Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures
Donghoon Lee1, David Marcinek1, 2
1Department of Radiology, University of Washington, 2Department of Bioengineering, University of Washington
Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.
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Part 1: Magnet Safety
Both MRI and MRS use a strong magnetic field that requires extreme caution. For example, the 4.7 T instrument that we used for the present work has a magnetic field approximately 90,000 times the earth magnetic field. The high magnetic field is always on even when the MR scanner is not being used. Any metallic object that comes into contact with such a high magnetic field will be strongly and rapidly attracted by the magnet. It is extremely dangerous if an experimental subject or operator is located in the projectile path of a metallic object flying into the magnet. Therefore, personnel who conduct MR experiments should be careful to remove any metallic objects from their clothing before entering the proximity of the instrument and also maintain the surrounding environment free from such objects. More detailed information on magnet safety appears in the literature1 and the following webpage: http://www.imrser.org/ . The presence of metallic material can not only cause the aforementioned safety issue but interfere with experimental results by inducing imaging/spectroscopic artifacts. The metallic material present near or inside the imaging object can alter the magnetic field in the vicinity and so generate artifacts on acquired images or broaden line-width of spectra.
Accordingly, leave wallet, keys, pens, etc. outside the magnet if you handle animals for MR procedures.
Part 2: In Vivo MRI of Mouse in a Horizontal Bore Magnet
Animal Preparation for MRI
- All the animal procedures should be approved by the institutional animal care and use committee (IACUC) prior to executing any types of animal handling.
- We use vaporized isoflurane to anesthetize animals for MRI experiments. Anesthesia of animals can be achieved by other anesthetics such as avertin (2,2,2-tribromoethanol or TBE) and a cocktail of ketamine and xylazine. Dosage information of each anesthetic is found in Table 1.
- Line an induction chamber with plastic backed absorbent pad (blue pad or chuck). Place a mouse (or multiple mice for multi-mouse imaging) in the induction chamber.
- Adjust the flowmeter of the isoflurane vaporizer to 0.8 1.5 L/min. Then adjust the isoflurane vaporizer to 4 % for about 2 3 minutes.
- After reaching the surgical plane of anesthesia (i.e. no toe pinch response), place the mouse on an animal holder with its nose inserted into a nose cone (or mask). A head restrainer can be used for head imaging and a body holder can be used for body imaging.
- Animal holders are used to prevent potential motion: there are several types of commercial holders. Also, a custom designed holder can be fabricated to accommodate any special requirements of an experimental setup. For a custom designed holder, make sure to use only nonmagnetic material.
- During the imaging period, adjust the flowmeter to 0.4 0.8 mL/min and reduce the isoflurane vaporizer to 1.2 1.5 %. The expired gas coming from the mouse nose cone is collected by a pump and removed into an in-house vacuum.
- The eyes of the animal will be kept moist with a sterile eye lubricating ointment. The animal will be kept at 35-37° C during the experiment within a warm water circulation system. Other types of heat source can be used such as heated pads and warm air blown into the radiofrequency (RF) coil.
- An animal monitoring system is in place to monitor body temperature, respiration/cardiac cycle, and synchronize respiration/cardiac gating with image acquisitions.
- A standard sample made of agarose is placed next to the animal to monitor abrupt signal change. This standard agar sample is particularly useful for multi-slice and multi-time point imaging. When an unexpected signal change is detected in a slice from the acquired images, the slice with the unexpected signal change can be eliminated. Also, its signal intensities can be adjusted based on the signal change of the standard sample during post-image analysis.
- After securely placing the animal and monitoring components on the animal holder, position the animal holder at the center of a RF coil.
- Move the RF coil to the magnet room and insert the RF coil into the warm water circulation system placed inside the magnet. Figure 2 displays several components of an MRI scanner observed from the front of the magnet bore.
- Tune the RF coil to the 1H resonance frequency and match the characteristic impedance of the coil to 50 Ohm using the tuning panel in the MR scanner. This is to achieve optimum conditions of signal reception. Most human MRI scanners do not require a separate process of tune/match except in MRS procedures.
- Conduct a shimming process using a single pulse sequence. An MR signal relies on the environmental magnetic field homogeneity. The shimming process enables the magnetic field in the region of interest to be as homogeneous as possible. Each MR scanner has its own way to perform the shimming process, including automatic fast shimming processes such as fast map and gradient shimming.
- Optimize the RF pulse by maximizing one dimensional image profile. RF pulse powers can be arrayed while keeping the pulse length constant and a long enough TR (recycle delay) that is approximately 3 - 5x the T1 of tissue.
- Acquire scout images along three orthogonal orientations to create axial, coronal and sagittal images. A fast image acquisition sequence (i.e. gradient echo or fast spin echo imaging sequence) can be used to acquire the scout images. The acquired images will be used to plan for actual imaging with determination of imaging planes.
- Change to spin echo sequence. Select proper sequence parameters: TR (recycle delay) should be three to five times the tissue T1 to acquire fully relaxed images such as proton density or T2 weighted images. TE (echo time) is the time duration between the first RF pulse and the center of echo signal. A TE value can be selected depending on image contrast as summarized in Table 2. Figure 2 shows in vivo images acquired with different relaxation effects of T1, T2 and T2* for a nude mouse with a xenograft tumor on its back.
- T2 measurements can be done either using multiecho imaging or single echo imaging with multiple TE values.
- Subsequent to the MRI/MRS in vivo experiment, animals should be monitored throughout the recovery process. After MR imaging, the animals will be taken out of the RF coil and monitored to assure full recovery when returned to the cage. Heat loss is rapid in anesthetized mice. Keep the animals warm by covering them with gauze pads or towels and/or providing a heat source until the animals are recovered from anesthesia.
- Review acquired images on the MR console and transfer selected data to a post-processing computer.
- We typically use ImageJ (http://rsbweb.nih.gov/ij/) for analyzing images. The image analysis includes image scaling/filtering, calculations of T1, T2 and diffusion, tumor volume measurements and segmentation of tumors.
Part 3. In Vivo MRS for Mouse Hindlimb Skeletal Muscle in a Vertical Bore Magnet
Construction of Cuff for Inducing Reversible Ischemia
- Start with a piece of PVC pipe that is approximately 5-7 mm wide with an i.d. of 12-15 mm. Drill a small hole through the wall of this piece and thread.
- Cut a piece of balloon so it is open on both ends (helium quality balloons work best). Insert this piece through the PVC piece and wrap back around and tape ends together on the outside wall of the PVC piece.
- Use shrink wrap to seal the balloon ends around the tube. You should have a cuff with a solid outer wall and an inflatable inner