in vivo endoluminal ultrasound biomicroscopic imaging in a mouse model of colorectal cancer
TRANSCRIPT
In Vivo Endoluminal UltrasoundBiomicroscopic Imaging in a Mouse
Model of Colorectal Cancer
Kelly Z. Alves, DSc, RossanaC. Soletti, PhD,Marcelo A. P. deBritto,MD,MSc, DyannaG. deMatos, BS,Monica Soldan, MD, DSc, Helena L. Borges, PhD, Jo~ao C. Machado, PhD
Ac
FrthSc(DCde31TeanFoco
ªht
90
Rationale and Objectives: The gold-standard tool for colorectal cancer detection is colonoscopy, but it provides only mucosal surface
visualization. Ultrasound biomicroscopy allows a clear delineation of the epithelium and adjacent colonic layers. The aim of this study wasto design a system to generate endoluminal ultrasound biomicroscopic images of the mouse colon, in vivo, in an animal model of
inflammation-associated colon cancer.
Materials andMethods: Thirteenmice (Musmusculus) were used. A 40-MHzminiprobe catheter was inserted into the accessory channelof a pediatric flexible bronchofiberscope. Control mice (n = 3) and mice treated with azoxymethane and dextran sulfate sodium (n = 10)
were subjected to simultaneous endoluminal ultrasound biomicroscopy and white-light colonoscopy. The diagnosis obtained with endo-
luminal ultrasound biomicroscopy and colonoscopy was compared and confirmed by postmortem histopathology.
Results: Endoluminal ultrasound biomicroscopic images showed all layers of the normal colon and revealed lesions such as lymphoid
hyperplasias and colon tumors. Additionally, endoluminal ultrasound biomicroscopy was able to detect two cases of mucosa layer thick-
ening, confirmed by histology. Compared to histologic results, the sensitivities of endoluminal ultrasound biomicroscopy and colonoscopy
were 0.95 and 0.83, respectively, and both methods achieved specificities of 1.0.
Conclusions: Endoluminal ultrasound biomicroscopy can be used, in addition to colonoscopy, as a diagnostic method for colonic lesions.
Moreover, experimental endoluminal ultrasound biomicroscopy inmousemodels is feasible andmight be used to further develop research
on the differentiation between benign and malignant colonic diseases.
Key Words: Ultrasound biomicroscopy; animal model; diagnostic imaging; colonic neoplasm.
ªAUR, 2013
olorectal cancer (CRC) has a high incidence in the Polyps and flat lesions in the mucosa are mostly benign and
Cworld, being the third most common cancer and
the third leading cause of cancer-related mortality
in the United States, irrespective of gender (1). Ninety per-
cent of malignant tumors can be cured if diagnosed in the early
stages of localized disease (1), and this motivates great interest
in the development and design of new tools for the early
detection and staging of CRC. The gold-standard tool for
CRC detection as well as for neoplastic alterations such as pol-
yps and flat lesions in the mucosa is colonoscopy (1). How-
ever, it provides only mucosal surface visualization.
ad Radiol 2013; 20:90–98
om the Biomedical Engineering Program, COPPE (K.Z.A., R.C.S., J.C.M.),e Post-Graduation Program in Surgical Sciences, Department of Surgery,hool of Medicine (M.A.P.B., J.C.M.), the Biomedical Science Institute.G.M., H.L.B.), and the Division of Gastroenterology, Endoscopy Unit,lementino Fraga Filho University Hospital (M.S.), Federal University of RioJaneiro, Rio de Janeiro, RJ, Brazil. Received April 2, 2012; accepted July, 2012. This work was supported by the National Council for Scientific andchnological Development, by the Brazilian Federal Agency for Supportd Evaluation of Higher Education, and by the Carlos Chagas Filhoundation for Research Support of the State of Rio de Janeiro. Addressrrespondence to: J.C.M. e-mail: [email protected]
AUR, 2013tp://dx.doi.org/10.1016/j.acra.2012.07.013
can often be adequately resected endoscopically (2,3).
Nevertheless, differentiation from carcinomatous lesions that
invade the muscularis mucosa is paramount to provide the
correct approach (4). Regarding malignant tumors, the deter-
mination of their penetration depth through the colonic layers
is also important for accurate lesion staging and treatment
strategy (5,6). Therefore, some cases may require the
colonoscopic results to be complemented with additional
information obtained with a diagnostic technique able to
determine tumor penetration depth through the colonic
wall. In this context, the use of endoscopic ultrasonography
in the diagnosis and determination of the malignant
potential and depth of colonic lesions has been proposed by
some authors (7–11). For the rectum, endoscopic
ultrasound staging has already been a standard for several
years, along with magnetic resonance imaging (MRI) (12,13).
Imaging the gastrointestinal tract with 20-MHz ultrasound
(7,14) provides data on the correct depth of lesions through
the intestinal layers and accurately determines if tumors are
restricted to the mucosa and submucosa, with clear
delineation of the epithelium and muscularis. Such results
were compared to those obtained with magnifying
colonoscopy (15) and optical coherence tomography (OCT)
Academic Radiology, Vol 20, No 1, January 2013 HIGH-FREQUENCY ULTRASOUND IMAGING OF MOUSE COLON
(16) and demonstrated advantages regarding lesion staging,
such as a better determination of small CRC invasion depth.
Ultrasound with higher frequencies (40–50 MHz), usually
denominated ultrasound biomicroscopy, provides images of
living biologic tissues with near microscopic resolution (17).
The benefits of ultrasound biomicroscopy include resolution
with typical values of 30 mm (axial) and 72 mm (lateral) at
40 MHz (18).
The field of knowledge of endoscopic high-frequency
ultrasonography is yet to be fully developed, and a reproduci-
ble and feasible animal model has the power to further develop
the potential for the technique. CRC mouse models (19,20)
can be used to understand the pathogenic mechanisms and
to establish therapeutic and preventive measures related to
human chronic intestinal inflammation and colon cancer.
Additionally, important advantages of mouse models include
relatively easy breeding and the possibility of using
syngeneic mouse strains.
Despite the great number of investigations related to
high-frequency ultrasonic imaging in small animal models
(17,21–24), very few ultrasound biomicroscopic (UBM)
images acquired in vivo from the murine colon have been
presented in the literature. Chiou et al (25) used a 20-MHz
ultrasound intravascular miniprobe, connected to a standard
echocardiographic system, for transrectal assessment of the
mouse aorta and iliac arteries. The ultrasonic images presented
by those investigators do not clearly elucidate a detailed colon
wall, and the reason seems to involve the low ultrasonic fre-
quency and corresponding insufficient resolution to image
the colon. Alternatively, our research group has used sector-
scan UBM instrumentation, operating at 45 MHz, for
in vitro UBM imaging of the dissected mouse colon (26).
The results obtained by Alves et al (26) demonstrated the fea-
sibility of ultrasound biomicroscopy to identify the layers of
the colon from mice with adequate contrast among them
and with enough resolution.
The present work was motivated by the previous investiga-
tion carried out by Alves et al (26) and includes in vivo mouse
colon imaging with endoluminal examination as the main
novelty. Endoluminal ultrasound biomicroscopy, based on
an ultrasound miniprobe catheter inserted into the accessory
channel of a bronchofiberscope, was associated with colono-
scopy to generate simultaneous colon images from a mouse
model of CRC in vivo.
MATERIALS AND METHODS
Animals
The animals weremaintained at room temperaturewith appro-
priate circadian cycle and diet. TheGuide for the Care and Use of
Laboratory Animals of the National Institutes of Health was also
considered. The procedure to induce colon tumor was con-
ductedunder a protocol (DAHEICB042) approved by theAni-
mal Care and Use Committee of the Biological Science
Institute/Federal University of Rio de Janeiro. Studies involv-
ing colon imaging, with endoluminal ultrasound biomicro-
scopy associated with colonoscopy, were conducted under a
protocol (71/08) approved by the Ethics Committee for Labo-
ratory Animal Research/Federal University of Rio de Janeiro.
Thirteen mice (Mus musculus), three females and 10 males,
p53+/+ and p53+/� (heterozygous for tumor suppressor gene
TrP53), with an average age of 51.19 � 9.24 weeks and an
average weight of 24� 4 g, were used. The animals were orig-
inally purchased from The Jackson Laboratory (Bar Harbor,
ME), kept in 129/SvJ background, and genotyped as
described in Jacks et al (27).
Ten animals were treated for tumor induction, and the
other three (untreated males) were used as negative controls.
Mouse TrP53mutations, in combination with specific gene
mutations, accelerate tumorigenesis in several tissues (28),
including colon cancers (29). In addition, p53+/� mice have
increased susceptibility, relative to control strains, to the rapid
development of neoplasia by mutagenic carcinogens (30).
Azoxymethane (AOM) and Dextran Sulfate Sodium(DSS) Carcinogenesis Protocol
AOM and DSS were used to induce colon tumors in the mice
(31). AOM, a colon-specific carcinogen, associated with DSS,
a mucosal-irritant agent, mimics an inflammation-associated
colon carcinogenesis (31). The animals were subjected to a
single intraperitoneal injection of AOM (A5486; Sigma
Aldrich, St Louis, MO) at a concentration of 12.5 mg/kg.
One week following AOM administration, the mice were
fed with water containing 3% DSS salt, 36,000 to 50,000
Da (02160110; Sigma Aldrich), during 1 week. All animals
received solid food and water ad libitum, with regular water
given after DSS intake. Water consumption was monitored
and found to be similar among all mice.
Endoluminal UBM (eUBM) System
The eUBM imaging system used in the present work func-
tions as a conventional B-mode imaging instrument used
for medical diagnosis. The main difference is the higher ultra-
sound frequency used with the eUBM system.
A 3.6-F, 40-MHz miniprobe catheter (Atlantis SR Pro
Coronary Imaging Catheter; Boston Scientific Corporation,
Natick, MA), designed for intravascular imaging, was used
in the present work to transmit and receive ultrasonic pulses.
The miniprobe consists of two main assemblies: the imaging
core and the catheter body. The imaging core contains a
radial-looking 40-MHz ultrasonic transducer at the distal
tip. The catheter body is formed by the telescoping, the prox-
imal single, and the distal luminal sections. These luminal sec-
tions constitute the catheter’s working length (135 cm), with
outer diameters of 1.18 mm (3.6 F) and 0.83 mm (2.5 F) for
the proximal and distal single luminal sections, respectively.
The miniprobe imaging core was mechanically driven by a
motor-drive unit (MD5; Boston Scientific Corporation) and
rotated 360� around its axis, inside the catheter body, to
91
ALVES ET AL Academic Radiology, Vol 20, No 1, January 2013
provide ultrasound images from a plane region scanned circu-
larly and perpendicular to the probe axis. The motor-drive
unit also contained the front-end electronics to excite the
ultrasonic transducer and to amplify the receiving echoes
that were band-pass filtered (25–70 MHz), digitized by an
8-bit analog-to-digital digitizing board (sampling frequency,
250 MHz) installed in a microcomputer. The digitized echoes
were processed by the microcomputer that performed loga-
rithmic compression, scan conversion, and image storage
plus display on a video monitor. Each image framewas formed
from 256 scan A-lines and displayed at a rate of 3.8 frames/s.
According to the miniprobe catheter manufacturer, it emits an
ultrasonic pulse with duration of 58 ns, which grants the
eUBM images range resolution on the order of 40 mm (32).
All parts of the eUBM system, except the miniprobe and
the motor-drive unit, were designed and implemented in
our laboratory by our research group.
Figure 1. Cross-sectional view of the endoscope tip containing the
ultrasonic (US) miniprobe (US transducer and the catheter) inserted
into the accessory channel of a pediatric flexible bronchofiberscope.The light channel and the objective lens are also seen.
Simultaneous eUBM and Endoscopic ImageAcquisition
Endoluminal ultrasound biomicroscopy was performed simul-
taneously with white-light colonoscopy, with the ultrasound
miniprobe inserted into the accessory channel of a pediatric
flexible bronchofiberscope (FB120P; Fujinon, Tokyo, Japan).
The bronchofiberscope has a total length of 920 mm and outer
diameters of 2.8 and 2.7mm for the flexible and distal-end por-
tions, respectively. Its accessory channel is 1.2 mm in diameter.
The miniprobe manufacturer suggests the use of a catheter
guide with an internal diameter of 1.63 mm, which is larger
than the bronchofiberscope accessory channel diameter. To
preclude this limitation, the miniprobe catheter luminal sec-
tions were removed, and solely the miniprobe imaging core
(diameter, 0.5 mm) was introduced into the bronchofiber-
scope accessory channel. A small piece of the catheter luminal
proximal section was introduced backward into the accessory
channel distal extremity and involving the tip of the minip-
robe imaging core to prevent it from spinning.
The miniprobe telescoping shaft section was used to
advance and retract the imaging core, allowing the ultrasonic
transducer, at the imaging core tip, to be placed outside the
distal-end accessory channel extremity and still as close as pos-
sible to the bronchofiberscope extremity (Fig 1). Therefore,
the regions of interest for endoluminal ultrasound biomicro-
scopy and colonoscopy were coincident.
During image acquisition, the animal was anesthetized with
isoflurane (Crist�alia, S~ao Paulo, Brazil) at 1.5% in 1.5 L/min
oxygen, using a laboratory animal anesthesia system (EZ-
7000; Euthanex, Palmer, PA). The animals were tape secured
in a supine position over a mouse/rat stainless steel heated sur-
gical waterbed kept at 37�C using the T/Pump System
(Gaymar, Orchard Park, NY).
With the animal positioned, a clyster was performed with 1
mL of water. Subsequently, the flexible bronchofiberscope
containing the ultrasound miniprobe catheter was introduced,
through the anus, into the descending colon. Finally, eBUM
92
and colonoscopic images were captured simultaneously and
stored every time a lesion was detected by colonoscopy or
the eUBM image revealed a modified colon wall anatomy.
During the procedure, the colon was irrigated with water,
injected through a flush port of the miniprobe catheter, to
act as the ultrasound coupling medium between the trans-
ducer and the colon wall and to avoid the presence of air bub-
bles or feces in the investigated area. The degree of abdominal
distention was visually monitored to avoid excessive colon
insufflation, to prevent respiratory distress. The waterbed
was kept tilted, keeping the mouse’s head elevated, to allow
air bubbles inside the descending colon to move upward
and away from the bronchofiberscope extremity.
Histologic Analysis
Once the imaging acquisition was complete, the anesthetized
mousewas euthanizedbycervical dislocation, and thedistal colon
was excised, cleaned, and fixed in 4% formaldehyde during 16
hours for paraffin wax embedding. The paraffin-embedded tis-
sues were cross-sectioned (5 mm) stepwise transversally to the
colon’s longitudinal axis and stainedwith hematoxylin and eosin.
All stained sections of treated animalswere analyzed using light
microscopy and compared to the ultrasonic imageswhose frames
were obtained from the same lesions observed with endoluminal
ultrasound biomicroscopy and/or white-light colonoscopy.
RESULTS
All 10 animals treated with AOM and DSS had their colons
inspected simultaneously using endoluminal ultrasound bio-
microscopy and colonoscopy, in vivo. For every epithelial
Figure 2. Endoluminal ultrasound biomicroscopic (eUBM) (left) and colonoscopic (center) images obtained simultaneously in vivo from a
healthy portion of a mouse colon and the corresponding hematoxylin and eosin–stained histologic section (right) (40 � magnification). TheeUBM image displays the ultrasound catheter miniprobe (Mp) at the center of the lumen and moving away from the miniprobe the hyperechoic
mucosa (Mu) layer, a second hypoechoic layer corresponding to themuscularismucosae (Mm), and a third hyperechoic layer, submucosa (Sm),
followed by the forth hypoechoic muscularis externa (Me) layer. The endoscopic image reveals a clean lumen and the miniprobe tip at the top.
The layers identified in the ultrasound image are well correlated with histology.
Academic Radiology, Vol 20, No 1, January 2013 HIGH-FREQUENCY ULTRASOUND IMAGING OF MOUSE COLON
anatomic change detected with the two techniques, as well as
the anatomic changes inside the colon wall detected by endo-
luminal ultrasound biomicroscopy, images were acquired and
compared to the corresponding histologic specimens obtained
from the same lesion sites. A perfect match between eUBM
images and corresponding histologic images was completely
impossible. It is very difficult to match both eUBM and histo-
logic image planes, and in addition, tissue detachment may
occur during microtome cutting and cause morphologic
changes in the sliced colon.
Figure 2 depicts an example of the cross-sectional eUBMand
endoscopic images acquired simultaneously from a healthy por-
tion of a mouse colon and the corresponding histology. The
center of the lumen is occupied by the ultrasound miniprobe,
represented by a gray circle that is surrounded by a bright area
corresponding to the ultrasonic pulses multireflected between
the transducer and the catheter wall. Moving away from the
miniprobe, the first and most superficial hyperechoic circular
layer is the mucosa, followed by the second hypoechoic layer
corresponding to the muscularis mucosae. The third hypere-
choic layer is the submucosa, followed by the muscularis
externa, which is the fourth hypoechoic circular layer. The
endoscopic image reveals a clean lumen and the miniprobe
tip at 2 o’clock. The folds and the four layers displayed in the
ultrasound image are identified in the histologic specimen.
An eUBM image of a lymphoid hyperplasia lesion, repre-
sented by a hypoechoic region between the mucosa and mus-
cularis mucosae layers, in the colon of one animal is presented
in Figure 3 with the corresponding histologic image. In this
case, colonoscopy was unable to detect this lesion, because
it was located at the submucosa.
Two examples of interrelated eUBM, endoscopic, and his-
tologic images are presented for a single tumor (Fig 4) and two
synchronic ones (Fig 5). These tumors, confirmed by histol-
ogy as an adenocarcinoma (the single tumor) and adenomas
(the synchronic tumors), have their interfaces with surround-
ing tissues outlined in the colonoscopic image. The ultrasonic
miniprobe tip is observed at 6:30 (Fig 4) and at 12 o’clock (Fig
5) in the endoscopic images.
An example of a colonic region containing amucosal thick-
ened area is displayed in Figure 6. The thickened mucosa,
undetected during colonoscopic examination, was always
clearly seen on the eUBM images.
The findings obtained with endoluminal ultrasound biomi-
croscopy and colonoscopy are presented in Table 1, together
with the corresponding histologic results. Regarding the
whole group of animals, most lesions were adenomas or lym-
phoid hyperplasias.
All animals had at least one colon lesion, denoted in Table 1
as Li-j, in which indexes i and j represent the lesion and animal
numbers, respectively. Lesion L1-7 was undetected by colono-
scopy, because of fecal material in the lumen during the
examination. With the exception of only one lesion, all
remaining lesions were detected by endoluminal ultrasound
biomicroscopy.
Concerning eUBM and colonoscopic examinations of the
distal colon from negative control animals, none revealed any
false-positive result if findings such as lymphoid hyperplasia,
tumor, or thickened mucosa were considered.
Eighteen representative stained sections, six from each
extremity and six from the middle part of distal colon, from
each negative control animal were analyzed by light
93
Figure 3. Endoluminal ultrasound biomi-
croscopic (eUBM) image (left) obtained
in vivo and the corresponding hematoxylinand eosin–stained histologic section (right)
(40 � magnification) of a mouse colon con-
taining a lymphoid hyperplasia in the colonic
wall. The eUBM image displays the ultra-sound catheter miniprobe (Mp), the hypere-
choic mucosa (Mu) layer, a hypoechoic
layer corresponding to the muscularis mu-
cosae (Mm), the hyperechoic submucosalayer (Sm), and a hypoechoic lymphoid hy-
perplasia (Lh) lesion. Both colonic layers
and the lymphoid hyperplasia are clearlyseen on the histologic image, which includes
the muscularis externa (Me) layer.
Figure 4. Endoluminal ultrasound biomicroscopic (eUBM) (left) and colonoscopic (center) images obtained simultaneously in vivo from a
mouse colon containing a tumor (Tu) and the corresponding hematoxylin and eosin–stained histologic section (right) (40 � magnification).
The endoscopic image reveals a large protruded lesion. The eUBM image displays the ultrasound catheter miniprobe (Mp) at the center ofthe lumen and the hyperechoic mucosa (Mu) and hypoechoic muscularis externa (Me) layers. The tumor boundaries are outlined at the endo-
scopic image, and the miniprobe tip is at the bottom. The tumor identified in the eUBM image is well correlated with the histologic image.
ALVES ET AL Academic Radiology, Vol 20, No 1, January 2013
microscopy, and the outcomes (a total of 54) were considered
as the true-negative results for histology.
The data on lesion detection from treated (Table 1) and
untreated animals reveal sensitivity of 0.95 (18 of 19) and spe-
cificity of 1.0 for endoluminal ultrasound biomicroscopy and
sensitivity of 0.83 (15 of 18) and specificity of 1.0 for colono-
scopy. Lesion L1-7 was not considered in the sensitivity calcu-
lation for colonoscopy, because it was impossible to examine
the colon because of stool.
DISCUSSION
The endoscopic procedure to collect eUBM and endoscopic
colon images simultaneously was undertaken with relative
ease, keeping the mice anesthetized under gas inhalation.
94
The resulting eUBM images resemble those already
obtained by our group from the mouse colon (26), in vitro,
and using a UBM system operating at 45 MHz. The ultra-
sound images obtained by Alves et al (26) have superior quality
compared to those in this present work, mainly because of
transducer specification differences, with a spherical focus
and a larger aperture (5 mm) in the previous study compared
to no focus and a smaller aperture (0.5 mm) in the present
work. The transducer with reduced size was necessary to be
inserted into the endoscope’s accessory channel.
Like the ultrasound images in Alves et al (26), the eUBM
images in the present work also present the morphologic
details of the normal colon, including the mucosa, muscularis
mucosae, submucosa, and muscularis externa layers (Fig 2).
The eUBM images also reveal lymphoid hyperplasia (Fig 3)
Figure 5. Endoluminal ultrasound biomicroscopic (eUBM) (left) and colonoscopic (center) images obtained simultaneously in vivo from amouse colon containing two synchronic tumors (Tu) and the corresponding hematoxylin and eosin–stained histologic section (right) (40�mag-
nification). The eUBM image displays the ultrasound catheter miniprobe (Mp) at the center of the lumen and the hyperechoicmucosa layer (Mu).
The endoscopic image reveals the two synchronic protruded tumors with outlined boundaries and the miniprobe tip at the top. The two tumors
are seen on the histologic examination from the same colonic site.
Figure 6. Endoluminal ultrasound biomi-
croscopic (eUBM) image (left) obtainedin vivo and the corresponding hematoxylin
and eosin–stained histologic section (right)
(40 � magnification) of a mouse colon con-taining a mucosal thickened area. The
eUBM image displays the ultrasound cathe-
ter miniprobe (Mp), the hyperechoic mucosa
layer (Mu), a hypoechoic layer correspond-ing to the muscularis mucosae (Mm), and
the hypoechoic muscularis externa (Me)
layer. Both colonic layers and the mucosal
thickness are clearly seen on the histologicimage.
Academic Radiology, Vol 20, No 1, January 2013 HIGH-FREQUENCY ULTRASOUND IMAGING OF MOUSE COLON
and colon tumor lesions as well as tumoral invasion through
the colon (Figs 4 and 5). The eUBM system was able to detect
lesions with maximal transverse diameters as small as 0.25 mm.
According to the data in Table 1, endoluminal ultrasound
biomicroscopy was able to detect 18 of 19 findings confirmed
by histopathologic analysis as lymphoid hyperplasia (eight
cases), tumor (nine cases), or thickened mucosa (two cases).
Only endoluminal ultrasound biomicroscopy was able to
detect the two cases of mucosa layer thickening, confirmed
by histology. Seven of 10 animals had colon tumors, most of
them diagnosed as adenomas and depicted as hyperechoic
regions in the eUBM images.
The number of animals with colon tumors (70%) was about
the same as previously reported in a study (33) using AOM
and DSS to induce colon tumors following a protocol quite
close to the one in our work. Although AOM is widely
used to induce CRC in rodents, tumor incidence varies
from 0% to 100% (34). The effectiveness on colon tumor inci-
dence in animals treated with AOM depends on the carcino-
gen dosage, duration, and frequency, as well as on the routing
and timing of administration. In addition, the effectiveness of
AOM also depends on sex, age, species, and animal strain (35).
From the data in Table 1, the histologic findings reveal that
lymphoid hyperplasia also developed in seven of 10 animals.
This represents the normal response of lymphoid tissue to var-
ious stimuli, commonly observed in a number of clinical situa-
tions and without any pathologic significance (36,37).
With the constant development of minimally invasive tech-
niques, both surgical and endoscopic, for the treatment of
colonic lesions in patients, local tumor staging has proven to
95
TABLE 1. Mouse Colon Findings Detected Simultaneously by Endoluminal Ultrasound Biomicroscopy and Colonoscopy and theCorresponding Histologic Diagnosis
Animal Lesion
Findings
Endoluminal Ultrasound Biomicroscopy Colonoscopy Histology
Yes No Yes No
Lymphoid
Hyperplasia Tumor
Thickened
Mucosa
1 L1-1 U U U
1 L2-1 U U U
2 L1-2 U U U
2 L2-2 U U U
2 L3-2 U U U
3 L1-3 U U U
4 L1-4 U U U
4 L2-4 U U U
5 L1-5 U U U
5 L2-5 U U U
6 L1-6 U U U
7 L1-7 U * * U
7 L2-7 U U U
7 L3-7 U U U
8 L1-8 U U U
9 L1-9 U U U
10 L1-10 U U U
10 L2-10 U U U
10 L3-10 U U U
*Impossible to examine because of stool.
ALVES ET AL Academic Radiology, Vol 20, No 1, January 2013
be relevant in the past few years (8,9,38). In this sense,
diagnostic methods for the prediction of malignancy have
been developed to differentiate benign and superficial
neoplasias from invasive and malignant lesions. The main
techniques discussed in the literature are magnifying
colonoscopy and endoscopic ultrasound (10,15), which
provide complementary information concerning the
decision for local versus surgical therapy.
Through the advent of mouse models to simulate or at least
provide plausible pathophysiologic mechanisms of CRC in
human, tumor sizing techniques based on micro–computed
tomographic colonography (37), high-resolution chromoen-
doscopy (39), and endoscopy (33) have been tested. The effort
made to size CRC tumors in mice is supported by the direct
correlation of polyp size with human colon cancer, as
adenomatous polyps with high-grade dysplasia, villous histol-
ogy, and sizes >1 cm have greater malignant potential.
In addition, other imaging modalities clinically available
and already used to investigate colon diseases in rodents
include OCT (40), MRI (41), single-photon emission com-
puted tomography (SPECT) (42), SPECT combined with
x-ray computed tomography (microSPECT/CT) (43), and
near-infrared imaging (44). Although colonoscopy presents
images with excellent resolution and is considered a gold
standard, it is a technique limited to mucosal surface visualiza-
tion. On the other hand, OCT has excellent resolution, about
3.2 and 4.4 mm for axial and lateral resolution, respectively,
while maximum tissue penetration is kept on the order of 1
96
mm. Regarding MRI, its resolution is on the order of 100
mm, and improvements to make its resolution <100 mm are
possible if cardiac gating and respiratory synchronization are
implemented. Besides being noninvasive techniques, OCT
and MRI also use no ionizing radiation. The other imaging
techniques, such as SPECT, micro–computed tomographic
colonography, and microSPECT/CT, do not offer image res-
olution comparable to that of OCT, and they use ionizing
radiation. Nevertheless, these three imaging techniques allow
full organ imaging, and in addition, SPECT and micro-
SPECT/CT provide molecular imaging. In comparison to
the mentioned imaging technologies for small animals, endo-
luminal ultrasound biomicroscopy has an intermediate resolu-
tion between that obtained with MRI and OCT and the
advantages of not using ionizing radiation, low cost, rapid
imaging speed of the whole colon cross-section, and
portability.
The outcomes of clinical applications of ultrasound and
colonoscopy are operator dependent (45,46), and with these
diagnostic tools applied experimentally, as in the present
work, there is no reason to believe that it would be
different. However, improvements may occur when
ultrasound is combined with other diagnostic techniques,
such as the joint diagnostic yield of mammography and
ultrasound, which has been shown to be greater than that of
mammography alone (47). When performing simultaneous
endoluminal ultrasound biomicroscopy and colonoscopy,
the ultrasound images may help the operator diagnose a simple
Academic Radiology, Vol 20, No 1, January 2013 HIGH-FREQUENCY ULTRASOUND IMAGING OF MOUSE COLON
elevation in the mucosa layer, which can represent a lymphoid
or a mucosal hyperplasia, from tumoral lesions. This is the case
pictured in the eUBM images (Figs 3–5), in which the
lymphoid hyperplasia is a hypoechoic region surrounded at
the top by a hyperechoic layer representing the mucosa.
Regarding tumors, they are presented as hyperechoic areas
with a lack of normal epithelial colon structure. The eUBM
visualization of the mucosa layer may become a
differentiation between nontumoral and tumoral lesions and
aid in treatment decision making and in clinical evaluation.
Also, the characteristic echogenicity of each lesion type may
be used to distinguish tumoral from nontumoral colon
lesions through eUBM images. In addition, normal (Fig 2)
and thickened (Fig 6) mucosa can be easily distinguished on
eUBM images. Simultaneous acquisition of eUBM and colo-
noscopic images of mouse colon may improve the measure-
ment of tumor and flat lesion sizes and also may enable the
determination of lesion penetration depth through the colon
wall, which is important for tumor staging.
Therefore, eUBM images allow the operator to analyze the
morphology of colonic layers and contribute to decrease the
operator-dependent diagnosis of colonoscopy when this
technique is combined with endoluminal ultrasound
biomicroscopy.
The instrumentation used in the present study has some
limitations that should be overcome to improve the quality
of the results. In this context, better colonoscopic results
would be obtained if the flexible bronchofiberscope were
replaced by a video endoscope. This depends of future tech-
nological developments to provide a flexible video endoscope
with an external diameter close to 2.5 mm and an accessory
channel with adequate diameter to pass the ultrasound minip-
robe. In addition, a proper match between the ultrasound
miniprobe and endoscope accessory channel diameters would
facilitate the handling of the miniprobe, avoiding removal of
the catheter luminal sections to allow the sole introduction
of the miniprobe imaging core into the endoscope accessory
channel. Keeping the miniprobe intact will guarantee more
protection for this delicate part of the instrumentation.
Future improvements of the proposed approach may
include three-dimensional visualization of colon lesions,
such as in thework of Kim et al (48), who used an intravascular
ultrasound probe, similar to the one in the present work, com-
bined with automatic positioning and localization of the
probe tip using the method proposed by Conversano et al
(49) to provide catheter self-localization with respect to
selected anatomic structures. In addition, ‘‘real-time virtual
biopsy’’ could be obtained with ultrasound contrast agent, tar-
geted to vascular endothelial growth factor receptor–2,
coupled to an eUBM system to form a molecular imaging
technique to diagnose, stage, and monitor colon tumor mor-
phology and vasculature in a CRC mouse model.
One major fact that withholds scientific development in
clinical areas is the lack of an experimental model, preferably
in vivo, to test novel techniques and interventions. The
eUBM technique used to visualize mouse colon tumors
in vivo adds more diagnostic information to existing methods,
and the eUBM technique may also be applied to distinct areas
of animal models of colon inflammatory and ischemic dis-
eases. The results so far obtained in the present work are pre-
liminary, and more research must be conducted, including
horizontal studies of tumor, inflammatory, and ischemic dis-
ease development in the colon. These future studies should
focus, for instance, on the early detection of malignant tumors
of the colon to form a basis for clinical translation of the pro-
posed approach.
CONCLUSIONS
The results obtained in the present work suggest that endolu-
minal ultrasound biomicroscopy could be used, in addition to
colonoscopy, as a diagnostic method for colonic lesions.
Moreover, experimental studies using endoluminal high-
resolution ultrasound methods in the mouse are feasible and
might be used to further develop research on the differentia-
tion between colonic benign and malignant diseases.
REFERENCES
1. American Cancer Society. Colorectal Cancer Facts & Figures 2011-2013.
Atlanta, GA: American Cancer Society, 2011.
2. Hurlstone DP, Sanders DS, Atkinson R, et al. Endoscopic mucosal
resection for flat neoplasia in chronic ulcerative colitis: can we
change the endoscopic management paradigm? Gut 2007; 56:
838–846.
3. Lim TR, Mahesh V, Singh S, et al. Endoscopic mucosal resection of color-
ectal polyps in typical UK hospitals. World J Gastroenterol 2010; 16:
5324–5328.
4. Atkinson RJ, Shorthouse AJ, Hurlstone DP. Novel colorectal endoscopic
in vivo imaging and resection practice: a short practice guide for interven-
tional endoscopists. Tech Coloproctol 2007; 11:7–16.
5. Greene FL, Stewart AK, Norton HJ. A new TNM staging strategy for node-
positive (stage III) colon cancer: an analysis of 50,042 patients. Ann Surg
2002; 236:416–421.
6. Puppa G, Sonzogni A, Colombari R, et al. TNM staging system of colorec-
tal carcinoma: a critical appraisal of challenging issues. Arch Pathol Lab
Med 2010; 134:837–852.
7. Waxman I, Saitoh Y, Raju GS, et al. High-frequency probe EUS-
assisted endoscopic mucosal resection: a therapeutic strategy for
submucosal tumors of the GI tract. Gastrointest Endosc 2002; 55:
44–49.
8. Stergiou N, Haji-Kermani N, Schneider C, et al. Staging of colonic neo-
plasms by colonoscopic miniprobe ultrasonography. Int J Colorectal Dis
2003; 18:445–449.
9. Hunerbein M, Handke T, Ulmer C, et al. Impact of miniprobe ultrasonogra-
phy on planning of minimally invasive surgery for gastric and colonic tu-
mors. Surg Endosc 2004; 18:601–605.
10. Hurlstone DP, Brown S, Cross SS, et al. High magnification chromoscopic
colonoscopy or high frequency 20MHz mini probe endoscopic ultrasound
staging for early colorectal neoplasia: a comparative prospective analysis.
Gut 2005; 54:1585–1589.
11. Nguyen-Tang T, Shah JN, Sanchez-Yague A, et al. Use of the front-view
forward-array echoendoscope to evaluate right colonic subepithelial le-
sions. Gastrointest Endosc 2010; 72:606–610.
12. Kwok H, Bissett IP, Hill GL. Preoperative staging of rectal cancer. Int J Col-
orectal Dis 2000; 15:9–20.
13. Schizas AM, Williams AB, Meenan J. Endosonographic staging of lower
intestinal malignancy. Best Pract Res Clin Gastroenterol 2009; 23:
663–670.
14. Hurlstone DP, Cross SS, Sanders DS. 20-MHz high-frequency endoscopic
ultrasound-assisted endoscopic mucosal resection for colorectal submu-
cosal lesions: a prospective analysis. J Clin Gastroenterol 2005; 39:
596–599.
97
ALVES ET AL Academic Radiology, Vol 20, No 1, January 2013
15. Matsumoto T, Hizawa K, Esaki M, et al. Comparison of EUS and magnify-
ing colonoscopy for assessment of small colorectal cancers. Gastrointest
Endosc 2002; 56:354–360.
16. Das A, Sivak MV Jr, Chak A, et al. High-resolution endoscopic imaging of
the GI tract: a comparative study of optical coherence tomography versus
high-frequency catheter probe EUS. Gastrointest Endosc 2001; 54:
219–224.
17. Foster FS, Zhang MY, Zhou YQ, et al. A new ultrasound instrument for
in vivo microimaging of mice. Ultrasound Med Biol 2002; 8:1165–1172.
18. Foster FS, Pavlin CJ, Harasiewicz KA, et al. Advances in ultrasound biomi-
croscopy. Ultrasound Med Biol 2000; 26:1–27.
19. Corpet DE, Pierre F. Point: from animal models to prevention of colon
cancer. Systematic review of chemoprevention in min mice and choice
of the model system. Cancer Epidemiol Biomarkers Prev 2003; 12:
391–400.
20. Taketo MM, Edelmann W. Mouse models of colon cancer. Gastroenterol-
ogy 2009; 136:780–798.
21. Foster FS, Zhang MY, Duckett AS, et al. In vivo imaging of embryonic de-
velopment in mice mouse eye by ultrasound biomicroscopy. Invest Oph-
thalmol Vis Sci 2003; 44:2361–2366.
22. Jolly C, Jeanny JC, Behar-Cohen F, et al. High-resolution ultrasonography
of subretinal structure and assessment of retina degeneration in rat. Exp
Eye Res 2005; 81:592–601.
23. Martin-McNulty B, Vincelette J, Vergona R, et al. Noninvasive measure-
ment of abdominal aortic aneurysms in intact mice by a high-frequency ul-
trasound imaging system. Ultrasound Med Biol 2005; 31:745–749.
24. Xu X, Sun L, Cannata JM, et al. High-frequency ultrasound Doppler system
for biomedical applications with a 30-MHz linear array. Ultrasound Med
Biol 2008; 34:638–646.
25. Chiou AC, Chiu B, Oppat WF, et al. Transrectal ultrasound assessment of
murine aorta and iliac arteries. J Surg Res 2000; 88:193–199.
26. Alves KZ, Borges HL, Soletti RC, et al. Features of in vitro ultrasound bio-
microscopic imaging and colonoscopy for detection of colon tumor in
mice. Ultrasound Med Biol 2011; 37:2086–2095.
27. Jacks T, Remington L, Williams BO, et al. Tumor spectrum analysis in p53-
mutant mice. Curr Biol 1994; 4:1–7.
28. Sherr CJ, Weber JD. The ARF/p53 pathway. Curr Opin Genet Dev 2000;
10:94–99.
29. Borges HL, Bird J, Wasson K, et al. Tumor promotion by caspase-resistant
retinoblastoma protein. Proc Natl Acad Sci U S A 2005; 102:15587–15592.
30. French J, Storer RD, Donehower LA. The nature of the heterozygous Trp53
knockout model for identification of mutagenic carcinogens. Toxicol
Pathol 2001; 29(suppl):24–29.
31. Tanaka T. Colorectal carcinogenesis: review of human and experimental
animal studies. J Carcinog 2009; 8:1–19.
32. Cobbold RSC. Ultrasound Imaging Systems: Design, Properties, and
Applications. Toronto, ON, Canada: Oxford University Press, 2007.
492–607.
98
33. Hensley HH, Merkel CE, Chang WC, et al. Endoscopic imaging and size
estimation of colorectal adenomas in the multiple intestinal neoplasia
mouse. Gastrointest Endosc 2009; 69:742–749.
34. Kobaek-Larsen M, Thorup I, Diederichsen A, et al. Review of colorectal
cancer and its metastases in rodent models: comparative aspects with
those in humans. Comp Med 2000; 50:16–26.
35. Heijstek MW, Kranenburg O, Borel Rinkes IH. Mouse models of colorectal
cancer and liver metastases. Dig Surg 2005; 22:16–25.
36. Pearce CB, Martin H, Duncan HD, et al. Colonic lymphoid hyperplasia in
melanosis coli. Arch Pathol Lab Med 2001; 125:1110–1112.
37. Pickhardt PJ, Halberg RB, Taylor AJ, et al. Microcomputed tomography
colonography for polyp detection in an in vivo mouse tumor model. Proc
Natl Acad Sci U S A 2005; 102:3419–3422.
38. Sano Y, Iwadate M. The importance of the macroscopic classification of
colorectal neoplasms. Gastrointest Endosc Clin N Am 2010; 20:461–469.
39. Becker C, Fantini MC, Wirtz S, et al. In vivo imaging of colitis and colon
cancer development in mice using high resolution chromoendoscopy.
Gut 2005; 54:950–954.
40. Hariri LP, Qiu Z, Tumlinson AR, et al. Serial endoscopy in azoxymethane
treated mice using ultra-high resolution optical coherence tomography.
Cancer Biol Ther 2007; 6:1753–1762.
41. Ericsson AC, Myles M, Davis W, et al. Noninvasive detection of
inflammation-associated colon cancer in a mouse model. Neoplasia
2010; 12:1054–1065.
42. Shi J, Jia B, Liu Z, et al. 99mTc-labeled bombesin(7-14)NH2 with favorable
properties for SPECT imaging of colon cancer. Bioconjug Chem 2008; 19:
1170–1178.
43. Chang YJ, Chang CH, Yu CY, et al. Therapeutic efficacy andmicroSPECT/
CT imaging of 188Re-DXR-liposome in a C26 murine colon carcinoma
solid tumor model. Nucl Med Biol 2010; 37:95–104.
44. Alencar H, Funovics MA, Figueiredo J, et al. Colonic adenocarcinomas:
near-infrared microcatheter imaging of smart probes for early detec-
tion—study in mice. Radiology 2007; 244:232–238.
45. Berg WA, Blume JD, Cormack JB, et al. Operator dependence of
physician-performed whole-breast US: lesion detection and characteriza-
tion. Radiology 2006; 241:355–365.
46. Baxter NN, Sutradhar R, Forbes SS, et al. Analysis of administrative data
finds endoscopist quality measures associated with postcolonoscopy col-
orectal cancer. Gastroenterology 2011; 140:65–72.
47. Flobbe K, Bosch AM, Kessels AG, et al. The additional diagnostic value of
ultrasonography in the diagnosis of breast cancer. Arch Intern Med 2003;
163:1194–1199.
48. Kim H, Moody MR, Laing ST, et al. In vivo volumetric intravascular ultra-
sound visualization of early/inflammatory arterial atheroma using targeted
echogenic immunoliposomes. Invest Radiol 2010; 45:685–691.
49. Conversano F, Casciaro E, Franchini R, et al. A quantitative and automatic
echographic method for real-time localization of endovascular devices.
IEEE Trans Ultrason Ferroelectr Freq Control 2011; 58:2107–2117.