Name: Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures (Video) | JoVE Journal
Uploaded: 2009-10-20T14:00:00.000+00:00
Duration: 12 min 27 s
Description: Learn noninvasive MRI and MRS procedures for small animal imaging using anesthetized mice. This method demonstrates RF coil tuning, pulse sequence optimization, and metabolic analysis for neuroscience research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This study was supported in part by NIH/NIBIB R21EB008166 to DL and NIA AG028455 and NIA AG022385 for DJM. We thank Dr. James Olson at the Fred Hutchinson Cancer Research Center for providing their D282 tumor mice.

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: <a href="http://www.imrser.org/">http://www.imrser.org/</a> . 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
<ol>
 <li>All the animal procedures should be approved by the institutional animal care and use committee (IACUC) prior to executing any types of animal handling.</li>
 <li>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. </li>
 <li>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. </li>
 <li>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.</li>
 <li>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. </li>
 <li>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. </li>
 <li>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.</li>
 <li>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.</li>
 <li>An animal monitoring system is in place to monitor body temperature, respiration/cardiac cycle, and synchronize respiration/cardiac gating with image acquisitions.</li>
 <li>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.</li>
 <li>After securely placing the animal and monitoring components on the animal holder, position the animal holder at the center of a RF coil.</li>
 <li>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. </li>
</ol>
MRI Experiment
<ol>
 <li>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. </li>
 <li>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. </li>
 <li>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. </li>
 <li>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. </li>
 <li>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.</li>
 <li>T2 measurements can be done either using multiecho imaging or single echo imaging with multiple TE values.</li>
 <li>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.</li>
</ol>
Image Processing
<ol>
 <li>Review acquired images on the MR console and transfer selected data to a post-processing computer.</li>
 <li>We typically use ImageJ (<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>) for analyzing images. The image analysis includes image scaling/filtering, calculations of T1, T2 and diffusion, tumor volume measurements and segmentation of tumors.</li>
</ol>
Part 3. In Vivo MRS for Mouse Hindlimb Skeletal Muscle in a Vertical Bore Magnet
Construction of Cuff for Inducing Reversible Ischemia
<ol>
 <li>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. </li>
 <li>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.</li>
 <li>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.</li>
 <li>Cut out 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 cm piece of non-ferrous metal (i.e. brass) into the hole in the PVC. This allows you to inflate the cuff. Seal the area with 5 minute epoxy.</li>
 <li>Fix this cuff in place next to the RF coil in the MRS probe and connect to an external sphygmomanometer.</li>
</ol>
Custom Respiration Monitor
<ol>
 <li>We use a custom made respiration monitor that is made to be compatible with the restricted space and access to the bore of the magnet. Several commercial models are also available.</li>
 <li>Tie a small balloon to the end of stretch resistant tubing fed into the probe.</li>
 <li>Attach the other end of the tubing to a pressure transducer.</li>
 <li>Be sure the line and balloon are free of air bubbles. The bubbles will attenuate the signal from compression of the balloon caused by movement of the mouse body due to respiration.</li>
</ol>
Mouse Positioning in MRS Probe
<ol>
 <li>Anesthetize the mouse with 5% Avertin (0.010 ml/g body weight).</li>
 <li>After reaching the surgical plane of anesthesia, position the mouse in the MRS probe by placing the mouse on its back on the mouse support. Place the fluid-filled balloon on the ventral side of the mouse and secure in place with mouse support straps.</li>
 <li>Position mouse and body support in the MRS probe.</li>
 <li>Pull one hindlimb through the ischemic cuff and MRS coil. The leg should be centered in the coil as much as possible. This arrangement allows the mouse body to be positioned horizontally in a vertical bore magnet (Figure 3). </li>
 <li>Fix leg in place by taping foot to rigid support.</li>
 <li>Place thermocouple subcutaneously in hindlimb outside the region sampled by the coil.</li>
 <li>Moisten the eyes with the eye lubricating ointment to prevent the eyes from drying. Cover mouse eyes and face to prevent rubbing or irritation from the wall of the probe.</li>
 <li>Other monitoring probes can be added depending on the specific needs of the experiment.</li>
</ol>
Setting up MRS Experiment
<ol>
 <li>Connect airflow to heating element on the MRS probe.</li>
 <li>Set the variable temperature control unit on VNMR software to maintain leg temperature at 35-37 °C.</li>
 <li>Tune coil frequency and match impedance using the tuning panel in the MRS software for both 1H and 31P resonances.</li>
 <li>Adjust shimming circuits to optimize the homogeneity of the B1 magnetic field in the region of interest using 1H spectroscopy.</li>
 <li>Switch to 31P frequency to determine the RF pulse width to yield maximum signal from one free induction decay (FID) (90° time).</li>
 <li>Collect high signal to noise fully relaxed spectra (FRS) to determine the ratios of inorganic phosphate (Pi) and phosphocreatine (PCr) to ATP under fully relaxed conditions. These spectra are collected using the 90° time with time between FID acquisitions (TR) of approximately 5x the T1 of PCr (20 sec. at 7 T). These will be used for quantification of PCr and Pi levels from MR spectra. </li>
</ol>
Ischemic Experiment
<ol>
 <li>A simple ischemic perturbation allows for the determination of resting and maximal mitochondrial ATP production by measuring changes in phosphocreatine during and immediately following ischemia.</li>
 <li>Set-up array to collect multiple spectra using a 45° pulse width (i.e. 0.5 x the 90° time) and a TR of 0.5x the T1 (~1.5 sec.). We typically collect 4 FIDs for every spectra (number of averaging (na) = 4 in VNMR software) for a time resolution of approximately 6 seconds. This time resolution is sufficient to accurately determine resting and maximal mitochondrial ATP production.</li>
 <li>Collect resting spectra for approximately 5 minutes.</li>
 <li>Induce ischemia by inflating the cuff to 270-300 mmHg for 10-12 minutes.</li>
 <li>Release the cuff and collect recovery spectra for 5 minutes.</li>
 <li>Remove the probe from magnet and the mouse from the probe. Allow the mouse to recover from anesthesia under appropriate conditions. Experiments can be repeated on subsequent days. Following the final spectroscopy experiment mouse leg muscles are removed and immediately freeze-clamped in liquid N2 for analysis of ATP concentrations by HPLC.</li>
</ol>
Data Analysis
<ol>
 <li>Data is analyzed offline using one of several spectral analysis programs for NMR spectra. Our laboratory typically uses fit to standard 2 and jMRUI (http://www.mrui.uab.es/mrui/mrui_Overview.shtml) for quantifying peak areas.</li>
<li>Initial PCr breakdown rates during ischemia are a measure of the mitochondrial ATP production under normoxic conditions as described in several papers 3-5. PCr recovery rates can be used to determine the maximal capacity for mitochondrial ATP production based on the approach described previously 4,6.</li>
</ol>
<img border="0" src="/files/ftp_upload/1592/1592fig1tmb.jpg" alt="Animal warming system setup; vacuum, isoflurane tubes; thermal control; experiment equipment." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. In vivo MRI setup viewed from the front of the magnet bore. The setup comprises an RF coil, animal warming system (or warm water circulation system) and gradient insert. All these components are inserted into a horizontal magnet. Warm water is heated in a water reservoir outside the magnet room and introduced into the animal warming system through a Tygon tube (green tape). After circulating in the cylinder, water is pulled from the warming system back to the water reservoir to be heated. An isoflurane tube and vacuum tube are used to anesthetize mice during MRI experiments. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig1.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig2tmb.jpg" alt="MRI imaging comparison, brain and whole-body, mouse study, preclinical results." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. In vivo images for a nude mouse with xenograft tumor (D282 tumor) on its back (arrow) with different relaxation effects: A. T1 weighed image (TR/TE=500/14.2ms). B. T2 weighed image (TR/TE=2s/40ms). Both T1 and T2 weighted images were acquired by spin echo sequence. C. T2 map processed with 4 sets of images acquired by different TE values ranging from 20 to 80 ms. D. T2* weighed image (TR/TE=180/7.39ms, flip angle = 20 degrees) acquired by gradient echo sequence. Field of view of 35 x 35 mm2 is for all MR images. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig2.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig3tmb.jpg" alt="Rodent surgery setup diagram; ischemia unit, anesthesia, respiratory balloon, solenoid coil detailed." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3. Illustration of mouse positioned in horizontal body holder with leg secured in RF coil. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig3.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig4tmb.jpg" alt="NMR spectrum chart of ATP, PCr, and Pi peaks; phosphorous metabolism analysis; spectral data." data-altupdatedat="1787630408000" data-alt="Figure 4"> Figure 4. In vivo 31P spectra through one ischemia reperfusion cycle. Data were collected on a 7 T vertical bore magnet and line broadened with a 20 Hz exponential filter. TR=1, na=4, every 20 spectra are plotted. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig4.jpg">click here</a> to see a larger version of this figure. 
Table 1. Dosage of anesthetic for mouse MRI/MRS.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Animal 
 Species</td>
 <td>Anesthetic 
 Agent </td>
 <td>Dose 
 (mg/kg for injectables)</td>
 <td>Route</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Isoflurane gas</td>
 <td>4.0% for 2-3 min (induction), then 1.2 to 1.5% continuously (maintenance)</td>
 <td>Inhaled via nose cone</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Avertin</td>
 <td>5%, 10ml/kg body weight</td>
 <td>intraperitoneal (IP)</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Ketamine/Xylazine</td>
 <td>100 mg/kg and 10 mg/kg</td>
 <td>IP</td>
 </tr>
</tbody>
</table>
 Table 2. Image with relaxation weightings
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Image Weighting</td>
 <td>TR (recycle delay)</td>
 <td>TE (echo time)</td>
 </tr>
 <tr>
 <td>T1</td>
 <td>Short (Shorter than T1)</td>
 <td>Short (Shorter than T2)</td>
 </tr>
 <tr>
 <td>T2</td>
 <td>Long (3 ~ 5 times T1)</td>
 <td>Long (around T2)</td>
 </tr>
 <tr>
 <td>PD (proton density)</td>
 <td>Long (3 ~ 5 times T1)</td>
 <td>Short (shorter than T2)</td>
 </tr>
</tbody>
</table>
T1: spin lattice relaxation (or longitudinal relaxation) time 
 T2: spin spin relaxation (or transverse relaxation) time

<ol><li>Stecco, A., Saponaro, A., Carriero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patient+safety+issues+in+magnetic+resonance+imaging%3A+state+of+the+art%5BTitle%5D)+AND+%22Radiol+Med%22%5BJournal%5D)">Patient safety issues in magnetic resonance imaging: state of the art.</a> Radiol Med. 112, 491-491 (2007).</li>
<li>Heineman, F. W., Eng, J., Berkowitz, B. A., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NMR+spectral+analysis+of+kinetic+data+using+natural+lineshapes%5BTitle%5D)+AND+%22Magn+Reson+Med%22%5BJournal%5D)">NMR spectral analysis of kinetic data using natural lineshapes.</a> Magn Reson Med. 13, 490-490 (1990).</li>
<li>Amara, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+function+in+vivo%3A+spectroscopy+provides+window+on+cellular+energetics%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Mitochondrial function in vivo: spectroscopy provides window on cellular energetics.</a> Methods. 46, 312-312 (2008).</li>
<li>Blei, M. L., Conley, K. E., Kushmerick, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separate+measures+of+ATP+utilization+and+recovery+in+human+skeletal+muscle%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Separate measures of ATP utilization and recovery in human skeletal muscle.</a> J Physiol. 465, 203-203 (1993).</li>
<li>Marcinek, D. J., Schenkman, K. A., Ciesielski, W. A., Conley, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+coupling+in+vivo+in+mouse+skeletal+muscle%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Mitochondrial coupling in vivo in mouse skeletal muscle.</a> Am J Physiol Cell Physiol. 286, C457-C457 (2004).</li>
<li>Paganini, A. T., Foley, J. M., Meyer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linear+dependence+of+muscle+phosphocreatine+kinetics+on+oxidative+capacity%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Linear dependence of muscle phosphocreatine kinetics on oxidative capacity.</a> Am J Physiol. 272, 501-501 (1997).</li>
<li>Sun, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+MRI+detection+of+gliomas+by+chlorotoxin-conjugated+superparamagnetic+nanoprobes%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes.</a> Small. 4, 495-495 (2008).</li>
<li>Marcinek, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+mitochondrial+coupling+in+vivo+alters+cellular+energetics+in+aged+mouse+skeletal+muscle%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle.</a> J Physiol. 569, 467-467 (2005).</li>
</ol>

The pre-acquisition steps of tune/match and shimming are critical to acquire high spatial resolution and a high signal-to-noise ratio (SNR). It is also important to monitor animal conditions with an animal monitoring system to maintain a stable physiological state for animals during signal acquisitions to comply with humane animal handling and to prevent any potential artifactual measurements. The procedures explained in the protocol can be modified to acquire additional information including diffusion, perfusion and flo...

<table><tbody><tr><td>Inova 200 MR scanner/4.7 T</td><td>Varian Inc., Agilent</td><td></td><td>Used for mouse MRI</td></tr><tr><td>Inova 300 NMR spectrometer/7 T</td><td>Varian Inc., Agilent</td><td></td><td>Used for MRS of mouse skeletal muscle</td></tr></tbody></table>

noninvasive in vivo small animal mri and mrs: basic experimental procedures

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

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.

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

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.

noninvasive-vivo-small-animal-mri-mrs-basic-experimental

Research

JoVE Journal

Biology

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.<br...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

An in vivo animal model of injury is described.  The method takes advantage of the subcutaneous position of the fibular nerve.  Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software.  Post injury changes are monitored in vivo using MR imaging/spectroscopy.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI and PET in Mouse Models of Myocardial Infarction

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the naïve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis.  The data indicate that the technique can be used to monitor the disease-progression in live animals.

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Learn noninvasive MRI and MRS procedures for small animal imaging using anesthetized mice. This method demonstrates RF coil tuning, pulse sequence optimization, and metabolic analysis for neuroscience research.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Explore More Videos