Delayed Treatment With AM-36, a Novel Neuroprotective
Agent, Reduces Neuronal Damage After
Endothelin-1–Induced Middle Cerebral Artery Occlusion
in Conscious Rats
Jennifer K. Callaway, PhD; Melissa J. Knight, BSc; Dianne J. Watkins, BSc;
Philip M. Beart, DSc; Bevyn Jarrott, PhD
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Background and Purpose—AM-36 is a novel arylalkylpiperazine with combined antioxidant and Na1 channel blocking
actions. Individually, these properties have been shown to confer neuroprotection in a variety of in vitro and in vivo
animal models of stroke. Preliminary studies have shown that AM-36 is neuroprotective in vivo. The purpose of the
present study was to assess the neuroprotective and behavioral outcome after delayed administration of AM-36 in an
endothelin-1–induced, middle cerebral artery model of cerebral ischemia in conscious rats.
Methods—Conscious male hooded Wistar rats were subjected to middle cerebral artery occlusion by perivascular
microinjection of endothelin-1 via a previously implanted cannula. AM-36 (6 mg/kg IP) or vehicle was administered
intraperitoneally 30, 60, or 180 minutes after middle cerebral artery occlusion. Functional outcome was determined 24,
48, and 72 hours after stroke by neurological deficit score, motor performance, and sensory hemineglect tests. Rats were
killed at 72 hours, and infarct area and volume were determined by histology and computerized image analysis.
Results—Endothelin-1–induced middle cerebral artery occlusion resulted in marked functional deficits and neuronal
damage. AM-36 significantly reduced cortical damage when administration was delayed until 30, 60, or 180 minutes
after stroke. Interestingly, neuronal damage was time-dependently reduced, with the greatest protection found when
AM-36 was administered 180 minutes after stroke. Striatal damage was significantly reduced after treatment with
AM-36 at 180 minutes after stroke. Functional outcome paralleled histopathology. Rota-rod performance, sensory
hemineglect, and neurological deficit scores returned to preischemia levels in AM-36 –treated rats by 72 hours after
stroke when administration was delayed by 180 minutes after stroke.
Conclusions—AM-36 potently protects against both neuronal damage and functional deficits even when administered up
to 180 minutes after induction of stroke. In fact, the greatest protection was found when administration was delayed by
180 minutes after stroke. The possible mechanisms of action of AM-36 are discussed. The present findings suggest that
AM-36 may have great promise in the acute treatment of human stroke. (Stroke. 1999;30:2704-2712.)
Key Words: cerebral ischemia, focal n endothelins n neuroprotection n rats
A
M-36 is a novel arylalkylpiperazine with combined
antioxidant and Na1 channel blocking activity within a
single compound.1,2 The activity of AM-36 has been tested in
in vitro assays, in which it was found to inhibit lipid
peroxidation in the modified thiobarbituric acid reactive
substances assay with activity comparable to that of known
antioxidants such as 3,5-tert-butyl-4-hydroxytoluene (BHT),
Trolox, and LY231617.3 In binding assays, AM-36 inhibits
[3H]batrachatoxinin binding to site 2 Na1 channels in rat brain
homogenates. Furthermore, in cultured cerebellar granule
cells, AM-36 potently inhibits veratridine-induced cell death
with an IC50 of 1.3 mmol/L.4 We have recently demonstrated
preliminary evidence that AM-36 is neuroprotective after
See Editorial Comment, page 2712
acute stroke4,5 and believe that both its free radical scavenging and Na1 channel blocking activity contribute to its
neuroprotective activity.2
Individually, antioxidants and Na1 channel antagonists
have been shown to be neuroprotective in animal models of
cerebral ischemia.6,7 Substantial evidence suggests that the
generation of reactive oxygen species during cerebral ischemia is an important contributor to neuronal damage.6 Studies
have indicated dramatic increases in levels of oxygen radicals
during reperfusion, which remain high for up to 2 hours and
then increase again at 24 hours.8,9 Loss or blockade of
Received August 2, 1999; final revision received September 2, 1999; accepted September 2, 1999.
From the Department of Pharmacology, Monash University, Clayton, Australia.
Correspondence to Jennifer K. Callaway, PhD, Department of Pharmacology, Monash University, Wellington Rd, Clayton 3168, Australia. E-mail
[email protected] Fax: 61 3 9905 5851 Ph: 61 3 9905 5753
© 1999 American Heart Association, Inc.
Stroke is available at http://www.strokeaha.org
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AM-36 Reduces Neuronal Damage After Focal Ischemia in Rats
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endogenous antioxidants worsens ischemic injury, and mice
overexpressing Cu/Zn superoxide dismutase are protected
from ischemia.10 Antioxidants and inhibitors of lipid peroxidation attenuate neuronal damage in animal models of cerebral ischemia.11–13 Additionally, the spin trap reagent
a-phenyl-tert-butyl nitrone (PBN) reduced brain infarct volume in a middle cerebral artery (MCA) occlusion model of
stroke when administered up to 3 hours after ischemia.11
Blockade of Na1 channels has also been proposed as a
possible neuroprotective mechanism by reducing energy expenditure in compromised tissue.7 Since a large part of the
energy expenditure of excitable cells is used to maintain Na1
and K1 gradients across cell membranes,14 blockade of Na1
channels may constitute an effective neuroprotective mechanism.7 A number of experimental findings strongly suggest
that administration of inhibitors of voltage-sensitive Na1
channels is beneficial even when it is delayed after stroke.7,15
This action may constitute an important mechanism since a
number of structurally unrelated neuroprotective drugs share
the property of downmodulating Na1 channels.7 Compounds
possessing Na1 channel blocking action such as lamotrigine,
BW619C89, and riluzole are neuroprotective in MCA models
of ischemia.15–17
In view of the delay in the time taken to hospitalize and
diagnose a stroke victim, it is now considered important to
demonstrate significant neuroprotection after delayed administration of a compound. Hence, the purpose of the present
study was to determine the effectiveness of the novel neuroprotective agent AM-36 when treatment commenced at various intervals after the onset of stroke. This study used the
endothelin-1 (ET-1) model of MCA occlusion, which is less
invasive than some other models, incorporates reperfusion,
and has the advantage that rats are conscious during
stroke.18,19 The use of anesthesia during stroke may confound
experimental findings since it has been reported to be
neuroprotective20 –22 and to have interactions with other pharmacological agents (eg, MK801),18 and it may also potentially affect free radical production.22 Our present results
indicate that AM-36 potently protects against neuronal damage and improves functional outcome after delayed
administration.
Materials and Methods
Surgical Preparation
All procedures used in this study were performed in accordance with
the Prevention of Cruelty to Animals Act 1986. Male hooded Wistar
rats (weight, 280 to 320 g) were anesthetized with a 50:50 mixture of
pentobarbital/methohexital sodium in a volume of 0.6 mL (30 mg/kg
and 30 mg/kg IP, respectively). According to the method of Sharkey
and colleagues,18 a 23-gauge stainless steel guide cannula was
stereotaxically implanted into the piriform cortex 2 mm dorsal to the
right MCA. The stereotaxic coordinates were modified for this rat
strain (0.2 mm anterior, 25.2 mm lateral, and 26.1 mm ventral,
according to a stereotaxic atlas).23 The cannula was secured with
dental acrylate cement, and 2 small screws were inserted into the
skull. The scalp was closed with sutures. Animals were housed
individually and allowed to recover for 3 days before induction of
stroke.
Rats were divided into drug or vehicle groups before induction of
stroke. Occlusion of the right MCA was induced in conscious rats by
administration of ET-1 (120 pmol in 6 mL of saline over 6 minutes)
2705
via a 30-gauge injector that protruded 2 mm beyond the end of the
previously implanted guide cannula. The injector was held in place
by a poly tubing cuff, and the animal was placed in a clear Plexiglas
box for observation during ET-1 injection. Stroke was characterized
by counterclockwise circling, clenching, or failure to extend the
contralateral forelimb. These behaviors occurred within 2 to 10
minutes of the beginning of the ET-1 injection. Rats that did not
show any behavioral signs were deemed not to have had a stroke and
were excluded from further study. Sham-injected rats underwent
cannula implantation but did not receive any ET-1 injection.
AM-36 hydrochloride was dissolved in water, and dose was
expressed as the free base. The dose of AM-36 used here was chosen
on the basis of preliminary investigations that evaluated 1.8 and 6
mg/kg IP. Whereas 1.8 mg/kg produced some neuroprotection, a
dose of 6 mg/kg considerably reduced the size of the infarct. The first
dose of AM-36 (6 mg/kg IP) or vehicle (water) was administered
either 30, 60, or 180 minutes after the beginning of ET-1 injection.
All rats then received the second and third doses at 24 and 48 hours
after stroke. Hence, AM-36 –treated rats received a total of 336
mg/kg IP. Rectal temperatures were taken with a thermistor probe
before stroke and at 30- or 60-minute intervals for 3 hours after
stroke and 2 hours after drug administration.
Assessment of Functional Outcome
All behavioral tests were conducted before any procedures (presurgery, day 1); after recovery from cannula implantation and immediately before ET-1–induced MCA occlusion (preischemia, day 4); and
24, 48, and 72 hours after ET-1–induced MCA occlusion (days 5, 6,
and 7, respectively). Each rat acted as its own control.
Neurological abnormalities were evaluated with the use of a
neurological deficit score based on detection of abnormal posture
and hemiplegia, as described by Yamamoto and colleagues24 and De
Ryck and colleagues.25 Abnormal posture was assessed by suspending rats by the tail and observing twisting of the thorax and extension
of forelimbs. Presence of thorax twisting and absence of contralateral
forepaw extension were scored at 1 each. Hemiplegia was evaluated
by placing rats on a raised platform. When there is a deficit, the
contralateral hindlimb slips off the edge of the platform (score51),
and the contralateral forelimb slips off when the snout and whiskers
lose contact with the surface (score51). Thus, when the scores were
summed, the maximum neurological deficit score was 4. A score of
0 was considered normal.
Motor impairment was assessed with the use of the accelerating
rota rod (Ugo Basile, model 7750). Rats were given 2 training
sessions 10 minutes apart before surgery. Latency to fall off the rota
rod was then determined before induction and 24, 48, and 72 hours
after stroke. Rats not falling off within 5 minutes were given a
maximum score of 300 seconds.
Sensory hemineglect was evaluated by a test developed by
Schallert and Whishaw26 that measures sensitivity to simultaneous
forelimb stimulation. This test is based on observations of behavior
in humans with unilateral brain damage.27–29 If 2 stimuli are
presented simultaneously, 1 on each side of the body, the contralateral stimulus appears to be masked (“extinguished”), and either
remains undetected until the ipsilateral stimulus is removed or feels
subjectively weaker. In rats, the test consists of placing adhesive
tapes (Avery adhesive labels, 1-cm circles) on the distal-radial region
of each wrist. Placement of the first tape was randomized between
contralateral and ipsilateral limbs. The tape on both forepaws was
touched simultaneously before the animal was placed in a Plexiglas
cage, and both the latency to touch and the latency to remove each
stimulus from the contralateral and ipsilateral forepaws were measured with a stopwatch. The test was terminated at 120 seconds if the
tapes had not already been removed.
Spontaneous activity of individual rats was measured for 10
minutes at the same time each day with an Animex type S activity
meter (FARAD Electronics). Rats were equilibrated in the apparatus
for a period of 30 minutes before the first testing time on presurgery
day 1.
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Figure 1. Effect of delayed administration of
AM-36 or vehicle on infarct area in cortex and
striatum. Data are presented as mean6SEM of
infarct area measured at 8 predetermined coronal planes through the brain. The first dose of
AM-36 (6 mg/kg IP) was administered 30 (A),
60 (B), or 180 minutes (C) after ET-1–induced
stroke. All AM-36 –treated rats were given the
second and third doses (both 6 mg/kg IP) at 24
and 48 hours. *P,0.05, **P,0.01 compared
with vehicle-treated control rats (ANOVA followed by Duncan’s test). n54 to 6 rats in each
treatment group.
Quantification of Ischemic Damage
Materials
Rats were decapitated 72 hours after ischemia; their brains were
removed and frozen in liquid nitrogen and stored at 280°C. Coronal
cryostat sections (18 mm thick) were cut at 8 predetermined planes
throughout the brain from 23.2 to 6.8 mm anterior to the interaural
line. The sections were then fixed in paraformaldehyde vapor for 30
minutes at 60°C and then overnight at room temperature before they
were stored at 280°C. Infarct area was measured in unstained
sections with the use of our recently reported novel method,5 which
employs an image analysis system (MCID M4 image analyzer,
Imaging Research Inc) to trace the areas of damage in each brain
section. Total infarct volume was calculated by integrating the
cross-sectional area of damage at each stereotaxic level and the
distances between the levels according to the method of Osbourne
and colleagues.30
AM-36 [1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis(1,1dimethyl)-4-hydroxyphenyl)methylpiperazine] was designed and
synthesized in conjunction with AMRAD Operations Pty Ltd.1 ET-1
was obtained from the American Peptide Company, Inc.
Statistical Analyses
Values are presented as mean6SEM unless stated otherwise. Physiological data and infarct area data were analyzed by ANOVA
followed by the Duncan test. Infarct volumes were compared by
1-way ANOVA followed by trend analysis. Neurological deficit
scores were analyzed by Kruskal-Wallis nonparametric ANOVA and
the Mann-Whitney test for analysis of individual differences. Rotarod performance and spontaneous activity were expressed as a
percentage of presurgery performance for each rat and analyzed by
TABLE 1. Effects of AM-36 (336 mg/kg IP) or Vehicle at Different Times of Administration
After Ischemia on Cortical and Striatal Infarct Volume
Cortex
Striatum
Time After
Stroke, min
n
Vehicle Control
n
AM-36
n
Vehicle Control
n
AM-36
30
4
111.265.9
4
60.4615.7*
4
31.862.6
4
32.261.9
60
6
152.9638.9
5
43.967.3*
6
31.967.9
5
20.866.9
180
4
107.4647.7
4
18.9610.5*
4
33.5610.4
4
10.769.5*
Values are volume of infarction (mean6SEM), expressed in cubic millimeters.
*Significant compared with vehicle control (P,0.05, Student’s t test).
Callaway et al
AM-36 Reduces Neuronal Damage After Focal Ischemia in Rats
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TABLE 2. Change From Presurgery Body Weight in Sham-Injected,
Vehicle-Treated, and AM-36 –Treated Rats Measured After Surgery and 24, 48,
and 72 Hours After Ischemia
Hours After Ischemia
Treatment
n
0
24
48
72
8
3.662.0
25.462.5
26.163.8
26.063.4
11
0.161.8
212.263.5*
212.365.0*
211.565.3*
30 min after ET-1
4
25.560.3
225.069.3*‡
229.8611.7†‡
226.068.8*‡
60 min after ET-1
5
24.463.5
215.863.1†
212.462.3†
212.263.3†
180 min after ET-1
4
23.064.6
210.564.9*
210.564.6*
213.364.9†
Sham
Vehicle control
AM-36
Values are change in body weight (mean6SEM), expressed in grams.
*P,0.05, †P,0.01 compared with 0 hours (ie, before ET-1–induced stroke).
‡P,0.05 compared with sham (ANOVA followed by Duncan’s test).
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ANOVA for repeated measures, followed by Student-NewmanKeuls test. Sensory hemineglect data were analyzed by the Friedman
repeated-measures test, and individual differences were analyzed by
the Wilcoxon signed rank test or Mann-Whitney test. A value of
P,0.05 was considered significant.
Results
Histopathological Analyses
Rats showed neurological deficits indicative of stroke (but not
stress or vocalization) within 5 minutes of injection of ET-1,
validating the correct placement of the cannula. These deficits
included circling in the direction contralateral to the occlusion
and failure to extend the contralateral forepaw. Some rats
showed loss of righting reflex on the side contralateral to MCA
occlusion. Rats showing no neurological deficits were deemed
not to have had a stroke and were excluded from the study.
Histopathology performed on the brains 72 hours after injection
of ET-1 showed a pattern of damage similar to that found by
Sharkey and colleagues18 and included consistent lesions in the
parietal and insular cortex, variable degrees of damage in the
frontal cortex, and infarction most often in the dorsolateral
striatum but sometimes extending throughout the corpus striatum. Sham-injected rats showed only localized damage associated with the tract of the guide cannula.
Infarct area in the cortex was significantly reduced at
several stereotaxic levels by AM-36 (336 mg/kg IP) when
administration of the first dose was delayed by either 30, 60,
or 180 minutes after MCA occlusion (Figure 1). AM-36
TABLE 3.
significantly reduced damage in striatum, but only when
administration of the first dose was delayed by 180 minutes
after the induction of stroke (Figure 1). Cortical infarct
volume was significantly reduced by AM-36 irrespective of
the time of administration after stroke induction (Table 1).
There was a significant linear trend for the degree of
protection to increase as the time delay in drug administration
increased (F2,1056.45, P50.029). Infarct volume was reduced
by 45%, 71%, and 82% when drug administration began 30,
60, or 180 minutes after stroke induction, respectively. A
similar trend was seen when striatal infarct volume was
calculated, with 0%, 35%, and 68% reductions when drug
treatment began 30, 60, and 180 minutes after stroke, respectively. This trend was not quite statistically significant
(F2,1054.52, P50.059).
All rats, including sham-injected animals, lost weight after
surgery (Table 2). Weight loss was greater in rats after
ET-1–induced stroke. Rats treated with AM-36 at 30 minutes
after stroke had significantly greater weight loss than shamoperated animals. Weight loss in rats given AM-36 at 60 or
180 minutes after stroke did not differ significantly from
vehicle-treated controls. Core (rectal) temperatures, measured
3 hours after stroke and 1 and 2 hours after administration, did
not differ significantly between treatment groups (Table 3).
Functional Assessment
Vehicle-treated control rats exhibited significantly higher
neurological deficit scores than sham-injected controls (Table
Change in Core (Rectal) Temperatures Before and After Ischemia and Before and After AM-36 Administration
Time After Ischemia, min
Treatment
Before Ischemia*
30
60
120
Time After AM-36, min
180
Before AM-36*
60
120
Sham
37.860.2
0.360.4
0.660.3
0.460.4
0.360.4
Vehicle control
37.260.3
0.260.3
0.360.2
20.160.3
20.260.2
30 min after ET-1
38.460.3
20.660.8
20.460.4
21.160.4
21.460.7
37.860.6
0.560.5
0.960.1
60 min after ET-1
37.060.1
0.360.6
20.160.4
0.060.3
20.160.3
37.360.3
20.160.3
20.060.3
180 min after ET-1
38.160.3
0.260.3
20.360.2
20.760.2
20.860.3
37.360.1
0.260.1
0.060.1
AM-36
Values are change in temperature (mean6SEM) (°C), unless otherwise indicated (n54 –5 for AM-36 –treated rats, n58 for vehicle control rats,
and n511 for sham rats). There were no significant differences between treatments or between time intervals within treatments (P.0.05, ANOVA).
*Basal temperature.
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TABLE 4. Neurological Deficit Score 24, 48, and 72 Hours After Ischemia in
Sham and Vehicle-Treated Rats and After Delayed Administration of AM-36
Hours After Ischemia
Treatment
Sham
n
24
48
72
8
0.060.0
0.060.0
0.060.0
11
2.360.3‡
2.160.4‡
2.260.4‡
30 min after ischemia
4
3.860.3†§
3.560.3†
3.060.4†
60 min after ischemia
5
1.660.7*
0.860.1
1.060.8
180 min after ischemia
4
2.361.8†
0.560.3§
0.560.9§
Vehicle control
AM-36
Values are neurological deficit score (mean6SEM).
*P,0.05, †P,0.01, ‡P,0.001 compared with sham; §P,0.05 compared with vehicle control
(Kruskal-Wallis nonparametric ANOVA and Mann-Whitney test for analysis of individual differences).
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4). Treatment with AM-36 at 30 minutes after stroke did not
improve neurological outcome. Neurological deficit scores
were significantly higher than those of vehicle controls in this
group 24 hours after stroke but had improved at 72 hours.
Rats treated with AM-36 at 60 or 180 minutes after stroke
showed deficits at 24 hours. By 48 and 72 hours, neurological
scores were improved and no longer significantly differed
from those of sham-injected animals. Rats in the group
treated with AM-36 at 180 minutes had statistically significantly better neurological deficit scores than vehicle-treated
controls at 48 and 72 hours after stroke.
In the accelerating rota-rod test, each animal acted as its
own control, and performance was compared with preischemia (0 hours after ischemia) results (Figure 2). Surgery had
no effect on performance since there was no significant
difference in presurgery and prestroke (postsurgery) performance in any of the treatment groups. In sham-injected rats,
rota-rod performance did not significantly alter over time
(Figure 2). Vehicle-treated control rats showed significant
impairments in performance 24, 48, and 72 hours after stroke
compared with prestroke performance (Figure 2). Rats treated
with AM-36 at 30 minutes after stroke showed significant
impairment at 24 and 48 hours compared with postsurgery
scores. Variability was high at 72 hours, and no statistically
significant differences were detected. When administration of
AM-36 was delayed until 60 or 180 minutes after stroke,
performance no longer differed from postsurgery performance when assessed at 24, 48, and 72 hours after stroke.
Spontaneous locomotor activity did not significantly differ
between control, drug-treated, or sham-operated rats at any of
the times tested (data not shown).
Latency to touch and remove an adhesive tape from the
contralateral forepaw was consistently and significantly increased in control vehicle-treated rats (Figures 3 and 4). In
comparison, there was no change compared with before
stroke in the time taken to touch or remove a tape simulta-
Figure 2. Effects of delayed administration of AM-36 on rota-rod performance after MCA occlusion in rats. Vehicle (A) or AM-36 was
administered 30 (B), 60 (C), or 180 minutes (D) after ET-1–induced stroke. Sham-injected rat data are shown in E. Performance before
ET-1 injection (0 hours) is shown for comparison. Data are mean6SEM for rota-rod performance expressed as a percentage of presurgery performance for each individual rat. *P,0.05, **P,0.01 compared with 0 hours (ANOVA followed by Newman-Keuls test).
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AM-36 Reduces Neuronal Damage After Focal Ischemia in Rats
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Figure 3. Effects of delayed administration of AM-36 on latency to touch a stimulus on the contralateral compared with the ipsilateral
forelimb assessed 24, 48, and 72 hours after ET-1–induced stroke. Data are mean6SEM of time taken to touch each stimulus. Each rat
acted as its own control, and results after stroke were compared with presurgery scores (0 hours after ischemia). *P,0.05 compared
with 0-hour score in the same forelimb; 1P,0.05, 11P,0.01 compared with opposite forelimb; #P,0.05 compared with result at 24
hours in the same forelimb (Friedman nonparametric repeated-measures ANOVA with individual differences determined by Wilcoxon
signed rank test [control and sham group] or Mann-Whitney test when sample size was below n56 [AM-36 treatment groups]).
neously placed on the ipsilateral forepaw; that is, control
vehicle-treated rats showed sensory hemineglect on the side
contralateral to the MCA occlusion after ET-1–induced
stroke. AM-36 had no significant effects on the increases in
latency to touch or remove tapes when the first dose was
administered 30 minutes after stroke. However, when the first
dose was delayed until either 60 or 180 minutes after stroke,
the difference in latency between contralateral and ipsilateral
forepaws was reduced, and the latency to touch and remove
tapes returned to presurgery times by 72 hours after stroke.
Discussion
This study demonstrates that the novel neuroprotective compound AM-36 greatly attenuates both cortical and striatal
Figure 4. Effects of delayed administration of AM-36 on latency to remove a stimulus on the contralateral compared with the ipsilateral
forelimb assessed 24, 48, and 72 hours after ET-1–induced stroke. Data are mean6SEM of time taken to remove each stimulus. Each
rat acted as its own control, and results after stroke were compared with presurgery scores (0 hours after ischemia). *P,0.05 compared with 0-hour score in the same forelimb; 1P,0.05, 11P,0.01 compared with opposite forelimb (Friedman nonparametric
repeated-measures ANOVA with individual differences determined by Wilcoxon signed rank test [control and sham group] or MannWhitney test when sample size was below n56 [AM-36 treatment groups]).
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damage and also improves functional outcome after MCA
occlusion in rats. Interestingly, the effects of this compound
on both histological and functional outcome were most
evident when administration of the first dose was delayed by
60 or 180 minutes after stroke. In fact, a significant linear
trend for greater histological improvement with increased
delay in time of administration of AM-36 was found with
respect to cortical damage. A similar but not statistically
significant trend was observed for striatal damage. However,
a marked reduction in striatal damage was found in rats
treated with AM-36 beginning 180 minutes after stroke. The
striatum is generally considered to be the core of the ischemic
lesion31 and previously has proved relatively refractory to
neuroprotection.
Unlike some N-methyl-D-aspartate antagonists, which have
a very narrow window of opportunity in focal ischemia
models (eg, MK801 is not effective if not administered within
30 minutes of stroke32,33 ), Na1 channel antagonists and
antioxidants appear to have a longer window of opportunity.
For example, PBN reduced brain infarct volume in a rat MCA
model of stroke when administered up to 3 hours after
ischemia,11 and the Na1 channel antagonist lamotrigine was
neuroprotective when administered 30 minutes or 24 hours
after MCA occlusion in rats.15
Studies have indicated dramatic increases in levels of
oxygen radicals on reperfusion, with levels remaining high
for 2 hours and then increasing again at 24 hours.8,9 A recent
study in a photochemical-induced model of stroke in rats
indicates that the period of hydroxyl radical formation most
critical for brain damage occurs between 3 and 6 hours after
ischemia.34 The present finding of a greater neuroprotective
effect after delayed administration of AM-36 may reflect its
greater effectiveness against higher free radical production at
the beginning of reperfusion in this model. Reperfusion most
likely occurs after at least 3 hours in this model.18 ET-1 (120
pmol in 3 mL) resulted in blood flows of ,25 mL/100 g per
minute (,20% of normal), which were still evident in the
striatum and sensory cortex at 3 hours after injection.18
Hence, it is possible that the greater effectiveness of this
compound at 180 minutes may reflect the greater production
of reactive oxygen species associated with reperfusion at this
time. Alternatively, it could be speculated that maintenance
of ionic homeostasis through the ability of AM-36 to block
Na1 channels may reach a critical period at approximately 60
to 180 minutes. Previous studies showing effectiveness of
delayed administration of Na1 channel antagonists support
this notion.7,15
Rectal temperatures did not differ between treatments
groups after ischemia, and AM-36 administration had no
effect on temperature, excluding the possibility that AM-36
exerted its effects by lowering body temperature. Cerebral
blood flow was not measured in this study; however, studies
in this laboratory have indicated that AM-36, at higher doses
than those used in the present experiments, had no cardiovascular effects in conscious normotensive rats (J.K. Callaway,
P.M. Beart, B. Jarrott, R.E. Widdop, unpublished data, 1999).
While an effect of AM-36 on cerebral blood flow cannot be
ruled out without direct measurement, it is unlikely that AM-36
would have a specific effect on other vascular beds.
Recent studies have emphasized the importance of demonstrating improvement in functional outcome as well as histological improvements when neuroprotective agents are assessed, since embolic stroke in humans leads to lesions and
consequent behavioral deficits, including language and motor
dysfunction.35 The importance of choosing functional tests
that result in a correlation between functional outcome and
histological outcome has been emphasized.36,37 The accelerating rota rod is a well-established procedure for testing
coordination and balance aspects of motor performance in
rats.38 Deficits in motor performance on the rota rod have
been reported after both permanent and transient focal ischemia and are directly related to histological outcome 24 hours
after occlusion.36,37 Deficits have been reported to still be
apparent 2 months after focal cerebral ischemia in the
rat.24,39,40 In the present study, significant deficits in performance were evident 24, 48, and 72 hours after ET-1–induced
MCA occlusion. In contrast, performance was no longer
different from prestroke levels in AM-36 –treated rats (60and 180-minute delayed administration groups) at 72 hours
after stroke. This result parallels the trend for greater histological improvement with increased delay in time of administration of AM-36 and indicates protection against motor
impairments induced by MCA occlusion.
Sensory hemineglect is a phenomenon that has been
reported in humans during the course of recovery from
stroke,29 in patients with damage to the striatum and surrounding white matter,27,28 and in patients with right parietal
lobe infarction.29 If 2 stimuli are presented simultaneously, 1
on each side of the body, the contralateral stimulus appears to
be masked (extinguished) and remains undetected until the
ipsilateral stimulus is removed. Patients report that they can
feel both stimuli, although the contralateral stimulus feels
subjectively weaker than the ipsilateral stimulus. Sensory
hemineglect was evaluated in the present study with a test
developed for rats by Schallert and Whishaw.26 Vehicletreated control rats showed a consistent sensory hemineglect
on the side contralateral to the MCA occlusion, as indicated
by an increased latency to both touch and remove a stimulus
(an adhesive disk) placed on the contralateral forelimb.
Latency to touch and remove a stimulus simultaneously
placed on the ipsilateral side was unaffected by ischemia.
Treatment with AM-36 either 60 or 180 minutes after stroke
effectively removed the hemineglect observed in control rats
and returned touch and remove latency to presurgery levels.
This effect was most apparent at 48 and 72 hours after stroke.
The lack of effect of AM-36 when administered 30 minutes
after stroke may reflect the lack of protection found in the
striatum. The greatest improvement in this test occurs in those
groups that show the greatest reductions in volumes of
infarction. Thus, the present results correlate well with
histological outcome. Improvement in neurological deficit
scores paralleled the findings in the hemineglect test.
Vehicle-treated control rats showed significant neurological
deficits that were reduced by delaying administration of
AM-36 by 60 or 180 minutes but not 30 minutes after stroke.
Recent reports have shown that neurological deficit scores
and rota-rod performance are sensitive indicators of behavioral deficits after stroke in experimental animals.36,37 Results
Callaway et al
AM-36 Reduces Neuronal Damage After Focal Ischemia in Rats
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from the present study support these findings and provide
further evidence that the sensory hemineglect test is a simple
test capable of demonstrating a phenomenon in ischemic rats
that is known to occur in human stroke patients. This test is
also sensitive enough to demonstrate a neuroprotective effect
of a compound. No differences in spontaneous activity were
observed in sham-operated rats or rats subjected to cerebral
ischemia with or without AM-36 treatment. Previous studies
have also reported a lack of effect of ischemia on spontaneous
activity.24 Nevertheless, this simple test serves to indicate that
differences in ability in motor performance and sensory
neglect tasks are unrelated to an effect of stroke or drug
treatment on activity. Weight loss has previously been reported after ischemia and has been shown to increase with
duration of ischemia.36 The greater weight loss compared
with vehicle in rats treated with AM-36 at 30 minutes after
stroke is difficult to explain since no differences between
vehicle and 60-minute and 180-minute AM-36 groups was
found. It is possible that the lack of functional improvements
in this group of rats may be related to greater weight loss.
In conclusion, we have now shown that the novel neuroprotective compound AM-36 potently protects against both
cortical and striatal neuronal damage in the ET-1–induced
MCA occlusion model of focal ischemia in conscious rats.
The effectiveness of this compound increased with the delay
in time after administration, a finding that may reflect
increased free radical production associated with reperfusion.
This compound also appears to provide some protection
against motor impairments, sensory hemineglect, and neurological deficits. These findings with AM-36 when taken with
the extent of neuroprotection noted, which also includes the
normally refractory striatum, suggest that the bifunctional
activity of the arylalkylpiperazines, incorporating both Na1
channel blockade and free radical scavenging activity, represents a unique strategy for the management of stroke injury.
Acknowledgments
We gratefully acknowledge AMRAD Operations Pty Ltd for funding
this project, and we thank Dr Alan Robertson of AMRAD for his
contributions to the project.
References
1. Jarrott B, Beart PM, Jackson WR, Kenche VB, Robertson A, Collis MP,
inventors. Arylalkylpiperazine compounds as antioxidants. Patent Cooperation Treaty No. WO97/43259. AMRAD Operations Pty Ltd, 1999.
2. Jarrott B, Callaway JK, Jackson, WR, Beart PM. Development of a novel
arylalkylpiperazine compound (AM-36) as a hybrid neuroprotective drug.
Drug Devel Res. 1999;46:261–267.
3. Callaway JK, Beart PM, Jarrott B. A reliable procedure for comparison of
antioxidants in rat brain homogenates. J Pharmacol Toxicol Methods.
1998;39:155–162.
4. Callaway JK, Giardina SF, Beart PM, Jarrott B. The arylalkylpiperazine
AM36 inhibits veratridine-induced toxicity and apoptosis in cultured
cerebellar granule cells. Proc Aust Neurosci Soc. 1999;10:222. Abstract.
5. Callaway JK, Knight MJ, Watkins DJ, Beart PM, Jarrott B, Delaney PM.
A novel computerised method for quantitation of neuronal damage and its
use to demonstrate neuroprotection by AM-36, a novel neuroprotective
agent. J Neurosci Methods. In press.
6. Kuroda S, Siesjo BK. Reperfusion damage following focal ischemia:
pathophysiology and therapeutic windows. Clin Neurosci. 1997;4:
199 –212.
7. Urenjak J, Obrenovitch TP. Pharmacological modulation of voltage-gated
Na1 channels: a rational and effective strategy against ischaemic brain
damage. Pharmacol Rev. 1996;48:21– 67.
2711
8. Matsuo Y, Onodera H, Shiga Y, Shozuhara H, Ninomiya M, Kihara T,
Tamatani T, Miyasaka M. Role of cell adhesion molecules in brain injury
after transient middle cerebral artery occlusion in the rat. Brain Res.
1994;656:344 –349.
9. Morimoto T, Globus MYT, Busto R, Martinez E, Ginsberg MD. Simultaneous measurement of salicylate hydroxylation and glutamate release in
the penumbral cortex following transient middle cerebral artery
occlusion. J Cereb Blood Flow Metab. 1996;16:92–99.
10. Kinouchi H, Epstein CJ, Mizui T, Carlson EJ, Chen SF, Chan PH.
Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A. 1991;
88:11158 –11162.
11. Zhao Q, Pahlmark K, Smith M-L, Siesjo BK. Delayed treatment with the
spin trap a-phenyl-N-tert-butyl nitrone (PBN) reduces infarct size following transient middle cerebral artery occlusion in rats. Acta Physiol
Scand. 1994;152:349 –350.
12. Umemura K, Wada K, Uematsu T, Mizuno A, Nakashima M. Effect of
21-aminosteroid lipid peroxidation inhibitor U74006F, in the middle
cerebral artery occlusion model. Eur J Pharmacol. 1994;251:69 –74.
13. Takasago T, Peters EE, Graham DI, Masayasu H, Macrae IM. Neuroprotective efficacy of ebselen, an anti-oxidant with anti-inflammatory
actions, in a rodent model of permanent middle cerebral artery occlusion.
Br J Pharmacol. 1997;122:1251–1256.
14. Ereckinska M, Silver IA. ATP and brain function. J Cereb Blood Flow
Metab. 1989;9:2–19.
15. Rataud J, Debarnot F, Mary V, Pratt J, Stutzmann J-M. Comparative
study of voltage-sensitive sodium channel blockers in focal ischemia and
electric convulsions in rodents. Neurosci Lett. 1994;172:19 –23.
16. Smith SE, Meldrum BS. Cerebroprotective effect of lamotrigine after
focal ischaemia in rats. Stroke. 1995;26:117–122.
17. Smith SE, Hodges H, Sowinski P, Man C-M, Leach MJ, Sinden JD, Gray
JA, Meldrum BS. Long-term beneficial effects of BW619C89 on neurological deficit, cognitive deficit and brain damage after middle cerebral
artery occlusion in the rat. Neuroscience. 1996;77:1123–1135.
18. Sharkey J, Ritchie IM, Kelly PAT. Perivascular microapplication of
endothelin-1: a new model of focal cerebral ischemia in the rat. J Cereb
Blood Flow Metab. 1993;13:865– 871.
19. Hunter AJ, Green AR, Cross AJ. Animal models of acute ischemic stroke:
can they predict clinically successful neuroprotective drugs? Trends
Pharmacol Sci. 1995;16:123–128.
20. Simeone FA, Frazer G, Lawner P. Ischemic brain edema: comparative
effects of barbiturates and hypothermia. Stroke. 1979;10:8 –12.
21. Tamura A, Asano T, Sano K, Tsumagari T, Nakajima A. Protection from
cerebral ischemia by a new imidazole derivative (Y-9179) and pentobarbital: a comparative study in chronic middle cerebral artery occlusion
in cats. Stroke. 1979;10:126 –134.
22. Hagerdal M, Welsh FA, Keykhah M, Perez E, Harp JR. Protective effects
of combinations of hypothermia and barbiturates in cerebral hypoxia in
the rat. Anaesthesiology. 1978;49:165–169.
23. Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. 2nd ed.
New York, NY: Academic Press Inc; 1986.
24. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K. Behavioural
changes after focal cerebral ischemia by left middle cerebral artery
occlusion in rats. Brain Res. 1988;452:323–328.
25. De Ryck M, Van Reempts J, Borgers M, Wauquier A, Janssen PAJ.
Photochemical stroke model: flunarizine prevents sensorimotor deficits
after neocortical infarcts in rats. Stroke. 1989;20:1383–1390.
26. Schallert T, Whishaw IQ. Bilateral cutaneous stimulation of the somatosensory system in hemi-decorticated rats. Behav Neurosci. 1984;98:
518 –540.
27. Ferro JM, Kertesz A, Black SE. Subcortical neglect: quantitation, anatomy, and recovery. Neurology. 1987;37:1487–1492.
28. Healton EB, Navarro C, Bressman S, Brust JCM. Subcortical neglect.
Neurology. 1982;32:776 –778.
29. Daffner KR, Ahern GL, Weintraub S, Mesulam M-M. Dissociated
neglect behaviour following sequential strokes in the right hemisphere.
Ann Neurol. 1990;28:97–101.
30. Osbourne KA, Shigeno T, Balarsky A-M, Ford I, McCulloch J, Teasdale
GM, Graham DI. Quantitative assessment of early brain damage in a rat
model of focal cerebral ischemia. J Neurol Neurosurg Psychiatry. 1987;
50:402– 410.
31. Fisher M, Garcia JH. Evolving stroke and the ischemic penumbra. Neurology. 1996;47:884 – 888.
2712
Stroke
December 1999
32. Hiroshi Y, Ginsberg MD, Watson BD, Prado R, Dietrich WD, Kraydieh
S, Busto R. Failure of MK-801 to reduce infarct volume in thrombotic
middle cerebral artery occlusion in rats. Stroke. 1993;24:864 – 871.
33. Margaill I, Parmentier S, Callebert J, Allix M, Boulu RG, Plotkine M.
Short therapeutic window for MK-801 in transient focal cerebral
ischemia in normotensive rats. J Cereb Blood Flow Metab. 1996;16:
107–113.
34. Takamatsu H, Kondo K, Ikeda Y, Umemura K. Hydroxyl radical generation after the third hour following ischemia contributes to brain
damage. Eur J Pharmacol. 1998;352:165–169.
35. Gresham GE. Rehabilitation of the stroke survivor. In: Barnett HJM,
Mohr JP, Stein BM, Yatsu FM, eds. Stroke: Pathophysiology, Diagnosis
and Management. 2nd ed. New York, NY: Churchill Livingstone; 1992:
1189 –1201.
36. Rogers DC, Campbell CA, Stretton JL, Mackay KB. Correlation
between motor impairment and infarct volume after permanent and
37.
38.
39.
40.
transient middle cerebral artery occlusion in the rat. Stroke. 1997;28:
2060 –2065.
Hunter AJ, Mackay KB, Rogers DC. To what extent have functional
studies of ischaemia been useful in the assessment of potential neuroprotective agents? Trends Pharmacol Sci. 1998;19:59 – 66.
Jones BJ, Roberts DJ. The quantitative measurement of motor incoordination in naive mice using an accelerating rotarod. J Pharm Pharmacol.
1968;20:302–304.
Markgraf CG, Green EJ, Hurwitz BE, Morikawa E, Dietrich WD,
McCabe PM, Ginsberg MD, Schneiderman N. Sensorimotor and cognitive consequences of middle cerebral artery occlusion in rats. Brain Res.
1992;575:238 –246.
Okada M, Tamura A, Urae A, Nakagomi T, Kirino T, Mine K, Fujiwara
M. Long-term spatial cognitive impairment following middle cerebral
artery occlusion in rats: a behavioural study. J Cereb Blood Flow Metab.
1995;15:505–512.
Editorial Comment
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It is refreshing to see that some groups are concerned with the
time window for therapeutic intervention as one of the major
factors for successful intervention in ischemic stroke. Gone
are the days when drugs that show neuroprotective effects
when given before or a short period after MCA occlusion can
be considered relevant for therapy of ischemic stroke. Several
agents from this group have been evaluated clinically and
failed. Other than unacceptable side effects, most of the
clinical failures with new therapeutic agents are likely attributable to the fact that when the agent was administered, the
targeted event that the therapeutic agent was designed to
block had already taken place. If acute neuroprotection is ever
to be realized successfully, agents that inhibit critical lateoccurring pathophysiological events will have to be developed and administered within a few hours after the stroke.
In the accompanying article, Callaway et al succeeded in
developing a novel neuroprotective agent, AM-36, that has a
time window for therapeutic intervention of at least 3 hours.
It is impressive that efficacy seems to improve as the interval
between MCA occlusion and AM-36 administration increases. This feature is unlike most of the previously reported
neuroprotective agents in studies of experimental stroke.
Future studies will have to be conducted to determine the
maximum time window for intervention with this compound.
The proposed mechanism of action, Na1 channel blockade
and antioxidant activity, may account for part of its efficacy,
but activation of Na1 channels and generation of reactive
oxygen species usually occur within the first 3 hours, and this
is suggestive that other activities may have a role in the
neuroprotective effect of AM-36. Other compounds, such as
basic fibroblast growth factor1 and the calpain inhibitor
MDL-28,170, have been reported to have expanded time
windows.2 In experimental focal stroke, the time window for
basic fibroblast growth factor was approximately 3 hours,
whereas the time window for MDL-28,170 had an unusually
long 6-hour time window for therapeutic intervention. Basic
fibroblast growth factor is being evaluated clinically. However, it is surprising that MDL-28,170, with a 6-hour time
window, is not being pursued further and has not been
replicated experimentally. Thus, if acute neuroprotection
from stroke is attainable, agents with expanded time windows, such as AM-36, will likely have an important role in
achieving this goal.
James A. Clemens, PhD, Guest Editor
Department of Neuroscience Research
Eli Lilly and Company
Indianapolis, Indiana
References
1. Ren JM, Finklestein SP. Time window of infarct reduction by intravenous
basic fibroblast growth factor in focal cerebral ischemia. Eur
J Pharmacol. 1997;327:11–16.
2. Markgraf CG, Nelson L, Velayo BS, Johnson MP, McCarty DR, Medhi S,
Koehl JR, Chmielewski PA, Linnik MD. 6-hour window of opportunity for
calpain inhibition in focal cerebral ischemia in rats. Stroke. 1998;29:
152–158.
Delayed Treatment With AM-36, a Novel Neuroprotective Agent, Reduces Neuronal
Damage After Endothelin-1−Induced Middle Cerebral Artery Occlusion in Conscious Rats
Jennifer K. Callaway, Melissa J. Knight, Dianne J. Watkins, Philip M. Beart and Bevyn Jarrott
Downloaded from http://stroke.ahajournals.org/ by guest on June 16, 2017
Stroke. 1999;30:2704-2712
doi: 10.1161/01.STR.30.12.2704
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1999 American Heart Association, Inc. All rights reserved.
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