864
Nerve Terminal Damage in Cerebral Ischemia:
Protective Effect of alpha-Methyl-para-Tyrosine
JESSE WEINBERGER, M . D . ,
JULIA NIEVES-ROSA, B . S . ,
AND GERALD COHEN, P H . D .
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SUMMARY Mongolian gerbils were treated with alpha-methyl-para-tyrosine methyl ester (AMPT, a
tyrosine hydroxylase inhibitor), in order to decrease brain levels of catecholamines. Six hours later,
unilateral ischemic stroke was induced by ligation of the left common carotid artery. The delayed degeneration of nerve terminals was studied sixteen hours later by measuring the high-affinity uptake of radiolabeled transmitters by isolated synaptosomes. Dopamine, serotonin and glutamate terminals were studied.
AMPT-treated gerbils were compared to untreated (no AMPT) animals; 220 gerbils were studied. AMPT
pretreatment (100, 250 and 400 mg/kg) produced a dose-dependent protection of all three types of nerve
terminals. In the absence of AMPT pretreatment, the uptake of radiolabeled transmitters by the ischemic
hemisphere, expressed as a percentage of that seen in the contralateral (unaffected) side of the brain, was as
follows (mean ± SEM): 27.3 ± 5.2% for dopamine terminals, 49.5 ± 6.2% for serotonin terminals, and
42.7 ± 5.3% for glutamate terminals. Protection was essentially complete at a dose of 400 mg AMPT per
kg. The number of animals with significant damage to nerve terminals was reduced from 38.5% in
untreated animals to 11.1% in animals treated with AMPT 400 mg/kg. Although the nerve terminals were
protected, gerbils still showed the behavioral signs of unilateral stroke due to the permanent occlusion of the
left carotid. These results indicate that endogenous dopamine may play a significant role in ischemic damage
to nerve terminals in the cerebrum.
Stroke Vol 16, No 5, 1985
THE MEASUREMENT of high affinity uptake of radiolabeled neurotransmitters by isolated synaptosomes
can be used to evaluate damage to nerve terminals in
experimental animals with cerebral ischemia.1-2 In the
Mongolian gerbil, unilateral cerebral ischemia can be
induced by unilateral carotid ligation in about 40% of
the animals.3-4 These animals have an incomplete Circle of Willis. Thus, they do not receive adequate collateral circulation from the contralateral carotid artery
and they suffer a hemispheric stroke.3-4
The uptake and accumulation of neurotransmitter
against a concentration gradient is a complex neuronal
function that requires energy and the structural integrity of the axonal membrane. The neuronal cell type is
specified by the high-affinity binding of neurotransmitter to the axonal transport system. Prior studies
have determined that the catecholamine nerve terminals that take up dopamine (DA) and norepinephrine
(NE) are damaged to a significantly greater degree by
cerebral ischemia than terminals incorporating serotonin (5-HT), GABA or glutamate.1-2 All nerve terminals were able to function normally in vitro when synaptosomes were isolated for up to eight hours after
carotid ligation.1 However, by sixteen hours, severe
reduction in uptake had occurred. Since the potassium-stimulated release of radiolabeled transmitter
proceeded normally, the reduction in uptake appeared
to be due to loss of terminals, rather than to reduced
uptake in existing terminals. This reduction in uptake
probably represents irreversible damage to the nerve
terminals because function cannot be restored by incubation in vitro with adequate supplies of substrate and
oxygen.1
From Mount Sinai School of Medicine, New York, New York.
This work was presented in part at the 108th Annual Meeting of the
American Neurological Association, New Orleans, October 2-5, 1983.
Address correspondence to: Jesse Weinberger, M.D., Department of
Neurology, The Mount Sinai School of Medicine, One Gustave L. Levy
Place, New York, New York 10029.
Received August 2, 1984; revision # 2 accepted February 6, 1985.
Measurement of the levels of transmitters in the
ischemic hemisphere of gerbils with irreversible unilateral carotid occlusion have shown a similar selective
reduction of catecholamines relative to the other transmitters.5 The reduction occurred by one hour after carotid ligation5 and was attributed to release of the catecholamine transmitters due to depolarization of
membranes in ischemia.6"8 The loss of catecholamines
has been corroborated by histofluorescence studies,
which have shown depletion of catecholamine-derived
fluorescence from the ischemic striatum in gerbils with
stroke.9
The present study was instituted to determine
whether the selective sensitivity of catecholamine
nerve terminals to ischemia could reflect a toxic action
mediated by the physical presence of the catecholamine transmitters. Alpha-methyl-para-tyrosine methyl
ester (AMPT) was administered to gerbils in order to
deplete the brain catecholamine stores. AMPT is a
tyrosine hydroxylase inhibitor that prevents synthesis
of catecholamines.10 AMPT reduces catecholamine
levels because catecholamines turned over in the
course of neurotransmission and metabolism cannot be
replaced by resynthesis.10 Uptake of the transmitters
DA, 5-HT and glutamate was measured in synaptosomes isolated from gerbils at sixteen hours after unilateral carotid artery ligation. Animals pretreated with
AMPT at six hours prior to unilateral carotid artery
ligation were compared to animals that were not pretreated. In this way, the role of endogenous catecholamines in nerve terminal damage could be assessed.
Materials and Methods
Studies were conducted with 220 gerbils. Male gerbils weighing 50 to 70 gm were injected i.p. with
AMPT in dosages of 100 mg/kg, 250 mg/kg or 400
mg/kg six hours prior to carotid ligation. Untreated
animals were injected with an equivalent volume of
saline. The 400 mg/kg dose has been shown to reduce
catecholamines from the cerebrum of gerbils 4 hours
NERVE TERMINAL DAMAGE IN CEREBRAL ISCHEMIA/Weinberger
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after injection.8 Time course studies in guinea pigs
injected with 80 mg AMPT/kg i.p. show a peak effect
on reduction of brain catecholamine levels at 8 hours,
with resynthesis occurring at 24 hours.10 Gerbils were
anesthetized with approximately 40 mg methohexital/kg (i.p.), with the dosage titrated to the stage of
surgical anesthesia for each animal. Animals could
breathe spontaneously without a respirator. The left
common carotid artery was exposed in the paratracheal
region by blunt dissection so that there was no blood
loss. After the animals exhibited partial recovery from
anesthesia to the extent that they were responsive to leg
pinch, the carotid artery was ligated with two ties. This
method has been employed by Lust et al5 to provide
animals in whom behavioral changes associated with
clinical stroke could be readily observed.
The behavioral criteria for designation of stroke
were similar to those described by Kahn:3 paucity of
movement or splaying of the contralateral extremities
(hemiparesis), torsion of the body and circling to the
left, obtundation and a comatose-like motionless state.
Animals that appeared otherwise normal but exhibited
mild circling behavior generally associated with some
lethargy were classified as "circling." The "circling"
category included some gerbils with such mild behavioral changes that thefinalcategorization was difficult.
At sixteen hours after carotid ligation, the gerbils
were decapitated with a guillotine. The cerebrum was
removed and separated into left and right hemispheres,
which were immediately immersed in ice cold isotonic
(0.9%) saline. This procedure took approximately 30
seconds. Each hemisphere was weighed and then homogenized in 10 ml of 0.32M sucrose. Centrifugation
was performed in a Sorvall RC 2B refrigerated centrifuge with a SS 34 head. The homogenates were centrifuged at 3000 rpm (1100 x g), for 10 minutes, to
remove blood and cellular debris. The supernatant was
centrifuged at 15,000 rpm (27,000 x g) for 30 minutes and the pellet retained. The pellet was then resuspended at a concentration of 150 mg wet weight of
original tissue per ml in Krebs-Ringer phosphate buffer, pH 7.4, containing 0.05 mM pargyline and 1.7 mM
ascorbic acid. This preparation has been used previously to provide a synaptosomal suspension which
contains mitochondria, but is relatively free of glial
elements and other subcellular particles.'-2- "•l2
Uptake measurements'- n were based on prior studies by other investigators'3"15 and were carried out in
triplicate. Aliquots of 200 fil of the synaptosomal
preparation in 5 ml of the Krebs-Ringer phosphate
buffer were preincubated for 10 minutes at 37°C in a
Precision Scientific GCA shaker bath. Radiolabeled
neurotransmitters were added to a final concentration
of 5 X 10-9M for DA and 5-HT and 8 X 10-6M for
glutamate. After 10 minutes, one ml aliquots were
removed from replicate flasks and passed through a
0.65 /x Millipore filter by means of suctionfiltrationto
isolate the synaptosomes. The filters with adhering
synaptosomes were washed quickly with 1.0 ml saline.
They were then suspended in 10 ml Scintiverse solution for scintillation counting of the radioactivity; ra-
865
dioactivity measurements were made 8-24 hours later
in a Packard Model 2450 Tricarb Scintillation Spectrometer. The uptake of radiolabeled transmitters, corrected for radioactivity retained on the filter papers,'
was expressed as the DPM per mg wet weight of original tissue divided by the DPM in an equivalent volume
of incubation medium, that is, a tissue-to-medium ratio. The uptake of transmitter in the ischemic left side
versus the control right side was compared by expressing results as a ratio of left/right (L/R ratio).
Statistical comparisons were made by paired Student "t" test for uptake of transmitters in each animal.
The unpaired Student "t" test and the Mann-Whitney
rank order U Test were employed for comparison of
uptake between AMPT-treated and untreated animals.
Chi square analysis was employed to determine if there
were any significant behavioral differences in the various treatment groups. Significant reduction in uptake
in the ischemic hemisphere of an individual animal
was defined as an uptake value more than two standard
deviations below the mean uptake in the left hemisphere of unaffected animals not treated with AMPT.
Radiolabeled neurotransmitters were obtained from
New England Nuclear (Boston, Mass.): 3H-dopamine
(22 Ci/mmol), 3H-5-HT (26.5 Ci/mmol) and 14C-glutamate (290 mCi/mmol). 14C-Glutamate was diluted
with unlabeled glutamate to a specific activity of 0.18
mCi/mmol. Unlabeled L-glutamic acid was obtained
from Sigma Chemical (St. Louis, MO). AMPT methyl
ester was obtained from Regis Chemical Company,
Morton Grove, Illinois. Scintiverse was from Fisher
Scientific (Springfield, N.J.). Gerbils were obtained
from Tumblebrook Farms (West Brookfield, Mass.).
Results
Neurotransmitter uptake was measured in synaptosomes prepared from the ischemic left cerebral hemisphere and control right cerebral hemisphere of gerbils
subjected to left carotid ligation (table 1). Uptake was
measured at 16 hours after carotid ligation. Animals
were separated into the following groups: those with
no behavioral change (unaffected), those with mild
behavioral changes such as slight lethargy and intermittent circling (circle), and those with hemiparesis
and rotational movement (stroke). The behavioral
characteristics of the animals are described in table 2.
Animals pretreated with AMPT were generally
slightly lethargic six hours later when carotid ligation
was performed. By 16 hours after ligation, animals
treated with AMPT 100 mg/kg and 250 mg/kg that did
not have a stroke were behaviorally indistinguishable
from untreated animals without stroke. Animals without stroke that were pretreated with AMPT 400 mg/kg
were hypokinetic, slightly lethargic, and tremulous 16
hours after carotid ligation, but would walk when
prodded and could stand on their hind legs when disturbed.
Animals that were pretreated with AMPT 100
mg/kg and suffered a stroke after carotid ligation displayed the same behavioral activity as untreated animals with stroke (see methods). Animals with stroke
pretreated with AMPT 400 mg/kg showed a somewhat
STROKE
866
VOL 16, No 5, SEPTEMBER-OCTOBER
1985
TABLE 1 Uptake of Dopamine, Serotonin (5-HT) and Glutamate Into Synaptosomes Prepared from Cerebral Hemispheres of Gerbils
Treated with Varying Doses of AMPT 6 Hours Prior to Left Carotid Ligation
5-HT
Dopamine
Glutamate
Right
Left/
right
ratio
Left
Right
Left/
right
ratio
Condition
n
Left
Right
Left/
right
ratio
No treatment
Stroke
10
1.03
(0.28)
1.32
(0.18)
3.25
(0.24)
3.39
(0.37)
1.95
(0.09)
3.20
(0.68)
0.273
(0.052)
0.663
(0.066)
1.02
(0.02)
1.23
(0.28)
3.06
(0.58)
2.77
(0.48)
2.34
(0.37)
3.69
(0.71)
2.72
(0.46)
0.495
(0.062)
0.862
(0.054)
1.01
(0.02)
2.91
(0.43)
3.41
(0.48)
5.73
(0.32)
6.70
(0.36)
4.49
(0.27)
5.47
(0.31)
0.427
(0.053)
0.742
(0.079)
1.05
(0.02)
1.63
(0.56)
3.85
(0.28)
3.46
(0.47)
4.05
(0.21)
4.33
(0.33)
3.65
(0.72)
0.409
(0.127)
0.895
(0.40)
0.960
(0.052)
0.735
(0.113)
1.88
(0.24)
1.90
(0.46)
1.89
(0.55)
1.96
(0.32)
1.83
(0.41)
0.525
(0.093)
1.00
(0.047)
0.988
(0.038)
2.99
(0.62)
6.87
(0.24)
5.41
(0.64)
5.74
(0.56)
6.58
(0.24)
5.31
(0.52)
0.532
(0.104)
1.01
(0.03)
1.00
(0.05)
2.19
(0.62)
2.95
(0.63)
3.84
(0.50)
3.91
(0.19)
3.49
(0.83)
3.71
(0.50)
0.542
(0.147)
0.848
(0.014)
1.03
(0.08)
1.28
(0.20)
2.05
(0.09)
2.15
(0.38)
2.02
(0.13)
2.31
(0.09)
2.18
(0.40)
0.675
(0.130)
0.892
(0.004)
0.995
(0.105)
3.50
(0.66)
5.66
(0.49)
5.99
(0.71)
5.70
(0.19)
5.73
(0.19)
5.84
(0.66)
0.637
(0.132)
0.976
(0.085)
1.03
(0.10)
3.12
(0.39)
3.96
(0.33)
4.05
(0.16)
4.05
(0.29)
4.08
(0.40)
4.10
(0.16)
0.818
(0.108)
0.979
(0.033)
0.996
(0.037)
2.32
(0.77)
1.47
(0.88)
0.638t
(0.038)
2.54
(0.54)
1.57
(0.79)
0.626t
(0.049)
0.885
(0.079)
0.954
(0.036)
1.07
(0.08)
4.78
(0.55)
4.54
(0.28)
5.30
(0.24)
5.50
(0.36)
4.06
(0.24)
5.18
(0.33)
0.879
(0.091)
1.13
(0.09)
1.04
(0.02)
Circle
Unaffected*
AMPT 100 mg/kg
Stroke
7
22
9
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Circle
8
Unaffected*
8
AMPT 250 mg/kg
Stroke
Circle
Unaffected*
AMPT 400 mg/kg
Stroke
Circle
Unaffected*
7
2
10
11
4
12
Left
The uptake of DA, 5-HT and glutamate into synaptosomes from the ischemic (left) hemisphere and control (right) hemisphere is shown at 16
hours after left carotid ligation in gerbils pretreated with varying doses of AMPT. Uptake is expressed as the tissue to medium ratio: the
disintegrations per minute (dpm)/mg wet weight of original tissue divided by the dpm in an equivalent volume of incubation medium. Figures
in parentheses are the S.E.M.
*For animals showing no behavioral change after left carotid ligation (unaffected groups), uptake was measured in 22 out of 45 control
animals (no AMPT), 8 out of 34 animals treated with 100 mg AMPT/kg, 10 out of 12 animals treated with 250 mg AMPT/kg, and 12 out of 20
animals treated with 400 mg AMPT/kg.
tThere is an apparent decrement in uptake of 5-HT in unaffected animals treated with AMPT 400 mg/kg. However, several untreated
animals studied at the same time as controls had similar tissue to medium uptake ratios. The lower 5-HT uptake in these animals (both with and
without AMPT) reflects decomposition of the 3H-5-HT tracer, rather than an effect of AMPT on uptake. Expression of the data as Left/Right
ratio compensates for the effect of radiotracer decomposition and permits evaluation of the effects of ischemia in the various subgroups. It
should be noted that earlier studies did not show bilateral change in uptake of neurotransmitters.24- 25
different behavior. Eight of these animals were curled
in a ball, hypotonic and lying at the bottom of the cage
because they could not support weight with their right
extremities. Three animals that were standing, but
hunched over, were classified as having a stroke because they had spastic extension of the right extremities when lifted by the tail and could not maintain
standing balance when pushed. Four animals that also
curled in a ball but had normal responses when lifted
by the tail and good righting reflexes when pushed
were put into the intermediate classification (circling).
In these four animals, it could not be distinguished
whether the paucity of movement was secondary to
cerebral ischemia or to the metabolic effects of AMPT.
None of the animals with stroke in the 400 mg
AMPT/kg group exhibited rotational movement as
seen in untreated animals. Lack of rotation may have
been due to depletion of dopamine by AMPT in the
unaffected striatum.
Two stroke animals pretreated with AMPT 250
NERVE TERMINAL DAMAGE IN CEREBRAL ISCHEMIA/Weinberger
867
TABLE 2 Behavioral Changes in Gerbils Following Left Carotid Ligation after Pretreatment with Varying Doses of
AMPT
Condition
Stroke
Mild circling
Unaffected
Intraoperative death
Deaths due to stroke
Total
No treatment
10
7
45
3
11
76
(13%)
(9%)
(59%)
(4%)
(14%)
AMPT
100 mg/kg
AMPT
250 mg/kg
AMPT
400 mg/kg
9
8
34
2
17
70
7
2
12
5
1
27
11 (23%)
4 (9%)
20 (43%)
7 (15%)
5 (11%)
47.
(13%)
(11%)
(49%)
(3%)
(24%)
(26%)
(7%)
(44%)
(19%)
(4%)
The number of gerbils affected with stroke or mild circling, or unaffected, after left carotid ligation is shown at varying
dosages of AMPT. Intraoperative deaths were due to respiratory or cardiac arrest during surgery. An apparent increase in
intraoperative deaths at the higher doses of AMPT may have been the result of compromise of sympathetic tone due to
depletion of catecholamines; however, the difference was not statistically significant by Chi-square analysis. Most of the
deaths due to stroke occurred 2-3 hours after carotid ligation and were secondary to seizures. The gerbils were not
continuously observed for the whole 16 hour period following carotid ligation. Therefore, the number of gerbils having
seizures in the AMPT-treated and untreated groups could not be quantitatively compared. There were no significant
differences in the total number of animals with stroke (i.e. stroke plus death due to stroke) in any of the treatment groups
by Chi-square analysis. No other differences were significant.
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mg/kg behaved in a similar fashion to the eight stroke
animals pretreated with AMPT 400 mg/kg. Five stroke
animals pretreated with AMPT 250 mg/kg behaved
like untreated stroke animals.
Inspection of the brains of animals pretreated with
AMPT 250 mg/kg and 400 mg/kg that exhibited stroke
behavior revealed evidence of tissue necrosis in the
cortex, but less softening of subcortical structures than
untreated animals with stroke. This was clearly evident
on removing the cerebrum from the cranium and separating the hemispheres. The diminution in the extent of
necrosis was present even in animals where uptake of
transmitter was impaired.
Uptake of DA into synaptosomes prepared from the
ischemic (left) hemisphere of 10 untreated gerbils with
stroke was significantly more impaired than uptake of
5-HT or glutamate (table 1), (p < 0.001). These results confirm earlier observations1'2 on the selective
sensitivity of DA neurons. Uptake of DA was 27.3 ±
5.2% of the control hemisphere, (see left/right ratio)
while uptake of 5-HT was 49.5 ± 6.2% and uptake of
glutamate was 42.7 ± 5.3%. Uptake of each transmitter was significantly reduced relative to control (p <
0.001).
In nine gerbils with stroke that were pretreated with
the lower dose of AMPT (100 mg/kg), uptake of DA
was 40.9 ± 12.7% of the control side. Uptake of 5-HT
was 52.5 ± 9.3% and uptake of glutamate was 53.2 ±
10.4%. Uptake of all three transmitters was significantly lower in synaptosomes from the ischemic hemisphere than from the control hemisphere (DA p <
0.005, 5-HT p < 0.05, glutamate p < 0.025). However, the differences between relative uptake of the three
transmitters were no longer statistically significant.
In seven gerbils with stroke treated with the intermediate dose of AMPT (250 mg/kg), uptake of DA into
synaptosomes from the ischemic hemisphere was 54.2
± 14.7% of control. Uptake of 5-HT was 67.5 ±
13.0% and uptake of glutamate was 63.7 ± 13.2%.
Again, uptake of all three transmitters was significantly reduced compared to the control side (p < 0.05), but
the differences between DA and the other transmitters
did not achieve statistical significance.
In eleven gerbils with stroke that had been treated
with the high dose of AMPT (400 mg/kg), uptake of
DA into synaptosomes from the ischemic hemisphere
was 81.8 ± 10.8% of control, uptake of 5-HT was
88.5 ± 7.9% and uptake of glutamate was 87.9 ±
9.1 %. At this dosage of AMPT, uptake of each transmitter was not significantly different from its respective control. Moreover, AMPT provided significant
protection for all three types of nerve terminals (DA p
< 0.001, 5-HT/? < 0.005, glutamate/? < 0.001 by ttest; p < 0.01 by Mann-Whitney U Test).
There was no significant difference between untreated and AMPT-treated animals in the uptake of
transmitters in unaffected animals (table 1). Thus,
AMPT did not appear to have a direct effect on uptake.
The reduction in the endogenous concentration of DA
by AMPT does not alter the accumulation of 3H-DA by
synaptosomes because the concentration added to the
medium is well below the Km of uptake for DA.15 In
this range, transport is proportional to the concentration used.
The uptake of DA (fig. la), 5-HT (fig. lb) and
glutamate (fig. lc) into synaptosomes prepared from
each individual stroke animal in the various treatment
groups can be seen in the accompanying figures. Seven
of the ten control (not pretreated with AMPT) animals
with stroke showed significant reduction in uptake of
DA, 5-HT and glutamate, with significant selective
reduction in uptake of DA (cf. table 1).
In stroke animals pretreated with 100 mg AMPT/kg,
five of nine showed this same significant reduction in
transmitter uptake with accentuation of damage to DA
terminals. The remaining animals showed considerable preservation of DA nerve terminal function. Inspection of tabular data for individual animals (not
shown), revealed that when uptake of DA was preserved, uptake of 5-HT and glutamate was also increased relative to control stroke animals.
In stroke animals pretreated with 250 mg AMPT/kg,
STROKE
868
« o.s
i
o
a.
o
o
lOOmg/kg
25Omg/kg
4 0 0 mg/Vg
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Dosage od-MPT
0
a
I
Control
100 mg/kg
2 5 0 mg/kg
400 mgfkg
Dosage <*-MPT
0 0.5
o
O
lOOmgAg
250 mg/kg
Dosage oi-MPT
400mgAg
VOL 16, No 5, SEPTEMBER-OCTOBER 1985
only two of seven animals showed a significant reduction in DA uptake. For three animals, DA uptake approached equality with uptake in the non-ischemic
right hemisphere. In these animals, uptake of 5-HT
and glutamate also were elevated to the level seen in
the control hemisphere.
In stroke animals that had been pretreated with 400
mg AMPT/kg, only two of eleven animals had significant reduction in DA uptake into synaptosomes from
the ischemic hemisphere, and one animal had intermediate reduction. These three animals correspond to the
lowest data points for 5-HT and glutamate uptake in
Figs, lb and lc. In the remaining eight animals, uptake
of DA, 5-HT and glutamate in the ischemic hemispheres were all in the same range as in the corresponding non-ischemic hemispheres.
We considered the possibility that the sedative effect
of AMPT 400 mg/kg may have caused animals with
mild stroke to appear as if they had a more severe
stroke. In such an event, mildly affected animals
would be placed into the "stroke" category and the
mean uptake value in the stroke category would given
an appearance of protection. However, this possibility
can be excluded: Only 3 of 27 animals treated with
AMPT 400 mg/kg had significant impairment of uptake of DA in the ischemic hemisphere. For animals
not pretreated with AMPT, all 10 with stroke and 5 of 7
with mild circling behavior had significant reduction
of uptake of DA in the ischemic hemisphere. A comparison of the AMPT-pretreated and untreated groups
shows that the total number of animals with significant
damage to nerve terminals in the pretreated group
(3/27 11.1%) was significantly lower than among untreated animals (15/39 38.5%) (p < 0.01), irrespective
of the extent of stroke behavior.
In summary, when there was prevention of damage
to DA nerve terminals by AMPT, there was also prevention of damage to 5-HT and glutamate terminals.
Protection by AMPT was dose-dependent upon the
administered amount of AMPT: Both the mean uptake
values for animals in the stroke category (table 1), and
the number of animals exhibiting preservation of neurotransmitter uptake (fig. 1), increased with increasing
dose of AMPT. Although AMPT protected against the
loss of neurotransmitter uptake, it did not prevent behavioral change associated with stroke after permanent
ligation of the carotid artery (table 2).
With regard to the possibility of selective protection
of DA terminals by AMPT, the simultaneous effects
FIGURE 1. a) The uptake ofdopamine into synaptosomes from
the ischemic left hemisphere compared to the control right
hemisphere is expressed as the ratio of left/right in gerbils
exhibiting signs of stroke after left carotid ligation. The left/
right ratio is shown for each animal with no pretreatment and
for animals pretreated with 100 mglkg, 250 mglkg or 400 mglkg
AMPT i.p. at 6 hours prior to left carotid ligation. The horizontal bars represent the mean uptake for each group of animals.
b) The uptake of 5-HT is shown as per the legend to figure la.
c) The uptake of glutamate is shown as per the legend to figure
la.
NERVE TERMINAL DAMAGE IN CEREBRAL ISCHEMIA/Weinberger
on DA, 5-HT, and glutamate terminals makes a clear
evaluation difficult. It is noted that for the lowest dose
of AMPT (100 mg/kg), where selectivity in action
might be most readily assessed, DA uptake remained
less than that for 5-HT or glutamate (table 1, L/R ratio,
stroke category), but differences between DA and the
other transmitters were no longer significant statistically. Increments in mean uptake of DA and glutamate
with increasing AMPT were apparent (table 1, DA
from 0.27 ± 0.05 to 0.40 ± 0.12, and glutamate from
0.42 ± 0.05 to 0.53 ± 0.10), while the mean uptake
of 5-HT was relatively unchanged (from 0.49 ± 0.06
to 0.52 ± 0.09). J h e increase in SEM for each transmitter (table 1, L/R ratio, 100 mg AMPT/kg), compared to no pretreatment, relates to the greater spread
of data, due in large measure to the strong protection
seen in some animals (Cf. figs, la, b, c).
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Discussion
Treatment of gerbils with AMPT prior to carotid
ligation reduced the amount of ischemic damage to the
uptake mechanism of DA nerve terminals, as well as to
those of 5-HT and glutamate terminals. The number of
animals with significant impairment of uptake was reduced from 38.5% in untreated animals to 11.1% in
animals treated with AMPT 400 mg/kg. This reduction
in damage was significant (p < 0.01). Depletion of the
catecholamines did not completely prevent stroke,
since animals still exhibited severe behavioral abnormalities. There was also damage to tissue in the cerebral hemispheres on gross inspection, though perhaps
to a lesser extent than untreated animals. Since AMPT
depletes cerebral catecholamines, these results suggest
that the catecholamines are in some way deleterious to
nerve terminals after an ischemic insult.
An initial goal of this study was to assess whether or
not AMPT could prevent the accentuated damage to
DA nerve terminals. As noted, however, the main
finding was that AMPT protected all three of the terminal types that were studied. The simultaneous action
on DA, 5-HT and glutamate makes it difficult to evaluate any selective degree of protection for DA nerve
terminals.
Other authors have reported release of endogenous
catecholamines from the brain, shortly after the onset
of ischemia. '•6"9 This could result in vasospasm, and in
a consequent worsening of the ischemic insult. Furthermore, there is some evidence that extraneuronal
catecholamines may be toxic to neuronal structures:
When norepinephrine is injected into the capillaries of
a pial window, regional necrosis of cerebral tissue
occurs.16 Necrosis does not occur when 5-HT is injected, even with an equivalent amount of regional ischemia.16
Acute release of endogenous catecholamine does
not explain the delayed damage to nerve terminals that
occurs after eight hours of ischemia, though it may be
involved in the initial damaging process. Indeed, histoflourescence studies have shown almost complete
depletion of catecholamine from the ipsilateral striatum early after carotid occlusion in the gerbil.9 At this
869
time, the structure of the striatum and the function of
nerve terminals remain intact.1-9
Even though there is depletion of catecholamine in
the ischemic hemisphere, there is evidence that catecholamine metabolism and turnover proceed. The levels of catecholamines fall initially and do not rise. 5
However, AMPT 400 mg/kg administered two hours
prior to the carotid ligation produces a further decrement in catecholamine level two hours after ligation.8
Similarly, the administration of the monoamine oxidase (MAO) inhibitor pargyline 75 mg/kg two hours
prior to ligation produces a rise in catecholamine levels
in the ischemic hemisphere two hours after ligation.8
These observations also imply that some circulation
and oxygen are present in the ischemic hemisphere,
since both tyrosine hydroxylase and MAO require molecular oxygen to function.8
Disruption of nerve terminal function by an ischemic insult appears to be a delayed process analogous to
the histologic finding of ripening of ischemic infarction, which occurs in a similar time frame.17 Some
acute injury does occur to neuronal membranes. This
can be seen ultrastructurally as a breakdown of ribosomal units and swelling of rough endoplasmic reticulum and Golgi complexes.18 This initial vacuolization
may cause leakage of catecholamine transmitters either into the cytoplasm or extracellularly. Extracellular
catecholamines may penetrate into the cytoplasm of
neurons and glia. Cytoplasmic catecholamines would
be exposed to MAO which produces H 2 O 2 as a product
of oxidative deamination.19 H 2 O 2 is a known neurotoxin and could possibly be involved in the ripening if
ischemic damage. Extracellular catecholamines may
also exert toxic effects via autoxidation or other
mechanisms. Prior depletion of catecholamines and
inhibition of catecholamine resynthesis in the ischemic
hemisphere may lessen or prevent this damage.
Synaptic activity appears to be necessary to mediate
the death of hypoxic neurons in tissue culture.20 When
young hippocampal neurons without synaptic activity
are exposed to sodium cyanide or hypoxia, no effect is
noted. Death occurs only in more mature neurons that
have developed synaptic activity. This hypoxic death
can be blocked by MgCl 2 , which inhibits synaptic activity.20
Our data indicate that catecholamine transmitters
are involved in the delayed degeneration of nerve terminals in the ischemic hemisphere of Mongolian gerbils. Depletion of the catecholamines by pretreatment
with AMPT exerted a strong protective effect at 16
hours after carotid ligation. The toxicity of catecholamines may be due to a direct action or to some byproduct of catecholamine metabolism. Glutamate and 5HT nerve terminals were protected along with DA
terminals, suggesting that catecholamines may be involved in the spread of ischemic damage to adjacent
nerve terminals.
Acknowledgments
The authors wish to acknowledge the assistance of Ms. Diane Davis
in preparing the manuscript and Ms. Marilyn Ilvento in preparing the
figures.
870
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J Weinberger, J Nieves-Rosa and G Cohen
Stroke. 1985;16:864-870
doi: 10.1161/01.STR.16.5.864
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