666
Hypothermia Prevents Ischemia-Induced
Increases in Hippocampal Glycine
Concentrations in Rabbits
Andrew J. Baker, MD; Mark H. Zornow, MD; Marjorie R. Grafe, MD, PhD;
Mark S. Scheller, MD; Stephen R. Skilling, DVM, PhD;
David H. Smullin, PhD; and Alice A. Larson, PhD
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We subjected 10 New Zealand White rabbits to 10 minutes of global cerebral ischemia under
either normothennic (37°C) or moderately hypothermic (29°C) conditions. Hippocampal
concentrations of glutamate, aspartate, and glycine were monitored using in vivo microdialysis.
Outcome was assessed by both neurological and neuropathologic criteria. Hypothermia
afforded nearly complete protection from ischemic injury. Ischemia-induced increases in the
concentrations of glutamate, aspartate, and glycine in the nonnothermic group (3, 12, and 3
times baseline) were strikingly attenuated in the hypothermic group. In addition, the prolonged
postischemic elevation of glycine levels seen in the normothennic group was absent in the
hypothermic group. These results suggest that the neuroprotective properties of hypothermia
may reside, in part, in their ability to prevent increases in the extracellular concentrations of
amino acids that enhance the activity of the /V-methyl-D-aspartate receptor complex. (Stroke
1991;22:666-673)
I
t has been known for decades that hypothermia
confers protection against the neuronal injury
produced by episodes of transient cerebral ischemia. The mechanism by which this protection is
achieved is not fully understood. Hypothermia is known
to decrease the cerebral metabolic rate, and there is
good evidence that at least some of the neuroprotective
properties of hypothermia are due to the associated
decrease in metabolic demand.1 Similar degrees of
metabolic suppression produced by barbiturates or
isoflurane, however, have shown inconsistent benefits
in terms of neurological outcome and degree of neuronal injury.2"4 Furthermore, recent studies have
shown that while modest hypothermia (33°C) does not
preserve high-energy phosphates (e.g., adenosine triphosphate, phosphocreatinine) or prevent the accumulation of metabolic wastes (e.g., lactate),5 it does confer
histopathologic protection from ischemia.6
From the Departments of Anesthesiology (AJ.B., M.H.Z.,
M.S.S.) and Pathology (M.R.G.), University of California, San
Diego, San Diego, Calif, and the Department of Veterinary
Biology (S.R.S., D.H.S., A.A.L.), University of Minnesota, St.
Paul, Mian.
Supported in part by United States Public Health Service grants
NIDA 04090, 04190, and 00124 (to A.A.L.) and NINDS NS RO 1
29403 (to M.H.Z).
Address for correspondence: Dr. M.H. Zornow, Neuroanesthesia Research M-029, University of California, San Diego, La Jolla,
CA 92093.
Received October 15, 1990; accepted January 31, 1991.
Considerable evidence has now accumulated from
both neuronal cultures and in vivo experiments that
excitatory amino acids (particularly glutamate and
aspartate) play an important role in the evolution of
ischemic brain damage.7-9 It appears that these excitatory amino acids are released into the brain's
extracellular space during normal neurotransmission
as well as in response to a variety of insults including
hypoxia,10'11 ischemia,12-13 and hypoglycemia.14 Excitatory amino acids are thought to produce neuronal
injury by activation of the Af-methyl-D-aspartate
(NMDA) receptor-gated ion channels, which results
in a massive increase of the intracellular calcium
concentration. Activation of the NMDA receptor by
glutamate has recently been shown to require the
presence of glycine at a strychnine-insensitive binding site.15-16
Busto et al5 have reported that modest hypothermia (33° and 30°C) was associated with a decrease in
the extracellular levels of glutamate during cerebral
ischemia in rats. These data suggest that hypothermia's beneficial effects may be mediated, at least in
part, by attenuation of the release of excitatory
amino acids. There has recently been an increased
interest17 in the role of glycine (a known allosteric
upregulator of the NMDA receptor) as a mediator of
ischemic neuronal injury. The effects of hypothermia
on extracellular glycine concentrations during transient cerebral ischemia have not been previously
Baker et al Hypothermia and Brain Ischemia
reported. The present study was designed to examine
the effects of moderate hypothermia on 10 minutes of
transient global cerebral ischemia in rabbits. Measurement of the extracellular concentrations of glutamate, aspartate, and glycine by in vivo brain microdialysis was combined with assessments of the
animals' postischemic neurological status and histopathologic evidence of neuronal ischemia.
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Materials and Methods
The protocol was reviewed and approved by the
Animal Care Committee of the University of California, San Diego. Ten New Zealand White rabbits
weighing 2.75 ±0.09 (mean±SEM) kg were anesthetized in a Plexiglas box with 4% halothane in oxygen.
Following intubation of the trachea with a 4.0 uncuffed wire-reinforced endotracheal tube, the animals were mechanically ventilated with 1-2%
halothane in oxygen, resulting in a PacOa of 35.6±1.1
mm Hg. Body temperature was monitored with an
esophageal thermistor. Femoral arterial and venous
catheters were inserted, and an ear vein was cannulated for the administration of maintenance fluids
(0.9% saline at 4 ml/kg/hr). Monitored variables
included mean arterial blood pressure, central venous pressure, heart rate, arterial blood gases (uncorrected for temperature), hematocrit, blood glucose concentration, and the electroencephalogram
(EEG). The rabbit's head was positioned in a stereotactic frame, and a pneumatic tourniquet was secured
loosely around the neck. The cranium was exposed,
and a burr hole was made over the right dorsal
hippocampus 4 mm posterior and 4 mm lateral to the
bregma for the insertion of a microdialysis probe. A
second burr hole was made 4 mm anterior and 3 mm
lateral to the bregma over the right hemisphere for
the insertion of a temperature probe into the epidural space. Bilateral frontoparietal needle electrodes
were placed into the scalp for the continuous recording of the EEG. Following completion of this surgical
preparation, the inspired halothane concentration
was decreased to 1%.
Prior to insertion, recovery rates for each microdialysis probe were determined using standard concentrations of glutamate in vitro. The dura over the
dorsal hippocampus was then incised, and microdialysis probes of concentric design18 (fiber length 4
mm, diameter 0.25 mm) were inserted vertically to a
depth of 7 mm using micromanipulators. The probes
were perfused with artificial cerebrospinal fluid (147
mM NaCl, 2.3 mM Caa 2 , 0.9 mM MgCl2, 4.0 mM
KC1) at a rate of 2 ^1/min. After implantation into
the brain, the probes were perfused for at least 30
minutes prior to collecting baseline samples of brain
tissue microdialysate.
The rabbits were randomly assigned to either a
normothermic (n=5) or a hypothermic (n=5) group.
While baseline microdialysis samples were being
collected, the animals assigned to the hypothermic
group were slowly cooled by the application of ice
bags to the body surface. Surface cooling continued
667
TABLE 1. Rabbit Neurological Deficit Grading Scale
Maximum
score
Assessment
Level of conciousness
Normal
Clouded
Stuporous
Comatose
Respirations
Normal
Abnormal
Cranial nerves
Vision
Light reflex
Oculocephalic
Corneal
Facia] sensation
Auditory
Gag reflex
Motor and sensory function
Flexor response to pain
Front
Rear
Righting reflex
Gait
Normal
Minimal ataxia
Moderate ataxia
Able to stand
Unable to stand
No purposeful movement
Behavior
Grooming
Eating/drinking
Exploratory
Total
0
5
10
25
25
0
5
5
1
1
1
1
1
1
1
7
2
2
10
14
0
5
10
15
20
25
25
4
10
10
24
100
until an epidural temperature of 29°C was achieved.
The rabbits assigned to the normothermic group
received surface warming with a heating pad and
lamp, which were servocontrolled to an esophageal
temperature of 38°C. In both groups, the mean
epidural temperature never differed by >1CC from
the esophageal temperature.
To induce global cerebral ischemia, the mean
arterial blood pressure was lowered to <50 mmHg
using trimethaphan boluses and the application of
positive end-expiratory airway pressure. The neck
tourniquet was then inflated to a pressure of 20 psi
for 10 minutes. Global cerebral ischemia was verified
in each rabbit by observation of an isoelectric EEG
:s40 seconds after tourniquet inflation. Immediately
upon deflation of the tourniquet, a bolus and then an
infusion of phenylephrine was administered to restore the mean arterial blood pressure to >75
mm Hg. Samples of microdialysate from the dorsal
hippocampus were collected as follows: three base-
668
Stroke Vol 22, No 5 May 1991
TABLE 2. Neoropathologic Scoring Criteria
Score
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Neocortex
0
1
2
3
Hippocampus
0
1
2
3
Cerebellum
0
1
2
3
TABLE 3. Summary of Physiological Data for Rabbits
Description
Normal
Rare ischemic neurons (<10%)
Frequent ischemic neurons (10-50%)
Majority of neurons ischemic (>50%)
Normal
Rare ischemic pyramidal or granule cells
Focal ischemic damage
Severe, diffuse ischemic damage
Normal
Rare ischemic Purkinje cells
10-50% ischemic Purkinje cells
>50% ischemic Purkinje cells
line samples (each of 20 minutes' duration) prior to
ischemia, followed by two ischemia samples (each of
5 minutes' duration), then two immediate reperfusion samples (each of 5 minutes' duration), and
finally three reperfusion samples (each of 20 minutes'
duration). Starting with reperfusion, the hypothermic
animals received surface warming in the same fashion as the normothermic rabbits.
At the end of the study period, the microdialysis
and temperature probes were removed and bone wax
was inserted into the burr holes. All monitoring lines
were removed, and the scalp and groin incisions were
sutured closed. The rabbits were allowed to recover
in a warmed Plexiglas box that contained supplemental oxygen (Fic>2 of 40%) and were allowed free
access to water and food. Intravenous fluids were
provided for those animals too neurologically impaired to eat or drink. At 24 and 48 hours after the
insult, the rabbits were neurologically assessed by an
observer unaware of the treatment group using the
grading scale in Table 1. Neurological deficit scores
could range from 0 (normal) to 100 (severely impaired). After completion of the neurological deficit
scoring at 48 hours, the animals were euthanized with
a 120 mg/kg i.v. bolus of pentobarbital and then
perfused with 10% formalin. The brains were removed for histopathologic assessment and grading.
The dialysate from the dorsal hippocampus was
analyzed for glutamate, aspartate, and glycine concentrations using high-performance liquid chromatography with o-phthaldialdehyde derivatization and
a reverse-phase C-18 column. Derivatives were detected fluorometrically, and peak areas were integrated and quantified based on linear calibration
with known amino acid standards. This method has
been shown to be sensitive to amounts as small as 50
fmol glutamate and 50 fmol aspartate.19-20
Coronal sections of the frontal cortex at the level
of the anterior basal ganglia, the parietal cortex, and
the dorsal hippocampus along with a transverse
Group
Variable
Time to flat electroencephalogram (sec)
Epidural temperature (°C)
Initial
Preischemic
Postischemic
1 hour post
Esophageal temperature (°C)
Initial
Preischemic
Postischemic
1 hour post
Mean arterial blood
pressure (mm Hg)
Initial
PTeischemic
Postischemic
1 hour post
Heart rate (beats/min)
Initial
Preischemic
Postischemic
1 hour post
pH
Initial
Preischemic
Postischemic
1 hour post
PacOj (mm Hg)
Initial
Preischemic
Postischemic
1 hour post
PaO} (mm Hg)
Initial
Preischemic
Postischemic
1 hour post
Blood glucose (mg%)
Initial
Preischemic
Postischemic
1 hour post
Trimethaphan dose (g/kg)
Phenylephrine dose (jig/kg)
Normothermic Hypothermic
(»-5)
(»-5)
26±5.1
30±4.2
36.4±0.6
36.6±0.5
36.8±0.2
36.6±0.2
35.5 ± 0 3
28.7±0.4*
28.5±0.4*
30.1 ±0.5*
37.2±0.3
373 ±0.3
37.8±0.1
37.6 ±.03
36.6±0.2
29.3 ±0.4*
29.4±0.5'
31.2±0.4»
62.8+3.0
68.6±3.1
82.2±1.4
75.6±1.8
58.6±4.7
57.0±4.1
83.2±3.3
78.6±2.5
280±6
260±14
220±8
205 ±21
244+15
162+7*
136±4'
150±9
7.362±0.024
7.354±0.010
7.348±0.025
7.324±0.007
7.350±0.022
7.414+0.029
7.410+0.037
7.350±0.052
33.2±1.0
36.6±1.6
35.2±1.8
34.2±1.2
38.0±1.2
31.8±2.6
32.2±2.4
34.4±3.2
506±6
496±7
479 ±6
500+11
499±20
572±12*
580±13*
525±12
98.8±3.4
102.0±3.2
107.0±3.5
107.0±6.6
0.014 ±0.007
496 ±201
114.8+9.8
142.0±14.3
121.8+11.6
0.016±0.009
274+. 126
92.6±7J5
Values are mean±SEM.
*p<0.01 different from normothermic group by two-way
ANOVA.
section of the cerebellum were examined using light
microscopy for evidence of neuronal injury. The
tissue blocks were embedded in paraffin, sectioned at
Baker et al
Hypothermia and Brain Ischemia
669
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FIGURE 1. Top panel: Photomicrograph of central CAl region of hippocampus from hypothermic (2SPC) rabbit demonstrating
normal morphology of pyramidal cells. Bottom panel: Central CAl region from normothermic (37 °C) animal. Note numerous
ischemic neurons. Bar=50 am.
6 yxa., and stained with heraatoxylin and eosin. The
histological sections were scored by a neuropathologist without knowledge of the experimental groups,
according to the criteria in Table 2. Ischemic neurons
were defined by cytoplasmic shrinkage and eosino-
philia with homogenization of the nucleus or karyorrhexis. Specified regions of the left frontal cortex, left
parietal cortex, left dorsal hippocampus (corttralateral to the dialysis probe), and bilateral cerebellar
hemispheres were evaluated. The total neuropatho-
670
Stroke
Vol 22, No 5 May 1991
logic score for each rabbit was obtained by summing
the scores for each individual region (no ischemic
injury=0, greatest ischemic injury=12).
The mean and SEM of the concentrations of glutamate, aspartate, and glycine were calculated for each
time period. The concentrations were analyzed statistically by examining the change over time using analysis of variance (ANOVA) for repeated-measures
followed by Dunnett's test comparing baseline concentrations with those during the ischemic and reperfusion periods. The neurological deficit and histopathologjc scores were tabulated and compared using
the Mann-Whitney U test for nonparametric data.
The physiological data were tabulated and compared
using two-way ANOVA (group versus repeated measure for each variable) followed by multiple comparison tests when indicated. Differences associated with
p<0.05 were considered significant.
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Results
Physiological data are shown in Table 3. Prior to
the onset of ischemia, the hypothermic group had a
lower heart rate (162 ±7 versus 260 ±14 beats/min)
and a higher Pao 2 (572±12 versus 496 ±14 mm Hg)
than the normothermic group. These differences
were most likely due to the well-known effects of
hypothermia on heart rate and oxygen solubility in
the blood. Other than the intended difference in
body temperature, there were no other significant
differences in physiological variables between the
groups. In vitro testing of the microdialysis catheters
prior to insertion into the brain revealed a mean
recovery rate of 8%.
The neurological deficit and histopathologic scores
for each rabbit are shown in Table 4. Statistical
TABLE 4. Neurological Deficit and Neuropathologic Scores
in Rabbits
Neurological deficit score
24 hours
Neuropathologic
score
48 hours
N
H*
N
Ht
0
26
37
69
77
0
0
0
5
14
9
28
22
71
60
0
0
0
1
0
N
5
7
3
4
2
Ht
1
1
1
4
0
N, normothermic group; H, hypothermic group.
*p<0.02 and tp<0.04 different from N by Mann-Whitney {/test.
analysis revealed the hypothermic group to have
significantly better neurological deficit scores than
the normothermic group. Four of the hypothermic
animals were neurologically normal (score of 0) at 48
hours, while ah1fiveof the normothermic rabbits were
significantly impaired (score in the range 9-71).
The neuropathologic scores demonstrated significantly less injury in the hypothermic group. In both
groups, only minimal injury was seen in the frontal
and parietal cortex (scores of 0 or 1). The major
neuropathologic changes were found in the hippocampus and cerebellum. In the hippocampus of the
normothermic rabbits, the entire range of injury was
observed, from normal to severe, diffuse damage. In
contrast, the hippocampus of three hypothermic animals was normal and that of the other two displayed
only rare ischemic pyramidal cells. The histological
differences between the groups can be easily seen by
comparing a photomicrograph of the central CA1
30-
F I G U R E 2. Graph of mean
±SEM glutamate concentrations
in diafysate over time for normothermic (m, n=5) and hypothermic (O, n=5) rabbits. Solid bar
on abscissa indicates duration of
ischemic episode. §p<0.01 different from baseline (0 minutes),
*p<0.05 different from baseline
by repeated-measures ANOVA.
iij
<
<
3
ioH
-60
-40
-20
0
20
TIME (mln)
40
BO
SO
Baker et al Hypothermia and Brain Ischemia
671
FIGURE 3. Graph of mean
±SEM aspartate concentrations
in diafysate over time for normothermic (m, n=5) and hypothermic (o, n=5) rabbits. Solid bar
on abscissa indicates duration of
ischemic episode. §p<0.07 different from baseline (0 minutes),
*p<0.05 different from baseline
by repeated-measures ANOVA.
ui
<
a.
a.
w
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-80
region from a hypothermic rabbit (Figure 1, top) with
that of an animal maintained at 37°C during the
peri-ischemic period (Figure 1, bottom). Similar differences between groups were found in the cerebellum. All normothermic rabbits had moderate or severe ischemic injury in the cerebellum (score of 2 or
3), whereas three hypothermic animals had no ischemic Purkinje cells, one had rare ischemic Purkinje
cells, and one had moderate damage (score of 2).
In the normothermic group the hippocampal extracellular concentrations of glutamate and aspartate
were significantly elevated during ischemia (Figures 2
and 3); these levels returned to baseline ^10 minutes
after reperfusion. In contrast, in the hypothermic
group hippocampal glutamate concentrations did not
increase during or following ischemia. Hippocampal
aspartate concentrations increased to a small (twice
baseline) but statistically significant degree only in
the first reperfusion sample at time (=15 minutes.
In the normothermic group the extracellular concentrations of glycine increased during ischemia and
continued to rise during reperfusion (Figure 4).
Glycine levels remained significantly greater than
baseline throughout the 70 minute postischemic observation period. In the hypothermic poup, no increase in glycine concentration was seen during ischemia. A transient rise was seen only in the second
reperfusion sample. Glycine concentrations subsequently remained at baseline levels in the hypothermic rabbits during the remainder of reperfusion.
Discussion
We make the novel observation that hypothermia
prevents the peri-ischemic increase in the extracellular glycine concentration. We also confirm in this
rabbit model the recent observation that elevations in
glutamate concentrations during ischemia are significantly reduced under conditions of moderate hypothermia.5 Finally, our study confirms the significant
neurological protection afforded by hypothermia
against ischemic injury.
There is a large body of evidence indicating that an
increase in the extracellular glutamate concentration
is an important mediator of ischemic neuronal injury.21 Thus, any intervention that results in a reduction
of such elevated levels may be expected to lessen the
ischemic injury. Clearly, there are other mechanisms
by which hypothermia may have a protective effect on
neuronal injury, including decreased production of
free radicals, preservation of energy stores, and prevention of the accumulation of toxic metabolic
wastes.
The significance and role of glycine in the evolution of ischemic neuronal injury has yet to be fully
elucidated. In vitro work in embryonic mouse neurons has demonstrated that glycine can facilitate the
agonist action of glutamate at the NMDA receptor.22
More recently, the intrathecal injection of glycine has
been shown to potentiate strychnine-induced seizures by action at the NMDA receptor complex.23
This suggests that there is a glycine receptor functionally linked to the NMDA receptor and that this
receptor is not normally saturated in vivo since
exogenous glycine produces a facilitating effect. Furthermore, it has been demonstrated that not only
does glycine increase the maximal glutamate response, the presence of glycine at the NMDA receptor is essential for the functional activation of the
receptor by glutamate.151<> Thus, it appears that
672
Stroke
Vol 22, No 5 May 1991
20
FIGURE 4. Graph of mean
±SEMgfycine concentrations in
diatysate over time for normothermic (m, n=5) and hypothermic (O, n=5) rabbits. Solid bar
on abscissa indicates duration of
ischemic episode. §p<0.01 different from baseline (0 minutes),
*p<0.05 different from baseline
by repeated-measures ANOVA.
I
S
u
io
o
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-BO
-40
-20
0
20
60
80
TIME (min)
glycine may play an important role in the current
theory of glutamate neurotoxicity secondary to ischemia.
While there is evidence to suggest ongoing toxicity
of glutamate during the postischemic period, our
data confirm the observation that the extracellular
concentration of glutamate returns quickly to baseline levels during reperfusion.24 Glycine's ability to
facilitate the actions of glutamate at the NMDA
receptor, and its persistent elevation during the
postischemic period, may help to explain glutamate's
apparent ongoing toxicity. The ability of hypothermia
to block a sustained elevation of the extracellular
glycine concentration in response to ischemia may
reflect an important component of the protective
mechanism of hypothermia. It is tempting to speculate that the ability of hypothermia to decrease
glycine levels may be at least as important as its effect
on glutamate concentrations.
In summary, we studied the effects of moderate
whole-body hypothermia on brain ischemia. We
confirmed that hypothermia confers protection as
assessed by neurological deficit and histopathologic
scoring. In the same rabbits, we demonstrated an
attenuation of the ischemia-induced increase of
extracellular glutamate and aspartate concentrations in the hippocampus. Hypothermia completely
prevented the postischemic rise in glycine concentrations that were seen in the normothermic group.
Given that glycine can facilitate the activity of
excitatory amino acids at the NMDA receptor and
that glycine's extracellular concentrations remain
elevated during the postischemic period in normothermic animals, we speculate that a component of
the beneficial effects of hypothermia may be medi-
ated by decreasing the postischemic extracellular
concentration of glycine.
Acknowledgments
The authors gratefully acknowledge the technical
expertise of Mr. Paul Kelly and Ms. Muriel Spooner,
without whom this work would not have been possible, and the invaluable support of Dr. Tony Yaksh.
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KEY WORDS
• rabbits
amino acids • cerebral ischemia • hypothermia
Hypothermia prevents ischemia-induced increases in hippocampal glycine concentrations in
rabbits.
A J Baker, M H Zornow, M R Grafe, M S Scheller, S R Skilling, D H Smullin and A A Larson
Stroke. 1991;22:666-673
doi: 10.1161/01.STR.22.5.666
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