Brain Research 864 (2000) 6068

www.elsevier.com / locate / bres

Research report

Quantitative evaluation of extracellular glutamate concentration in

postischemic glutamate re-uptake, dependent on brain temperature, in
the rat following severe global brain ischemia
Satoshi Asai a , *, Heng Zhao a , Tadashi Kohno b , Yasuo Takahashi a , Toshihito Nagata a ,
Koichi Ishikawa a
b

a
Department of Pharmacology, Nihon University School of Medicine, Oyaguchi-Kami Machi, Itabashi-ku, Tokyo 173, Japan
Biomedical Research Team, Frontier Technology Research Institute, Tokyo Gas Co., Ltd., Suehoro-cho, Tsurumi, Yokohama 230, Japan

Accepted 7 February 2000

Abstract
Changes in brain temperature are known to modulate the marked neuronal damage caused by an approximately 10-min intra-ischemic
period. Numerous studies have suggested that the extracellular glutamate concentration ([Glu] e ) in the intra-ischemic period and the initial
postischemia period is strongly implicated in such damage. In this study, the effects of intra-ischemic brain temperature (32, 37, 398C) on
[Glu] e were investigated utilizing a dialysis electrode combined with ferrocene bovine serum albumin (BSA), which allows oxygenindependent real-time measurement of [Glu] e . This system allowed separate quantitative evaluation of intra-ischemic biphasic glutamate
release from the neurotransmitter and metabolic pools, and of postischemic glutamate re-uptake in ischemiareperfusion models. The
biphasic [Glu] e elevation in the intra-ischemic period did not differ markedly among intra-ischemic brain temperatures ranging from 32 to
398C. Intra-ischemic normothermia (378C) and mild hyperthermia (398C) markedly inhibited [Glu] e re-uptake during the postischemic
period, although the intra-ischemic [Glu] e elevation did not differ from that during intra-ischemic hypothermia (328C). It was assumed
that normothermia or mild hyperthermia in the intra-ischemic period influences intracellular functional abnormalities other than the
intra-ischemic [Glu] e elevation, thereby inhibiting glutamate re-uptake after reperfusion rather than directly modulating intra-ischemic
[Glu] e dynamics. 2000 Elsevier Science B.V. All rights reserved.
Themes: Disorders of the nervous system
Topics: Ischemia
Keywords: Dialysis electrode; Real-time monitoring; Ferrocene; Glutamate release; Glutamate re-uptake; Reversed uptake; Ischemia; Normothermia;
Hypothermia; In vivo

1. Introduction
The importance of mild to moderate variations in brain
temperature in the pathological and functional outcome has
been demonstrated in various models of brain injury and
ischemia [5,6,10,15,26]. While a 238C decrease in intraischemic brain temperature can be neuroprotective, mild
brain hyperthermia significantly worsens outcome [11],
when the intra-ischemic period is brief, e.g., approximately
10 min [27]. Although no such sensitive temperature*Corresponding author. Tel.: 181-3-3972-8111, ext. 2246; fax: 181-35995-5914.
E-mail address: satoshi@med.nihon-u.ac.jp (S. Asai)

dependence was observed in the severity of energy metabolism disturbance [27] or the extent of damage to membrane lipids [7], in vivo microdialysis studies of extracellular glutamate dynamics have indicated that hypothermic
protection attenuates the ischemia-induced increase in
extracellular level of striatal glutamate in global brain
ischemia [4,28,38]. Therefore, it is assumed that reduced
glutamate release into the extracellular space is an important mechanism underlying the cytoprotective effect of
hypothermia [14].
The glutamate concentration in the extracellular space
([Glu] e ) appears to be largely dependent not only on the
glutamate release system [2,19,31,40] but also on its reuptake system [2,18,39]. Thus, [Glu] e during ischemia is

0006-8993 / 00 / $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02151-X

S. Asai et al. / Brain Research 864 (2000) 60 68

measured as the balance between glutamate release from

neurons and glia and its re-uptake. It would thus be very
interesting to determine whether the [Glu] e reduction in
response to hypothermic protection depends mainly on
[Glu] e output from the intracellular space or inhibition of
re-uptake into the intracellular space. However, the relationship between [Glu] e dynamics and the neuroprotective
effect derived from lowering the intra-ischemic or postischemic brain temperature is as yet poorly understood,
because of sampling problems diminishing the time resolution of the microdialysis method.
Recently, we reported the application of an oxygenindependent real-time technique for monitoring glutamate
levels in the extracellular space during in vivo ischemia
and hypoxia, using a dialysis electrode [2224,30]. This
dialysis electrode technique allows detailed analysis of the
in vivo dynamics of not only severe acute ischemia
produced by transecting the bilateral carotid arteries [2],
but also those of an ischemiareperfusion model [1,38,39].
In particular, during the ischemiareperfusion period, the
mechanisms of glutamate release are assumed to involve
three sequential processes [29]: (1) neurotransmitter, i.e.,
glutamate, release from synaptic vesicles (first phase of
[Glu] e ); (2) reversed uptake of glutamate from the metabolic pool in neuronal cells (second phase of release); and
(3) re-uptake into the intracellular space by normalization
of the glutamate uptake carrier system during the postischemic period (re-uptake phase of [Glu] e ). Therefore, we
deemed it worthwhile to examine the effects of temperature on glutamate release by analyzing the dynamics of
[Glu] e elevation via these three postulated mechanisms.
The aim of the present study was to investigate these three
mechanisms, focusing on the relationship between temperature and [Glu] e quantitative elevation, in the Smith
ischemiareperfusion model. This study was designed to
analyze the in vivo dynamics of biphasic [Glu] e output
from the intracellular space, as well as reduced re-uptake
of [Glu] e into the intracellular space following 10 min of
ischemia in terms of the three above-mentioned [Glu] e
compartments. Furthermore, the relationship between
changes in brain temperature and focal cerebral blood flow
in the Smith ischemiareperfusion model was studied, to
provide supporting data.

61

sulfonic acid (MES) from Dojindo Laboratories (Kumamoto, Japan); and Dulbeccos phosphate-buffered saline
(PBS(1)) (product No. 21300) from Gibco-BRL (Grand
Island, NY, USA). All other chemicals used were of
analytical grade.

2.2. Preparation of dialysis electrode for in vivo

experiments

2. Materials and methods

Glutamate oxidase was dissolved in PBS(2) and

dialyzed against PBS(2) three times on ice. The solution
of glutamate oxidase was stored at 2808C until use. The
dialysis electrodes (Microdialysis Biosenser: type general,
10-2-2) employed in this study were purchased from
Sycopel International (Boldon, Tyne and Wear, UK). The
principle of the dialysis electrode used for the measurement of glutamate was as previously described [22,23].
Briefly, the dialysis electrode was filled with Dulbeccos
phosphate-buffered saline (PBS(2) supplemented with 1.3
mM CaCl 2 (PBS(1)). The perfusate entered the fluid inlet
tube at a rate of 0.5 ml / min via an EP70 perfusion pump
(Eicom, Kyoto, Japan). The electrode was prepared by
filling it with PBS(1) and immersing the membrane in a
beaker of 5 mM O-phenylenediamine in PBS(1) bubbled
with 100% N 2 for 15 min while constantly stirring the
PBS(1). The dialysis electrode was connected to an EPS800 potentiostat (Eicom), and a voltage clamp was
switched on at 1650 mV for 15 min to carry out
electropolymerization with continuous bubbling and stirring. Upon completion of electropolymerization, the potentiostat was switched off. The dialysis electrode membrane
was removed from the O-phenylenediamine solution, and
the membrane portion was stored in PBS(1). After 20
min, the solution in the dialysis electrode was replaced
with fresh PBS(1) by the perfusion pump, and the current,
set at 1650 mV, was allowed to stabilize. Finally, the
mixed solution of ferrocene-conjugated BSA and glutamate oxidase (1:1) was introduced into the dialysis electrode and perfused at a constant flow rate (0.5 ml / min)
with a syringe pump. Ascorbate calibration was then
carried out while the bulk solution was being stirred, and a
small amount of concentrated ascorbate solution (up to 300
mM) was added. This procedure tested the efficacy of
O-phenylenediamine coverage in preventing such compounds from being oxidized electrochemically by the
working electrode.

2.1. Reagents

2.3. Implantation in rat striatum

Glutamate oxidase (EC 1.4.3.11) was purchased from

Yamasa Corporation (Chiba, Japan). Ferrocene carboxylic
acid was obtained from Tokyo Kasei Kogyo, Tokyo,
Japan; BSA (RIA grade) and O-phenylenediamine from
Sigma (St. Louis, MO, USA); l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)
from Pierce (Rockford, IL, USA); 2-(N-morpholino)ethane

Adult male WistarKyoto rats (Shizuoka Laboratory

Animal Center, Hamamatsu, Japan), weighing between 270
and 325 g, which had been allowed food and water ad
libitum, were used in all experiments. The rats were
anesthetized with an intraperitoneal injection of urethane
(1.25 g / kg). After tracheotomy, the rats were orotracheally
intubated and artificially ventilated employing a small-

S. Asai et al. / Brain Research 864 (2000) 60 68

62

animal ventilator (Rodent Ventilator, Ugo Basile, Italy)

and an appropriate stroke volume to maintain normocapnia. Arterial pCO 2 was maintained between 30 and 40
mmHg, and was monitored with an i-STAT 200A (i-STAT
Corporation, NJ, USA). After the induction of ischemia,
the arterial pCO 2 was continuously monitored at 15 min in
the postischemic period. The head was fixed in a stereotactic frame, and rectal temperature was maintained at 36.5
378C by means of a heating pad. The skull was exposed,
2-mm bilateral holes were drilled through the skull, and the
dura was carefully pierced. An electroencephalogram
(EEG) electrode attachment was placed on the brain
surface. The dialysis electrode was carefully implanted
stereotactically, in the striatum. The laser Doppler blood
flow monitor FLO-1 attachment (Omega Wave, Tokyo,
Japan) includes a heliumneon laser, an electronic processor and a contact-type fiber-optic probe (0.5 mm od).
The tip of the temperature sensor probe (Physitemp
Instruments, Clifton, NJ, USA) (0.5 mm O.D.) was in
contact with the outer tip of the laser probe, 1 mm above
the lowest portion. The unit with the laser Doppler probe
and the temperature probe were carefully implanted
stereotactically, separately, in the other side of the striatum
to the insertion of the electrode. The exposed brain surface
was covered with aluminum foil to prevent a Doppler

response from warm light. All signals were monitored

continuously using Mac Lab System (AD Instruments,
Castle Hill, Australia) and analyzed after the experiment.
Brain temperature (BT) was maintained manually throughout the experiment with red light irradiation heating and a
cold blower. Brain temperature was preconditioned for 10
min prior to ischemic insult to maintain the target temperature (32 and 398C), to avoid brain temperature variation in
the intra-ischemic period. Both common carotid arteries
were isolated. Induction of acute brain ischemia, known as
the Smith model [33], for investigating brain ischemia
(328C (n510), 378C (n510) or 398C (n510)) and reperfusion (328C (n510), 378C (n510) or 398C (n510)),
consisted of a combination of bilateral carotid artery
clamping and rapidly reducing mean arterial blood pressure (BP) to approximately 30 mmHg by blood withdrawal
through a jugular vein. At the end of the 10-min occlusion
period, the occlusion was released to allow reperfusion via
a combination of resumption of blood flow and reinfusion
of the withdrawn blood, which had been maintained at
378C in a water bath. After each in vivo monitoring
procedure, the dialysis electrode was removed from the
brain and immediately recalibrated in vitro to restore the in
vivo glutamate concentration. For the Smith ischemia
reperfusion model (Table 1), glutamate calibration for

Table 1
Changes in [Glu] e as demonstrated by real-time monitoring with a dialysis electrode, with BT maintained near 32, 37 or 398C, during ischemia (n510) and
reperfusion (n510)a

Temperature
(8C)

32
37
39
a

Time (s)
after inducing ischemia

D[Glu] e (mM)
after inducing ischemia

D[Glu] e (mM)
after start of reperfusion

To

Tp

D[Glu] p

D[Glu] 5

D[Glu] 10

D[Glu] 5 onset

RD[Glu] 5

RD[Glu] 15

RD[Glu] 30

134625**
71613
4868*

240619**
164642
136620

272661
217643
201619

173615
177616

187628
214614
209635

167626
179611
182617

Baseline**
183621
192613

Baseline**
60625
154632**

Baseline
Baseline
90645**

Average plots of the [Glu] e time course at 5-min intervals in an ischemiareperfusion model are shown. Average plots of the [Glu] e time course were
evaluated for 40 min, at 5-min intervals, after inducing ischemia (D[Glu] 5 , D[Glu] 10 ), and from the onset of the [Glu] e increase in the first phase (D[Glu] 5
min.onset, D[Glu] 10 min.onset). D[Glu] p is the peak value in the first phase. T o is the time from ischemia induction until the onset of [Glu] e elevation. T p is
the time from the induction of ischemia until the [Glu] e peak. The [Glu] e dynamics after reperfusion were represented by RD[Glu] 5 , RD[Glu] 15 and
RD[Glu] 30 . Glutamate calibration for the ischemic and reperfusion phases in [Glu] e dynamics was carried out in different groups. Data represent
mean6S.D. of 10 samples.
**P,0.01; *P,0.05 (in comparison with normothermic intra-ischemia maintained at 378C; ANOVA with Fisher LSD).

S. Asai et al. / Brain Research 864 (2000) 60 68

monitoring of intra-ischemic [Glu] e dynamics was carried

out immediately after the end of reperfusion at 32, 37 or
398C, which were the same temperatures as those in the
intra-ischemic period. Calibration for the reperfusion phase
was carried out approximately 30 min after reperfusion at
378C. The value of 100% CBF was calculated by subtracting the value in the postmortem condition after completion
of the experiment from baseline data under normal conditions.

2.4. Statistical analysis

Data are presented as mean6S.D. Statistical comparisons between groups of samples were made by analysis of
variance (ANOVA) with the Fisher least-significant difference test.

3. Results

3.1. Ischemiareperfusion in the Smith model

Maintaining the mean BP under 30 mmHg creates a
reproducible, very low CBF condition, only slightly above
the minimum level during the intra-ischemic period. After
the induction of severe ischemia, a highly reproducible
biphasic pattern of [Glu] e was observed in nearly all
animals, and the intra-ischemic [Glu] e change in the
reperfusion model approximated that in the carotid artery
transection model. As shown in Fig. 1A, real-time [Glu] e
monitoring revealed the events occurring when brain
temperature was kept constant at 328C during the ischemic
period while body temperature was kept nearly constant at
around 378C, and the events occurring when bilateral
carotid artery clamping and rapid reduction of mean
arterial BP were used in combination under the latter
temperature conditions, i.e., a rapid decrease in brain
temperature with immediate flattening of the EEG pattern.
In our data, most animals with a decrease in BP to
approximately 30 mmHg in the intra-ischemic period
demonstrated very low CBF, i.e., less than 5% of the
baseline value, which showed no relation to intra-ischemic
brain temperature (data not shown). A sharp and rapid
elevation of glutamate release took place (first phase), and
this elevation shifted, continuing to rise throughout the
10-min ischemic period (second phase). Following reperfusion, the brain temperature recovered to 378C and 90% of
the [Glu] e increase produced by the ischemic insult was
cleared within 5 min despite poor EEG recovery over 20
min at 378C following reperfusion. As shown in Fig. 1A,
CBF under the above three conditions decreased to approximately 5% of the baseline level when ischemia was
induced. Following reperfusion, CBF immediately recovered to the baseline value, followed by compensatory
upregulation above the baseline. Fig. 1B shows the data
obtained from real-time monitoring of glutamate with the

63

dialysis electrode, with the brain temperature kept constant

at 378C throughout the experimental period. Although the
first phase of [Glu] e elevation began approximately a
minute earlier than the change in temperature, which was
328C during the intra-ischemic period (Fig. 1A), the
biphasic [Glu] e elevation curve was similar. As shown in
Fig. 1C, real-time [Glu] e monitoring revealed the events
occurring when brain temperature was maintained at a
constant 398C. The biphasic [Glu] e elevation curve was
similar to those shown in Fig. 1A,B, although the first
phase of [Glu] e elevation began approximately 30 s later
than the change in brain temperature when brain temperature was raised to and then maintained at 378C for the
remainder of the experimental period. In the postischemic
period, the [Glu] e re-uptake phase was markedly inhibited
for more than 30 min. Table 1 presents detailed [Glu] e
dynamics data with statistical comparison of the average
plots of the [Glu] e time course at 5-min intervals in the
Smith ischemiareperfusion model at different brain temperatures.
Fig. 2 shows the CBF change in the postischemic period
with BT maintained at approximately 328C (n510), 378C
(n510) or 398C (n510) during ischemia. CBF decreased
to less than 5% of the baseline level in the intra-ischemic
period. When brain temperature was kept constant at 32 or
378C during the intra-ischemic period, CBF was upregulated after starting reperfusion and eventually reached the
baseline level. When brain temperature was kept constant
at 398C during the intra-ischemic period, following reperfusion, CBF did not completely recover to the baseline
and there was no compensatory upregulation as seen at
other brain temperatures, but there was recovery to more
than 80% of baseline CBF in most animals during the
postischemic period. When arterial pCO 2 was monitored
for 15 min in the postischemic period, changes in pCO 2
were within the normal range (3040 mmHg) in the three
groups (data not shown).
Table 1 shows the changes in [Glu] e with real-time
monitoring using the dialysis electrode, when BT was
maintained near 32, 37 or 398C, during ischemia and
reperfusion. The average [Glu] e of 10 rats was plotted at
5-min intervals in this ischemiareperfusion model produced by the Smith method. BT was maintained near 32,
37 or 398C during the intra-ischemic period. When BT was
changed in this model, the onset of glutamate release was
delayed in a temperature-dependent manner. The amount
of glutamate released in the first phase decreased as BT
rose. When BT was maintained near 328C in the intraischemic period, we could not plot [Glu] e at 5 min,
because the onset of the [Glu] e elevation in the first phase
was delayed at this BT and [Glu] e did not consistently
reach the initial point of the second phase by 5 min. We
could not divide [Glu] e elevation into first and second
phases, which are thought to represent different mechanisms of glutamate release. Thus, it was not possible to
plot overrange [Glu] e values when BT was kept constant at

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S. Asai et al. / Brain Research 864 (2000) 60 68

Fig. 1. Representative [Glu] e response, EEG, BP and CBF, with BT maintained near 328C (A), 378C (B), or 398C (C), during severe global
ischemiareperfusion. Simultaneous recordings of [Glu] e , EEG, BT, BP and CBF were obtained under all ischemiatemperature conditions. Real-time
monitoring of glutamate with the dialysis electrode was started at 10 min after induction of acute severe ischemia, with mean BP maintained under 30
mmHg, followed by reperfusion. The dialysis electrode and laser Doppler probe with temperature sensor probe were carefully implanted into the brain
stereotactically, on each side of the striatum. An EEG electrode attachment was placed on the brain surface. The brain temperature was maintained with red
light irradiation under the prescribed conditions and rectal temperature was maintained at approximately 378C by means of a heating pad. BP was
monitored via a catheter in the femoral artery.

328C, hence, the absence of these data from Table 1. The

[Glu] e values at 10 min did not differ in the 32398C
temperature range in this complete ischemia model, produced by transection of both common carotid arteries with
scissors, as previously reported [2]. The [Glu] e values 5,
15 and 30 min after start of reperfusion after the ischemia
at 378C (n510) or 398C (n510) did not decrease, although
the [Glu] e 5 value at 328C (n510) recovered to the
baseline level. The [Glu] e 30 value decreased to the

baseline level at 378C, but did not decrease to the baseline

at 398C.

4. Discussion
In the present study, detailed analysis of [Glu] e dynamics, as reflected by intra-ischemic changes in BT, was
performed in relation to increased release and inhibited

S. Asai et al. / Brain Research 864 (2000) 60 68

65

Fig. 2. CBF changes in the postischemic period with BT maintained at approximately 328C (n510), 378C (n510) or 398C (n510) during ischemia. CBF
decreased to less than 5% of the baseline level in the ischemic period. The value of 100% CBF was calculated by subtracting the value in the postmortem
condition after completion of the experiment from baseline data under normal conditions. Values are mean6S.D.

re-uptake of glutamate. It is well known that [Glu] e and

brain damage during ischemia are critically dependent on a
threshold decrease in CBF [32,34]. Furthermore, our
previous study showed CBF threshold for [Glu] e elevation
during ischemia was changed by brain temperature [39]. In
order to equalize the conditions of acute ischemic induction in the ischemiareperfusion model as much as possible, we used a model of severe global ischemia, the
Smith ischemiareperfusion model, in which BP decreases
by 30 mmHg in the intra-ischemic period, for evaluating
glutamate re-uptake. During the ischemiareperfusion
period, the mechanisms of glutamate release are assumed
to involve three sequential processes; glutamate release
during the intra-ischemic period (first phase), reversed
uptake of glutamate from the metabolic pool in the intraischemic period (second phase), and glutamate re-uptake
by normalization of the glutamate uptake carrier system in
the postischemic period (re-uptake phase) [1,2,38]. We
studied the effects of BT during these three phases in two
severe acute ischemia models, in detail.
Glutamate from the massive release in the first phase
binds to postsynaptic glutamate receptors and excites cells.
It also binds to receptors and induces nerve excitation. The
resultant rapid increase in energy consumption leads to

energy depletion and disruption of the ionic gradients

responsible for reversal of glutamate transport (reversed
uptake of glutamate) [3]. We found that [Glu] e 5 and 10
min after the start of ischemia showed no marked differences in response to BT changes within the 32398C range
(Table 1). Taken together with the previous observations
of the second phase of [Glu] e dynamics [2], different BT
does not appear to affect [Glu] e in the plateau phase in the
setting of severe acute ischemia. It is reasonable to
speculate that the brain is under anaerobic conditions for
several minutes due to diminished ATP production and
supply to the tissue [20]. Therefore, this second phase of
glutamate release, which is considered to be from the
metabolic pool, could be ATP-independent. The reversed
uptake mechanism operating in the second phase of [Glu] e
elevation is reportedly based on electrogenic stoichiometry. According to a numerical formula for glutamate
electrogenic stoichiometry, BT had little effect on [Glu] e
changes in the second phase of ischemia [3]. Our results,
together with our previous study [2], suggest that changes
in intra-ischemic brain temperature have little effect on the
second phase of [Glu] e elevation in severe acute ischemia.
We evaluated the dynamics of [Glu] e re-uptake in an
ischemiareperfusion model produced by the Smith meth-

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S. Asai et al. / Brain Research 864 (2000) 60 68

od. It is well known that subtle differences in experimental

conditions during the ischemic period, i.e., changes in
mean BP, BT and occlusion time, are important determinants of [Glu] e dynamics. In the Smith model, [Glu] e
dynamics also changed markedly depending on the maintenance of mean BP during the ischemic period (data not
shown) [16,32]. In our previous study, when ischemia was
efficiently severe at CBF below 15% of the baseline level,
the [Glu] e elevation was not obviously influenced by brain
temperature (Table 1) [2], and CBF threshold for [Glu] e
elevation decreased as brain temperature was reduced [39].
Therefore, we produced severe ischemia in this reperfusion
model by reducing the mean BP to 30 mmHg during the
ischemic period. As shown in Table 1, in this severe
ischemiareperfusion model, glutamate release was the
same regardless of BT, i.e., the results are similar to those
of irreversible ischemia produced by transecting the bilateral carotid arteries, as previously reported [2]. As reuptake may be affected by glutamate release, we compared
only the uptake phase in the ischemic period under
different BT conditions. Although the effects on [Glu] e of
altering BT at the start of reperfusion have little relation to
the intra-ischemic BT, intra-ischemic normothermia and
mild hyperthermia markedly inhibited [Glu] e re-uptake
during the postischemic period. Our results suggest that
intra-ischemic normothermia and mild hyperthermia markedly inhibit [Glu] e re-uptake during the postischemic
period.
The following summarizes the effects of intra-ischemic
BT and the mechanisms of glutamate release by the three
sequential processes. Mild cerebral hypothermia during the
ischemic period did not inhibit [Glu] e elevation in the first
and second phases, which are due to massive glutamate
release. Nonetheless, postischemic glutamate re-uptake
was markedly inhibited in a temperature-dependent manner. The disruption of the re-uptake system after reperfusion when the temperature is maintained above 378C and
the inability of hypothermia during the postischemic
period, in contrast to the intra-ischemic period, to prevent
this disruption [9,13,37] suggest that raising BT in the
ischemic period may lead to intracellular functional abnormalities other than intra-ischemic [Glu] e elevation, thereby
inhibiting glutamate re-uptake after reperfusion rather than
directly modulating intra-ischemic [Glu] e dynamics. Although the mechanism underlying disruption of the glutamate re-uptake system during the ischemic period is
obscure, persistence of this high [Glu] e in the postischemic
period probably promotes further cell injury and initiates a
vicious cycle following reperfusion.
It is not surprising that CBF recovery in the postischemic period is very important for ATP production and
for minimizing the extent of damage in the postischemic
brain (Figs. 1 and 2). We monitored CBF for the purpose
of analyzing this parameter in detail during the time course
of [Glu] e re-uptake. As we previously reported [38],
prolonged arterial hypotension did not contribute to the

prolonged period of glutamate re-uptake seen during

normothermia. From these observations, intra-ischemic
normothermia can be considered to inhibit glutamate
uptake by a mechanism that has little effect on CBF
recovery. On the other hand, when the brain temperature
was maintained at a mildly hyperthermic 398C during
ischemia, re-uptake of [Glu] e in the postischemic period
deteriorated markedly, requiring more than 30 min for
[Glu] e to reach the baseline. Although approximately 80%
CBF recovery was seen at the initiation of reperfusion,
there were no compensatory increases in CBF and BP
comparable to those associated with normothermia and
mild hypothermia (Figs. 1 and 2). The mechanism of
suppression of CBF recovery in the ischemic period, at a
constant 398C, is not clear. This poor recovery of both BP
and CBF may contribute to delayed tissue recovery and
energy supply in the postischemic period. Another possibility is suggested by our previous study [39], which
showed that the minimum CBF threshold for [Glu] e
elevation was approximately 80% of normal CBF value
and the [Glu] e uptake mechanism was preserved at more
than 30% of normal CBF value in the intra-ischemic
period at 398C. Thus, inhibition of the glutamate uptake
mechanism by mild hyperthermia possibly plays a critical
role in intracellular regulation of [Glu] e re-uptake by
glutamate transporters [35], such as arachidonic acid [36],
and in calcium overload [25]. Furthermore, mild variations
in temperature also reportedly produce alterations of the
bloodbrain barrier [12]. Altered bloodbrain barrier
function could have a significant effect on [Glu] e dynamics
and postischemic reperfusion. Thus, under normothermic
and mild hyperthermic conditions, as opposed to mild
hypothermic conditions, a far longer exposure is needed to
produce neurotoxic effects [17]. This prolonged exposure
to high [Glu] e presumably exacerbates cell damage.
It is established that [Glu] e plays a critical role in
neuronal cell death, whether in association with acute,
subacute or chronic brain damage [8,21]. Our findings
support the concept that variations in brain temperature are
not important in the initial biphasic [Glu] e elevation, while
being crucial to glutamate re-uptake during the reperfusion
phase after an ischemic insult. The mechanism of injury,
which is sensitive to temperature, may not actually lead to
massive release of glutamate in the intra-ischemic period,
but rather appears to impair the [Glu] e re-uptake system,
such that recovery of the glutamate transporter system and
restoration of depleted ATP stores cannot occur during the
ischemic and postischemic periods. It is well known that
cooling during ischemia provides permanent protection,
while cooling just after ischemia is less protective
[9,13,37]. Investigating the process by which the glutamate
re-uptake system operates may reveal key steps in the
process of cell damage [30]. Taken together with the
results of our previous study [2], these observations
suggest that intra-ischemic mild hypothermia might have
relatively little influence on the initial glutamate release

S. Asai et al. / Brain Research 864 (2000) 60 68

during the ischemic period, while playing a major role in

protecting the glutamate re-uptake system. Our data suggest that enhancing re-uptake, by activation of glutamate
transporters pharmacologically or hypothermically, may
shorten exposure to high [Glu] e in the postischemic period
and thereby decrease its deleterious excitotoxic effect on
neuronal cells. Thus, our results provide insights into
reducing the neuronal damage associated with brain ischemia.

Acknowledgements
We would like to thank Dr Bierta Barfod and Dr Wendy
Gray for assistance in preparation of the manuscript and
Mr Yuichi Matsumoto for his excellent technical advice
regarding the dialysis electrode. This work was supported
in part by Grants-in-Aid from the Ministry of Education,
Science, Sports and Culture of Japan for the High-Tech
Research Center (Nihon University) and for Science
Research (C) No. 11680766 and a Special Research in
Health Science Research Grants from The Ministry of
Health and Welfare.

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