Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534& 2011 ISCBFM All rights reserved 0271-678X/11 $32.00
www.jcbfm.com

Development of an H215O steady-state method
combining a bolus and slow increasing injection
with a multiprogramming syringe pump
Masato Kobayashi1,2, Yasushi Kiyono1, Rikiya Maruyama1, Tetsuya Mori1, Keiichi Kawai1,2and Hidehiko Okazawa1,3
1

Biomedical Imaging Research Center, University of Fukui, Fukui, Japan; 2School of Health Sciences,
College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan;
3
Research and Education Program for Life Science, University of Fukui, Fukui, JapanAn 15O-labeled water (H215O) steady-state method for quantitative measurement of cerebral bloodflow (CBF), which is less stressful to small animals with a few point blood sampling, was developed.
After a simulation using a dose meter to achieve stable H215O radioactivity in the blood with amultiprogramming syringe pump programmed for slowly increasing injection volume, 10 rats werestudied with the injection method. Arterial blood was sampled every minute during 6-minutepositron emission tomography (PET) scans. After the PET scan, N-isopropyl-p-[125I]-iodoamphetamine (125I-IMP) was injected into the same rat to measure CBF using the autoradiography method
based on a microsphere model. Regions of interest were placed on the whole brain in H215O-PET and
125
I-IMP-autoradiography images, and CBF values calculated from both methods were compared.
Radioactivity in the dose meter achieved equilibrium B1 minute after starting the H215O injection. In
rat studies, radioactivity in the blood and brain rapidly achieved equilibrium at 2 minutes afteradministration. The correlation of CBF values of H215O PET (49.2±5.4 mL per 100 g per minute) andthose of 125I-IMP autoradiography (49.1±5.2 mL per 100 g per minute) was excellent (y = 1.01x0.37,
r2 = 0.97). The H215O steady-state method with a continuously increasing injection is useful for CBF
measurement in small animal studies, especially when multiple scans are required in the same
animal.
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534; doi:10.1038/jcbfm.2010.122; published online
28 July 2010
Keywords: cerebral blood flow; H215O PET; multiprogramming syringe pump; N-isopropyl-p-[125I]iodoamphetamine;
steady-state method

Introduction
Small animal models for cerebrovascular diseases
have recently been studied using 15O positronemission tomography (PET) (Temma et al, 2006).
15
O-labeled water (H215O) and 15O-gas PET for
measurement of cerebral blood flow (CBF) andoxygen metabolism usually requires multipoint
arterial blood sampling (Mintun et al, 1984). However, the number of blood samplings is limited due tothe small blood volume in animals, especially when

Correspondence: Dr H Okazawa, Biomedical Imaging ResearchCenter, University of Fukui, Japan 23-3 Matsuoka-Shimoaizuki,
Eiheiji-cho, Fukui 910-1193, Japan.
E-mail: okazawa@u-fukui.ac.jp
This study was partly funded by a Grant-in-Aid for Scientific
Research from Japan Society for the Promotion of Science(17209040, 19790861).
Received 18 April 2010; revised 14 June 2010; accepted 20 June
2010; published online 28 July 2010

multiple scans are required in the same animal. To
avoid the stress caused by blood sampling, several
techniques have been proposed to obtain an input
function without arterial blood sampling in small
animal PET studies using 18F-FDG (18F-fluorodeoxyglucose) or 11C-acetate (Laforest et al, 2005; Wu et al,
1996; Kim et al, 2006). However, the region ofinterest-based method with dynamic PET data wasless accurate because of a partial volume effect andother errors and estimates (Laforest et al, 2005).
Although a factor analysis technique successfully
extracted the time–activity curve of blood fromdynamic PET images in mice and rats (Wu et al,
1996; Kim et al, 2006), these techniques depended on
the spatial resolution of PET scanners to obtain clear
cardiac ventricle images. Because of high radioactivity in the lung, it is difficult to apply thesemethods to 15O-gas PET studies.
The steady-state method with 15O-labeled carbon
dioxide (C15O2) and 15O-labeled oxygen (15O2) was
developed to calculate the cerebral hemodynamic

H2 15O steady-state method for animal studies
M Kobayashi et al528state with a few arterial blood samplings as a simple
and practical method in clinical studies (Frackowiak
et al, 1980; Lammertsma et al, 1983). Jones et al(1985) applied the steady-state method to H215O PET
with continuous intravenous tracer infusion tomeasure CBF in a clinical PET study because arterialradioactivity during C15O2 inhalation is unstable dueto the condition of subject’s breathing. However, thesteady-state H215O method has not been applied toanimal studies because the total volume of tracer
injected is substantial even though the injection
speed is very slow.
The purpose of this study was to develop a new
steady-state method with a slowly increasing H215O
injection using a multiprogramming syringe pump tomeasure CBF in rats. The CBF values obtained fromthe H215O steady-state method (CBFH O) were compared with CBF measured by autoradiography using
the N-isopropyl-p-[125I]-iodoamphetamine (125I-IMP)microsphere method (CBFIMP) to evaluate the accuracy of the PET measurement of CBF in rats.

2.5

Velocity (mL/min)

2.0

1.5

1.0

0.5

0.0
0

1

2

Syringe2

67CatheterSyringe pump
Vial

15

Preparation of H2 O

Evaluation of the Program for the H2 15O Injection
The program for a multiprogramming syringe pump
(FP-2000, Melquest, Toyama, Japan) was designed to
combine the bolus and slowly increasing (B/SI) the H215O
injection by using a PE-50 tube (i.d. 0.5 mm, o.d. 0.9 mm,
length 60 cm) as a venous line. In this method, the injection
speed was controlled rapidly for the first 5 seconds to fill
the dead volume of the venous line with a small amount ofovershoot, the rate was changed moderately for 40 seconds
to increase blood radioactivity, followed by a slow but
gradually increased administration rate to compensate forthe decay of blood 15O radioactivity (Figure 1A). The rapid
injection rates in the initial two phases were programmed
to accelerate equilibrium of radioactivity in the rat’s body.
To compensate for the decay of blood radioactivity in theslowly increasing injection velocity, the injection rate wascontinuously changed under the assumption of an inverse
decay function as expressed by the following equation:
ð1Þ

where Y (mL/min) is the injection velocity for the syringe
pump, l (/min) is the decay constant of 15O, and t (minute) istime after H215O injection. a (mL/min) and b (dimensionless)are the constants for this injection method. The velocity

Dose meter
25
Radioactivity (MBq)15
O2 gas was produced by a 15N (p, n) 15O nuclear reaction
with a 2.5% O2/N2 gas target at an 18-mA proton current
accelerated for B10 minutes using an in-house cyclotron
Eclipse HP/RD (Siemens, Knoxville, TN, USA). The H215O
was synthesized by reduction of 15O2 with H2 gas. The
synthesized H215O gas was trapped in a saline solution andthen passed though a 0.22-mm millipore filter before
experimental use.

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

5

LeadshieldMaterials and methods

Y ¼ a exp ðb l tÞ

34Time (min)

20
15
10
5
0
0

1

2

3
Time (min)

4

5

6

Figure 1 Program of the bolus and slowly increasing (B/SI)
injection method and 15O-labeled water (H215O) radioactivity
measured with a dose meter. (A) The H215O was injected rapidly
for 5 seconds (32.0 mL/sec) to fill the dead volume of the venous
line with a small amount of overshoot, then for 40 seconds toincrease blood radioactivity until reaching 20 MBq in the body,followed by a slow but gradual increase in injection velocity
to compensate for the decay of 15O radioactivity in the blood.
(B) A diagram of the dose meter simulation for the steady-state
condition in the rat’s body. Automatically controlled H215O
injection system was connected to a vial in a dose meterand radiotracer was collected in the vial. (C) Radioactivityof H215O achieved equilibrium at B20 MBq in a dose meter.
Total radioactivity reached a plateau B1 minute after starting theinjection, which was kept within a 5% difference.
gradually increases according to reciprocal function of the
decay of 15O in the body, and the initial velocity of a isestimated from the radioactivity concentration at the end of

H2 15O steady-state method for animal studies
M Kobayashi et al529

phase 2. To determine the two constants in Eq. (1) for thesteady-state condition, radioactivity in the rat’s body wassimulated by collecting the tracer into an empty vial, which
was connected to the injection system with prefilled H215O.
The vial was put in a dose meter (Capintec Inc., Ramsy, NJ,
USA) and total radioactivity without decay correction wasmeasured as a function of time (Figure 1B).

Animal Positron Emission Tomography andAutoradiography
Animal studies were approved by the Animal Care
Committee at the University of Fukui and conducted inaccordance with the international standards for animal
welfare and institutional guidelines. Adequate measures
were taken to minimize pain or discomfort. Male SpragueDawley rats from Japan SLC Inc. (Hamamatsu, Japan) werehoused for 1 week under a 12-hour light/12-hour darkcycle with free access to food and water. The rats were
fasted with no food overnight with water supplied
ad libitum.
Ten rats (303.0±17.0 g) were anesthetized with intraperitoneal injection of chloral hydrate (0.4 mg/g bodyweight, intraperitoneally). A PE-50 catheter was inserted
into the femoral artery for blood sampling and the femoral
vein for H215O administration. The PET studies were
performed using a small animal PET scanner (SHR-41000,
Hamamatsu Photonics, Hamamatsu, Japan) (Yamada et al,2008). The scanner acquires 213 slices covering an axial
length of 160 mm, with a three-dimension mode andachieving a resolution of B2.0 mm full width at half
maximum in the transaxial direction and 2.8 mm full widthat half maximum in the axial direction. The rats wereplaced in a supine position on the scanner bed, and thelimbs were fixed using surgical tape. The orientation of
the cranial position was determined using a laser beam on
the scanner. Before emission scans, a transmission scan
was performed for attenuation correction using a 68Ge/68Ga
external source. A measure of 9 mL of B555 MBq H215O
was filled in a 10-mL syringe and set up on the syringepump.
A 6-minute list-mode PET scan was started withintravenous administration of H215O using a syringe pumpwith a B/SI injection program. During the PET scan, rats
were heated using an electric lamp to maintain the bodytemperature. Approximately 50 mL of arterial blood was
sampled every 1 minute, and radioactivity in the blood
samples was immediately measured with a well scintillation counter (ARC380, Aloka, Tokyo, Japan), and radioactivity concentration of each sampled blood was corrected
for the decay from the sampling time. Fifteen minutes
after the H215O-PET scan, 125I-IMP (1.11 MBq) was injected
into the same rat and arterial blood was withdrawn for
10 minutes after tracer administration with a constant rateof 100 mL/min using the same syringe pump. To obtain thearea under the curve of the true input function, collectedblood was put into 5 mL of octanole and a lipophilic
fraction was extracted (Kuhl et al, 1982). Rats were killedusing a guillotine at 10 minutes after the tracer injection.
The brains were immediately removed, frozen in iso-pentane, and sliced into 30 mm thickness slices in acryostat at approximately 201C. The brain slices were
mounted on glass slides and exposed to an imaging platewith 125I standards for 24 hours to obtain autoradiography.
Exposed imaging plates were scanned by a fluoro-image
analyzer (FLA-7000, Fuji Film, Tokyo, Japan). Body temperature was monitored during the experiment using animal
body temperature control (ATC-1000, World Precision
Instruments, Sarasota, FL, USA). Arterial blood gas andother physiological parameters were measured before the
H215O and 125I-IMP injection using a blood gas analyzer(ABL555, Radiometer, Copenhagen, Denmark) and a hemooximeter (OSM3, Radiometer, Copenhagen, Denmark).

Cerebral Blood Flow Calculation
The H215O-PET images were reconstructed using the Fourier rebinning-filtered back projection method with attenuation correction, and calibrated by the cross-calibration
factor. The CBFH O using the steady-state method was
calculated from PET data and arterial blood radioactivity
concentration using the following equation:
2

CBFH2 O ¼lðCa =Ct Þ  ð1=rÞð2Þwhere l (/min) is the decay constant of 15O, Ca (Bq/mL)
is the mean arterial H215O concentration, Ct (Bq/mL) is
the average brain radioactivity concentration for 2 minutes
calculated from 3 to 5 minutes dynamic data without decay
correction. r is a brain–blood partition coefficient forwater, which was fixed as 0.91 mL/g (Herscovitch andRaichle, 1985).
In 125I-IMP-autoradiography studies, CBFIMP using the
microsphere method was calculated according to thefollowing equation:
CBFIMP ¼

C IMP ðtÞRt i
E 0 CaIMP ðtÞdt

ð3Þ(t) (Bq/mL) is the total 125I-IMP radioactivity
where CIMP
iconcentration in the brain on autoradiography images andE is the first-pass extraction fraction. CIMP(t) (Bq/mL) is theainput function obtained from the arterial radioactivity
concentration of 125I-IMP and its area under the curve
was obtained using the radioactivity concentration of
the octanol extraction as mentioned above. The counts
for CIMP(t) were calibrated by using agar standard includingimeasured 125I radioactivity, which was exposed simultaneously on the imaging plate. For E of 125I-IMP, 0.92 wasused from a previous report (Di-Rocco et al, 1993).

Data Analysis
Magnetic resonance imaging (MRI) scans were taken underchloral hydrate anesthesia (0.4 mg/g body weight, intraperitoneally) using a 3.0-Tesla MR scanner (Signa Horizon,
GE Medical Systems, Milwaukee, WI, USA) before the PET
scans. The rats were placed and fixed between a pairof surface coils during the MR scan. T2-weighted images of
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

H2 15O steady-state method for animal studies
M Kobayashi et al530

the rat’s brain were acquired using a fast spin echo method(repetition time 5050 milliseconds, echo time 85 milliseconds, 256  256 matrix, slice thickness 1.0 mm, field ofview 6.0 cm, phase-field of view 4.8 cm, number of
excitations 3). The MRI and H215O images were coregistered using the Dr View software (AJS, Tokyo, Japan). The
MRI slices were used to draw region of interests. Threecoronal slices of 125I-IMP-autoradiography images wereused for drawing region of interests on the autoradiography
at the following four slice levels of corresponding MRI and
H215O-PET images, referring to the rat brain atlas: frontal
cortex, sensorimotor cortex, visual cortex, striatum, andthalamus (see Figure 5). The region of interests wereapplied to H215O and 125I-IMP images to compare global
and regional CBF values.
The CBFH O and CBFIMP values were compared using theStudent’s t-test. Blood gas data in the rats were compared
for the two experiments using a paired t-test. A P value of< 0.05 was considered to be statistically significant.
2

the rat body was B20 MBq, which was below thesaturation count of our small animal PET scanner.
Figure 3 shows representative H215O-PET
images. The H215O accumulation in the brain at 2 to
4 minutes was little different from that at 3 to
5 minutes. The body temperature of all rats was kept
between 321C and 381C, and the mean CBFH O andCBFIMP values in the whole brain were 49.2±5.4 and49.1±5.2 (mL per 100 g per minute), respectively.
The CBFH O values were well correlated with CBFIMP
as given in Figure 4A (y = 1.01x0.37, r2 = 0.97).
Figure 4B shows changes in CBFH O as a function ofbody temperature. The CBFH O values were closely
associated with body temperatures in rats. Table 1
shows mean values of arterial blood gas and otherphysiologic data in all rats measured before H215O
and 125I-IMP injection. None of these data weresignificantly different between two phases (P > 0.05),
and they were not associated with rat body temperature. Figure 5 shows the corresponding coronal slice
images of MRI, H215O PET, and autoradiography of125
I-IMP. The CBFH O in the regional cerebral areas is
given in Table 2. The CBFH O values were corrected
into cCBFH O using the regression line in Figure 4B toadjust CBFH O from 321C to 381C to 371C in termsof the rat’s body temperature. Each cCBFH O in theregional cerebral areas showed a small s.d. comparedwith CBFH O.
2

2

2

2

2

Results

2

Radioactivity measured by a dose calibrator using
the B/SI injection method is given in Figure 1C.
The increasing injection velocity was controlled
to achieve a steady state and the constants of a andb in Eq. (1) were determined to be a = 117 (mL/min)and b = 1.0  102 in our setting of the syringeand catheter. Total radioactivity reached a plateau
B1 minute after starting the injection, which was
kept within a 5% difference. In application of the
B/SI injection method, the H215O radioactivity concentration rapidly achieved equilibrium in the whole
blood and brain B2 minutes after H215O administration (Figure 2). The radioactivity after equilibrium in

2

2

2

2

Discussion
In this study, the H215O steady-state method wasused to measure CBF in the rat brain and the quantitative values were evaluated by the consecutive CBF
measurements using the 125I-IMP-autoradiographic
method. The steady-state method is ideal for a small
animal study because it requires only a few sampling

300

Radioactivity (kBq/mL)

250
Transaxial

200(kBq/mL)
150
R

L

150
100
Sagittal
50
0
0
0

1

2

3
Time (min)

4

5

6

Figure 2 Time–activity curve of the whole blood and wholebrain using the 15O-labeled water (H215O)-SS method with bolus
and slowly increasing (B/SI) injection. Radioactivity of H215O
achieved equilibrium in the whole blood (K) rapidly and thewhole brain (J) at B2 minutes after H215O injection.
Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

2-4 min

3-5 minFigure 3 Comparison of 15O-labeled water (H215O)-positronemission tomography (PET) images in the transaxial and coronal
direction at 2 to 3 minutes and 3 to 5 minutes. Cerebral
accumulation in the H215O images at 2 to 3 minutes was little
different from that at 5 to 6 minutes.

H2 15O steady-state method for animal studies
M Kobayashi et al

Table 1 Arterial blood gas data at H2 15O and 125I-IMP injection

60

CBFH2O (mL/100 g/min)

55

pH
PCO2 (mm Hg)
PCO2 (mm Hg)
Hct (%)
O2 Sat (%)
Hb (g/dL)

50

Before H2 15O

Before 125I-IMP

Difference

7.31±0.04
49.7±3.994.7±9.950.2±5.795.0±1.915.4±2.2

7.30±0.04
51.6±4.493.8±9.547.4±5.8
94.1±3.2
15.2±2.0

NS
NS
NS
NS
NS
NS

531

Hb, hemoglobin; Hct, hematocrit; H2 15O, 15O-labeled water; O2 Sat, arterial
oxygen saturation; PCO2, arterial carbon dioxide tension; PO2, arterial oxygen
tension; 125I-IMP, N-isopropyl-p-[125I]-iodoamphetamine.
NS, not significantly different between the two conditions.

45

40

35
35

40

45
50
CBFIMP (mL/100 g/min)

55

60

60

CBFH2O (mL/100 g/min)

55

50

45

40

35
32

34
36
Body temperature (°C)

38

Figure 4 Correlation between the cerebral blood flow (CBF)H O
and CBFIMP values and relationship between CBFH O and rat bodytemperature. (A) The CBFH O values were well correlated with
CBFIMP (y = 1.01x0.37, r2 = 0.97). (B) Change in CBFH O as afunction of body temperature. The CBFH O values were closely
associated with body temperatures in rats (y = 3.0x55.4,
r2 = 0.83).
2

2

2

2

2

points with a small sampling blood volume afterradioactivity has achieved equilibrium, which is lessinvasive to animals. If C15O2 gas is applied as a tracer
for CBF measurement, nasal radioactivity from thetracer gas affects the brain radioactivity count insmall animal PET studies, and the brain radioactivity
is also affected by respiratory conditions. We applied
the B/SI injection of H215O for the steady-statemethod, and achieved rapid radioactive equilibriumin the brain and blood at 2 minutes after the H215O

injection (Figure 2). The small injection volume of
H215O of only a 1.5-mL net increase by subtracting
the sampling volume from the injection volume
during a 6-minute scan is not stressful for rats
(Morton et al, 1997), and only slightly affects
physical parameters.
The steady-state method for CBF measurement wasdeveloped in clinical 15O-gas studies with continuous administration of a tracer (Frackowiak et al,
1980; Lammertsma et al, 1983). Jones et al (1985)
applied continuous H215O injection for the steadystate method. An approach with a multiprogramming
syringe pump has been implemented in 11C or 18FPET studies by using a bolus infusion followed byconstant infusion to achieve a steady-state concentration curve in tissue and plasma (Carson et al,
1993, 1997, 2000). Since the half-life of O-15 is
too short to achieve steady-state radioactivity in thebrain with a small injection volume, these approaches have not been applied to small animal 15O-PET
studies. In the B/SI injection method, the injection
velocity of the first phase was set at 32.0 mL/sec tofill the dead volume (B118 mL) of the venous line
tube (Figure 1A), followed by the moderate velocity
phase to increase the rat’s total body radioactivity
to be 20 MBq. In the final phase, the velocity
was gradually increased to maintain equilibriumby compensating the decay of blood 15O radioactivity
with a reciprocal function of the exponential decay
curve. The constants of a and b were obtained from adose calibrator simulation (Figure 1B), because total
radioactivity in the body, as well as blood radioactivity concentration, should be constant in thesteady-state method. Achieving overall equilibriumof radioactivity in the rat’s body is important to keeparterial input at a constant value. After the simulation, equilibrium of radioactivity in the rat’s bodywas successfully achieved at B2 minutes after thestart of injection with a small injection volume.
The CBFH O values from H215O PET were closely
correlated with the CBFIMP values obtained by the
autoradiographic method, which is considered to be
the gold standard for CBF measurement because of
fewer errors such as partial volume effects or otherestimates. Several previous studies using different
methods showed similar mean CBF values to our2

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

H2 15O steady-state method for animal studies
M Kobayashi et al
532

A

B

C

D

MRI

H215O

125I-IMP

Figure 5 Magnetic resonance imaging (MRI), 15O-labeled water (H215O), and N-isopropyl-p-[125I]-iodoamphetamine (125I-IMP)
images at the same slices. Region of interests were placed on the whole brain, the regions of frontal cortex (A), striatum(B), sensorimotor cortex (C), thalamus (C), and visual cortex (D) using MRI. The same region of interests was applied on the H215O
and 125I-IMP images at the same slice levels.

Table 2 CBFH O and cCBFH O (mL per 100g per minute) in the regional cerebral areas
2

CBFH OcCBFH O2

2

2

Whole brain

Frontal cortex

Sensorimotor cortex

Visual cortex

Striatum

Thalamus

49.2±5.454.4±2.249.5±5.954.9±2.4

51.2±4.856.5±1.849.4±6.4
54.6±2.951.4±5.6
56.7±2.353.8±4.3
59.1±1.2

cCBFH O, CBFH O at a body temperature of 321C to 381C was corrected to 371C.
2

2

CBFH O mean (Magata et al, 1995, 2003; Temma et al,
2006; Tiwari et al, 2010). The CBF values are wellknown to be affected by anesthesia, but the rat’s bodytemperature also closely associated with CBFH O
values (Rosomoff and Holaday, 1954). Our results
were also varying according to body temperature
between 321C and 381C as shown in Figure 4B.
Although the PCO2 is reported to be correlated withthe body temperature (Hägerdal et al, 1975), PCO2
values in the present study were not significantly
correlated, and thus, the correlation between CBFH O
and the body temperature was not caused by changes
in PCO2 . Lower body temperature may reduce brain
function, which induces decrease in CBF. The s.d. ofcCBFH O, in which CBFH O was corrected to be 371C
by using linear regression of body temperature, wassmaller than that in previous studies (Table 2). This
correction for body temperature would be a good
method for precise CBF evaluation using model
animals.
Another advantage of the H215O steady-state methodin our study is a better image quality obtained fromstable radioactivity in the brain averaged for 2 to
3 minutes from a 6-minute scan, as well as fewer blood
samples showing fewer errors caused by handlingand counting many sequential samples. Furthermore, handling of an injectable radiotracer is easierthan radioactive gas. As the B/SI injection method
increases the injection dose as a function of time, thelargest volume is injected in the last minute. If the
2

2

2

2

2

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

PET scanning time is minimized to 5 minutes from thestart of H215O injection to use a 2-minute average
image from a 3 to 5-minute dynamic scan, the method
can reduce the injection volume, and the stress to rats
could be minimized, allowing repeated CBF measurements in different conditions.
The bolus injection method usually requires
multipoint blood sampling to obtain time–activity
curves for the input function. Frequent multipoint
blood sampling is also stressful for small animals,
and radioactivity in the blood may include substantial errors because of busy handling of blood
samples. However, the steady-state method requires
fewer arterial samples of 3 to 4 points during thescan, which is less invasive for rats. As the total
volume of arterial blood sampling must be limited
to B10% of the total blood volume (Scipioni et al,1997), it should be < 2.0 mL for 300 g rats.
However, the steady-state method requires highspecific radioactivity of H215O ( > 62 MBq/mL), as
well as a high-performance and wide-range multiprogramming syringe pump because decay of 15O
in the blood should be compensated using a slowly
increasing injection rate. The mechanical performance of the syringe pump used in this study
achieved accuracy of ±0.1% and a variable velocityof 0.001 to 150 mm/min. The program using highspecific radioactivity resulted in high reproducibility
and accuracy. Eriksson et al (2008) developed a regulation system for the target tissue concentration of

H2 15O steady-state method for animal studies
M Kobayashi et al
11

C-flumazenil using a computer-controlled infusion
pump. However, the pump could not rapidly andcontinuously maintain a steady-state level radioactivity concentration in the whole brain. As a stableinjection protocol is needed for stable measurementof CBF, H215O radioactivity at the initial setting of thesyringe pump should be constant to avoid correctionof the injection program for every experiment.
The steady-state method may induce systemic
underestimation because of tissue heterogeneity
between gray and white matter as compared withthe bolus injection method (Herscovitch and Raichle,
1983; Herscovitch et al, 1983). This underestimation
is usually observed in the regions of relatively greater
CBF, especially under the conditions of hypercapnia
or acetazolamide administration. However, our results
showed regional values corresponding to CBFIMP in
the normal stable condition, as well as those ofprevious studies, indicating that this is a reliable
method for animal studies with model animals.
In conclusion, an H215O steady-state method forsmall animal PET was established using the B/SI
injection method, which can provide precise andstable CBFH O values. This method is useful for
repeated measurement of CBF in small animals withlower stress because of the smaller injection volume
with only a few blood samples. The CBFH O values
calculated from this method were well correlatedwith CBFIMP values from autoradiography. The s.d. ofthe cCBFH O was also smaller than that in previous
studies.
22

2

Acknowledgements
The authors thank the staff of the Biological Imaging
Research Center, University of Fukui for theirtechnical help.

Disclosure/conflict of interest
The authors declare no conflict of interest.

References
Carson RE, Channing MA, Blasberg RG, Dunn BB,
Cohen RM, Rice KC, Herscovitch P (1993) Comparisonof bolus and infusion methods for receptor quantitation:
application to [18F]cyclofoxy and positron emission
tomography. J Cereb Blood Flow Metab 13:24–42
Carson RE, Breier A, de Bartolomeis A, Saunders RC,
Su TP, Schmall B, Der MG, Pickar D, Eckelman WC(1997) Quantification of amphetamine-induced changes
in [11C]raclopride binding with continuous infusion.
J Cereb Blood Flow Metab 17:437–47
Carson RE (2000) PET physiological measurements using
constant infusion. Nucl Med Biol 27:657–60
Di-Rocco RJ, Silva DA, Kuczynski BL, Narra RK,
Ramalingam K, Jurisson S, Nunn AD, Eckelman WC(1993) The single-pass cerebral extraction and capillarypermeability-surface area product of several putative
cerebral blood flow imaging agents. J Nucl Med 34:641–8
Eriksson O, Josephsson R, Långstrom B, Bergström M(2008) Positron emission tomography and targetcontrolled infusion for precise modulation of brain drugconcentration. Nucl Med Biol 35:299–303
Frackowiak RS, Lenzi GL, Jones T, Heather JD (1980)
Quantitative measurement of regional cerebral bloodflow and oxygen metabolism in man using 15O andpositron emission tomography: theory, procedure, andnormal values. J Comput Assist Tomogr 4:727–36
Hägerdal M, Harp JR, Siesjö BK (1975) Influence ofchanges in arterial PCO2 on cerebral blood flow andcerebral energy state during hypothermia in the rat.
Acta Anaesthesiol Scand Suppl 57:25–33
Herscovitch P, Raichle ME (1983) Effect of tissue heterogeneity on the measurement of cerebral blood flow withthe equilibrium C15O2 inhalation technique. J Cereb
Blood Flow Metab 3:407–15
Herscovitch P, Markham J, Raichle ME (1983) Brain bloodflow measured with intravenous H15
2 O. I. Theory anderror analysis. J Nucl Med 24:782–9
Herscovitch P, Raichle ME (1985) What is the correct
value for the brain-blood partition coefficient for water?
J Cereb Blood Flow Metab 5:65–9
Jones SC, Greenberg JH, Dann R, Robinson Jr GD, Kushner M,
Alavi A, Reivich M (1985) Cerebral blood flow with thecontinuous infusion of oxygen-15-labeled water. J Cereb
Blood Flow Metab 5:566–75
Kim J, Herrero P, Sharp T, Laforest R, Rowland DJ, Tai YC,
Lewis JS, Welch MJ (2006) Minimally invasive methodof determining blood input function from PET images inrodents. J Nucl Med 47:330–6
Kuhl DE, Barrio JR, Huang SC, Selin C, Ackermann RF,
Lear JL, Wu JL, Lin TH, Phelps ME (1982) Quantifying
local cerebral blood flow by N-isopropyl-p-[123I] iodoamphetamine (IMP) tomography. J Nucl Med 23:
196–203
Laforest R, Sharp TL, Engelbach JA, Fettig NM, Herrero P,
Kim J, Lewis JS, Rowland DJ, Tai YC, Welch MJ (2005)
Measurement of input functions in rodents: challenges
and solutions. Nucl Med Biol 2:679–85
Lammertsma AA, Wise RJ, Heather JD, Gibbs JM, Leenders KL,
Frackowiak RS, Rhode CG, Jones T (1983) Correction forthe presence of intravascular oxygen-15 in the steady-statetechnique for measuring regional oxygen extraction ratio in
the brain: 2. Results in normal subjects and brain tumourand stroke patients. J Cereb Blood Flow Metab 3:425–31
Magata Y, Saji H, Choi SR, Tajima K, Takagaki T, Sasayama
S, Yonekura Y, Kitano H, Watanabe M, Okada H (1995)
Noninvasive measurement of cerebral blood flow andglucose metabolic rate in the rat with high-resolution
animal positron emission tomography (PET): a novel invivo approach for assessing drug action in the brains ofsmall animals. Biol Pharm Bull 18:753–6
Magata Y, Temma T, Iida H, Ogawa M, Mukai T, Iida Y,
Morimot T, Konishi J, Saji H (2003) Developmentof injectable O-15 oxygen and estimation of rat OEF.
J Cereb Blood Flow Metab 23:671–6
Mintun MA, Raichle ME, Martin WR, Herscovitch P (1984)
Brain oxygen utilization measured with O-15 radiotracers and positron emission tomography. J Nucl Med
25:177–87
Morton D, Safron JA, Rice DW, Wilson DM, White RD(1997) Effects of infusion rates in rats receiving repeatedlarge volumes of saline solution intravenously. Lab
Anim Sci 47:656–9

533

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

H2 15O steady-state method for animal studies
M Kobayashi et al534

Rosomoff HL, Holaday DA (1954) Cerebral blood flow
and cerebral oxygen consumption during hypothermia.
Am J Physiol 179:85–8
Scipioni RL, Diters RW, Myers WR, Hart SM (1997) Clinicaland clinicopathological assessment of serial phlebotomy in the Sprague Dawley rat. Lab Anim Sci 47:293–9
Temma T, Magata Y, Kuge Y, Shimonaka S, Sano K, Katada Y,
Kawashima H, Mukai T, Watabe H, Iida H, Saji H (2006)
Estimation of oxygen metabolism in a rat model of permanent ischemia using positron emission tomography with
injectable 15O-O2. J Cereb Blood Flow Metab 26:1577–83
Tiwari VN, Kiyono Y, Kobayashi M, Mori T, Kudo T,
Okazawa H, Fujibayashi Y (2010) Automatic labeling

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 527–534

method for injectable 15O-oxygen using hemoglobincontaining liposome vesicles and its application formeasurement of brain oxygen consumption by PET.
Nucl Med Biol 37:77–83
Wu HM, Huang SC, Allada V, Wolfenden PJ, Schelbert HR,
Phelps ME, Hoh CK (1996) Derivation of input function
from FDG-PET studies in small hearts. J Nucl Med
37:1717–22
Yamada R, Watanabe M, Omura T, Sato N, Shimizu K,
Takahashi M, Ote K, Katabe A, Moriya T, Sakai K,
Yamashita T, Tanaka E (2008) Development of a small
animal PET scanner using DOI detectors. IEEE Trans
Nucl Sci 55:906–11