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-
3.1. Physiological parameters Table 1 depicts physiological parameters at the resting
period, 10 min of ischemia, and 80 min of recirculation. Mean arterial blood pressure increased by approximately
30 mmHg during carotid artery occlusion, and recovered to the resting values during recirculation. Each rat developed
respiratory alkalosis during ischemia. There was no signifi- cant difference in any physiological parameters among the
groups. Changes in CBF to the hippocampus are present in Fig. 1. CBF before ischemia was 46.362.2 ml 100 g min,
44.465.0 and 47.764.7 in the 36, 33 and 308C group, respectively. Brain hypothermia for 20 min did not alter
the blood flow. Bilateral carotid artery occlusion for 20 min reduced CBF to less than 12 ml 100 g min in all
groups. CBF increased from 85 to 160 of the resting value immediately after recirculation, followed by mild
Fig. 1. Changes in hippocampal blood flow in the 368C n57, 338C
hypoperfusion 60–80 of the resting value in all groups.
n56 and 308C n56 groups. Data are expressed as mean6S.E.M.
There was no significant difference in hippocampal blood
Bilateral carotid artery occlusion reduced the blood flow to less than 12
flow during and after brain ischemia among the groups
ml 100 g min in all groups. b P,0.05, a P,0.01 compared with the
F 2,1650.89.
basal value.
26 H
though concentrations of GABA were not detectable at the 3.4. Inhibitory amino acids
resting periods, the basal values of the other amino acids did not differ among the three groups, and changes in
Twenty min after induction of hypothermia, concen- concentrations of amino acids were shown as percentages
trations of taurine were significantly reduced to 80 of the of basal values.
basal value. In the 368C group, taurine increased to a greater extent 16-fold than glutamate and aspartate
during and soon after cerebral ischemia Fig. 3. On the 3.3. Excitatory amino acids
other hand, changes in taurine levels in the 308C group were only 3-fold and not significant compared with basal
Concentrations of glutamate before ischemia were not levels. The elevation of taurine in the 338C group, how-
altered by brain hypothermia. The extracellular glutamate ever, was still marked 10-fold, P,0.05. Concentrations
significantly increased approximately 6-fold P,0.01 of GABA increased to 171.0632.3 nM during ischemia in
during bilateral carotid artery occlusion in the 368C group five of seven rats in the 368C group. In contrast, only one
Fig. 2. Concentrations of glutamate returned to the basal of six rats in each hypothermic group showed detectable
level after 20 min of recirculation. Ischemia-induced GABA concentrations 338C group, 88.5 nM; 308C, 58.8
elevation of glutamate was markedly attenuated F 2,165 nM.
6.17; P,0.02 to less than 2-fold the basal values in both hypothermia groups. The time course of extracellular
3.5. Other amino acids aspartate was similar to glutamate Fig. 2, showing 5-fold
increases by ischemia in the normothermic group, but no The ischemia-induced increase of glycine was relatively
significant elevation in hypothermic groups. small 2-fold but significant in the normothermic group
Fig. 2. Changes in concentrations of glutamate, aspartate and glycine in the dialysate in the 368C n57, 338C n56 and 308C n56 groups. Data are expressed as mean6S.E.M. of percentages of the resting value. Differences among the groups were significant in the concentrations of glutamate
F 2,1656.17; 0.02, aspartate F 2,1654.55; P,0.05 and glycine F 2,16513.0; P,0.001 by two-way repeated measured analysis of variance. P,0.05,
w w w
P,0.01, difference of 338C from 368C, P,0.05,
P,0.01, difference of 308C from 368C by one-way analysis of variance and Dunnett’s t-test. The ischemia-induced increases in glutamate, aspartate and glycine were significant only in the 368C group. a P,0.01 compared with the basal value by
one-way analysis of variance and Dunnett’s t-test.
H . Ooboshi et al. Brain Research 884 2000 23 –30
27
Fig. 3. Changes in concentrations of taurine and alanine in dialysate in the 368C n57, 338C n56 and 308C n56 groups. Data are expressed as mean6S.E.M. of percentages of the resting value. Differences among the groups were significant in the concentrations of taurine F 2,1655.59; P,0.02
w
and alanine F 2,16512.2; P,0.001 by two-way repeated measured analysis of variance. P,0.05, P,0.01, difference of 338C from 368C, P,0.05,
w w
P,0.01, difference of 308C from 368C by one-way analysis of variance and Dunnett’s t-test. The ischemia-induced increases in taurine and alanine were significant in the 368C and 338C groups. b P,0.05, a P,0.01 compared with the basal value by one-way analysis of variance and Dunnett’s t-test.
Fig. 4. Photomicrographs of the CA1 subfield of the hippocampus of SHR 7 days after transient cerebral ischemia. In the 368C rat a, majority of pyramidal cells revealed shrunken cytoplasm and picnotic nuclei associated with perinuclear vacuolation. Nuclei of glial cells were also increased in
number. There were a few degenerative pyramidal cells in the 338C rat b. No apparent ischemic damage was observed in the 308C rat c. Paraffin section with hematoxylin and eosin stain. Magnification, 3300.
28 H
Fig. 2. The extracellular glycine decreased by 20 at 20 tamate, aspartate and inhibitory taurine, GABA amino
min after induction of hypothermia, which lasted during acids in the hippocampus, which was for the first time
ischemia and recirculation. Concentrations of alanine in demonstrated in the aged ischemic model.
the normothermic rats significantly elevated 3-fold during Because most of ischemic stroke occur in the elderly
ischemia, and remained at high values during recirculation populations [35] and the age-related vulnerability to is-
Fig. 3. The alanine level was reduced in rats with chemia are reported [12,37], it is important to examine the
hypothermia during and after cerebral ischemia F 2,165 pathophysiology of brain ischemia and to explore effective
12.2; P,0.001. treatment with aged models [23]. Those studies are,
however, limited [11,12,37]. Therefore, our study using the 3.6. Histological examination
aged hypertensive animals would provide useful infor- mation for treatment of brain ischemia in aged populations.
Photomicrographs of the CA1 subfield of the hippocam- Because CBF and other physiological parameters were
pus 7 days after cerebral ischemia are shown in Fig. 4. In similar among normothermic and hypothermic animals in
rats of the normothermic group, most pyramidal cells our study, factors other than circulation are suggested to
revealed shrunken cytoplasm with acidophilic changes and contribute to the protection of hippocampal neurons. One
pyknotic nuclei associated with perinuclear vacuolation. possible mechanism is the altered effluxes of excitatory
Glial cells also increased in number. In contrast, degenera- amino acids, because ischemia-induced effluxes of gluta-
tive pyramidal cells in the 338C group were scattered, and mate and aspartate are regarded to play crucial roles in the
ischemic damages were markedly attenuated in the 308C development of ischemic neuronal damages in the adult
group. Scores for ischemic damages Fig. 5 were sig- animals [10,31,32,34]. Interestingly, our results revealed
nificantly attenuated in both mild 1.360.5 and moderate- complete inhibition of effluxes of glutamate and aspartate
ly 0.860.3 hypothermic groups as compared with the in the hippocampus by mild decreases of brain temperature
normothermic group 3.460.4. by 38C. In the previous reports with adult animals
[22,24], mild brain hypothermia approximately 38C reduc- tion provided moderate reductions of the ischemia-in-
4. Discussion duced effluxes in the hippocampus. Therefore, mild brain
