Impact of animal handling on the results of 18F-FDG PET studies in mice.
ABSTRACT Small-animal PET scanning with (18)F-FDG is increasingly used in murine models of human diseases. However, the impact of dietary conditions, mode of anesthesia, and ambient temperature on the biodistribution of (18)F-FDG in mice has not been systematically studied so far. The aim of this study was to determine how these factors affect assessment of tumor glucose use by (18)F-FDG PET and to develop an imaging protocol that optimizes visualization of tumor xenografts.
Groups of severe combined immunodeficient (SCID) mice were first imaged by microPET with free access to food, at room temperature (20 degrees C), and no anesthesia during the uptake period (reference condition). Subsequently, the impact of (a) fasting for 8-12 h, (b) warming the animals with a heating pad (30 degrees C), and (c) general anesthesia using isoflurane or ketamine/xylazine on the (18)F-FDG biodistribution was evaluated. Subcutaneously implanted human A431 epidermoid carcinoma and U251 glioblastoma cells served as tumor models.
Depending on the study conditions, (18)F-FDG uptake by normal tissues varied 3-fold for skeletal muscle, 13-fold for brown adipose tissue, and 15-fold for myocardium. Warming and fasting significantly reduced the intense (18)F-FDG uptake by brown adipose tissue observed under the reference condition and markedly improved visualization of tumor xenografts. Although tumor (18)F-FDG uptake was not above background activity under the reference condition, tumors demonstrated marked focal (18)F-FDG uptake in warmed and fasted animals. Quantitatively, tumor (18)F-FDG uptake increased 4-fold and tumor-to-organ ratios were increased up to 17-fold. Ketamine/xylazine anesthesia caused marked hyperglycemia and was not further evaluated. Isoflurane anesthesia only mildly increased blood glucose levels and had no significant effect on tumor (18)F-FDG uptake. Isoflurane markedly reduced (18)F-FDG uptake by brown adipose tissue and skeletal muscle but increased the activity concentration in liver, myocardium, and kidney.
Animal handling has a dramatic effect on (18)F-FDG biodistribution and significantly influences the results of microPET studies in tumor-bearing mice. To improve tumor visualization mice should be fasted and warmed before (18)F-FDG injection and during the uptake period. Isoflurane appears well suited for anesthesia of tumor-bearing mice, whereas ketamine/xylazine should be used with caution, as it may induce marked hyperglycemia.
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ABSTRACT: This study aims at using 18F-FDG microPET to monitor the brown adipose tissue (BAT) glucose metabolism in obese and diabetic mouse models under different interventions, and study the therapeutic potential of BAT activation for weight loss and lowering of blood glucose in these models. Obese mice were established by a high-fat diet for eight weeks, and diabetes mellitus(DM) models were induced with Streptozocin in obese mice. 18F-FDG microPET was used to monitor BAT function during obese and DM modeling, and also after BRL37344 (a β3-adrenergic receptor agonist) or levothyroxine treatment. The BAT function was correlated with the body weight and blood glucose levels. Compared with the controls, the obese mice and DM mice showed successively lower 18F-FDG uptake in the interscapular BAT (P = 0.036 and <0.001, respectively). After two-week BRL37344 treatment, the BAT uptake was significantly elevated in both obese mice (P = 0.010) and DM mice (P = 0.004), accompanied with significantly decreased blood glucose levels (P = 0.023 and 0.036, respectively). The BAT uptake was negatively correlated with the blood glucose levels in both obese mice (r = -0.71, P = 0.003) and DM mice (r = -0.74, P = 0.010). BRL37344 treatment also caused significant weight loss in the obese mice (P = 0.001). Levothyroxine treatment increased the BAT uptake in the control mice (P = 0.025) and obese mice (P = 0.013), but not in the DM mice (P = 0.45). The inhibited BAT function in obese and DM mice can be re-activated by β3-adrenergic receptor agonist or thyroid hormone, and effective BAT activation may lead to weight loss and blood glucose lowering. Activating BAT can provide a new treatment strategy for obesity and DM.PLoS ONE 12/2014; 9(12):e113742. · 3.53 Impact Factor
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ABSTRACT: PET (positron emission tomography) imaging researches of functional metabolism using fluorodeoxyglucose (18F-FDG) of animal brain are important in neuroscience studies. FDG-PET imaging studies are often performed on groups of rats, so it is desirable to establish an objective voxel-based statistical methodology for group data analysis.PLoS ONE 09/2014; 9(9):e108295. · 3.53 Impact Factor
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ABSTRACT: Besides its application in oncology, 18F-FDG PET-CT imaging is also useful in the diagnosis of certain lung infections, inflammatory diseases, and atherosclerotic plaques. Myocardial uptake of 18F-FDG may hamper visualization of the lesions caused by these diseases. Two approaches have been proposed for reducing myocardial uptake in preclinical studies, namely, calcium channel blockers (verapamil) and high-fat diets such as commercial ketogenic diets and sunflower seed diets. The objective of this study was to compare the efficacy of these approaches in reducing myocardial uptake of 18F-FDG in mice.PLoS ONE 09/2014; 9(9):e107999. · 3.53 Impact Factor
Impact of Animal Handling on the Results of
18F-FDG PET Studies in Mice
Barbara J. Fueger1, Johannes Czernin1, Isabel Hildebrandt1, Chris Tran2, Benjamin S. Halpern1, David Stout1,
Michael E. Phelps1, and Wolfgang A. Weber1
1Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California; and
2Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California
Small-animal PET scanning with18F-FDG is increasingly used
in murine models of human diseases. However, the impact of
dietary conditions, mode of anesthesia, and ambient tempera-
ture on the biodistribution of18F-FDG in mice has not been
systematically studied so far. The aim of this study was to deter-
mine how these factors affect assessment of tumor glucose use
18F-FDG PET and to develop an imaging protocol that
optimizes visualization of tumor xenografts. Methods: Groups
of severe combined immunodeficient (SCID) mice were first im-
agedbymicroPET withfreeaccess to food, atroomtemperature
(20?C), and no anesthesia during the uptake period (reference
condition). Subsequently, the impact of (a) fasting for 8–12 h,
anesthesia using isoflurane or ketamine/xylazine on the18F-FDG
biodistribution was evaluated. Subcutaneously implanted hu-
man A431 epidermoid carcinoma and U251 glioblastoma cells
served as tumor models. Results: Depending on the study con-
ditions,18F-FDG uptake by normal tissues varied 3-fold for skel-
etal muscle, 13-fold for brown adipose tissue, and 15-fold for
myocardium. Warming and fasting significantly reduced the in-
tense18F-FDG uptake by brown adipose tissue observed under
the reference condition and markedly improved visualization
of tumor xenografts. Although tumor18F-FDG uptake was not
demonstrated marked focal18F-FDG uptake in warmed and
fasted animals. Quantitatively, tumor18F-FDG uptake increased
4-fold and tumor-to-organ ratios were increased up to 17-fold.
Ketamine/xylazine anesthesia caused marked hyperglycemia
and was not further evaluated. Isoflurane anesthesia only mildly
increased blood glucose levels and had no significant effect on
tumor18F-FDG uptake. Isoflurane markedly reduced18F-FDG
uptake by brown adipose tissue and skeletal muscle but in-
creased the activity concentration in liver, myocardium, and kid-
ney. Conclusion: Animal handling has a dramatic effect on
18F-FDG biodistribution and significantly influences the results
ofmicroPET studiesintumor-bearing mice.Toimprovetumorvi-
sualization mice should be fasted and warmed before18F-FDG
injection and during the uptake period. Isoflurane appears
well suited for anesthesia of tumor-bearing mice, whereas
ketamine/xylazine should be used with caution, as it may
induce marked hyperglycemia.
Key Words:18F-FDG; microPET; SCID mice; study conditions;
brown adipose tissue
J Nucl Med 2006; 47:999–1006
PETwith the glucose analog18F-FDG (18F-FDG PET) is
increasingly used in murine models of human diseases.
Specifically,18F-FDG PET is rapidly gaining importance
for monitoring progression and transformation of tumors
in mice (1), biologic characterization of tumor tissue (2),
and studying the effectiveness of anticancer drugs (3,4).
Furthermore,18F-FDG is frequently used as a reference stan-
dard when evaluating other imaging agents in mice (3–9).
For human18F-FDG PET studies, standard protocols have
been established that optimize contrast between tumor and
normal tissues (10,11). Protocols for animal imaging, on the
other hand, vary widely (1–9,12).
Despite their rapid growth, malignant tumor xenografts
frequently exhibit only modestly higher18F-FDG uptake
than most normal tissues. For example, we found in a re-
cent study that A431 tumor xenografts (volume doubling
time ,1 wk) were only barely visible in18F-FDG PET studies
(4). Upon more careful review we realized that glucose met-
abolic activity of various background tissues was high, thereby
possibly masking glucose metabolic activity of the tumors. We
hypothesized that the dietary state, ambient temperature, or
muscle activity might influence tumor detectability in small
animals by changing18F-FDG uptake of normal tissues. These
factors are known to significantly affect the biodistribution of
18F-FDG in humans. Because mice have approximately 7-fold
higher basal metabolic rates per body weight than humans
(13), the effect of dietary state and ambient temperature on
18F-FDG biodistribution may be even more pronounced than
in humans. Preliminary studies by Akhurst et al. (14) have
suggested that isoflurane anesthesia and fasting may improve
biodistribution of18F-FDG for tumor imaging. However, to
our knowledge, no systematic studies on this issue have been
published so far. The aim of this study was therefore to
investigate how18F-FDG biodistribution and tumor detectabil-
ity could be manipulated in mice by altering dietary state,
ambient temperature, and mode of anesthesia.
Received Nov. 2, 2005; revision accepted Mar. 10, 2006.
For correspondence or reprints contact: Wolfgang A. Weber, MD, Nuclear
Medicine, AR-264 CHS, UCLA School of Medicine, 10833 Le Conte Ave., Los
Angeles, CA 90095-6942.
ANIMAL HANDLING FOR18F-FDG PET STUDIES • Fueger et al.999
MATERIALS AND METHODS
All animal handling was performed in accordance with and
approved by the University of California Animal Research Com-
mittee guidelines. Eight- to 10-wk-old male severe combined
immunodeficient (C.B.-17 Scid/Scid) mice were obtained from
The human epidermoid carcinoma cell line A431 (15) was
acquired from the American Type Culture Collection. The human
glioma cell line U251 (16) was obtained from Dr. Charles Sawyer’s
laboratory, Department of Medicine, UCLA, Los Angeles, CA. Both
were cultivated in Dulbecco’s modification of Eagle’s medium
supplemented with 10% fetal bovine serum. All animal manipu-
lations were performed under sterile conditions. Cells growing
exponentially in vitro were trypsinized, resuspended in phosphate-
buffered saline and Matrigel (Collaborative Research), and in-
jected subcutaneously into the right shoulder area of SCID mice
(;106cells per mouse). Mice were imaged when tumor diameter
was at least 5 mm.
Measurement of Physiologic Parameters
Rectal temperature was measured with a thermistor probe. One
group of animals was kept under isoflurane anesthesia and on a
heating pad for 60 min. The heating pad we used is a plastic pad
(41 · 31 cm), with water-filled chambers (Baxter Healthcare
Corp.). Warm water of a defined temperature is continuously
being pumped through the chambers. Another group of animals
was kept under isoflurane anesthesia at room temperature. To
avoid severe hypothermia, this experiment was stopped in this
group after 30 min. Serum glucose levels were assayed in fasted
and nonfasted conscious mice before and after isoflurane and
ketamine anesthesia for 60 min. Blood samples (;10 mL per
mouse) were collected from the tail vein and glucose concentra-
tion (2 samples per condition) was measured using the Freestyle
glucose meter by TheraSense.
Influence of Animal Preparation and Handling on
To determine the impact of dietary state, ambient temperature,
and anesthesia on the biodistribution of18F-FDG, groups of 3–6
mice each were studied under the experimental conditions sum-
marized in Table 1. At the time of PET mice were 10–12 wk old
with an average body weight 6 SD of 24.2 6 2.4 g.18F-FDG (7.4
MBq [200 mCi] in 0.2 mL) was injected intraperitoneally after a
short (;5 min) isoflurane (2% in 100% oxygen) anesthesia period
unless otherwise indicated in Table 1. PET was started 60 min
after18F-FDG injection. As a reference condition, we imaged the
mice with no special preparation—that is, mice not fasted and kept
conscious at room temperature—during the uptake period. The
biodistribution of18F-FDG during all other conditions (Table 1)
was compared with this reference condition. For the fasting
condition, mice were deprived of food for 8–12 h before18F-
FDG injection. Mice had access to drinking water at all times.
Warming was achieved by placing the entire cage, including 5 or 6
animals, on the heating pad kept at 30?C. Warming was started at
least 30 min before18F-FDG injection and continued throughout
the uptake and imaging period. To evaluate the influence of
anesthesia on18F-FDG biodistribution, mice were either conscious
during the uptake period or anesthetized by either isoflurane
inhalation anesthesia (2% in 100% oxygen, IsoFlo; Abbott Lab-
oratories) or intraperitoneal injection of a ketamine/xylazine solu-
tion (200 mg/kg ketamine and 10 mg/kg xylazine; Fort Dodge
Animals Health, Division of Wyeth).
microPET was performed with the P4 microPET scanner
(Concorde Microsystems Inc.). The characteristics of this device
have been reported previously (17). In brief, the device has a ring
diameter of 26 cm and a 7.8-cm axial field of view. The intrinsic
spatial resolution ranges from 1.56 to 2.01 mm, with a mean of
1.75 mm. The reconstructed resolution is 1.8-mm full width at half
maximum in the center of the field of view and 3 mm at 4-cm
radial offset. For the PET scans the mice were kept under
isoflurane anesthesia and placed in an imaging chamber with an
ambient temperature of 30?C (18).18F-FDG was synthesized by a
previously described method (19) that is routinely used in our
facility. Quality control procedures were similar to the ones given
by Hung (20).
Images were reconstructed using filtered backprojection with-
out scatter or attenuation correction. We chose a ramp filter with a
cutoff frequency of 0.5 and a zoom of 5 to give a voxel size of
0.379 mm3. For cross-calibration of the dose calibrator and the
microPET scanner, a 3.5-cm cylinder phantom filled with a known
concentration of18F-FDG was imaged. From this scan a system
calibration factor was derived by dividing the known activity
concentration in the phantom by the measured mean counts per
voxel in the reconstructed PET images.
Quantitative Image Analysis
Regions of interest were manually drawn over the following
organs: brain, brown adipose tissue, heart, liver, paraspinal mus-
cle, kidney, Harderian glands, and subcutaneous tumors. Tracer
uptake by various organs was quantified as standardized uptake
values (SUVs) using the formula: SUV 5 tissue activity concen-
tration (Bq/mL)/injected dose (Bq) · body weight (g).
Intravenous Versus Intraperitoneal Injection of18F-FDG
Because of the very small caliber of the murine tail veins,
partial paravenous injection is common if18F-FDG is adminis-
tered by tail vein injection (intravenous). This could have signif-
icantly biased our comparison of the biodistribution of18F-FDG
under various conditions. Therefore, we used intraperitoneal in-
jection of18F-FDG for our experiments evaluating the influence of
animal handling on
18F-FDG biodistribution. To compare the
Summary of Study Conditions
nFastingWarmingAnesthesia during uptake period*
No, also conscious during18F-FDG
*No anesthesia indicates reference condition.
n 5 number of animals per experiment.
1000THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 6 • June 2006
biodistribution of18F-FDG after intravenous or intraperitoneal
injection, a dynamic PET scan of 60-min duration (12 · 5 s, 4 ·
1 min, 1 · 5 min, 5 · 10 min) was acquired in 12 fasted and
warmed mice bearing U251 xenografts; half had18F-FDG injected
intravenously and half had intraperitoneal injections. In a second
experiment we compared intravenous and intraperitoneal injection
of18F-FDG in not-fasted and not-warmed mice that were not kept
under anesthesia during the uptake period. In these animals, we
acquired 10-min static images 60 min after injection of18F-FDG.
Thus, comparison of intravenous and intraperitoneal injection of
18F-FDG was performed for the 2 most diverse experimental
conditions studied (fasted, warmed, and anesthesia vs. not fasted,
not warmed, and no anesthesia).
Results are presented as mean 6 1 SD. Differences among the
experimental groups in the SUVs of the various tissues and tumor-
to-organ ratios were statistically evaluated by ANOVA and
Bonferroni post hoc tests. Statistical significance was established
at the 95% level.
Changes in Physiologic Parameters During Anesthesia
When animals were kept under anesthesia for 30 min at
room temperature, the mean rectal temperature dropped from
32.1?C 6 0.8?C to 24.1?C 6 0.3?C. This dramatic decline
of body temperature was avoided when mice were kept on a
heating pad (body temperature after 30 and 60 min of
anesthesia, 35?C 6 0.7?C and 35?C 6 0.7?C, respectively).
Serum glucose levels averaged 122 6 21 mg/dL in the
nonfasted state and 73 6 34 mg/dL in the fasted state. One
hour of isoflurane anesthesia caused a modest increase of
blood glucose levels to 147 6 33 mg/dL. A similar effect
was observed in fasted animals (blood glucose, 104 6 49
mg/dL after 1 h of anesthesia). Anesthetizing the mice with
ketamine/xylazine markedly increased the serum glucose
level in the fasted as well as in the nonfasted animals (335 6
73 mg/dL and 363 6 59 mg/dL, respectively).
Influence of Warming and Fasting on Biodistribution of
18F-FDG in Mice Without Anesthesia During Uptake
Figure 1 shows typical examples for PET scans acquired
under the various conditions in nonanesthetized animals.
The results of the quantitative data analysis are summarized
in Figure 2. Under the reference condition (no warming and
no fasting, Fig. 1E), the highest18F-FDG uptake was seen
in brown fat (SUV, 4.9 6 1.1), Harderian glands (SUV, 2.2 6
0.5), skeletal muscle (SUV, 2.0 6 0.26), and myocardium
(SUV, 1.7 6 0.5).
Warming of the animals (Fig. 1A) reduced18F-FDG up-
take of brown fat by 56% (P 5 0.0001) and in skeletal
muscle by 28% (P 5 0.0001, Fig. 2). Fasting (Fig. 1B) re-
duced18F-FDG uptake of skeletal muscle by 31% (P 5
0.001) and of brown fat by 32% (P 5 0.006). Combining
warming and fasting (Fig. 1C) reduced18F-FDG uptake of
brown fat by 67% (P 5 0.0001) and of skeletal muscle by
47% (P 5 0.0001) as compared with the reference condi-
tion.18F-FDG uptake by brown fat was significantly lower
after combined warming and fasting than after fasting alone
(P 5 0.001). Conversely,18F-FDG uptake by skeletal mus-
cle was significantly lower after combined warming and
fasting than after warming alone (P 5 0.04). The effects of
warming and fasting on18F-FDG uptake by skeletal muscle
were offset when no anesthesia was used during18F-FDG
injection. Under this condition (Fig. 1D),18F-FDG uptake
by muscle was not significantly different from the reference
condition (SUV, 2.2 6 0.4, P 5 0.23). As expected, there
was also a significant increase in cerebral18F-FDG uptake
when no anesthesia was used for18F-FDG injection (SUV,
3.3 6 0.9, P 5 0.04).
18F-FDG uptake by Harderian glands was not signifi-
cantly influenced by warming or fasting but was markedly
increased when no anesthesia was used for18F-FDG injec-
tion (SUV, 8.6 6 1.9, P , 0.001). None of the other
analyzed organs (kidney, myocardium, liver) showed sig-
nificant differences in18F-FDG uptake in conscious mice.
For myocardium and kidney, this was caused mainly by a
large interindividual variability of tracer uptake within all
study conditions. In contrast, mean liver18F-FDG uptake
showed little variability within and across the different
study conditions (Fig. 2).
18F-FDG Biodistribution in Anesthetized Mice
Isoflurane Anesthesia. Isoflurane anesthesia in nonfasted
(Fig. 1G) mice caused a 5.5-fold increase of myocardial
18F-FDG uptake, when compared with the reference con-
dition (P 5 0.0001, Fig. 3). Isoflurane anesthesia also caused
a significant increase in18F-FDG uptake by liver and kid-
neys (P , 0.002, Fig. 3). In contrast,18F-FDG uptake in
brown fat and skeletal muscle was markedly reduced
(292% and 267%, respectively, P 5 0.0001).
When mice were fasted before isoflurane anesthesia
(Fig. 1H),18F-FDG uptake by the myocardium was reduced
by a factor of 3 compared with nonfasted animals, but still
remained higher (1.7-fold, P 5 0.03) than in the reference
condition (no anesthesia, no fasting). Under isoflurane anes-
thesia, fasting had no significant effect on18F-FDG uptake
by brown fat and muscle (Fig. 3).
Ketamine/Xylazine Anesthesia. Ketamine/xylazine anes-
thesia (Fig. 1I) had a similar effect as isoflurane on the
18F-FDG uptake in brown fat, skeletal muscle, kidneys, and
liver (Fig. 3). Interestingly, ketamine/xylazine had the
opposite effect on myocardial18F-FDG uptake than isoflu-
rane. Isoflurane increased myocardial18F-FDG uptake up
to 5.5-fold, whereas ketamine/xylazine decreased myocar-
dial18F-FDG uptake by 62%.
Comparison of Intravenous Versus Intraperitoneal
Figure 4 shows the time course of18F-FDG accumula-
tion by various tissues after intraperitoneal and intrave-
nous injection in fasted and warmed mice anesthetized by
ANIMAL HANDLING FOR18F-FDG PET STUDIES • Fueger et al.1001
isoflurane (n 5 6 per group). Though tracer uptake is
slower after intraperitoneal injection, all organs and the
U251 tumors reach comparable activity concentrations within
60 min after injection. Similarly, no significant differences
were found for tissue18F-FDG uptake of not-fasted and
not-warmed mice that were not kept under anesthesia dur-
ing the uptake period (Table 2, n 5 4 per group). Thus,
these data indicate that at 60 min after injection18F-FDG
biodistribution is comparable for intravenous and intraperi-
Impact of Warming, Fasting, and Anesthesia on Tumor
In summary, our data in nontumor–bearing mice indi-
cated that warming, fasting, and isoflurane anesthesia were
likely to improve the biodistribution of18F-FDG for tumor
imaging. We therefore evaluated whether fasting, warming,
and isoflurane anesthesia significantly improve visualization
of A431 and U251 tumor xenografts by reducing back-
ground activity. Groups of 3 (A431) and 6 (U251) tumor-
bearing mice each were imaged under the reference con-
dition as well as after combined warming and fasting with
and without isoflurane anesthesia. Because of the marked
increase in blood glucose levels caused by ketamine/xylazine,
no further experiments were performed with this form of
anesthesia. At the time of imaging tumor size was 5–11 mm
and did not show a significant difference among the 3
groups of animals. Figure 5 illustrates the biodistribution of
18F-FDG in mice bearing A431 xenografts. Though tumors
did not show focal18F-FDG above background activity
under the reference condition, they demonstrated marked
18F-FDG uptake, when animals were fasted and
warmed. The average SUV of the tumors under reference
conditions was 0.5 6 0.1. In fasted, warmed, and not-
anesthetized animals the tumor SUV was 3.7 times higher
(1.8 6 0.6, P 5 0.008). Keeping fasted and warmed mice
under isoflurane during the uptake period increased tumor
SUV to the same level (1.60 6 0.47, P 5 0.04 for com-
parison with the reference condition).
tribution of18F-FDG under various con-
ditions. Images show sagittal sections
through mice. A contrast-enhanced
microCT scan is shown for anatomic
reference. (A) Not fasted, warmed, no
anesthesia. (B) Fasted, not warmed, no
anesthesia. (C) Fasted, warmed, no an-
esthesia. (D) Fasted, warmed, no anes-
thesia, conscious injection. (E) Reference
conditions: not fasted, not warmed, no
anesthesia. (F) microCT, sagittal view for
anatomic reference. (G) Not fasted,
warmed, isoflurane. (H) Fasted, warmed,
isoflurane. (I) Fasted, warmed, ketamine.
Typical examples of biodis-
1002THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 6 • June 2006
To assess image contrast, we calculated various tumor-
to-organ ratios as shown in Table 3. Tumor-to-organ ratios
were lowest under the reference condition. Warming and
fasting (without isoflurane anesthesia) significantly im-
proved the tumor-to-muscle ratio (7.9-fold, P 5 0.008)
and the tumor-to-brown fat ratio (17.4-fold, P 5 0.01). A
similar increase in18F-FDG uptake was seen in warmed,
fasted, and anesthetized animals. Under this condition, the
tumor-to-muscle ratio increased 8.3-fold (P 5 0.01) and the
tumor-to-brown fat ratio increased 15.5-fold (P 5 0.04).
Experiments with mice bearing U251 xenografts provided
similar results (Table 3). There was a highly significant
increase in the tumor-to-muscle and tumor-to-brown-fat
ratios in fasted and warmed mice (P , 0.001 in anes-
thetized and in not-anesthetized mice).
This study demonstrates that animal handling has a
profound impact on the biodistribution of18F-FDG in mice
and significantly influences the visualization of xenotrans-
planted tumors. Varying fasting state, body temperature,
and mode of anesthesia caused .10-fold changes in the
18F-FDG uptake of normal organs. Tumor18F-FDG uptake
varied by a factor of 3.7. Depending on the conditions used
for animal handling, tumors were either barely visible in
the microPET studies or demonstrated marked focal18F-
The influence of blood glucose and insulin levels on18F-
FDG biodistribution is well known from human PET
studies and previous studies in rats using tissue sampling
to assess regional18F-FDG uptake (21–23). Because18F-
FDG competes with glucose for intracellular transport and
18F-FDG uptake decreases with
increasing blood glucose levels. Furthermore, insulin mark-
edly increases18F-FDG uptake by skeletal muscles and
myocardium though it has generally no effect on18F-FDG
uptake of cancer cells. Therefore, tumor18F-FDG uptake
and image contrast are lower in the nonfasted state (high
insulin and glucose levels) than in the fasted state (low
insulin and glucose levels).
More recently the effect of ambient temperature on the
18F-FDG uptake by brown adipose tissue has been de-
scribed in patients (24). Our data show that the ambient
temperature has a much more pronounced effect on18F-
FDG biodistribution in mice. For mice the so-called zone of
thermoneutrality lies between 30?C and 34?C (25). At this
temperature body temperature is controlled by heat con-
vection and no active processes are needed to maintain
body temperature. At room temperature (21?C) mice need
to generate heat by activation of brown adipose tissue and
muscle activity to maintain a stable body temperature.
Accordingly, metabolic rates have been shown to be 67%
higher at room temperature (15 W/kg) than at the zone of
thermoneutrality (9 W/kg) (25). Consistent with these
previous observations, mice that were kept at 30?C showed
markedly lower18F-FDG uptake by brown fat and muscle
in our study (Fig. 2). Because the zone of thermoneutrality
varies between different mouse strains (25) and we were
unable to find specific data for SCID mice, we arbitrarily
selected an ambient temperature of 30?C for our experi-
ments. At higher temperatures a further reduction in18F-
FDG uptake by brown adipose tissue might have been
achieved. However, keeping mice above the zone of
thermoneutrality represents a considerable heat stress. For
example, C57BL/6J mice exposed to 39.5?C for 4 h dem-
onstrate dehydration, hypoglycemia, and renal tubular ne-
crosis (26). Therefore, we selected the lower end of the
zone of thermoneutrality for our experiments.
18F-FDG uptake by brown fat was also reduced by fast-
ing the animals overnight (Figs. 1 and 2). It is known from
previous studies that feeding increases the metabolic activ-
ity of brown fat in rodents. This is considered to represent a
mechanism for stabilization of body weight: excess caloric
intake is converted to heat by the brown adipose tissue.
Conversely, overnight fasting has been shown to decrease
anesthetized during uptake period for various studied condi-
tions. Error bars show SD.
Biodistribution of18F-FDG in mice that were not
anesthetized during uptake period. Reference condition (not
fasted, not warmed, no anesthesia during uptake period) is
shown as a comparison. Error bars show SD.
18F-FDG in mice that were
ANIMAL HANDLING FOR18F-FDG PET STUDIES • Fueger et al.1003
perfusion of brown adipose tissue as well as heat produc-
In addition to its effect on18F-FDG uptake by normal
tissues, warming and fasting lead to a .3-fold increase in
tumor18F-FDG uptake. This finding is likely explained by
a combination of lower plasma glucose levels and de-
creased18F-FDG uptake by normal organs.
The effect of anesthesia on18F-FDG biodistribution has
very recently been studied by Lee et al. for ketamine/
xylazine and pentobarbital (28). In the present study we
extended these observations to isoflurane. Both xylazine and
isoflurane are known to suppress insulin secretion (28–30).
However, the effects of xylazine appear to be much more
pronounced, as xylazine induced marked hyperglycemia
(.300 mg/dL) even when mice were fasted for at least 8
h. Fasting mice for 20 h before18F-FDG injection has been
shown to attenuate xylazine-induced hyperglycemia (28).
However, prolonged fasting leads to weight loss and may
therefore be impractical, when animals need to be repeatedly
imaged within a short period of time—for example, for
treatment monitoring. In contrast to xylazine, isoflurane
anesthesia caused only a modest increase in blood glucose
levels in fasted and nonfasted animals, suggesting that its
effect on insulin secretion is relatively mild.
injection of18F-FDG (n 5 6 per group). Error bars shown as SEs of the mean.
(A–D)18F-FDG uptake of various normal tissues and U251 xenografts after intravenous (iv) and intraperitoneal (ip)
18F-FDG Uptake of Various Tissues 60 Minutes After Intravenous and Intraperitoneal Injection of18F-FDG (n 5 4 per group)
Injection Brown fatBrain Muscle Kidney Myocardium Liver
5.7 6 0.5
6.5 6 1.9
1.7 6 0.2
2.1 6 0.7
2.3 6 0.02
3.5 6 0.8
0.9 6 0.08
1.3 6 0.9
2.2 6 0.05
2.7 6 0.09
0.53 6 0.04
0.60 6 0.2
Data are expressed in g/mL (SUV). No anesthesia was used during uptake period.
1004THE JOURNAL OF NUCLEAR MEDICINE • Vol. 47 • No. 6 • June 2006
In addition to affecting insulin secretion, anesthetic drugs
also have specific effects on the glucose use of various
tissues. Brown adipose tissue has a dense sympathetic
innervation and its metabolic activity is regulated by b3
and a2receptors. Activation of b3receptors increases and
activation of a2receptors decreases perfusion and meta-
bolic activity. Norepinephrine binds to both types of
receptors, but its effect on b3 receptors is predominant
and norepinephrine markedly stimulates metabolic activity
(31). Accordingly, ketamine, which increases norepineph-
rine plasma levels, has been shown to stimulate metabolic
activity and18F-FDG uptake of brown adipose tissue (32).
However, our data indicate that during anesthesia with a
combination of ketamine and xylazine the effects of a2
receptor stimulation by xylazine are predominant and lead
to a marked decrease in18F-FDG uptake.
Isoflurane anesthesia also decreased18F-FDG uptake by
brown adipose tissue, which is consistent with its inhibiting
effect on thermogenesis by brown adipose tissue (33). As
observed in a previous study for BALB/c mice, isoflurane
markedly (up to 5.4-fold) increased18F-FDG uptake by the
myocardium (12). The mechanisms underlying the high
myocardial18F-FDG uptake during isoflurane anesthesia
are currently unknown, but
significantly decreased by fasting of the animals and using
isoflurane only during18F-FDG injection.
18F-FDG uptake could be
On the basis of our data we propose the following
protocol for imaging tumor xenografts with18F-FDG. Mice
should be fasted the night before the18F-FDG PET scan
and warmed on a heating pad before and after18F-FDG
injection. This approach markedly reduces18F-FDG uptake
by brown adipose tissue and skeletal muscle, which other-
wise significantly interferes with the visualization of tumor
tissue.18F-FDG should be administered under anesthesia to
further decrease skeletal muscle uptake. Keeping the mice
under isoflurane anesthesia for the whole uptake period not
only minimizes18F-FDG uptake by skeletal muscle and
brown fat but also decreases blood clearance resulting in
higher activity concentrations in liver and kidneys. Whereas
isoflurane anesthesia demonstrates a favorable effect on
18F-FDG biodistribution, ketamine/xylazine anesthesia
should be used with caution as it induces marked hyper-
glycemia. Standardization of animal handling and anesthe-
sia will be essential to ensure that reproducible and
comparable data are obtained from18F-FDG PET scans
of mice performed at different institutions.
We thank Waldemar Ladno and Judy Edwards for their
support with animal handling and imaging. This research
was supported by the UCLA Center for In-Vivo Imaging in
Cancer Biology (National Institutes of Health [NIH] grant
P50 CA86306), the UCLA Institute of Molecular Medicine
(Department of Energy grant DE-FC03-87E60615), UCLA
Lung Specialized Programs of Research Excellence (NIH
grant P50 CA90388), and the Austrian Science Fund
through an Erwin Schroedinger Scholarship.
Coronal and axial sections are shown. White lines in the coronal
sections indicate position of axial sections. (A) Not fasted, not
warmed, no anesthesia. (B) Fasted, warmed, no anesthesia. (C)
Fasted, warmed, isoflurane anesthesia. Red arrow indicates
tumor; brown arrow indicates brown fat; white arrow indicates
paraspinal muscle; yellow arrow indicates myocardium.
Tumor18F-FDG uptake under various conditions.
Tumor-to-Organ Ratios for Groups of Mice (n 5 3–6) Imaged Under 3 Different Conditions
TumorNot fasted, not warmed, no anesthesiaFasted, warmed, no anesthesiaFasted, warmed, isoflurane anesthesia
0.25 6 0.07
0.95 6 0.49
1.37 6 0.41
0.14 6 0.04
1.97 6 0.71*
1.83 6 1.12
3.57 6 1.47
2.44 6 1.23*
2.08 6 0.49*
0.69 6 0.13
2.21 6 0.80
2.17 6 0.68*
0.30 6 0.07
1.08 6 0.27
2.02 6 0.48
0.12 6 0.02
1.90 6 0.39*
1.86 6 0.49*
4.61 6 2.23*
2.58 6 0.47*
1.83 6 0.42*
0.94 6 0.28
1.45 6 0.38
2.21 6 0.53*
*P , 0.05 for comparison with reference condition (Bonferroni post hoc test).
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