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Gliomas: Motexafin Gadolinium-enhanced Molecular MR Imaging and Optical Imaging for Potential Intraoperative Delineation of Tumor Margins

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भाषा:
english
पत्रिका:
Radiology
DOI:
10.1148/radiol.2015150895
Date:
November, 2015
फ़ाइल:
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Original Research

Longhua Qiu, PhD, MD
Feng Zhang, PhD, MD
Yaoping Shi, PhD, MD
Zhibin Bai, MD
Jianfeng Wang, PhD, MD
Yonggang Li, MD, PhD
Donghoon Lee, PhD
Christopher Ingraham, MD
Xiaoyuan Feng, PhD, MD
Xiaoming Yang, PhD, MD

Purpose:

To investigate the possibility of using motexafin gadolinium (MGd)–enhanced molecular magnetic resonance
(MR) imaging and optical imaging to identify the true
margins of gliomas.

Materials and
Methods:

The animal protocol was approved by the institutional animal care and use committee. Thirty-six Sprague-Dawley
rats with gliomas were randomized into six groups of six
rats. Five groups were euthanized 15, 30, 60, 120, and
240 minutes after intravenous administration of 6 mg/kg
of MGd, while one group received only saline solution as
a control group. After craniotomy, optical imaging and
T1-weighted MR imaging were performed to identify the
tumor margins. One-way analysis of variance was used to
compare optical photon intensity and MR imaging signalto-noise ratios. Histologic analysis was performed to confirm the intracellular uptake of MGd by tumor cells and
to correlate the tumor margins delineated on both optical
and MR images.

Results:

Both optical imaging and T1-weighted MR imaging showed
tumor margins. The highest optical photon intensity (2.6
3 108 photons per second per mm2 6 2.3 3 107; analysis
of variance, P , .001) and MR signal-to-noise ratio (77.61
6 2.52; analysis of variance, P = .006) were reached at
15–30 minutes after administration of MGd, with continued tumor visibility at 2–4 hours. Examination with confocal microscopy allowed confirmation that the fluorescence
of optical images and MR imaging T1 enhancement exclusively originated from MGd that accumulated in the
cytoplasm of tumor cells.

Conclusion:

MGd-enhanced optical and MR imaging can allow determination of glioma tumor margins at the optimal time of
15–120 minutes after administration of MGd. Clinical appl; ication of these results may allow complete removal of
gliomas in a hybrid surgical setting in which intraoperative
optical and MR imaging are available.

1

From the Image-Guided Biomolecular Intervention
Research, Department of Radiology, University of Washington School of Medicine, 850 Republican St, Seattle,
WA 98109 (L.Q., F.Z., Y.S., Z.B., J.W., Y.L., D.L., C.I., X.Y.);
and Department of Radiology, Huashan Hospital, Fudan
University, Shanghai, China (L.Q., X.F.). Received April 22,
2015; revision requested June 10; revision received August
4; accepted August 11; final version accepted August 27.
Address correspondence to X.Y. (e-mail: xmyang@u.
washington.edu).

q

n Experimental Studies

Gliomas: Motexafin Gadoliniumenhanced Molecular MR Imaging
and Optical Imaging for Potential
Intraoperative Delineation of Tumor
Margins1

RSNA, 2015

L.Q. and F.Z. contributed equally to this work.
q

RSNA, 2015

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EXPERIMENTAL STUDIES: Motexafin Gadolinium-enhanced Molecular MR and Optical Imaging in Gliomas

G

liomas are the most common
primary brain tumors. The prognosis of patients with high-grade
gliomas remains extremely poor, with a
median survival of only 12–15 months
from the time of diagnosis (1,2). Current treatment strategies for gliomas
include surgical resection and combined radiation therapy and chemotherapy, which only extend survival
by a few months (3). The extent of
gross total resection of gliomas with
the goal of achieving maximal tumor
resection with the least postoperative
neurologic deficits has been a critical
factor in survival (3–5). However, gliomas have a high propensity to infiltrate along white matter tracts, sometimes to considerable distances from
the edge of the gross tumor (6,7).
This factor can make complete resection of tumors extremely challenging
with conventional imaging-based microsurgical techniques (5). This difficulty
is in part due to limitations of conventional imaging techniques to accurately

Advances in Knowledge
nn Glioma tumor cells take up
motexafin gadolinium (MGd) in a
time- and concentration-dependent fashion, with the earliest
intracellular accumulation at
15–30 minutes and an optimal
concentration of 100 mg/mL after
the addition of MGd.
nn Both optical and MR imaging
demonstrated the highest photon
signal intensity (2.6 3 108 photons per second per mm2 6 2.3
3 107; analysis of variance, P ,
.001) and MGd enhancement
(signal-to-noise ratio, 77.61 6
2.52; analysis of variance, P =
.006) at 15–30 minutes after
MGd administration, with continued visibility lasting for 2–4
hours.
nn Histologic analysis allowed confirmation that the fluorescence of
optical images and MR imaging
T1 enhancement originated exclusively from MGd that accumulated in the cytoplasm of tumor
cells.
2

distinguish glioma infiltration from
normal brain tissue (5). Therefore, it
is imperative to develop intraoperative
imaging-guided techniques that can ensure identification of tumor margins to
allow complete tumor removal (8).
Recent advances in imaging-guided
techniques for glioma resection include
intraoperative magnetic resonance
(MR) imaging (9–12) and three-dimensional ultrasonographic (US) imaging
(13). Both of these imaging techniques
can provide cross-sectional and threedimensional information and depict the
depth of the tumor under the cortical
surface. This improves tumor visualization during surgery and allows for
more complete tumor resection (13).
Fluorescence-guided resection with
5-aminolevulinic acid also has been
used as a promising tool in localizing tumors and guiding resection (5,13–15).
However, each of these modalities has
inherent limitations and disadvantages.
Three-dimensional US can easily create
artifacts, causing its accuracy to decrease as the surgery progresses (13).
Intraoperative MR imaging is typically
performed after the administration of
paramagnetic contrast agent, which is
an extracellular agent. This poses the
potential problem of obscuring tumor
margins if the contrast agent leaks from
a vessel that has been opened surgically
(10). As an alternative, visualization
with fluorescence is essentially a surface imaging technique, with limited
depth penetration (13,16). Therefore,
recent efforts have been made to integrate intraoperative MR imaging with
optical imaging in one surgical setting,
combining the advantages of two imaging modalities: high soft-tissue contrast
and spatial resolution with MR imaging
and high sensitivity and real-time data
acquisition with optical imaging (17).

Implication for Patient Care
nn Intraoperative integration of
MGd-enhanced molecular optical
imaging and MR imaging allows
for the potential clinical benefit
of guiding complete removal of
human gliomas in one hybrid surgical setting.

Qiu et al

The feasibility of this combination is
reliant on the availability of an intracellular MR and optical imaging marker.
Once a reliable imaging marker is available, simultaneous intraoperative dualmodality imaging can guide the resection of complex brain tumors, such as
gliomas.
Motexafin gadolinium (MGd), a
porphyrin-like molecule that has been
used in clinical trials, functioning as a
radiation and chemotherapy sensitizer,
an intracellular T1-weighted MR imaging enhancer, and a red fluorescence
emitter (18). Results of previous studies
(19–22) have confirmed that MGd can
be internalized exclusively in metabolically active tissues, such as atherosclerotic plaques and different tumors. It
also can cross the disrupted blood-brain
barrier associated with brain tumors,
but it is not detectable in normal brains
(18,23,24). The aim of this study was
to investigate the possibility of using
MGd-enhanced molecular MR imaging
and optical imaging to identify the true
margins of gliomas.

Materials and Methods
MGd was provided by Pharmacyclics
(Sunnyvale, Calif). The authors had full

Published online before print
10.1148/radiol.2015150895 Content codes:
Radiology 2016; 000:1–10
Abbreviation:
MGd = motexafin gadolinium
Author contributions:
Guarantors of integrity of entire study, L.Q., F.Z., Z.B., J.W.,
Y.L., X.Y.; study concepts/study design or data acquisition
or data analysis/interpretation, all authors; manuscript
drafting or manuscript revision for important intellectual
content, all authors; approval of final version of submitted
manuscript, all authors; agrees to ensure any questions
related to the work are appropriately resolved, all authors;
literature research, L.Q., Z.B., J.W., C.I., X.F.; clinical studies, Z.B., J.W.; experimental studies, L.Q., F.Z., Y.S., Z.B.,
J.W., Y.L., D.L.; statistical analysis, L.Q., F.Z., Z.B., J.W., X.Y.;
and manuscript editing, L.Q., F.Z., Z.B., J.W., D.L., C.I., X.Y.
Funding:
This research was supported by the National Institutes of
Health (grant RO1EBO12467).
Conflicts of interest are listed at the end of this article.

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control of the data and the submitted
information.

Study Design
This study was performed between December 2013 and February 2015. Our
study included two phases: (a) in vitro
experiments to investigate the feasibility of using optical imaging and molecular MR imaging to confirm and quantify the intracellular uptake of MGd
by glioma cells, while determining the
optimal concentration of MGd and incubation time; and (b) in vivo animal
experiments to validate the feasibility of
using combined molecular MR imaging
and optical imaging to assess the real
margins of MGd-illuminated gliomas in
rat brains.
In Vitro Experiments
Cell culture.—Rat C6 glioma cells were
seeded (5 3104 per well) into fourwell chamber plates (Laboratory Tek
II; Thermo Fisher Scientific, Rochester, NH). Cells were maintained in
Delbecco’s modified Eagle’s medium
(HyClone Laboratories, Logan, Utah)
supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, Mo) in
an incubator at 37°C with a 5% carbon
dioxide atmosphere.
Confocal microscopy of MGd-treated cells.—As cells were cultured to
reach 80% confluence, MGd was added
to yield final concentrations of 0, 25, 50,
75, 100, 125, 150, and 200 µg/mL. Cells
were then cultured for an additional 24
hours. Six wells were prepared for each
concentration. MGd-treated cells were
then washed twice with phosphatebuffered saline to remove free MGd,
were fixed in 4% paraformaldehyde,
and were dried at room temperature.
Cell plates were counterstained with
4’, 6-diamidino-2-phenylindole (DAPI;
Vector Laboratories, Burlingame, Calif)
and then imaged with a laser confocal
microscope (A1R; Nikon, Tokyo, Japan) to detect and compare the MGdemitted red fluorescence in the cells at
different MGd concentrations. To verify
the time required for uptake of MGd by
the cells, additional glioma cell groups
were treated with MGd at 100 mg/mL
for 15 and 30 minutes and 1, 2, 4, 12,
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24, and 48 hours for the respective
concentration levels. A nontreated cell
group served as a control group.
Optical imaging and MR imaging of
MGd-treated cells.—Additional groups
of cells were cultured and grown on 10mm culture plates to reach 80% confluence, and then treated with MGd at 0,
25, 50, 75, 100, 125, 150, and 200 mg/
mL for 24 hours. Six plates were created for each concentration. Cells were
then collected and rinsed twice with
phosphate-buffered saline. MGd-treated cells (5 3 106) were diluted with 0.1
mL of phosphate-buffered saline and
were dispersed into 0.2-mL Eppendorf
tubes containing 1% agarose.
Fluorescent optical imaging of
cell-containing agarose tubes was performed with an in vivo small animal optical and x-ray imaging system (Bruker
Biospin; Bruker, Billerica, Mass). Cellcontaining tubes were imaged with an
excitation wavelength at 470 nm, emission wavelength with a 750 nm filter, a
field of view of 19 3 19 mm, and exposure time of 30 seconds.
MR imaging of cell-containing agarose tubes was performed by using a
3.0-T MR imaging system (Achieva;
Philips Healthcare, Best, the Netherlands) and a wrist coil (SENSE wrist
four-element coil; Philips Healthcare).
T1 mapping of cells was performed
by using the Look-Locker sequence:
repetition time msec/echo time msec,
6.9/4.4; flip angle, 10°; field of view,
100 mm; section thickness, 0.8 mm;
matrix, 434 3 434; and number of signals acquired, four. T1-weighted images were acquired in the sagittal and
transverse planes by using a spin-echo
sequence, 550/12; flip angle, 90°; field
of view, 100 mm; section thickness, 1
mm; matrix, 500 3 498; and number of
signals acquired, eight.
To evaluate MGd uptake times, cells
cultured in 10-mm petri dishes were
treated with MGd at a concentration of
100 mg/mL at varying incubation times
of 15, 30, and 60 minutes and 2, 4, 12,
24, and 48 hours. MGd-treated cells
(5 3 106) were collected, dispersed in
1% agarose tubes, and then imaged by
using the optical imaging system and
3.0-T MR imager by using the same

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Qiu et al

imaging sequences and parameters as
those previously described.
Imaging analysis.—Fluorescent signal intensity of MGd-treated cells was
quantified by placing a 3-mm2 region
of interest at the center of cell-containing agarose tubes to measure photon quantities. The signal intensity of
MGd-containing cells was plotted with
MGd concentrations and incubation
times to determine the optimal MGd
dose and incubation time (F.Z., with 4
years of experience in optical imaging).
MR images of MGd-treated cells were
analyzed by measuring the signal intensity of cell-containing tubes. The signal
intensity of each tube was measured
by using the Digital Imaging and Communication in Medicine viewer (Philips
DICOM Viewer R2.5 Version 1 Level 1;
Philips Healthcare). A region of interest
of 3 mm2 was placed at the center of
each tube by one radiologist (L.Q., with
5 years of experience in MR imaging).

Animal Experiments
Creation of rat models with brain gliomas.—The animal protocol was approved by our institutional animal care
and use committee. Thirty-six male
Sprague-Dawley rats (mean weight, 160
g 6 20) were anesthetized by means of
inhalation of 1%–3% isoflurane (Piramal Health care, Andhra, India) in
oxygen. By means of a stereotactic
approach, 1.5 3 106 glioma cells were
injected into the right caudate nucleus
(2.8 mm lateral and 1.0 mm anterior
to the bregma, at a depth of 4.0 mm)
through a 10-mL Hamilton syringe. Tumors were then allowed to grow for 2
weeks. Twelve non–tumor-bearing rats
served as negative controls.
Optical imaging.—All tumor-bearing rats were randomized into six groups
of six rats. After injection of MGd (6
mg/kg) via the tail veins into the animals of five groups, the animals in these
groups were euthanized at 15, 30, 60,
120, and 240 minutes. The remaining
group of rats (tumor bearing) was injected with only saline solution to serve
as controls. Twelve normal rats (without brain tumors) were randomized
into two control groups of six rats. One
group received 6 mg/kg of intravenous
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EXPERIMENTAL STUDIES: Motexafin Gadolinium-enhanced Molecular MR and Optical Imaging in Gliomas

MGd and the other group received saline solution only. Tumor-free animals
were euthanized at 15 minutes after
MGd or saline solution administration.
By means of craniotomy, whole-brain
optical imaging (with 470/750-nm excitation and emission filter sets, 19 3
19-cm field of view, 1.1 f-stop, 4 3 4
bin, and 30-second exposure time) was
performed with the optical/x-ray imaging system to delineate the tumors.
MR imaging.—Whole rat brains
were placed into 15-mL tubes filled with
perfluoropolyether (Fomblin; Solvay
solexis, West Deptford, NJ). Wholebrain axial T1-weighted MR imaging
(T1-weighted and T2-weighted imaging
were performed by using a 14-T vertical
wide-bore MR Spectrometer with a microimaging coil [Bruker]). T1-weighted
images were acquired by using a low–
flip angle gradient-echo sequence with
the following parameters: 213.9/2.0;
field of view, 190 3 128 mm; matrix,
190 3 96; section thickness, 0.5 mm;
and number of signals acquired, 20. T2weighted images were acquired by using a multisection multiecho sequence:
3000/5.7; field of view, 190 3 128 mm;
matrix, 190 3 96; section thickness,
0.5 mm; and number of signals acquired, one.
Imaging analysis.—For optical imaging, images were analyzed (F.Z., with
4 years of experiences in optical imaging) by using imaging analysis software
(Bruker MI 7.1 SE; Bruker). Regions
of interest were drawn manually to
include the entire red fluorescence–illuminated tumors, while in the control
group, regions of interest were drawn
to include right frontal lobes. Photons
in the tumors were then quantified. For
molecular MR imaging, all images were
analyzed (L.Q., with 5 years of MR
imaging experience) by using Digital
Imaging and Communications in Medicine viewer software (RadiAnt DICOM
viewer 1.9; Medixant, Poznan, Poland).
Signal intensity of tumors was measured
by drawing four regions of interest (25
pixels each) over the peripheral zone
of the tumors at the largest cross-sectional diameters. Then, the average signal intensity of each tumor (SIT) was
calculated. The signal intensity (SIN)
4

and standard deviation (SDN) of the
background noise were also measured
by placing a region of interest on the
background of each image. Signal-tonoise ratios were calculated by using
the following equation: (SIT – SIN)/SDN.
Optical photon intensity and MR imaging signal intensity were plotted with
the time-to-create photon and signal
intensity time curves.
Histologic evaluation.—After optical and MR imaging were performed,
the brain was snap-frozen and cryogenically sectioned with sections 8–10
mm thick. Sections of tumors were then
taken at the point of largest diameter of
the mass. These were then stained with
either 4’, 6-diamidino-2-phenylindole
for confocal microscopy to confirm exclusive intracellular uptake of MGd by
tumor cells or hematoxylin and eosin to
define the histologic margins of tumors.
These sections were correlated with
their corresponding optical and MR images of the tumors at the largest mass
diameters.

Statistical Analysis
All values including the average tumor
sizes, fluorescent signal intensity, and
signal-to-noise ratios were depicted as
means 6 standard error of the mean.
Statistical comparison for the photon
and signal intensity of the tumors and
tumor sizes or control region of interest among different study groups was
performed by using one-way analysis
of variance (SigmaStat 3.5; Systat Software, Chicago, Ill). When analysis of
variance showed significance, comparisons between means were performed
with a two-tailed indirect Student t test.
Results
In Vitro Experiments: Time- and Dosedependent Uptake of MGd by Glioma Cells
Confocal microscopy showed that MGdtreated cells emitted red fluorescence
in the cytoplasm, which was not seen in
the control cells (those not treated with
MGd) (Fig 1). For the group of cells
treated with different concentrations
of MGd, ranging from 25 to 200 mg/
mL, we found that, in the concentration

Qiu et al

range of 252100 mg/mL, as the concentration of MGd increased, fluorescent
signal intensity of cells also increased,
while MR imaging T1 values decreased.
When MGd concentration was greater
than 100 mg/mL, the fluorescence signal intensity and MR imaging T1 values
plateaued (Fig 2). Confocal microscopy
allowed confirmation of quick intracellular uptake of MGd after 15–30 minutes of incubation (Fig 3). Quantification of cells by means of optical imaging
showed an increase of fluorescence
signal intensity as incubation times increased, which plateaued after the 24hour time point (Fig 3).

Animal Experiments
All tumor-bearing rats survived to the
time of euthanasia. Statistical comparisons of the tumor sizes among groups
showed that there was no significance
(4.5 mm 6 0.5, 4.5 mm 6 0.3, 4.9 mm
6 0.7, 5.7 mm 6 0.6, 5.9 mm 6 0.7,
5.2 mm 6 0.6, for the groups, respectively; one-way analysis of variance, P
= .525). Both optical imaging and MR
imaging allowed delineation of MGdilluminated tumors and definition of the
margins of these tumors, which correlated well with pathologic findings with
hematoxylin and eosin staining (Fig 4).
The exclusive accumulation of MGd in
tumor cells further confirmed that MGd
could be selectively taken up by tumor
cells and thus could be used to outline
the distinct boundary between the tumor and normal brain parenchyma.
In the evaluation of the kinetics of
MGd throughout a 4-hour period, both
optical and MR imaging showed that
the highest photon signal (2.6 3 108
photons per second per mm2 6 2.3 3
107; analysis of variance, P , .001) and
MGd enhancement occurred at 15–30
minutes after MGd administration (MR
imaging signal-to-noise ratio, 77.61 6
2.52, analysis of variance, P = .006)
(Fig 5). Even though MGd-enhancing
tumors demonstrated visibility for up to
4 hours, tumor margins at optical imaging became indistinct and tumor enhancement at MR imaging attenuated
during the observation time course.
No statistical differences were found
between the 15-minute and the 2-hour

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time points for either fluorescence or
MR imaging signal intensity. Imaging
findings correlated with results of confocal microscopy and hematoxylin and
eosin–stained microscopy of the tumor
tissues (Fig 6), which confirmed that
tumor enhancement at optical and MR
imaging originated from the intracellular location of MGd in tumor cells.

Figure 1

Discussion

Figure 1: Confocal microscopic images of rat C6 glioma cells treated with MGd at different concentrations show that MGd-emitting red fluorescence signal intensity becomes more intense as concentration of
MGd increases, which indicates that intracellular uptake of MGd is concentration dependent (magnification, 320).

Figure 2

Figure 2: Quantitative analysis of cells treated with MGd at different concentrations. A, MR imaging T1
map of glioma cells and, B, fluorescent optical image of cells at different concentrations from 0 to 200 mg/
mL show that T1 value decreases and fluorescent signal (SI ) increases as MGd concentrations increase. C,
Graph shows T1 values and fluorescent signal compared with MGd concentration, further confirming that
intracellular uptake of MGd increases as concentrations of MGd increase from 0 to 100 mg/mL and then
maintains stable level as MGd concentration increases further.
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In this preclinical study, we investigated the use of simultaneous MGdbased fluorescent optical imaging and
MR imaging to outline the histologic
margins of C6 gliomas in rats and to
determine the optimal time window to
visualize the tumors. Through in vitro
experiments, we found the best concentration of MGd to be 100 mg/mL,
which allowed for the maximal internalization of MGd by cells, with the
earliest uptake noted at 15–30 minutes
after the addition of MGd into the cell
culture medium. For our in vivo experiments, we used a rat C6 glioma cell
line to create the orthotopic glioma tumors, which tend to be morphologically
similar to glioblastoma multiforme, in
Sprague-Dawley rats. Fluorescent optical imaging and MR imaging were used
to evaluate MGd-enhancing gliomas at
different time points after the intravenous administration of MGd. Histologic analysis with confocal microscopy
and hematoxylin and eosin staining
microscopy on a section-by-section
basis showed that at the boundary
between normal brain tissue and tumor tissue, MGd had exclusively accumulated in the cytoplasm of tumor
cells. The results from our animal experiments confirmed that fluorescent
signal intensity and the enhancement
on T1-weighted MR images peaked at
15–30 minutes after MGd injection.
During the 4 hours when tumors were
observed, tumors maintained their visibility with decreasing margin distinctions at both optical and MR imaging.
These imaging findings were confirmed
by subsequent pathologic correlations.
High-grade gliomas are among the
most serious primary brain malignancies, which account for most brain
5

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Qiu et al

Figure 3

Figure 3: Optical imaging of cells treated with MGd at concentration of 100 mg/mL at various incubation durations. A, Optical image shows that signal intensity (SI ) of MGd-emitting fluorescence increases as MGd incubation time increases. B,
Graph shows time to fluorescence signal intensity curve in which signal intensity increases from 0 to 24 hours and then
plateaus for following 24 hours. C, Confocal microscopy confirms early uptake of MGd by cells after MGd incubation for
15 minutes, D, while no intracellular red fluorescence was seen in nontreated control cells (magnification, 320).

tumor–related deaths worldwide (1,2).
To achieve the most durable long-term
survival benefit for patients with gliomas, complete eradication of tumors
while sparing the normal brain parenchyma remains the most challenging
task for neurosurgeons (3,6,14,15).
Because of this daunting task in patients with gliomas, numerous intraoperative imaging–guided surgical
techniques have been developed in an
effort to extend survival and improve
quality of life (5,6,12,25,26). Use of
a single imaging modality to guide
surgery has inherent limitations and
disadvantages. Surgery performed on
the basis of the 5-aminolevulinic acid
signal without functional data—especially in the vicinity of eloquent brain
areas—could result in excess resection at the cost of increased risk of
postoperative neurologic deficits (3).
To date, intraoperative MR imaging
involves the use of a conventional
contrast agent such as gadopentetate
dimeglumine (11,12). However, this
gadolinium chelate may exude through
6

a glioma-compromised blood-brain
barrier and accumulate in the extracellular space, which may lead to the
pseudoenhancement of the adjacent
normal brain tissues and thereby
lead to further, unnecessary resection
(9,18). To resolve these potential issues, new dual-modality MR imaging
and optical imaging probes were developed to bridge these two distinctive
but complementary imaging modalities with the intent of improving intraoperative imaging–guided surgery
(9,27). In this context, MR imaging
offers three-dimensional visualization
of brain anatomy at high spatial resolution appropriate for presurgical
planning, while optical imaging provides real-time intraoperative imaging
for precise outlining of the margins
of tumors being resected. However,
these MR imaging and optical imaging probes still remain in their in vitro
and in vivo experimental stages.
MGd has been used in clinical trials as a radiation and chemotherapy
sensitizer (23,28–30), an intracellular

T1-weighted MR imaging contrast
agent, and a red fluorescence emitter
(19–21). In the current study, we attempted to investigate the application
of MGd for intraoperative imaging–
guided complete glioma resection. The
results of our study show that MGd can
allow delineation of the glioma tumor
margins at an early time point after
intravenous administration. Tumor visibility can last up to 4 hours at both
fluorescent optical imaging and MR
imaging because of the exclusive accumulation of MGd in tumor cells. The
results of this study suggest that MGd
has the promising potential to function
as an ideal intracellular contrast agent
for use in intraoperative optical and MR
imaging with tumor specificity and intracellular localization. We also found
that the peak enhancement of tumors
occurred at 15–30 minutes after MGd
administration. As observation time
increased, the visible margins of the
tumors decreased. As time progressed,
the tumors became less visible because
of the slow clearance of MGd from

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Figure 4

Figure 4: Representative optical and MR images of MGd-enhanced rat glioma mass, with pathologic correlation and confirmation. A, Wholebrain white light image of tumor (arrow) and, B, overlay image of fluorescent tumor on white-light image clearly show margin of tumor outlined
by MGd-emitting fluorescence. C, Axial non-contrast-enhanced T2-weighted (T2WI ) MR image shows tumor with intermediate signal intensity
(arrow). D, MGd-enhanced T1-weighted (T1WI ) MR image shows heterogeneous and significant internal enhancement of tumor with clear
margin. E, Whole-brain photograph displays tumor extruding (arrow) from surface of brain. F, Cross-sectional view of hematoxylin and eosin
(H&E )–stained brain section shows tumor located in frontal caudate area (arrow) and, G, glioma tumor is confirmed by means of microscopy. H,
Confocal microscopic image shows exclusive intracellular accumulation of MGd (pink spots in H) in glioma tumor cells.

the tumors. However, the total uptake
of MGd was limited. To achieve maximum visibility of tumor margins, continuous intravenous infusion of MGd is
necessary to compensate for the gradual loss of MGd from the tumors. Thus,
the results of this study are encouraging
for the development of an intraoperative imaging technique with MGd as an
optical and MR imaging marker to establish the true tumor margin, thereby
facilitating the complete removal of
gliomas.
Our current animal model had limitations. We could only perform ex vivo
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n

imaging of brain tumors from the rat
model because of the inherent difficulty
in achieving hemostasis at the ruptured
cerebral venous sinus in these small
animals after craniotomy. This limitation also prevented longitudinal imaging
follow-up of each animal. In addition,
although we could have performed in
vivo MR imaging first to evaluate the
enhancement patterns of tumors at
different time points, this would have
been at the expense of optical imaging.
During the initial 1–2-hour MR imaging time, MGd might have been cleared
from the tumors before optical imaging.

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However, the results of our study have
established the groundwork that will
allow for further advances in commercializing a hybrid imaging modality that
integrates both optical and MR imaging. The use of MGd-enhanced intraoperative imaging for precise resection
of gliomas will ultimately substantially
improve and affect patient care for
the population with this challenging
condition.
In conclusion, MGd-enhanced optical imaging and molecular MR imaging
can allow accurate definition of rat glioma tumor margins within the optimal
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Qiu et al

Figure 5

Figure 5: Optical imaging and MGd-enhanced T1-weighted imaging of rat glioma tumors collected at different time points after MGd administration.
Optical images (top row) and overlay optical images (second row) show that tumors have highest signal intensity at 15–30 minutes after administration
of MGd, and signal intensity becomes weak during observation times of 4 hours. Axial T1-weighted MR images (third row) show strongest enhancement
of tumors at 15 minutes, with signal intensity of MGd-enhanced tumors decreasing during observation times. Cross-sectional view of hematoxylin and
eosin–stained sections (fourth row) confirms presence of tumors in brains. Graphs show time-to-photon intensity (left) and time-to-signal-to-noise ratio
(SNR ) curves (right). Steep increase in signal intensity was seen at 15 minutes, followed by gradual signal intensity decline on both optical and MR
images. T1WI = T1-weighted imaging.
8

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Figure 6

Figure 6: Confocal microscopic images of MGd-treated tumors show that MGd is specifically taken up by glioma cells (intracellular pink spots on G–K  ), and highest signal intensity is demonstrated at 15 minutes. There is clear margin between MGd-containing tumor cells (T ) and normal brain cells (NB ).

time window ranging from 15 minutes
to 2 hours after MGd administration.
These findings present a potential clinical application for complete resection
of gliomas in a hybrid surgical setting
with intraoperative optical and/or MR
imaging.
Acknowledgment: We thank Pharmacyclics for
providing motexafin gadolinium.
Disclosures of Conflicts of Interest: L.Q. disclosed no relevant relationships. F.Z. disclosed
no relevant relationships. Y.S. disclosed no relevant relationships. Z.B. disclosed no relevant
relationships. J.W. disclosed no relevant relationships. Y.L. disclosed no relevant relationships. D.L. disclosed no relevant relationships.
C.I. disclosed no relevant relationships. X.F. disclosed no relevant relationships. X.Y. disclosed
no relevant relationships.

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