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(Stroke. 1999;30:1472-1477.)
© 1999 American Heart Association, Inc.
Original Contributions |
The Neuroprotective Effect of the Novel AMPA Receptor Antagonist PD152247 (PNQX) in Temporary Focal Ischemia in the Rat
From the Departments of Neuroscience Therapeutics (G.P.S., N.C.K., P.A.B.), Chemistry (C.F.B.), and Pharmacokinetics, Dynamics Metabolism (D.F.W.), Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, and Department of Surgery (Neurosurgery), University of Michigan (G.P.S.), Ann Arbor, Mich.
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Abstract |
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Background and Purpose—Evidence suggests that glutamate contributes to ischemic brain damage through activation of the
-amino-3-hydroxy-5-methyl-4-isoxazole
propionate (AMPA) receptor. We tested the novel, selective AMPA
receptor antagonist PD152247 (PNQX) in a model of temporary
focal ischemia to determine the dose-response relationship and
to investigate the contribution of drug-induced hypothermia to the
neuroprotective action of AMPA receptor antagonists. Methods—Temporary focal cerebral ischemia was induced in Sprague-Dawley rats by occluding the middle cerebral artery and both carotid arteries for 3 hours. Body temperature was monitored by telemetry. PNQX was administered intraperitoneally or by intravenous infusion with various doses for 6 hours. Lesion volume was determined after 3 days by stereological methods.
Results—PNQX reduced the lesion volume by 51% after intraperitoneal administration. The intravenous dose-response study demonstrated that the lowest effective dose of PNQX was 1.0 mg/kg per hour, which corresponded to a steady state plasma level of 685 ng/mL. Neuroprotection was demonstrated at PNQX plasma concentrations that did not lower body temperature over the entire course of the experiment.
Conclusions—AMPA receptor activation plays an important role in the development of ischemic brain damage. Thus, novel AMPA receptor antagonists may be useful for the treatment of stroke in humans.
Key Words: cerebral ischemia, focal • glutamate antagonists • middle cerebral artery occlusion • neuroprotection • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Elevation of extracellular glutamate after cerebral ischemia is thought to play a major role in the pathophysiological processes leading to death of ischemic brain tissue. Early studies demonstrated that glutamate killed neurons in culture by a calcium-dependent process that was blocked by selective antagonists of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors.1 2
-Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), the ligand
for a distinct non-NMDA glutamate receptor subtype, also results in
cell death in cultured neurons with a slower time course.3
The observation that extracellular brain glutamate levels, measured by
microdialysis, are increased in ischemic tissue4 5
suggested that the excitotoxicity demonstrated in culture may be
relevant to ischemic cell death in vivo. Subsequently, it was
demonstrated that blockers of the NMDA receptor reduce brain damage in
several animal models of cerebral
ischemia.6 7 8 9 10 More recently, several antagonists of the AMPA subtype of the glutamate receptor have been discovered and shown to be neuroprotective in a variety of models of cerebral ischemia.11 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), a quinoxalinedione, was the first selective, competitive AMPA antagonist demonstrated to be effective in reducing ischemic brain damage.12 Unlike NMDA receptor antagonists, the AMPA receptor antagonist NBQX reduces selective neuronal death after global forebrain ischemia.13 14 15 In models of focal ischemia, AMPA antagonists including NBQX16 17 18 19 and more recently discovered antagonists LY215490,20 YM90K,21 YM872,22 and GYKI5246623 have been reported to reduce the volume of necrotic tissue. Additional evidence that AMPA receptor activation plays a role in ischemia is the recent finding that transgenic mice that overexpress the AMPA receptor subunit GluR2-flip have increased ischemic brain damage.24
Recently, a novel quinoxalinedione PD152247 (PNQX) was discovered.25 PNQX binds with high affinity to the AMPA receptor (IC50, 90 nmol/L) and with modest affinity to the kainate receptor and the glycine site of the NMDA receptor. PNQX antagonizes effects of AMPA in AMPA-induced cytotoxicity in primary neuronal cultures and protects against AMPA and maximal electroshock-induced seizures in mice (unpublished data, Boxer and Probert, 1999).
The present study was performed to determine whether PNQX is neuroprotective in a model of temporary focal ischemia. The first rationale for this study is that evaluation of novel AMPA antagonists with varying profiles of in vitro and in vivo activity and water solubility is important because the solubility-related renal toxicity of NBQX18 has prevented its clinical testing. Second, since it has been suggested that drug-induced hypothermia is responsible for the neuroprotective effects of AMPA antagonists,26 the possible contribution of hypothermia to the neuroprotective action of PNQX was addressed. Finally, the relationship between plasma concentrations of PNQX and the degree of neuroprotection in this model was evaluated.
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Materials and Methods |
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All procedures were within the guidelines of the Institutional Animal Care and Use Committee of Parke-Davis.
Surgery
Male Sprague-Dawley rats (Charles River Laboratories), weighing
275 to 325 g, were anesthetized with 2% isoflurane
(Anaquest) balanced with air and oxygen and allowed to breath
spontaneously. Rats receiving intravenous treatments had
femoral vein catheters (Micro-Renathane, Braintree Scientific)
implanted before middle cerebral artery occlusion (MCAO). Both common
carotid arteries were isolated through a small neck incision and
occluded with vascular clips (Microserrefine No. 3, FST Inc) just
before MCAO. A vertical incision was made between the left eye and ear,
the temporalis muscle was partially excised, and a
craniotomy was performed at the site where the middle
cerebral artery (MCA) meets the rhinal fissure. The dura over the MCA
was opened and reflected to allow occlusion of the vessel with a Sundt
AVM Micro Clip No. 1 (Codman) at the point where it meets the rhinal
fissure. Anesthesia was discontinued until the time of
reperfusion. All vascular clips were removed after 3 hours of
ischemia, and reperfusion of the MCA was verified by
examination of the site of occlusion. An absorbable gelatin sponge
(Gelfoam, Upjohn) was placed in the area of excised muscle and
incisions sutured with 4-0 silk. Rats receiving intravenous
infusions (study 2) were returned to their restrainers for the
remaining 2 hours of infusion. After drug or vehicle administration,
rats were placed in plastic isolator cages and allowed free access to
food and water. Rats were killed 3 days after initiation of
ischemia, and their brains were removed and frozen in
isopentane over dry ice at -40°C and stored at -80°C.
Temperature Monitoring
Rats receiving intraperitoneal treatments
(study 1) were placed in isolators after MCAO and kept warm with a
heating pad for 5 hours. In study 2, body temperature was maintained at
37.5°C during surgery, drug infusion, and recovery (6 hours) by
individual feedback controllers and heating pads. In both studies,
after the rats were returned to their cages, core body temperatures
were monitored by telemetry with the use of a temperature-sensing radio
transmitter (Data Sciences International) previously placed in the
peritoneal cavity. The radio signal was converted to body temperature
(Dataquest IV 2.10, Data Sciences International) and recorded every
minute for 72 hours. Temperatures were averaged over 1-hour intervals
for analysis and plotted at 5-hour intervals for illustration.
Drug Delivery
In study 1, drug or vehicle was administered by 3
intraperitoneal injections given at 0.5-hour
intervals beginning 30 minutes after initiation of ischemia.
The dose of PNQX was 30 mg/kg x3 injections (total of 90 mg/kg)
(concentration, 15 mg/mL; volume, 2 mL/kg per injection). PNQX was
suspended in water, and pH was adjusted with
methanesulfonic acid. In study 2, drug or vehicle was
administered by continuous intravenous infusion (3 mL/kg
per hour) for 5 hours, beginning 15 minutes after the initiation of
ischemia. PNQX was dissolved in water with a small amount of
85% lactic acid; the pH was adjusted to 4.0 with sodium hydroxide and
filtered before administration. Rats were randomly assigned to 1 of 5
groups: PNQX at 5.0, 2.5, 1.0, or 0.1 mg/kg per hour or vehicle.
Histology
Brains were sectioned at -17°C on a cryomicrotome.
Twenty-micrometer coronal sections were collected at
600-µm intervals and stained with hematoxylin and eosin. Lesion
volume was measured with the aid of the stereological software package
GRID (Olympus, Denmark AS). A grid of points was randomly superimposed
over a video image of each tissue section, and the number of points
overlying an area of interest was counted. Total lesion volume was
estimated by the equation V= txA(p)x
P, where V is lesion volume, t
is the distance between sections analyzed (1200 µm),
A(p) is the surface area associated with 1 grid point, and P is the
total number of grid points associated with an area of interest in all
of the sections examined.
PNQX Analytical Methods
Plasma PNQX levels were determined by UV
high-performance liquid chromatography. In
brief, matrix standards, quality control samples, and unknown samples
were mixed with internal standard, diluted with 0.1 mol/L sodium
phosphate buffer (dibasic), pH 6.0, and extracted through C18
solid-phase cartridges with 1% trifluoroacetic acid in acetonitrile.
Samples were then evaporate to dryness and reconstituted with mobile
phase and injected onto a reverse-phase C3 column. The mobile phase
consisted of 90% 20 mmol/L sodium phosphate (dibasic), pH 2.75,
and 10% methanol. Detection was by ultraviolet absorption at 210 nm,
and quantitation was by peak height ratios.
Statistical Methods
Data are expressed as mean±SD. Lesion volumes were compared by
ANOVA and a Tukey-Kramer multiple comparisons test. Temperature data
from study 1 were analyzed with 1-way ANOVA to compare control
with the drug-treated animals at each time point. In study 2, ANOVA
with linear contrasts to test for a relationship between dose and
temperature was performed. P<0.05 after correction for
multiple comparisons was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Effect of PNQX on Infarct Volume
In this model of temporary focal ischemia, the histological lesion, defined by the area of pallor at 72 hours, is large and primarily limited to the cortical tissue. In study 1, rats were treated intraperitoneally with PNQX (30 mg/kg at 30, 60, and 90 minutes after MCAO), a dosing paradigm used in the original studies with NBQX. The drug-treated group had a mean lesion volume 51% smaller than vehicle-treated rats (287±110 versus 142±87 mm3; P<0.001) (Figure 1
, top). When lesion
volumes were calculated by the indirect method,27 28
a similar reduction was seen (171±72 versus 73±57
mm3; P<0.001), demonstrating that the
effect of PNQX on stroke size was not due solely to a reduction in
brain swelling. There was a modest (1°C to 1.25°C) reduction in
body temperature, which was statistically significant between 4 and 19
hours (P<0.05) after reperfusion in the PNQX-treated rats
(Figure 1, bottom).
|
) rats during the reperfusion period after 3 hours
of MCAO. Data are plotted at 5-hour intervals. Body temperature was
significantly lower in the PNQX-treated rats between 4 and 19 hours and
at 30 hours of reperfusion. *P<0.05.
Relation of PNQX Plasma Concentration to Infarct Reduction
To determine the relationship between plasma levels of PNQX and
its neuroprotective action, a 5-hour intravenous
dose-response study was performed. Rats treated with PNQX at 5, 2.5, or
1 mg/kg per hour but not 0.1 mg/kg per hour had significantly smaller
lesions than vehicle-treated controls (Figure 2, top). Figure 2 (bottom)
illustrates that there was a slight but statistically significant
reduction in body temperature (0.5°C, between 2 and 6 hours;
P<0.05) in rats receiving 5 mg/kg per hour PNQX. At the
other neuroprotective doses of PNQX (2.5 and 1 mg/kg), body
temperatures were not reduced compared with vehicle-treated rats at any
time over the entire course of the experiment. Thus, in this
reperfusion model of focal ischemia, the neuroprotective action
of PNQX is not due to hypothermia. The plasma concentration of PNQX at
the end of the 5-hour infusion was 685±534 ng/mL at the lowest
neuroprotective dose (1.0 mg/kg per hour), and values for the other
groups were roughly proportional to dose. The Table
shows the concentrations of PNQX in the
plasma sampled at the end of the 5-hour infusion, mean lesion volumes,
and percent protection at each dose.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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This study demonstrates that treatment with the AMPA receptor antagonist PNQX results in a dose-dependent reduction in cortical infarction in a rodent model of transient focal ischemia. Importantly, PNQX is neuroprotective at plasma drug concentrations that do not lower body temperature.
Previous studies with both competitive antagonists and noncompetitive allosteric modulators of the AMPA receptor have established the neuroprotective potential of blocking the action of this ionotropic glutamate receptor. The quinoxalinedione NBQX has been shown to reduce lesion size in both permanent and temporary rat focal ischemia models.16 17 18 19 More recently, a 31% reduction in infarct volume in the rat was reported after intravenous administration of the decahydroisoquinoline AMPA receptor antagonist LY-215490.20 LY-215490 is the racemate of LY-243558, which was found to be neuroprotective in an acute (6-hour) cat MCAO study.29 YM-90K, another quinoxalinedione, was similarly efficacious in cats under conditions in which physiological variables, including brain temperature, were monitored or controlled.21 The noncompetitive AMPA antagonist GYKI-52466 reduced the ischemic lesion volume by 68% and 48% when given immediately or 1 hour after MCAO.23 Unlike NMDA antagonists, AMPA antagonists also reduce selective neuronal loss in global forebrain ischemia models.13 14 30 Interestingly, in this setting NBQX is protective when administration is delayed for up to 12 hours after the ischemic period.31 However, in focal ischemia, the therapeutic time window for AMPA antagonists has not been well defined. Both NBQX16 and PNQX (data not shown) are effective when given intraperitoneally 90 minutes after initiation of MCAO. A recent report32 suggests that the window could be as long as 5 to 6 hours for this class of compounds.
The mechanisms by which AMPA antagonists provide neuroprotection in models of cerebral ischemia are not completely understood. One possibility is that reduced lesion size is secondary to drug-induced hypothermia, since reduction in body temperature in rodents can ameliorate ischemic neuronal damage in both focal33 and global34 35 models. In a study of global forebrain ischemia in the gerbil, NBQX given at 30 mg/kg (x3) caused mild (1°C to 1.5°C) hypothermia that persisted for days.26 When the postischemic temperature was controlled, neuroprotection was lost. In focal ischemia in rats, mild to moderate hypothermia (30°C to 32°C) reduces lesion volume when the body temperature is lowered during the ischemia (in permanent MCAO),33 as well as during the reperfusion phase (in temporary MCAO).36 Typically, studies that have evaluated the neuroprotective effects of AMPA receptor antagonists (see above) used body temperature monitoring and control only during the surgical procedure and drug administration, leaving open the possibility that body temperature was reduced during the remainder of the survival period. In our study, we first demonstrated that a high dose of PNQX administered into the peritoneal cavity reduced lesion volume but also lowered body temperature up to 1.25°C for an extended time. This is likely due to prolonged high-plasma concentrations of PNQX resulting from this dosing paradigm (data not shown). To address the possibility that this extended mild hypothermia might be responsible for the smaller lesions, we performed an intravenous dose-response study in which we controlled body temperature during the 5-hour drug infusion and monitored temperature by telemetry every minute for 67 hours (until death). Body temperature was slightly but significantly reduced for 2 to 6 hours after the drug infusion in rats receiving the highest PNQX dose (5 mg/kg per hour). However, body temperature in rats treated with the 2 lower efficacious doses did not differ from vehicle-treated rats at any time during the experiment. Thus, even though PNQX can cause hypothermia at high doses, the reduction in infarct volume by PNQX at 1.0 and 2.5 mg/kg per hour cannot be a result of even mild or prolonged hypothermia. Data from acute studies in cats21 29 are consistent with the conclusion that AMPA antagonists can be neuroprotective without inducing hypothermia.
AMPA receptor antagonists may protect the brain by reducing neuronal depolarization in the ischemic territory and thus limiting calcium entry into neurons. Excessive glutamate acting on the AMPA receptor depolarizes neurons, and in this depolarized state, the magnesium block of the NMDA channel is lost. Thus, calcium may enter neurons through the open NMDA channels and voltage-dependent calcium channels, as well as the subtype of AMPA-gated channels, which are permeable to calcium.11 The heteromeric AMPA receptor, which does not contain a GluR2 subunit, has increased calcium permeability. After transient forebrain ischemia, the expression of GluR2 mRNA is reduced in vulnerable brain regions,37 38 suggesting a shift to the more calcium-permeable receptors. Excessive intracellular calcium activates pathological processes that contribute to the death of the neurons.39 Additionally, evidence is accumulating that repeated waves of depolarization in the periphery of the ischemic zone (cortical spreading depressions), which occur after the onset of focal ischemia, increase metabolic demand and contribute to the subsequent loss of viable tissue.40 NBQX has been shown to reduce the number of these direct current shifts, even though, unlike NMDA antagonists, it has no effect on chemical or electrical invoked spreading depressions.41 42
In summary, AMPA receptor blockade is a therapeutic strategy that is, with few exceptions,43 robust across several models, laboratories, and chemical classes and that holds promise of a clinically relevant time window. The present study demonstrates that the novel AMPA receptor antagonist PNQX ameliorates focal ischemic brain damage at plasma concentrations that do not produce hypothermia. This finding supports the concept that selective AMPA receptor antagonists will be useful for the treatment of stroke in humans.
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Acknowledgments |
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This study was supported by the Parke-Davis/Warner-Lambert Company. We thank Jerry Paulissen for his valuable statistical assistance.
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Footnotes |
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Reprint requests to Gerald P. Schielke, PhD, Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd, Ann Arbor, MI 48105.
This study was funded by the Parke-Davis/Warner-Lambert Company.
Received November 16, 1998; revision received March 30, 1999; accepted April 15, 1999.
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Editorial Comment
Department of Neurology, University of Minnesota, Minneapolis, Minnesota
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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This study reports that the new AMPA receptor inhibitor PNQX, administered in the initial postischemic period, reduces cerebral ischemic injury in a rat model of transient focal cerebral ischemia. Importantly, the effect occurs at doses that do not induce hypothermia. The data provide additional evidence in support of the idea that AMPA receptor inhibitors are promising candidates for the treatment of human stroke.
The mechanisms of the protective effect of PNQX remain unclear. Although PNQX is likely to act by blocking AMPA receptors, the cellular mechanisms linking AMPA receptor blockade to neuroprotection have not been defined. Central to the glutamate hypothesis of cerebral ischemic damage is that activation of glutamate receptors increases intracellular calcium, which in turn initiates a series of cytoplasmic and nuclear events leading to tissue damage.1,2 Unlike NMDA receptors, however, most AMPA receptors are not permeable to calcium in the normal state. Yet the toxicity mediated through AMPA receptors seems to be calcium dependent.3,4 One possibility is that ischemia, through downregulation of the GluR2 subunit of the receptor, leads to formation of calcium-permeable AMPA receptors (eg, Reference 5). This attractive hypothesis, however, needs to be tested experimentally in this particular model.
There are a number of practical issues that still remain to be addressed. First, the "time window" after ischemia in which PNQX administration is still effective remains to be defined. In the accompanying study PNQX was administered 15 or 30 minutes after induction of ischemia, whereas most stroke patients reach emergency rooms several hours after the onset of symptoms. Therefore, it would be important to study the effect of PNQX with longer intervals between induction of ischemia and onset of treatment. Second, it remains to be determined whether PNQX is also effective in species phylogenetically closer to humans. Many treatment modalities reduce stroke volume in rodents but not in higher species.6 Therefore, demonstration of efficacy in other species would increase the likelihood that the drug will be effective in humans. Third, it remains to be determined whether effective concentrations of PNQX can be safely reached in humans. These issues will have to be addressed in future studies. Overall, the well-controlled study of Schielke et al provides new and interesting data that, due to their potential clinical applicability, are likely to generate a great deal of interest.
Received November 16, 1998; revision received March 30, 1999; accepted April 15, 1999.
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