**2.1 MRI system**

#### **Magnet**

NMR machines to image small animals are becoming more and more widespread. The various manufacturers have developed systems for use in studies using small animals by adapting their components (magnet, gradients, antennae, monitoring system and animal handling solutions).

The main component of an MRI system is the magnet, which generates an intense static magnetic field. While for clinical imaging 1.5-T and 3-T magnets are used, for rats and mice a magnetic field between 4.7 T and 11.6 T is generally used. The magnets can be positioned either horizontally or vertically. This type of magnetic fields provides an increased signal-to-noise ratio (S/N), required for the acquisition of highly spatially resolved images. However, a theoretical increase in signal causes problems which must be considered when high-quality images are to be obtained. In this way, an increase in magnetic field leads to a reduced natural contrast between the tissues in T2 or T1 weighted scans. In addition, susceptibility effects are enhanced and may lead to artifacts with gradient-echo sequences.

Most of the images presented here were acquired at 4.7 T. This level of magnetic field provides a compromise between signal-to-noise ratio, contrast and sensitivity to susceptibility artifacts. The use of a more intense magnetic field will also be addressed.

#### **2.2 Gradient system**

The second essential component of an MRI system is the magnetic field gradient. Gradients allow the image to be spatially encoded. When imaging small animals, the spatial resolution required is much greater than for human imaging. For example, in anatomic imaging of rats, a voxel size of at least 400 µm is required in all three dimensions (3D); in mice, a size of at least 200 µm is needed.

In MRI, the digital resolution, i.e. pixel size, is inversely proportional to the gradient intensity, G, and application time, tG (Eq. [1]).

$$
\Delta \mathbf{y} = \frac{\mathbf{w}}{\mathbf{t} \mathbf{c} \cdot \mathbf{y} \mathbf{C}} \tag{1}
$$

The actual resolution is inversely proportional to the relaxation time, T2\*, and to the gradient intensity and application time (Eq. [2]).

$$\text{Resolution} = \frac{2}{\text{\textsuperscript{gGT}}} \tag{2}$$

The advantages of using intense magnetic field gradients are thus obvious, particularly at strong magnetic fields, with very short T2\*.

Commercial gradient systems have been developed with intensities of greater than 400 mT/m over 10 cm, up to 1 T/m over 3 cm. Because the target diameter of these gradient systems is spatially limited, they can only be used to study animals such as rats or mice.

Fig. 1. Photograph of a 1-T/m gradient system. The system can be inserted into a magnet equipped with a 12-cm-diameter tunnel and can contain an antenna up to 6 cm in diameter. The gradient is linear over a distance of approximately 3 cm.

#### **2.3 Coil**

126 Advances in the Biology, Imaging and Therapies for Glioblastoma

accurate 3D information is very important in longitudinal studies such as, for instance, the

An alternative to RARE T2 [6] or 3D contrast-enhanced T1 imaging is fully balanced SSFP imaging (also called bSSFP, TrueFISP or FIESTA) [7]. In fact, it has recently been shown that this sequence can be used at high field, in 3D, to detect tumors of very small size [8]. The advantage of this sequence is the ability to combine the speed of 3D gradient echo and

The purpose of this study is to demonstrate that a 3D TrueFISP MRI sequence is applicable, at high magnetic field, to glioma-bearing mouse models in longitudinal studies. Theoretical considerations of tumor contrast and signal-to-noise ratio as a function of sequence parameters (TE/TR/flip angle) were carried out and compared with experimental data. The 3D TrueFISP MRI sequence was also compared with the sequence most widely used in clinical applications: 2D RARE. Finally, the sequence was used to perform accurate

NMR machines to image small animals are becoming more and more widespread. The various manufacturers have developed systems for use in studies using small animals by adapting their components (magnet, gradients, antennae, monitoring system and animal

The main component of an MRI system is the magnet, which generates an intense static magnetic field. While for clinical imaging 1.5-T and 3-T magnets are used, for rats and mice a magnetic field between 4.7 T and 11.6 T is generally used. The magnets can be positioned either horizontally or vertically. This type of magnetic fields provides an increased signal-to-noise ratio (S/N), required for the acquisition of highly spatially resolved images. However, a theoretical increase in signal causes problems which must be considered when high-quality images are to be obtained. In this way, an increase in magnetic field leads to a reduced natural contrast between the tissues in T2 or T1 weighted scans. In addition, susceptibility effects are enhanced and may lead to artifacts

Most of the images presented here were acquired at 4.7 T. This level of magnetic field provides a compromise between signal-to-noise ratio, contrast and sensitivity to susceptibility artifacts. The use of a more intense magnetic field will also be addressed.

The second essential component of an MRI system is the magnetic field gradient. Gradients allow the image to be spatially encoded. When imaging small animals, the spatial resolution required is much greater than for human imaging. For example, in anatomic imaging of rats, a voxel size of at least 400 µm is required in all three dimensions (3D); in mice, a size of at

In MRI, the digital resolution, i.e. pixel size, is inversely proportional to the gradient

evaluation of a therapeutic treatment.

**2. Materials and methods** 

with gradient-echo sequences.

**2.2 Gradient system** 

least 200 µm is needed.

intensity, G, and application time, tG (Eq. [1]).

longitudinal measurements of glioma volumes in mice.

T1/T2 contrast.

**2.1 MRI system** 

handling solutions).

**Magnet** 

Image quality is also significantly affected by the choice of antenna. The S/N depends entirely on this component as it is proportional to the square root of the antenna's quality factor, Q. Thus, a well-designed antenna is recommended. In addition, the S/N is directly proportional to the filling factor for the antenna, represented by η in Eq. [3].

$$\mathbf{S}/\mathbf{N} \triangleq \langle \mathbf{Q} \rangle^{1/2} \boldsymbol{\eta} \tag{3}$$

Optimal image quality will therefore be obtained using an antenna with a size and shape perfectly adapted to the zone to be imaged. Several types of antennae have been developed and can be used to meet these criteria. Volumetric emission/reception antennae are the most common. They provide a very homogeneous radio-frequency field, often required when

3D TrueFISP MRI Provides Accurate Longitudinal Measurements of Glioma Volumes in Mice 129

fairly reproducible position. In particular, it must allow imaging to be applied repeatedly

A tooth bar assembly is generally used to position the animal's head based on the position of its teeth. Ear bars may be added but are not always necessary. This type of stereotactic

The animal is monitored by measuring its respiratory rate using an air balloon placed on the abdomen or back of the animal and connected to a pressure sensor linked to a computer via an optical fiber during imaging sessions. The respiratory rate can thus be followed on a

Isoflurane inhalation-based anesthesia is used, with adaptation of the percentage of isoflurane mixed with air to maintain the animal's respiration rate at greater than 70 and less

The animal's body temperature is maintained constant either with an electric blanket, or by

Fig. 3. Nude mouse in a home-made bed. A tooth-bar assembly holds the animal in position and a 1-2% air/isoflurane mixture is delivered in the region of the nose. A balloon is placed on the animal's back to measure its respiration rate. The SA Instruments system is linked to

U87 human brain tumor cells were implanted in nude mice (18-20 g, n = 20, Charles Rivers, L'Arbresle, France) by stereotactic injection into the striatum. Mice were anesthetized with isoflurane (1.5% in air) and secured in the stereotactic apparatus (Stoelting Europe, Dublin, Ireland). The scalp was cleaned with Betadine (MEDA Pharma, Paris, France) and the skull was exposed by midline scalp excision. A small hole (0.5 mm in diameter) was then drilled 0.1 mm posterior and 2.3 mm left to the bregma. Five hundred thousand U87 cells dissolved in 2 mL of Minimum Essential Medium were injected using a 10-mL Hamilton syringe into the left hemisphere at a depth of 3 mm below the brain's surface. On withdrawal of the injection needle, the hole in the skull was sealed with bone wax and the scalp was sutured.

a computer, where the animal's respiration can be visualized on-screen.

than 110 breaths/min. Isoflurane can be recycled through a capture system.

using circulating hot water to maintain the gradient system at 32 °C.

(approximately once a day) to particularly fragile animals.

positioning is often used for brain imaging in animals.

computer screen in real time.

**2.5 Animal model** 

using spin-echo sequences. However, their filling factor is relatively low, which can penalize the S/N. This sensitivity problem can be partially mitigated by using cross-polarized antennae. This type of volumetric antenna is the easiest to use, and was used for the images presented in this chapter.

Surface antennae can also be used, offering an excellent quality factor combined with a very high filling factor. This type of antenna therefore provides a higher S/N than the antennae described previously.

However, due to their configuration, the flip angle applied varies depending on the depth of the zone to be imaged within the sample, making the use of spin-echo sequences impossible. In addition, with this type of antenna, contrast can vary depending on the depth of the zone observed within the sample.

Finally, in clinical imaging, and increasingly for small-animal imaging, volumetric emission antennae coupled to an array of surface reception antennae are the most commonly used. This setup offers the advantages of the two types of antennae described above: excellent B1 emission homogeneity associated with an excellent signal-to-noise ratio.

Fig. 2. a) Cross-polarized 25-mm-diameter volumetric antenna; b) 16-mm-diameter surface emission/reception antenna; c) & d) antenna system combining volumetric emission (80 mm in diameter) and phased-array reception.

These three types of antennae are exclusively used for mouse brain imaging and were tuned to 200 MHz in the experiments described here.

#### **2.4 Animal handling/monitoring**

Finally, quality imaging in small animals requires a specific component to position and monitor the animal. This component must allow the animal to be maintained in a stable and fairly reproducible position. In particular, it must allow imaging to be applied repeatedly (approximately once a day) to particularly fragile animals.

A tooth bar assembly is generally used to position the animal's head based on the position of its teeth. Ear bars may be added but are not always necessary. This type of stereotactic positioning is often used for brain imaging in animals.

The animal is monitored by measuring its respiratory rate using an air balloon placed on the abdomen or back of the animal and connected to a pressure sensor linked to a computer via an optical fiber during imaging sessions. The respiratory rate can thus be followed on a computer screen in real time.

Isoflurane inhalation-based anesthesia is used, with adaptation of the percentage of isoflurane mixed with air to maintain the animal's respiration rate at greater than 70 and less than 110 breaths/min. Isoflurane can be recycled through a capture system.

The animal's body temperature is maintained constant either with an electric blanket, or by using circulating hot water to maintain the gradient system at 32 °C.

Fig. 3. Nude mouse in a home-made bed. A tooth-bar assembly holds the animal in position and a 1-2% air/isoflurane mixture is delivered in the region of the nose. A balloon is placed on the animal's back to measure its respiration rate. The SA Instruments system is linked to a computer, where the animal's respiration can be visualized on-screen.
