Myelin imaging compound (MIC) enhanced magnetic resonance imaging of myelination

Article abstract of DOI:10.1021/jm201010e

The vertebrate nervous system is characterized by myelination, a fundamental biological process that protects the axons and facilitates electric pulse transduction. Damage to myelin is considered a major effect of autoimmune diseases such as multiple scle

Full text of DOI:10.1021/jm201010e

Article
pubs.acs.org/jmc
Myelin Imaging Compound (MIC) Enhanced Magnetic Resonance
Imaging of Myelination
Luca Frullano,‡ Junqing Zhu,‡ Changning Wang,‡ Chunying Wu,‡ Robert H. Miller,§
and Yanming Wang*,‡
§
‡Department of Radiology, Case Center for Imaging Research, Division of Radiopharmaceutical Science and Department of
Neuroscience, Case Western Reserve University, Cleveland, Ohio 44106, United States
ABSTRACT: The vertebrate nervous system is characterized
by myelination, a fundamental biological process that protects
the axons and facilitates electric pulse transduction. Damage to
myelin is considered a major effect of autoimmune diseases
such as multiple sclerosis (MS). Currently, therapeutic
interventions are focused on protecting myelin integrity and
promoting myelin repair. These efforts need to be
accompanied by an effective imaging tool that correlates the
disease progression with the extent of myelination. To date,
magnetic resonance imaging (MRI) is the primary imaging
technique to detect brain lesions in MS. However, conventional MRI cannot differentiate demyelinated lesions from other
inflammatory lesions and therefore cannot predict disease progression in MS. To address this problem, we have prepared a Gd-
based contrast agent, termed MIC (myelin imaging compound), which binds to myelin with high specificity. In this work, we
demonstrate that MIC exhibits a high kinetic stability toward transmetalation with promising relaxometric properties. MIC was
used for in vivo imaging of myelination following intracerebroventricular infusion in the rat brain. MIC was found to distribute
preferentially in highly myelinated regions and was able to detect regions of focally induced demyelination.
that study, the magnitude of the treatment effect on MRI and
clinical outcomes is quantitatively different, with 38.9% of the
treated group demonstrating confirmed progression in
expanded disability status scale despite stabilization of total
lesion volume and a reduction in new lesion activity of 57.3%. It
concluded that “... the modest overall nature of the clinical−
MRI correlations suggests that it would be unwise to rely on
measurement based on T2-weighted or Gd-enhanced lesions
alone as the primary efficacy variables”.3
To address this problem, a number of advanced MRI
methods are being developed, which promise to increase
selectivity and specificity and to provide more detailed
information about MS pathology. Among them, magnetization
transfer (MT) has been shown to be sensitive to changes in
myelin content.4,5 Since MT is dependent on the specific pulse
sequence and hardware, “quantitative magnetization transfer”
methods6−9 have also been developed that allow the
quantification of the fractional size of the pool of protons
whose diffusion is restricted by myelin, which could be
ultimately related to the degree of myelination.10 Furthermore,
the quantitative measurement of the fraction of water that is
associated with myelin (myelin water fraction or MWF) has
been used to obtain an indirect assessment of the level of
myelination. The MR water signal in the nervous systems arises
from three components with distinctively different T2: (i)
INTRODUCTION
■
In the vertebrate nervous system, rapid and efficient signal
transduction of nerve impulses is fostered by the presence of
myelin sheaths, which wrap around axons and provide electrical
insulation. Myelin is composed of a complex mixture of lipids
and proteins, with the lipids accounting for 70−85% of its dry
weight and the remaining being composed of proteins.1
Disruption of myelination is a major event in many acquired
or inherited neurodegenerative diseases such as MS and various
leukodystrophies. MS is characterized by demyelination in the
central nervous system (CNS), which affects an estimated 350
000 people in the U.S. and 2 million people worldwide.2
Current diagnosis, prognosis, and therapeutic interventions of
MS intimately depend on the ability to assess myelin changes in
the brain. To date, MR imaging has been used as the first-line
modality for noninvasive detection of brain lesions in MS.
However, conventional MR imaging techniques do not provide
information about the myelination status of the brain. The
hyperintensity observed on T2 weighed images of MS lesions is
primarily related to increased water content and reflects a broad
spectrum of tissue damage, which may be caused by not only
demyelination but also inflammation, edema, Wallerian
degeneration, or axonal loss.
As a result, conventional MRI does not permit differentiation
between demyelination and inflammation. The lesion load
detected by conventional MRI is often dissociated from disease
progression. This dissociation was evidenced by a clinical study
of interferon-β measured by conventional MRI parameters. In
Received: July 28, 2011
Published: November 18, 2011
© 2011 American Chemical Society
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Figure 1. Reaction scheme for the preparation of compound 10, a myelin targeted MR contrast agent.
cerebrospinal fluid (>1.5 s), (ii) intracellular and extracellular
water (∼100 ms), and (iii) MWF (20−50 ms). Quantification
of the shortest T2 component fraction has been related to the
distribution of myelin;11 however, the measurement of the
MWF remains technically challenging. Moreover, the presence
of axon cytoskeletons and myelin membranes leads to
orientationally restricted diffusion of water molecules. This is
exploited in diffusion weighed imaging (DWI) and diffusion
tensor imaging (DTI) for the assessment of anomalies in white
matter diseases.12,13 In general, these new techniques have
improved sensitivity and specificity for the detection of lesions
with respect to traditional MR imaging and provide more
quantitative information to extend our knowledge of MS
processes. However, these new methods have yet to
demonstrate a higher sensitivity than traditional MR to the
detection of MS.
Alternatively, the development of MR contrast agents that
can efficiently and selectively label myelin fibers in living
organisms could improve the sensitivity of traditional and
advanced MR methods for the detection of MS lesions. Until
now, the diagnosis of demyelinating diseases like MS is
obtained following a set of criteria recommended by the
International Panel on MS Diagnosis, which combines a
number of clinical and paraclinical observations. MRI, although
playing a fundamental role in the diagnosis of MS, has not been
accepted as a primary measure of disease activity because of its
lack of specificity.
lesions. However, none of the clinical contrast agents exhibit
any affinity and specificity for myelin; lesion enhancement by
these agents is mainly indicative of disruption of the BBB. No
information can be extracted from these images regarding the
myelination status in detected lesions. Recently gadofluorine, a
fluorinated T1 MR agent, has been described. Currently, it is
being investigated for its ability to detect brain lesions with high
sensitivity in MR images.14,15 Gadofluorine binds extracellular
matrix proteins with high affinity. These proteins become
accessible because of BBB or blood to nerve barrier (BNB)
disruption resulting from inflammatory processes; thus,
gadofluorine enhancement does not reflect the tissue
myelination status. The lack of specificity for myelination
hampers the use of MR for the unequivocal diagnosis of
demyelinating diseases like MS. Subsequently, the use of MRI
as a primary measure of disease activity still has not been
accepted by the Federal Drug Administration (FDA). The
development of myelin-targeting contrast agents is crucial in
order to improve the myelin imaging specificity of MRI for
efficacy evaluation of novel myelin repair therapies currently
under development.
For this reason, we exploited the myelin-specific probes that
have been identified in our laboratory16−23 to develop a myelin-
targeting contrast agent through conjugation with a para-
magnetic Gd complex. Our previous structure−activity relation-
ship studies of coumarin derivatives led us to identify a lead
compound, named case imaging compound (CMC), that
readily crosses the BBB and selectively binds to myelin sheaths
in vivo.17 We found that the structure of CMC can be
selectively modified without adversely affecting its binding
affinity and specificity for myelin. To develop a contrast agent
for MR imaging, we explored the possibility of introducing a
linker in the 3-position so that CMC could be conjugated to a
To date, clinical contrast agents such as GdDTPA (DTPA =
diethylenetriamine pentaacetic acid) are widely used for MR
studies in MS patients. The use of these contrast agents is
possible because of the fact that the blood−brain barrier (BBB)
is often disrupted in MS patient. Thus, GdDTPA enhancement
increases the reliability and sensitivity of detecting active
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gadolinium complex based on a DOTA derived monoamide
macrocyclic ligand (DOTA = 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid). The choice of this ligand was guided
by the need to obtain a highly stable and inert gadolinium
complex to avoid the release of toxic gadolinium aquo ion.
Macrocyclic ligands derived from DOTA are known to provide
gadolinium complexes with a large formation constant and are
particularly inert toward transmetalation with endogenous
cations.24−26 Following these considerations, we have devel-
oped MIC (compound 10 Figure 1), the first Gd-based
contrast agent that specifically binds to myelin. Compound 10
was prepared by conjugation of a gadolinium DOTA
water protons would be negligible. For this reason, the
transmetalation reaction could be followed by monitoring the
proton longitudinal relaxation rate of the solution versus time.
The relaxation rate of a 2.5 mM solution of 10 in the presence
of 2.5 mM ZnCl2 in phosphate buffer (50 mM, pH 7)
incubated at 40 °C is approximately constant over a period of 4
days (Figure 2). This is comparable with the trend shown by
monoamide complex to CMC and exhibits promising MR
properties.27
We have recently shown that 10 accumulates preferentially in
highly myelinated regions in mouse brain tissue blocks. In this
report, we demonstrate the effectiveness of 10 to enhance
highly myelinated brain regions in T1 weighed MR imaging of
live rat models. Our studies show that 10 accumulates
preferentially in myelinated regions after stereotaxic injection
in the lateral ventricles and that T1 shortening can be readily
visualized through MR T1 mapping. Here, we report the full
account of the synthesis of compound 10 and its use as a MR
contrast agent for in vivo imaging of myelination.
Figure 2. Evolution of the relative water proton paramagnetic
longitudinal relaxation rate (R1(t)/R1(t=0)) of three commercial MR
agents: GdDOTA (circle), GdHPDO3A (triangle), and GdDTPA
(square) compared to 10 (diamond) (2.5 mM). All agents are in a
phosphate buffer solution (50 mM, pH 7.0) in the presence of ZnCl2
(2.5 mM). Figure is adapted in part from ref 29.
other clinical macrocyclic complexes such as GdDOTA and
GdHPDO3A (HPDO3A = 10-(2-hydroxypropyl)-1,4,7-tetraa-
zacyclododecane-1,4,7-triacetic acid).
Relaxometric Characterization. The relaxivity of 10 at
three magnetic field strengths was measured in water and
compared to the relaxivities of two clinical MR contrast agents,
GdDOTA and GdDTPA (Table 1). The longitudinal relaxivity
RESULTS
■
Chemical Synthesis. The synthesis of compound 10 is
illustrated in Figure 1. A Perkin condensation of 1 and 2
followed by acid hydrolysis afforded the coumarin derivative 4
in 27% yield. A three-carbon linker was introduced to the 3-
position of 4 by reaction of 4 with Boc-protected 3-
bromopropylamine in acetonitrile at 70 °C in the presence of
cesium carbonate, which afforded 5 in 79% yield. Boc
deprotection under acidic conditions afforded 6 in 93% yield
as a free amine after basic extraction. The lanthanide chelator
was then introduced by reacting DOTA-tris-tert-butyl ester (7)
with 6 using an HBTU-mediated coupling (HBTU = O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphate). The protected ligand 8 was obtained in 66%
yield. tert-Butyl deprotection in neat TFA provided the free
ligand 9 in quantitative yield as a trifluoroacetate salt, which was
then complexed with GdCl3 in water at pH 6 and 50 °C to give
10.
Transmetalation Stability. To determine the transmeta-
lation stability of 10, we used a method developed by Muller
and co-workers to assess the resistance of gadolinium
complexes against transmetalation in a highly competitive
environment.28,29 Thus, a solution of 10 and ZnCl2 in
phosphate buffer was incubated at 40 °C. Gadolinium
phosphate is characterized by a very low solubility product
(Ksp = 10−25.6 M2).30 In the event that Zn(II) displaced the
Gd(III) ions from the coordination cage, the Gd ions would
rapidly precipitate as insoluble phosphate and their contribu-
tion to the observed longitudinal relaxation rate of solvent
Table 1. Longitudinal Relaxivity with Relative Standard
Deviation of Compound 10 and Two Commonly Used Gd
Contrast Agents, GdDOTA and GdDTPA, at Three
a
Magnetic Field Strengths
magnetic field
strength (T)
10, r1 (s−1
GdDOTA, r1 (s−1 GdDTPA, r1 (s−1
mM−1 mM−1
mM−1
)
)
)
b
c
9.4
5.2 0.1
5.1 0.1
5.8 0.2
3.931 4.131
2.9 0.2 3.3 0.2
3.4 0.232 3.4 0.232
d
,
32
d32
,
1.41
0.47
c
a
Relaxivity values for GdDOTA and GdDTPA have been obtained
b
c
d
from the literature. T = 21 °C. T = 40 °C. 1.5 T, T = 37 °C.
(r1) of 10 was determined by measuring the longitudinal
relaxation rate of five solutions of compound 10 with
concentrations in the range 0−1 mM. The relaxation rates
were fitted to eq 1 to obtain the relaxivity of 10. The
longitudinal relaxation rate of the prepared solutions was
measured using a standard inversion−recovery technique,
paying attention to leaving a relaxation delay of at least 5T1
between each successive scan. The longitudinal relaxivity of 10
measured at 0.47 and 1.41 T at 40 °C is 1.5−1.7 times higher
than GdDOTA or GdDTPA. At 9.4 T and 20 °C, the relaxivity
of 10 is approximately 1.3 times higher than those of the other
Gd complexes as shown in Table 1.
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Figure 3. (A) Compound 10 staining of a mouse brain section in vitro showing all the myelinated regions, which was consistent with
immunohistochemical staining of MBP (B) and LFB (C) staining in an adjacent sections. (D) In vitro compound 10 staining of a shiverer mouse
brain section showing remarkably reduced myelination in the corpus callosum, as confirmed by immunohistochemical staining of MBP (E) and LFB
(F) staining in an adjacent section. For comparison, in vitro fluorescent tissue staining with CMC (G) and dMCMC (H) in wild-type mouse brain
sections is presented. Stained areas can be identified in part I: 1 = external capsule, 2 = corpus callosum, 3 = anterior commissure (anterior part), 4 =
caudate putamen (striatum). Scale bar = 1 mm. Reproduced with permission from The Mouse Brain in Stereotaxic Coordinates, 3rd ed.; Franklin, K. B.
J.; Paxinos, G., Copyright 2007 Elsevier.33
In Vitro Staining of Myelin. Compound 10 is a
fluorescent compound with maximal excitation and emission
at 340 and 423 nm, respectively. We thus evaluated myelin-
binding properties of 10 through fluorescent staining in mouse
brain tissue sections. Compound 10 selectively stains
myelinated fibers in myelin-rich white matter regions such as
the corpus callosum, striatum, and cerebellum after chemical
staining of freshly frozen mouse brain sections (Figure 3A).
Compound 10 staining was significantly lower in gray matter
than in myelin-rich white matter. The observed staining pattern
of compound 10 is consistent with that of immunohistochem-
ical staining for myelin basic protein (MBP) (Figure 3B) and
Luxol Fast Blue (LFB) (Figure 3C) staining of adjacent
sections, suggesting that 10 binds selectively to myelin
membranes.
The binding of 10 to myelin was found to be proportional to
the myelin content. This was revealed by staining brain tissue
sections of C3Fe.SWV-Mbpshi/J shiverer mice, an autosomal
recessive mutant with myelin deficiency in the CNS (Figure
3D−F). Compound 10 staining was consistent with the
hypomyelination that is characteristic of the shiverer mouse
brain. For example, the fluorescent intensity in the corpus
callosum region was remarkably lower than that in the wild-
type control mice. The hypomyelination was also confirmed by
immunohistochemical staining for MBP, suggesting that
compound 10 distributes in the brain in proportion to the
myelin content.
and dMCMC (Figure 3). This result suggests that structural
modification of our previously identified myelin-targeting
coumarin derivatives did not negatively alter the myelin-
binding properties. This is important as it allows us to exploit
small-molecule myelin-imaging probes to develop Gd-based
contrast agents for MR imaging of myelination.
In order to determine the potential of compound 10 to
monitor demyelination and remyelination processes, in vitro
staining was performed using L-α-lysophosphatidylcholine
(LPC) treated rat models and cuprizone treated mouse models,
two common animal models of demyelination and remyelina-
tion.
LPC is a toxic chemical that has been shown to induce focal
demyelination followed by remyelination in rodents.34 A LPC
solution was stereotaxically injected into the right external
capsules (ec) of six Sprague−Dawley rats (Figure 4, white
arrows). Three animals were sacrificed after 7 days, a time that
was sufficient for the development of a conspicuous
demyelinated lesion. Three animals were sacrificed after 30
days, a period sufficient for spontaneous remyelination to
occur. The brains were extracted, and frozen sections were
prepared. Chemical staining with compound 10 displays the
presence of a large demyelinated region in the external capsule
in rats that were sacrificed 7 days post-LPC injection. Rats that
were sacrificed 30 days post-LPC injection showed instead a
normal myelination pattern in correspondence of the injection
site. The demyelinated nature of the lesion was confirmed by
immunohistochemical staining for MBP and by chemical
staining with LFB on adjacent brain sections (Figure 4).
The ability of compound 10 to monitor demyelination and
remyelination phenomena was further confirmed using
cuprizone treated wild type mice. In this model of toxic
Compound 10 chemical staining of wild-type mouse brain
tissue sections was compared with the staining of the parent
myelin targeted agents, CMC, and the dimethylamine
derivative of CMC (dMCMC).17 The staining pattern observed
with 10 was consistent with the patterns observed with CMC
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Figure 4. Chemical staining with compound 10 of brain sections of
LPC treated rats during demyelination (A) and remyelination (B).
The correspondence of MIC fluorescence with myelin distribution
within the brain was confirmed by immunohistochemical staining for
MBP (C, demyelination; D, remyelination) and LFB staining (E,
demyelination; F, remyelination). Stained areas can be identified in
part G: 1 = external capsule, 2 = corpus callosum, 3 = anterior
commissure (anterior part), 4 = caudate putamen (striatum). Scale bar
= 1 mm. Reproduced with permission from The Rat Brain in
Stereotaxic Coordinates, 6th ed.; Paxinos, G.; Watson, C.; Copyright
2007 Elsevier.35
Figure 5. Chemical staining with compound 10 of brain sections of
cuprizone treated mice during demyelination (A) and remyelination
(B). The correspondence of MIC fluorescence with myelin
distribution within the brain was confirmed by immunohistochemical
staining for MBP (C, demyelination; D, remyelination) and LFB
staining (E, demyelination; F, remyelination). Stained areas can be
identified in part G: 1 = external capsule, 2 = corpus callosum, 3 =
internal capsule. Scale bar = 1 mm. Reproduced with permission from
The Mouse Brain in Stereotaxic Coordinates, 3rd ed.; Franklin, K. B. J.;
Paxinos, G.; Copyright 2007 Elsevier.33
demyelination, mice are fed with the copper chelator cuprizone,
which induces oligodendrocyte cell death and consequently
demyelination. In this model, remyelination start to occur only
few days after the suspension of the cuprizone treatment.
Chemical staining with compound 10 on brain sections
prepared from mice that were fed with cuprizone for 4
weeks, close to the peak of demyelination, showed a large
demyelinated area within the corpus callosum. The same region
showed almost no abnormality in brain sections prepared from
mice sacrificed 10 days after the withdrawal of the cuprizone
treatment (Figure 5).
In Vivo MR Imaging of Myelin after Intracerebroven-
tricular Administration. Compound 10 is a polar compound
that is not permeable across the BBB. For this reason, we
evaluated the brain biodistribution of 10 after intracerebroven-
tricular infusion to bypass the BBB. Sprague−Dawley rats were
anesthetized, and 10 (2−10 μL, 20−100 nmol) was
administered via stereotaxic injection to the lateral ventricles
(LV). After injection, the rats were allowed to recover. At 5−7
h after injection, the animals were imaged using a spin−echo
multiple TR saturation recovery method. T1 maps were
generated using the QuickVol II plugin in ImageJ.36 Compound
10 induced a dramatic shortening of T1 that was clearly visible
at a dose of 20 nmol (∼1 mg/kg).
The analysis of the T1 maps acquired 5−7 h after injection
reveals that 10 was selectively localized in the highly myelinated
corpus callosum and striatum following diffusion from the site
of injection (Figure 6). T1 maps acquired more than 24 h after
injection continued to show a conspicuously visible corpus
callosum, although characterized by a relatively longer T1,
indicating that compound 10 had largely been cleared. Animals
injected with 10 recovered fully after the injection procedure
and did not show any motor or behavioral deficiency for up to
3 weeks after injection. These studies suggest that, once
delivered to the brain, 10 can selectively localize in different
brain regions in proportion to myelin content.
In Situ Staining of Myelin. We studied the in situ staining
with compound 10 following intraventricular injection in wild-
type Sprague−Dawley rats. The rats that were used for MR
imaging were euthanized and perfused to remove the blood.
The brains were extracted and dissected to prepare frozen axial
sections. Fluorescent microscopy showed that 10 was
selectively localized to myelinated regions such as corpus
callosum and striatum (Figure 7A). The fluorescence of 10 in
the brain could be visualized up to 6 days after injection. The
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is conspicuously reduced in the demyelinated lesion region
(Figure 8A−C). After MR imaging the rat brain was extracted
and sectioned for fluorescent microscopy. As shown in Figure
8D, compound 10 binding was significantly reduced in the
demyelinated lesions, which resulted in significantly longer T1.
The sections containing the demyelinated lesions were also
subjected to immunohistochemical staining for MBP (Figure 8
E), which confirmed the presence of the demyelinated lesions.
DISCUSSION
■
In most imaging methodologies that make use of contrast
media, contrast is generated by the agent’s emission [e.g.,
optical imaging, positron emission tomography (PET), single-
photon emission computed tomography (SPECT)] or
absorption of energy [e.g., computed tomography (CT) and
ultrasound (US)]. In the case of MRI, contrast is generated by
a number of factors including the density of water protons and
their longitudinal (R1) and transverse (R2) relaxation times.
Paramagnetic agents can alter the proton relaxation rate of the
solvent water protons. Gd(III) ions, because of their high
magnetic moment and long electron relaxation time, are able to
efficiently shorten R1 and R2 of the solvent water protons.37
The effectiveness with which a paramagnetic agent is able to
shorten the relaxation rate of the solvent water protons is given
by a parameter called relaxivity (ri, i = 1, 2), which represents
the increase in relaxation rate of a water solution occurring after
the addition of the agent, normalized to a concentration of 1
mM (eq 1).37
Figure 6. T1 maps (spin−echo multiple TR saturation recovery, TE =
6.99 ms, TR = 385−12500 ms, spatial resolution = 0.195 mm/pixel ×
0.195 mm/pixel, matrix size = 256 × 256, 30 slices, slice thickness =
0.5 mm, 1 average) of a Sprague−Dawley rat brain proximal to the
injection site 5 h (A) and 28 h (B) after injection and relative
anatomical MR images (C, 5 h; D, 28 h) (RARE, TE = 11.3 ms, TR =
5000 ms, spatial resolution = 0.195 mm/pixel × 0.195 mm/pixel,
matrix size 256 × 256, 30 slices, slice thickness = 0.5 mm, 1 average).
1
Solv
R =
= r[Gd] + R
i
;
i = 1, 2
i
i
T
(1)
i
Gd-based contrast agents have been widely used in MR
imaging. However, the Gd(III) aqua ion is toxic because it
forms insoluble phosphate, carbonate, and/or hydroxide
complexes in blood at pH 7.4. Moreover, it has been shown
that Gd(III) is toxic to human cells in vitro38−40 and to rats41 in
vivo, likely because of the inhibition of transmembrane currents
through Ca(II) channels42,43 or by forming inorganic insoluble
salt aggregates with anions such phosphates.44,45
For this reason, Gd(III) must be chelated with ligands that
can provide complexes with a high thermodynamic and kinetic
stability in order to avoid the release of the toxic Gd aqua ion.
For the design of 10, we chose a macrocyclic ligand derived
from DOTA over linear ligands derived from DTPA.
Macrocyclic ligands are known to generate complexes that
couple excellent thermodynamic stability26 to a high kinetic
inertness toward dissociation and transmetalation, while linear
ligands, even when they provide complexes with a high
formation constant, are more prone to dissociation and
transmetalation.
A semiquantitative method was used for the evaluation of the
stability of MR contrast agents toward transmetalation with
Zn(II) ions, which has previously been developed by Laurent et
al.28,29 Among the various cations that could potentially
compete with Gd(III) in physiological media, Zn(II) is the
ion that is more likely to succeed because of its relatively high
concentration in plasma (55−125 μmol/L) and relatively high
formation constants with polyazapolycarboxylic ligands. Gd
complexes with linear ligands derived from DTPA are more
susceptible to transmetalation than macrocyclic ligands like
DOTA and HPDO3A. The longitudinal proton relaxation rate
of the solutions of these macrocyclic ligands in the assay
Figure 7. In situ staining with compound 10 of myelin sheaths in the
corpus callosum and striatum (A, blue) and MBP staining (B, red),
which colocalize in the same sections (C, purple): excitation, 330−380
nm; DM, 400 nm; BA, 400 nm; scale bar = 400 μm.
same sections were then subjected to immunohistochemical
staining for MBP. As shown in Figure 7, the pattern of staining
with compound 10 (blue) is consistent with the pattern of
MBP immunohistochemical staining (red). Compound 10
fluorescence was primarily retained in myelin-rich white matter
regions such as the corpus callosum and striatum. These data
suggest that 10 can selectively label myelin fibers in situ and
potentially be used to monitor myelin changes in the course of
disease progression.
Imaging of Demyelination. The sensitivity of 10-
enhanced MR to detect demyelinated lesions was evaluated
using an LPC-treated rat model. A LPC solution was
stereotaxically injected into the right and left external capsules
(ec) of the Sprague−Dawley rat brain. The demyelinated
lesions were allowed to develop between 11 and 18 days after
LPC injection, and compound 10 was stereotaxically injected
into the lateral ventricles. T1 maps were then acquired 24 h
after injection, which reveals that the retention of compound 10
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Figure 8. (A) Axial anatomical MR image of an LPC treated rat brain showing 11 day (arrow) and 18 day old (arrowhead) lesions in the external
capsule (ec). (B) T1 map generated using a multiple TR spin echo sequence and showing longer T1 and less binding of compound 10 in the
demyelinated lesion regions. (C) Overlay of MR images confirms the colocalization of the areas of longer T1 and the demyelinated lesions. (D) In
situ fluorescent microscopy of brain tissue sections following in vivo 10-enhanced MR imaging (excitation, 340−380 nm; DM, 400 nm; BA, 435−
485 nm). (E) Immunohistochemical staining of the same section for MBP (excitation, 548−580 nm; DM, 595 nm; BA, 600−660 nm). (F) Bright
field image. Scale bars in parts A−C are 2.5 mm. Scale bars in parts D−F are 400 μm.
conditions remains unchanged over the course of more than 4
days. Similarly, the relaxation rate of a compound 10 solution in
the same conditions showed no decrease, suggesting a high
kinetic stability toward transmetalation (Figure 2).
fluorescent intensity observed in myelinated white matter
regions such as the corpus callosum and the striatum and low
fluorescent intensity observed in myelin-deficient gray matter
regions (Figure 3A), which was consistent with immunohis-
tochemical staining of adjacent brain tissue sections (Figure
3B). The myelin-binding specificity of 10 was further
determined by fluorescent tissue staining in myelin-deficient
shiverer mice. Compared to staining in wild-type mouse brains,
staining with compound 10 in shiverer mouse brain was
significantly reduced because of dysmyelination in the brain
(Figure 3C,D).
An important property that a myelin probe needs to possess
is the ability to monitor demyelination and remyelination
processes. We examined compound 10 by using LPC rat model
and cuprizone mouse model of demyelination and remyelina-
tion. These are two common models used to study
demyelinating diseases. Both models develop demyelinated
lesions that are followed by spontaneous remyelination. LPC is
a neurotoxin that induces focal demyelination that peaks
around 7 days after stereotactic injection. Cuprizone is another
neurotoxin that induces characteristic demyelination in the
corpus callosum following oral uptake for a period between 4
and 6 weeks. Tissue staining of brain sections with maximum
demyelination showed that retention of compound 10 in the
lesion regions was significantly decreased relative to remyeli-
nation and healthy controls as visualized by fluorescence
microscopy. In contrast, tissue staining of brain sections with
subsequent remyelination showed that retention of compound
10 in the affected regions was restored to nearly normal levels.
In our previous report, we showed that 10 could be used to
provide in vitro myelin mapping of brain tissue blocks in MR
images.27 In order to test the ability of 10 for in vivo imaging of
myelination, we investigated the distribution of 10 within the
brain in live animals. Compound 10 is a hydrophilic, highly
water-soluble gadolinium complex with a molecular weight
(897 Da) that exceeds 500 Da, a value that is usually recognized
as the upper limit for BBB permeability by passive diffusion.
Small Gd complexes are normally classified as T1 agents
because, by virtue of the usually small ratio r2/r1 (r2/r1 ≈ 1−
2)46 when compared with T2 agents like superparamagnetic
iron oxides (r2/r1 > 4),47,48 they do not excessively broaden the
water proton resonance. For this reason, they can be used to
generate positive contrast images, where the image intensity is
directly proportional to the agent’s local concentration.
Relaxivity is not a constant, and it depends on a number of
external parameters, such as the applied magnetic field strength
and temperature, and on molecular parameters including the
number of coordinated water molecule(s) (q), the mean
residence time (τM) of the coordinated water molecule(s), and
the reorientational correlation time (τR). These molecular
parameters can be fine-tuned in order to generate contrast
agents with a much higher relaxivity than first generation agents
like GdDOTA or GdDTPA.
The optimal combination of these parameters is field and
temperature dependent. In general, the maximum achievable
relaxivity at high field (B0 > 1.5 T; r1 < 10 mM−1 s−1; q = 1) is
much lower than at low field (0.5−1.5 T; r1 > 100 mM−1 s−1; q
= 1), but the range of values that can produce high relaxivity
agents is much wider, making it easier to generate high field and
high relaxivity agents. The relaxivity of compound 10 measured
at three fields (0.47, 1.41, and 9.4 T) commonly used in clinical
and preclinical studies is higher than the relaxivities of
GdDTPA and GdDOTA, two commonly used MR contrast
agents (Table 1). This higher relaxivity is most likely due to the
higher molecular weight and the slower τR of 10.
In order to demonstrate that 10 can be used to image myelin,
we investigated its myelin targeting capability by fluorescent
tissue stainining on freshly frozen mouse brain sections. In vitro
tissue staining on wild type brain sections showed that staining
with compound 10 is related to myelin content, with high
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Thus, like other clinical MR contrast agents, 10 is not readily
permeable across the BBB. The ability of 10 to detect
demyelinated lesions in animal models of demyelinating
diseases would depend on the disruption of the BBB. It is
important to note that in MS the BBB is normally disrupted
where brain lesions are present. Therefore, we expect 10 to be
able to cross the compromised BBB similarly to GdDTPA. In
the present study, we examined the distribution of compound
10 in the brain of wild type rats with an intact BBB. For this
reason, we had to bypass the BBB in order to deliver 10 to
brain parenchyma. To date, a number of different drug delivery
methods have been devised such as trans-cranial brain delivery,
administration of hyperosmolar solutions, use of solvents like
DMSO and ethanol, or surfactants like Tween 80 or SDS,
among others.49 We opted for the intracerebroventricular
infusion route of delivery, which relies on the perfusion of the
agent within the brain from the site of injection. As evidenced
by T1 maps (Figure 6), compound 10 distributed preferentially
in the highly myelinated white matter regions such as the
corpus callosum and external capsule in a manner similar to
what we previously observed in the in vitro studies.
Subsequently, the rats that were used for MR imaging were
euthanized and the brains extracted and used to prepare frozen
sections. Fluorescent microscopy of these tissue sections
(Figure 7) showed that staining with compound 10 was
consistent with immunohistochemical staining for MBP, which
confirms that the distribution of 10 reflects the myelin content
in the brain.
To investigate the ability of 10 to detect demyelinated
lesions, we studied its distribution in the brain of wild type rats
with LPC induced lesions. LPC is a detergent able to induce
focal demyelination in the CNS and PNS, followed by
remyelination. In the present study, demyelinated lesions
were generated by injecting a 1% LPC solution in the external
capsule of wild type rat brains. T1 maps show that T1 values in
the demyelinated lesions are longer than in adjacent regions in
the external capsule, revealing a lower binding of 10 (Figure
8A−C). The lower concentration of 10 in the demyelinated
regions was confirmed by ex vivo fluorescent microscopy. The
brain of the LPC rat models was used to prepare frozen
sections that were stained with Cy3 labeled anti-MBP antibody.
As shown in Figure 8D,E, the demyelinated lesion, revealed by
immunostaining, is colocalized with the area of reduced
fluorescence of compound 10.
CONCLUSION
■
Myelin damage is a hallmark of many acquired and inherited
neurological diseases including MS and various leukodystro-
phies. To date, MRI has been the primary tool for diagnosing
and monitoring the progression of myelin diseases. However,
conventional MRI cannot differentiate demyelinated lesions
from other inflammatory lesions, and therefore, the use of MRI
as a primary measure of disease activity still has not been
accepted by the FDA. Currently, therapeutic interventions are
focused on protecting myelin integrity and promoting myelin
repair. These efforts need to be accompanied by an effective
imaging tool that correlates the disease progression with the
extent of myelination. Therefore, there is an urgent need to
improve the sensitivity of MRI to the myelination status in the
CNS and PNS. We previously reported MIC (compound 10), a
myelin specific MR contrast agent, and demonstrated its ability
to bind myelin and to produce MR renderings of myelin
distribution in mouse brains in vitro. In this report, we have
demonstrated that compound 10 is capable of labeling highly
myelinated fibers in the brain of living animals when
administered by intraventricular injection. Furthermore, com-
pound 10 can be used to detect areas of reduced myelination in
a LPC rat model of focal demyelination.
EXPERIMENTAL SECTION
■
Materials and Methods. All chemicals, unless otherwise stated,
were purchased from commercial sources and used without further
purification. All NMR spectra were acquired on an Inova 400 NMR or
on an INOVA 600 NMR system (Varian) equipped with a 5 mm
broadband probe. Analytical HPLC was performed on an Agilent 1100
series system equipped with a dual channel UV/vis detector using a
Phenomenex 5 μm C18(2) 100A (250 mm × 4.56 mm, 5 μm) column
(4.6 mm × 250 mm): eluent A, H2O/0.1% TFA; eluent B, MeOH/
0.1% TFA; elution, 10% B for 3 min, 10% B to 100% B in 15 min; flow
rate 1 mL/min. The purity of the tested compounds as determined by
analytical HPLC was ≥95%. Low resolution ESI mass spectra were
acquired on a Finnigan LCQ Deca. High resolution ESI mass spectra
were acquired on a Waters Qtof API US instrument at the Chemical
Instrumentation Center at Boston University, MA. Relaxivity was
measured at 9.4 T on a Varian Inova 400 NMR system equipped with
a 5 mm broadband probe at 21 °C and at 0.47 and 1.41 T on a Bruker
Minispec at 40 °C. The pH was measured using a PHM210 standard
pH meter (Radiometer Analytical) connected to a Symphony pH glass
electrode (VWR).
3-(4-(Dimethylamino)phenyl)-2-oxo-2H-chromen-7-yl Ace-
tate (3). Compound 1 (2.0 g, 14.5 mmol), compound 2 (2.6 g,
14.5 mmol), and sodium acetate (2.4 g, 29.0 mmol) were dissolved in
acetic anhydride (30 mL) and refluxed overnight. After cooling to
As expected for many Gd-chelated contrast agents,
compound 10 itself is very hydrophilic and does not penetrate
the intact BBB. In MS patients, however, the BBB is disrupted,
making it possible for compound 10 to enter the brain. Thus,
compound 10 could be used for clinical studies in a way similar
to GdDTPA that is currently used in humans. Lesions detected
by GdDTPA enhanced MR are not necessarily indicative of
myelin pathology. Because GdDTPA does not bind to myelin,
its uptake only reflects disruption of the BBB where lesions are
formed. Compound 10 differs from GdDTPA because of its
ability to bind myelin sheaths. Uptake of 10 could not only
potentially identify disruption of the BBB but also help
characterize the myelin content of the lesions. Because MIC
binds to myelin specifically, it could provide additional
information on the myelin integrity, which is urgently needed
for accurate diagnosis and efficacy evaluation of myelin repair
therapies currently under development.
room temperature, the mixture was neutralized with 1 M NaOH(aq)
.
The solid then was collected and washed with EtOAc. The solid was
transferred to an Erlenmeyer flask and suspended in EtOAc (20 mL).
The suspension was refluxed for 5 min. Upon cooling, the suspension
was filtered and the solid was dried under vacuum to give product 3
(1.7 g, 36%) as a yellow solid. 1H NMR (400 MHz, CDCl3): δ 2.35 (s,
CH3CO, 3H), 3.01 (s, NCH3, 6H), 6.74−6.79 (m, ArH, 2H), 7.04
(dd, ArH, J = 2.1, 8.4 Hz, 1H), 7.12 (d, ArH, J = 2.1 Hz, 1H), 7.51 (d,
ArH, J = 8.5 Hz, 1H), 7.65 (m, ArH, 2H), 7.70 (s, CCHAr, 1H). 13C
NMR (100 MHz, CDCl3): δ 21.07 (CH3CO), 40.22 (CH3N), 109.64
(CHarom), 111.80 (CHarom), 117.95 (Carom), 118.14 (CHarom), 121.87
(Carom), 127.42 (Carom), 127.97 (Carom), 129.23 (CHarom), 135.89
(CHarom), 150.67 (Carom), 151.88 (Carom), 153.30 (Carom), 160.58
(CO), 168.80 (CH3CO). HR ESI-MS (m/z): calcd, 346.1055; found,
346.1047 [M + Na]+.
3-(4-(Dimethylamino)phenyl)-7-hydroxy-2H-chromen-2-one
(4). A solution of 3 (3.0 g, 9.3 mmol) in MeOH (20 mL) and 2 N
HCl(aq) (10 mL) was refluxed for 4 h. After cooling to room
temperature, the mixture was neutralized with 1 M NaOH(aq). The
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solid was filtered and washed with EtOAc. The solid was transferred to
an Erlenmeyer flask and suspended in EtOAc (20 mL). The
suspension was refluxed for 5 min. Upon cooling, the suspension
was then filtered and the solid was dried under vacuum to give product
(CH2O), 81.65 (CMe3), 81.71(CMe3), 101.03 (CHarom), 111.88
(CHarom), 112.30 (CHarom), 113.65 (Carom), 122.50 (Carom), 124.38
(Carom), 128.51 (CHarom), 129.03 (CHarom), 137.23 (CHCCO), 150.38
(Carom), 154.36 (Carom), 161.16 (Carom), 161.30 (CCOO), 171.74
(CH2CO), 172.28 (CH2CO, broad), 172.48 (CH2CO).
1
4 (1.96 g, 75%) as a yellow solid. H NMR (400 MHz, DMSO-d6): δ
2.94 (s, NCH3, 6H), 6.66−6.83 (m, ArH, 4H), 7.48−7.63 (m, ArH,
3H), 8.00 (s, CCHAr, 1H). HR ESI-MS (m/z): calcd, 282.1130;
found 282.1186 [M + H]+.
[4,10-Bis-carboxymethyl-7-({3-[3-(4-dimethylaminophenyl)-
2-oxo-2H-chromen-7-yloxy]propylcarbamoyl}methyl)-
1,4,7,10-tetraazacyclododec-1-yl]acetic Acid (9). A solution of 8
(0.052 g, 0.058 mmol) in TFA (2 mL) was stirred at room
temperature for 24 h. TFA was removed under reduced pressure and
the residue dissolved in water (2 mL). The slightly cloudy solution was
filtered and the solvent was evaporated to give 9·TFA as a bright
yellow solid (0.048 g, quantitative). 1H NMR (400 MHz, D2O): δ 1.82
(m, CH2CH2CH2, 2H), 3.20 (s, NCH3, 6H), 2.7−4.3 (b, CH2
macrocycle, CH2 acetic arms, OCH2, NCH2, 28H), 6.57 (d, J = 2.3
Hz, ArH, 1H), 6.66 (dd, J = 2.3 Hz, J = 9.7 Hz, ArH, 1H), 7.27 (d, J =
8.8 Hz ArH, 1H), 7.55 (d, AB system, J = 8.9 Hz ArH, 2H), 7.62 (d,
AB system, J = 8.9 Hz, ArH, 2H), 7.68 (s, CCH, 1H). ESI-MS (m/
z): calcd, 725.35; found, 725.33 [M + H]+.
{3-[3-(4-Dimethylaminophenyl)-2-oxo-2H-chromen-7-
yloxy]propyl}carbamic Acid tert-Butyl Ester (5). To a solution of
4 (0.155 g, 0.55 mmol) and (3-bromopropyl)carbamic acid tert-butyl
ester (0.164 mg, 0.69 mmol) in dry acetonitrile (5 mL) was added
cesium carbonate (0.349 mg, 1.07 mmol). The reaction mixture was
stirred at 70 °C and monitored by TLC (MeOH/DCM 2/98, Rf =
0.76) until completion (∼20 h). The solvent was removed under
reduced pressure, and the residue was triturated and then decanted,
first with water (2 × 10 mL) and subsequently with diethyl ether (2 ×
5 mL). The residual solid was dried under reduced pressure to yield
1
compound 5 (0.186 g, 79%) as a yellow-orange solid. H NMR (400
Gadolinium(III) [4,10-Bis-carboxymethyl-7-({3-[3-(4-dime-
t h y l a m i n o p h e n y l ) - 2 - o x o - 2 H - c h r o m e n - 7 - y l o x y ] -
propylcarbamoyl}methyl)-1,4,7,10-tetraazacyclododec-1-yl]-
acetate (10). To a solution of 9 (0.042 g, 0.058 mmol) in water (10
mL) was added a solution of GdCl3 in water (5.3 mM, 12.5 mL, 0.067
mmol) while maintaining the pH at 6.0 by addition of NaOH(aq), 1 M.
The solution was stirred at 50 °C for 12 h. The pH was raised to 9.0.
The solution was filtered through a 0.2 μm filter, and the pH was
lowered to 7.0 by adding HCl. The solution was loaded on a Hypersep
C-18 cartridge, and the stationary phase was washed extensively with
water. The product was recovered after gradient elution with MeOH/
H2O from 1:9 to 3:2. The fractions containing the product were
evaporated under reduced pressure to give 10 (0.039 g, 68%) as a
yellow solid. HR-ESI-MS (m/z): calcd 880.2520; found 880.2501 [M
+ H]+. A correct Gd isotope pattern was observed.
Animal Handling. All animal experiments were performed in
accordance with guidelines approved by the Institutional Animal Care
and Use Committee of Case Western Reserve University (Protocols
2010-0006 and 2010-0007). Two-month-old Swiss-Webster R/J mice
were purchased from Harlan Laboratories, Indianapolis, IN. Two-
month-old C57BL/6 mice and C3Fe.SWV-Mbpshi/J shiverer mice
were obtained from The Jackson Laboratory, Bar Harbor, ME.
Sprague−Dawley rats were purchased from Harlan Laboratories,
Haslett, MI.
Preparation of Frozen Sections. Mice were deeply anesthetized
with isoflurane and perfused via the ascending aorta with 1× PBS
followed by 4% paraformaldehyde in PBS. Brains were removed and
incubated for 24 h in 4% paraformaldehyde in PBS at 4 °C and then in
30% sucrose at 4 °C until submerged. Frozen sections were used for
fluorescence microscopy. For preparation of fresh frozen sections, the
cryoprotected tissues were first frozen in OCT on dry ice before axial
sectioning (20 μm) with a cryostat at −20 °C.
In Vitro Staining. Frozen sections or floating sections mounted
on Superfrost slides (Fisher Scientific) were incubated with an
aqueous solution of compound 10 (100 μM) for 20 min at room
temperature. Excess compound 10 was removed by briefly rinsing the
sections in PBS before coverslipping with Vectashield mounting
medium (Vector Laboratories; Burlingame, CA). Sections were then
examined using epifluorescence microscopy with a Leica DM5000B
microscope equipped with an HCX PL FLUOTAR 1.25×/0.04
objective and using the A4 filter (360/40 nm band-pass excitation, 400
nm dichromatic mirror, 470/40 nm band-pass suppression). In some
cases, tissue sections were double stained with antimyelin basic protein
(MBP) monoclonal antibody (MAb) (see below), in which case the
coverslipping and Vectrashield were omitted.
MHz, CDCl3): δ 1.43 (s, CCH3, 9H), 1.99 (m, CH2CH2CH2, 2H),
2.97 (s, NCH3, 6H), 3.32 (m, NCH2, 2H), 4.03 (t, OCH2, J = 5.99 Hz,
2H), 4.88 (t b, NH, 1H), 6.71−6.82 (m, ArH, 4H), 7.35 (d, ArH, J =
8.49, 1H), 7.58−7.63 (m, ArCH2, and CCHAr, 3H). 13C NMR (100
MHz, CDCl3): δ 28.29 (CCH3), 29.29 (CH2CH2CH2), 37.59
(NCH2), 40.23 (NCH3), 66.05 (OCH2), 79.11 (CCH3), 100.71
(CH), 111.84 (CH), 112.53 (CH), 113.69 (C), 122.46 (C), 124.57
(C), 128.24 (CH), 129.01 (CH), 136.90 (CH), 150.34 (C), 154.42
(C), 155.89 (CO), 160.89 (C), 161.13 (CO). ESI-MS (m/z): calcd,
439.22; found 439.12 [M + H]+.
7-(3-Aminopropoxy)-3-(4-dimethylaminophenyl)chromen-
2-one (6). Compound 5 (0.25 g, 0.57 mmol) was dissolved in
trifluoroacetic acid and stirred at room temperature for 12 h. The
slightly cloudy solution was filtered to remove any insoluble impurity
before the solvent was evaporated under reduced pressure. The residue
was dissolved in dichloromethane (25 mL) and extracted with 0.2 M
NaOH(aq) (3 × 25 mL) to yield 6 (106 mg, 93%) as a glassy yellow
1
solid. H NMR (400 MHz, CD3OD): δ 2.19 (dt, CH2CH2CH2, J =
7.3, 5.8 Hz, 2H), 3.18 (t, NCH2, J = 7.3 Hz, 2H), 3.27 (s, NCH3, 6H),
4.2 (t, OCH2, J = 5.8 Hz, 2H), 6.91−6.96 (m, ArH, 2H), 7.57−7.62
(m, ArH, 3H), 7.85−7.90 (m, ArH, 2H), 8.04 (s, CCHAr, 1H). 13C
NMR (150 MHz, 60 °C, DMSO-d6): δ 26.38 (CH2CH2CH2), 35.84
(NCH3), 42.62 (NCH2), 65.44 (OCH2), 100.66 (CH), 112.68 (CH),
113.06 (CH), 116.34 (2 × C), 122.31 (C), 129.13 (CH), 129.31 (CH),
139.30 (CH), 146.13 (C), 154.30 (C), 159.68 (CO), 161.02 (CO).
ESI-MS (m/z): calcd, 339.17; found 339.19 [M + H]+.
[4,10-Bis-tert-butoxycarbonylmethyl-7-({3-[3-(4-dimethyla-
minophenyl)-2-oxo-2H-chromen-7-yloxy]propylcarbamoyl}-
methyl)-1,4,7,10-tetraazacyclododec-1-yl]acetic Acid tert-
Butyl Ester (8). A solution of 6 (0.075 g, 0.222 mmol), DOTA-
tris-(t-Bu) ester (7, 0.102 g, 0.177 mmol), HOBT (0.041 g, 0.222
mmol), HBTU (0.101 g, 0.266 mmol), and DIPEA (0.046 g, 0.355
mmol) in dry DMF (10 mL) was stirred at room temperature for 20 h
(TLC MeOH/DCM, 1/9, Rf = 0.66). The solvent was removed under
reduced pressure, and the residue was triturated with water and then
decanted (2 × 10 mL). The yellow residue was dissolved in DCM (15
mL) and the solution dried over Na2SO4. The solvent was removed
under reduced pressure and the residue dissolved in a minimum
amount of DCM and purified by preparative TLC (MeOH/DCM, 12/
88). After isolation, the product was recovered by rinsing the silica
with the same eluent. Evaporation of the solvent gave 8 (0.104 g, 66%)
1
as a yellow solid. H NMR (400 MHz, CDCl3): δ 1.44 (s, b, CCH3,
18H), 1.46 (s, CCH3, 9H), 2.04 (m, CH2CH2CH2, 2H), 3.00 (s,
NCH3, 6H), 3.40 (m, NCH2, 2H), 4.06 (t, OCH2, J = 6.3 Hz, 2H),
1.5−3.7 (b, CH2 macrocycle, CH2 acetic arms, 24 H), 6.62 (t, NH, J =
6.0 Hz, 1H), 6.74−6.78 (m, ArH, 3H), 6.87 (dd, ArH, J = 2.42, 8.62
Hz, 1H), 7.40 (d, ArH, J = 7.4 Hz, 1H), 7.62 (m, ArH, 2H), 7.67 (s,
CCH, 1H). 13C NMR (100 MHz, CDCl3): δ 27.81 (CCH3), 27.84
(CCH3), 28.73 (CH2CH2CH2), 36.48 (CONHCH2), 40.27 (NCH3),
48.13 (NCH2, ring, broad), 52.41, (NCH2, ring, broad), 55.45
(NCH2CO, broad), 55.57 (NCH2CO), 55.68 (NCH2CO), 66.36
Immunohistochemistry. For immunohistochemistry, sections
mounted on slides were incubated in a solution containing anti-
MBPMAb primary antibody (rat anti-MBP, 1:300; Chemicon,
Temecula, CA) diluted in 1% normal donkey serum overnight at 4
°C. Following three rinses with PBS, sections were incubated in
donkey anti-rabbit rhodamine red X-conjugated secondary antibody or
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goat anti-rat IgG Texas-red-conjugated secondary antibody (Jackson
ImmunoResearch Laboratories; West Grove, PA) (diluted 1:200 in
PBS with 1% normal donkey serum) for 1 h at 37 °C, then washed
three times for 5 min each with PBS. Sections were then examined
using epifluorescence microscopy with a Leica DM5000B microscope
equipped with an HCX PL FLUOTAR 1.25×/0.04 objective and using
the T×2 filter (560/40 nm band-pass excitation, 595 nm dichromatic
mirror, 645/75 nm band-pass suppression).
= 5000 ms, spatial resolution = 0.195 mm/pixel × 0.195 mm/pixel,
matrix size 256 × 256, 30 slices, slice thickness = 0.5 mm, 1 average),
for the identification of the region of interest (ROI). T1 measurements
were carried out using a spin−echo multiple TR saturation recovery
method with at least 10 TRs (TE = 6.99 ms, TR = 385−12500 ms,
spatial resolution = 0.195 mm/pixel × 0.195 mm/pixel, matrix size =
256 × 256, 30 slices, slice thickness = 0.5 mm, 1 average). T1 maps
were generated using the QuickVol II plug-in in ImageJ.36
Luxol Fast Blue Staining for Myelin. Frozen sections or floating
sections mounted on Superfrost slides (Fisher Scientific) were washed
in phosphate buffer saline (PBS) for 5 min, in 35% ethanol for 5 min,
and in 70% ethanol for 5 min. The sections were then incubated in a
LFB solution (0.1%) at 60 °C overnight. The sections were washed
sequentially with 95% ethanol, 70% ethanol, 35% ethanol, distilled
water, and a water solution of lithium carbonate (0.05%) for 5 min for
each step. The washing procedure was then repeated in reverse, and
the sections were washed with water, 35% ethanol, 70% ethanol, and
95% ethanol for 5 min for each step. The entire washing procedure
was repeated, if necessary, until gray matter is colorless and white
matter appears blue.
The sections were then washed three times for 5 min with fresh
95% ethanol, three times with 100% ethanol, and then twice with
xylenes. The stained sections were analyzed in bright field with a Leica
DM5000B microscope equipped with an HCX PL FLUOTAR 1.25×/
0.04 objective.
Intracerebroventricular Injection of Compound 10.
Sprague−Dawley rats were anesthetized by ip injection of 200−250
μL of rodent cocktail [9 parts ketamine (100 mg/mL) + 9 parts
xylazine (20 mg/mL) + 3 parts acepromazine (10 mg/mL) + 79 parts
sterile saline] and were fixed to a rat head restraining stereotaxic
surgical table. The skull was shaved, and a 3 cm longitudinal incision
was made on the scalp. A burr hole was created using the spherical
dental burr over the site of the intended injection. A water solution of
compound 10 (10 mM, 2−10 μL) was injected into the brain through
a 50 μL syringe equipped with a 28 gauge needle. After injection the
incision was sutured and the animals were allowed to recover
consciousness while being warmed over a heating pad. The injection
sites are located in the lateral ventricle. Typical coordinates for the
injection sites relative to the bregma are A = −0.5 0.1 mm, ML = 2.3
0.1 mm, DV = 3.5 0.1 mm.
Induction of LPC Lesions. One Sprague−Dawley rat was
anesthetized by ip injection of 225 μL of rodent cocktail [9 parts
ketamine (100 mg/mL) + 9 parts xylazine (20 mg/mL) + 3 parts
acepromazine (10 mg/mL) + 79 parts sterile saline]. The skull was
shaved, and a 3 cm longitudinal incision was made on the scalp. A burr
hole was created using the spherical dental burr over the site of the
intended injection. Then 6 μL of a 1% LPC solution was injected at a
rate of 0.25 μL/min. The injections coordinates were AP = 0.0 mm,
ML = 2.0 mm, DV = 3.2 mm, corresponding to the external capsule.
After the injections the incision was sutured and the animals were
allowed to recover while being warmed over a heating pad.
For in vitro chemical and immunohistochemical studies, the lesions
were allowed to develop for 7 days (demyelination stage) or 30 days
(remyelination stage). For MR imaging, the lesions were allowed to
develop for 11−18 days before the injection of compound 10.
Cuprizone Mouse Model. Brain sections from cuprizone mouse
models were kindly provided by Dr. Liping Liu (Cleveland Clinic,
OH). Briefly, C57BL/6J were fed with 0.2% cuprizone (w/w). Mice
used to prepare sections in the demyelinated stage were euthanized
after 4 weeks. Mice used to prepare sections in the remyelinating stage
were fed with cuprizone for 6 weeks, subsequently allowed to
remyelinate for 10 days, and then euthanized. The brains were used to
prepare floating sections (30 μm) according to standard procedures.
MR Imaging. All MR experiments were performed at 9.4 T
(Bruker “BIOSPEC”, Bruker, Germany) using a 60 mm diameter
volume coil. Anesthesia was maintained by mask inhalation of
isoflurane vaporized at concentrations of up to 4% in the induction
phase, at 1.5% during the imaging experiments. Prior to measurement
of the longitudinal relaxation time, axial images of the rat brain were
acquired, using a multislice RARE pulse sequence (TE = 11.3 ms, TR
AUTHOR INFORMATION
Corresponding Author
*Telephone: (216) 844-3288. Fax: (216) 844-8062. E-mail:
yanming.wang@case.edu.
■
Notes
Part of this work was reported as a communication in J. Am.
Chem. Soc., 2011.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support through grants
from the Department of Defense (Grant MS090082/
W81XWH-10-1-0842), National Multiple Sclerosis Society,
and NIH/NINDS (Grant R01 NS061837). We thank Drs.
Richard Ransohoff and Liping Liu (Cleveland Clinic, OH) for
providing floating sections of cuprizone mouse brains.
ABBREVIATIONS USED
■
BA, barrier filter; BBB, blood−brain barrier; BNB, blood to
nerve barrier; CMC, case myelin compound; CNS, central
nervous system; CT, computed tomography; DM, dichroic
mirror; DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-
acetic acid; DTPA, diethylenetriamine pentaacetic acid; DWI,
diffusion weighed imaging; ESI-MS, electrospray ionization
mass spectrometry; HBTU, O-benzotriazole-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate; HP-DO3A, 10-(2-hy-
droxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid;
HPLC, high-performance liquid chromatography; LFB, Luxol
Fast Blue; LPC, L-α-lysophosphatidylcholine; MBP, myelin
basic protein; MIC, myelin imaging compound; MRI, magnetic
resonance imaging; MS, multiple sclerosis; MT, magnetization
transfer; MWF, myelin water fraction; PBS, phosphate buffered
saline; PET, positron emission tomography; RARE, rapid
acquisition with refocused echoes; SPECT, single-photon
emission computed tomography; US, ultrasound
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