24 H
1 2
acids [30]. Therefore, the second goal in this study was to K , 155.5 mM Cl , pH 7.4 was infused in the hippocam-
determine effects of hypothermia on the effluxes of pus through the dialysis probe at a rate of 4.0 ml min with
excitatory and inhibitory amino acids in the aged rats. We a syringe pump Eicom Co., Kyoto, Japan. The perfusate
monitored the level of extracellular amino acids using a was collected every 10 min into a plastic tube and stored at
microdialysis method, and compared with the severity of 2808C for the later measurement of amino acids.
histopathological damages. Regional brain temperature in the hippocampus was satisfactorily controlled by the
2.4. Brain hypothermia specific thermoregulator [19].
Brain temperature was modulated by a selective brain thermoregulator metallic plate brain cooling device which
2. Materials and methods
we developed model BTC-100, Unique Medical Co., Tokyo, Japan. Briefly, an aluminum metallic plate 22
2.1. Animals mm316 mm31 mm consisted of two thermomodules
one for heating and the other for cooling using a water- Nineteen aged female SHR, 19–23 months old and
circulating system was placed on the surface of rat’s weighed 195–215 g, were used in this study. Rats were
scalp, and the thermocouple was inserted through an maintained in Animal Center, Kyushu University under
elliptocal center hole 8 mm36 mm in the plate. The specific pathogen-free conditions, and fed stock chow diet
device with a continuous-monitoring system can quickly and tap water ad libitum. All experimental procedures were
and precisely adjust the cerebral cortex at desired brain performed in accordance with the Physiological Society of
temperature [19]. Following a resting period of 90 min, the Japan Guiding Principles for the Care and Use of Animals
baseline CBF and arterial blood pressure were determined in the Field of Physiological Sciences. Female SHR were
with hippocampal temperature of 368C using the ther- chosen because of their low mortality rate under 30 at
moregulator. Then, the temperature of the hippocampus 20 months, while a half of male SHR expired between 15
was adjusted to 308C moderate hypothermia, n56 or and 20 months.
338C mild hypothermia, n56 or remained 368C nor- mothermia, n57 until 80 min after brain ischemia. Rectal
2.2. Surgery temperature was maintained at 378C using a heat pad
throughout the experiment. Both carotid arteries were Cerebral ischemia was produced by bilateral carotid
ligated for 20 min, followed by 80 min of recirculation. artery occlusion [13]. Briefly, each rat was anesthetized
CBF was determined at every 10 min of ischemia and with amobarbital 100 mg kg i.p. and breathed room air
recirculation. Arterial blood gases, pH and hematocrit were spontaneously. Both femoral arteries were cannulated, one
measured at the resting period, 20 min of ischemia and 80 for sampling blood to measure arterial blood gases and pH
min of recirculation. with an IL meter Model 1304 Instrumentation Laboratory
Inc., Lexington, MA, USA and the other for continuous 2.5. HPLC analysis
recording of blood pressure. Bilateral common carotid artery was exposed through a ventral incision in the neck,
Concentrations of amino acids were measured using carefully separated from the vagosympathetic trunks, and
HPLC combined with fluorescent detection after pre- loosely encircled with sutures for later retraction.
column derivatization. Each sample was automatically mixed with o-phthalaldehyde and 2-mercaptoethanol for 2
2.3. Microdialysis min, and then injected into the HPLC system, which
consisted of an Eicom pump Eicom Co., Kyoto, Japan at Concentrations of extracellular amino acids and cerebral
a flow rate of 1.0 ml min, a reverse phase column blood flow CBF in the hippocampal CA1 subfield were
Eicompac MA-5 ODS, 4.63250 mm, Eicom Co., Kyoto, simultaneously determined using a microdialysis technique
Japan and a fluorescent detector Shimadzu Co., Tokyo, and a hydrogen clearance method, respectively [29]. Each
Japan. The mobile phase was 0.1 M sodium phosphate rat was fixed in a head holder, and a small burr hole was
pH 6.0 containing 30 vol vol methanol. Concen- made in the parietal region. A dialysis probe with 1-mm
trations of glutamate, aspartate, glycine, taurine, GABA membrane CMA-10: Carnegie Medicine, Stockholm,
and alanine were determined by comparison of standard Sweden and a Teflon-coated electrode with micro-ther-
solution. mocouple 300 mm in diameter, 1-mm portion at its tip
uncoated for measurement of both CBF and brain tem- perature were placed stereotaxically in the right hippocam-
2.6. Histological examination pal CA1; 4.3 mm posterior and 1.5 mm lateral to the
bregma and 3.0 mm in depth from the surface of the brain. After 80 min of recirculation, the dialysis probe and
1 21
Ringer’s solution 147 mM Na , 2.3 mM Ca , 4 mM
electrodes were withdrawn, and bone wax was pasted on
H . Ooboshi et al. Brain Research 884 2000 23 –30
25 Table 1
the hole in the skull. The femoral arteries were ligated, and
Mean arterial blood pressure MABP, arterial gases, pH and hematocrit
incisions in the head, neck and leg were sutured. Each rat
at resting period, 10 min of ischemia and 80 min of recirculation
was brought back to the cage and freely fed water and
At rest Ischemia
Recirculaton
food. Seven days after forebrain ischemia, five rats in the 368C groups and four rats in each hypothermic group were
MABP mmHg 368C
16567 19764
14866
anesthetized with amobarbital 100 mg kg i.p.. Brains
338C 16568
19866 157611
were transcardially perfused with 4 paraformaldehyde in
308C 16963
19663 15766
1 15 M phosphate buffer pH 7.3 after a brief wash-out
PaCO mmHg
2
period with heparinized saline. Each brain was removed
368C 43.861.9
23.462.0 42.260.8
and fixed in 4 neutral formaldehyde for 7 days. Paraffin
338C 43.561.7
31.162.6 42.862.1
308C 44.461.2
29.363.2 45.362.2
sections were taken at the level of the hippocampus in each
PaO mmHg
2
rat and were stained with hematoxylin and eosin. Ischemic
368C 8262
10563 8161
neuronal damage of the hippocampal CA1 subfield in each
338C 7865
9865 8064
hemisphere was graded from 0 to 3 3, majority of neurons
308C 7963
10264 8563
damaged; 2, many neurons damaged; 1, a few neurons
pH 368C
7.4260.01 7.6160.03
7.4360.01
damaged; or 0, normal by a neuropathologist A.K.
338C 7.4160.01
7.5460.02 7.4260.02
without knowledge of the experimental conditions, and the
308C 7.4360.01
7.5760.03 7.4360.01
summed value of both hemispheres was regarded as
Hematocrit
ischemic score for each animal.
368C 41.561.2
44.162.6 44.460.9
338C 40.261.9
40.062.4 42.762.1
308C 45.161.8
47.461.1 44.262.2
2.7. Statistical analysis
Values are means6S.E.M. n56–7. Paired data were compared with control values by analysis of variance and Dunnett’s t-test P,0.05,
P,0.01. No significant difference was found among the groups.
All values were presented as mean6S.E.M. The statisti- cal differences among and within groups for physiological
parameter, CBF and concentrations of amino acids were 3.2. Basal amino acid level
analyzed by two-way repeated analysis of variance fol- lowed by Dunnett’s t-test. Ischemic scores for neuronal
Concentrations of the last three dialysates at the resting damages were compared among groups by nonparametric
period were averaged and regarded as basal values, which, Kruskal–Wallis’ h-test followed by Dunnett’s t-test.
in the 36, 33, or 308C group, were 5668 nM, 56613 or 4868 for glutamate, 2466 nM, 2464 or 2666 for
aspartate, 307646 nM, 395634 or 304628 for glycine, 243642 nM, 214636 or 219652 for taurine and 234627
3. Results nM, 301634 or 256645 for alanine, respectively. Al-
