Beruflich Dokumente
Kultur Dokumente
Research report
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
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
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.
2.1. Reagents
62
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).
3. Results
63
64
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.
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
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.
66
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.
References
[1] S. Asai, Y. Iribe, T. Kohno, K. Ishikawa, Real time monitoring of
biphasic glutamate release using dialysis electrode in rat acute brain
ischemia, NeuroReport 7 (1996) 10921096.
[2] S. Asai, H. Zhao, Y. Takahashi, T. Nagata, T. Kohno, K. Ishikawa,
Minimal effect of brain temperature changes on glutamate release in
rat following severe global brain ischemia: a dialysis electrode
study, NeuroReport 9 (1998) 38633868.
[3] D. Attwell, B. Barbour, M. Szatkowski, Nonvesicular release of
neurotransmitter, Neuron 11 (1993) 401407.
[4] A.J. Baker, M.H. Zornow, M.R. Grafe, M.S. Scheller, S.R. Skilling,
D.H. Smullin, A.A. Larson, Hypothermia prevents ischemia-induced
increases in hippocampal glycine concentrations in rabbits, Stroke
22 (1991) 666673.
[5] R. Bullock, A. Zauner, J. Woodward, H.F. Young, Massive persistent
release of excitatory amino acids following human occlusive stroke,
Stroke 26 (1995) 21872189.
[6] R. Busto, W.D. Dietrich, M.Y. Globus, I. Valdes, P. Scheinberg, M.D.
Ginsberg, Small differences in intraischemic brain temperature
critically determine the extent of ischemic neuronal injury, J. Cereb.
Blood Flow Metab. 7 (1987) 729738.
[7] R. Busto, M.Y. Globus, W.D. Dietrich, E. Martinez, I. Valdes, M.D.
Ginsberg, Effect of mild hypothermia on ischemia-induced release
of neurotransmitters and free fatty acids in rat brain, Stroke 20
(1989) 904910.
[8] D.W. Choi, S.M. Rothman, The role of glutamate neurotoxicity in
hypoxic-ischemic neuronal death, Annu. Rev. Neurosci. 13 (1990)
171182.
[9] H. Chen, M. Chopp, L.A. Vande, M.O. Dereski, J.H. Garcia, K.M.
Welch, The effects of postischemic hypothermia on the neuronal
injury and brain metabolism after forebrain ischemia in the rat, J.
Neurol. Sci. 107 (1992) 191198.
[10] M. Chopp, K.M. Welch, C.D. Tidwell, R. Knight, J.A. Helpern,
Effect of mild hyperthermia on recovery of metabolic function after
global cerebral ischemia in cats, Stroke 19 (1988) 15211525.
67
68
[32]
[33]
[34]
[35]