(Stroke. 1997;28:2244-2251.)
© 1997 American Heart Association, Inc.
Articles |
Effect of CP101,606, a Novel NR2B Subunit Antagonist of the N-Methyl-D-Aspartate Receptor, on the Volume of Ischemic Brain Damage and Cytotoxic Brain Edema After Middle Cerebral Artery Occlusion in the Feline Brain
From the Divisions of Neurosurgery (X.D., R.B., J.W.) and Radiation Biology (P.F., F.C.), Medical College of Virginia, Virginia Commonwealth University, Richmond, Va, and Pfizer Central Research, Groton, Ct (B.C., F.W.).
Correspondence to Ross Bullock, MD, PhD, Division of Neurosurgery, Medical College of Virginia, Virginia Commonwealth University, Box 980631, MCV Station, Richmond, VA 23298-0631.
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Abstract |
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Background and Purpose The purpose of this study was to test the hypothesis that the neuroprotective compound CP101,606 will ameliorate the increase in lactate, retard the development of cytotoxic edema, and decrease the infarct volume after ischemic stroke.
Methods Seventeen adult cats were allocated to control (n=7) and CP101,606-treated groups (n=10). Transorbital middle cerebral artery occlusion was performed under anesthesia. Extracellular fluid lactate by microdialysis as well as infarct volume measurement by triphenyltetrazolium chloride (TTC)–stained section, with and without neuroprotective agents, was used to determine the value of these potential "surrogate markers" of ischemic damage.
Results The control group showed an increased dialysate lactate (15.5% increase) at 30 minutes and a peak (332.0% increase) in dialysate lactate at 1 hour after middle cerebral artery occlusion compared with the drug-treated group. Significant differences between control and drug-treated groups were seen in the rate of fall of the apparent diffusion coefficient at both 1 and 5 hours. A close correlation was seen between the 1- and 5-hour apparent diffusion coefficient maps and the TTC-stained sections. There was a significantly smaller lesion in the CP101,606-treated group (62.9% reduction in infarct size compared with the control group; P<.001).
Conclusions CP101,606 ranks very highly among the current neuroprotection candidates for clinical trials, and its excellent safety record in both animals and phase II studies in conscious, moderate head injury patients suggests that it will be highly effective in human occlusive stroke.
Key Words: brain edema • glutamate antagonist • cats
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Introduction |
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Traditional approaches to the acute treatment of ischemic stroke focus on maintaining cardiac output and blood pressure, restoring cerebral blood flow, and preventing complications. Recently, attention has been focused on developing new therapies that are directed toward harmful biochemical events at excitatory synapses.1 2 3 4 Pharmacological blockade at the NMDA receptor ion channel complex prevents ischemic neuronal damage.3 5 6 7 8 Studies using specific pharmacological compounds that block the action of glutamate hold particular promise for treating stroke in humans. These include competitive antagonists at the NMDA glutamate binding site (for example, 2-amino-5-phosphonovalerate, AP5, CGS 19755, and D-CPP-ene), noncompetitive antagonists at the channel site (for example, MK-801, CNS 1102, dextromethorphan, and ketamine), and agents that might be directed at the glycine, zinc, and magnesium sites.9 10 11 12 13 CP101,606 is a novel and potent NMDA receptor antagonist. Its binding site is localized in forebrain, most notably in hippocampus and the outer layers of cortex. The functional selectivity for hippocampal neurons and forebrain localization of binding sites suggest that CP101,606 is an NMDA antagonist highly selective for NR2B subunit–containing receptors.12 14 15 Since CP101,606 is currently well established in phase II studies for neurotrauma and hemorrhagic stroke, we performed this study to assess the compound in an occlusive stroke model.
The purpose of this study was to test the hypothesis that the neuroprotective compound CP101,606 will reduce the volume of infarction, retard the rate of development of cytotoxic brain edema due to loss of energy homeostasis as measured by MR DWI, and ameliorate the increase in tissue lactate that has been shown to occur in both human stroke and animal ischemia models.16 17
The advantages of these acute studies are as follows: (1) They provide a rapid means of assessing potential antistroke efficacy in a gyrencephalic animal, with brain anatomy similar to that of humans. (2) They allow cross-comparison between this agent and several others in this model, evaluated in a similar paradigm. (3) They allow cross-comparison between an in vivo MRI end point and volumetric infarct size measurements postmortem.
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Materials and Methods |
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Seventeen adult cats of either sex were assigned to receive vehicle (n=7) or CP101,606 (n=10). Studies were approved by the Institutional Animal Care and Use Committee of Medical College of Virginia (IACUC No. 9401-1921).
Drug Preparation and Administration
CP101,606 was provided by Pfizer as a white powder, and the drug
was solubilized in sterile water to obtain a final drug concentration
of 15 mg/mL. The solution was then separated into a number of
aliquots and frozen. Individual aliquots were then thawed daily. Each
animal was administered a bolus of 1 mg/kg IV at 15 minutes
before MCA occlusion was initiated. Immediately after administration of
the bolus injection, a continuous intravenous infusion of
7.5 µg/min per kilogram was begun. For the control group,
similar volumes of sterile saline were administered throughout the
experiment. Drug/vehicle preparation was performed by a
technologist not involved in the conduct of the experiment or
analysis of results. Experiments were thus kept blinded.
General Preparation
Anesthesia was induced with an
intravenous injection of the short-acting steroid induction
agent Saffan (alphadalone/ alphaxalone). The cats were then orally
intubated and mechanically ventilated with a small animal ventilator
with the use of a nitrous oxide/oxygen mixture (70:30) and halothane
(0.75% to 1.5%). Both right femoral artery and vein were cannulated
for continuous monitoring of blood pressure, sampling for
arterial blood gas analysis, and
intravenous administration of the drug. Temperature was
monitored by a rectal thermistor probe, a temporalis muscle
thermocouple, and during the MRI studies by a mercury and glass rectal
thermometer. Temperature was maintained within the
normothermic range with either a heating blanket or by
blowing warm air over the cat in the MRI magnet bore. Ventilation was
adjusted to normocarbia by blood gas analysis every hour or
more frequently as needed.
MCA Occlusion
After general preparation, the animals were mounted in the
sphinx position in a purpose-built Plexiglas stereotaxic
apparatus designed to immobilize the head and
body within the MRI magnet. Through a supraorbital incision, the cavity
of the left orbit was entered and the contents of the orbit were
removed. The optic nerve and ophthalmic artery were coagulated, and the
posterior and superior walls of the orbit were drilled away with a
high-speed drill. The dura overlying the orbital surface of the sylvian
region was then opened to expose the MCA throughout its course from the
carotid at the base of the skull to beyond its trifurcation. After
administration of drug or vehicle bolus, the MCA was coagulated
throughout its length from the carotid bifurcation to distal to the MCA
trifurcation, and it was cut. Sketches were made of the anatomy
in each case.
Microdialysis
A CMA/20 flexible 10-mm microdialysis probe was axially
positioned in the territory of MCA occlusion though the orbital long
window. Microdialysis sampling was performed with sterile saline used
as the perfusate at a flow rate of 2.0 µL/min with 30-minute
sampling intervals throughout the experiment. After 1 hour of
stabilization, two samples for the baseline before and 10 samples after
MCA occlusion were collected for lactate measurement by HPLC. For
analysis of lactate, 10 µL of diluted dialysate (1:5 in
saline) was injected into a Hamilton PRP-X300 ion exclusion column. The
column was connected to a BAS 30A HPLC pump, and lactate was
analyzed by ultraviolet detection at a wavelength of 214 nm
(BAS UV116A). The mobile phase consisted of 0.5mN
H2SO4 solution and was pumped at a flow rate of
0.6 mL/min. Signals were compared against standard l-lactate solutions
(Sigma Chemical Co).
MRI
After MCA occlusion, a saddle-shaped volume coil (95 mm in
diameter), used for both signal transmission and reception, was placed
over the animal's head. The coil assembly was rigidly attached to the
stereotaxic assembly securing the cat's head, thereby
ensuring reproducible positioning between animals. Within 10 minutes
after occlusion, the animal was positioned in the 2.35-T,
40-cm-diameter magnet bore of the MR imager (Bruker Instruments), and
data acquisition was begun.
A sagittal T1-weighted image (repetition time, 700 ms; echo time, 18 ms; matrix size, 64x64), with a standard single-echo sequence, was acquired for correct positioning of the DWI. All DWIs were acquired from three parallel coronal slices (3-mm thickness, 1-mm gap between slices, 8-cm2 field of view), centered at a standard anatomic plane chosen to be 15 mm from the tip of the forebrain. DWIs were performed with the use of a spin-echo sequence18 (repetition time, 1500 ms; echo time, 23 ms; matrix size, 128x128), with the diffusion-sensitizing gradients applied along the frequency-encoding direction. Diffusion-weighting factors, or b values, of 10, 333, 667, and 100 s/mm2 were used (maximum diffusion gradient strength of 5.4 G/cm). ADC maps were generated on a pixel-by-pixel basis for each slice with the use of a least-squares fitting algorithm supplied by Bruker. The effective cross-terms between the diffusion-sensitizing and frequency-encoding imaging gradients were included in the ADC calculations.
Regions-of-interest were drawn by hand around the infarct zone and various anatomic structures (cortex, caudate nucleus, internal capsule, thalamus, hemisphere) visible in the ADC image. Regions of interest were mirrored between the infarcted and contralateral brain hemispheres.
Volumetric Histopathology
At the end of the 5-hour survival period after MCA
occlusion, the animal was removed from the MRI magnet, and
anesthesia was deepened with 4% halothane. The animal was
then killed by potassium chloride overdose (3 mL), and after cardiac
arrest the brain was removed. The forebrain was chilled in a -80°C
super freezer for 15 minutes and cut with the use of a cat-brain matrix
at 0.5-cm coronal intervals. These coronal slices corresponding to the
same slices acquired with the MRI were then immersion fixed in TTC
solution 2% in saline at 37°C. After 30 minutes of immersion, the
slices were transferred to a 10% formaldehyde solution, where they
were immersion fixed overnight. Each side of the brain slices was then
photographed, and the volume of ischemic damage, shown as a
zone of pallor of staining, was quantitated with an image-analyzing
computer (Imaging Research Inc, Broke University). This was done by an
observer blinded to the identity of the animals. TTC staining 5 hours
after MCA occlusion was identical to necrosis as seen on hematoxylin
and eosin staining in a pilot study, which accords with the finding by
Liszczak et al.19
Plasma Levels of CP-101,606
Before these experiments, plasma samples, drawn at hourly
intervals for 5 hours, were obtained from two normal cats under the
same anesthetic regimen as in these studies. The estimated plasma
half-life of the CP101,606 is 2.7 hours, and the systemic clearance is
25 mL/min per kilogram. Administration of 1 mg/kg and 7.5
µg/min per kilogram as an infusion was chosen to achieve a
steady state of 200 ng/mL, which is consistent with the
desired therapeutic dose level for adequate receptor binding.
Statistical Analysis
MRI ADC and area values and extracellular fluid microdialysis
lactate in each group were analyzed by ANOVA. Furthermore, a
t test was used for comparison of infarct volume as measured
by TTC staining between the two groups at a single time point.
Correlations between TTC staining and DWI in the volumetric
measurements were tested with Spearman's rank test and plotted by
linear regression. All values were expressed as mean±SD. A
statistically significant difference was indicated by a value of
P<.05.
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Results |
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Although there were significant differences in Po2, Pco2, pH, and core temperature between drug and control groups at certain time points, mean blood pressure did not differ between drug-treated and the control groups (Table 1). There was no significant difference in the comparison of cat weight between the two groups (mean±SD, 3.39±0.20 kg in drug-treated group versus 3.41±0.14 kg in controls).
ADC Values in Drug-Treated and Control Groups: 1 and 5 Hours After
MCA Occlusion
The mean ADC values (±SD) taken from the contralateral
(noninfarcted) hemisphere of all animals were 0.68±0.05 and
0.66±0.06 mm2/s at 1 and 5 hours, respectively, while
the ipsilateral (infarcted) hemisphere showed 0.49±0.11 and 0.45
mm2/s at same time points after MCA occlusion. The decrease
of ADC after MCA occlusion agrees well with values in the
literature.20 21 22
Separate split-plot ANOVAs (GroupxTime) were calculated for each
brain area (anterior, middle, and posterior). In the lesioned (left)
hemisphere, the ANOVA of ADC for the anterior section resulted in a
nonsignificant main effect of Group (F1,7=0.909,
P=.372). The main effect of Time was significant
(F1,7=15.192, P=.006), which indicated that
averaged over Groups, the ADC value decreased. The GroupxTime
interaction was not significant (F1,7=2.032,
P=.1970). The ANOVA on the ADC values from the middle
section produced a significant main effect of Group
(F1,7=16.501, P=.0019), which indicated that the
drug-treated animals had a higher ADC value than the control animals.
The main effect of Time was also significant (F1,7=77.123,
P=<0.0001), demonstrating that between 1 and 5 hours there
was an increase in ADC. The GroupxTime interaction was also
significant (F1,7=8. 191, P=. 0155). This
interaction indicates that ADC values decreased over time more in the
control animals that it did in the drug-treated animals. In the
posterior slice, the ANOVA did not produce a significant main effect of
Group (F1,11=4.439, P=.0589) or Time
(F1,11=4.838, P=.501) individually. The
significant GroupxTime interaction (F1,11=9.791,
P=.0096) indicates that ADC values decreased over time more
in the control animals than in the drug-treated animals (Fig 1
). There was no significant difference
between the two groups in ADC value of right hemisphere by ANOVA.
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Area of Ischemic Damage, at Predetermined Brain Slices, by
ADC Mapping
For each of three sets of brain sections through the infarct
(anterior, middle, and posterior slices), a significant difference was
seen in the lesioned area of middle and posterior slices by separate
split-plot analyses (GroupxTime) of ANOVA. The ANOVA of
ischemic area as measured by ADC mapping for the anterior
section resulted in a nonsignificant main effect of Group
(F1,13=2.347, P=.1495). The main effect of Time
was significant (F1,1=21.978, P=.004), which
indicated that on average over Groups the ischemic area
decreased. The ANOVA for the lesioned area from the middle section
showed a significant main effect of Group (F1,15=4.760,
P=.0454), which indicated that the control animals had a
larger lesioned area than the drug-treated animals. The main effect of
Time was also significant (F1,1=38.918,
P=<0.0001), demonstrating that there was a gradual increase
in ischemic area between 1 and 5 hours. In the posterior slice,
the ANOVA also produced a significant main effect of Group
(F1,13=6.495, P=.0243) and Time
(F1,1=17.166, P=.0024)
(F1,11=4.838), indicating that control animals had a
significantly larger ischemic area than drug-treated animals in
the posterior series after MCA occlusion (Fig 2).
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Volume of Ischemic Brain Damage as Measured by TTC
Staining
Significantly smaller lesions were noted in the CP101,606-treated
group (62.9% reduction in infarct size compared with the control
group; P<.001) (Fig 3).
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Relationship Between DWI Measurement of Infarct Area and Volume of
Infarcted Tissue by TTC Staining
As shown in Figs 4 and 5, a close correlation was seen between
the 1- and 5-hour ADC maps and the TTC-stained sections. A very close
topographic relationship was seen between DWI at 1 and 5 hours and the
corresponding TTC-stained brain slices. When this relationship was
tested by linear regression analysis, a significant correlation
was seen at both 1 and at 5 hours in both control and drug-treated
groups. (Fig 6). However, when the
difference in the slopes of the four regression lines (1 and 5 hours,
drug versus control) were tested, no significant difference was found
statistically.
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Dialysate Lactate Concentration
The control group showed an increased dialysate lactate (15.5%
increase) at 30 minutes and a peak (332.0% increase) in dialysate
lactate at 1 hour after MCA occlusion compared with the drug-treated
group. However, this difference in dialysate lactate between the two
groups only became significant at 2 hours after MCA occlusion. This is
because dialysate lactate in the control group varied very widely
compared with the much less variable levels in the drug group.
Significantly higher levels were maintained throughout the experiment
compared with the control group (Fig 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study has demonstrated the following: (1) CP101,606 significantly reduced the volume of the ischemic lesion by 62.9% by TTC staining at 5 hours in the drug-treated group compared with the control group. (2) CP101,606 reduced the tendency for the ADC to fall after MCA occlusion by 23% and 40% at 1 and 5 hours, respectively, compared with the control group. (3) CP101,606 abolished the increase in dialysate lactate concentration, seen in controls, after ischemia in the infarcted hemisphere. (4) In common with other clinical and laboratory studies, MRI DWI provides an early surrogate marker for ischemic tissue, and the difference between the DWI size and the final TTC-stained lesion size may represent a zone of potentially salvageable "penumbral" tissue, which may be influenced by "neuroprotective" strategies.23 24
This study was designed to test the acute effects of CP101,606 on tissue salvage. We used DWI, ADC, and dialysate lactate as possible surrogate early measures of ischemia in this study, as well as the volume of ischemically damaged tissue at 5 hours, to explore possible mechanistic effects of the drug. The reduced volume of the ischemic lesion in the drug-treated group was achieved mainly by reduction of the volume of tissue showing ischemic change within the ectosylvian and temporal gyri. The neuroprotective effect was most obvious when total hemisphere volumes were compared as either percentage of hemisphere infarcted or infarct volume.
It is clearly seen that the percent hemispheric lesion areas measured
by DWI correlated well with the area measurements from the TTC-stained
sections for both drug-treated and control groups (Fig 6
). However, DWI
uniformly overrepresented the lesion areas compared with
histopathological necrosis stained by TTC (Figs 4, 5, and 6). This
suggests that the penumbral zone around the pannecrosis area, which is
abnormal by DWI but not by TTC staining, is a zone of salvageable
tissue for neuroprotectants (particularly ion channel blockers, for
example), which will retard cytotoxic edema formation.
ADC imaging and measurements reflect the degree to which water protons
are free to diffuse in tissue. Extracellular fluid protons move up to
60 µm over millisecond periods, but intracellular protons are
constrained and move much less—only 3 to 4 µm. Thus, falling
ADC values may reflect a shift of water into cells in response to ion
flux, secondary to ion channel opening, in
ischemia.24 25 26 27 28 CP101,606 markedly reduced this
tendency for the ADC to fall, as shown in Table 2 and Figs 5 and 6.
Temperature has been reported to have an effect on the ADC.29 In this study a small but significant difference in temperature was seen between the drug-treated and control groups. However, the difference was most marked before MCA occlusion, and this was only significant for 1.5 hours after occlusion. Core temperature was similar in both groups for the majority of the experiment. Moreover, review of the literature suggests a 2.4% change in ADC per 1°C temperature change. Thus, only approximately 5% of the 20% to 50% ADC change seen between the two groups can accounted for by temperature. Mean arterial blood pressure, which is a much more important factor in determining ischemic brain damage, showed no significant difference between the two groups throughout the experiment.
We hypothesize that the difference in the slope of the regression lines
between the drug-treated and the control groups (as seen in Fig 6) may
represent an index of the neuroprotective effect of CP101,606.
In the drug-treated group, the difference between the "low-ADC
zone" and the ischemic zone, obtained by TTC staining, is
greater than in the controls. This suggests that in the drug-treated
group a larger tissue zone may develop cytotoxic edema, yet remain
viable, as judged by later TTC staining, at 5 hours.
CP101,606 acts by blocking the NR2B subunit of the NMDA receptor, thus preventing activation of this ion channel by free glutamate, which is known to be released in circumstances of acute focal ischemia.12 14 15 This prevention of ion channel opening may then retard potassium efflux and sodium and particularly calcium influxes, thus preventing cytotoxic swelling. Since TTC stain uptake depends on an intact mitochondrial reductase system, and excessive calcium entry is a major factor in deactivation of the these reductase systems, our findings are in agreement with this concept of drug-induced reduction of calcium entry.30 Furthermore, the significant reduction in lactate accumulation in the ischemic tissue in the drug-treated animals, as measured by microdialysis, is in agreement with a reduction in anaerobic metabolism in the CP101,606-treated group of animals, or retained mitochondrial metabolism, in the "protected" tissue.31 32 33 Clearly, a "posttreatment" paradigm to evaluate the compound over longer periods, such as 1 to 2 days after onset of ischemia, in other models suitable for survival studies is necessary to establish the long-term effect of the drug.
These studies were performed in an "experimenter-blinded" paradigm. The magnitude (62.9%) of the neuroprotective effect of CP101,606 was very large and was seen across several end points. This magnitude of neuroprotection exceeds that of most of the other compounds tested in this model in which a similar paradigm was used.3 5 6 7 8 34 35 These compounds include CGS19755 (42.0%), MK-801 (both pretreated [50%] and postocclusively treated [55.6%]), and the oxygen transport enhancer RSR13 (36.4%) and D-CPP-ene (66.7%). Thus, this compound ranks very highly among the current neuroprotection candidates for clinical trials, and its excellent safety record, which we have established in phase II studies in conscious moderate head injury patients, suggests that it will be highly effective in human occlusive stroke.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from Pfizer, Groton, Ct, and by National Institutes of Health grant NS-12587. We are grateful to M.L. Giebel for image analysis, to Dr J.J. Woodward for assistance with HPLC measurement of lactate, and to D. Sung Choi, PhD, for advice on statistical analysis.
Received January 24, 1997; revision received August 1, 1997; accepted August 1, 1997.
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