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The mouse is anesthetized either by i.p. injection of an anesthetic agent such as Ketamine/Xylazine (Ketamine 80 mg/kg bodyweight [bw] and Xylazine 8 mg/kg bw) or by inhalational anesthesia with Isoflurane or similar. For induction with Isoflurane, the animal is placed in a chamber with a constant flow of 2–3 l/min 2-3% Isoflurane in medical air (21% O2). Induction usually takes less than a minute, after which the animal is transferred to the heated imaging pad. The paws are gently taped to the ECG electrodes to register the animal’s vital signs during imaging (ECG electrode gel might be needed to establish electrical contact) and the animal’s nose is placed into the Isoflurane/air nozzle. The Isoflurane concentration can be reduced to 0.5–1% at this point and must be adjusted if the animal shows signs of arousal or extensively deep anesthesia. Protective eye ointment should be applied.
Hair can be removed at this point (hair can also be removed one day before image acquisition to accelerate the scanning procedure). The hair should be removed from a ventral area rostral to the anus and as far as the xiphoid process to ensure that the whole colon can be imaged (Figure 1b). An electric hair clipper can be used to remove long hair, followed by hair removal cream (under no circumstances should the recommended incubation times for chemical hair removal be extended as murine skin can be very sensitive. Creams without fragrances should be used). After hair removal, the skin can be cleaned carefully with physiological saline solution or water to avoid artifacts.
2D Imaging in Brightness (B)-Mode
US gel is applied generously to the abdomen. Care should be taken not to introduce air bubbles and it is particularly important when using photoacoustic imaging that the gel is free of bubbles. Optionally, the gel can be centrifuged briefly to remove bubbles. The transducer is then carefully lowered under US guidance towards the lower abdomen of the mouse until the first structures below the abdominal musculature become visible. Intraluminal contrast can be used to facilitate assessment of polypoid lesions. To this end, a 1 ml syringe is filled with US gel (a Luer-Lok syringe avoids the accidental disconnection of syringe and gavage tube). The syringe is connected to the plastic feeding tube (Figure 1c) and a small amount of US gel is pushed out to moisten the tip. Subsequently, the tube is inserted carefully into the anus of the animal while soft pressure is applied to the plunger to gently open the lumen. The tube should become visible in the live US image, although the imaging depth or the position of the transducer might need to be adjusted to visualize the tube. Once the tube is visible, US gel is injected to carefully inflate the lumen (Figure 2a). Depending on the position of the colon in the abdomen and the possible presence of feces that might interfere with the distribution of the gel, the amount of gel needed to inflate individual regions varies. Care should be taken not to cause a rupture (a very rare event that only occurred once every several hundred imaging sessions in our hands). A ruptured colonic wall may be identified by the presence of US gel in the abdominal cavity, and is immediately recognizable under B-mode µUS as a black, hypoechoic area (Figure 2b), so that the animal can be euthanized upon diagnosis while under anesthesia. Intra-abdominal US gel can be differentiated from blood, which also appears hypoechoic, by using the Doppler function, which is described in more detail below. While intra-abdominal US gel does not show any Doppler signal, adjacent vessels depict signals characteristic of blood flow (Figure 2c).
Feces can easily be distinguished from tumor tissue by their ragged shape and strong acoustic shadowing (Figure 2d). To further aid in the differentiation of feces from tumors, the Doppler function can be used to ensure that no blood flow is present (Figure 2e and below).
To count the number of tumors, a video loop can be recorded from the rectum rostrally 13. While recording a video, the scanning table is slowly moved manually towards the investigator to image the colon completely. In most cases, a single scan is sufficient to determine the total tumor count. A series of video loops may be required in cases where overlying anatomical structures, bends in the colon, or inconsistent fillings with intraluminal contrast, compromise imaging. Applying gentle manual pressure to the abdomen, or lowering the rostral end of the imaging table may help to remove gas-filled loops in the small intestine from the imaging plane. In male mice, the testes and the penis may impair the view of the most distal portion of the colon; however, this problem can often be overcome by tilting the imaging table to obtain an oblique view. In most cases, a full view of the rectum can be achieved. We find a 38 mm tube in most cases sufficient to inflate and image the area in which tumors in the model used predominantly arise (chemically induced with azoxymethane [AOM] and dextran sulfate sodium [DSS]).
2D-Color Doppler Imaging and Pulse-Wave (PW) Doppler
In addition to anatomical images acquired in B-mode, functional information on vascularization can be obtained during the same imaging session. The tumor vasculature can be visualized by switching to Color Doppler mode once the transducer is positioned over a tumor.
Color Doppler images represent a function of the mean blood flow through the arteries and veins that supply the tumor. Conventionally, vessels with flow towards the transducer are colored red, while those with flow away from the transducer are blue (note that this does not relate to veins and arteries, but to the direction of flow relative to the transducer). Alternatively, Power-Doppler can be used, which has a higher sensitivity for signal detection, but does not differentiate between the directions of blood flow. For Doppler mode in our setup, we commonly employ the Vevo probe MS550D (40 MHz center frequency, 40/90 µm axial/lateral resolution), which can also be used to acquire B-mode images so that it is not necessary to switch between probes. This allows changing back and forth between Doppler mode and B-mode in order to obtain optimal positioning of the transducer. Non-vascularized tissue (e.g. necrotic tissue) is clearly visible using this method as Doppler signal is lacking 14.
For reliable inter- and intra-individual comparisons of blood flow and vessel density, anesthesia depth should be tightly controlled to generate comparable hemodynamic conditions. Isoflurane may be advantageous to Ketamine/Xylazine for this application, as Ketamine/Xylazine anesthesia has a marked effect on blood flow, at least in the brain 17, and is known to reduce cardiac output 18. Hemodynamic alterations also occur when using Isoflurane, but these can be controlled for to some extent by keeping the anesthesia depth as shallow as possible 19. It is worth noting that anesthesia with Ketamine/Xylazine seems to have a strong effect on glucose metabolism, which may significantly affect any additional positron emission tomography (PET) imaging with fludeoxyglucose, for example 20.
Optimal heart rates and respiratory rates may vary among mouse strains, as well as between males and females 21. Generally, we found a heart rate >450 beats/min and a respiratory rate >100 breaths/min suitable standard parameters for C57BL/6J and FVBN/J mice.
Depending on the US system used, a multitude of technical settings can be adjusted to improve imaging quality, for example, varying the imaging depth, resolution, or dynamic range of the input signal strength. Generally, it is important to maintain the same settings between individual measurements. The system used here allows all preferences to be saved and reloaded in the next imaging session. To acquire steady images devoid of breathing artifacts, respiratory gating can be used when acquiring Doppler images. Additionally, image averaging can be performed to further reduce artifacts (this increases acquisition time, particularly when used in 3D, see below).
Color or power Doppler images visualize the vasculature of the tumors and the velocity of the blood flood through the vessels. For single vessels, the measurements can be quantified using pulsed-wave (PW) Doppler (Figure 2g). From the Color Doppler window, PW Doppler can be selected, which then displays a scout window to locate the respective region of interest for PW measurements; the beam angle is then adjusted to match the flow direction of the vessel of interest (Figure 2g).
3D Image Acquisition
A particular asset of µUS is the straightforward acquisition of 3D volumetric data, which allows for measurements of tumor size at high resolution. It is worth noting that 3D imaging can be combined with B-mode, Doppler mode, or photoacoustic imaging.
The z dimension is computed using a 3D-motor, to which the imaging probe is attached (Figure 1a). Z-axis resolution can be as low as 76 µm, while the scanning time in B-mode does not exceed a few seconds. For 3D scanning, the transducer is placed exactly in the middle of the tumor of interest and the z-axis length, which includes the axial tumor diameter from its proximal to distal borders, is then set (typically 2–10 mm). To avoid breathing artifacts in the 3D images, physiological settings should be adjusted accordingly, resulting in steady 3D-images of tumors of different sizes (Figures 3a–c). Volumetric measurements made by several investigators were generally consistent with limited absolute deviations (Figure 3d), while in our hands, relative differences between two independent raters were highest for smaller tumors (Figure 3e).
The 3D-Doppler imaging process allows for concise visualization of the tumor vasculature, for example, 3D Color Doppler µUS reveals AOM/DSS-induced colonic tumors to be highly vascularized (Figures 4a-c and ref. 14). Note that 3D imaging with Color Doppler mode cannot be achieved within the scanning time of a few minutes per mouse, as correction for breathing artifacts and averaging is crucial to acquire steady images, and this prolongs the imaging time considerably.
In addition, 3D images can be acquired with the Vevo US system photoacoustic add-on 14. The basic principle of photoacoustics is the optical excitation of fluorophores, which induces a thermoelastic acoustic wave that can be detected with the US array element 16. Previously, we have used this method to detect tissue oxygenation in situ in xenografts and autochthonous colon tumors. We observed high levels of oxygenated hemoglobin in both tumor types, which is in line with the excellent vascularization of these tumors as detected with Doppler imaging 14.