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Magnetic resonance microscopy of hamster olfactory bulbA histological correlation.

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THE ANATOMICAL RECORD 242:132-135 (1995)
Magnetic Resonance Microscopy of Hamster Olfactory Bulb:
A Histological Correlation
Institute of Biomedical Sciences, Academia Sinica, Taipei, ROC (C.C.) and Worcester
Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545 (T.J.),
Background: Magnetic Resonance Imaging (MRI) has been
widely used as a noninvasive diagnostic tool for obtaining morphological,
metabolic, and functional information from tissue. However, its potential
application in observing detailed structure comparable to that of the light
microscope has not yet been fully explored. In order to evaluate the usefulness of MR microscopy, a high resolution three-dimensional (3-D) technique was applied to observe the laminar structure of the mammalian olfactory bulb (OB).
Methods: Adult male hamsters (Mesocrecitus auratus) were used as an
animal model. Hamster OB and the attached anterior olfactory nucleus
were removed from the skull for the MRI examinations. The images were
performed with a Bruker AMX-400 system equipped with microimaging
accessories. T2 weighted 3-D spin echo sequence was used with a field of
view of 9 mm and data matrix of 128*128*128.The in-plane resolution was
70*70*70 pm. Histological preparation, including vibratome sectioning at
40 pm and Nissl staining, were used for light microscopic evaluations and
Results: Five distinct layers from the superficial to the center of the OB
were distinguished in the MR images of coronal, sagittal and horizontal
slices. As compared to the histological sections at the corresponding cutting planes, the laminar structure of the OB displayed in the MR microscopic images correlated well with its counterparts.
Conclusions: MR microscopy is capable of detecting cellular variation of
unsectioned and unstained tissue. It can also be easily applied to obtain
spatial information with good resolution. It appears to provide a great potential for diagnostic pathology. o 1995 Wiley-Liss, Inc.
Key words: Magnetic resonance imaging, MR microscopy, Hamster, 01factory bulb
The applications of the magnetic resonance imaging
(MRI) have progressed rapidly in recent years. MRI
became a n established diagnostic tool to reveal morphological information of tissues and organs for its superior soft tissue contrast in truly non-invasive and
three-dimensional (3-D) manner (Morris, 1986; Stark
and Bradley, 1992). It draws research interests for its
ability to obtain biological parameters such as oxygen
consumption, energy metabolism, and blood flow and
volume of tissues (Shulman, 1993). In addition, MR
microscopy with potential to visualize the cellular
structures gradually emerges as a major imaging technique and is expected to have a considerable impact on
biomedical sciences (Aquayo et al., 1986; Callaghan,
1991). MR microscopy has several advantages over the
conventional histological preparation, including 1) its
3-D data are able to visualize tissue slices in multiplane and -orientation, e.g., coronal, sagittal, and horizontal planes, 2) isotropic resolution, and 3) non-destructive specimen prepara5on. These competitive
edges drive the present study to focus on the feasibility
of MR microscopy by comparing the cytoarchitecture of
a biological structure revealed by MRI to that of being
processed with conventional histology procedures. In
the present study, 9.4 Tesla (T), 8.9 cm vertical-bore
superconducting magnet was used and isotropic 3-D
data acquisition was applied to evaluate the microscopic structure of the olfactory bulb (OB). The OB was
chosen to illustrate the use of MR microscopy for its
distinctively layered structure as well as its apparent
variations in the cellular organization of each layer
(Allison, 1953; Mori, 1987; Shepherd, 1990). These microscopic features provide a good pattern and criteria
to validate the sub-millimeter spatial resolution of the
Received September 22, 1994; accepted January 3, 1995.
Address reprint requests to Dr. Chen Chang, Ph.D. Institute of
Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Olfactory bulb sampling and histology preparations
Male adult Syrian hamsters (Mesocricetus aurutus)
were used. Animals were deeply anesthetized with intraperitoneal injection of sodium pentobarbital (80
mg/kg body weight) followed by perfusion through the
heart with either 10% phosphate buffered formalin, or
with the mixture of 2% paraformaldehyde and 1%formalin, at pH7.4 for 30 min. At the completion of perfusion, the brain was removed immediately and immersed in the same fixative for at least 3 additional h.
The pair of OB, including a part of the frontal lobe, was
incised coronally a t a level caudal to the anterior olfactory nucleus. The OB was then positioned rostra1
end up and glued onto a stage made of a role of
parafilm. The parafilm stage was approximately 1 cm
in height. The OB together with the parafilm stage was
inserted into a 10 mm NMR tube for further MR imaging. In the histological procedure, the animals were
perfused and their OB were removed in the same manner as for the MRI preparation. Serial sections in the
coronal, sagittal, and horizontal planes of the OB were
cut with a Vibratome a t a thickness of 40 pm and were
stained with Nissl (thionin) stain.
MR Imaging
The MR imaging experiments were performed with a
Bruker AMX-400 spectrometer (8.9 cm Oxford verticalbore superconducting magnet) equipped with microimagining accessory. Spin echo version of isotropic
Three Dimensional Fourier Transform (3DFT) pulse
sequence was used (Edelstein, et al., 1980; Suddarth
and Johnson, 19911, consisting of a hard 90" radiofrequency (rf) pulse and hard 180" refocussing rf pulse
with two phase encoding gradients between the period
of 90" and 180" rf pulses. The 3DFT isotropic volume
images were obtained by using the acquisition parameters of an echo time (TE, time between the center of
90" rf pulse and the center of echo) of 80 ms to enhance
the spin-spin relaxation effect, a repetition time of 2.5
Fig. 1. Coronal sections of the hamster OB. a: The MRI coronal slice
sec, and a field of view of 9 mm with data matrix of showing
the cytoarchitectural layers of the OB as repeated bright and
128*128*128. The resulting inplane resolution was dark bands surround the OV-PVZ. b: Nissl stained section of the OB
70*70*70 pm which is comparable to the histological at approximately the same plane as (a).
sections. A saddle type rf coil with a diameter of 10 mm
was used and its longitudinal axis parallel t o the static
magnetic field. The raw data were processed on a
Bruker X32 data station with the 3-D image display tology sections cut a t approximately the same plane.
Coronal, sagittal, and horizontal sections of the hamand analysis package.
ster OB processed with MRI and conventional histology
are illustrated in Figures 1, 2, and 3, respectively. The
The cytoarchitectural layers of the hamster OB ob- three cutting planes of the OB prepared with histologserved in the MRI slices correlated well to that of his- ical procedure (Figs. lb, 2b, and 3b) were obtained from
three animals, whileas the MRI of the three corresponding planes (Figs. l a , 2a, and 3a) were done with a
single pair of OB a t one performance of 3DFT imaging
The MRI contrast was based on the differences in
accessory olfactory bulb
external plexiform layer
spin-lattice (Tl) and spin-spin (T2) relaxation times,
glomerular layer
and proton density of water in different tissues. NuGRL
granule cell layer
clear relaxation is due to several different mechanisms
internal plexiform layer
combined mitral cell body and external plexiform layers and has been reviewed extensively in the literature
mitral cell body layer
(Bloembergen et al., 1948; Bottomley et al., 1984; EdelOB
olfactory bulb
et al., 1983; Solomon, 1955; Stark and Bradley,
olfactory nerve layer
1992). No attempt was made in the present study to
olfactory ventricle
periventricular zone
specify the density-weighted, TI weighted, or T2
Fig. 2. Sagittal sections of the hamster OB. a: MRI slice of the OB at
the sagittal plane. b: Nissl stained OB through the same plane. Note
the AOB located at the dorso-posterior region (parenthesis) in both (a)
and (b).
Fig. 3. Horizontal sections of the hamster OB. a: MRI horizontal
slice of the OB. The photograph was selected at rather dorsal aspect to
show the AOB (arrow). b Nissl stained OB at the same plane. AOB is
indicated by a n arrow.
weighted image. The acquisition parameters were chosen to highlight the best contrast among the different
organizations of the OB a t the field strength of 9.4 T.
The OB is well known for its neat layering and orderly repetitive cytoarchitecture (Mori, 1987; Shepherd, 1990). Figure l b shows the Nissl stained coronal
section of the hamster OB. From the superficial to deep
region, five layers were recognized based on their cellular organization, olfactory nerve layer (ONL), glomerular layer (GL), external plexiform layer (EPL),
mitral cell body layer (MBL), internal plexiform layer
(IPL), and granule cell layer (GRL). The center or the
innermost area of the OB was occupied by a narrow
band composed of the olfactory ventricle (OV) and
periventricular zone (PVC, Mori, 1987; Shepherd,
1990). Figures 2b and 3b are the sagittal and horizontal sections of the OB, respectively. The same laminar
arrangement was recognized in these sections. Since
the OB has greater length in both dorso-ventral and
antero-posterior axes than in the medio-lateral axis,
the OV as well as each surrounding layer in the coronal
and horizontal sections exhibited roughly a n ovalshaped profile (Figs. 1 and 3). In addition, the accessory
olfactory bulb (AOB) at the postero-dorsal region of the
OB was visible in the sagittal and horizontal section
(parentheses in Fig. 2a,b, and arrows in Fig. 3a,b).
Figures l a , 2a, and 3a are 3DFT MRI slices of the OB
in the coronal, sagittal, and horizontal planes, respectively. As compared to the histology sections, the MR
images displayed a n equally apparent layered-configuration. With the exception of the MBL and IPL, each
layer observed in the histological sections was easily
distinguished and assigned to a corresponding layer in
the MRI slices. The MBL was a single-cell layer, and
the IPL was a very thin layer of dendrites and axons
(less than 100 pm in the hamster). Other layers found
in the MRI slices of the OB appeared to be orderly
repeated bright and dark bands surround the narrow
OV and PVZ. This characteristic lamination is particularly obvious in the coronal and horizontal MRI slices
(Figs. l a and 3a). The OV and PVZ were located in the
center of the OB, and together they represented a narrow and elongated dark band in the MRI slices. The
GRL and EPL could respectively be recognized as a
bright and a dark band that surrounded the OV and
PVZ. The GL was found as the sequentially outer
bright layer, and the ONL as the outermost, bright,
and discontinued layer. With a similar layer-to-layer
corresponding assignment, each layer of the OB in the
MRI sagittal slice matched its counterpart in the histological sections very well (Fig. 2a,b). In general, the
layer with more cellular elements was represented as a
brighter area in the MRI slice. The GL and GRL possessed numerous small periglomerular cells and granule cells, respectively, and they were recognized as a
bright bands in the MRI slices. On the contrary, the
layer dominated by dendrites and axons such as
the EPL was represented by a dark band. Owing to the
postero-dorsal location of the AOB, it was shown only
in the sagittal and horizontal planes (parenthesis in
Fig. 2b and arrow in Fig. 3b). As seen in these histology
sections, the AOB also displayed lamination. The most
superficial layer of the AOB was the vomeronasal
nerve layer (VNL), immediately bellowed was the GL,
followed by the combined mitral cell and external plexiform layers (MBEPL). Deep to the MBEPL was the
IPL. The deepest cellular region was occupied by the
GRL (Mori, 1987; Shepherd, 1990). Despite the small
size, the AOB was very evident in the MRI slices as
well (parenthesis in Fig. 2a and arrow in Fig. 3a). In
the MRI sagittal and horizontal slices, the most striking feature of the AOB was the IPL. The IPL is predominantly composed of myelinated axons and therefore was represented as a very dark band in the MRI
The present study utilized rodent OB, a well-known
layered structure as a model to validate the suitability
of MR microscopy to visualize the different cellular
components of brain structure. Our data have shown
that the high resolution MRI is able to distinguish cytoarchitectural variations in unsectioned and unstained brain tissue. As compared to the histology sections of the OB, the cellular differences displayed with
MRI correlated well with that of light microscopy. Furthermore, the MR isotropic 3-D volume acquisition is
able to acquire one data set visualizing the entire sample and its serial sections a t any selected planes or
angles, and to obtain various specimen volume measurements all in one single acquisition. The same principle can be applied to other brain regions. A layer-tolayer assignment on the MRI slices of the hamster OB
in all three different cutting planes, as described in our
study, has demonstrated that the MR microscopy was
comparable to histological sections. The MRI can
“slice” the brain tissue and collect its serial section
images without involving tedious histological procedures. Most importantly, it provides images with quality to reveal sub-millimeter structure differences. Although as where detailed sub-cellular structure
differences. Although where detailed subcellular organization of a tissue is required, an autopsy using conventional histological processing with light microscopy
still is the procedure of choice to characterize pathological changes. Improvement of MR hardware and imaging processing software in the future would enhance
the resolution and availability of MR microscopy.
In conclusion,the capabilities of MR microscopy complement conventional histology and enable investigators to avoid massive animal sacrifice and time consuming tissue preparation procedures. It provides a
quick and convenient way to visualize loci of interest
buried in the brain and at the same time make morphological observations in serial sections as well as in
3-D structure.
This work is supported by a grant from the National
Science Council of the Republic of China (NSC83-0203B001-102).
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correlation, hamster, microscopy, magnetic, bulb, resonance, olfactory, histological
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