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From anatomy to the targetContributions of magnetic resonance imaging to preclinical pharmaceutical research.

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THE ANATOMICAL RECORD (NEW ANAT.) 265:85–100, 2001
RESEARCH ARTICLE
From Anatomy to the Target: Contributions of
Magnetic Resonance Imaging to Preclinical
Pharmaceutical Research
NICOLAU BECKMANN,* THOMAS MUEGGLER, PETER ROLAND ALLEGRINI, DIDIER LAURENT,
AND
MARKUS RUDIN
In recent years, in vivo magnetic resonance (MR) methods have become established tools in the drug discovery and
development process. In this article, the role of MR imaging (MRI) in the preclinical evaluation of drugs in animal
models of diseases is illustrated on the basis of selected examples. The individual sections are devoted to
applications of anatomic, physiologic, and “molecular” imaging providing, respectively, structural-morphological,
functional, and target-specific information. The impact of these developments upon clinical drug evaluation is also
briefly addressed. The main advantages of MRI are versatility, allowing a comprehensive characterization of a disease
state and of the corresponding drug intervention; high spatial resolution; and noninvasiveness, enabling repeated
measurements. Successful applications in drug discovery exploit one or several of these aspects. Additionally, MRI
is contributing to strengthen the link between preclinical and clinical drug research. Anat Rec (New Anat) 265:85–100,
2001. © 2001 Wiley-Liss, Inc.
KEY WORDS: biomedical imaging; imaging; magnetic resonance imaging; MRI; functional MRI; fMRI; pharmacologic MRI;
pharmaceutical research; drug development
MRI IN THE DRUG DISCOVERY/
DEVELOPMENT PROCESS
The rapid advance of imaging techniques such as magnetic resonance
Dr. Allegrini earned his PhD in Biochemistry at the University of Bern, Switzerland. He was a postdoctoral fellow at
the Biocenter of the University of Basel
from 1981 to 1985. He now heads an
MRI laboratory at Novartis Pharma. In
1995, during a sabbatical leave, he
spent 6 months at the Max-Planck-Institute for Neurological Research in Cologne. Dr. Beckmann received his BSc
in Physics and MSc in Applied Physics
at the University of São Paulo, Brazil,
and from there was an Alexander von
Humboldt Fellow at Siemens Medical
Systems in Erlangen, Germany. After receiving his PhD in Biophysics at the Biocenter of the University of Basel in 1990,
he was appointed Director of Physics of
the MR Center of the University of
Basel. Since 1993, he has headed an
MRI laboratory at Novartis Pharma. Dr.
Laurent received a PhD in biology at the
University of Grenoble, France. He conducted postdoctoral research in the
area of in vivo MRI and NMR spectroscopy at Ciba-Geigy Pharmaceuticals
in Basel, and eventually at Yale University. Since 1999, he has headed an MRI
laboratory at Novartis Pharma. Mr.
Müggler received his BSc in Biology
from the University of Bern in 1999.
© 2001 Wiley-Liss, Inc.
imaging (MRI; see Box 1 for abbreviations) has made them indispensable
tools in clinical diagnosis. Whereas
the drive for method development was
clearly given by clinical applications,
recently similar approaches became
available for experimental studies in
research animals as well. The major
issues that have to be addressed in
animal imaging are the demands for
high spatial resolution and the associ-
He is currently doing PhD work in Biophysics at the Biocenter of the University of Basel. Dr. Rudin received his PhD
in Physical Chemistry in 1981 at the
Federal Polytechnic Institute (ETH) in
Zurich. He then worked on a joint
project of the ETH Zurich and the Swiss
Institute for Water Pollution Control,
and thereafter at the Biocenter of the
University of Basel. He became head of
an MRI laboratory at Novartis Pharma in
1983. He is currently head of the Analytical and Imaging Sciences Unit at Novartis Pharma and Associate Professor
for Biophysics at the University of Basel.
*Correspondence to: Dr. Nicolau Beckmann, Novartis Pharma Ltd., Core Technologies Area, Analytics and Imaging
Sciences Unit, WSJ-386.209, CH-4002
Basel, Switzerland. Fax: ⴙ41-613243889; E-mail: nicolau.beckmann@
pharma.novartis.com
ated issues of improved sensitivity.
From a technologic point of view, the
status of clinical and animal MRI today can be considered equivalent. It is
precisely the link between preclinical
and clinical applications that renders
MRI so attractive in pharmacologic
research. In this review, we illustrate
the role of MRI in the preclinical evaluation of drugs in animal models of
diseases.
Animal models are used in several
phases of the drug development process. The first steps are the identification and validation of a potential drug
target. Genetically engineered animals are being increasingly used in
this early phase. Later in the process,
the safety and efficacy of drug candidates are evaluated in animal models
of human diseases. Ideally, in order
that study paradigms be easily transferable to clinical drug development,
questions concerning drug absorption, distribution, efficacy, metabolism, and elimination should be addressed in a noninvasive manner with
high sensitivity and spatial resolution.
For most drugs, average tissue con-
86 THE ANATOMICAL RECORD (NEW ANAT.)
RESEARCH ARTICLE
Box 1. Abbreviations used in this article
ACPL ⫽ antibody-conjugated paramagnetic liposomes, ADC ⫽
apparent diffusion coefficient, BAL ⫽ broncho-alveolar lavage,
BOLD ⫽ blood-oxygenation-level-dependent, CA ⫽ contract
agent, CBF ⫽ cerebral blood flow, CBV ⫽ cerebral blood volume,
DWI ⫽ diffusion-weighted imaging, EAE ⫽ experimental autoimmune encephalitis, ECG ⫽ electrocardiogram, ETR ⫽ engineered
transferrin receptor, fMRI ⫽ functional magnetic resonance imaging, ICAM-1 intercellular adhesion molecule-1, Gd ⫽ gadolinium,
ITP ⫽ interphalangeal, i.v. ⫽ intravenous, mBSA ⫽ methylated
bovine serum albumin, MCA ⫽ middle cerebral artery MION ⫽
centration is too low to be detected by
using MR methods. Only nuclear
medicine techniques, like gamma
scintigraphy, single-photon emission
computer tomography (SPECT) or
positron emission tomography (PET)
possess the required sensitivity for exposure and pharmacokinetic studies.
However, these methods are limited
by relatively poor resolution (ⱖ1 mm)
and lack of chemical specificity, i.e.,
the inability to distinguish whether
the emitting radionuclide is bound to
the parent drug molecule or to a metabolite thereof.
The principal assets of MRI are high
spatial resolution, of the order of 100
␮m for rodent studies, and the excellent soft tissue contrasting capabilities. The MR signal is governed by
several parameters, e.g., proton density, relaxation times (T1, T2, T2*),
proton exchange rates, water diffusion, macroscopic motion (blood
flow), which depend on the biophysical properties of the tissue (see Mori
and Barker, 1999 for a general technical explanation of the principles of
MRI, including relaxation times). This
wealth of information renders MRI a
valuable tool for diagnosis, tissue
staging, and in vivo morphometry, for
obtaining physiologic and functional
readouts, and for deriving metabolic
and target-specific tissue characteristics.
In view of the increased life expectancy in industrialized countries the
prevalence of neurodegenerative (Parkinson and Alzheimer diseases) and
cardiovascular disorders, diabetes,
cancer, or bone and joint diseases
may also increase. The use of noninvasive imaging techniques to detect
monocrystalline iron oxide nanoparticles, MPTP ⫽ 4-phenyl1,2,3,6-tetrahydropyridine hydrochloride, MR ⫽ magnetic resonance, MRA ⫽ magnetic resonance angiography, MRI ⫽ magnetic
resonance imaging, MRS ⫽ magnetic resonance spectroscopy,
OA ⫽ ovalbumin, PcommA ⫽ posterior communicating artery,
PET ⫽ positron emission tomography, pMCAO ⫽ permanent MCA
occlusion, RA ⫽ rheumatoid arthritis, SPECT ⫽ single-photon
emission computer tomography, TE ⫽ echo time, TOF ⫽ time-offlight
and monitor disease progression and
to evaluate therapy response in animal models of these diseases will become increasingly important for the
pharmaceutical industry. Because repetitive measurements are feasible,
the inter-individual variance can be
reduced by using each animal as its
own control, thereby enhancing the
statistical power of the experiments.
The use of MR methods in pharmaceutical research has been already extensively reviewed (Rudin et al., 1995,
1999; Beckmann et al., 2000). The primary role of MR methods in pharmacologic research is to study drug effects
on tissue morphology, physiology
and (endogenous) biochemistry. The
strengths of the approach are versatility, allowing a comprehensive tissue
characterization, high spatial resolution, and noninvasiveness. The poor
sensitivity of MR largely prevents its use
for pharmacokinetic studies. Here we
will illustrate the use of MRI methods
in pharmacologic research on the basis
of selected examples. The individual
sections are devoted to applications of
anatomic, physiologic, and “molecular”
imaging, providing structural-morphologic, functional, and target-specific information. The impact of these developments upon clinical drug evaluation
will also be briefly addressed.
ANATOMIC IMAGING: TISSUE
STAGING AND IN VIVO
MORPHOMETRY
MRI is inherently a three-dimensional
(3D) technique providing cross-sectional images as well as full 3D data of
body regions (Mori and Barker, 1999).
Depending on the spatial resolution
required and the imaging protocol applied, acquisition times for one image
range from typically 80 ms to 1 hr. It
is evident that longer acquisition
times are necessary to improve the
spatial resolution. Because animals
need to be kept immobilized and anesthetized, total investigation times including preparation are usually set to
1 hr at maximum.
Anatomic imaging constitutes the basis of pharmacologic applications of in
vivo MR techniques. However, rather
than being competitive to histology,
MRI provides complementary information. Histology is and will remain the
gold standard of anatomy in most pharmaceutical preclinical studies. Anatomic MRI becomes indispensable
when noninvasiveness is an essential
requirement, e.g., in longitudinal studies in chronic diseases, especially in
those involving the use of transgenic
animals and higher species. Moreover,
due to the multiparametric signal dependence, MRI provides a comprehensive characterization of soft tissue pathologies. With this in mind, we will
present some examples to illustrate the
benefits of using anatomic MRI for the
characterization of disease models and
therapeutic interventions.
Degenerative Joint Diseases
Chronic diseases affecting the joints,
such as rheumatoid arthritis (RA) and
osteoarthritis, are a major medical
challenge with the aging of the population, and an efficient therapy has yet
to emerge. Due to its excellent softtissue contrast, allowing differentiation of bone, cartilage, tendons, synovium, muscle, and adipose tissue,
RESEARCH ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 87
plified the location of the lesions, as
well as the comparison with histology.
The main advantage of applying
MRI in the first example was the fact
that one parameter, the joint gap volume, served as biomarker to monitor
joint destruction and the effect of
drug treatment. Additionally, assessments could be made in several joints
of the hind paw. Quantitative assessment of individual joints by using histologic methods would have been very
labor-intensive and time consuming.
Alternatively, assessing paw swelling
by measuring the external thickness
of the hind paws as indicator of RA
was of limited use, because swelling
receded before significant structural
changes were observed in the joints
(Beckmann et al., 1995, 1998). In the
second example, histologic assessment was carried out at the end of the
Figure 1. Collagen-induced arthritis in the rat. High-resolution magnetic resonance image
of interphalangeal joints of mid toes from an untreated and a Neoral- (cyclosporine-)
treated rat. Images extracted from 3D data sets acquired 60 days after collagen administration (pixel size, 94⫻81⫻60 ␮m3; acquisition time, 54.6 min). Adapted from Beckmann et
al. (1998) with permission of the publisher.
MRI has become the method of choice
in the diagnosis of joint diseases in
humans. (For information on MRand CT-microscopic imaging of bone
in osteoporosis, see Borah et al. 2001.)
MRI has also been used in animal
models of RA (Waterton et al., 1993;
Beckmann et al., 1995, 1998; Dawson
et al., 1999). Animal studies, especially in small rodents, demand high
spatial resolution and reproducibility
of joint positioning in different imaging sessions. 3D imaging protocols allow reproducible monitoring of the
complex joint architecture necessary
for longitudinal studies. Proper coregistration of the images is feasible by
postprocessing. In addition, 3D images are less affected by partial volume artifacts due to the smaller slice
thickness achieved, improving the anatomic definition in the images.
Figure 1 shows results obtained on
a model of RA in which rats were immunized with type II bovine collagen
(Beckmann et al., 1998). The images
on the left side, extracted from 3D
data sets acquired 60 days after collagen injection, depict the proximal interphalangeal (ITP) joints of the mid
toes of hind paws of untreated and
treated rats (Neoral 15 mg/kg per day
p.o. for 42 days, starting 14 days after
immunization). Volumes of the joint
gaps, used as a marker to assess joint
destruction by the arthritic process,
are shown on the right side. Pathomorphologic changes associated with
the collagen-induced arthritic process, e.g., increase of joint space and
cartilage and bone erosion, have been
observed in the control group. In contrast, the joint architecture remained
unaltered in drug-treated rats.
In the second model, RA was induced by intra-articular injection of
methylated bovine serum albumin
(mBSA) into the knee of rabbits that
had been presensitized to the same
antigen. High-resolution 3D MRI has
been applied to investigate chronic
joint damage in this model (Dawson
et al., 1999). Inflammatory changes,
such as soft-tissue swelling (edema
formation), have been observed initially. Hard tissue (cartilage and bone)
damage occurred at later time points
(Figure 2). Use of 3D MRI was particularly important because the position
of the lesions differed from animal to
animal; proper selection of the orientation of the slices by using landmarks
in the 3D data sets significantly sim-
The primary role of
MR methods in
pharmacologic research
is to study drug effects
on tissue morphology,
physiology, and
(endogenous)
biochemistry.
study, with the position of the histologic sections being chosen based on
the 3D MR images. This was especially important in preparing histologic sections of the damaged joints.
The excellent correlation between histology and MRI indicated that the
technique could be reliably applied to
follow the disease process in this
model of RA in rabbits, allowing to
address questions concerning the effectiveness of therapy over extended
treatment periods.
Lung Inflammation
From the MRI standpoint, the living
lung is one of the most challenging organs to image. Because of the low water
density of approximately 20 –30% (Harris and Heath, 1986), the proton density
is significantly lower than in other tissues. Consequently, it is harder to ob-
88 THE ANATOMICAL RECORD (NEW ANAT.)
RESEARCH ARTICLE
Figure 2. Antigen-induced arthritis in the rabbit knee. Two sagittal sections extracted from high-resolution three-dimensional magnetic
resonance image data sets (acquisition time, 31 min; pixel size, 219⫻219⫻250 ␮m3) acquired from the right knee before (day 0) and at
several time points relative to intra-articular injection of methylated bovine serum albumin. F, femur; T, tibia; M, meniscus; Se, sesamoid
bone; Ef, synovial fluid effusion; Er, hard tissue erosion. Adapted from Dawson et al. (1999) with permission of the publisher.
tain good imaging data. Moreover, due
to the difference in magnetic susceptibilities between lung tissue and the air
that comprises approximately 80% of
pulmonary volume, local inhomogeneities in the magnetic field are produced,
resulting in very short T2 and T2* relaxation times. In addition, cardiac and
respiratory movements can cause severe image artifacts. These problems
are more evident in rodents because of
the higher cardiac and respiratory rates
and the necessity to acquire images of
improved resolution.
To detect signal from lung parenchyma, especially at high fields, nonstandard techniques need to be used
for considerably reducing the echo
times (TEs) (Bergin et al., 1991). Furthermore, respiratory and cardiac
motion has to be taken into account.
For instance, by using projection encoding MRI protocols combined with
scan-synchronous ventilation, highresolution images of the rat (Gewalt et
al., 1993) and the guinea pig lung
(Shattuck et al., 1997) have been obtained.
An easier approach based on the
gradient-echo sequence was used recently for the detection of lung inflammation after allergen challenge in the
rat (Beckmann et al., 2001a). Potential image artifacts caused by cardiac
and respiratory movements were suppressed by averaging. Neither respiratory nor ECG triggering was applied,
and the rats could respire freely during data collection. At a TE of 2.7 ms,
the signal from lung parenchyma was
too weak to be detected at 4.7 T. The
absence of detectable lung parenchyma signal in combination with a
background free of artifacts provided
high contrast-to-noise ratio for the detection of edema associated with pulmonary inflammation. This is clearly
evident in Figure 3A, which reveals
edema formation detectable 6 hr after
intratracheal challenge with 0.3
mg/kg ovalbumin (OA). The edema
progressed to a maximum at 48 hr after
OA exposure and was clearly receding
by 72 hr. The mean time course of
edema formation after challenge with
saline or different doses of OA is shown
in Figure 3B. A dose-related response
was observed, since higher doses of OA
produced larger edema and, after 1
mg/kg OA, the maximum edema occurred at 24 hr, whereas for lower doses
the maximum appeared later, around
48 hr after challenge. With the doses
used, edema was observed for approximately 100 hr. No edema was detected
after application of saline. MRI also revealed that budesonide, a potent glucocorticosteroid, could significantly accelerate edema resolution if given 24 hr
after allergen challenge (Figure 3C).
An important finding in this study
was that changes in edema volume
measured by MRI and protein concentration (a marker for plasma extravasation into the lungs) in the lung
RESEARCH ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 89
fluid obtained by bronchoalveolar lavage followed a similar time course
(Beckmann et al., 2001a). Thus, after
the pulmonary edema formation,
noninvasive MRI provides a reliable
means for assessing pulmonary inflammation after an allergen challenge. Furthermore, therapy response
can be evaluated in individual animals, illustrating the potential of MRI
for the profiling of anti-inflammatory
drugs both in animal models of
asthma and in the clinic.
Phenotyping Transgenic
Animals
The study of the effects of specific
gene products on the pathophysiology
has been enabled by the availability of
knockout/transgenic technology in
the mouse, allowing manipulation of
the expression of specific genes and
their particular translational products
in this species. In the context of modern pharmacologic research and drug
development, transgenic mice are of
importance both as disease models
and for target validation. Phenotyping
of genetically engineered animals is
essential, preferentially by using a
noninvasive method. MR techniques
are being increasingly used to assess
phenotypes in models of neurodegenerative disorders (Cohen et al., 1999;
Ferrante et al., 2000), cardiovascular
disorders (Franco et al., 1999; Chacko
et al., 2000; Omerovic et al., 2000;
Wiesmann et al., 2000), metabolic disorders (Clapham et al., 2000), and
stress (Beckmann et al., 2001b). Currently, several centers are being established worldwide with the aim of phenotyping transgenic mice by using
high throughput MR techniques. The
following example illustrates the advantages of using MRI in this area.
Numerous genes are induced by ce-
Figure 3. Lung inflammation in the rat due
to allergen challenge. a: Representative
sections through the rat chest, obtained
previous to and at various time points after
i.t. instillation of 0.3 mg/kg ovalbumin (OA).
Mean edema volume (⫾ SEM) as a function
of time after i.t. challenge with (b) saline or
various doses of OA, (c) 0.3 mg/kg OA in
untreated animals and animals treated i.t.
with budesonide, administered 24 hr after
challenge. Adapted from Beckmann et al.
(2001a) with permission of the publisher.
90 THE ANATOMICAL RECORD (NEW ANAT.)
Figure 4. Coronal maximum intensity projections of three-dimensional angiograms (16.4min acquisition time) of the brains of mice from three different strains. The middle cerebral
arteries (MCAs) are indicated by red arrows, whereas the anastomoses at the level of the
posterior communicating artery (PcommA) are indicated by yellow arrows. Although the
CD1 mice showed well-developed, albeit unilateral, PcommA, this anastomosis was less
well developed in C57BL/6 and SV-129 mice. The MCAs in the SV-129 strain were significantly
shorter than in other strains. Adapted from Beckmann (2000) with permission of the
publisher.
rebral ischemia, such as genes encoding the heat shock protein hsp72 (Kinouchi et al., 1993), the cytokines
interleukin-1, interleukin-6, and tumor necrosis factor-␣ (Buttini et al.,
1996; Wiessner et al., 1993), and the
immediate-early genes c-fos, c-jun,
and junB (Kinouchi et al., 1994).
Transgenic mice are being used to investigate ischemia-induced genomic
responses in the brain, to clarify the
mechanisms of inherent degenerative
or protective processes, and to define
targets for specific modulations of
these cellular responses. Several approaches exist to induce focal cerebral
ischemia in mice, e.g., permanent occlusion of the middle cerebral artery
(MCA) and transient occlusion of the
MCA with an intraluminal thread. The
last is of special interest because of
the possibility of studying the consequences of reperfusion. In this
method, a thread is advanced from
the internal carotid artery into the anterior cerebral artery, until its tip covers the MCA. Because many factors
can lead to inconsistent vascular occlusion, e.g. variable thread diameter
and vessel size, as well as biological
variability, reproducible ischemia requires precise standardization of experimental conditions.
Differences in vascular anatomy
have to be taken into account when
cerebral ischemia is produced in a
transgenic or knockout mouse (Kitagawa et al., 1998; Maeda et al., 1998).
In particular, it has been shown that
the patency of the posterior communicating artery (PcommA), which connects the superior cerebellar and the
posterior cerebral arteries, is a crucial
determinant of ischemia in the
PcommA territory after intraluminal
suture occlusion (Kitagawa et al.,
1998). This patency, although being
always present in rats, is highly variable and strain-dependent in mice
(Barone et al., 1993). Furthermore,
the supplying territory of the MCA,
which determines the size of the infarcted area after intraluminal occlusion, has also been shown to be straindependent (Maeda et al., 1998).
Recently, high-resolution MR angiography (MRA) has been used to verify
the success of transient MCA occlusion
and reperfusion in rats (Reese et al.,
1999) and mice (Beckmann et al.,
RESEARCH ARTICLE
1999). By using 3D time-of-flight (TOF)
techniques (based on the suppression
of stationary tissue by saturation of the
longitudinal magnetization; the degree
of signal enhancement strongly depends upon the velocity of the blood
and the interval between radio-frequency pulses), the cerebral arterial
vascular structure could be detected
with high contrast in both species in
acquisition times ranging between 8
and 25 min, without the application of
contrast agents. Also, MRA revealed
highly variable arterial cerebrovascular
structures in mice from different strains
and within the same strain (Beckmann,
2000). Of special interest was the detection of differences at the level of the
PcommA. Furthermore, SV-129 mice
showed significantly shorter MCAs
compared with other strains (Figure 4).
C57BL/6 and SV-129 mice have
been the most widely used parent
strains for the production of mutants.
Because of the different vascular architectures of these strains, differences in infarct volume after targeted
gene mutation could well be confused
with differences in the genetic background of the parent strains. Therefore, it is important to confirm that
independent variables, as differences
in vascular architecture, do not interfere with the result of gene targeting.
Visualization of the vascular architecture by MRA provides a means of detecting and refuting the existence of
such interactions. Also, noninvasively
assessing whether and on which side
of the circle of Willis the PcommAs
are present could be of help in selecting the animals and/or the side for
performing the MCA occlusion. Presurgery MRA evaluation could potentially help to improve the reproducibility of murine cerebral infarction
studies.
In summary, anatomic MRI has become an indispensable tool for qualitative and quantitative tissue characterization, including the identification
of pathomorphologic changes. The
method is inherently 3D and yields
highly accurate morphometric data.
PHYSIOLOGIC MRI: DYNAMIC
ASSESSMENT OF FUNCTIONAL
PARAMETERS
Physiologic MRI refers in a broader
sense to the mapping of physiologic
RESEARCH ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 91
nal-to-noise ratio of the signal of the
gas in the lungs, also that the background signal is nonexistent.
Pharmacologic Stimulation of
the Central Nervous System
Figure 5. Hyperpolarized 3He coronal slice of the rat lungs (right) with an enlarged view of
the right upper lobe from the same image (left). This radial projection was acquired during
multiple breath holds after full inspiration. Courtesy of G.A. Johnson, Duke University.
parameters such as lung ventilation,
tissue perfusion and oxygenation,
myocardial motion, tracer clearance
and uptake, metabolism, and neural
activity. Its relevance for the characterization of pathologies lies in the
fact that changes in physiologic parameters generally precede morphologic alterations. Performing functional studies in small rodents is
particularly challenging due to the demands on spatial and temporal resolution as physiologic processes (e.g.,
heart rate, ventilation, bolus passage
in perfusion studies) are considerably
faster than in humans. The following
examples should illustrate that, nonetheless, functional studies with MRI
are playing an increasing role in pharmacologic research.
Lung Ventilation
Gas exchange is the major function of
the lung. Matched distribution of the
regional pulmonary blood flow (perfusion) and ventilation is necessary for
this process to occur efficiently. The
large amount of ventilation in the
lungs is matched by a correspondingly
high perfusion. The balance between
pulmonary perfusion and ventilation
can be altered in disease states. Accurate assessment of perfusion and ventilation is especially important in the
evaluation of patients with suspected
pulmonary embolism, a common and
life-threatening disorder that can be
treated by anticoagulants.
Recently, MRI of the lungs after in-
halation of hyperpolarized gases (Albert et al., 1994; Middleton et al.,
1995; Black et al., 1996; Kauczor et al.,
1996; Wagshul et al., 1996; de Lange
et al., 1999) has aroused interest
among the medical community because ventilation defects can be
clearly identified on the images. In addition, dynamic ventilation images,
which provide regional information
about the gas dynamics in the lungs,
can be obtained with high temporal
resolution with this technique (Johnson et al., 1997; Saam et al., 1999;
Viallon et al., 1999, 2000). Information obtained from dynamic ventilation images are complementary to
static, breath-hold ventilation images.
These methods are of interest in the
context of pharmacologic research
also, in models of pulmonary diseases
like asthma and emphysema in small
rodents (Hedlund et al., 1999). To perform such studies, mechanical ventilation becomes mandatory to maintain normal gas exchange and to
control breathing motion. Furthermore, ventilators must be able to deliver hyperpolarized gases in synchrony with the imaging process
without contributing to unnecessary
depolarization. Figure 5 shows a hyperpolarized 3He coronal slice of the
lungs of a rat, obtained by triggering
the image acquisition by using an MRcompatible ventilator (Hedlund et al.,
2000). The main advantage of using
hyperpolarized gases is the good sig-
In normal mammalian brain, activity
is closely coupled to metabolic rates
of oxygen and glucose and to cerebral
blood flow (CBF). These couplings are
the basis for methods used to noninvasively assess regional brain activity,
such as PET and functional MRI
(fMRI). The major focus of fMRI is
presently directed toward human
brain mapping (Zeineh et al., 2001).
However, the high spatial and temporal resolutions achievable make fMRI
an attractive tool to study brain function in laboratory animals as well. Local activation in sensory cortical areas
has been demonstrated in healthy,
anesthetized rats after electrical, somatosensory stimulation during normocapnia (Gyngell et al., 1996; Kerskens et al., 1996; Yang et al., 1996)
and hypercapnia (Bock et al., 1998),
and to monitor functional recovery after cardiac arrest (Schmitz et al.,
1998).
In another attractive application
of fMRI in the rat brain, hemodynamic changes reflecting local and
temporal changes of brain activities
after administration of CNS stimulatory or inhibitory drugs can be measured. Such studies may contribute
to a better understanding of the sites
and mechanisms involved in the action of CNS drugs. Brain stimulations by physical or pharmacologic
means, once elaborated and characterized, could be used for both differential diagnosis of neurologic or
psychiatric disorders, as well as for
evaluating the efficacy of therapeutic
interventions.
The feasibility of this approach has
been demonstrated by several groups.
Stein et al. (1998) used fMRI to demonstrate the acute CNS effects of nicotine in smokers. Nicotine induced a
dose-dependent increase in neuronal
activity in a distributed system of
brain regions, including the nucleus
accumbens, amygdala, cingulate, and
frontal lobes. In a rat model of Parkinson disease, in which the striatum was
lesioned unilaterally by injection of
6-OH-dopamine, no response to am-
92 THE ANATOMICAL RECORD (NEW ANAT.)
Figure 6. Regional activation in the rat brain by bicuculline. Changes in cerebral blood
volume as a function of time for cortex, caudate putamen, thalamus, and cerebellum.
Bicuculline infusion at a dose of 1 mg/kg per min for the first 2 min (gray bar) and 0.1 mg/kg
per min for the subsequent 10 min (light gray bar). Adapted from Reese et al. (2000a)
with permission of the publisher.
phetamine injected systemically was
observed in the lesioned area in contrast to the unlesioned striatum, an
observation that was paralleled by
PET data (Chen et al., 1997). A related
study was carried out in rhesus monkeys with unilateral striatal 4-phenyl1,2,3,6-tetrahydropyridine hydrochloride (MPTP) lesions showing altered
responses to stimulation with levodopa in the affected area (Chen et al.,
1996).
Recently, Reese et al. (2000a) described transient changes in local cerebral blood volume (CBV) in the rat
brain caused by i.v. administration of
the GABAA antagonist bicuculline.
Upon infusion of bicuculline, regionspecific increases in CBV occurred, reflecting brain activity (Figure 6). During the first 2 min, increased CBV was
predominant in the cortex, followed
by increases in other brain areas. Ten
minutes after the start of the infusion,
a dominant response was observed in
the thalamus, whereas CBV in the
caudate putamen showed a biphasic
response pattern (Reese et al., 2000a).
The magnitude of the signal responses
in all brain regions was dependent on
the bicuculline dose. Similar results
were obtained for the mouse brain
(Mueggler et al., 2001). These rodent
studies show that pharmacologic
fMRI, displaying brain function at the
highly specific level of drug-receptor
interaction, should foster our understanding of normal and pathologic
brain function (see also “Mapping of
Ligand-Receptor Interactions,” below). However, because anesthesia
can affect the results, e.g., when probing cortical responses in the brain due
to somatosensory stimuli, the complex pharmacology of anesthetics as a
result of their interaction with several
different receptor systems (Franks
and Lieb, 1999) has to be considered
when testing compounds in fMRI experiments.
Functional Studies in Stroke
Research
Cerebral infarction is one of the leading causes of mortality and morbidity
in industrialized countries. Despite
major research efforts in both academia and industry, there is to date no
generally accepted therapy.
Several animal models of infarct do
exist. The application of various MRI
approaches using the multiparametric character of MRI contrast mechanisms allows a comprehensive characterization of the pathophysiologic
cascade (Figure 7). Determination of
the link between the observed changes
in MRI contrast parameters and physiologic events is essential to address
mechanistic aspects. As an example,
consider the permanent MCA occlusion (pMCAO) model in rats (Tamura
et al., 1981), which is routinely used to
test drugs with various mechanisms
aimed at reducing the extent of cellu-
RESEARCH ARTICLE
lar damage in the acute phase of
stroke (Sauter and Rudin, 1986). MRA
allows to assess the success of vascular
occlusion (Beckmann et al., 1999; Reese et al., 1999). Perfusion-weighted
MRI provides information on changes
in CBF (Rudin and Sauter, 1991;
Kohno et al., 1995; Rudin et al., 1997),
whereas methods exploiting blood-oxygenation-level– dependent (BOLD) contrast assess relative tissue oxygenation
(Roussel et al., 1995). Diffusionweighted imaging (DWI) enables early
(within minutes after MCA occlusion)
detection of cerebral changes preceding
infarction (Moseley et al., 1991; Mori
and Barker, 1999). A significant decrease of the apparent diffusion coefficient (ADC) of water is observed in the
ischemic region. The observed changes
in ADC have been associated with the
formation of cytotoxic edema: as the
intracellular ADC value is smaller than
the extracellular, an increase of the intracellular volume fraction leads to a
reduction of the total ADC (Benveniste
et al., 1992). With T2-weighted sequences, a high contrast between infarcted (edematous) and healthy tissue
is obtained at 24 or 48 hr after MCA
occlusion, allowing quantitative analysis of infarct volumes. fMRI studies involving, e.g., electrical or pharmacologic stimulation are used to probe the
functional recovery of infarcted tissue
after treatment. The time course of
these parameters in a model of MCA
occlusion in the rat has been discussed
in detail by Hoehn-Berlage et al. (1995)
and Rudin et al. (2001).
In the pMCAO model of stroke, the
effectiveness of a therapy is commonly judged from infarct size measurements, assuming that a reduction
in infarct size results in reduction of
the functional deficits. Reese et al.
(2000b) evaluated the concept that
structural integrity translates into
functional integrity during the acute
post-stroke period (24 hr). The functional integrity of the somatosensory
cortex was assessed by detecting
changes in local CBV after electrical
stimulation of the forepaws and pharmacologic stimulation by bicuculline
(see previous section). In sham-operated rats, simultaneous electrical
stimulation of both forepaws led to
similar activation of both somatosensory cortices, whereas in pMCAO animals, only the contralateral cortex
RESEARCH ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 93
Figure 7. Middle cerebral artery (MCA) occlusion model of stroke in the rat. The multiparametric nature of MRI is explored to follow the
course of pathophysiology in this model. Brain activity mapping was derived from experiments involving electrical stimulation of both
forepaws. FMRI, functional MRI.
94 THE ANATOMICAL RECORD (NEW ANAT.)
RESEARCH ARTICLE
showed an fMRI response. Similarly,
in pMCAO rats treated with the calcium antagonist isradipine, functional activation after bilateral electrical stimulation was only detected in
the contralateral somatosensory cortex, despite the normal appearance of
the ipsilateral cortex in T2-weighted
and diffusion-weighted images. Furthermore, fMRI responses to pharmacologic stimulation with bicuculline
were virtually absent in the ipsilateral
somatosensory cortices both in vehicle- and isradipine-treated animals
(Reese et al., 2000b). These results
correlated well with behavioral studies (beam-walking test) carried out
with the same rats. Thus, structural
integrity in the somatosensory cortex
revealed by anatomic MRI does not
necessarily translate into preservation
of function in the acute (24 hr) postocclusion period. Probing functional
integrity by fMRI in addition to structural assessments by MRI could become an integral part of routine drug
testing in stroke models.
Kidney Transplantation
Organ transplantation is currently the
preferred procedure in end-stage organ
disease; although, because the introduction of cyclosporine immunosuppressive therapy acute rejection can be
well managed, chronic rejection remains the main complication. This rejection involves both the reaction of the
host to the graft (mainly T-cell mediated and also antibody — B-lymphocyte-driven) and reactions in the graft
toward injury (vasculopathy or graft
vessel disease in, e.g., kidney and heart
grafts, bronchiolitis obliterans in lung
grafts). The first reaction asks for improved immunosuppressants including
tolerance-inducing approaches, the second one for interventions to protect the
graft (e.g. endothelial cells) from damage like ischemia/reperfusion injury or
for treatment of vessel disease (Olsen,
1991; Cook et al., 1998).
An important animal model of kidney
transplantation is orthotopic kidney allografting in rats by using the strain
combination of DA into Lewis. This
strain combination is generally considered to be very stringent, because of the
major histocompatibility mismatch between donor and recipient, and the
strong alloreactive potential of Lewis
Figure 8. Noninvasive assessment of graft function in a DA to Lewis model of kidney
transplantation (tx) by MRI. a: Perfusion MRI. A series of MR images is acquired sequentially
with a time resolution of 800 ms per image. After acquiring baseline images, a contrast
agent containing iron nanoparticles is injected i.v. during 1 sec. The signal attenuation is
proportional to the perfusion in the organ. b: Clearance of an extracellular contrast agent
(Gd-DOTA). Similarly to perfusion MRI, images are acquired sequentially (time resolution of
6 sec per image). After acquisition of baseline images, the contrast agent is injected i.v.
during 2 sec. Signal enhancement for native, normal kidneys (left) and for a rejected kidney
(right). Adapted from Beckmann et al. (2000) with permission of the publisher.
rats. If left untreated, animals with
acute rejection die at around day 11 due
to the loss of graft function. Long-term
survival of the transplant (up to 130
days) is achieved in recipients treated
with cyclosporine starting from the day
of transplantation until day 14 (Borel,
1989). With the aim of determining the
efficacy of immunosuppressive treatments, anatomic and perfusion MRI as
well as MR renography were used as a
means to assess noninvasively graft status in this model (Beckmann et al.,
1996, 2000; Schuurman et al., 1996;
Baumann and Rudin, 2000).
Perfusion MRI involving the i.v. ad-
RESEARCH ARTICLE
THE ANATOMICAL RECORD (NEW ANAT.) 95
procedures to analyze spectra of body
fluids, e.g., of urine, to evaluate the
nephrotoxic effects of drugs (Anthony
et al., 1994; Holmes et al., 1995).
MOLECULAR MRI: IMAGING THE
TARGET
Figure 8. (Continued)
ministration of an intravascular contrast agent (CA) reveals that tracer
concentration in a rejected kidney is
significantly lower than that in native,
normal kidneys (Figure 8A), demonstrating a compromised perfusion of
the graft due, e.g., to vasculopathy.
Furthermore, applying MR renography techniques (Baumann and Rudin,
2000), signal enhancements in the
cortex and medulla of a rejected kidney after i.v. application of an extracellular CA were significantly smaller
than those observed in native organs
for the same amount of CA administered. In particular, almost no signal
enhancement occurred in the pelvis,
demonstrating that the clearance of
the extracellular contrast agent was
severely compromised in the rejected
graft (Beckmann et al., 2000). Of importance is that changes in perfusion
and tracer clearance occurred before
anatomic changes. These functional
parameters are, therefore, sensitive
markers of early kidney rejection.
Physiologic MRI allows the assessment
of pathophysiological alterations. As
these changes frequently precede structural pathologies, physiologic MR readouts have been shown to serve as early
biomarkers of the disease process and of
therapy response. Although not yet systematically pursued, anatomic and functional MRI, as well as MR spectroscopy
readouts, may also add value to identifying toxicologic effects of drugs. An interesting approach in this regard is
high-resolution MR spectroscopy in
combination with pattern recognition
MRI has evolved over the past decade
from a radiologic technique to a
method that provides information on
physiologic tissue parameters, e.g., tissue blood flow or oxygenation. Presently, MRI is evolving toward molecular or target-specific imaging, i.e., the
mapping of ligand-target interactions.
Target-specific imaging providing direct insight into biological processes at
the molecular or cellular level is an important aid in addressing fundamental
questions in biomedical research. Because of its high sensitivity and wider
availability, PET is currently (and for
the foreseeable future) the method of
choice in the area of molecular imaging. However, PET has the drawback of
requiring the design and synthesis of a
suitable ligand, which has to be prepared in situ because of the short halflife of the positron-emitting nuclei.
Thus, in certain instances, MRI with its
superior spatial resolution might be exploited either by following the PET concept of using magnetically labeled ligands or by mapping the consequences
of a ligand-receptor interaction on
physiological tissue parameters. Both
avenues are going to be illustrated here.
A strength of MRI is its ability to allow
direct comparison of anatomic and molecular images.
Magnetically Labeled Ligands
Paramagnetic gadolinium (Gd) complexes such as Gd-DTPA, which penetrate the extracellular space and primarily alter the T1 values of tissues, and
superparamagnetic iron oxide preparations, which exhibit a susceptibility that
is two to three orders of magnitude
larger than that of paramagnetic complexes, are the contrast agents commonly used in MRI. By coupling them
to target-specific ligands, receptor-specific CAs have been synthesized. For instance, a liver-specific CA was obtained
by coupling iron nanoparticles to asialofetuin, a ligand for the hepatic asialoglycoprotein receptor (Reimer et al.,
1991). Coupling to secretin yielded pref-
96 THE ANATOMICAL RECORD (NEW ANAT.)
Figure 9. In vivo magnetic resonance image of a mouse with engineered transferrin receptorpositive (ETR⫹, left) and ETR⫺ (right) flank tumors. Composite image of a T1-weighted spin-echo
image with superimposed contrast changes after Tf-MION administration. Inserts: Histological
analysis of ETR expression, showing iron staining in ETR⫹ (left) and ETR⫺ (right) tumor cells.
Courtesy of R. Weissleder, Harvard University. Adapted from Weissleder et al. (2000) with
permission of the publisher.
erential accumulation of the CA in the
pancreas because of the high affinity to
the secretin receptors, which are abundant in exocrine pancreatic cells (Shen
et al., 1996).
Lewin et al. (2000) labeled stem and
progenitor cells ex vivo with superparamagnetic iron oxide nanopar-
ticles that had been coated with dextran covalently bound to peptide
sequences from the HIV-derived Tat
protein. The cells were injected into
immunodeficient NOD/SCID mice.
The Tat-derived sequences enabled
the paramagnetic label to be efficiently internalized into target cells,
RESEARCH ARTICLE
which were tracked by MRI in harvested bone marrow and liver.
Similarly, Bulte et al. (1999) tracked
ex vivo cell migration and myelination
in the spinal cord of myelin-deficient
rats, into which magnetically labeled
oligodendrocyte progenitor cells were
transplanted. Cell migration could be
easily detected by using 3D MRI, with
a close correlation between the areas
of contrast enhancement and the
achieved extent of myelination. By allowing the direct imaging of cell trafficking, this approach may not only
provide information for the optimization of stem cell-based therapies, but
also advance our basic understanding
of developmental biology, pathology,
and general pharmacology.
Real-time imaging of gene expression
in vivo at high spatial resolution has
been a long-lasting goal in molecular
research. If such techniques were available, both endogenous and exogenous
(e.g., gene therapy) gene expression
could be studied in live animals and
potentially in the clinic. The feasibility
of such an approach was demonstrated
recently by Weissleder et al. (2000),
who used an engineered transferrin receptor (ETR) as a reporter that shuttled
targeted superparamagnetic nanoparticles into cells. The internalizing receptor used was a human transferrin receptor that lacked the iron-regulating
region and mRNA destabilization mo-
Figure 10. Comparison of functional magnetic resonance imaging-derived maps of change in cerebral blood volume with the density of
GABAA binding sites as derived from autoradiography (values taken from Chu et al., 1990). Adapted from Reese et al. (2000a). Reproduced
with permission of the publisher.
RESEARCH ARTICLE
tifs in the 3⬘ untranslated region and,
therefore, constitutively overexpressed
high levels of the receptor in the cell
(Casey et al., 1988). The receptor-targeted MR reporter consisted of 3-nm
monocrystalline iron oxide nanoparticles (MION), sterically protected by a
layer of low-molecular-weight dextran
(Shen et al., 1993) to which human
holo-transferrin was covalently conjugated (Tf-MION). To demonstrate that
ETR expression correlated with increases in internalized Tf-MION, nude
mice were implanted with both stably
transfected (ETR⫹) and control transfected (ETR⫺) 9L gliosarcoma cells
(Weissleder et al., 2000). Only transfected tumors (ETR⫹) overexpressed
the engineered transferrin receptor and
had more iron. This accumulation of
iron was detected in vivo by T2- and
T2*-weighted sequences, reflecting the
increased transverse relaxation rate after cellular internalization (Figure 9).
A further example is the visualization
by MRI of leukocyte adhesion molecules in the intact brains of mice with
experimental autoimmune encephalitis
(EAE). Antibody-conjugated paramagnetic liposomes (ACPLs) targeted to intercellular adhesion molecule-1 (ICAM1), an endothelial leukocyte receptor
up-regulated on cerebral microvasculature during EAE expression, were administered to diseased mice (Sipkins et
al., 2000). High-resolution MRI of
mouse brains ex vivo demonstrated that
ACPL binding conferred significant enhancement compared with control images, suggesting that ACPLs can be
used as CAs to visualize specific molecules expressed on vascular endothelium during disease.
Another approach is the use of
switchable CAs (Moats et al., 1997),
which consist of a chelator with highaffinity binding to Gd and a galactopyranose residue positioned to block the
remaining coordination site on the Gd
ion from water. In this water-inaccessible conformation, the CA is inactive and
does not strongly modulate T1. The enzyme ␤-galactosidase cleaves the sugar
residue from the chelate, causing the
irreversible transition of the CA to an
active state, in which it increases relaxivity. By using switchable CAs, ␤-galactosidase mRNA expression in living X.
laevis embryos was detected by MRI
(Louie et al., 2000). By using genetic
engineering approaches, ␤-gal can be
THE ANATOMICAL RECORD (NEW ANAT.) 97
coupled to any promoter sequence;
thus, in principle, the method described
by Louie et al. (2000) could be a generic
tool for imaging of gene expression. The
problems that remain to be solved are
cellular uptake of the CA and potential
cytotoxicity.
Mapping of Ligand-Receptor
Interactions
As mentioned previously, another avenue of molecular imaging consists in
mapping the consequences of a ligand-receptor interaction on physiological parameters. The example already discussed above, namely the
visualization of brain activation by
bicuculline, is used as an illustration
of this approach. Figure 10 presents a
map representing the integrated CBV
changes in the rat brain in the 20 min
In pharmaceutical
research, only MR
applications that offer
substantial advantages
over existing
technologies will
be successful in
the long term.
after i.v. administration of bicuculline
(Reese et al., 2000a). The density of
GABAA binding sites taken from Chu
et al. (1990) is also displayed for comparison. There was a good correlation
between the degree of CBV changes
and the receptor densities in the midand forebrain. Both values were high
in frontal and parietal cortex as well
as in thalamic nuclei, medium in the
caudate putamen and lateral septal
nucleus, and low in the hippocampal
regions and other midbrain nuclei.
Some discrepancy was observed in
the cerebellum, where intermediate
density of binding sites was reported
for the molecular layer and very high
density for the granule cell layer. By
using this approach, the complex pharmacology of the ionotropic GABA receptor system (Bormann, 2000) would
be amenable to an in vivo analysis.
It is clear that molecular imaging
will have a significant impact on biomedical research as well as on drug
discovery and development (Rudin,
2000). Imaging the target; its location
in cells, tissues, or in the body; its
interaction with other molecules (e.g.,
substrate-target interactions, receptor
occupancy); and the functional consequences thereof will be important not
only in preclinical research but also in
clinical work. The key success factor
in such studies is the design of the
appropriate labeled ligand rather than
the imaging technology itself. The
concerted interdisciplinary efforts of
chemists, molecular biologists, and
imaging experts are essential for making full use of the advantages offered
by molecular imaging techniques. For
MRI in particular, the main issue is
coupling nanoparticles to bioactive
peptidic ligands without significantly
altering their receptor affinity and
specificity. This method should be feasible in favorable cases as shown by
the previous examples; the study of
general drug-receptor interactions,
however, is a major challenge.
The principal limitations of molecular MRI are the inherent low sensitivity
and the requirement of relatively bulky
para- or superparamagnetic reporter
groups. Despite that exciting MRI applications have been demonstrated to
date, target-specific imaging will be the
domain of more sensitive imaging modalities (e.g., PET and optical imaging).
OUTLOOK
MR methods have become an established tool in biomedical and pharmaceutical research, covering a broad
range of applications from noninvasive morphologic measurements to
comprehensive studies of tissue physiology and metabolism. Both in the
clinical environment and in animal
experimentation, MR methods are in
competition with alternative, highly
developed analysis tools. In pharmaceutical research, only MR applications that offer substantial advantages
over existing technologies will be successful in the long term.
The strengths of MR methodologies
are noninvasiveness and the multiparametric character of the approach.
This has been illustrated, for instance,
with the stroke study (Figure 7), i.e.,
98 THE ANATOMICAL RECORD (NEW ANAT.)
information on local brain perfusion,
local CBV, oxygenation deficit, cytotoxic and vasogenic edema, and functional responsiveness can be obtained
with high spatial and temporal resolution in the same animal. Many other
applications that exploit this versatility will be forthcoming.
A prerequisite for studying chronic
diseases both in humans and animals is
noninvasiveness. In preclinical research, apart from the obvious statistical advantages when monitoring disease progression and therapy response
in an individual, animal welfare and
economical aspects need also to be
taken into account. In long-term studies
of chronic disorders — for which animals are expensive — it is economically
attractive to use a noninvasive readout.
The number of animals per study is reduced and the information obtained is
refined, because additional studies can
be performed in the same animal. This
is especially important when working
with higher species, e.g., nonhuman
primates. Noninvasive technologies are
also highly desirable when dealing with
transgenic animals. Given the relevance
of transgenics to modern biomedical
and pharmaceutical research, the number of studies involving MR methods in
the characterization of novel phenotypes in these animals will rapidly increase.
The development of biomedical MR
methods has been mainly driven by
their clinical applications. In this context, preclinical research is lagging behind, mainly because the need for using noninvasive techniques is less
imperative. However, a major advantage of using noninvasive technologies in animal research is the possibility of directly linking preclinical to
clinical findings through relevant imaging biomarkers. This link will become even more important, as there is
a strong need to further validate animal models of human disorders, e.g.,
multiple sclerosis, stroke, and dementias.
The link between preclinical characterization of development compounds in animal models and clinical
testing of drugs is also becoming
tighter. In particular, there is a generalized strong tendency toward early
pharmacodynamic studies in man, to
assess whether new compounds are
exerting the same pharmacologic ef-
fects in patients as in animals. Such
proof-of-concept studies aim at guiding the dose selection for the extensive
and expensive clinical efficacy trials.
Importantly, the safety of human subjects would not likely be compromised
by these imaging methodologies. In
this sense, it is foreseeable that preclinical studies involving MR methods
and MR-derived biomarkers will play
an increasing role in the validation and
optimization of clinical protocols. MR
methods will strengthen the link between the two intimately connected —
however, in practice, often too distant
— areas of preclinical and clinical research. This should ultimately be one of
the main contributions of MR in pharmaceutical research.
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