Nuclear Magnetic Resonance Spectroscopy and Imaging in Animal Research
John C. Chatham and Stephen J. Blackband
Abstract
Nuclear magnetic resonance (NMR) spectroscopy and imag-
ing can be used to investigate, noninvasively, a wide range of
biological processes in systems as diverse as protein solu-
tions, single cells, isolated perfused organs, and tissues in
vivo. It is also possible to combine different NMR tech-
niques enabling metabolic, anatomical, and physiological
information to be obtained in the same experiment. This
review provides a simple overview of the basic principles of
NMR and outlines both the advantages and disadvantages of
NMR spectroscopy and imaging. A few examples of poten-
tial applications of NMR spectroscopy and imaging are pre-
sented, which demonstrate the range of questions that can be
asked using these techniques. The potential impact of using
NMR techniques in a biomedical research program on the
total number of animals required for specific investigations,
as well as the number of animals used in research, are dis-
cussed. The article concludes with a personal perspective on
the impact of continuing improvements in NMR technology
for future applications in animal research.
Key Words: magnetic resonance imaging; MRI; NMR spec-
troscopy; nuclear magnetic resonance spectroscopy
Introduction
uclear magnetic resonance (NMR1) spectroscopy
N
and imaging are arguably the most versatile tech-
niques in use in biomedical research today. The
John C. Chatham, D.Phil., is Associate Professor at the Center for NMR
Research and Development, Department of Medicine, University of Ala-
bama at Birmingham. Stephen J. Blackband, Ph.D., is Associate Professor
in the Department of Neuroscience and Director of the Advanced Magnetic
Resonance Imaging and Spectroscopy Facility at the University of Florida,
Gainesville, and Associate Director of the National High Magnetic Field
Laboratory, Tallahassee, Florida.
'Abbreviations used in this article: ATP, adenosine triphosphate; CT,
computer-assisted tomography; ECG, electrocardiogram; fMRI, functional
magnetic resonance imaging; MR, magnetic resonance; MRI, magnetic
resonance imaging; NAA, N-acetyl aspartate; NCRR, National Center for
Research Resources; NIH, National Institues of Health; NMR, nuclear mag-
netic resonance; PCr, phosphocreatine; PET, positron emission tomography;
Pj, inorganic phosphate; TCA cycle, tricarboxylic acid cycle.
Volume 42, Number 3 2001
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phenomenon of NMR was first discovered in the 1940s and
was primarily the domain of physicists (Bloch et al. 1946;
Purcell et al. 1946). During the next 50 yr or so, applications
of NMR developed rapidly and were used first by chemists.
The use of NMR to study the structure of proteins and other
biological molecules was markedly improved in the late
1960s with the development of superconducting magnets and
the implementation of Fourier transform NMR. However, it
was not until the mid-1970s that the first applications of
NMR to the study of metabolism in living biological systems
were reported (Berden et al. 1974; Burt et al. 1976a,b; Hoult
et al. 1974; Moon and Richards 1973; Sequin and Scott
1974). At approximately the same time, it was demonstrated
that the use of magnetic field gradients could be used to
encode NMR signals spatially (Lauterbur 1973), and thus the
concept of magnetic resonance imaging (MRI1) was born.
Soon thereafter, MR images of the human body were obtained
(Andrew et al. 1977; Hinshaw et al. 1977), and as early as
1980, the evaluation of MRI as a clinically useful imaging
modality had started.
Currently, with continuing advancements in magnet and
gradient technology coupled with increasingly powerful
computers, NMR spectroscopy and imaging can be used to
investigate a wide range of biological processes in systems
as diverse as a single cell, isolated perfused organs, and
tissues in vivo. It is also possible to combine different NMR
techniques enabling metabolic, anatomical, and physiologi-
cal information to be obtained in the same experiment. In
light of the many potential applications of NMR imaging and
spectroscopy in biomedical research, it is impossible to
provide a comprehensive review within the context of this
article. Therefore, the aim of this article is to provide an
overview of the technique and to demonstrate the potential
utility of NMR-based methods in biomedical research.
We begin with a brief description of the fundamental
principles of nuclear magnetic resonance. This description is
followed by a summary of the advantages and disadvantages
of NMR spectroscopy and imaging compared with other
techniques. We also provide details regarding access to
NMR systems. The primary focus of the article is to demon-
strate the utility of NMR imaging and spectroscopy. To
accomplish this goal, we present a selection of examples that
we believe best captures the diversity of questions that can
be asked using these techniques. We conclude with a per-
sonal perspective regarding the future of NMR spectroscopy
and imaging in biomedical research.
189
Basic Principles of NMR and MR I
The following section is by necessity a general overview of
the basic principles of NMR presented from a classical point
of view. We provide this broad, albeit simplified, descrip-
tion because we believe it includes enough depth to make the
subsequent discussions intelligible without confusing the
reader with the details of NMR theory. A more complete
classical description as well as a quantum mechanical under-
standing of NMR and MRI can be found in the following
literature: (1) general (Blackband 1995; Gadian 1995);
(2) NMR (Abragam 1961; Ernst et al. 1987; Farrar and
Becker 1971; Harris 1986; Schlicter 1978); and (3) MRI
(Foster and Hutchinson 1987; Mansfield and Morris 1982;
Partain et al. 1988; Stark and Bradley 1999; Wehrli et al.
1988).
Nuclear Magnetization
The nuclei in the atoms in materials are spinning and charged
and, as such, most form magnetic dipoles (hence the nuclear
magnetic in NMR). As a simple explanation, these dipoles
are often compared with little bar magnets with north and
south poles, and we use this tool herein (Figure 1A). Normally
these nuclear bar magnets are randomly oriented. The result
is that no net magnetic field arises from them and thus no
NMR signal can be generated. However, when placed in a
strong magnetic field, B o , on average some of the nuclei
"align" with B o . The result is a net magnetic moment, M,
from the sample (Figure IB).
The net magnetization, M, may be "pushed" away from
alignment with B o by using a second magnetic field, B l s
applied perpendicularly to B o with an excitation coil. When
the second magnetic field is turned off, M quickly returns to
align with B o and, as it does so, it generates a signal at
radiofrequencies in a detector coil placed around the sample
(Figure 1C). It is this signal that forms the basis of MR
imaging and spectroscopy. To be effective, the applied mag-
netic field Bj must oscillate at the same frequency at which
the nuclei oscillate in B o so that there is an efficient energy
transfer. The condition that the excitation field must be at
the same frequency at which the sample oscillates is called
the resonance condition, hence the name nuclear magnetic
resonance. Most often the signal excitation and detector coil
are the same physical coil, which consists of an array of
wires designed to generate B x and detect the signal homoge-
neously over the region of interest in the sample.
The nuclear bar magnets oscillate as they realign along
B o , and the signal detected thus has a characteristic fre-
quency. The exact frequency depends on which nucleus is
examined (e.g., hydrogen, sodium, phosphorus) and the
strength of the magnetic field, B o . The signal decays due to
energy loss from the excited nuclei to the surroundings, a
sort of NMR "friction." This energy loss is called "relax-
ation" and is different for differing materials with different
surroundings. A full description of the relaxation requires
190
two relaxation rates, T{ and T2, which represent different
energy loss mechanisms. Tj represents a loss of energy to
the molecular lattice, whereas T2 represents an exchange of
energy between neighboring spins. Consequently, Tx and T2
can provide separate information about the nuclear environ-
ment. It is here that the unique capabilities of NMR can be
appreciated. In other words, by placing a sample in a mag-
netic field and exciting it with a second magnetic field, infor-
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mation regarding the intrinsic nuclear content and the envi-
ronment of those nuclei can be gained noninvasively and
nondestructively.
NMR Spectroscopy
Nuclei in differing chemical environments (different chemi-
cal structures) bond to other nuclei by the sharing of elec-
trons; thus, different chemical groups are subsequently sur-
rounded by different electron shells. These electrons also
have associated magnetic fields that have the effect of shield-
ing the nuclei from the applied B o by slightly differing
amounts, depending on the number of electrons in the shell.
Consequently, nuclei in different chemical groups will reso-
nate at slightly different frequencies, an effect referred to as
the chemical shift. When excited, complex chemical samples
thus generate multiple signals at different frequencies corre-
sponding to all of the distinct chemical groups that are
detected simultaneously in the signal. A technique called the
Fourier transform is used to work out the frequencies of the
signal, generating a NMR spectrum. This is the basis of
NMR spectroscopy, and the information contained within
the NMR spectrum can be used to determine the chemical
makeup and structure of materials.
For example, the hydrogen (1H) NMR spectrum of
ethanol would contain three frequencies corresponding to
the three hydrogen groups, with peak heights proportional to
the number of YH nuclei in each chemical group (Figure 2).
Larger molecules such as proteins have much more compli-
cated spectra (literally hundreds of peaks), which can be
used to determine its three-dimensional solution structure.
Consequently, NMR is now an invaluable tool in the deter-
mination of biomolecular structures. Thus, by placing a
sample in a magnetic field and exciting a signal, the chemi-
cal content and structure of the sample can be determined
noninvasively and nondestructively.
Several factors contribute to the line widths of the reso-
nance signals in a NMR spectrum. In the context of biologi-
cal systems, an important issue relates to the fact that small
freely moving molecules typically yield narrow lines, whereas
large or relatively immobile molecules tend to result in very
broad signals. As a result, NMR spectra of tissues and cells
consist predominantly of narrow line-width resonances origi-
nating from small nonbound molecules, which are often
important metabolites such as adenosine triphosphate (ATP1),
lactate, and N-acetyl aspartate (NAA1). In contrast, the broad
signals from membranes, phospholipids, and proteins cannot
typically be resolved.
WAR Journal
 A)
N

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B, M
B)

apply magnetic equivalent to a

field B, net magnetization, M

Random orientation Align with applied

magnetic field

C)
A
B, M

i) Bj tips M away from B o ii) When Bj is removed, M ii) As M returns to align

returns to equilibrium with B o , signal is
generated

Figure 1 (A) Nuclear dipole equated to a bar magnet. (B) When placed in a magnetic field, B o , the nuclear moments align with B o and
generate a net magnetic moment, M. (C) Bj pushes M away from B o . When Bj is removed, M returns to alignment with B o and generates an
oscillating signal.

Volume 42, Number 3 2001 191


HO
Fourier
SUE,
transform
time
Figure 2 Distinct chemical groups emit signals at different frequencies. Signals with multiple frequency components can be decoded into
component frequencies using the Fourier transform.
MR Imaging
To progress from NMR spectroscopy to NMR imaging, or
MRI, required approximately 30 yr. (The nuclear in NMRI
is usually dropped to make patients less nervous because no
conventional nuclear radiation is employed in NMR.) Imag-
ing is achieved through the inspirational step of applying a
linear magnetic field gradient on top of B o .
As illustrated in Figure 3 A, a simple sample of water
placed in B o will result in a single ! H peak from hydrogen
atoms in the water molecules. When the linear gradient is
applied, different parts of the sample are in different mag-
netic fields and thus signals at different frequencies are
observed. The frequency obtained is linearly proportional to
the resultant magnetic field (Bo + gradient), and the frequency
is thus linearly proportional to the position along the sample.
In this way, a one-dimensional projection of the object is
formed. This is the basis of spatial encoding in one dimension
and can be extended to provide two- or three-dimensional
imaging techniques.
When an unknown object is placed in the magnet, the
gradient may be rotated electronically and a series of projec-
tions of the object obtained at different angles (Figure 3B).
A mathematical technique, called projection-reconstruction
(Herman 1980), can be used to take this series of projections
and create an image of the object. There are in fact many
ways in which two-dimensional encoding can be accom-
plished (most being variants of the use of phase encoding),
making MR the most versatile of the radiological imaging
techniques.
192
Ethanol Relative
-CH,— CPL intensities
3
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frequency
Advantages and Disadvantages of NMR
Imaging and Spectroscopy
Advantages
Noninvasiveness and Lack of Ionizing Radiation
The greatest asset of MR techniques is their noninvasiveness,
which includes studies of isolated tissues and cells where the
MR measurement on that sample is noninvasive. The fact
that both biochemical (spectroscopy) and spatial information
(imaging) can be obtained without destroying the sample is
obviously a great asset for in vivo studies. An additional
advantage of NMR methods for both imaging and spectros-
copy versus comparable techniques is the lack of ionizing
radiation. The majority of non-NMR-based techniques used
for imaging or for in vivo studies of metabolism involve
ionizing radiation in one form or another. For example,
computer-assisted tomography (CT1) uses x-rays, and positron
emission tomography (PET1) involves the administration of
radioactive tracers. Even traditional studies of metabolic
processes in intact cells and organs use compounds labeled
with radioactive isotopes of hydrogen and/or carbon. There-
fore, the use of NMR imaging techniques instead of x-ray-
based methods avoids the exposure of both the investigators
and the subject being studied to x-rays, thus eliminating any
exposure to potentially damaging ionizing radiation. The
ability to use stable isotopes such as carbon-13 to measure
metabolic fluxes with NMR spectroscopy, instead of the
traditional radioisotope techniques, not only minimizes the
IIAR Journal
 A)

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sample B,
B,

gradient

spectrum

frequency
frequency= space

B)

Projections

Multiple
projections used
samples DPI
to reconstruct an
image

Projections
Projections

Figure 3 (A) Application of a linear field gradient causes different parts of the sample to resonate at different frequencies. If the gradient is
linear, then the frequency spread is linear with space. Hence, the spectrum is a one-dimensional projection of the sample. (B) Left, a series of
projections are collected at different projection angles, which, at right, are used to reconstruct the image of the sample. Generally, M
projections are required to form an M x N digital image, where N is the number of points sampling the signal.

Volume 42, Number 3 2001 193

exposure of the investigator to radioactivity but also elimi-
nates the need to dispose of radioactive tissues, carcasses,
and other ancillary materials that might be contaminated with
radioactivity during an experiment. Thus, the use of NMR
techniques can improve the safety of personnel as well as
reduce experimental costs due to elimination of the disposal
of radioactive material.
Flexibility
The most attractive features of NMR techniques are the wide
range of biological processes that can be investigated using
these methods and the variety and versatility of the specific
MR techniques that can be applied. For example, it is pos-
sible to study glucose metabolism in isolated neuronal cells
in culture, in vivo in the brain of an animal, and in humans
using the same basic techniques (Alves et al. 1995; Gruetter
et al. 1998a,b; Manor et al. 1996; Sibson et al. 1998a,b;
Sonnewald et al. 1996). This ability to encompass studies of
cells, animals, and humans should greatly enhance the trans-
lation of knowledge obtained in basic biomedical research to
humans. As another example of flexibility, NMR imaging
can provide not only outstanding images, especially of soft
tissues, but also physiological information such as blood flow
or cardiac function. Furthermore, NMR spectroscopy and
imaging can be combined, thus making it possible to obtain
metabolic, physiological, and anatomical data in a single
experiment. The ability to obtain such a wide range of infor-
mation in a single experiment is not possible with any other
modality. For example, PET can be used to measure metabo-
lism or blood flow noninvasively; however, the spatial reso-
lution of the images is relatively poor so that correlation of
the results with anatomy is difficult without the use of other
techniques. Similarly, although CT can provide rapid, rela-
tively high-resolution cross-sectional images, it cannot pro-
vide metabolic or physiological information.
Disadvantages
Sensitivity
The greatest disadvantage of NMR spectroscopy and imag-
ing compared with other modalities is the intrinsic insensi-
tivity of the methods. The signal that can be generated in the
NMR experiment is small and, for practical purposes, most
strongly coupled with the concentration of the nuclei in the
sample. For example, the human body is composed of -70%
water, and thus a relatively large signal can be obtained from
the ! H nucleus in water that is effectively at a concentration
in the tens of molar range. Thus, it is possible to measure
signals from cubes (voxels) of tissue as small as =0.3mm on
a side from the human brain, generating the high-quality
images used in clinical MRI. Other nuclei in metabolites
found in vivo are typically at much lower concentrations.
For example, the 31P nucleus in ATP in tissues is at a concen-
tration ~104 to 105 orders of magnitude less than water,
194
which, when combined with lower sensitivity of the 31P
nucleus relative to *H, results in a total decrease in sensitivity
of ~106. Because 1 million is 100 x 100 x 100, the cubic
volume needed to obtain the same signal will be 100 times
larger on each side, or =3cm. This calculation is a broad
approximation, and other factors contribute to differences in
sensitivity between different nuclei. For example, the use of
higher field strengths and small surface coils placed closer to
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the tissue of interest, as is often the case in animal studies,
will make it possible to sample much smaller volumes of
tissue. Nevertheless, regardless of the field strengths and
size of the coil, the NMR signals from water will always be
detectable at resolutions approximately two orders of magni-
tude greater than those of other NMR-sensitive nuclei. Thus,
compounds present in submillimolar and certainly micro-
molar concentrations cannot practically be detected directly
in tissues. (However, it should be noted that exogenous
contrast agents can be detected indirectly at much lower con-
centrations through their effects on the relaxation of the water
signal.)
The stronger the magnetic field, Bo, the more nuclei on
average that align with this magnetic field. Thus, as B o
becomes stronger, a larger signal can be generated. This
effect is the main reason for the current drive in NMR to
build stronger magnets, inasmuch as more signal generally
means that higher spatial resolution images and better spectra
can be obtained. The strength of the magnetic field that can
be generated depends on the open aperture (or bore size) of
the magnet required. Smaller bore magnets can be made
stronger. Hence, present clinical (human sized, i.e., 1 m
diameter bore) magnets range up to 3 T (with 4- to 8-T
instruments under evaluation); and smaller bore (as small as
6 cm diameter bore) presently reach more than 20 T, with a
range of field strengths and bore sizes available between
these extremes. As a result, the sample size generally dic-
tates the choice of magnet and field strength; thus, the smaller
the sample, the more sensitive the experiments.
Working in a High-Magnetic-Field Environment
An inevitable consequence of carrying out NMR investiga-
tions is the need to work in a high-magnetic-field environ-
ment. No known intrinsic risks are associated with high
magnetic fields; however, the presence of the magnetic field
can affect equipment routinely used in animal research. For
example, electronic monitors and computer-controlled
devices may function improperly or not at all. The handling
of standard surgical equipment close to NMR systems can
also be difficult and potentially hazardous. One of the very
few dangers associated with NMR is the potential for ferro-
magnetic objects that are not held in place to be attracted to
the magnet. Due to the nature of the forces involved, the
result can be a scalpel, a pair of scissors, or even a gas
cylinder becoming a flying object. Fortunately, an increas-
ing amount of monitoring equipment is now available that is
designed to function in relatively close proximity to NMR
magnets. There is also a growing number of surgical instru-
WAR Journal
ments that can be obtained that are nonferromagnetic, thus
reducing the potential difficulties associated with working in
a high-magnetic-field environment. Additionally, a steel
passive shield or an active shield may be placed around the
magnet to reduce the magnet fringe fields and minimize the
risks. This reduction can be particularly important when the
space available to site the instrument is limited.
Motion Sensitivity
Most MR techniques are motion sensitive, especially when
spatial encoding using gradients is employed. This sen-
sitivity leads to signal distortions that are visually most evi-
dent in artifacts on images or more subtly in quantitative
measurements. Some MR techniques such as functional MRI
(fMRI1) are particularly sensitive to motion artifact, thus
great care must be taken not to minimize the distorting effects
of motion and thus minimize misinterpretation of data. In
animal studies, anesthesia is usually essential to avoid gross
movement of the animal during the study. Imaging of the
body often requires specific sequences designed to minimize
motion. For most studies of the heart, it is necessary to time
image acquisition to the cardiac cycle. This process is nor-
mally achieved by recording the electrocardiogram (ECG1)
signal from the subject, which is then used to trigger the MR
console and thus control the timing of the image acquisition.
This method is called cardiac gating, and by adjusting the
timing of image acquisition relative to the ECG signal, it is
possible to obtain images of the heart at any point in the
cardiac cycle. Cardiac gating may be necessary even when
imaging other organs such as the brain because of motion
caused by the pulsatile blood flow. In some cases, such as
lung imaging, it may also be necessary to gate to respiratory
motion or, alternatively, the subject may be controlled via
mechanical ventilation.
Availability and Access to NMR Systems
The purchase of a NMR system is normally beyond the
means of a single project or investigator. At the time of this
writing, a relatively simple system that can be used for imag-
ing and spectroscopy of small animals such as mice costs
approximately $1 million. Costs increase with the size of the
magnet, allowing investigations of larger animals and with
higher magnetic fields that provide increased sensitivity. A
state-of-the-art system that can accommodate animals such
as dogs or small pigs will cost at least $1.5 to $3 million.
Costs associated with renovation and siting requirements can
easily total hundreds of thousands of dollars. Once installed,
the basic operating costs will be at least $50,000 to $150,000
per year to cover cryogens required to maintain the super-
conducting magnet. In most cases, it is also necessary to
employ an individual full time to manage and maintain the
instrument.
As a result of these expenses, the majority of NMR sys-
tems are often purchased by a consortium of investigators
Volume 42, Number 3 2001
via mechanisms such as the Shared Instrumentation Grant
Program supported by the National Center for Research
Resources (NCRR1) at the National Institutes of Health. The
objective of this program is to make available to institutions
expensive research instruments that can be justified only on
a shared-use basis, and it is designed to support investigators
to obtain commercially available, technologically sophisti-
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cated equipment costing more than $100,000. Users of
instruments obtained through such funding mechanisms are
typically charged an hourly rate that covers the operating
costs of the facility. These rates can range from as little as
$10/hour to several hundred dollars per hour depending on
the instrument and administrative policies of the institution
where the facility is located. Many NMR research facilities
are supported by regional resource grants awarded by the
NCRR, which help offset some of the operational costs and
support research and technical development.
Therefore, investigators who are interested in using NMR
spectroscopy and imaging in their research program can
usually gain access to the necessary equipment without hav-
ing to purchase their own instrument. A list of NCRR-funded
NMR Centers is provided in Table 1. Due to the dynamic
nature of research funding over time and because support for
established centers may cease and new centers may be
funded, the information in Table 1 should be used only as a
guide. (NCRR usually funds 5-yr programs.) There are other
major NMR research centers that are not supported by the
NCRR; these are often located at major medical centers such
as Johns Hopkins, Harvard, and University of California-San
Francisco.
Examples of NMR Spectroscopy and
Imaging
Spectroscopy
Several different NMR-sensitive nuclei can be used in the
study of biological systems, and the most common are 31 P,
]
H, 13C, 23Na, and 19F. 31P, 1H-, and 13C-NMR spectroscopy
are typically used to investigate cellular metabolism and
bioenergetics, whereas 23Na NMR studies usually focus on
issues related to ion transport and regulation of ion pumps
(Askenasy et al. 1996; Kohler et al. 1991; Van Emous et al.
1998). Fluorine does not occur naturally in biological sys-
tems; however, it is a very sensitive NMR nucleus. There-
fore, fluorine-labeled compounds can be introduced into cells
and used as an indicator of a cellular process such as calcium
concentration (Kusuoka et al. 1991; Marban et al. 1987;
Steenbergen et al. 1993; Yanagida et al. 1996). 19F-NMR
spectroscopy has also been used to follow the metabolism of
drugs, such as 5-fluorouracil (Koutcher et al. 1991; Prior et
al. 1990). In addition to the diverse cellular process that can
be studied using NMR spectroscopy, another advantage of
the technique is the wide range of biological systems that can
be investigated. NMR spectroscopic studies are routinely
carried out on isolated cells, both in culture (Bhakoo et al.
195
Table 1 NMR imaging and spectroscopy research centers funded by the National Center for Research
Resources (NCRR)a

Biomedical Magnetic Resonance & Technology Center, University of Illinois at Urbana-Champaign

Principal Investigator: Paul C. Lauterbur, Ph.D. TEL: 217-244-0600; FAX: 217-244-1330
E-mail: bmrl@bmrl.med.uiuc.edu; Web site: bmrl.med.uiuc.edu:8080/
Center for Advanced Magnetic Resonance Technology, Stanford University Medical Center
Principal Investigator: Gary H. Glover, Ph.D. TEL: 650-723-7577; FAX: 650-723-5795

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E-mail: gary@s-word.stanford.edu; Web site: www-radiology.stanford.edu/research/RR.html
Clinical MR Studies at 4.1T: A Research Resource, University of Alabama at Birmingham
Principal Investigator: Gerald M. Pohost, M.D. TEL: 205-934-9736; FAX: 205-975-1952
E-mail: nmrcenter@uab.edu; Web site: www.cnir.uab.edu
The Center for In Vivo Microscopy, Duke University Medical Center
Principal Investigator: G. Allan Johnson, Ph.D. TEL: 919-684-7755; FAX: 919-684-7122
E-mail: egf@orion.mc.duke.edu; Web site: wwwcivm.mc.duke.edu/
Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon University
Principal Investigator: Chien Ho, Ph.D. TEL: 412-268-3395; FAX: 412-268-7083
E-mail: chienho@andrew.cmu.edu; Web site: info.bio.cmu/NMR-Center/NMR.html
Center For Magnetic Resonance Research, University of Minnesota
Principal Investigator: Kamil Ugurbil, Ph.D. TEL: 612-626-2001; FAX: 612-626-626-2004
E-mail: kamil@geronimo.drad.umn.edu; Web site: www.cmrr.drad.umn.edu
Resource for Magnetic Resonance Research & Optical Research, University of Pennsylvania
Principal Investigator: John S. Leigh, Ph.D. TEL: 215-898-9357; FAX: 215-573-2113
E-mail: jack@mail.mmrrcc.upennn.edu; Website: www.mmrrcc.upenn.edu
Center for Magnetic Resonance, Massachusetts Institute of Technology
Principal Investigator: Robert G. Griffin, Ph.D. TEL: 617-253-5597; FAX: 617-253-5405
E-mail: rgg@mit.edu; Website: web.mit.edu/fbml/cmr/
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison
Principal Investigator: John L. Markley, Ph.D. TEL: 608-263-9349; FAX: 608-262-3759
E-mail: markely@nmrfam.wisc.edu; Website: www.nmrfam.wisc.edu
Rogers Magnetic Resonance Facility; University of Texas Southwestern Medical Center
Principal Investigator: Craig R. Malloy, M.D. TEL: 214-648-5886; FAX: 214-648-5881
E-mail: jthach@mednet.swmed.edu; Web site: www.swmed.edu/home_pages/rogersmr
a
From the 1998 Research Resources Directory published by the NCRR. The Directory contains additional information regarding the capabili-
ties of these resources and is available on-line at the NCRR web site: www.ncrr.nih.gov.

1996; Sonnewald et al. 1995,1996) and under perfused con-
ditions (Gamcsik et al. 1995; Lohmeier-Vogel et al. 1995;
Papas et al. 1999); in isolated perfused organs, such as
kidney, liver, heart, and vascular smooth muscle (Burns et al.
1999; Chatham et al. 1990; Cohen et al. 1998; Colet et al.
1998; Dowd and Gupta 1993; Freeman et al. 1989; Gupta et
al. 1989; Hardin et al. 1995; Leach et al. 1998; Raine et al.
1993); and on tissues and organs in vivo, such as the heart,
brain, liver, skeletal muscle, and tumors (Barker et al. 1993,
1994; Bhujwalla et al. 1994, 1996; Chavin et al. 1999;
Constantinidis et al. 1991; Cortez-Pinto et al. 1999; Jucker et
al. 1999; Kushmerick 1995; Laughlin et al. 1992; Zhang et
al. 1999).
The examples described below focus on 31 P-, 13C-, and
!
H-NMR spectroscopy because these are the most commonly
used in the study of biological systems. We have concen-
trated above on a few examples we believe demonstrate the
range of potential questions rather than provide a compre-
hensive survey of all possible applications. It is worth noting
that the limitations of applying NMR techniques to biologi-
cal systems is more a function of the ability to maintain a
physiologically viable preparation for the duration of the
experiment than it is related to any limitations inherent in the
technique itself.
The isolated perfused heart was one of the first biological
systems to be studied in detail using 31P-NMR spectroscopy
(Ackerman et al. 1980; Garlick et al. 1977; Hollis et al. 1977;
Jacobus et al. 1977). A 31P-NMR spectrum of an isolated
perfused mouse heart is shown in Figure 4. The spectrum
consists of a total of six peaks or resonances, one each from

196 ILAR Journal

 PCr
10 0 -5
Figure 4 31P nuclear magnetic resonance (NMR) spectrum of an isolated perfused mouse heart collected on a 500-MHz NMR spectrometer.
The spectrum required approximately 5 min to acquire. Pj, inorganic phosphate; PCr, phosphocreatine; ATP, adenosine triphosphate; PPM,
parts per million.
the a-, p%, and y-phosphates of ATP, one from phospho-
creatine (PCr1), and two from inorganic phosphate (Pj1). The
area under each resonance is directly proportional to the con-
centration of each of the metabolites; thus, from a single
spectrum such as this, it is possible to determine the concen-
trations of ATP, PCr, and Pj. A unique feature of 31P-NMR
spectroscopy is indicated by the presence of two resonances
from inorganic phosphate, namely, its ability to determine
pH. The position of the P; resonance, known as its chemical
shift, is dependent on pH, as first described by Moon and
Richards (1973) in their 31P-NMR studies of red blood cells.
Thus, the two P{ resonances in Figure 4 indicate the presence
of two compartments containing Pj with slightly different
pH. In this case, the two compartments represent the extra-
cellular medium, which has a pH of approximately 7.4, and
the intracellular space, which has a pH of approximately 7.0.
It is important to note that although it is possible to
measure the concentrations of ATP and PCr using traditional
techniques, these techniques involve destroying the sample
under investigation. By necessity, such methods provide
information at only a single point in time, in contrast to 31P-
NMR spectra, which can be collected repeatedly over a pro-
longed period of time and depend only on the viability of the
system under investigation. It is therefore possible to inves-
tigate the consequences of interventions on the bioenergetics
of the tissue of interest. In studies of the heart, one of the
most common interventions is the induction of ischemia
followed by reperfusion (e.g., Cave et al. 2000; Portman et
al. 1997; Schwartz et al. 1992a,b). In such experiments, the
changes in ATP, PCr, P;, and intracellular pH can be
measured as a function of time during ischemic period and
after reflow. The time required to obtain a spectrum with
Volume 42, Number 3 2001
ATP
P
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-10 -15 PPM
adequate signal to noise depends on many factors but can be
5 min or less. Thus, in an experiment in which the protocol
consists of 20 min of normal perfusion, 20 min of ischemia,
and 20 min of reperfusion, 12 separate measurements of the
high-energy phosphate content of a single sample can be
obtained. Using traditional biochemical techniques, 12 dif-
ferent samples must be frozen and extracted at each time
point. We have used the isolated perfused heart as an example
here; however, as noted above, similar studies have been
carried out in other isolated perfused organs such as liver
(Vidal et al. 1998) and kidney (Dowd and Gupta 1995), and
in animal studies in which the organ of interest has been
surgically exposed and a NMR coil was placed directly adja-
cent to the organ (Chavin et al. 1999; Portman and Ning
1990; Schwartz et al. 1992a,b; Sorensen et al. 1998).
There is no doubt that 31P-NMR spectroscopy is a very
valuable tool for investigating tissue bioenergetics; however,
the examples cited above have not exploited the truly non-
invasive feature of NMR because they focused on isolated
organs or surgically exposed tissues. In many NMR studies
of tissue metabolism, it is necessary to harvest samples at the
end of the experiment for additional biochemical or histo-
logical examination. For this reason, the drive to implement
noninvasive localized spectroscopic techniques for animal
studies has been relatively limited, and much of the driving
force for these methods has been their use in studies of meta-
bolic processes in humans (Bottomley 1989). In animal
studies, noninvasive spectroscopic techniques are clearly
valuable for investigating the evolution of tissue injury over
a prolonged period of time or for determining adaptive or
developmental changes in response to a physiological stress.
This application is especially valuable when the animals
197
under investigation are rare or expensive (e.g., primates)
(Brownell et al. 1998; Dautry et al. 1999, 2000; Kinoshita et
al. 1997; Richards et al. 1995).
With recent growth in the development of transgenic
mice models of human disease, there is growing interest in
the use of NMR spectroscopy for the noninvasive evaluation
of tissue metabolism in mice. Because transgenic mice are
2000; Wiesmann et al. 2000; Weiss et al. 2000). Thus, in a
single noninvasive study, it is possible to determine function,
anatomy, and metabolism. In this example, we have shown
data on the mouse heart, which technically is the most chal-
lenging of the major organ systems to investigate in this
manner because it is moving and the left ventricular free wall
is only a few millimeters thick. Thus, similar techniques

expensive to produce, it would be preferable in characteriz-
ing a new line of animals to be able to monitor the develop-
ment of phenotypic changes in individual animals over a
period of time. The alternative is to sacrifice animals at
arbitrarily selected time points. As a result, there has been
interest in applying the techniques of localized NMR spec-
troscopy to mice. An example of this application is shown in
Figure 5, in which a 31P-NMR spectrum of a mouse heart is
shown alongside the accompanying 'H-NMR image of the
heart and the ECG trace from the same animal. The spec-
trum is in essence the same as that shown in Figure 4,
although due to the lower field strength of the NMR system
used for these experiments, only a single Pj resonance is
observed. This limited view is due primarily to the lower
field strength of the NMR system used for these experiments.
The *H-NMR image of the heart was collected during the
same experiment. By collecting multislice images covering
the whole heart at both systole and diastole, it is possible to
determine cardiac function and cardiac mass (Chacko et al.
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could be used for the study of other organ systems in mice,
such as the liver or brain. Nevertheless, to our knowledge,
no investigations of that nature have been published at the
time of this writing.
The NMR-sensitive isotope of phosphorus, 31 P, is natu-
rally occurring and represents 100% of the phosphate found
in biological systems. Therefore, in 31P-NMR spectroscopy
studies of metabolism, the resonances originate from the
phosphorus-containing compounds that naturally occur in
the tissues. However, the NMR-sensitive isotope of carbon,
13
C, represents only 1.1% of the carbon atoms that occur in
nature. The remainder is carbon-12, which is not NMR sen-
sitive. Thus, a 13C-NMR spectrum of a biological sample
will yield relatively little metabolic information consisting
of signals from components such as triglycerides, which have
many chemically equivalent carbon atoms. Despite this
potential handicap, 13C-NMR spectroscopy has been used to
investigate metabolism in cells and tissues for almost as long
as 31P-NMR spectroscopy. If the sample of interest is pro-

PCr

20 PPM -10 •20

Figure 5 [ H- nuclear magnetic resonance (NMR) image of the thorax of a mouse through the heart at end diastole. Selected 31P-NMR spectra
containing the myocardium (A) and a 31 P standard adjacent to the chest (B). The spectra and images were obtained using electrocardiogram
(ECG) gated imaging sequences. (C) ECG tracing of the mouse during the study. Reprinted with permission from Chacko VP, Aresta F,
Chacko SM, Weiss RG. 2000. MRI/MRS assessment of in vivo murine cardiac metabolism, morphology, and function at physiological heart
rates. Am J Physiol Heart Circ Physiol 279:H2218-H2224.

198 WAR Journal

vided with substrates such as glucose or fatty acids, which
are highly enriched with carbon-13,13C-NMR spectroscopy
can be used to monitor the transfer of the 13C-labeled carbons
from the substrates into a variety of metabolic intermediates.
Thus, it is possible to use 13C-NMR spectroscopy to measure
the rate of metabolism through a variety of different meta-
bolic pathways. This process is similar to using traditional
radioisotope techniques (i.e., 14C- or3H-labeled substrates),
except that no radioactivity is involved, the measurements
can be made without the necessity of separating and isolating
the intermediates of interest, and information about several
pathways often can be obtained in a single experiment. As
with all NMR techniques, the principal limitation is sensitiv-
ity; thus, the metabolic intermediates that are of sufficient
concentration to be observed in most biological systems are
glycogen, lactate, alanine, glutamate, and, to a lesser extent,
glutamine and glutathione.
13
C-NMR spectroscopy has been used to investigate
glycogen metabolism in liver (Cohen et al. 1981,1998), heart
(Laughlin et al. 1993, 1994), smooth muscle (Hardin et al.
1995), and skeletal muscle (Jucker et al. 1997, 1999). Al-
though not directly involved in energy metabolism, glutamate
is in equilibrium with the tricarboxylic acid (TCA1) cycle;
thus, the appearance of 13C-labeling in glutamate from r e -
labeled glucose or fatty acids can be used as an index of
oxidative metabolism and flux through the TCA cycle. In a
variety of studies, primarily limited to brain (Hyder et al.
1997; Sibson et al. 1997, 1998) and heart (Chatham et al.
1995; Weiss et al. 1992; Yu et al. 1995), the time course of
13
C-enrichment of glutamate has been used noninvasively to
determine TCA cycle flux and oxygen consumption in vivo.
One limitation of such studies is the necessity of using math-
ematical modeling to convert the kinetic NMR data into the
required metabolic fluxes. Several different models have
been published to date (Chatham et al. 1995; Mason et al.
1995; Weiss et al. 1992; Yu et al. 1995) and, as yet, there is
no consensus as to the most appropriate model.
13
C-NMR glutamate isotopomer analysis, pioneered by
Malloy and colleagues at the University of Texas-Southwestern
(Malloy et al. 1990), is a powerful method for the study of
substrate selection and utilization in biological systems. It is
also based on measurements of 13C-label incorporation from
13
C-labeled substrates into glutamate. However, instead of
determining the time course of enrichment, the method is
based on analyzing the complex line-splitting pattern of 13C-
glutamate resonances observed in high-resolution 13C-NMR
spectra of tissue or cell extracts. This approach is clearly
invasive in that it requires the preparation of tissue extracts;
however, it has several advantages over traditional radio-
isotope methods, including the fact that the contributions of
up to four different substrates to energy production can be
determined in a single experiment. The analysis of spectra
from simple experiments can be carried out by hand readily.
The analysis of more complex experiments is simplified by
software developed by Mark Jeffrey from the University of
Texas-Southwestern and is available free of charge. (For
more information, their Web site is listed in Table 1.) This
Volume 42, Number 3 2001
approach has been used to study intermediary metabolism in
a variety of systems, including cells in culture (Hall et al.
2001), heart (Chatham and Forder 1997; Chatham et al.
1999a,b; Jeffrey et al. 1995), skeletal muscle (Bertocci and
Lujan 1999; Bertocci et al. 1997), and vascular smooth
muscle (Allen and Hardin 1998).
Although the focus of many 13C-NMR studies is on
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glycolytic and oxidative metabolism, it is possible to investi-
gate other metabolic pathways by the appropriate choice of
13
C-labeled precursors. For example, as shown in Figure 6,
glutathione synthesis was measured in a line of adriamycin-
resistant human mammary adenocarcinoma cells (MCF7adr).
The cells had been grown on a collagen sponge matrix and
perfused as previously described (Gamcsik et al. 1996). After
switching to perfusion medium containing [3,3'-13C2]-
cystine, resonances from 13C-labeled glutathione began to
appear, which reflected the metabolism of the 13C-labeled
cystine through the gamma-glutamyl cycle and the resulting
synthesis of glutathione. From such experiments, it is pos-
sible to determine the rate of glutathione synthesis and turn-
over. Because glutathione is a major intracellular antioxidant
that plays a central role in the protection of cells from oxidative
stress, it is possible that alterations in glutathione metabolism
may contribute to different sensitivities to drug therapy or
radiation. Thus, by judiciously choosing I3C-labeled pre-
cursors, it is possible to use 13C-NMR spectroscopy to deter-
mine metabolic fluxes noninvasively through a variety of
different metabolic pathways.
The most sensitive NMR nucleus is the proton lH. Given
the large number of metabolites that contain protons, one
would anticipate that !H-NMR spectroscopy would be widely
used to study metabolism in biological samples; however,
several factors have limited its widespread use. The main
obstacle in the use of 'H-NMR spectroscopy is that the reso-
nances from water and lipids are several orders of magnitude
greater than that of the potential metabolites of interest. The
first step in obtaining a JH-NMR spectrum of metabolites in
biological systems is the suppression of the very intense
water signal. A variety of methods for achieving this sup-
pression have been documented (Duyn et al. 1993; Ernst and
Hennig 1995; Moonen et al. 1989); however, these methods
are usually motion sensitive and thus limited to stationary
samples. Even with excellent water suppression, the pres-
ence of large lipid signals can also obscure much of the
spectral region of interest. Consequently, 'H-NMR spec-
troscopy has been applied primarily in the brain (Anderson
et al. 1994; Barker et al. 1993, 1994; Monsein et al. 1993)
and prostate (Kurhanewicz et al. 1993; Narayan and
Kurhanewicz 1992; Narayan et al. 1989), which are rela-
tively stationary and have negligible endogenous lipids. To
a lesser extent, 'H-NMR spectroscopy has also been used in
metabolic studies of perfused cells (Pilatus et al. 1997) and
tumors (Bhujwalla et al. 1996; Shungu et al. 1992). Among
the few investigations of the heart that have used ^-NMR
spectroscopy, most have focused on investigations of lipid
metabolism (Balschi et al. 1992, 1997). However, a few
reports have documented the ability to measure other
199
 13
C-glutathione

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13
C-cystine
\

(B)

(A)

1
40 30 ppm
Figure 6 Portion of the C nuclear magnetic resonance spectrum of 9 x 107 perfused MCF7adr cells (A) in Dulbecco's modified Eagle's
13

medium/fetal bovine serum; (B) immediately after switching to medium containing [3,3'-13C-]-cystine; and (C) 8 hr after switching to the
labeled medium. The resonances from cystine (at 39.9 ppm) and glutathione (26.5 ppm) are readily detectable. Each spectrum was acquired
in 40 min. Unpublished data courtesy of Dr. Michael P. Gamcsik, The Cancer Center, Duke University, Chapel Hill, North Carolina.

metabolites in the heart with this technique (Bottomley et al.
1997; Ugurbil et al. 1984).
In lH spectra, besides the large water resonance, which is
usually suppressed, the major resonances observed in the
brain are NAA, creatine, and choline. Lactate is evident in
many diseases and can be detected very early in stroke
models (Monsein et al. 1993). NAA in the brain is thought to
be present predominantly in neuronal cell bodies, where it
acts as a neuronal marker; and choline increases are indica-
tive of increased membrane synthesis. Future work may

200 WAR Journal

result in detection of smaller resonances arising from lower-
concentration metabolites, and the limits of detection are
being explored at increasingly higher level field strengths.
Imaging
with increasingly high levels of field strength. Ancillary
hardware, which includes radiofrequency and gradient coils
used for spatial encoding, are still undergoing improvements;
consequently, the applications of MRI continue to grow and
evolve.
Well-established MRI

As with spectroscopy, the range of studies used for MRI is
very large. In this section, examples of these capabilities are
given in the context of this review. For the most part, the
applications of MRI on animals have paralleled similar devel-
opments in clinical studies, and both have been intimately
tied to technological developments. Magnetic resonance
(MR1) technology is still evolving and continues to operate
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Like clinical MR studies, MR on animal models is immedi-
ately effective as a standard radiological technique that, com-
pared with other modalities, provides exquisite anatomical
pictures of soft tissues. The application of these images for
assessing tissue structure and damage is obvious. The images
shown in Figure 7 indicate this potential. It is important to

Figure 7 Selection of 'H images demonstrating the utility of magnetic resonance imaging for assessing tissue structure. (A) Single neuron
from Aplysia californica (bright central nucleus surrounded by dark cytoplasm). (B) Isolated perfused rat hippocampal brain slice. (C)
Formalin-fixed rat brain (courtesy of Dr. Beneviste, Duke University). (D) Formalin-fixed rabbit heart (courtesy of Dr. Forder, University of
Alabama at Birmingham). In vivo (E) axial mouse (courtesy of Dr. Salmon, The University of Florida), (F) saggital rat, (G) axial cat, and (H)
saggital monkey brain images. Note that the fields of view (size of the image) increase from -500 um in (A) to -16 cm in (H). (I) Live image
of a normal 11-day-old rat pup, and (J) a similar animal with hydrocephalus (note enlarged ventricles). (K) Live image of a normal rat brain,
with (L) a diffusion-weighted image after inducement of a stroke, which shows hyperintense region on the left.

Volume 42, Number 3 2001 201

restate that the size of the radiofrequency coil used to detect
the NMR signal dictates the signal-to-noise ratio that can be
attained; thus, higher levels of resolutions are feasible on
smaller samples. Additionally, animal studies have often
utilized excised tissues to provide improved access and con-
trol. The examples in Figure 7 indicate the wide applicabil-
ity of MRI on samples as small as single cells (spatial resolu-
tions of 10-20 |0m) through excised (fixed or perfused) tissues
(with resolutions of 20-100 |am), through live whole animal
models (resolutions of 100-300 |im). The figure also includes
two examples of pathology in rats. The choice of animal
model of course is dependent on the biological questions
being addressed. Mice, rats, and rabbits are commonly used;
however, in principle, any animal can be imaged. This prin-
ciple has also led to MRI's use as a diagnostic tool in clinical
veterinary medicine; however, this potential is beyond the
scope of this article and is therefore not explored.
Signal differences (contrast) observed in images between
tissues are caused by variations in tissue water content, T{ and
T2. By varying the imaging sequence, the image contrast can
be controlled and varied, allowing MRI to discern a wide range
of pathologies, including stroke (ischemia) from blood vessel
rupture or blockage, cancer, multiple sclerosis, and muscle
atrophy. Image contrast can also be improved with the use of
injectable contrast agents, mainly Gd-diethylenetriamine
penta-acetic acid, which has the effect of changing the local
magnetic field (hence, T{ or T2 in the tissue), leading to
changes in signal and contrast. Contrast agents, in particular,
are especially effective in highlighting regions in the brain
where the blood brain barrier has broken down, thus identify-
ing tumors that would otherwise be difficult to see. By using
multislice or volume acquisition MR imaging sequences,
three-dimensional information may be obtained. Thus, the
size and extent of normal or pathological tissues can be
extracted and followed over time in multiple studies.
The amount of information required will dictate the data
acquisition time required. The higher the spatial resolution
required, the larger the sample volume and number of differ-
ent sequences with different contrast required, and the longer
a single study will be. These choices are modulated by the
temporal resolution required and the ability to maintain the
animal under anesthesia (which in turn is related to the health
of the animal). Although the length of studies can range
from minutes to many hours, depending on the specific nature
of the experiment, typical total data acquisition times are
approximately 1 hr. The sensitivity of the animal to repeated
anesthesia will also affect the time difference and frequency
of subsequent studies on the same animal.
Evolving MRI Techniques
MR signals can be modulated to encode information other
than T,, T2, and proton density in many ways, and these
methodologies are in various stages of development. Although
some are as close to becoming routine on animal systems as
they are on clinical instruments, we believe that with MR
technology still improving, the extent to which these tech-
202
niques can be applied is still evolving and is, in fact, the
focus of much of the current developmental research. Fur-
thermore, because of commercial issues, the drive to develop
animal machines is understandably not as strong as the drive
to develop human clinical instrumentation; thus, technology
for animal applications lags behind. Although the increase
in field strengths helps improve the signal-to-noise ratio, this
increase entails new challenges that must also be met. Tj
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and, to a smaller extent, T2 change with field strength so that
the image sequence timings are not the same and must be
reevaluated. Artifacts from magnetic field inhomogeneities
and sample-induced inhomogeneities (called susceptibility
effects) become significant at higher fields and cause image
distortions that must be addressed. Radiofrequency coil
design and power issues also require much further investiga-
tion. In these areas, we are still in a growing phase of MR
technology, and we are certain there are surprises yet to come.
MR as described above is rarely quantified in human
studies, although some degree of quantification is more
prevalent in animal research. Additionally, we do not yet
understand the origins of the signals and how they change in
tissues—the development of mathematical and physical
models is an area of strong research at present. This research,
especially using a variety of physical models, will be most
effectively pursued on animals systems in which real con-
trols are feasible, and the system may be more accurately
perturbed and controlled. It is our long-term hope that MR
may to a large extent become quantitative, ultimately leading
to improved sensitivity and specificity for the technique. An
overview of evolving techniques applied to novel research
areas can be found in Gilles (1994), and references to the
techniques described briefly below can be found in this and
the MRI texts referred to in the Introduction.
As the stability of MR technology has improved, so has
the sensitivity of the measurements that can be made. One of
the first advances was the realization that moving spins cause
the MR signals to change (a spin that moves is not properly
rephased by the imaging sequence), which leads to a signal
loss that is proportional to the distance the spin has moved.
One of the largest (and hence easiest to measure) "movement
of spins" arises from blood flow. Images can be sensitized to
the blood flow to generate MR angiograms. MR angiography
is relatively straightforward, and its clinical use is increas-
ing, particularly as a screening technique. However, it is not
yet preferred over the gold standard of invasive x-ray
angiography. One of the limiting factors is that spatial reso-
lution restricts the examination to the larger vessels. The
method is also sensitive to motion artifacts; thus, application
of MR angiography to the heart is problematic. Neverthe-
less, MR angiography is gaining wider acceptance and is
being applied not only to the brain, but also to limbs and the
body.
In principle, any moving spin can be encoded in this
way; however, the smaller the motion, the less the signal
change and the greater the susceptibility to error that will
arise from larger confounding motions (such as the animal
moving), which could be misinterpreted. Despite these diffi-
ILAR Journal
culties, the MR signal can be made sensitive to water diffu-
sion. An early application of the technique involved an
animal model of stroke in which an ischemic region is clearly
well defined in a diffusion-weighted image before any other
signal changes can be detected (Lebihan 1995; Mosley et al.
1990). Furthermore, with appropriate encoding, the prefer-
ential direction of water diffusion can be quantified, leading
to diffusion tensor imaging. These diffusion images are cur-
rently being intensively investigated because the anisotropic
diffusion thus measured in some way represents the under-
lying tissue structure. At the time of this writing, the research
goal is to identify these structures. However, in more simple
cases, the cause of the diffusion anisotropy is clear, leading
to the generation of three-dimensional maps of nerve fibers
that can be used to track fiber bundles in the central nervous
system. Diffusion tensor MRI has also been used to measure
fiber organization in the isolated perfused heart and has dem-
onstrated distinct advantages over traditional histological
methods for obtaining similar information (Scollan et al.
1998).
MR images also can be sensitized to other physical pro-
cesses and lead to the generation of, for example, tempera-
ture maps (Jolesz et al. 1988; Mulkern et al. 1998; Wang and
Plewes 1999), current density maps (Beravs et al. 1997;
Bodurka et al. 1999; Joy et al. 1989, 1999), and pH maps
(van Sluis et al. 1999). However, great care must be taken in
the collection and interpretation of these data. Essentially,
they all rely on the same information change in the NMR
signal (phase changes); thus, it must be clear that the phase
changes result from the variable of interest and not other
factors (in particular, motion). For the most part, the utility
of these methods is still being evaluated.
The development of so-called functional MRI (fMRI)
has generated great interest and has stimulated a flurry of
activity in the neurological sciences. During the earlyl990s,
when MR systems were sufficiently stable and sensitive, it
was realized that MR signals were sensitive to local mag-
netic field changes induced by the change of oxyhemoglobin
to deoxyhemoglobin, compounded by blood flow changes
when the brain tissue is required to work. Surprisingly, these
signal changes can be observed in the brain in local tissues
that undergo activation; for example, image contrast is gen-
erated in the occipital cortex when the eyes are visually
stimulated. Similar localized brain MR signal changes can
be observed after a variety of stimulations, including motor
(Baudendistel et al. 1996; Deiber et al. 1999; Miezin et al.
2000; Sadato et al. 1997), verbal (Ojemann et al. 1998;
Reichle et al. 2000), acoustic (Belin et al. 2000; Giraud et al.
2000), and emotional stimulation (Schmahmann and
Sherman 1998; Sprengelmeyer et al. 1998; Teasdale et al.
1999). These observations have ignited the neurological
sciences, offering the potential to visualize brain function
directly, noninvasively, and repeatedly. However, the inter-
pretation of the subtle signal changes, a few percentages at
best, is fraught with danger; and the development of animal
models of fMRI is thus desirable, if not essential. However,
the use of animal models in fMRI studies is difficult in terms
Volume 42, Number 3 2001
of developing meaningful stimulation paradigms, especially
because the animal most often must be anesthetized, which
often negates the activation effects. Consequently, although
fMRI development in animal models offers great potential
for elucidating the mechanisms of basic brain function, as
well as validating fMRI against invasive techniques, it is still
a "work in progress" rather than a fully developed technique
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in routine use.
As stated above, the ! H nucleus is most often used in
MRI because the relative signal-to-noise ratio is very large
(water in molar quantities) compared with signals from other
nuclei (millimeters or less). Consequently, the spatial reso-
lution that can be achieved using other nuclei is very poor.
Nevertheless, there have been some attempts to use 23Na,
31
P, and 13C with imaging techniques. Although we are not
yet aware of any significant application of these methodolo-
gies, it is still early. Several investigators have speculated
that very high fields may have their most significant applica-
tions through the studies of nuclei other than the hydrogen
nucleus.
Finally, differences between NMR spectroscopy and MRI
become less distinct when it is realized that spatially local-
ized spectroscopy is feasible. Although many techniques
exist, the most effective techniques involve using imaging
methodologies to localize single or multiple regions of tissue
spatially and to collect spectra from them. More often it is
realized that MR techniques offer the possibility of obtaining
structural (MRI), physiological (e.g., diffusion, flow, fMRI),
and metabolic (spectroscopy) information on the same tissue,
if not simultaneously, at least in an interleaved fashion. The
subsequent ability to look at the relations between function,
structure, metabolism, and mechanics demonstrates both the
flexibility and great potential of MRI.
Impact of NMR Spectroscopy and Imaging
on Animal Welfare
Simply stated, the use of NMR spectroscopy and imaging as
part of a biomedical research program can significantly
reduce the number of animals required for a specific project.
For example, consider a study in which the goal is to deter-
mine the changes in concentration of ATP every 6 min in an
isolated perfused organ in response to a physiological stress
such as ischemia during a period of 1 hr. Assume the aim is
to investigate some new therapy or treatment designed to
minimize the effects of the stress, and there will be two
experimental groups—control and treatment. Traditional
biochemical methods would require measuring individual
samples at each time point. Thus, if we require a sample size
of five in each of the two experimental groups, we need a
total of 100 animals for the study (2 groups x 5 samples x 10
time points = 100). In contrast, if we use 31P-NMR spectros-
copy, we require only 10 animals because all of the time
points can be measured in each sample (2 groups x 5 samples
= 10). This total would represent a reduction of 90% in the
number of animals required for that project.
203
 The same argument can be used when considering the
use of NMR imaging. Consider as an example the impact of
knocking out a specific gene on a specific organ system in
mice. Using traditional methods, it would be necessary to
sacrifice a series of animals at several arbitrary time points to
harvest, fix, and examine histologically the organ of interest.
However, using MR imaging techniques, it would be possible
to monitor the mice as often as one wished without ever
having to sacrifice a single animal. Clearly, in most cases,
the organs or tissues of interest would be harvested for more
detailed analysis; however, by using MRI as a noninvasive
imaging tool, it is possible to select specific time points that
will yield the most information. Currently, ultrasound is the
only other method that is routinely used to image mice
noninvasively; and although it is a relatively cheap and quick
method, it does not provide the detailed anatomical informa-
tion that can be obtained with MRI.
The examples cited above have focused on the use of
isolated organs or small animals; however, NMR imaging
and spectroscopy may have even greater impact in stud-
ies on larger animals. The cost of purchase and husbandry
of larger animals such as dogs and primates has increased
dramatically. Clearly, the ability to perform repeated,
noninvasive measurements of structure, function, and
metabolism in such valuable animals would represent a
major cost savings. Furthermore, when using NMR methods,
the measurements are made repeatedly in the same sample;
thus, the statistical power of the study should also improve.
Consequently, in cases in which differences between
groups are small, the use of NMR techniques would decrease
the number of samples required to obtain statistical signifi-
cance compared with more traditional methods. Therefore,
by including NMR techniques in a biomedical research
program, it should be possible to reduce significantly the
number of animals used in research, thereby also decreasing
experimental costs.
Future Directions
The development of stronger NMR magnets will continue at
least in the near future. For example, during 2000, a horizon-
tal 11.7-T, 40-cm bore NMR system (the first in the world)
and a 17.6-T, 9-cm bore magnet (one of three in the world)
have been installed at the Advanced Magnetic Resonance
Imaging and Spectroscopy facility at the University of Florida,
Gainesville, Florida. In addition, the National High Mag-
netic Field Laboratory in Tallahassee, Florida, is presently
constructing the world's first 21.1-T, 11-cm bore magnet.
Although the widespread use of such specialized instruments
is limited, they will provide a platform for improving our
understanding of the fundamental biophysical processes that
underlie the characteristics of NMR signals in biological sys-
tems. Technological developments, particularly in radio-
frequency coil design and high field imaging techniques,
will be required to use these instruments effectively so that
their potential can be fully realized.
204
Some potential exists for the development of so-called
niche magnets and systems for animal research. Although
more of an issue in human studies, real-time interventional
studies require easier access to the sample under study
than presently feasible with conventional closed cylinder
geometry. Open magnet geometry's (e.g., General Electric's
[Milwaukee, Wisconsin] double doughnut-shaped system,)
Downloaded from https://academic.oup.com/ilarjournal/article/42/3/189/778963 by Korea national university of transportation user on 06 November 2020
or other C-shaped magnets afford some degree of open access
and may be applied in animal studies. Additionally, Bruker
Instruments Ltd. (Billerica, Massachusetts) has determined a
need for high-throughput and low-cost MR studies for the
rapid MR evaluation of relatively large numbers of animals
aimed at the pharmaceutical industry. In response, they have
developed a mid-field low cost (approximately $500K) sys-
tem designed to perform basic MR scans on rats and mice in
just a few minutes. Although more sophisticated and time-
consuming MR measurements are thus precluded, the hope
is that this use will promote the viability of using MR for
drug testing. The practicality and utility of this approach are
being evaluated.
Probably the largest expansion in the use and utility of
MR will be explored through a multimodality approach.
Small-scale CT and PET instruments (called microPET;
Cherry and Gambhir 2001) have become available, and there
is considerable interest in combining these imaging modali-
ties with MR. The superior spatial resolution of MR already
provides localization for PET, CT, and radiation therapy
through image overlays and stereotactic procedures. Further
integration may be warranted through the construction of
multimodality instruments.
We believe the development and application of MR tech-
niques will continue along the lines indicated in this review,
particularly through efforts to increase the functional infor-
mation MR can provide and to examine relations between
structure, function, and metabolism. However, some of these
areas require the following additional comments.
Our knowledge regarding the regulation of gene expres-
sion and our ability to manipulate gene expression in cells
and animals are increasing rapidly. As a result, there will be
an escalating need to evaluate the relation between alter-
ations in gene and protein expression with cellular function.
Such investigations will necessitate the quantitative mea-
surement of a wide range of physiological and metabolic
processes in intact biological systems. Thus, in the future,
the demands for noninvasive, quantitative measurements of
anatomy, physiology, and metabolism in transgenic animals
will provide additional impetus for the development and
application of novel NMR techniques.
We expect that fMRI techniques, which offer great
potential for understanding brain function and connectivity,
will continue to be investigated. The successful develop-
ment and exploration of animal models will be key to an
understanding of these issues because they can be perturbed
in a controlled fashion. Coupled with access to animal brain
atlases presently being developed for availability on the Web,
the relations between structure and function can be accu-
rately explored.
WAR Journal
 Targeted agents are being developed for a variety of
applications, and the most exciting are those developed to
highlight a particular cell or tissue type (Aime et al. 2000;
Kayyem JF et al. 1995; Loui et al, 2000; Sipe et al. 2000;
Sipkins et al. 2000). These so-called "smart" contrast agents
offer great potential for improving the specificity of MR
techniques. However, intrinsic difficulties remain in making
the agents nontoxic and particularly in the delivery of the
agent. Most agents are restricted to the extracellular space
that limits the information they can provide. Efforts are
under way to construct agents that can enter particular cells,
offering many possibilities for assessing function and detect-
ing abnormal cell populations. Aspects of these studies lead
to the concept of molecular imaging with MRI in which
contrast agents are attached to molecules such as antibodies
targeted to specific molecules and receptors (Sipkins et al.,
2000). This kind of molecular imaging is analogous to that
used in other diagnostic techniques such as PET (Phelps
2000a,b).
Summary
It is clear that MR is still an evolving technology, and a few
more surprises are undoubtedly around the corner. Already,
MRI is the most versatile and widely applicable imaging
modality available today. The combination of MRI and
NMR spectroscopy enables us to obtain information from
the molecular level through cells, tissues, animal models,
and humans. The necessity of animal models in research
dictates the development of dedicated animal research sys-
tems. With proper use, MR offers the potential for greatly
reducing the number of animals that would otherwise be
required to move forward in our understanding of intact bio-
logical systems.
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Acknowledgments
We thank Drs. Weiss and Gamcsik for providing Figures 5
and 6, respectively, and Drs. Forder, Beveniste, and Salmon
for their contributions to Figure 7. This work has been sup-
ported in part by National Institutes of Health (NIH) grants
HL48789 and HL67464 and an American Heart Association
grant-in-aid to J.C.C., and NIH grant NS36992 to S.J.B.
Dr. Blackband also gratefully acknowledges the support of
the University of Florida Brain Institute and the National
High Magnetic Field Laboratory.
ILAR Journal