez-Villaseñor L, Merchant SN, Tsuji K, Glynn RJ,
Wall C III, Rauch SD. Temporal bone studies of the human
peripheral vestibular system. Normative Scarpa’s ganglion cell data. Ann Otol Rhinol Laryngol Suppl. 2000;181:
14–19.
21. O’Malley JT, Burgess BJ, Jones DD, Adams JC, Merchant
SN. Techniques of celloidin removal from temporal bone
sections. Ann Otol Rhinol Laryngol. 2009 Jun;118(6):435–441.
22. 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
Neurootol. 2009;14(2):78–87
23. Nadol JB Jr, O’Malley JT, Burgess BJ, Galler D. Cellular
immunologic responses to cochlear implantation in the
human. Hear Res. 2014;318:11–17.
24. O’Malley JT, Nadol JB Jr, McKenna MJ. Anti CD163+,
Iba1+, and CD68+ cells in the adult human inner ear:
Normal distribution of an unappreciated class of macrophages/microglia and implications for inflammatory
otopathology in humans. Otol Neurotol. 2016;37(1):99–108.
25. Nadol JB Jr. Electron microscopic findings in presbycusic degeneration of the basal turn of the human cochlea.
Otolaryngol Head Neck Surg. 1979;87(6):818–836.
26. Nadol JB Jr. Reciprocal synapses at the base of outer hair
cells in the organ of corti of man. Ann Otol Rhinol Laryn-
gol. 1981;90(1 Pt 1):12–17.
27. Thiers FA, Nadol JB Jr, Liberman MC. Reciprocal synapses between outer hair cells and their afferent terminals: evidence for a local neural network in the mammalian cochlea. J Assoc Res Otolaryngol. 2008;9(4):477–489.
28. Sergeyenko Y, Lall K, Liberman MC, Kujawa SG. Agerelated cochlear synaptopathy: an early-onset contributor
to auditory functional decline. J Neurosci. 2013 21;33(34):
13686–13694.
29. Merchant SN, Burgess BJ, Adams JC, Kashtan CE, Gregory MC, Santi PA, Colvin R, Collins B, Nadol JB Jr. Temporal bone histopathology in alport syndrome. Laryngo-
scope. 2004;114(9):1609–1618.
Соседние файлы в папке Библиотека им академика М.И. Перельмана