ORIGINAL ARTICLE
In vivo measurement of superoxide in the cerebral cortex
during anoxia-reoxygenation and ischemia-reperfusion
Keiji Yamaguchi, Daisuke Uematsu, Yoshiaki Itoh, Shigeru Watanabe and Yasuo Fukuuchi
Department of Neurology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on June 26, 2002)

Abstract. The exact time proﬁle of superoxide generation during anoxia-reoxygenation and ischemiareperfusion was assessed in the feline cerebral cortex in vivo using a chemiluminescence techniquewith a probe speciﬁc for superoxide, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin3-one (MCLA). MCLA solution was superfused on the cortex throughout the protocol, and MCLA
chemiluminescence was measured using a newly developed photon counting system. Reﬂectance at 398
nm was simultaneously measured to compensate for hemodynamic artifacts resulting from cerebral
blood volume changes. In 19 animals, a 90-second anoxia was induced by the inhalation of 100% nitrogen followed by a 40-minute reoxygenation. The chemiluminescence decreased during the period ofanoxia (p H 0.01) and then exceeded the baseline level at 15 and 20 minutes after reoxygenation(p H 0.05). In six animals, superoxide dismutase (SOD) was continuously superfused and anoxiareoxygenation was performed in the same manner. The chemiluminescence decreased during theperiod of anoxia (p H 0.05) but did not exceed the baseline level during the reoxygenation period,
indicating that an increase in superoxide production was the main cause of the chemiluminescence increase. In eight animals, a 15-minute forebrain ischemia was induced by the occlusion of the bilateralcommon carotid arteries with systemic hypotension (systolic blood pressure less than 50 mmHg) followed by a 30-minute reperfusion. The chemiluminescence decreased during the period of ischemia(p H 0.01) and then increased at 20 and 25 minutes after reperfusion (p H 0.05). These results indicatethat superoxide generation decreases during anoxia and ischemia and then increases within 20 minutes
after reoxygenation or reperfusion. (Keio J Med 51 (4): 201–207, December 2002)
Key words: SOD, cat, photon counting, cranial window

Introduction
During cerebral ischemia or cardiac arrest, the oxygen and glucose supplies to the brain are severely
reduced, resulting in an energy crisis.1 Energy depletion causes irreversible tissue damage and thus theearly restoration of blood ﬂow is pivotal to reduce theinjury; however, injuries are known to progress evenafter reperfusion.2 Lipid peroxidation in brain tissue
occurs mainly during the post-ischemic reperfusion period. Reactive oxygen species (ROS) are considered
important mediators in reperfusion injury.3
Among the ROS, superoxide (O2  ) is the principal
reduction product of oxygen. Though relatively inactive
itself, its metabolites, peroxynitrite and hydroxyl radical, are highly reactive and can cause tissue injury.4,5Many previous reports have shown that O2  might play
an important role in the progression of tissue injury
during cerebral ischemia or reperfusion: brain injury
was attenuated by superoxide dismutase (SOD) administration6 and was suppressed in transgenic mice overproducing SOD,7–14 while it was more severe in SODdeﬁcient mice.15,16 Clarifying the temporal course ofO2  production is essential to understand the mechanism of ROS-related reperfusion injury. However, theexact timetable of O2  production in cerebral ischemia
and reperfusion in vivo remains unknown.
The short half-life of O2  makes it difﬁcult to measure and only a few techniques can be utilized: SODinhibitable nitroblue tetrazolium,17 chemiluminescence(CL),18,19 electron spin resonance spectroscopy,20 andcytochrome c-coated platinized carbon electrodes.21

Reprint requests to: Dr. Keiji Yamaguchi, Department of Neurology, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku,
Tokyo, 160-8582, Japan, e-mail: kei-yama@sc.itc.keio.ac.jp
201

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Yamaguchi K, et al: Oxidative stress in cerebral ischemia and anoxia

Among them, the CL technique is very reliable, has abetter time resolution, and is less invasive. This technique is also suitable for continuous in vivo measurements and has been used to assess ROS production.18,19 However, speciﬁc CL probes for the detectionof O2  have not been used to investigate cerebral
ischemia. Furthermore, the presence of hemodynamic
artifacts was not excluded in previous studies using CL.
The aim of the present study was to develop a newin vivo CL technique for assessing the exact time proﬁleof O2  generation in the feline cerebral cortex duringanoxia-reoxygenation and ischemia-reperfusion phases.

composition of the aCSF was as follows: Na, 140 mEq;
K, 2.5 mEq; Ca, 1.5 mEq; Mg, 1.0 mEq; Cl, 130 mEq;
HCO3 , 14.5 mEq; glucose, 3.3 mEq; and HEPES, 10
mEq (pH 7.35 at 37 C).

Methods
Animal preparation
All procedures were performed in compliance with
the Experimental Animal Guidelines of Keio University School of Medicine and were approved by the Experimental Committee of Keio University. A total of
33 adult male cats, weighing 2.5 to 4.5 kg, were used inthis study. The cats were anesthetized with an intraperitoneal injection of 50 mg/kg of a-chloralose (Sigma
Chemical Co.) and 500 mg/kg of urethane (Sigma
Chemical Co.). The rectal temperature was maintained
at 37.0 G 0.5 C by a warming pad. An incision wasmade in the neck and the animals were tracheotomized
and artiﬁcially ventilated (Harvard Respirator, Model
662). A catheter (3 Fr) was placed in the left lingual
artery to enable the injection of a small amount ofsaline; this step was necessary to obtain the cerebrovascular hemodilution curves. The bilateral carotid
arteries were isolated and loops made from polyethylene catheter (PE-10) were carefully passed around thearteries for later occlusion and release. Catheters (5 Fr)
were inserted into one femoral vein and both arteries: acatheter was inserted into one femoral artery to monitor the arterial blood pressure using an electronic recorder (Rikadenki Kogyo, Model R-66), another catheter was inserted into the other femoral artery to obtainblood gas analysis samples (arterial pH, PaO2 , andPaCO2 ), and a third catheter was inserted into a femoral vein for blood withdrawal and re-infusion during theischemia-reperfusion protocol. The head of each animal
was ﬁxed in a stereotactic frame (Narishige Co. Ltd.).
After the skin on the head was incised and the muscles
were removed from the skull, a burr hole was trephinedin the left temporoparietal region of the skull and thedura was carefully incised. A cranial window (10 mm indiameter) with inlet and outlet tubes was sealed into
the burr hole using dental cement. An infusion pump
was connected to the inlet tube with a catheter and artiﬁcial cerebrospinal ﬂuid (aCSF) was superfused beneath the cranial window at a rate of 5.0 ml/hour. The

MCLA chemiluminescence
2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA: Tokyo Kasei Co. Ltd.), a
Cypridina luciferin analog, is a highly sensitive andspeciﬁc probe for detecting O2  . The light emitted by
the probe has a maximum intensity at a wavelength of
465 nm in the presence of O2  , which has been widelyused to detect O2  both in vivo22–25 and in vitro.26–28
Since the probe partially penetrates into the cell membrane,29 it reﬂects the O2  concentration in both theextracellular and intracellular spaces.
Photon counting system
Optical signals were measured using the system
illustrated in Fig. 1. A microscope equipped with adichroic beam splitter and two photomultiplier tubes(R647-04, Hamamatsu Photonics K.K.) was focused ona cortical region devoid of large vessels. A xenon-arc
lamp (Model PS-X500, Japan Spectroscopic Co., Ltd)
set at a wavelength of 398-nm was used to light the areawith a constant intensity. The 398-nm reﬂectance and
MCLA CL from the cortex were measured simultaneously using the system and the dichroic beam splitter
was used to separate the two optical signals. MCLA CL(465 nm) passing through bandpass ﬁlter 1 (BPF 1:
465 G 10 nm) was detected using photomultiplier tube 1(PMT 1), while the 398-nm reﬂectance passing throughbandpass ﬁlter 2 (BPF 2: 398 G 10 nm) was detectedusing photomultiplier tube 2 (PMT 2). The signals weretransferred to, processed by, and then visually displayedin real time on a personal computer. These measurements were performed in a dark room where the background photon count was less than 50 counts/sec.
Correction of hemodynamic inﬂuence
When light passes through a tissue containing blood,its intensity is attenuated by hemoglobin absorption.30,31 Because the MCLA CL intensity measured invivo is inﬂuenced by the changes in cerebral blood volume (CBV), hemodynamic artifacts were corrected toassess the true changes in MCLA CL. This method of
hemodynamic artifact correction has been described
elsewhere.32–34
During the resting state, a small amount of saline(1.0 to 2.0 ml) was injected through the lingual arteryinducing hemodilution and causing a transient increasein the apparent MCLA CL and 398-nm reﬂectance. The

Keio J Med 2002; 51 (4): 201–207

203

Fig. 1 Photon counting system. A xenon-arc lamp (398 nm) is projected on the cerebral cortex. The 398 nm reﬂectance (398 nm) and MCLA
chemiluminescence (460 nm) are collected with a microscope, are separated with a dichroic beam splitter, and detected by photomultiplier tube
1 (PMT 1) and photomultiplier tube 2 (PMT 2) with bandpass ﬁlter 1 (BPF 1: 465 G 10 nm) and bandpass ﬁlter 2 (BPF 2: 398 G 10 nm), respectively. The signals are then analyzed by a personal computer and monitored on a display. All solutions are superfused from an inlet tube
over the cortex and excreted through an outlet tube, using an infusion pump.

resulting dilution curve was utilized to obtain the
hemodynamic correction factor (k factor) for each animal. The k factor can be expressed as follows:
k ¼ DCL=DR
where DCL is the difference between the peak intensity
and the basal intensity of the apparent MCLA CL during hemodilution and DR is the difference between thepeak intensity and the basal intensity of the 398-nm
reﬂectance during hemodilution. The corrected MCLA
CL can, therefore, be expressed as:cCL ¼ CL  k  R
where cCL represents the change in the corrected
MCLA CL intensity, CL represents the change in theapparent MCLA CL intensity, and R represents thechange in the 398-nm reﬂectance intensity.
Experimental protocol
MCLA CL and 398-nm reﬂectance were simultaneously measured throughout the procedure. Before
the following experimental protocols were performed,
the background photon level was measured under aCSF
superfusion. Then, aCSF containing 100 mM of MCLA
(MCLA solution) was superfused at a rate of 5.0 ml/
hour. The baseline MCLA CL and 398 nm reﬂectancelevels were designated as the points where theirintensities became constant, after about 30 minutesof MCLA superfusion in the non-SOD and ischemiareperfusion groups. On the other hand, the baseline of
the SOD-group was designated as the steady state after
SOD superfusion. The value of photon counting at thebaseline was assigned to 100% CL in the following
analysis.
1) Anoxia-reoxygenation (n ¼ 25)a) Non-SOD group (n ¼ 19)
Anoxia was induced by 100% nitrogen inhalation for
90 seconds and then followed by 40 minutes of reoxygenation with room air.
b) SOD group (n ¼ 6)
MCLA solution containing 1000 units/ml SOD (from
Bovine Kidney, Sigma Chemical Co.) was superfused
onto the cortical surface at a rate of 5.0 ml/hour
throughout the protocol. After 30 minutes of SOD
superfusion, when the intensity of both the MCLA CL
and 398-nm reﬂectance had stabilized, anoxia andreoxygenation were induced as in the non-SOD group.
2) Ischemia-reperfusion (n ¼ 8)
A 15-minute period of forebrain ischemia was induced by occluding the bilateral common carotid arteries. In addition, hypotension was induced by thewithdrawal of venous blood (50 to 100 ml) from thefemoral vein so that the systolic arterial blood pressure
was maintained at less than 50 mmHg. The ischemia
was then followed by a 30-minute reperfusion period.

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Table 1

Yamaguchi K, et al: Oxidative stress in cerebral ischemia and anoxia
Physiological Parameters and k Value
Anoxia/Reoxygenation

Body weight (kg)Systolic blood pressure (mm Hg)
Diastolic blood pressure (mm Hg)
pH
PaO2 (mm Hg)
PaCO2 (mm Hg)
k value

Non-SOD
group (n ¼ 19)

SOD group(n ¼ 6)

3.5 G 0.7
173 G 17
121 G 15
7.375 G 0.085
131 G 1729 G 60.27 G 0.19

3.5 G 1.0
172 G 10124 G 117.381 G 0.055
136 G 24
25 G 50.32 G 0.22

Ischemia/
Reperfusion(n ¼ 8)3.3 G 0.8
170 G 19113 G 18
N.A.
N.A.
N.A.
0.34 G 0.23

N.A.: not available.

Data analysis
All values are expressed as the mean G S.D. The
corrected CL data were statistically compared with thebaseline levels obtained before the induction of anoxia
or ischemia. For the statistical analysis, we carried out aFisher’s protected least signiﬁcant difference test. The
level of statistical signiﬁcance was deﬁned as p < 0.05.
Results
Physiological parameters
Physiological parameters under the resting conditions are given in Table 1. Before anoxia or ischemia
was induced, the physiological parameters of all animals were within the normal ranges. There was no statistical signiﬁcance between the SOD and the non-SOD
groups.
k factor
The k factor for the non-SOD, SOD, and ischemiareperfusion groups were 0.27 G 0.19, 0.32 G 0.22, and0.34 G 0.23, respectively. There was no statistical signiﬁcance among the three groups (Table 1).
Optical signals in the basal level
Because photon counts reached a steady state after
30 minutes of MCLA superfusion, we deﬁned this condition as the basal level of the optical signals. The
MCLA CL and 398-nm reﬂectance intensities were1111 G 543 counts/sec and 1241 G 553 counts/sec, respectively (n ¼ 33).
1) Anoxia/reoxygenation (n ¼ 25)a) Non-SOD group (n ¼ 19)

Fig. 2 Representative recording of optical signals obtained during
the anoxia and reoxygenation phases in non-SOD group (see text).

Fig. 2 shows representative MCLA CL and 398-nm
reﬂectance recordings. Several seconds after the initiation of 100% nitrogen inhalation, the observed
MCLA CL began to decrease markedly. Thirty seconds
after reoxygenation, the MCLA CL started to increase
sharply, overshooting the baseline level within 3
minutes and remaining high throughout the remainderof the protocol. The 398-nm reﬂectance also decreased
during the period of anoxia (indicating an increase in
CBV). Several seconds after reoxygenation, the 398-nm
reﬂectance began to increase and returned to the baseline level within a few minutes. In contrast to the
MCLA CL recordings, the 398-nm reﬂectance recordings gradually decreased thereafter until the end of the
protocol.
Fig. 3 shows the percent changes of the corrected
MCLA CL to the baseline level during the anoxia
and reoxygenation phases. The corrected MCLA CL
decreased signiﬁcantly at the end of the anoxia phase(75.1 G 23.9%: p < 0.01), and increased signiﬁcantly atKeio J Med 2002; 51 (4): 201–207

Fig. 3 Changes in MCLA chemiluminescence during the anoxia andreoxygenation phase. The percent changes in the corrected MCLA
chemiluminescence compared to the baseline level decreased signiﬁcantly at the end of the anoxic phase in both the non-SOD group(75.1 G 23.9%; p < 0.01) and the SOD group (83.9 G 13.4%; p < 0.05).
After reoxygenation, the corrected MCLA chemiluminescenceincreased signiﬁcantly at 15 and 20 minutes in the non-SOD group(110.3 G 10.3%; p < 0.05 and 109.1 G 11.7%; p < 0.05, respectively),
but did not increase in the SOD group. *: p < 0.05, **: p < 0.01 to thecorrected MCLA chemiluminescence levels at the baseline level(Fisher’s protected least signiﬁcant difference test).

15 and 20 minutes after reoxygenation (110.3 G 10.3%
and 109.1 G 11.7%, respectively: p < 0.05).
b) SOD group (n ¼ 6)
When MCLA solution containing SOD was superfused instead of MCLA solution without SOD, opticalsignals began to decrease and stabilized within 30
minutes; MCLA decreased from 1086 G 464 counts/sec
in the basal level to 468 G 170 counts/sec after SOD
superfusion and the 398-nm reﬂectance decreased from1517 G 673 counts/sec to 1284 G 522 counts/sec. Thus,
the corrected MCLA CL became 51.1 G 9.0% of
the basal level after SOD superfusion. The anoxiareoxygenation protocol was then carried out. Percent
change in corrected MCLA intensity was calculated bycomparing the signal intensity after the anoxia to that
before anoxia. As shown in Fig. 3, the percent changesof the corrected MCLA CL level decreased signiﬁcantly
at the end of the anoxia phase (83.9 G 13.4%: p < 0.05).
The corrected MCLA CL level almost recovered during
reoxygenation but did not exceed the baseline level.
2) Ischemia/reperfusion (n ¼ 8)
As shown in Fig. 4, the percent changes of thecorrected MCLA CL decreased signiﬁcantly during
the ischemia phase (87.0 G 14.0%, 87.9 G 10.4%, and82.8 G 9.8% after 5, 10, and 15 minutes of ischemia;
p < 0.01) and then increased to surpass the baseline

205

Fig. 4 Changes in MCLA chemiluminescence during the ischemia
and reperfusion phase. The percent changes in the corrected MCLA
chemiluminescence decreased signiﬁcantly during the ischemia phase(87.0 G 14.0%, 87.9 G 10.4%, and 82.8 G 9.8% after 5, 10, and 15
minutes of ischemia; p < 0.01) and then increased to surpass thebaseline level at 20 and 25 minutes after reperfusion (113.9 G 8.5%
and 113.4 G 8.4%, respectively; p < 0.05). *: p < 0.05, **: p < 0.01 to
the corrected MCLA chemiluminescence levels at the baseline level(Fisher’s protected least signiﬁcant difference test).

level at 20 and 25 minutes after reperfusion (113.9 G
8.5% and 113.4 G 8.4%, respectively; p < 0.05).
Discussion
We have developed a novel photon counting system
for the simultaneous assessment of MCLA CL and 398nm reﬂectance in the feline cortex during anoxia andreoxygenation. After correcting for hemodynamic
inﬂuences by measuring the 398-nm reﬂectance, CLchanges in the cortex were assessed. This study can besummarized as follows: 1) O2  production was assessedby a CL method with excellent time resolution andminimal invasiveness using MCLA, an O2  sensitiveand speciﬁc CL probe and 2) hemodynamic inﬂuenceswere taken into account to precisely assess O2  production.
Because detected light intensity decreases by the
inverse square of the distance between the objective
lens and the tissue, movement of the cortical surface
may inﬂuence the light intensity in this experiment.
However, we evaluated the motion artifact by observing the cortex covered with a cranial window through a
microscope in a preliminary experiment. The surface
image, in fact, never went out of focus during the procedure. Furthermore, we used an objective lens with along working distance (50 mm) in order to minimize themotion artifact if present. Therefore, we believe thatthe movement of the cortical surface is negligible inour experiment. Thus, the exact in vivo time proﬁle of206

Yamaguchi K, et al: Oxidative stress in cerebral ischemia and anoxia

O2  production in the feline cortex during anoxiareoxygenation and ischemia-reperfusion was successfully assessed.
Since gender difference in cerebral blood ﬂow has
been reported,35–38 we used only male animals.
SOD superfusion reduced the corrected MCLA CL
to half of the basal level, indicating that superoxideis spontaneously produced in the steady state before
the insult of anoxia/ischemia. Furthermore, because themembrane permeability of SOD is low, some part of the
MCLA CL that was not suppressed by the SOD superfusion may arise from the intracellular space. A smallpart of basal MCLA CL may also arise from autooxidation due to oxygen itself.
The MCLA CL increased within 20 minutes ofreoxygenation and reperfusion. On the other hand, theincrease in MCLA CL after reoxygenation was notdemonstrated when SOD was superfused throughout
the protocol. The SOD-induced suppression of theenhancement in CL during reoxygenation indicatesthat enhanced O2  production was the main cause of
the increase in CL. Considering the low membrane
permeability of SOD, this result also indicates thatthe enhancement in O2  production is likely to occur
mainly in the extracellular space. Some investigators
have studied the site of O2  production during reperfusion in a transient cerebral ischemia model using
electron microscopic histochemistry and Karnovsky’s
Mn 2þ /diaminobenzidine (DAB) technique.39,40 Kontos
et al. reported that O2  reaction products were produced during reperfusion primarily in the extracellularspace around the blood vessels and that the products
were eliminated by pretreatment with SOD superfusion.39 Mori et al. reported that O2  reaction products were detected within the arteries, capillaries, andvenular lumens during reperfusion.40 Furthermore, theextracellular space around blood vessels has beenreported to be a possible site of ROS production invarious organs. Suematsu et al. studied the site of ROS
production in vivo; they found that ROS production
occurred in rat mesenteric microvascular beds throughan endothelium-granulocyte interaction using the CLtechnique41 and in hypoperfused rat liver utilizing using
ﬂuorescence technique.42 These ﬁndings are compatible with our results obtained using the CL technique.
We found that O2  production decreased duringanoxia and severe forebrain ischemia. The supply of O2
to the brain tissue is reduced during anoxia or severe
forebrain ischemia, which may limit O2  production.
Furthermore, Marklund et al. reported that the half-lifeof O2  becomes shorter as the pH level decreases.43
Brain pH has also been reported to decrease during
severe ischemia44–46 and cardiac arrest,47 which may
cause the reduction in O2  concentration during anoxia
or severe forebrain ischemia.

In conclusion, we have established a new CL technique for the in vivo assessment of O2  generation in
the feline cortex. O2  generation decreased during a
90-second period of anoxia and severe transient forebrain ischemia and increased within 20 minutes ofreoxygenation and reperfusion. The increase in O2 
production during reoxygenation or reperfusion may
occur mainly in the extracellular space.
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