©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2682 | VOL.9 NO.11 | 2014 | nature protocols

IntroDuctIonColorectal cancer is a highly preventable cancer, yet it is the third most common cause of cancer-related mortality in the US1. Mouse models of colorectal cancer are important for understanding the biology of cancer progression and for testing preventive measures. Spontaneous and inducible mouse colorectal cancer models are available; however, it can be difficult to monitor adenomatous or carcinomatous growth without killing these animals or using invasive procedures such as optical endoscopy. Conventional microendoscopes are currently available and they are capable of providing high-quality images2,3. However, the major drawbacks of this technology are the risk of perforating the colon during routine endoscopy and obstruction of the image because of bleed-ing associated with colitis2. Moreover, specialized equipment and experienced operators are required to perform the endoscopy procedure properly.

Minimally invasive imaging methods for use with small animals are now available; these methods include X-ray computed tomographic (CT) and MRC, in which mouse tumors can be monitored in a manner similar to tumors in humans. Micro-CT colonography, for example, has been used in small living animals for detecting colonic tumors and estimating their volumes4–6. Although this method has demonstrated high spatial resolution, good tissue contrast and high throughput, the main concern is exposure to ionizing radiation. In mice, the high X-ray exposure required for CT colonography could lead to radiation-induced changes in the model or to cumula-tive effects on its immune system, especially in longitudinal studies requiring multiple imaging sessions7,8. These concerns have encouraged the development of MRC as a noninvasive, radiation-free imaging method. Suitable techniques for distend-ing the bowel to avoid artifacts and to enable identification of nascent polyps need to be developed.

Visualization of colon lesions in clinical MRCIn clinical MRC, two conventional methods for colon distension and improved visualization of the colon lesions are broadly used: those that induce bright signal or those that induce dark signal in the colonic lumen. In the bright lumen method, the colon is filled with a gadolinium (Gd)-water solution to create a posi-tive-contrast, high-intensity signal on T1-weighted (T1w) images and to render the polyps protruding from the colonic wall as dark structures9. This approach has some limitations in detect-ing small polyps, and it needs improvement for distinguishing the tumor masses from air bubbles or residual fecal matter. The dark lumen technique is considered a standard methodology for clinical MRC10. This technique is based on using water or gas distension, followed by i.v. contrast (Gd-chelate)-enhanced T1w MRI sequence. Although CO2 or air can be used11,12, as in CT, to insufflate the colon and to create a dark background on the MR images, the water enema remains the most commonly used method in MRC for patients10,13. Water creates various signals depending on the pulse sequence (dark on T1w and bright on T2-weighted (T2w) images). As an alternative approach to the water and gas, a fecal tagging technique based on the use of oral barium to reduce the signal intensity of fecal matter, as well as a barium enema to create a dark lumen, has been applied14. In comparison with the bright and dark lumen methods, this technique does not require bowel cleansing.

In rodents, most of the MRC studies have been performed using identifiable markers for the colon15, and the use of enemas has been uncommon16. This has hampered the accuracy of lesion detection and the estimation of the degree of inflammation, because proper bowel distention is necessary to provide sufficient contrast and to differentiate the colonic wall from the luminal contents. Although researchers have tried fecal tagging17 and

In vivo MRI virtual colonography in a mouse model of colon cancerLilia V Ileva1, Marcelino Bernardo2, Matthew R Young3, Lisa A Riffle1, James L Tatum4, Joseph D Kalen1 & Peter L Choyke5

1Small Animal Imaging Program, Laboratory Animal Sciences Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA. 2Molecular Imaging Program, Laboratory Animal Sciences Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA. 3Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland, USA. 4Cancer Imaging Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. 5Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA. Correspondence should be addressed to L.V.I. (ileval@mail.nih.gov).

Published online 23 October 2014; doi:10.1038/nprot.2014.178

We have developed a reliable noninvasive method for monitoring colonic tumors and mucosal inflammation in a mouse model of colon cancer using magnetic resonance colonography (Mrc). after a mild cleansing enema, the colon is filled with Fluorinert, a perfluorinated liquid that does not produce a proton Mr signal. the mouse is placed in a dedicated volume Mr receiver coil, and high-resolution images are acquired in three planes. the Fluorinert enema distends the mouse colon, creating an artifact-free black homogeneous background, allowing clear delineation of the inflamed colonic wall and visualization of luminal tumors in various stages of development. a gadolinium-based contrast agent can be administered i.v. to the animal to detect mural inflammation or tumor vascularity. this technique is useful for serial monitoring of the effects of preventive or therapeutic strategies on tumor development without killing the animal or requiring invasive endoscopies. the animal preparation and imaging can be completed in ~1.5 h.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.9 NO.11 | 2014 | 2683

water enema18 in small animals as well, a water enema creates a bright lumen on T2w images, making it difficult to distinguish the normal bowel wall from inflamed colonic mucosa, which also appears hyperintense.

T2w imaging is an essential component of colonic imaging, because it can be used on its own for the evaluation of bowel wall inflammation19 and as a fast screening method for providing high-throughput MRI in studying mouse colonic inflammation16. It is therefore important to develop an imaging protocol that maintains a dark lumen on both T1w and T2w imaging.

Protocol developmentTo define the most effective approach, we performed a pilot com-parison study exploring four different media for colon distention: mineral oil, air, water and Fluorinert. Some essential results of this study are illustrated in Figure 1, which presents sagittal, coronal and transverse views of the 3D T1w images acquired with dif-ferent enema media. Although the T1w gradient echo sequence demonstrates a high-intensity signal from the colon filled with mineral oil, the presence of air bubbles and fecal debris could be mistaken for colonic polyps. Moreover, the hyperintense inflamed colonic wall cannot be differentiated from the bright lumen in cases of bowel inflammation (Fig. 1a). The air-insufflated colon provides good contrast between the lumen and mucosa. However, the difference in the magnetic susceptibility between the colon wall tissue and air (having almost zero magnetic susceptibility) causes a magnetic field distortion that leads to the induction of a susceptibility artifact that is more apparent in the transverse view (Fig. 1b). Although the water creates a dark lumen, there is still residual signal arising from the colon that limits the signal-to-noise ratio (SNR) to 6.5 (Fig. 1c). As a nonhydrogenous liquid, Fluorinert provides a minimal SNR of 0.9, indicating that the signal from the lumen is approximately equal to the background of both T1w and T2w scans20 (Fig. 1d). On the basis of the results of this pilot study, we have developed a versatile methodology for MR colonography using a Fluorinert enema.

Experimental designHere we describe our experience with MRC, which should be applicable to a wide range of mouse colon disease models. We have used MRC to visualize inflammation and to follow tumor progression in a mouse model of colorectal cancer. In this model, tumorigenesis is initiated with azoxymethane (AOM) i.p. injection, and one or two cycles of dextran sodium sulfate (DSS)-induced inflammation drives tumor promotion21,22.

Before imaging, animals are deprived of food for 12 h, but they are allowed to consume water. After a mild cleansing of the colon, a tail-vein catheter is inserted, the animals are anesthe-tized and placed in a supine position in a dedicated MR receiver coil (Supplementary Fig. 1 shows the basic components of the experimental setup). One milliliter of Fluorinert is administered as an enema to distend the colon and to demarcate the colon lumen. 3D high-resolution T2w spin echo and T1w gradient

echo images are acquired on a 3-T clinical scanner. Gd chelate (0.2 mmol/kg) administered i.v. after the first T1w acquisition enhances the signal intensity of bowel wall inflammation and tumor vascularity. The described method generates artifact-free, high-resolution T1w and T2w images that clearly depict the inflammation of the colon epithelium and the tumor protrud-ing from the colon wall. In addition, the postimaging analysis yields a quantitative assessment of the degree of inflammation and colonic tumor burden. This technique can be used to assess inflammatory and neoplastic diseases in various mouse models without killing the animal.

We used the procedure described here to monitor tumor growth, and we were able to detect tumors as small as 1.2 mm3 and to measure changes in tumor size by serial imaging over 8 weeks with no adverse effects to the mice20. In our second study, we used serial MRC to visualize the attenuation of colitis in mice fed an isorhamnetin diet23. The quantitative results obtained from the MR images have been validated by ex vivo measurements of colon tissue collected just after imaging. The whole-mount H&E-stained sections of colon tissue during the acute and resolved inflammation correlated with the changes of contrast-to-noise ratio (CNR), whereas the tumor growth measured by MRI was verified with ex vivo caliper measurement of the tumor volumes20. An antiperistaltic agent administered for the prevention of bowel spasms and motion artifacts in clinical MRC was found to be unnecessary in this type of mouse study, as the T2w-gated images and nongated T1 gradient echo images yield a good SNR.

The use of Fluorinert as an enema in small rodent studies has been reported recently from other authors as well24,25. Fluorinert has been used as an enema in a multimodality imaging study (MRC and endoscopy) for quantitative monitoring of rapamycin therapy24 in a mouse model of colonic adenomas, as well as for anatomic and functional (dynamic contrast-enhanced (DCE)-MRI) assessment of colonic polyps in multiple intestinal neo-plasia (Min) mice25.

Applications of the methodAn important advantage of MRC with a Fluorinert enema is that it creates an artifact-free dark lumen on all MRI sequences, including both T1w and T2w imaging sequences, thereby enabling a sharp contrast between the colon wall and the bowel lumen and

a b c dMineral oil Air Water Fluorinert

Sagittal view

Coronal view

Transverseview

Figure 1 | Sagittal, coronal and transverse views of the 3D T1w images acquired with different enema media. (a) Mineral oil; the red arrow points to the air bubbles. (b) Air; the red arrow points to the susceptibility artifact. (c) Water. (d) Fluorinert. The red line indicates the level in the abdomen where the transverse views are acquired. White arrows show the colon.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2684 | VOL.9 NO.11 | 2014 | nature protocols

generating a high SNR. In addition, the Fluorinert enema is well tolerated by the mice. We have imaged more than 200 mice, some of them up to five times, and all have survived the procedure with no adverse effects.

The proposed MRC method has broad applications in research involving colonic diseases. It can be used in a wide

range of mouse colon disease models, as well as in rats or any other laboratory animals. Moreover, this advanced method’s fea-tures, along with the excellent distention and delineation of the colon and the inert nontoxic character of the Fluorinert enema, suggest the potential for the transfer of this methodology into clinical applications.

MaterIalsREAGENTS

Mice. In our laboratory, we use FVB/N strain male and female mice, 6 weeks of age from National Cancer Institute (NCI)-Frederick Animal Production Area ! cautIon Appropriate institutional regulatory board guidelines must be followed. For our work, all experimental animals were maintained in accordance with the Institutional Animal Care and Use Committee (IACUC), and the US National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All procedures performed on mice were approved by the ACUC of the Frederick National Laboratory for Cancer Research.AIN-96G purified diet from Harlan Teklad and drinking water ad libitumAzoxymethane (AOM; Sigma-Aldrich, cat. no. A2853)Dextran sulfate sodium (DSS) 36,000–50,000 Da (MP Biomedicals) dissolved in normal drinking waterFluorinert FC-77 (Sigma-Aldrich, cat. no. F4758). See Box 1 for more informationFluorinert FC-770 (Sigma-Aldrich, cat. no. F3556)Gadopentetate dimeglumine (Gd-DTPA), Magnevist (Schering)Dulbecco’s PBS (BioWittaker)Isoflurane (Abbott, Animal Health)Tissue adhesive (3M Vetbond)

EQUIPMENTMRI scanner: Intera Achieva 3.0T clinical scanner (Philips Medical Systems; see Equipment Setup)Mouse solenoid receiver coil, 40-mm (Philips Medical Systems)Plastic chamber around the coil, made in-houseAnimal physiology monitoring, triggering and air heating system for a single animal (Small Animal Instruments, Inc. (SAII))Custom-built (3D-printing) imaging bedSyringe pump BS-9000-8, for contrast medium infusion (Braintree Scientific)Syringe pump BS-8000, for enema infusion (Braintree Scientific)Tygon microbore tubing, inner diameter, 0.01 inches (VWR International)Needles, 30-gauge and 1-cc syringes (Becton Dickinson)Animal anesthesia system (Summit Anesthesia Solution)Plastic tubing ¼-inch, for anesthesia and waste gas (Fisher Scientific)

•

•••

•

•••••

•

•••

•••••••

Biosafety cabinet for venting waste gas during the animal preparation (The Baker Company)Mouse tail illuminator constrainer (Braintree Scientific)Disposable respiratory sensor–connector V467349 (CareFusion)Stainless steel curved feeding (gavage) needle, 20 gauge for initial cleansing (Harvard Apparatus, cat. no. 524157)In-house catheter system (Fig. 2a)In-house applicator for enema delivery (Fig. 2b)

MR analysis softwarePhilips Achieva, release 3.2 softwareImageJ: ImageJ is a public domain Java image-processing program developed by the NIH (http://rsbweb.nih.gov/ij)Osirix software for the fly-through movie (http://www.osirix-viewer.com/)

REAGENT SETUPTumorigenesis To initiate tumorigenesis, inject 6-week-old mice i.p. with AOM at a dose of 10 mg per kg of body weight in 0.1 ml of saline. One week later, to promote tumor growth, start the mice on 1–2% (wt/vol) DSS dissolved in reverse osmosis–purified normal drinking water for 5 d. A second cycle of 5 d of DSS is initiated after 16 d of normal drinking water. Dilute the MRI Gd chelate (e.g., Magnevist, Schering) with PBS to a final concentration of 80 mmol Gd, to be injected at a dose of 0.2 mmol/kg.EQUIPMENT SETUPMRI scanner We use a Philips Intera Achieva 3.0T clinical scanner equipped with dual-mode gradient system for imaging with a small field of view, a gradient amplitude of 80 mT/m with a slew rate of 100 mT/m/ms and a gradient amplitude of 40 mT/m with a 200 mT/m/ms slew rate (Supplementary Fig. 1a). The same results can be obtained using similar human or dedicated rodent MRI scanners.Physiology monitoring system A SAII system is used for respiratory-triggered acquisition to reduce the artifacts and blurring caused by breathing motion.Plastic chamber The plastic chamber is connected through a thermo-isolated hose to the heating system that keeps the air around the imaging bed warm during the imaging (Supplementary Fig. 1b).

•

•••

••

••

•

Box 1 | Fluorinert Perfluoro-compound FC-77 or Fluorinert FC-77 is a perfluorocarbon (PFC) with a molecular formula of (C8F18)n.(C8F16O)m. The PFC is a synthetic organic compound in which all of the hydrogen atoms are replaced with fluorine atoms. As the MRI signal is produced by the hydrogen nuclei of the imaged subject, Fluorinert, which has no hydrogen atoms, creates no signal in the MR images. The PFC is biologically inert, and therefore it does not pose a toxicological risk from metabolic degradation26. Because of its oxygen-carrying capacity, Fluorinert has been used for attenuation of acute lung injury in rodents27,28, for partial liquid ventilation29,30, in clinical trials as a blood substitute26,31, and in in vivo molecular imaging32–34. The distribution of Fluorinert FC-77 was discontinued by Sigma-Aldrich and replaced with Fluorinert FC-770 (perfluorotri-n-butylamine), with a molecular formula of C12F27N. Several studies that used FC-770 in human prostate cancer MR imaging35,36 have been reported recently. Chemical structures of Fluorinert FC-77 (left images) and FC-770 (right image; http://www.chemspider.com) are shown below.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.9 NO.11 | 2014 | 2685

The temperature in the chamber is measured using a small thermistor probe (SAII, part no. TPEC-109-12), which provides a feedback to the heating system via a fiber-optic temperature module. The thermistor probe and fiber-optic temperature module are components of the physiology monitoring system (SAII).Imaging bed The imaging bed was designed on 3D CAD software (SolidWorks) and fabricated with polycarbonate using an open-source 3D printer. The two channels on the bottom of the bed supply the nose cone with anesthesia and remove waste gas. The bed has output from the pulmonary pad for monitoring, and is covered with an outer plastic tube to isolate the animal from the surrounding environment (Supplementary Fig. 1c).Syringe pumps Two syringe pumps are needed: one for remote infusion of the Fluorinert enema and a second for IV administration of the imaging contrast. Tygon microtubing lines connect the pumps in the operator room with the animal positioned in the magnet. An infusion pump for enema delivery is an essential technical feature of the method, because it allows

control over colonic distension during the MR imaging. A second pump with associated microtubing delivers the imaging contrast agent to the mouse tail vein catheter. The pump system facilitates the enema and contrast infusion without moving the animal during imaging (Supplementary Fig. 1d).

Needle with removed huba

b

c

Needle with blunt tip

Figure 2 | Tools setup. (a) Catheter for contrast media administration. (b) Applicator construction for enema delivery. (c) 20-gauge curved feeding needle for initial cleansing attached to a syringe filled with saline.

proceDurepreparation of contrast medium injection ● tIMInG ~7 min1| Construction of i.v. catheter: Hold a 30-gauge needle using needle nose pliers, bend and break its plastic hub, and insert the blunt end into a 20-cm-long plastic Tygon microtubing. Attach the hub of a second 30-gauge needle to a 1-cc syringe filled with PBS. Break its tip with the pliers or use a blunt needle if available. Insert the blunt tip into the 20-cm microtubing and fill the section with PBS (Fig. 2a).

2| Load the syringe pump with another 1-cc PBS-filled syringe with a blunt needle tip and connect it to a microtubing long enough to reach the center of the magnet. In our case, the syringe pump is located outside the magnet room at ~6 m distance from the magnet to avoid the metal attraction. Attach a second blunt needle with a removed hub to the other end of the 6-m-long tubing in the magnet room. Flush the tubing line with PBS.

3| Dip the blunt needle tubing end located in the magnet room into a vial with 80 mmol of Gd Magnevist solution. Calculate the amount of the contrast needed to be injected in the mouse per animal body weight (0.2 mmol/kg). Program the syringe pump with this volume, and then press the withdraw button. The end of the tubing line will be filled with the necessary amount of Magnevist.

preparation for enema infusion ● tIMInG ~8 min4| To make an applicator construction, cut the connecting tip of a disposable respiratory sensor, leaving ~5-mm-long blue tubing (Fig. 2b). Insert a 3-mm-long blue tubing into the other end of the tip. Insert ~15 mm of a 25-cm-long Tygon microtubing into it. Glue the tubing to the plastic tip. Tissue 3M Vetbond works well, but any plastic material adhesive can be used. The butterfly shape of the applicator is essential for preventing the Fluorinert leakage and for keeping the colon distended during the imaging.

5| Repeat Step 2 using a second syringe pump to deliver Fluorinert enema to the animal in the magnet room. In this case, however, the syringe and microtubing line should be filled with Fluorinert instead of PBS. Connect the blunt needle at the end of the 6-m-long microtubing to the applicator prepared as described in Step 4.? trouBlesHootInG

animal preparation ● tIMInG ~15 min6| Before the MRC procedure, deprive animals of food for 12 h to clean the colon. During the fasting period, the animals may have access to drinking water.

7| Weigh the mouse. The body weight is necessary for calculation of the dose of the imaging contrast agent.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2686 | VOL.9 NO.11 | 2014 | nature protocols

8| Colon cleansing: place the mouse in the constrainer and hold the mouse tail. Gently insert the 20-gauge gavage needle (Fig. 2c) into the rectum until resistance is felt, which is the point at which the colon makes a 90° turn (at a depth of ~3 cm). Begin injecting 1 ml of saline into the colon as the gavage needle is slowly removed from the colon. Repeat the procedure one or two more times if necessary to remove any residual fecal material from the colon.

9| While holding the mouse tail, insert the catheter needle in the lateral tail vein. Secure the position of the needle with a drop of tissue adhesive, and anesthetize the mouse with a nose cone that delivers oxygen with 3% (vol/vol) isoflurane.

10| Position the animal on the imaging bed in a supine position and immobilize the legs, tail and needle with a plastic tape. Decrease the anesthesia level to 2% (vol/vol).

11| Insert the 5-mm-long plastic applicator into the mouse’s rectum and secure it with tape.

12| Position the respiratory sensor on the abdomen area and secure it with tape.

13| Insert the imaging bed loaded with the animal into the plastic isolation tube and slide the tube into the coil.

14| Disconnect the syringe from the i.v. catheter and connect the catheter end to the blunt needle of the microtubing line, whose end is already filled with Magnevist.

15| Move the coil to the isocenter of the magnet.

16| Before starting the imaging session, adjust the anesthesia chamber temperature to 33 °C using SAII software. This maintains the body temperature at 37.0 ± 0.5 °C (the correlation between chamber and body temperatures was previously determined) (L.V.I., M.R.Y., L.A.R. and J.D.K., unpublished observations).

17| Monitor the respiratory signal and the chamber temperature at all the times. crItIcal step Maintain the level of isoflurane between 1.5 and 2.0% to keep the breathing rate at 40 ± 5 breaths per minute. It was empirically determined as an optimal breathing rate that generates minimal motion noise.

MrI ● tIMInG ~1 h18| Start filling the colon with Fluorinert at an infusion rate of 200 µl/min. After 3 min, acquire a fast field echo (FFE; gradient echo) survey scan in the three planes to visualize the colon and to ensure that it is optimally distended. If necessary, increase the flow rate to 250 µl/min for one more minute and repeat the survey. Stop the infusion pump when the colon is sufficiently filled. Do not exceed a total amount of 1.5 ml of infused enema.! cautIon Do not overfill the colon, as this can create substantial pressure on the lumen walls and can harm the animal. The diameter of the distended colon should not exceed 4 mm. crItIcal step It is important to achieve a well-distended colon. A survey scan in three planes—sagittal, coronal and transverse—must be used for a good plan of the 3D coronal slab for T2w and T1w images. Adjust the center of the 3D slice package to try to capture the entire colon in a single slice. Sometimes the colon is very tortuous and this is impossible.? trouBlesHootInG

19| (Optional) In case of tortuous colon, perform an additional fast 2D T2w nongated image in sagittal view (Fig. 3).

20| Acquire a high-resolution respiratory-gated 3D T2w turbo spin echo (TSE) sequence. table 1 illustrates the parameters of the MR imaging sequences used in the protocol. crItIcal step As the T2w TSE sequence is extremely susceptible to motion artifacts, it is essential to perform gating on the animal pulmonary signal in order to achieve a good-quality image. A time repetition (TR) of two beats with a heart rate of 60 b.p.m. results in TR of ~2,000 ms. Note that when small rodents are imaged with the clinical scanner, the animal’s putlmonary signal is connected to the scanner’s cardiac input (supplementary Fig. 1a).

a b

Figure 3 | Planning of the 3D slab. (a) In addition to the survey scan, a fast 2D T2w nongated image in sagittal view assists in planning the colon image. (b) The central slice of the 3D imaging slab in a T1w postcontrast image.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.9 NO.11 | 2014 | 2687

21| Apply 3D T1w FFE precontrast sequence with the same geometry parameters as the T2w image. No gating is necessary, as the TR is very short (15 ms) and the breathing motion does not have a substantial effect on the image quality.? trouBlesHootInG

22| Once the precontrast scan is completed, start the infusion pump for the Gd contrast administration. Inject a volume of 100 µl at a rate of 150 µl/min. In our experiments, we calculated that the amount of PBS in the tail vein catheter is 10 µl and the amount of Magnevist at the end of the microtubing line is ~50 µl (as the body weight of the mouse was 20–25 g), and thus we infuse 100 µl to be sure that the entire dose of contrast medium is injected and the tubing is flushed.

23| Repeat the T1w FFE scan from Step 18 to acquire the postcontrast image.? trouBlesHootInG

24| After completion of the imaging procedure, place the mouse on a heated (37 °C) blanket to recover from anesthesia. Figure 4 shows representative images of a healthy control mouse (first row) and a mouse with tumor-bearing colon (second row) acquired before and after infusion of Fluorinert enema. As the time for animal preparation and MR imaging only allows an average of five mice to be imaged per day, this method does not allow for high throughput and it is not suitable for imaging a large cohort of mice at once. To avoid this limitation, the tumor initiation should be staggered, which will guarantee sufficient MR imaging time.

Data analysis: quantitative assessment of the degree of inflammation ● tIMInG ~30 min crItIcal In this section, we use the multiplanar reformat (MPR) package of the Philips interactive software to generate transverse and sagittal views of the colon from the T1w and T2w coronal 3D image slabs. These views provide additional visual information and can be used for evaluating the degree of inflammation. The same data analysis can be performed using the analytical tool of any human or dedicated rodent MRI scanners. As T2w signal intensity and T1w contrast enhancement are linked to the presence of edema in colonic mucosa, a CNR calculated from the T2w images and the T1w contrast enhancement together with the colon wall thickness is used for quantitative evaluation of the inflammatory response.

25| In the Basic View mode of the Philips interactive software, select the scan and click on the MPR button. The source screen will be loaded with the image from the selected study. The two remaining orthogonal projections will be displayed in subscreens (Fig. 5a).

Healthymouse

Tumor-bearing mouse

T2wa b c d

T2wT1w

precontrastT1w

postcontrast

Figure 4 | MR images of the mouse colon of healthy and tumor-bearing mice. (a) Coronal T2w image before infusion of Fluorinert enema. (b–d) After Fluorinert enema: T2w image (b); T1w precontrast (c); and T1w postcontrast image (d). Scale bar, 5 mm.

taBle 1 | MRI parameters.

sequencetr

(ms)te

(ms)Fa

(degrees)Imaging

slab (mm) resolution (mm) nsaacquisition time (min)

Survey 2D T1w FFE 14 2.6 50 100 × 100 × 29 0.39 × 0.78 × 6.00 2 0.5 Nongated

2D T2w TSE 1,200 65 90 50 × 35 × 9 0.22 × 0.22 × 0.50 1 1.1 Nongated

3D T2w TSE 2,400 65 90 50 × 35 × 9 0.195 × 0.195 × 0.10 2 17 Gated

3D T1w FFE 15 3.3 25 50 × 35 × 9 0.195 × 0.195 × 0.10 8 17 NongatedTR, repetition time; TE, echo time; FA, flip angle; NSA, number of signals averaged; FFE, fast field echo; TSE, turbo spin echo.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2688 | VOL.9 NO.11 | 2014 | nature protocols

26| Choose the orientation of the new slices (sagittal or transverse) and position the slice package in three different locations on the rectum, sigmoid colon and descending colon, which are ~5 mm, 15 mm and 25 mm from the anus, respectively.

27| Choose the slice thickness, number of slices and slice gap. The results of the input values are displayed on the ‘preview’ screen. Click Submit to submit the job. Exit the package. The newly generated images can be seen in the Basic View mode.

28| Use the transverse slices (Fig. 5b) to manually draw a region of interest (ROI) on the colon on the middle slice of the package for each of the three levels. The same ROI is used for the T2w, T1w precontrast and T1w postcontrast image. Right-clicking on the image will show the statistics of the ROI.

29| Acquire the ROI’s area, mean signal intensity and s.d. The mean area from the three levels is used for estimation of the colon wall thickness.

30| Draw an ROI on the muscle of the same slice and acquire its mean signal intensity and s.d. Draw a 3 × 3 mm square in the left lower corner of every image (i.e., outside the mouse body) to acquire background signal intensity and its s.d.

31| Use the values of the parameters acquired in Steps 25–30 to calculate the CNR from the T2w images using the following equation:

CNR SNR SNRcolon muscle= −

where SNR is the mean signal intensity S of the corresponding ROI divided by the s.d. of the noise signal σnoise:

SNR S noise= s

32| Calculate the T1w contrast enhancement as the ratio of the mean T1w signal obtained after the Gd contrast injection divided by the T1w signal precontrast, both normalized to the corresponding muscle tissue signal. Use the same ROI as for the T2w images.

Data analysis: quantitative assessment of tumor burden using ImageJ ● tIMInG ~20 min33| Import the raw file into ImageJ and choose Image in the main menu.

a Slicepackage

DC SC Rb

DC

SC

R

T2w T1w precontrast T1w postcontrast

Figure 5 | Multiplanar reformat package generates transverse and sagittal views of the colon. (a) A selected coronal image with slice package on sigmoid colon. (b) The sagittal colon (top) shows the positions of the transverse slices on the rectum (R), sigmoid colon (SC) and descending colon (DC). The resulting transverse slices (bottom) are generated from the T1w and T2w coronal 3D image slabs.

ROI on the tumor

BrightnessContrast

Results

Figure 6 | Calculation of the volumes of individual tumors with ImageJ. Scale bar, 5 mm.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.9 NO.11 | 2014 | 2689

34| In the drop-down menu, choose Adjust → Brightness/Contrast. Adjust the brightness and contrast of the image automatically or by using sliders for manual adjustment.

35| Choose the Polygon selection as a drawing option to draw an ROI around the tumor (Fig. 6).

36| Repeat drawing of the ROI for every slice with visible tumor. Click ‘Control’ and the ‘M’ key simultaneously to save the ROI statistics. The total tumor area summed and multiplied by the slice thickness results in tumor volume. Although this procedure is time-consuming, it is the most precise way to calculate the tumor volumes. The cluster character of the tumors and the wall thickening prevent the use of an automatic algorithm.

Data analysis: digital image subtraction ● tIMInG ~5 min37| Use digital subtraction of the precontrast and postcontrast T1w images to demonstrate enhancement in tumors while removing the signal from residual fecal matter and improving the sensitivity of the method by reducing background. Import the raw files of the T1w precontrast and postcontrast images in the ImageJ. In the menu, click on Process → Image Calculator. Choose the postcontrast image as Image 1 and the precontrast image as Image 2, and then select Operation Subtract. The example in Figure 7 demonstrates an enhancement of the tumor signal and elimination of the signal from the feces in the colon on the subtraction image.

Data analysis: fly-through movie ● tIMInG ~40 min38| The 3D image data allow the reconstruction of a virtual colonoscopy fly-through movie that enables tumor searching inside the walls of the colon. Refer to Box 2 for advice on how to do this. See also supplementary Video 1.

T1w Pre T1w Post Subtractiona

b

Figure 7 | The digital subtraction of the precontrast and postcontrast T1w images enhances the tumor vascularity, removes the signal from residual stools and improves the sensitivity of the method. (a) The red arrowheads show enhanced tumors on the subtraction image. (b) The yellow arrowheads point to dark residual feces on the subtraction image. Scale bar, 5 mm.

Box 2 | Fly-through movie reconstruction Osirix is an open-source picture archiving and communication system (PACS) workstation digital imaging and communications in medicine (DICOM) viewer that includes the capability of rendering virtual 3D endoscopy views from tomographic images. This option is available in the 3D Viewer menu after opening the 2D view of the 3D T1w FFE image.

procedure1. Before using the Endoscopy Viewer, it may be necessary to reduce the noise in the image. To do this, use the ‘Basic Smooth 5×5’ filter in the ‘Convolution Filters’ choice in the ‘2D Viewer menu’.2. Once the Endoscopy Viewer is open, select the window tool for the 3D view located on the upper right and change the window level to be around the signal intensity of the colon wall and adjust the window width to be slightly less than twice the window level to get a good rendering of the walls. The window is adjusted by dragging the mouse with the left button pressed on the 3D view in the up and down direction for the window level and in the left and right direction for the window width.3. Because of the way the animal is positioned in the clinical scanner, it may be preferable to rotate the 3D view by 90° using the rotate tool so that the left-right direction is horizontal. The position and direction of the Volume Rendering Camera is shown on the three MPR views as a purple line with a solid circle at one end. The solid circle indicates the focal point vector and the opposite end of the line indicates the camera position. The position of the camera can be changed by scrolling any of the views or by clicking the cursor tool on one of the views or using the 3D Mouse zoom tool (right mouse button). The direction of the camera can be rotated using the 3D rotate tool or by dragging the focal point in the MPR views. A convenient method to position the camera is to select the 3D Mouse rotate tool with the left mouse button. This way, dragging the mouse upward with the right button down will move the camera in the direction of the focal point, and dragging the mouse with the left button down will rotate the focal point. Dragging the mouse with the middle button will move the camera laterally.4. To generate a fly-through movie, click on the ‘Fly Thru’ button. In the ‘Fly Thru’ window, click the ‘+’ button for each camera position and orientation desired, then select the ‘Movie’ tab, set the desired number of frames and click ‘Compute’. The ‘Play’ and ‘Save’ buttons will appear for previewing and saving the movie.

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2690 | VOL.9 NO.11 | 2014 | nature protocols

? trouBlesHootInGTroubleshooting advice can be found in table 2.

● tIMInGSteps 1–17, preparation of contrast medium injection: ~30 minSteps 18–24, MRI: ~1 hSteps 25–32, data analysis: quantitative assessment of the degree of inflammation: ~30 minSteps 33–36, data analysis: quantitative assessment of tumor burden by manually segmenting and calculating tumor values using ImageJ: ~20 minStep 37, data analysis: digital image subtraction: ~5 minStep 38, data analysis: fly-through movie: ~40 min

antIcIpateD resultsThe proposed Fluorinert enema–based MRC method provides a quantitative assessment of the severity of mural inflammation, as well as accurate detection and quantification of colonic tumors.

As an example of the type of results that can be obtained, we calculated CNR and colon wall thickness from the T2w images, as well as colonic wall enhancement from the T1w images for three mice by following Steps 25–32 of the PROCEDURE. Mouse 1 was treated with two cycles of 1% (wt/vol) DSS, Mouse 2 was treated with two cycles of 2% (wt/vol)

taBle 2 | Troubleshooting table.

step problem reason solution

5 Air bubbles in the lumen could cause susceptibility artifacts

Air pockets formed in the microtubing junctions

Before the insertion of the applicator to the animal’s rectum, flush the Fluorinert through the microtubing line

18 Colon is not fully distended, which limits the visibility

Large tumor or feces in the way of Fluorinert flow

Pull the animal from the magnet isocenter, remove the plastic applicator, insert an additional amount of Fluorinert with the gavage needle and tape the applicator back to the animal’s rectum

The Fluorinert is leaking out of the rectum

Re-tape the applicator; ensure that it seals the animal’s rectum well

21 Uneven expansion of the colon between the three imaging sequences: T2w, T1w precontrast and T1w postcontrast

Slow leakage from the applicator

Observe the images after every scan. If the lumen is visibly losing volume, start the infusion pump again and check the fullness of the colon with a survey image before continuing with the next scan of the protocol

23 Artifact around the bladder in the T1w postcontrast image obstructs the vision of the colon

Accumulation of Gd in the urinary bladder causes susceptibility artifact

Plan the slices through the colon so as to avoid the having the bladder in the field of view

taBle 3 | Quantitative assessment of inflamed mouse colon.

Mouse t2w cnrt1w contrast enhancement

Wall thickness (mm2)

rectum sigmoid Descending average

Control 6.85 ± 3.56 1.80 ± 0.57 3.56 4.25 4.67 4.16 ± 0.45

1% (wt/vol) DSS

First cycle Mouse 1 16.21 ± 6.71 1.84 ± 1.04 4.54 3.22 4.99 4.25 ± 0.75

Second cycle Mouse 1 17.46 ± 9.55 1.89 ± 0.79 5.63 5.65 4.71 5.33 ± 0.48

2% (wt/vol) DSS

First cycle Mouse 2 27.96 ± 8.95 2.02 ± 0.80 10.22 3.9 5.72 6.61 ± 2.65

Second cycle Mouse 2 36.21 ± 10.50 2.21 ± 0.84 11.25 3.67 4.37 6.43 ± 3.42

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

nature protocols | VOL.9 NO.11 | 2014 | 2691

DSS and the third mouse was a control (no AOM, no DSS treatment). The MR images were acquired 2 d after the DSS exposure ended. The results are presented in table 3.

By performing the procedure at multiple time points on the same mouse, it is possible to monitor the growth of tumors. As an example of this, we show the growth of five individual tumors in a single mouse, as measured at two time points, 35 and 58 d after AOM and after two cycles of DSS (Fig. 8). Tumor shape and distance from the anus were used for the identification of the same tumor at two time points. The animal was euthanized after the last MRI session and the tumor volume was determined post necropsy. A correlation with a coefficient of determination of R2 = 0.969 between the MRI-estimated tumor volumes with those meas-ured with a caliper after necropsy is shown in Young et al.20. A follow-up necropsy with tumor measurements and pathology is useful for validating interpretations made from the MRI data. In the late stage of tumor development, an overlapping between multiple large tumors could limit the MRI ability to accurately measure the individual tumor volumes. In this case, however, the total tumor mass can be estimated.

35

30

25

20

15

10

5

0

Tumor growth

Tum

or v

olum

e, m

m3

Tumor number1 2 3 4 5

35 d58 d

Figure 8 | Tumor growth rate of five individual tumors measured in the same mouse at two time points: 35 and 58 d after AOM and after two cycles of DSS.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

acknoWleDGMents This project has been funded in whole or in part with federal funds from the National Cancer Institute, US National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government.

autHor contrIButIons L.V.I. performed the MRC experiments and prepared the manuscript; M.B. designed the imaging protocol, assembled the imaging tools, reconstructed and described the fly-through movie and edited the manuscript; M.R.Y. designed the animal study and edited the manuscript; L.A.R. provided animal support; J.L.T. advised and discussed on the imaging methods; J.D.K. supervised the project and edited the manuscript; P.L.C. developed the project concept and edited the manuscript.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Jemal, A., Siegel, R., Xu, J. & Ward, E. Cancer statistics, 2010. CA Cancer J. Clin. 60, 277–300 (2010).

2. Becker, C., Fantini, M.C. & Neurath, M.F. High-resolution colonoscopy in live mice. Nat. Protoc. 1, 2900–2904 (2006).

3. Becker, C. et al. In vivo imaging of colitis and colon cancer development in mice using high-resolution chromoendoscopy. Gut 54, 950–954 (2005).

4. Durkee, B.Y., Weichert, J.P. & Halberg, R.B. Small animal micro-CT colonography. Methods 50, 36–41 (2010).

5. Pickhardt, P.J. et al. Microcomputed tomography colonography for polyp detection in an in vivo mouse tumor model. Proc. Natl. Acad. Sci. 102, 3419–3422 (2005).

6. Boll, H. et al. Double-contrast micro-CT colonoscopy in live mice. Int. J. Colorectal Dis. 26, 721–727 (2011).

7. Krupnick, A.S. et al. Quantitative monitoring of mouse lung tumors by magnetic resonance imaging. Nat. Protoc. 7, 128–142 (2012).

8. Amundson, S.A. et al. Induction of gene expression as a monitor of exposure to ionizing radiation. Radiat. Res. 156, 657–661 (2001).

9. Saar, B. et al. Prospective study on bright lumen magnetic resonance colonography in comparison with conventional colonoscopy. Br. J. Radiol. 80, 235–241 (2007).

10. Ajaj, W. et al. Dark lumen magnetic resonance colonography: comparison with conventional colonoscopy for the detection of colorectal pathology. Gut 52, 1738–1743 (2003).

11. Zijta, F.M. et al. Feasibility of using automated insufflated carbon dioxide (CO2) for luminal distension in 3.0T MR colonography. Eur. J. Radiol. 81, 1128–1133 (2012).

12. Keeling, A.N. et al. Intravenous, contrast-enhanced MR colonography using air as endoluminal contrast agent: impact on colorectal polyp detection. Eur. J. Radiol. 81, 31–38 (2012).

13. Graser, A. Magnetic Resonance colonography. Radiol. Clin. N. Am. 51, 113–120 (2013).

14. Lauenstein, T.C., Goehde, S.C. & Debatin, J.F. Fecal tagging: MR colonography without colonic cleansing. Abdom. Imaging 4, 410–417 (2002).

15. Hensley, H.H., Chang, W.C. & Clapper, M.L. Detection and volume determination of colonic tumors in Min mice by magnetic resonance micro-imaging. Magn. Reson. Med. 52, 524–529 (2004).

16. Melgar, S., Gillberg, P.G., Hockings, P.D. & Olsson, L.E. High-throughput magnetic resonance imaging in murine colonic inflammation. Biochem. Biophys. Res. Commun. 355, 1102–1107 (2007).

17. Larsson, A.E. et al. Magnetic resonance imaging of experimental mouse colitis and association with inflammatory activity. Inflamm. Bowel Dis. 12, 478–485 (2006).

18. Herborn, C.U. et al. Dark lumen magnetic resonance colonography in a rodent polyp model: initial experience and demonstration of feasibility. Invest. Radiol. 39, 723–727 (2004).

19. Breynaert, C. et al. Unique gene expression and MR T2 relaxometry patterns define chronic murine dextran sodium sulphate colitis as a model for connective tissue changes in human Crohn’s disease. PLoS ONE 8, e68876 (2013).

20. Young, M.R. et al. Monitoring of tumor promotion and progression in a mouse model of inflammation-induced colon cancer with magnetic resonance colonography. Neoplasia 11, 237–246 (2009).

21. Tanaka, T. et al. A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate. Cancer Sci. 94, 965–973 (2003).

22. Neufert, C., Becker, C. & Neurath, M.F. An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression. Nat. Protoc. 2, 1998–2004 (2007).

©20

14 N

atu

re A

mer

ica,

Inc.

All

rig

hts

res

erve

d.

protocol

2692 | VOL.9 NO.11 | 2014 | nature protocols

23. Saud, S. et al. Chemopreventive activity of plant flavonoid isorhamnetin in colorectal cancer is mediated by oncogenic Src and β-catenin. Cancer Res. 73, 5473–5484 (2013).

24. Miller, S.J. et al. Multimodal imaging of growth and rapamycin-induced regression of colonic adenomas in Apc mutation–dependent mouse. Transl. Oncol. 5, 313–320 (2012).

25. Quarles, C.C. et al. Functional colonography of Min mice using dark lumen dynamic contrast-enhanced MRI. Magn. Reson. Med. 60, 718–726 (2008).

26. Flaim, S.F. Pharmacokinetics and side effects of perfluorocarbon-based blood substitutes. Artif. Cells Blood Substit. Immobil. Biotechnol. 22, 1043–1054 (1994).

27. Chu, S.J. et al. Systemic administration of FC-77 dampens ischemia-reperfusion-induced acute lung injury in rats. Inflammation 36, 1383–1392 (2013).

28. Chang, H. et al. Intravascular FC-77 attenuates phorbol myristate acetate-induced acute lung injury in isolated rat lungs. Crit. Care Med. 36, 1222–1229 (2008).

29. Zhu, Y.B. et al. Partial liquid ventilation decreases tissue and serum tumor necrosis factor-α concentrations in acute lung injury model of immature piglet induced by oleic acid. Chin. Med. J. (Engl) 125, 123–128 (2012).

30. Hirayama, Y. et al. Partial liquid ventilation with FC-77 suppresses the release of lipid mediators in rat acute lung injury model. Crit. Care Med. 32, 2085–2089 (2004).

31. Maevsky, E., Ivanitsky, G. & Bogdanova, L. Clinical results of Perftoran application: present and future. Artif. Cells Blood Substit. Immobil. Biotechnol. 33, 37–46 (2005).

32. Kaneda, M.M., Caruthers, S., Lanza, G.M. & Wickline, S.A. Perfluorocarbon nanoemulsions for quantitative molecular imaging and targeted therapeutics. Ann. Biomed. Eng. 37, 1922–1933 (2009).

33. Giraudeau, C. et al. 19F molecular MR imaging for detection of brain tumor angiogenesis: in vivo validation using targeted PFOB nanoparticles. Angiogenesis 16, 171–179 (2013).

34. Liao, A.H. et al. Evaluation of 18F-labeled targeted perfluorocarbon-filled albumin microbubbles as a probe for micro-US and micro-PET in tumor-bearing mice. Ultrasonics 53, 320–327 (2013).

35. Turkbey, B. et al. Is apparent diffusion coefficient associated with clinical risk scores for prostate cancers that are visible on 3-T MR images? Radiology 258, 488–495 (2011).

36. Turkbey, B. et al. Documenting the location of systematic transrectal ultrasound-guided prostate biopsies: correlation with multi-parametric MRI. Cancer Imaging 11, 31–36 (2011).