THE ANATOMICAL RECORD 259:229 –236 (2000)
High Resolution Imaging of the
Mouse Inner Ear by
Microtomography: A New Tool in
Inner Ear Research
M.P. VAN SPAENDONCK,1,2 K. CRYNS,3 P.H. VAN DE HEYNING,2
D.W. SCHEUERMANN,1 G. VAN CAMP,3 AND J.-P. TIMMERMANS1*
1
Laboratory of Cell Biology and Histology, University of Antwerp,
B-2020 Antwerpen, Belgium
2
Dept. of Otolaryngology, University of Antwerp, B-2020 Antwerpen, Belgium
3
Dept. of Medical Genetics, University of Antwerp, B-2020 Antwerpen, Belgium
ABSTRACT
A newly developed desktop microtomograph was used to evaluate
whether it is suitable for visualizing the three-dimensional (3D) morphology
of the mouse inner ear (at a micrometer level) and whether it is applicable
as a fast screening tool to detect hereditary abnormalities in this organ. To
this end, the epistatic circler, a mutant mouse showing abnormal circling
behaviour, was used as a model. The inner ears were dissected out, formaldehyde-fixed, and scanned at maximal resolution along the longitudinal
axis. After segmentation, stacks of tomographic images were used for 3D
reconstruction of the bony labyrinth. Finally, the obtained data were correlated with subsequent conventional histological examination. The spatial
resolution (8 ␮m) achieved by this instrument, was found to be far superior
to that obtained by conventional computer tomography (CT) and magnetic
resonance (MR)-imaging equipment. The technique provides detailed tomographic images of the bony labyrinths and enables an adequate 3D reconstruction of the inner ear structures in this small mammal. In addition, it
allows a screening for pathologic specimens prior to the more time- and
labour-consuming histological techniques, which are still essential to
gather information at a (sub)cellular level. This imaging technique can be
regarded as a valuable tool in future research on hereditary inner ear
abnormalities. Anat Rec 259:229 –236, 2000. © 2000 Wiley-Liss, Inc.
Key words: computer tomography; inner ear; vestibular development; three-dimensional reconstruction; epistatic circler; mouse
Mouse models play a key role in today’s otovestibular
genetic research, either as existing mutants or as transgenic or knockout models. In order to adequately examine
whether their otovestibular labyrinths show a normal
morphology, three-dimensional (3D) reconstruction has
proven to be extremely useful.
To this end, a number of medical imaging techniques,
such as computer tomography (CT) and magnetic resonance (MR), which nowadays are commonly used to assess
human inner ear morphology and clinical diagnosis, are
applied (Reisser et al., 1996; Isono et al., 1997). However,
these instruments still suffer from a limited resolution,
which becomes a severe drawback when extremely small
©
2000 WILEY-LISS, INC.
structures like murine inner ears have to be investigated.
Therefore, the classical way to obtain 3D reconstructions
of the latter structures has mainly been based on the
conventional labour-intensive histological serial sectioning (Harada et al., 1990). Apart from the typical limita-
*Correspondence to: J-P Timmermans, Laboratory of Cell Biology & Histology, University of Antwerp (RUCA), Groenenborgerlaan 171, B-2020 Antwerpen, Belgium.
E-mail: [email protected]
Received 25 May 1999; Accepted 24 February 2000
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VAN SPAENDONCK ET AL.
stripped of excessive bone and adherent structures such
as muscle and paraflocculus. Subsequently, all inner ears
were visualized under the stereomicroscope using transillumination, a technique which renders the bony capsule
slightly transparent.
Microtomography
Fig. 1. Schematic drawing of the principle design of the desktop
X-ray-microtomograph.
tions of this technique regarding uneven slice thickness,
loss or damage of consecutive sections and shrinkage, an
additional difficulty lies in a proper orientation and alignment of consecutive sections, which is essential to obtain
accurate 3D reconstructions (Henson et al., 1994). Recently, new imaging technologies producing a much
higher resolution have become available for laboratory
purposes. A newly developed desktop CT scanner (Sasov
and Van Dyck, 1998) was used to study the inner ear
structure of the epistatic circler mouse, a pre-existing
mutant with an unknown otovestibular morphology. Possible harmful effects on tissue morphology were evaluated
at the light microscopical level by comparison with inner
ears that were not scanned.
MATERIALS AND METHODS
Epistatic Circler Mice
A small proportion of the F2 generation in the offspring
of the cross between C57L/J and SWR/J mice shows abnormal circling behaviour (Doolittle, 1963; Taylor, 1976).
This abnormal phenotype is believed to be due to homozygosity for two genes, viz. Ecl and Ecs. The circling F2
individuals are referred to as epistatic circlers. The inner
ears of three circling F2 individuals were scanned by the
microtomograph, as were those of three normally behaving SWR/J mice serving as controls. All scanned inner
ears were afterwards further processed for histology (including those of one epistatic circler and one SWR/J mouse
that were not scanned).
Dissection and Tissue Handling
All animal handling was done as prescribed by European Community Directive 86/609/EEC. The mice were
sacrificed by cervical dislocation after which the temporal
bones were quickly removed. The bony labyrinths were
immediately dissected out of the temporal bones by separating them from the middle ear along a suture line.
Ice-cold 4% buffered paraformaldehyde was gently perfused through the round and oval windows. Following
further immersion fixation in the same fixative for 48 hr at
4°C, the specimens were transferred to phosphate-buffered saline (0.01M, pH: 7.4) in which the inner ears were
This newly developed desktop X-ray microtomograph
(Skyscan威, Aartselaar, Belgium) consists of a compact microfocus X-ray tube, a slow scan CCD camera, and a Pentium computer processor (Sasov and Van Dyck, 1998). It is
based on the same working principles as a conventional
CT scanner, but uses instead of parallel X-rays a conical
X-ray beam generated by an X-ray tube with a very small
focus at an energy level of 10 –100 keV (maximal current
100 ␮A, maximal voltage 80 kV). The object is transversed
by this conical X-ray beam and the resulting signals are
recorded by a two-dimensional CCD camera (512 ⫻ 512
pixels), producing an enlarged radiograph of the object.
The magnification can be altered by changing the distance
between the source, object, and camera (Fig. 1). Using
small samples, a spatial resolution as high as 6 ␮m can be
achieved. Optimal contrast is obtained by tuning the energy of the X-ray tube to the density of the tissue, characterized by the attenuation length.
Unlike conventional CT scanners the object to be
scanned is rotated instead of the X-ray source. In this way,
radiographic projections from different viewing angles are
obtained, recorded, and stored. A 3D reconstruction is
calculated from these projections using the filtered backprojection algorithm. From this three-dimensional dataset, tomographic images are generated. The technique
works under ambient conditions, does not require any
specimen preparation nor staining, and no harmful heating of the specimen occurs.
Before being scanned, the inner ears were covered with
Vaseline威 to prevent damage by dehydration and were
mounted on the stage of the scanner using Plasticine威.
Each inner ear (dimensions 3.5 ⫻ 2 ⫻ 2.5 mm) was positioned in a longitudinal orientation and scanned at maximal resolution, depending on the sample size, i.e., the
enlarged radiograph should maximally cover but at the
same time be kept within the scope of the CCD camera in
all viewing angles. Total scanning time was 30 min. After
scanning, the bony labyrinths were further processed for
histology.
Morphometric Analysis of the Dimensions of
the Semicircular Canals
Based on the calibrated tomographic images of the left
and right inner ears of three control (SRW/J) and three
epistatic circler mice, the diameter of each of the semicircular canals was determined. In all cases the point for
measurement was taken at the top of each semicircular
canal opposite to the vestibule. For statistical analysis, a
non-parametric Mann Whitney-U test was performed.
3D Reconstruction
Segmentation was performed on the outer limits of the
bony labyrinth thus revealing after 3D reconstruction the
contours of the fluid-filled space inside the bony capsule
containing both the perilymphatic and endolymphatic
structures. Segmentation and subsequent 3D reconstructions based on these tomographic images were performed
using commercially available software (Surfdriver 2.5.5威).
HIGH RESOLUTION IMAGING WITH MICROTOMOGRAPHY
231
Fig. 2. a– h: A selection from a stack of 442 tomographic images
from the left inner ear of an epistatic circler mouse showing a distinct
narrowing at the level of its lateral semicircular canal. Image-level is
indicated on the scout view at the left upper corner. Cochlear structures
(a– c), vestibular structures (d), semicircular canals (e– h, overleaf). The
severely narrowed aspect of the lateral semicircular canal (CSL) is indicated by an open arrow in f (cross section at level of maximum reached
diameter). The arrows in g and h show the normal maximum diameter of
the anterior semicircular canal (CSA) and the posterior semicircular canal
(CSP). AOA, ampulla ossea anterior; AOL, ampulla ossea lateralis; COC,
crus osseum commune; CSA, canalis semicircularis anterior; CSL, canalis semicircularis lateralis; CSP, canalis semicircularis posterior; FMAI,
fundus meatus acusticus internus; FV, fenestra vestibuli (ovale); LSO,
lamina spiralis ossea; MOD, modiolus; VEST, vestibulum.
Histological Processing
orthogonally to the axis of the modiolus. Under these
circumstances, the spatial resolution (8 ␮m) is 100 times
higher than that obtained by currently used clinical CT
scanners. Both the bony cochlear structures (the osseous
spiral lamina, the thin wall of the modiolus and the secondary osseous spiral lamina at the basal portion of the
cochlear duct) and the bony vestibular structures (the
vestibule with its recesses, the slight widening of the
ampullae of the semicircular canals and the common crus)
can be studied in sufficient detail (Fig. 2a– h). The soft
membraneous tissues such as the stria vascularis and the
limbus are visible but not so sharply delineated since
contrast was such that bony structures were easily distinguishable from the surrounding soft tissues and fluids
which greatly facilitated the segmentation process. The
contours of the bony labyrinth (the fluid-filled space inside
the bony capsule containing both the perilymphatic and
endolymphatic structures) were delineated in the com-
Evaluations of inner ear morphology usually are based
on sections parallel to the modiolus, i.e., orthogonal to the
plane of the images generated by the microtomograph.
The temporal bones were completely decalcified in 5%
EDTA (1 week), dehydrated in a graded alcohol series, and
embedded in paraffin. Serial 10 ␮m-thick sections were
cut parallel to the modiolus. All sections were stained with
hematoxylin-eosin.
RESULTS
Serious abnormalities of the bony labyrinth such as
severe distortion of the lateral semicircular canal were
detected by means of transillumination. However, less
conspicious deformations such as a slight narrowing of the
canal could not be discerned in sufficient detail. In general, scanning of a mouse inner ear at a slice thickness of
8 ␮m resulted in about 440 tomographic images oriented
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VAN SPAENDONCK ET AL.
Figure 2. (Continued.)
plete set of consecutive images by segmentation thus revealing its shape after 3D reconstruction (Fig. 3a– d).
Whereas the bony labyrinths including the semicircular
canals of the normally behaving SWR/J mice showed a
normal morphology, a variety of abnormalities at the level
of the lateral semicircular canals of the epistatic circler
mice (ranging from slight narrowing to complete interruption) were revealed on both the tomographic images and
3D reconstructions.
Figures 2a– h and 3c,d show the tomographic images
and the 3D reconstuction of the same left bony labyrinth of
an epistatic circler. The narrowing at the level of the
lateral semicircular canal which is observed on the twodimensional tomographic images (Fig. 2f) is clearly visible
on the 3D reconstructions (Fig. 3c,d).
Morphometric analysis of the anterior semicircular canal revealed a mean diameter of 193 ⫾ 19 ␮m (SD) for the
control group and 177 ⫾ 15 ␮m (SD) for the epistatic
circler. Similar values were obtained for the posterior
semicircular canal (control: 186 ⫾ 21 ␮m [SD]; epistatic
circler: 185 ⫾ 5 ␮m [SD]). The mean diameter for the
lateral semicircular canal in the control group amounted
to 179 ⫾ 14 ␮m (SD), whereas in the epistatic circler group
a mean value of 14 ⫾ 22 (SD) was observed. It should also
be noted that the latter value includes a number of cases
(n ⫽ 4) where this canal was completely interrupted (Fig. 4).
After microtomography and histological processing, conventional midmodiolar sections were cut from the decalcified paraffin-embedded inner ears (Fig. 5a– c). The preceding CT procedure did not seem to have affected the microscopic structure. No differences could be noted between
hematoxylin eosin-stained paraffin sections of the decalcified samples scanned in the tomograph compared to
samples not previously scanned. Abnormalities at the lateral semicircular canals of the epistatic circler mice were
also visible on the histological sections. Figure 5c shows
the same narrowing of the left lateral semicircular canal
previously depicted in Figures 2f and 3c,d. The orientation
of the serial sections is orthogonal to that of the tomographic images, explaining the different appearance of
Figures 2f and 5c.
DISCUSSION
Mouse models of inner ear diseases have proven a very
valuable tool in otovestibular research. Genetic and morphological research based on animal models with inner
ear abnormalities not only greatly contributes to a better
understanding of inner ear ontogenesis and physiology
HIGH RESOLUTION IMAGING WITH MICROTOMOGRAPHY
Fig. 3. a: 3D reconstruction of the bony labyrinth (left inner ear of an
epistatic circler mouse). b: 3D image (to be viewed with conventional 3D
red/green spectacles), of the same 3D reconstruction as a. c,d: 3D
reconstruction of the left inner ear of another epistatic circler. The
restricted diameter of the lateral semicircular canal (CSL) is clearly visible
(red arrow). White arrows indicate normal dimensions of the anterior
semicircular canal (CSA) and the posterior semicircular canal (CSP).
233
(Same specimen as depicted in Fig. 2 and 4.) AOA, ampulla ossea
anterior; AOL, ampulla ossea lateralis; AOP, ampulla ossea posterior;
COC, crus osseum commune; COCH, cochlea; COS, crus osseum
simplex; CSA, canalis semicircularis anterior; CSL, canalis semicircularis
lateralis; CSP, canalis semicircularis posterior; FC, fenestra cochleae
(rotunda); FV, fenestra vestibuli (ovale); LSS, lamina spiralis secundaria;
VEST, vestibulum.
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VAN SPAENDONCK ET AL.
Fig. 4. Diagram representing the mean diameters of the semicircular
canals of the control (SWR/J) and epistatic circler mice. Error bars refer
to standard errors. Statistically significant differences (P ⫽ 0.002) are
marked by an asterisk.
but also provides insight into pathophysiological processes
in hereditary and non-hereditary inner ear diseases in
humans (Probst et al., 1998; Vahava et al., 1998). These
mouse models with inner ear defects (pre-existing mutants as well as transgenic and knockout mice) are numerous, and abnormalities vary from severe otovestibular
dysmorphogenesis to developmental or degenerative (sub)
cellular abnormalities (Deol, 1980). Anatomical as well as
functional abnormalities caused by mutations often vary
considerably across animals and may be asymmetric between the ears in one individual animal. It is therefore
necessary to investigate the complete bony and membraneous labyrinths of several mice before any conclusions
can be drawn (Bohne and Harding, 1997). Investigations
defining the morphology of the overall labyrinth are thus
complementary to other methods demonstrating (sub)cellular morphology or function.
Severe dysmorphogenesis can be detected by close inspection of the temporal bone, for instance, by means of
transillumination. This technique however does not provide sufficient detail and photographs obtained in this
way are difficult to interpret. For animal studies on embryologic development paint-filling of the membraneous
labyrinths can be performed (Martin and Swanson, 1994).
This method renders the morphological features of the
developing labyrinth but fails to preserve the neuro-epithelial tissues.
Three-dimensional reconstruction is generally considered to be the most indicated way to examine the complete
structure of the inner ear. Minute abnormalities that cannot be detected by two-dimensional investigation alone
can be revealed because functionally relevant spatial relationships of the distinct inner ear components can be
evaluated more accurately (Isono et al., 1997). Such a
detailed 3D examination is of utmost importance in case of
dysmorphogenesis caused by gene defects.
The classical way to investigate the complete labyrinth
in the mouse consists of histological serial sectioning after
decalcification. However, 3D reconstructions based on
classical serial sections are very time-consuming and are
hampered by practical difficulties such as loss of or damage to the slices. In addition a proper alignment of consecutive slices is difficult and this serial sectioning obviously limits the possibilities for further ultrastructural
and molecular biological research on the same tissue.
Three-dimensional reconstruction based on imaging
techniques such as CT and MR scanning constitutes
a rapid and convenient way to study the morphology of the
labyrinth. Medical imaging is a rapidly developing field in
which improving techniques and resolutions have contributed to a lot of progress over the last years (Valvassori,
1994; Casselman, 1996). Clinical CT and MR imaging
with 3D reconstructions of human inner ears have already
resulted in clinical implications (Reisser et al., 1996; Isono
et al., 1997). However, the resolution of clinically used CT
and MRI equipment is insufficient to study the inner ear
structure of small laboratory animals such as mice. Recently, new imaging technologies producing much higher
resolutions have become available for laboratory purposes. Micro computer tomography (Micro CT; Sasov and
Van Dyck, 1998), magnetic resonance microscopy (MR
microscopy; Henson et al., 1994), micro positron emission
tomography (micro PET; Cherry et al., 1997), and micro
single photon emission computer tomography (micro
SPECT; Weber et al., 1994) are new tools that allow observation of both functional and morphological effects of
mutations without impeding possibilities for further research.
In this study the applicability of such a micro-CT device
as a rapid non-destructive screening tool to study the
morphology of the murine labyrinth was tested. The inner
ears from SWR/J and epistatic circler mice were scanned
at a resolution of 8 ␮m at a scanning time of 30 min, which
is still markedly less compared to micro-MR-devices (Henson et al., 1994).
Comparison of the diameter of both the anterior and
posterior semicircular canals between the control SWR/J
mice and the epistatic circler mice revealed no significant
differences. The diameter of the lateral canal in the control group was comparable to the anterior and posterior
ones. The diameter of the lateral semicircular canal of the
epistatic circler mice, however, was significantly smaller
(P ⫽ 0.002).
Stacks of detailed tomographic images of the inner ear,
which were further processed for subsequent 3D reconstructions, disclosed both subtle and marked deformations
at the level of the lateral semicircular canals of the epistatic circler mice. Light microscopical examination of histological sections of these scanned samples indicated that
this tomographic imaging procedure does not cause additional tissue artifacts and inherently does not impede
further processing for microscopical investigations.
In the future, MR microscopy and micro CT technology
will almost certainly find widespread use for high resolution morphological investigation of all kinds of organs and
tissues. Other new imaging technologies such as micro
PET and micro-SPECT reach much lower resolutions and
will be preferentially used for functional studies. The combination of these non-destructive new imaging technologies with other morphological and functional investigation
techniques will probably prove to be a very valuable tool in
inner ear research in the near future.
In conclusion, the unprecedented high resolution of this
CT-imaging method provides quick and detailed informa-
HIGH RESOLUTION IMAGING WITH MICROTOMOGRAPHY
235
Fig. 5. a– c: Histological sections (haematoxylin eosin). Magnification: 57⫻. (Same specimen as Fig. 2 and 3c,d.) a: Midmodiolar section
showing the cochlea and the vestibule containing the macula utriculi and
the macula sacculi. b: Section through the anterior semicircular canal
(CSA) (bony labyrinth) showing a normal diameter (black arrow). The
anterior semicircular duct (DSA; membranous labyrinth) is clearly visible.
c: Section showing the restricted aspect of the lateral semicircular canal
(CSL; bony labyrinth; open arrow). The lateral semicircular duct (DSL;
membranous labyrinth) is clearly discontinuous. The black arrow shows
the normal outlook of the posterior semicircular canal (CSP) and posterior semicircular duct (DSP). AOP, ampulla ossea posterior; CSA, canalis
semicircularis anterior; CSL, canalis semicircularis lateralis; CSP, canalis
semicircularis posterior; DSA, ductus semicircularis anterior; DSL, ductus semicircularis lateralis; DSP, ductus semicircularis posterior; MS,
macula sacculi; MU, macula utriculi; OC, organum spirale (Corti); SM,
scala media; ST, scala tympani; SV, scala vestibuli; VEST, vestibulum.
tion about the morphology of mouse inner ear structures
without hampering subsequent investigations. This technique allowed detection of subtle abnormalities at the
level of the semicircular canals of hereditarily impaired
mice i.e. the epistatic circler, and appears to be a very
valuable tool in inner ear research especially in view of
transgenic and knockout mouse models.
from the Flemish government) and G.V.C. holds a postdoctoral research position with the FWO.
ACKNOWLEDGMENTS
We thank Dr. D. Van Dyck and Dr. N. De Clerck for
giving access to the microtomograph; and Dr. W. Decraemer for helping with the 3D reconstructions. The technical
assistance provided by D. De Rijck, R. Van Beeck and D.
Vindevogel is also greatly acknowledged. K.C. holds a
pre-doctoral research position (financed by an IWT grant
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