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: jcm@peb.ufrj.br

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.

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