(Stroke. 1997;28:1255-1263.)
© 1997 American Heart Association, Inc.
Articles |
Glycine Site Antagonist Attenuates Infarct Size in Experimental Focal Ischemia
Postmortem and Diffusion Mapping Studies
From the Department of Neurology, The Medical Center of Central Massachusetts and University of Massachusetts Medical School (K.T., T.T., A.G.B., M.F.), and the Department of Biomedical Engineering, Worcester Polytechnic Institute (J.E.F., R.A.D.C., C.H.S.), Worcester, Mass; Zeneca Pharmaceuticals, Wilmington, Del (L.M.P., T.M.B.); and the Department of Neurology, Helsinki University Central Hospital, Finland (T.T.).
Correspondence to Marc Fisher, MD, Memorial Health Care and University of Massachusetts Medical School, 119 Belmont St, Worcester, MA 01605-2982.
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Abstract |
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Background and Purpose The glycine site on the N-methyl-D-aspartate (NMDA) receptor complex offers a therapeutic target for acute focal ischemia, potentially devoid of most side effects associated with competitive and noncompetitive NMDA antagonists.
Methods A novel glycine receptor antagonist, ZD9379, was studied in 70 Sprague-Dawley rats using the suture occlusion model of permanent middle cerebral artery occlusion (MCAO). In the first experiment, 20 rats received an initial bolus of vehicle or 10 mg/kg ZD9379 (n=10 in each group) 30 minutes after MCAO, followed by a continuous infusion of the same dose per hour for 4 hours. Diffusion-weighted MRI with echo-planar acquisition was used to generate maps of the apparent diffusion coefficient (ADC) of water. In a second experiment, 50 rats were assigned to five groups: vehicle and 10, 5, 2.5, and 1 mg/kg ZD9379 (n=10 in each group) with the same dosing protocol but no imaging. In both experiments, infarct volume was determined by 2,3,5-triphenyltetrazolium chloride staining.
Results In the first experiment, before therapy was begun, there was no significant difference in ADC-derived ischemic lesion volume between the two groups. Over time, the 10-mg/kg ZD9379–treated rats had a significant delayed regional recovery of reduced ADC values in the peripheral parietal cortex (P=.0156). Postmortem corrected infarct volume at 24 hours after MCAO was significantly smaller in the group treated with 10 mg/kg ZD9379 than in the vehicle group (119.2±52.2 versus 211.2±50.0 mm3 [mean±SD]; P=.0008; a reduction of 43.6%). In the second experiment, postmortem corrected infarct volumes in rats receiving 10, 5, and 2.5 mg/kg ZD9379 were significantly smaller than in those receiving vehicle, a reduction of 42.6%, 51.4%, and 42.9%, respectively (P=.0001).
Conclusions This study demonstrates that 2.5- to 10-mg/kg doses of ZD9379 initiated 30 minutes after MCAO significantly reduced infarct size. Diffusion mapping disclosed a delayed treatment effect of this glycine antagonist in focal ischemia, confirmed by the postmortem study.
Key Words: ischemia • magnetic resonance imaging • middle cerebral artery occlusion • rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.1 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 most side effects associated with competitive and noncompetitive NMDA antagonists.2 Recent investigations using the partial agonist L-687,414 or full antagonists, such as 7-chlorokynurenic acid and its derivatives and ACEA-1021, ACEA-1031, have demonstrated efficacy in experimental focal ischemia models.3 4 5 ZD9379 is a soluble, potent, bioavailable full antagonist at the glycine site that was used in the present studies.6
Recent advances in DWI technology allow investigators to observe ischemic lesions in vivo during very early stages of ischemic stroke, not only in experimental models but also in humans.7 8 9 10 11 12 13 Measurement of the ADC of water is sensitive to changes in cellular structure, ie, shrinkage of the extracellular space due to intracellular water accumulation, based on Brownian diffusion of water in tissues.7 8 9 14 The failure of energy-dependent cell membrane pumps during ischemia causes intracellular sodium and water accumulation (cytotoxic edema) that presumably reduces the absolute ADC value in the ischemic tissue, resulting in hyperintensity on DWI or hypointensity on ADC mapping.7 8 15 DWI also allows for early in vivo estimation of therapeutic efficacy with neuroprotective drugs.16 17 Multislice ADC mapping with echo-planar acquisition allows regional and spatial analyses in the brain to further examine therapeutic intervention during acute ischemia in experimental stroke models.18 The aim of the present study was to investigate the effect of a novel glycine antagonist of the NMDA receptor-channel complex on experimental focal ischemia evaluated by ADC mapping in vivo and at postmortem.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal Preparation and Focal Brain Ischemia
The present study was approved by the Animal Research Committee of the University of Massachusetts Medical School (protocol A-643). Seventy male Sprague-Dawley rats weighing 300 to 370 g were used in this study. Anesthesia was induced by intraperitoneal injection of chloral hydrate (400 mg/kg body wt). PE-50 polyethylene tubing was cannulated into the left femoral artery for continuous monitoring of MABP (78905A, Hewlett-Packard) and measurement of pH, PaCO2, and PaO2 (Corning 170-pH Blood Gas Analyzer) and into the left femoral vein for infusion of ZD9379 or vehicle. Rectal (core) temperature was continuously monitored with a rectal probe (inserted to a 4-cm depth from the anal ring) and maintained at 37.0°C with a thermostatically controlled heating lamp (model 73ATD, YSI Inc) positioned 30 to 35 cm from the head; the room temperature was controlled at 25°C to 27°C throughout the study.
Focal brain ischemia was induced by the intraluminal-suture MCAO method, modified from the original method of Koizumi et al.19 20 Briefly, the common carotid artery and the carotid bifurcation were exposed through a ventral midline incision in the neck. Thirty-five mm of a 4-0 monofilament nylon suture, whose tip had been rounded by flame heating and coated with silicon (Provil L, Bayer Dental), was used as the intraluminal occluder. After ligation of the proximal portion of the right common carotid and the origin of the external carotid arteries with a 3-0 silk suture, the intraluminal occluder was introduced through an arteriectomy of the right common carotid artery. The occluder was carefully advanced intracranially, approximately 17 mm from the carotid bifurcation.20
MRI Measurements
Pulsed field gradient nuclear MR was used to noninvasively
measure brain water diffusion rates in each rat on a pixel-by-pixel
basis. In tissue, an ADC14 21 is defined as
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is the observation time, and
M0 is the equilibrium magnetization at k=0. MRI
experiments were conducted using a General Electric CSI-II 2.0-T/45-cm
imaging spectrometer (General Electric Medical System) operating at
85.56 MHz for 1H equipped with self-shielding gradient
coils (15-cm bore) and capable of delivering a maximum gradient
strength of ±20 G·cm–1. An eight-slice, multislice
diffusion-weighted echo-planar imaging pulse sequence was used to
generate ADC maps with an echo time of 92 milliseconds, and
half-sine–shaped diffusion gradients were used along the z
axis (anterior-posterior) of the brain.15 Images were
64x64 pixels with an in-plane resolution of 400 µm, a 1.5-mm
slice thickness in the axial plane, and a 25.6x25.6-mm field of view.
The echo-planar data acquisition time of 65 milliseconds with two
signal averages per image was obtained. Eight contiguous slices were
acquired using an interleaved slice-acquisition pattern to avoid signal
contamination from adjacent slices. Ten b-values (k2)
ranging from 63 to 1898 s/mm2 were applied in generating
eight-slice ADC maps. The ADC maps had a repetition time of 4 seconds.
Data were transferred from the nuclear MR spectrometer to a workstation
(100 MHz Iris Indigo R4000, Silicon Graphics Inc) for data processing.
A linear regression was performed on a pixel-by-pixel basis using the
above equation to calculate the ADC value in each pixel.
The threshold value to define abnormal lesion volume on ADC maps was
evaluated as follows: to define abnormal diffusion values of water in
the brain, we compared each pixel in the ischemic hemisphere
with its homologous pixel in the normal hemisphere. Ischemic
lesion volume determined by an interhemispheric difference in ADC
values of 
29% was previously shown to highly correlate with
postmortem infarct size.20 Therefore, in this study, a
ADC value of –29% was used to define abnormal ischemic
pixels. Different threshold values of ADC were also examined to
observe the effects of treatment on various degrees of the
ischemic insult.
ROI on ADC Mapping
A quantitative ROI data analysis was performed on the
serial ADC maps obtained from the slice at the optic chiasm. Three ROIs
of 2x2 pixels were respectively examined bilaterally in the
peripheral parietal cortex close to the hind limb and
forelimb area, the central parietal cortex close to insular cortex, and
the caudoputamen and expressed as the mean absolute ADC
value for the four pixels. These ROIs in the right hemisphere were used
to estimate regional therapeutic effects of the glycine site
antagonist ZD9379 and in the left nonischemic
hemisphere for noninvasive evaluation of the effects of ZD9379 on
regional brain temperature.22
Calculation of the Infarct Volume
At 270 minutes after MCAO, the arterial and venous
catheters were removed, the infusion was discontinued, and the animals
were allowed to recover from the anesthesia and to eat and
drink freely; 24 hours later, neurological function was evaluated using
a six-point scale: 0, no neurological 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; and 5,
death.16 The rats were then killed, and the brains were
quickly removed, inspected to confirm appropriate placement of the
intraluminal occluder, and sectioned coronally into six slices each
with a 2-mm thickness. The six brain slices were stained with TTC, and
the areas of the uncorrected infarcted area and the total areas of both
hemispheres were calculated for each coronal
slice.18 20 22 The uncorrected infarct volume in the
cortex and caudoputamen was calculated by multiplying the
area by the slice thickness and summing the volumes. A corrected
infarct volume was calculated to compensate for the effect of brain
edema and then related to the ADC-derived lesion
volume.18 23 24 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.
Drug Characteristics and Preparation
ZD9379 (Fig 1
) is a 2-aryl substituted derivative
of a series of pyridazinoquinolinediones6 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,25 ZD9379 penetrates into
the brain as illustrated by the relatively rapid complete inhibition of
NMDA-induced firing of rat red nucleus neurons (L.M.P., T.M.B.,
unpublished data, 1996).
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The ZD9379 was dissolved in distilled water. The solution was then made isotonic by the successive additions of required amounts of concentrated solutions of dextrose and sodium chloride. One half of the isotonicity was provided by dextrose, while the other half was provided by the 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 0.1 N hydrochloric acid. The concentration of ZD9379 in the isotonic solution was adjusted by dilution with isotonic dextrose/saline, resulting in a final volume of 0.3 mL for dosing regimens between 1.0 and 10 mg/kg ZD9379 or vehicle. ZD9379 or vehicle was given as a 0.3-mL loading bolus (for 3 minutes) and subsequently as 0.3 mL per hour by continuous intravenous infusion (for 4 hours).
Study Protocol
Experiment A
Immediately after completion of MCAO, the head of the animal was
fixed inside a 1H "birdcage" imaging coil, and
anesthesia with 1% isoflurane (delivered in air at 1.0
L/min) was initiated. Body temperature was continuously monitored using
a rectal probe with 0.1°C resolution (T-type thermocouple, Omega
Engineering Inc) and was maintained at 37.0°C by means of a
thermostatically regulated heated air flow system. Rats were randomly
and blindly assigned to receive vehicle (control group) or a loading
dose of 10 mg/kg ZD9379 (treated group). The ADC mapping was performed
30 minutes after MCAO, just before the commencement of ZD9379 or
vehicle administration, and at 60, 120, 180, and 210 minutes after
MCAO.
Experiment B
To further examine dose-dependent effects of ZD9379, 50 animals
were randomly and blindly assigned into five groups that received
vehicle (control group) or ZD9379 10 mg/kg loading dose, 5 mg/kg, 2.5
mg/kg, and 1 mg/kg (n=10 in each group). The protocol for treatment was
the same as experiment A. During the infusion, rats were
anesthetized with three additional
intraperitoneal injections of chloral hydrate (100
mg/kg body wt) beginning 1.5 hour after the initiation of
anesthesia.
Statistical Analyses
Values are presented as mean±SD. The statistical
analyses were performed with the Student's t test
for unpaired continuous variables and Mann-Whitney U
test for nonparametric variables in experiment A.
One-factor ANOVA and subsequent post hoc Scheffé's test were
used for continuous variables in experiment B. Repeated measures
ANOVA was applied for serial changes in
physiological variables, ADC-derived
ischemic lesion volume, and regional absolute ADC values in
experiments A and B. The Kruskal-Wallis H test was applied
for neurological examination in experiment B. Linear regression
analysis was used to correlate the abnormal lesion volume
calculated by ADC value and the infarct volume measured by TTC
staining. A two-tailed value of P<.05 was considered
significant.
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Results |
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Experiment A
Physiological Parameters
Physiological parameters from experiments A and B are summarized in Tables 1
and 2. In experiment A, there were no significant
differences in body weight or core temperature between the control and
treated groups (data not shown). Other
physiological parameters from
experiment A are shown in Table 1. The arterial pH and
PaO2 at baseline and 60 and 210 minutes after
MCAO did not differ between the two groups. In the 10-mg/kg ZD9379
group, the average PaCO2 value increased at 60
and 210 minutes after MCAO (during ZD9379 administration) in comparison
with the baseline, although the change in PaCO2
was not statistically significant. A transient decline of MABP (20 to
30 mm Hg) after the initial bolus infusion that recovered to
baseline levels within 5 minutes occurred in 4 of 10 animals receiving
10 mg/kg ZD9379 but not in any rat with vehicle. No significant changes
were noted over the course of the MRI experiment between the two
groups, although there was a trend toward a lower MABP in the treated
group between 150 and 270 minutes after MCAO.
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Neurological Examination
In the control group, one rat died 20 hours after MCAO under
observation. In the 10-mg/kg ZD9379 group, one rat died within 12 hours
after MCAO, and three rats died between 12 and 24 hours after MCAO.
These animals that died prematurely were immediately subjected to TTC
staining. Although there was no significant difference in the
neurological grading scale between the control and treated groups
(3.8±1.0 and 3.3±0.7, respectively; P=.2579), the
premature mortality rate in the treated group (40%) was
insignificantly higher than in the control group (10%). The recovery
interval after anesthesia to full consciousness was delayed
for 2 hours in all treated animals but not in any control rat. No
abnormal behavior was observed in any animal after recovery from
anesthesia.
TTC-Derived Infarct Lesion Volume
At postmortem, all animals showed appropriate positioning of
the inserted suture in the MCA. Subarachnoid hemorrhage
was absent in all rats. ZD9379 significantly reduced corrected infarct
volume compared with vehicle: 119.2±52.2 versus 211.2±50.0
mm3 (P=.0008), a reduction of 43.6% (Table 3). Corrected infarct volume excluding the five animals
that died prematurely was also significantly smaller in the treated
group (122.1±48.0 mm3, n=6) than the control group
(211.1±53.0 mm3, n=9; P=.0057).
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The uncorrected infarct volume was significantly reduced in the treated group (157.2±62.2 mm3) versus the control group (275.6±56.4 mm3; P=.0003). Cortical infarct volume was significantly attenuated in the treated group (97.2±46.1 mm3) compared with in the control group (191.3±62.6 mm3; P=.0012). There was, however, no significant difference in the infarct volume in the caudoputamen between the control group (39.2±7.6 mm3) and the treated group (33.6±6.9 mm3; P=.1021).
Changes in Ischemic Lesion Volume by ADC Value
The results of the ADC mapping in experiment A are
presented in Fig 2. The initial ADC mapping
using a –29% ADC threshold value to define ischemia
(comparing ADC values in homologous pixels in the ischemic and
normal hemispheres throughout the brain), 30 minutes after MCAO and
before the commencement of treatment, showed no significant difference
in the ADC-derived ischemic lesion volume between the control
and treated groups (122.3±36.1 and 105.2±62.4 mm3,
respectively). The ADC-derived ischemic lesion volume then
increased similarly over time in each group, as shown in Fig 2. There
was no significant difference in the serial changes on the ADC maps
between the two groups during the 3.5-hour MRI protocol, although the
ADC-derived ischemic lesion volume at the final imaging time
point, 3.5 hours after MCAO, was insignificantly smaller in the
ZD9379-treated group (199.9±79.7 mm3) than in the
control group (265.2±55.4 mm3;
P=.0777).
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) and 10
mg/kg ZD9379 (
). There is no significant difference in the
ADC-derived ischemic lesion volume at 25 minutes after MCAO,
just before the treatment began. Although the difference in the serial
changes of the ADC-derived ischemic lesion volume between both
groups is not significant, there is a trend toward decreasing
ADC-derived ischemic lesion volume in the ZD9379-treated group
at 210 minutes after MCAO (180 minutes after the initiation of
infusion) (P=.0777).
The ADC-derived lesion volume defined by the ADC threshold value of
–29% at 210 minutes after MCAO was significantly related to
postmortem TTC-derived infarct volume in the control group
(r=.72, P=.0461) but not in the ZD9379-treated
group (r=.58, P=.1317), confirming that a ADC
of –29% at 210 minutes after MCAO is a reliable predictive threshold
to differentiate normal tissue from ischemic brain tissue when
compared with the postmortem infarct volume in untreated animals and
that the development of the ischemic lesion between 3.5 and 24
hours after MCAO was different in the two groups.
Brain Temperature Assessed by ADC Value
Serial changes in the average values of absolute ADC values
obtained in the peripheral parietal cortex, the central
parietal cortex, and the caudoputamen in the
nonischemic hemisphere were not significantly different between
the two groups during the MRI protocol (data not shown), implying that
brain temperature was not affected by ZD9379 administration.
ROI on ADC Mapping
Comparable ROIs on the ADC maps between the two groups are shown
in Fig 3A through 3C. Just before commencement of
treatment, there were no significant differences between the two groups
in the absolute ADC values in any ROI. In the ischemic right
peripheral parietal cortex (Fig 3A), the ADC value in the
treated group declined similarly to that in the control group between
30 and 180 minutes after MCAO and then increased at 210 minutes after
MCAO. The difference was significant when comparing the two groups at
this 210-minute time point (P=.0156), suggesting a regional
neuroprotective effect of ZD9379. There were no significant differences
in serial changes in ADC values between the two groups in the central
parietal cortex (Fig 3B) or the caudoputamen (Fig 3C). This
significant delayed improvement of ADC values in the
peripheral parietal cortex, along with the trend toward a
reduction of ADC-derived ischemic lesion volume at this time
point, suggests that ZD9379 has relatively late beneficial effects.
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Experiment B
Experiment A showed that 10 mg/kg ZD9379 significantly attenuated
infarct volume at postmortem. Experiment B was designed to examine the
most effective dose of ZD9379 on postmortem infarct volume,
physiological parameters, neurological
behavior, and mortality using a multiple-dose regimen compared with
another vehicle group.
Physiological Parameters
Body weight or rectal temperature did not differ significantly
among the five groups (data not shown).
Physiological parameters in experiment
B are summarized in Table 2
. There were no significant differences in
serial changes in arterial pH or
PaO2 over time among the five groups. The
alterations in the average values of PaCO2 were
not significantly different among the five groups at any sampling time
point. There was also no significant difference in serial MABP among
the five groups during the time of observation. Temporary hypotension
was again observed in 5 of 10 rats receiving 10 mg/kg ZD9379 but not in
the other four groups. The decreased MABP in the 10-mg/kg ZD9379 group
recovered within 5 minutes.
Neurological Examination
Of the 50 rats studied in experiment B, 5 died prematurely more
than 20 hours after MCAO under observation: 2 rats receiving 10 mg/kg
ZD9379 (20% mortality rate), 1 in the 5-mg/kg group (10%), and 2 in
the 2.5-mg/kg group (20%). No premature mortality was observed in rats
receiving 1 mg/kg ZD9379 or vehicle. The mortality rate was not
statistically different among the five groups. There was no significant
difference in the neurological grading scale among the five groups
receiving vehicle or 10, 5, 2.5, or 1 mg/kg ZD9379 (2.4±0.9, 2.9±1.3,
2.6±1.2, 2.8±1.4, and 2.4±0.8, respectively). Sedation during the
first 2 hours after recovery from the infusion and
anesthesia was again observed in all the rats receiving 10
mg/kg ZD9379 but not in any rats in the 5-, 2.5-, or 1-mg/kg ZD9379 or
vehicle groups. No abnormal behavior was seen in any rat at later time
points.
TTC-Derived Infarct Lesion Volume
At postmortem, all animals showed appropriate positioning of
the suture in the MCA without subarachnoid hemorrhage.
There were significant reductions of corrected TTC-derived infarct
lesion volume in the treated groups receiving 10, 5, and 2.5 mg/kg
ZD9379 (120.8±42.1, 102.3±32.6, and 120.2±33.1 mm3,
respectively) in comparison with the control group (210.5±42.4
mm3; P=.0001), a reduction of –42.6%,
–51.4%, and –42.9%, respectively (Table 3). Corrected infarct
volumes excluding the five rats that died prematurely were also
significantly reduced in the three treatment groups: 120.4±47.5
mm3 in 10 mg/kg ZD9379 (n=8), 105.3±33.2 in 5 mg/kg (n=9),
and 116.4±36.3 in 2.5 mg/kg (n=8), respectively (P=.0001).
The TTC-derived infarct volume did not significantly differ between the
control and 1-mg/kg ZD9379–treated groups (168.7±69.3
mm3). Uncorrected cortical infarct volume was significantly
smaller in the treated group receiving 10, 5, and 2.5 mg/kg ZD9379
(114.0±51.4, 103.0±41.5, and 113.1±39.8 mm3,
respectively) than in the controls (192.1±44.7 mm3;
P=.0006), a reduction of –40.7%, –46.4%, and –41.1%,
respectively. In contrast, there were no significant differences in the
infarct volume of the caudoputamen among the five groups
(P=.1703).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies concluded that glycine antagonists when given before or immediately after ischemia are neuroprotective in permanent focal ischemia3 5 and temporary focal ischemia,4 causing 34.0% to 60.5% reduction of infarct volume. The magnitude of the protective effect on infarct volume using glycine antagonists is similar to that provided by competitive or noncompetitive NMDA antagonists.16 26 27 28 29 30 31 32 33 34 35 36 37 The therapeutic time window with the NMDA antagonists is apparent with initiation of therapy up to 90 minutes after the induction of focal ischemia in rats.17 28 37 38 39 Chen et al3 reported a beneficial effect when therapy started 15 minutes after ischemia but not at 60 minutes after ischemia with a full glycine antagonist. This study demonstrates that treatment with ZD9379, a novel antagonist of the glycine site in the NMDA receptor-channel complex, initiated 30 minutes after permanent MCAO significantly attenuated postmortem infarct volume. A maximal 51.4% reduction of corrected infarct volume occurred with the 5-mg/kg dose. The protective effect was not seen in the caudoputamen with any dose of ZD9379, likely because the lenticulostriate arteries that supply the caudoputamen are occluded at their origin in this MCAO model.
No observation of ischemic lesion volume before or during treatment is available using traditional animal stroke models that only assess infarct volume at postmortem. DWI is a powerful tool for identifying ischemic lesions during their earliest phase7 8 9 10 11 and is useful for early in vivo estimation of therapeutic efficacy.16 17 In the present study, the ADC mapping demonstrated that there was no significant difference in the evolution of ischemic lesion volume in vivo between the control and treated animals during the first 3 hours of therapy. The in vivo observations of a trend toward delayed reduction of ADC-derived ischemic lesion volume, a delayed increase in regional absolute ADC values, and the reduction of infarct volume at postmortem with ZD9379 suggest a delayed therapeutic effect with this glycine site antagonist. The serial measurement of absolute ADC values in the peripheral parietal cortex demonstrated a delayed improvement of ADC values with ZD9379, while the ADC values remained reduced and unchanged over time in the temporal cortex and caudoputamen in rats receiving both ZD9379 and vehicle. The overall volumetric analysis, however, failed to demonstrate a significant reduction of the ADC-derived ischemic lesion volume in vivo. A significant reduction of ischemic lesion volume might have been detected with a longer MRI protocol. A delayed therapeutic effect was observed by another group using similar MRI technology.40 The authors reported that a significant difference in the ischemic lesion volume between the ZD9379-treated and control groups occurred at 6 hours after treatment began but not at 2.5 hours, demonstrating a delayed therapeutic effect of ZD9379.40 ZD9379 appears to rapidly cross the blood-brain barrier as it inhibits neuronal firing in the red nucleus within 60 seconds after intravenous dosing (L.M.P., T.M.B., J.B.P., unpublished data, 1996). It is therefore unlikely that delayed brain penetration of ZD9379 explains the late treatment effect. It is presently uncertain whether this apparent delayed treatment effect is specific to ZD9379 or is generalizable to other full glycine site antagonists.
In contrast to the results with a glycine antagonist, previous studies using delayed treatment with competitive and noncompetitive NMDA receptor antagonists reported relatively rapid therapeutic efficacy on ischemic brain injury after focal ischemia,28 29 30 41 42 43 although no information about brain ischemia before therapy was available. Histopathological studies demonstrated rapid therapeutic efficacy within 3 hours after the commencement of MK-801 administration in permanent MCAO in rats.28 42 Recent investigations, however, suggested the possibility that NMDA antagonists may only postpone the progression of ischemic brain injury or that a considerable part of the tissue showing acute ischemic injury in untreated animals may recover spontaneously.32 39 41 44 45 In the present study, serial diffusion mapping initiated before therapy demonstrated that with ZD9379, such a delayed progression of brain ischemia is unlikely because the alterations of the ADC-derived ischemic lesion volume and the regional ADC values in the treated rats were similar to those in the control rats for the first 2.5 hours after the drug administration began. To explore the differences between glycine antagonists and other antagonists of the NMDA receptor complex, further study will be needed.
The hypothesis of a delayed therapeutic effect with ZD9379 is also
supported by a poor correlation between the ADC-derived
ischemic lesion volume defined by –29% of ADC threshold at
the final imaging time point and TTC-derived infarct volume in the
treated rats. A significant correlation was obtained in rats receiving
vehicle, suggesting that the ischemic lesion was further
reduced after the MRI protocol was completed in the treated group. We
have previously observed a similar significant correlation of
ischemic lesion size with TTC-derived postmortem infarct volume
and the ADC-derived ischemic lesion volume at 2 hours after
permanent MCAO with a ADC of –29%.20 A 43.6%
reduction of corrected infarct volume was observed in the 10-mg/kg
ZD9379–treated rats at postmortem, suggesting that the
ischemic lesion volume defined by ADC threshold value of
–29% at 3.5 hours after MCAO includes both reversible and
irreversible regions.8 16 17 46 Hoehn-Berlage et
al47 48 reported ADC threshold values of –23% using
the difference of homologous ADC values for defining the irreversible
ischemic region, –10% to –23% for defining the penumbral
zone at 2 hours after a similar MCAO suture model in Wistar rats, and
–20% for defining the irreversible ischemic region at 7 hours
after either a proximal or distal MCAO using electrocoagulation in
Fischer 344 rats. The ADC threshold values in their studies are
different from those of the present study. Such differences are
likely due to the MRI analyses (area or volume, imaging time
point), timing of histopathological analysis, stroke model, or
animal strain (Sprague-Dawley, Wistar, or Fischer 344), eg, the
treatable penumbral zone in focal ischemia may be larger in
Wistar than in Sprague-Dawley rats.49 Additional studies
will be needed to determine an appropriate threshold value for
identifying potentially reversible ischemic regions. The ADC
value that defines the irreversibly ischemic region likely
varies over time, but during the initial 60 minutes after stroke onset
it may not be possible to correlate the ADC defined ischemic
lesion with a pathological "gold standard" of irreversible
ischemic injury.
In experiment A, the lower PaCO2 value in controls versus the ZD9379 group could be considered a causative factor for the significant difference in ischemic lesion volume between the two groups. Because CO2 is a potent vasodilator, cerebral hemodynamics in the controls might be more restricted than in the treated animals. It is unlikely, however, because similar effects on postmortem infarct volume were obtained without any difference of PaCO2 among the groups in experiment B. With a loading dose of 10 mg/kg ZD9379, there was a trend toward elevation of PaCO2 and lowering of MABP over time and higher mortality in experiment A with prolonged anesthesia but not in experiment B. It is likely that such hypercarbic hypotensive trends and higher mortality were partly due to the experimental conditions in experiment A. The temporary hypotensive effect caused by the bolus injection of 10 mg/kg ZD9379, but not the other doses, implies a potential anesthesia-potentiating effect of this compound at higher doses. Such transient hypotension did not occur with 10- to 60-mg/kg intravenous bolus doses of ZD9379 in unanesthetized rats (L.M.P., T.M.B., unpublished data, 1995), supporting the potential interaction between ZD9379 and anesthetic agents. Toxic side effects with higher loading doses of ZD9379 will need to be further evaluated.
The optimum neuroprotective dosing regimen of ZD9379 appears to be 2.5 to 5 mg/kg because, in the present protocol, these doses resulted in the smallest TTC-derived infarct volume. A sedative side effect occurred only with the 10-mg/kg dose of ZD9379. Such a transient sedative effect also occurred with a full glycine antagonist.3 No abnormal behavior occurred in any animal after recovery from anesthesia, consistent with the studies using other glycine antagonists.3 4 5 These observations suggest a possible advantage of glycine antagonists compared with competitive or noncompetitive NMDA antagonists that have a similar magnitude of infarct size reduction.
A potential relationship between neuroprotective agents and lowering of brain temperature has been suggested.50 With use of a full antagonist of the glycine site, a significant suppression of the elevation of rectal temperature during focal ischemia and a significant relationship between increased rectal temperature and infarct volumes were observed.4 The authors claimed an important effect on brain temperature with glycine antagonists during focal ischemia. DWI technology allows for noninvasive thermometry based on the relationship between molecular diffusion and temperature.21 51 52 Brain temperature is an important determinant of ADC, and brain ADC values in normal tissue accurately reflect changes in brain temperature.22 In the present study, regional brain ADC values in the nonaffected contralateral hemisphere remained constant during vehicle or ZD9379 infusion, implying that brain temperature was not affected by ZD9379 administration in this protocol.
In conclusion, we observed that the full glycine antagonist ZD9379 reduced postmortem infarct volume at a dose ranging from 2.5 to 10 mg/kg. The DWI studies unexpectedly suggested that ZD9379 has a delayed therapeutic effect. The side effect profile of ZD9379 is favorable, especially at the 2.5- and 5-mg/kg doses. ZD9379 is a promising and novel neuroprotectant that appears to deserve consideration for further development.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by Zeneca Pharmaceuticals and the Harrington Neurological Research Fund. We gratefully thank Stephen J. Hadley, BS, Ronald A. Cohen, PhD, and Vanessa Brown for their technical assistance and thoughtful comments.
Received January 2, 1997; revision received February 21, 1997; accepted March 27, 1997.
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References |
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