非侵襲的インビボでの小動物MRIとMRS：基本的な実験手順

To begin this experiment, an anesthetized mouse is placed on an animal holder that is subsequently inserted to a radio frequency or RF coil. Prior to an MRI or MRS experiment, the RF coil is then tuned at the resonance frequency and matched to 50 ohm characteristic impedance. Next MRI or MRS acquisition is conducted after optimizing the pulse sequence, finally images or spectra are analyzed using analysis programs.

Hi, I'm Dawn Lee from the R Research Lab in the Department of Radiology at the University Of Washington. And I'm David Senek, also of the R Research Lab and the departments of radiology and bioengineering at the University of Washington. Today we'll show you a procedure for magnetic ance imaging and magnetic ance is spectroscopy in mice.

In our Lab, we use these procedures to study mouse tumor models, muscular dystrophy, and muscle metabolism. So let's get Started. This protocol does not intend to cover every aspect of small animal M-R-I-M-R-S, but rather introduce basic procedures of mouse M-R-I-M-R-S experiments.

The goal is to provide a better understanding of basic procedures for in vivo MR experiments on small animals so that researchers new to the MR field can better plan for non MR components of their studies. So in the end, both MR and non MR procedures could be seamlessly integrated. To begin preparations for the magnetic resonance imaging or MRI experiment, place an isof fluorine anesthetized mouse onto an animal holder with its nose inserted into a nose cone.

A head restrainer can be used for head imaging, and a body holder can be used for body imaging. Keep the eyes of the animal moist. With sterile eye lubricating ointment.

The animal should be kept at 35 to 37 degrees Celsius during the experiment within a warm water circulation system. An animal monitoring system is used both to monitor body temperature, respiration, and cardiac cycle, and also to synchronize respiration and cardiac gating with image acquisitions. Next place a standard agro sample next to the animal to monitor abrupt signal change.

This standard agri 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 can be eliminated. Once the animal is secure and all monitoring components are in place, position the animal holder at the center of a radio frequency or RF coil.

Move the RF coil into the magnet room and insert it into the warm water circulation system placed inside the magnet. Now begin MRI data acquisition. During the imaging period, adjust the flow meter to 0.4 to 0.8 milliliters per minute and reduce the isof fluorine vaporizer to 1.2 to 1.5%The expired gas coming from the mouse nose cone is removed into an in-house vacuum.

To optimize signal reception, tune the RF coil to the proton H one resonance frequency and match the characteristic impedance of the coil to 50 ohm. Using the tuning panel in the MR scanner, most human MRI scanners do not require a separate process of matching except in MRS procedures. Begin shimming using a single pulse sequence.

The shimming process optimizes the homogeneity of the magnetic field in the region of interest. Each scanner has its own way of performing the shimming process, including automatic fast shimming processes, such as fast map and gradient shimming. When shimming is complete, optimize the RF pulse.

By maximizing the one dimensional image profile. RF pulse powers can be arrayed while keeping the pulse length constant and maintaining a long enough or recycle delay. The TR should be approximately three to five times the T one of tissue acquire scout images along three orthogonal orientations to create axial coronal and sagittal images.

A fast image acquisition 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. During actual imaging, a spin echo sequence is used.

T two measurements can be done either using multi echo imaging or single echo imaging with multiple T values. With the MRI experiment to complete, let's look at in vivo magnetic resonance spectroscopy or MRS for mouse hind limb skeletal muscle, the resting and maximal mitochondrial. A TP production can be determined by using MRS to measure changes in phosphocreatine during and immediately following ischemia.

Begin by constructing a cuff to induce reversible ischemia. Start with a piece of PVC pipe that is approximately five to seven millimeters wide with an inner diameter of 12 to 15 millimeters. Drill a small hole through the wall, cut a piece of a balloon so that it's open on both ends.

Helium quality balloons work best. Insert this piece through the PVC piece and wrap it back around. Tape the ends together on the outside wall of the PVC piece.

Next, 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 wall. Cut an area of shrink wrap and balloon around the threaded hole.

Taking care to leave plenty of material between the hole and the edge of the PVC piece thread a 1.5 centimeter piece of non Ferris metal into the hole in the PVC. This will allow you to inflate the cuff. Seal the area with five minute epoxy.

Now fix the cuff in place next to the RF coil in the MRS probe and connect it to an external sig. Mom manometer. Now position an avert anesthetized mouse into the MRS probe by placing it face up on the mouse support.

Place the fluid filled balloon on the ventral side of the mouse and secure it in place with mouse support straps. Put the mouse and body support into the MRS probe. Pull one hind limb through the ischemic cuff and MRS coil.

The leg should be centered in the coil as much as possible. This arrangement allows the animal's body to be positioned horizontally. Inside a vertical bore magnet, place probe body into magnet, tune, match and shim to optimize MRS signal to noise as described for MRI except in MRS, the RF pulse is optimized by maximizing the spectrum amplitude.

Now use P 31 MRS to collect high signal to noise, fully relaxed spectra or FRS to determine the ratios of inorganic phosphate and phospho creatine to a TP under fully relaxed conditions. To perform an ischemic experiment, first, collect the resting spectra for approximately five minutes. Now induce ischemia by inflating the cuff to 270 to 300 millimeters mercury for 10 to 12 minutes.

Release the cuff and collect recovery spectra for five minutes. Experiments can be repeated on subsequent days. Now we'll show some representative results of in vivo MRI and MRS.

The nude mouse has a xenograft D 2 82 brain tumor on its back and was imaged using a conventional spin echo sequence with different relaxation effects. On the T one weighted image, the tumor appears at a similar intensity to that of a adjacent tissue. While it's corresponding T two image clearly visualizes the tumor as a hyperintense region.

Multip echo acquisition enables calculation of a T two map containing quantitative information of T two variation in the animal's body. Regional T two changes in the tumor region can be monitored longitudinally on the overlaid image. A fully relaxed P 31 MRS spectrum from a mouse hind limb is shown here.

This spectrum represents 16 summed fis. The order of peaks from left to right represent the following muscle metabolites, inorganic phosphate pi, phospho, creatine, PCR, and the phosphates of a TP gamma alpha and beta P 31 MRS spectra collected throughout the dynamic ischemia Reperfusion experiment are shown during ischemia, PCR declines and PI increases. The rate of decline in PCR during ischemia is equal to the resting mitochondrial A TP production.

The PCR recovery following ischemia can be used to calculate maximal mitochondrial A TP production. We've just shown you how to acquire in vivo mouse images and measure in vivo mouse muscle metabolism using magnetic resonance spectroscopy and magnetic resonance imaging. For the MRS, we've built a custom RF probe that allows the mouse body to be maintained in a horizontal position in a vertical bore magnet.

Thus, these experiments can be done on any wide bore vertical system. When doing this procedure, it's important to remember to conduct a procedure on the optimum conditions such as better tuning and matching on RF coil and improvement in magnetic field streaming. It is also important to monitor the animal's condition with an animal monitoring system to maintain a stable physiological state from an animal and to prevent any potential artifactual measurements.

So that's it. Thanks for watching and good luck with your experiments.