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(Stroke. 1998;29:190-195.)
© 1998 American Heart Association, Inc.

Original Contributions

A Glycine Site Antagonist, ZD9379, Reduces Number of Spreading Depressions and Infarct Size in Rats With Permanent Middle Cerebral Artery Occlusion

Turgut Tatlisumak, MD; Kentaro Takano, MD, PhD; Michael R. Meiler, MS; Marc Fisher, MD

From the Department of Neurology, Helsinki University Central Hospital (Finland) (T.T.); Department of Neurology, Medical Center of Central Massachusetts–Memorial and the University of Massachusetts Medical School, Worcester, Mass (T.T., K.T., M.F.); and Department of Biomedical Engineering, Worcester Polytechnic Institute (Mass) (M.R.M.).

	Abstract

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Background and Purpose—Spreading depressions (SDs) occur in experimental focal ischemia and contribute to lesion evolution. N-Methyl-D-aspartate (NMDA) antagonists inhibit SDs and reduce infarct size. The glycine site on the NMDA receptor complex offers a therapeutic target for acute focal ischemia, potentially devoid of many side effects associated with competitive and noncompetitive NMDA antagonists. We evaluated the effect of the glycine antagonist ZD9379 on SDs and brain infarction.

Methods—Male Sprague-Dawley rats (n=18) weighing 290 to 340 g undergoing permanent middle cerebral artery occlusion (MCAO) were randomly and blindly assigned to receive drug or placebo: group 1 (pre-MCAO treatment group; n=5), a 5-mg/kg bolus of ZD9379 over 5 minutes followed by 5 mg/kg per hour drug infusion for 4 hours beginning 30 minutes before MCAO; group 2 (post-MCAO treatment group; n=7), a 5-mg/kg bolus of ZD9379 30 minutes after MCAO followed by 5 mg/kg per hour drug infusion for 4 hours; and group 3 (control group; n=6), vehicle for 5 hours beginning 30 minutes before MCAO. SDs were monitored electrophysiologically for 4.5 hours after MCAO by continuous recording of cortical DC potentials and electrocorticogram. Infarct volume was measured 24 hours after MCAO by 2,3,5-triphenyltetrazolium chloride staining.

Results—Corrected infarct volume was 90±72 mm³ (mean±standard deviation) in group 1, 105±46 mm³ in group 2, and 226±40 mm³ in group 3 (P<.001). The corresponding numbers of SDs in the three groups were 8.2±5.8, 8.1±2.5, and 16.0±5.1, respectively (P<.01). When all animals (n=18) were analyzed, infarct volumes and the number of SDs were significantly correlated (r=.68, P=.002).

Conclusions—This study demonstrated that ZD9379 initiated before or after MCAO significantly reduced the number of SDs and infarct volume in a permanent focal ischemia model, implying that ZD9379 is neuroprotective and its neuroprotective effect may be related to inhibiting ischemia-related SDs.

Key Words: cerebral ischemia • middle cerebral artery occlusion • N-methyl-D-aspartate • spreading cortical depression • rats

	Introduction

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Leão's SD is a generalized and stereotyped response of the cerebral cortex to a variety of noxious stimuli and is characterized by a slowly moving, transient, and reversible depression of cortical electrical activity that spreads like ripples in a pond from the site of onset usually to the whole cortex of the ipsilateral brain hemisphere with a speed of 2 to 5 mm per minute.^{1 2} Experimental SD represents a propagating disturbance of membrane activity, accompanied by marked alterations in extracellular ion concentrations and a characteristic deflection of the DC potential caused by neuronal depolarization.^{2 3} A pathological SD associated with focal ischemia was first described by Nedergaard and Astrup,⁴ and several studies to date have demonstrated the contribution of repetitive pathological SDs in the peri-infarct border zone to the expansion of ischemic brain injury.^{5 6 7 8 9 10 11 12 13} SD is an energy-consuming process, and repetitive SDs play an important role in the evolution of ischemic injury into infarction by exhausting the energy reserves of ischemic penumbra.^{13 14} There is a positive, significant relationship between the number of SDs and the increasing size of experimental ischemic lesion volume after focal brain ischemia.^{6 7 8 10 12 13 15}

A role for the excitatory amino acid glutamate in the pathogenesis of focal and global ischemic injury is well established. Several therapeutic strategies both in vivo and in vitro inhibiting the NMDA receptor complex support this hypothesis.¹⁶ However, most competitive and noncompetitive NMDA antagonists have been limited by psychotomimetic side effects. Glycine is a coagonist of the NMDA receptor channel complex. The glycine site on the NMDA receptor complex may offer a therapeutic target for acute focal ischemia, potentially devoid of many side effects associated with competitive and noncompetitive NMDA antagonists.¹⁷ Recent studies concluded that partial or full glycine antagonists when given before or immediately after brain ischemia are neuroprotective in permanent and temporary focal ischemia, leading to a 34% to 60% reduction of infarct volume.^{18 19 20} The magnitude of the protective effect on infarct volume with the use of glycine antagonists is similar to that provided by competitive or noncompetitive NMDA antagonists. ZD9379 is a soluble, potent, bioavailable full antagonist at the glycine site.²¹ We previously examined the effect of ZD9379 on experimental focal ischemia and found that the drug reduced infarct volume in rats up to 51.4% when started 30 minutes after induction of MCAO.²² In the present study we extended our observations to the effect of this drug on SDs, since glutamate is a key molecule in the elicitation and propagation of SD^{5 23} and NMDA receptor antagonism was previously found effective in reducing SDs and infarct volume in experimental focal ischemia.^{5 6 7}

	Materials and Methods

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Animal Preparation
All experiments and surgical procedures were approved by the Animal Research Committee (ARC) of the University of Massachusetts Medical School (ARC protocol A-773). Eighteen male Sprague-Dawley rats weighing 290 to 340 g were used. Animals were housed under diurnal lighting conditions and allowed free access to food and water before and after the experiment. Anesthesia was induced by the intraperitoneal injection of chloral hydrate (400 mg/kg body wt) and repeated in 100-mg/kg doses, as required throughout the surgery. PE-50 polyethylene tubing was inserted into the left femoral artery for continuous monitoring of blood pressure (HP78304A blood pressure monitor, Hewlett Packard) throughout the study and for measuring arterial pH, PaO₂, and PaCO₂ (Corning 170-pH blood gas analyzer) at baseline and 60 and 180 minutes after the induction of focal ischemia. Another PE-50 catheter was inserted into the left femoral vein for drug infusions. Rectal (core) temperature was continuously monitored with a rectal probe inserted to a 4-cm depth from the anal ring and maintained at 37°C during the whole experiment with a thermostatically controlled heating lamp (model 73ATD, YSI Inc). Mean arterial blood pressure and body temperature were recorded every 30 minutes throughout the experiment.

Preparation for Measurement of SD
To record the ECoG and DC potential from the right frontoparietal cortex simultaneously, the frontoparietal cranium was exposed by a midsagittal incision, and two small burr holes of 1.5-mm diameter were made in the right frontoparietal cortex, 1 mm and 3 mm posterior, respectively, and 1.5 mm lateral of the bregma, a brain region outside the area of focal infarction but close to the penumbral zone in this stroke model.^{24 25 26} Care was taken to keep the dura intact. Two Ag/AgCl electrodes (E205A In Vivo Metric), 1 mm in diameter, were attached to a Delrin plate, 8 mm² by 1 mm thick, with the use of a "5-minute" two-part epoxy, leaving approximately 1 mm of electrode exposed beneath the plate. Before surgery, the electrodes were electrically shortened for 4 hours in saline to allow ionic equilibration. A small amount of electrogel (Nihon Kohden) was placed in each burr hole to provide electrical continuity, and the assembly was then fixed to the exposed cranium with a ceramic-based dental cement (DURELON, Espe Fabrik Pharmazeutischer Präparate Gmbh and Co KG). Two Ag/AgCl disk electrodes were attached to the subcutaneous tissue in the neck as a reference and ground. After the apparatus was in place, the incision was closed, and electrophysiological monitoring was started approximately 30 minutes before induction of focal ischemia.

Two homemade amplifiers with an adjustable gain were constructed on a ground-plane circuit board. The DC amplifier consisted of an instrumentation amplifier followed by a summing amplifier to correct for an offset potential at the electrodes and a second-order low-pass Butterworth filter with a corner frequency of 10 Hz to eliminate higher-frequency noise and power line interference. The ECoG amplifier consisted of an instrumentation amplifier in series with a second-order low-pass Butterworth filter having a corner frequency of 230 Hz followed by a summing amplifier to correct for offset potential and a 60-Hz notch filter to reduce power line interference. Both amplifiers had a constant gain over the measured frequency range.

Two biopotentials were monitored and recorded on a chart recorder (Recorder 2400S, Gould Inc); the potential between the two electrodes on the cranium provided a differential measurement of DC potential, and the potential between the implanted electrode and the subcutaneous reference electrode at the neck provided a measurement of ECoG. The DC amplifier stage was adjusted to provide a passband from 0 to 10 Hz and with a total gain of 175, while the ECoG amplifier stage was adjusted to have a passband from 0 to 230 Hz and a total gain of 615.

Focal Cerebral Ischemia
After we fixed the apparatus for electrophysiological measurements, the animal was placed in the supine position. Focal cerebral ischemia was induced by the suture occlusion of the middle cerebral artery model, as described previously.²⁴ Briefly, the right CCA and the right external carotid artery were exposed through a ventral midline neck incision. The proximal CCA and the origin of the external carotid artery were ligated. A 4–0 nylon monofilament suture (Ethilon nylon suture, ETHICON Inc) with its tip rounded by heating near a flame and then coated with silicone (Bayer Inc) was inserted into the right CCA through an arteriectomy of the right CCA approximately 3 mm below the right carotid bifurcation and advanced into the internal carotid artery approximately 17 mm from the carotid bifurcation. At that point, a slight resistance was felt, indicating that the occluder lodged into the anterior cerebral artery, thus occluding the orifice of the middle cerebral artery, the anterior cerebral artery, and the posterior cerebral communicating artery. After the induction of focal ischemia, anesthesia was maintained by 1% of isoflurane delivered in air at 1.0 L/min.

Drug Characteristics, Preparation, and Application
ZD9379, 7-chloro-4-hydroxy-2-(4-methoxy-2-methylphenyl)-1,2,5,10-tetrahydropyridanizo[4,5-b]quinoline-1,10-dione, sodium salt, is a 2-aryl substituted derivative of a series of pyridazinoquinolinediones²¹ that are potent selective antagonists at the glycine site of the NMDA receptor. ZD9379 displaces [³H]glycine binding to rat brain synaptic plasma membranes with an IC₅₀ of 75±42 mol/L and antagonizes binding of [³H] 1-[1-(2-thienyl)cyclohexyl]piperidine to the NMDA receptor in a glycine surmountable manner. In contrast to many previous glycine/NMDA antagonists with little or no in vivo activity presumably due to poor brain penetration,²⁷ ZD9379 penetrates into the brain as illustrated by the relatively rapid complete inhibition of NMDA-induced firing of rat red nucleus neurons.²²

The ZD9379 was dissolved in distilled water. The solution was then made isotonic by successive additions of concentrated solutions of dextrose and sodium chloride. One half of the isotonicity was provided by dextrose, and the other half was provided by sodium chloride. The pH of the resulting solution was then measured with a pH meter (LOG R Compensator model 611, Orion Research Inc) and adjusted to a pH of 9.0 with 0.1N hydrochloric acid. The concentration of ZD9379 in the isotonic solution was adjusted as 5 mg/mL.

The experiment was completely conducted in a blinded and randomized fashion with a complicated pattern of drug assignment: group 1 received a 5-mg/kg bolus of ZD9379 over 5 minutes, 30 minutes before MCAO followed by 5 mg/kg per hour continuous drug infusion for 1 hour. Thirty minutes after MCAO, the rats received a bolus of vehicle in the same volume, and drug infusion continued for 3 more hours followed by a 1-hour vehicle infusion. Group 2 received a bolus of vehicle and a 1-hour vehicle infusion, beginning 30 minutes before MCAO and ending 30 minutes after MCAO. At that point, these animals received a 5-mg/kg bolus of ZD9379 over 5 minutes followed by a continuous infusion of drug for 3 hours and another hour of drug infusion from a different syringe. Group 3 received a bolus of vehicle 30 minutes before MCAO, 1-hour infusion of vehicle, another bolus of vehicle 30 minutes after MCAO, 3 hours of vehicle infusion, and 1 more hour of vehicle infusion.

Calculation of Infarct Volume
After removal of the femoral catheters and electrodes and closure of the wounds, the animals were allowed to recover from the anesthesia in separate cages. Twenty-four hours after MCAO, the animals were examined neurologically with the use of a 6-point scale (0=no deficit, 1=failure to extend left forepaw fully, 2=circling to the left, 3=falling to the left, 4=no spontaneous walking with a depressed level of consciousness, 5=dead) modified from that previously described by Zea Longa et al.²⁸ The animals were then reanesthetized with chloral hydrate and killed. The brains were quickly removed, inspected for appropriate placement of the intraluminal occluder, and sectioned coronally into six slices each with a 2-mm thickness. The brain slices were incubated for 30 minutes in a 2% solution of TTC at 37°C and fixed by immersion in a 10% buffered formalin solution.²⁹ The unstained area was defined as infarcted tissue. All six brain slices per animal were photographed with a charge-coupled device camera (EDC-1000HR Computer Camera, ELECTRIM Corp).The areas of the infarcted tissue and the areas of both hemispheres were calculated for each coronal slice with an image analysis program (Bio Scan OPTIMAS). The corrected infarct volume was calculated to compensate for the effect of cerebral edema. Corrected infarct area in a slice was calculated by subtracting the area of normal tissue in the ipsilateral hemisphere from the total area of the contralateral hemisphere. Total corrected infarct volume was then calculated by multiplying the area by the slice thickness and summing the volumes from all slices.

Statistical Analyses
Values are presented as mean±standard deviation. One-factor ANOVA and subsequent post hoc Scheffé's test were used for continuous variables. Repeated-measures ANOVA was applied for serial changes in physiological variables. The Kruskal-Wallis H test followed by the Mann-Whitney U test was applied for neurological scores. Linear regression analysis was used to correlate the number of SDs, infarct volumes, and neurological scores. A two-tailed value of P<.05 was considered significant.

	Results

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There were no significant differences in body weight, body temperature, mean arterial blood pressure, or arterial blood gases among the three groups. These data are summarized in Table 1. We did not observe any side effects during the experiment. No abnormal behavior was observed after the animals recovered from anesthesia. Two animals in the control group and 1 animal from each treated group died prematurely (11, 12, 12, and 19 hours after MCAO). These animals were assigned a score of 5 on the neurological assessment scale and underwent immediate craniectomy and TTC staining for determination of infarct volume. At postmortem, all animals showed appropriate positioning of the inserted occluder without subarachnoid hemorrhage.

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters

As shown in Table 2, neurological scores were significantly better in group 2 and better, but not significantly different, in group 1 than in the control group (P=.022 by Kruskal-Wallis H test; P=.053 for control versus group 1 and P=.02 control versus group 2 by Mann-Whitney U test). Both treated groups showed significantly smaller corrected infarct volumes than controls (60% and 53% reduction in infarct volumes in groups 1 and 2, respectively; P<.001 by ANOVA; P<.05 for control versus group 1 and P<.0002 for control versus group 2 by post hoc Scheffé's test), and the number of SDs was also significantly smaller in the treated groups (P<.01 by ANOVA; P<.05 control versus group 1 and P<.005 control versus group 2 by post hoc Scheffé's test). When the animals that died prematurely were excluded from the statistical analyses, the significance in favor of the treated groups in the neurological scores (P<.005 by Kruskal-Wallis H test; P=.02 control versus group 1 and P<.01 control versus group 2 by Mann-Whitney U test), in infarct volumes (P<.005 by ANOVA; P<.01 control versus group 1 and P<.005 control versus group 2 by post hoc Scheffé's test), and in the number of SDs (P=.001 by ANOVA; P=.001 control versus group 1 and P=.001 control versus group 2 by post hoc Scheffé's test) persisted.

View this table: [in this window] [in a new window]   Table 2. Major Results of the Study

No SDs were seen before the induction of focal ischemia. The time lag from MCAO to the initial SD was 11.8±10.3 minutes, with a range of 3 to 36 minutes (n=18). When each group was analyzed separately, time lags were as follows: in group 1, 19.4±13.5 minutes (range, 7 to 36 minutes); in group 2, 11.3±9.3 minutes (range, 3 to 25 minutes); and in group 3, 6.0±2.0 minutes (range, 3 to 12 minutes). The difference between the groups was not significant (P=.092 by one-factor ANOVA).

The number of SDs varied between 3 and 23 per animal. The mean was 10.8±5.5. When all 18 animals were pooled, infarct volume and number of SDs showed a significant, positive correlation (r=.68, P=.002); larger infarcts were seen in animals with more SDs. Similarly, infarct volume and neurological score were well correlated (r=.59, P=.01), indicating that larger infarcts caused worse neurological scores. Number of SDs also correlated with neurological score (r=.7, P=.001). When the animals dying prematurely (n=4) were excluded, the correlation between infarct volume and number of SDs (r=.76, P<.002), the correlation between infarct volume and neurological score (r=.6, P=.02), and the correlation between number of SDs and neurological score (r=.64, P<.02) persisted.

The duration of a single SD varied between 1 to 3 minutes; most lasted close to 2 minutes. The amplitude, shape, and the duration of SDs in each animal were almost identical (Figure, panel A ). In one animal, we observed a series of 15 SDs in 65 minutes (Figure, panel B). SDs usually came in an irregular pattern. Occasionally, we observed SDs of different shape in the same animal (Figure, panel C).

View larger version (8K): [in this window] [in a new window]   Figure 1. DC recordings from three different animals. A, The amplitude, shape, and duration of SDs were mostly identical in each animal over time. B, A series of 15 SDs in 65 minutes in a control animal. C, Occasionally, SDs changed in amplitude, shape, and duration in the same animal.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
This study demonstrates that the glycine site antagonist ZD9379 led to a substantial decrease in infarct size and in the number of SDs and improved the neurological outcome in rats with permanent MCAO without causing any observable adverse effects or any significant changes in physiological parameters. The frequency, amplitude, and duration of SDs and the time lag between MCAO and the initial SD observed in this study were in concordance with several previous reports.^{4 6 7 8 11 12 13 15} The premature mortality rate in this study may have been related to the dual invasive surgery, prolonged anesthesia (8 to 9 hours), brain edema accompanying this stroke model, or other undetermined causes. The premature deaths were evenly distributed to all groups (two in the control group and one in each treated group), suggesting no relationship to the drug. All four animals that died prematurely had well-demarcated infarcts compatible in size and shape with those seen at 24 hours after focal ischemia. Reliability of TTC staining at 6 hours after focal ischemia has been demonstrated previously.³⁰ We analyzed our results with and without the animals that died prematurely (Table 2). However, inclusion or exclusion of those animals did not affect the study results.

Potassium ions and glutamate have been suggested to play a pivotal role in SD.^{31 32} After ischemia, the extracellular glutamate and potassium concentrations increase, activating the glutamate receptors and the voltage-dependent ion channels, respectively. The subsequent plasma membrane depolarization releases the voltage-dependent magnesium block of the NMDA receptor,³³ dramatically increasing the ion conductance of the plasma membrane, thereby initiating the SD.³⁴ Pharmacological antagonism of glutamate at the NMDA receptor has been shown to inhibit SD.^{6 7 34} The NMDA receptor has a binding site for glycine, in addition to a binding site for glutamate.¹⁶ For the NMDA ionophore to become permeable to calcium, a recognition site for glycine must be occupied simultaneously with the glutamate binding at the NMDA receptor.^{35 36} The mechanism whereby NMDA receptor antagonists prevent the elicitation of SD most probably resides in their prevention of glutamate binding to the receptor protein or to the blockade of the receptor-coupled ionophore.³⁴ Hypothermia can reduce or even completely inhibit the release of glutamate³⁷ and can abolish SD.⁸ We have previously shown that ZD9379 did not affect brain temperature during focal ischemia in rats.²² These data suggest that the glycine antagonist might inhibit SD by a mechanism similar to that of NMDA antagonists.

This study is the first to report inhibition of SDs and attenuation of infarct size simultaneously with a glycine site antagonist. In a previous study, Martin et al³⁸ reported that a glycine receptor antagonist, ACEA-1201, did not inhibit elicitation of SDs but reduced the SD propagation rate in a dose-dependent fashion. In their study, however, they used electrocortical stimulation in rats under general anesthesia and without ischemia.³⁸ The design of their study was much different than the present study.

The correlation between the number of the SDs and infarct volume may not reflect a causal relationship. The present study cannot answer the question of whether the inhibition of SDs led to neuroprotection or the glycine antagonist ZD9379 is itself neuroprotective by other mechanisms independent of inhibition of SDs and SD inhibition is only a epiphenomenon. However, other data strongly suggest that SDs are responsible for expansion of infarct volume in experimental ischemia.^{12 13} Back et al¹² and Takano et al¹³ induced SDs repetitively by KCl application onto the cerebral cortex in rats with induced focal ischemia. Back et al, with a postmortem analysis, and Takano et al, using both in vivo diffusion-weighted MRI and postmortem assessment, demonstrated a significant increase in ischemic lesion size associated with the occurrence of SD. Thus we conclude that glycine site inhibition in rats with focal brain ischemia led to neuroprotection, and this effect may be, at least in part, due to inhibition of SD elicitation.

	Selected Abbreviations and Acronyms

 

CCA = common carotid artery EcoG = electrocorticogram MCAO = middle cerebral artery occlusion NMDA = N-methyl-D-aspartate SD = spreading depression TTC = 2,3,5-triphenyltetrazolium chloride

	Footnotes

 
Reprint requests to Dr Turgut Tatlisumak, Department of Neurology, Helsinki University Central Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland.

Received May 28, 1997; revision received August 6, 1997; accepted September 29, 1997.

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Editorial Comment

W. Dalton Dietrich, PhD, Guest Editor

Department of Neurology, University of Miami School of Medicine, Miami, Florida

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Repetitive episodes of cortical spreading depression have been implicated in the pathophysiology of penumbral vulnerability following focal cerebral ischemia. This study evaluated the effect of the glycine antagonist ZD9379 on spreading depressions monitored by the recording of DC potentials and infarct volume 24 hours after permanent MCAO. Treated groups showed significantly smaller infarct volumes and decreased numbers of spreading depressions. The authors have previously reported that ZD9379 reduces infarct volume when treatment is started 30 minutes after the induction of MCAO. However, the present study asks the important question of whether protection is associated with decreased numbers of spreading depression. Thus, the present study provides a potential mechanism for this neuroprotective effect.

Recent studies have begun to use more chronic survival strategies to assess potentially neuroprotective agents. Several investigations have reported that certain procedures, including pharmacotherapy and brain cooling, confer acute but not chronic protection. It would therefore be important in future studies to determine whether chronic histopathologic and behavioral improvement can be demonstrated by use of this therapeutic strategy. Because reperfusion of ischemic regions is possible with thrombolytic therapy, it would be interesting to assess this novel agent in models of transient focal ischemia. A final point is whether more delayed treatment with this agent would also improve ischemic outcome. Obviously, the success of this agent in terms of clinical stroke will depend on whether delayed treatment can improve histopathologic and functional outcome months after the initial ischemic insult.

	Selected Abbreviations and Acronyms

 

CCA = common carotid artery EcoG = electrocorticogram MCAO = middle cerebral artery occlusion NMDA = N-methyl-D-aspartate SD = spreading depression TTC = 2,3,5-triphenyltetrazolium chloride

Received May 28, 1997; revision received August 6, 1997; accepted September 29, 1997.

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