(Stroke. 1999;30:1891-1899.)
© 1999 American Heart Association, Inc.
Original Contributions |
Combination Drug Therapy and Mild Hypothermia
A Promising Treatment Strategy for Reversible, Focal Cerebral Ischemia
From the Department of Neurosurgery (R.S.-E., S.Z., H.-J.R.) and the Institute for Surgical Research (E.H., A.B.), Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Germany.
Correspondence to Dr Robert Schmid-Elsaesser, Department of Neurosurgery, Ludwig-Maximilians-Universität, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany. E-mail Schmid-elsaesser{at}nc.med.uni-muenchen.de
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Background and Purpose—Hypothermia has been suggested to be the most potent therapeutic approach to reduce experimental ischemic brain injury identified to date, and mild hypothermia is increasingly used for neuroprotection during neurovascular surgery. We have recently demonstrated that combined administration of tirilazad mesylate and magnesium provides for an overall enhanced neuroprotective effect. The present study was designed to determine whether the efficacy of mild hypothermia (33°C) can be increased by combination pharmacotherapy with tirilazad and magnesium (MgCl2).
Methods—Forty Sprague-Dawley rats were subjected to transient, middle cerebral artery occlusion and were randomly assigned to 1 of 4 treatment arms (n=10 each): (1) normothermia+vehicle, (2) normothermia+tirilazad+MgCl2, (3) hypothermia+vehicle, or (4) hypothermia+tirilazad+MgCl2. Cortical blood flow was monitored by a bilateral laser-Doppler flowmeter, and the electroencephalogram was continuously recorded. Functional deficits were quantified by daily neurological examinations. Infarct volume was assessed after 7 days.
Results—Tirilazad+MgCl2, hypothermia, and hypothermia+tirilazad+MgCl2 reduced total infarct volume by 56%, 63%, and 77%, respectively, relative to controls. In animals treated with both hypothermia and combination pharmacotherapy, cortical infarction was almost completely abolished (-99%), and infarct volume in the basal ganglia was significantly reduced by 55%. In addition, this treatment provided for the best electrophysiological recovery and functional outcome.
Conclusions—The neuroprotective efficacy of hypothermia can be increased by pharmacological antagonism of excitatory amino acids and free radicals by using clinically available drugs. This treatment strategy could be of great benefit when applied during temporary artery occlusion in cerebrovascular surgery.
Key Words: cerebral ischemia • focal • drug therapy • hypothermia • laser-Doppler flowmeter
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Cell death after cerebral ischemia is mediated by a massive release of excitatory amino acids, generation of free radicals, and—a crucial step—calcium influx into cells.1 This knowledge has led to research into the pharmacological agents to antagonize excitatory amino acids, inhibit the production of free radicals, and prevent calcium influx into cells. The pathophysiological mechanisms leading to neuronal death are so complex that, apart from thrombolytic therapy, 1 agent alone has never been shown to be sufficient to significantly reduce cerebral infarction in humans. This has led to a number of recent articles and editorials regarding the need for clinical trials involving combinations of pharmacological agents.2 3 4 Combinations of agents may act synergistically, but it is also possible for undesirable interactions to occur. Therefore, clinical trials of combinations of drugs should be preceded by animal experiments that are capable of identifying interactions.5 6 We have recently shown that combined administration of tirilazad mesylate and magnesium provides for an overall enhanced neuroprotective effect in rats subjected to transient, focal cerebral ischemia.7 Tirilazad is a free-radical scavenger and lipid peroxidation inhibitor.8 Magnesium is a naturally occurring calcium antagonist and a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors.9 Both drugs have proven their beneficial effects in numerous animal studies of focal and global cerebral ischemia, subarachnoid hemorrhage (SAH), traumatic brain injury, and spinal cord ischemia.10 11 Furthermore, these drugs offer the advantage that they are clinically available without severe side effects at the neuroprotective dosage.11 12 13 Tirilazad (Freedox) is licensed for clinical use in Australia and in many European and other countries. Magnesium is an over-the-counter drug with an established safety profile. It is conceivable that this drug combination could be improved or supplemented by other more potent agents, especially compounds that affect glutamate receptors. However, most glutamate antagonists have severe psychomimetic and cardiorespiratory-depressant side effects and are not suitable for awake stroke patients or for those undergoing elective cerebrovascular surgery.14
Barone et al15 stated that cerebral hypothermia is the most potent therapeutic approach to reduce experimental ischemic brain injury identified to date. Multiple mechanisms for hypothermia-induced neuroprotection have been identified: (1) reduced metabolic rate and energy depletion, (2) decreased excitatory transmitter release or enhanced postischemic reuptake of glutamate, (3) decreased generation of free radicals, (4) improved ion homeostasis, and (5) reduced vascular permeability, blood-brain barrier disruption, and edema.16 17 18 In view of these multiple protective mechanism, it seems questionable whether the efficacy of hypothermia can be enhanced by pharmacological agents.
Because mild hypothermia is increasingly used for neuroprotection during neurovascular surgery,19 we conducted the present study to investigate whether the neuroprotective efficacy of hypothermia (33°C) could be improved by combined administration of tirilazad and magnesium.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
A total of 42 male, Sprague-Dawley rats (270 to 300 g body weight) were used for this study. Animals were purchased from Charles River Laboratory (Sulzfeld, Germany) and were cared for before and at all stages of the experiment in compliance with applicable institutional guidelines and regulations of the government of Bavaria.
Animal Preparation and Monitoring
Rats were fasted overnight before surgery with free access to
water. For the operative procedures, the animals received atropine (0.5
mg/kg SC), and anesthesia was induced with 4% halothane.
The animals were orally intubated and mechanically ventilated with
0.8% halothane in a mixture of 70% N2O and 30%
O2 to maintain normal arterial
blood gases. Temporalis muscle and rectal probes were used to monitor
temperature throughout the experiment. A thermostatically regulated,
feedback-controlled heating lamp and pad were used to maintain
temporalis muscle and rectal temperatures at 37.0°C in
normothermic animals. The tail artery and left femoral vein
were cannulated for blood sampling, monitoring of arterial
blood pressure, and administration of fluids and drugs.
Arterial blood gases, serum glucose, hemoglobin, and
hematocrit were measured before, during, and after
ischemia.
Laser-Doppler Flowmeter Measurement and
Electroencephalography
A continuous laser-Doppler flowmeter (LDF; model MBF3D, Moor
Instruments Ltd) was used to monitor local cortical blood flow (LCBF)
in the area of the cerebral cortex in each hemisphere supplied by the
middle cerebral artery (MCA). To allow placement of the LDF probe, a
burr hole (1-mm diameter) was drilled 5 mm lateral and 1 mm
posterior to the bregma on each side, with care being taken not to
injure the dura mater. The animals were placed in a supine position,
and the head was firmly immobilized in a
stereotaxic frame. Then a micromanipulator was used to
position a rectangularly bent LDF probe over each brain hemisphere.
LCBF was continuously measured (2-Hz sampling rate) from before the
onset of ischemia until 2 hours after reperfusion.
For continuous electroencephalographic (EEG) recordings (EEG-7109, Nihon Kohden Kogyo Ltd), silver electrodes were connected to both laser-Doppler probes, and a reference electrode was placed at the jaw bone. Bandpass was set at 0.15 to 45 Hz and amplitude at 1.2 mm/50 µV.
MCA Occlusion
All rats were subjected to 90 minutes of MCA occlusion by
insertion of a silicone-coated, 4-0 nylon monofilament via the external
carotid artery as previously described.20 In brief, the
filament was gently advanced until the LDF showed a sharp decrease of
the ipsilateral LCBF to 
20% of baseline, indicating adequate
occlusion of the MCA. Two animals were excluded from the study and
replaced because their LDF measurements showed a sharp drop in
contralateral LCBF after insertion of the filament, indicating SAH. SAH
was confirmed by autopsy. Reperfusion was achieved by withdrawing the
filament into the external carotid artery after 90 minutes.
Drug Administration and Treatment Arms
Rats were randomly assigned to 1 of 4 treatment arms (n=10
each): (1) vehicle-treated, normothermic controls at 37°C
(rectal and temporalis muscle temperatures); (2) tirilazad and
MgCl2 at 37°C; (3) vehicle-treated, hypothermic
controls at 33°C; or (4) tirilazad and MgCl2 at
33°C. In the vehicle-treated groups, 5 animals received 0.9% saline
and 5 received the same volume of 0.02 mol/L citric acid. Each dose of
tirilazad mesylate (Freedox, Upjohn) was 3 mg/kg and each dose of
MgCl2 (Sigma-Aldrich Chemie) was 1
mmol/kg. Isovolumetric doses of vehicle or drugs were administered
intravenously over 15 minutes. Each animal received 2 doses
of vehicle or drugs, with the first dose being administered before
ischemia and the second dose at reperfusion. In the hypothermic
groups, whole-body hypothermia was induced with the use of ice packs
until a temperature of 33°C in the rectal and temporalis muscle areas
was reached and maintained. Before induction of ischemia, an
interval of 20 minutes was allowed for
physiological stabilization. Rewarming (1°C/10
min) was started 30 minutes after reperfusion. A study flow diagram is
presented in Figure 1
.
|
Quantification of Ischemic Damage
Neurological deficits and infarct volume were assessed by a
colleague who was "blinded" to the animals' treatments.
Postoperatively, each animal's neurological function was evaluated
daily on a 6-point grading scale: 5, no apparent deficit; 4,
contralateral forelimb flexion; 3, lowered resistance to lateral push
without circling; 2, circling if pulled by tail; 1, spontaneous
circling; and 0, no spontaneous activity. In addition, each animal's
body weight was determined daily.
Seven days after transient cerebral ischemia, each rat was again anesthetized and perfused transcardially with isotonic heparinized saline, followed by 2% paraformaldehyde for fixation of tissues. The brain was removed, embedded in paraffin, and cut into 4-µm-thick coronal sections at 400-µm intervals. The brain slices were stained with hematoxylin and eosin. Twenty-four slices from each brain containing the entire infarct were used, and the infarct area on each slice was planimetrically determined (OPTIMAS 5.1, BioScan Inc). The total infarct volume (IT) expressed in mm3 was calculated to be the sum of the area of infarct on each slice (In), multiplied by the distance (400 µm) between successive slices (IT=0.4[I1+I2+... I24] mm3). The volumes of infarcts in the cortex and basal ganglia were determined by measuring the area of infarct in sections obtained 2.0, 3.6, 5.2, 6.8, and 8.4 mm from the frontal pole.
Statistical Analysis
Statistical analyses were performed with the use of
SigmaStat 2.0 Statistical Software (Jandel Scientific).
Physiological data for each time point and infarct
volumes were analyzed with 1-way ANOVA, laser-Doppler and
EEG data were analyzed with 2-way ANOVA for repeated measures,
and neurological function scores were analyzed with
Kruskal-Wallis ANOVA on ranks for each of the 7 postoperative days.
When multiple comparisons were indicated, Dunnett's test or the
Student-Newman-Keuls test for neurological function scores was applied.
Differences were considered significant at the P<0.05
level. Results are presented as mean±SD.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
Physiological Variables
There were no statistically significant differences in mean physiological values, cortical CBF, neurological deficits, or infarct volume between normothermic animals that received normal saline or citric acid and no significant differences between hypothermic animals that received drug vehicles. Therefore, data from all normothermic and all hypothermic rats receiving vehicle were included in a normothermic or hypothermic control group, respectively.
Blood pressure dropped by 10 to 15 mm Hg during drug infusion of MgCl2, but this effect did not significantly influence the mean values during and after ischemia. Furthermore, blood glucose levels increased after drug administration in rats that received MgCl2 and in hypothermic animals, while blood glucose levels decreased in vehicle-treated normothermic animals. This difference was significant in drug-treated hypothermic animals compared with vehicle-treated normothermic controls. There were no statistically significant differences among groups in any of the other mean values, but hypothermic animals had a trend toward a lower PaCO2, a higher PaO2, and higher pH values.
LDF Measurements
In normothermic animals, MCA occlusion resulted in an
immediate reduction of LCBF to 20% of baseline in the territory
supplied by the ipsilateral artery, while contralateral blood flow
remained unchanged throughout the experiment. After reperfusion, a
short period of postischemic hyperemia was followed
by a decrease in ipsilateral LCBF to 70% of baseline. Delayed
hypoperfusion persisted until the end of the 2-hour postreperfusion
observation period. There was no significant difference in cortical CBF
between normothermic vehicle-treated animals and
normothermic animals that received tirilazad and magnesium
(Figure 2).
|
) and contralateral
(•) local cortical blood flow (LCBF) measured by laser-Doppler
flowmeter from before induction of hypothermia and ischemia
until 2 hours after reperfusion. Flow values shown are averages of
measurements made during 1-minute periods every 10 minutes and
expressed as percent of baseline (mean±SD) for n=10 in each group. The
course of temporalis muscle temperature (Temp.) is indicated. Middle
cerebral artery occlusion significantly reduced ipsilateral LCBF to
20% of baseline in all groups (P<0.05 vs baseline; not
indicated). *P<0.05 vs ipsilateral flow in
normothermic vehicle-treated controls for each time point;
§P<0.05 vs contralateral flow in
normothermic vehicle-treated controls for each time point,
by 2-way ANOVA for repeated measures followed by Dunnett's test.
In the hypothermic groups, ipsilateral and contralateral LCBF decreased
to 80% of baseline during cooling. MCA occlusion resulted in an
immediate reduction of ipsilateral LCBF to 20% of baseline.
Reperfusion was followed by a prolonged period of
postischemic hyperemia, compared with
normothermic animals, before ipsilateral CBF gradually
decreased to 70% of baseline. Delayed hypoperfusion persisted until
the end of the 2-hour postreperfusion observation period. Contralateral
LCBF recovered to baseline values (Figure 2
) during
rewarming.
Electroencephalography
In normothermic animals, the ipsilateral EEG amplitude
decreased immediately after MCA occlusion to 40% of baseline. EEG
changes appeared, with a delay of 4 seconds, after the onset of LCBF
reduction as indicated by the LDF. After reperfusion, the amplitude
slowly recovered to 60% to 70% of baseline. There were no significant
differences between normothermic vehicle-treated animals
and normothermic animals that received tirilazad and
magnesium (Figure 3).
|
In the hypothermic groups, the ipsilateral and contralateral EEG
amplitude significantly decreased to 80% of baseline during
cooling. Immediately after MCA occlusion, the ipsilateral EEG amplitude
decreased to 40% of baseline. After reperfusion and rewarming, the
contralateral EEG amplitude recovered to baseline values. In animals
subjected to hypothermia alone, the ipsilateral EEG amplitude slowly
recovered to 80% of baseline, which was not quite statistically
significant. In hypothermic animals that received tirilazad and
magnesium, the ipsilateral EEG amplitude recovered faster to 80% of
baseline and more. This increase was significant when compared with
normothermic animals (Figure 3).
Functional Outcome and Weight Gain
Except for the 2 animals that experienced SAH under
anesthesia, there was no additional mortality.
Normothermic animals that received tirilazad and
MgCl2 had significantly (P<0.05) less
neurological deficits from postoperative days 4 to 7 compared with
normothermic vehicle-treated controls. Hypothermic animals
that received the drug vehicle had significantly fewer neurological
deficits from postoperative days 3 to 7. Hypothermic animals that
received tirilazad and MgCl2 had significantly
fewer neurological deficits from postoperative days 1 to 7 compared
with normothermic controls and fewer deficits from
postoperative days 1 to 3 compared with all other groups. None of the
animals in this group showed any residual functional deficit at the end
of the observation period (Figure 4).
|
P<0.05 vs hypothermia+tirilazad+MgCl2, by
Kruskal-Wallis ANOVA on ranks followed by the Student-Newman-Keuls
test.Hypothermic animals that received tirilazad and MgCl2 also exhibited the best weight gain from postoperative days 1 to 7, although this difference was not significant owing to interanimal variations.
Infarct Volume
There was no difference in brain size between the groups and
between the ipsilateral and contralateral hemispheric volumes of each
individual animal on postoperative day 7. Therefore, indirect
measurements of infarct volumes to correct for brain size or edema were
not necessary.21
The total infarct volume was 70.2±17.4 mm3
(mean±SD) in normothermic vehicle-treated controls,
30.8±9.6 mm3 in normothermic
animals that received tirilazad and MgCl2,
25.9±12.3 mm3 in hypothermic animals that
received the drug vehicle, and 15.8±8.2 mm3
in hypothermic animals that received tirilazad and
MgCl2. Compared with those in
normothermic vehicle-treated controls, total infarct
volumes were significantly (P<0.05) smaller in
normothermic animals treated with tirilazad and
MgCl2 (-56%), hypothermic vehicle-treated
animals (-63%), and hypothermic animals that received tirilazad and
MgCl2 (-77%) (Figure 5).
|
When infarct volume was determined separately for cortical brain tissue
and the basal ganglia, all treatment strategies were significantly
(P<0.05) effective in limiting cortical infarct volume. The
average cortical infarct volume was 36.2±12.2
mm3 in normothermic vehicle-treated
controls (mean±SD), 10.8±5.8 mm3 in
normothermic animals treated with tirilazad and
MgCl2 (-70%), 1.7±2.2
mm3 in hypothermic animals that received drug
vehicle (-95%), and 0.4±1.3 mm3 in
hypothermic animals that received tirilazad and
MgCl2 (-99%) (Figure 6).
|
Similarly, tirilazad and MgCl2 and the
combination therapy of tirilazad and MgCl2 plus
hypothermia significantly (P<0.05) reduced infarct volume
in the basal ganglia. Compared with normothermic
vehicle-treated controls (32.9±5.3
mm3, mean±SD), the infarct volume in the basal
ganglia in normothermic rats treated with tirilazad and
MgCl2 was 19.4±5.4
mm3 (-41%) and in hypothermic rats treated with
tirilazad and MgCl2, it was 14.9±7.6
mm3 (-55%). The reduction in basal ganglia
infarct volume in hypothermic rats that received drug vehicle
(24.2±10.4 mm3; -26%) was not significant
(Figure 6).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
The present study demonstrates that combination drug therapy with tirilazad and MgCl2 accompanied by mild hypothermia provides significant cerebroprotection to rats subjected to transient MCA occlusion. Even the most potent therapeutic approach, mild hypothermia, can be improved by additional pharmacotherapy. Animals treated with the drug combination and hypothermia had the best electrophysiological recovery, the fewest postoperative neurological deficits, the best weight gain, and the smallest infarct volume.
Cerebral Blood Flow
In accordance with the results of other studies, neither
tirilazad nor MgCl2 seems to exert its beneficial
effects by improving CBF.6 7 22 23 In contrast, Chi et
al24 found that magnesium sulfate increased CBF after
permanent MCA occlusion in rats. Magnesium is involved in the
regulation of smooth muscle tone and may induce cerebral
vasodilatation.9 One or more of several factors might
explain our failure to note a significant effect on LCBF with
administration of MgCl2. First, we measured CBF
in the somatosensory cortex near the core of the infarct, and it is
possible that improvements in CBF might have occurred in more
peripheral areas. Second, it is conceivable that a
different magnesium salt or a higher dosage of
MgCl2 than the one we used is required to
influence CBF,25 but high dosages of magnesium cause
arterial hypotension and hyperglycemia.
In the present study, mild hypothermia (33°C) decreased CBF and
the EEG amplitude to 80% of baseline. The average decrease in LCBF
per degree of temperature was 5% of control flow. This effect was
first described by Rosomoff and Holaday26 and later
confirmed in experiments with dogs, monkeys, cats, rabbits, and
rats.27 28 29 There was no difference in LCBF during
ischemia and delayed hypoperfusion between
normothermic and hypothermic animals, but hypothermia
resulted in a prolonged hyperemic phase. This effect was even
more pronounced in animals that additionally received tirilazad and
magnesium, possibly as a result of alterations in ion homeostasis
including calcium, potassium, and magnesium fluxes.30
Increased serum magnesium levels were found in animals subjected to
hypothermia.31 It is conceivable that a greater and more
prolonged elevation in magnesium levels may act to ameliorate
ischemic damage by virtue of magnesium's ability to block the
NMDA channel noncompetitively and to cause
vasodilatation.9 32
In accordance with our results, Baldwin et al33 and Oku et al34 found that transient hypothermia is associated with a more pronounced postischemic hyperemia but does not alter delayed hypoperfusion after global ischemia in dogs. Other authors failed to demonstrate any influence of brain temperature on CBF in rats subjected to global35 or focal36 ischemia. Interestingly, Kuluz et al37 observed in rats that during selective brain cooling, LCBF increased to 215% of baseline at a cortical brain temperature of 30.9°C and a rectal temperature of 37.5°C. They concluded that this probably occurs secondary to a decrease in cerebral vascular resistance, independent of the increased resistance of other vascular beds. It seems that the cerebrovascular response varies, depending on the depth of hypothermia, the method of cooling, the model of ischemia, and animal species. These differences may be the key factors in determining the local hemodynamic consequences of hypothermia.18
Morphological and Functional Outcome
Combination drug therapy with tirilazad and
MgCl2 reduced total infarct volume by 56%
compared with drug vehicle. This finding is consistent with the
results of an earlier study wherein we compared the neuroprotective
efficacy of tirilazad and MgCl2 alone and in
combination.7 The fact that tirilazad was more potent than
MgCl2 underlines the detrimental role that
reactive oxygen species play in brain damage after transient
ischemia. However, the combination of both drugs still resulted
in an overall enhanced neuroprotective effect with respect to infarct
volume and functional outcome. In the present study, hypothermia
was the most potent, single therapeutic approach, but the morphological
protection occurred mainly in the cortex and was not significant in the
basal ganglia. Also, the effect of the combined pharmacotherapy was
more prominent in the cortex than in the basal ganglia, which is
consistent with observations that antioxidants and
antiexcitotoxic drugs have maximal effects on the ischemic
penumbra, which is believed to be potentially
salvageable.38 39 Combined treatment with tirilazad,
magnesium, and hypothermia almost abolished cortical infarction and
reduced the infarct volume in the basal ganglia significantly by 55%.
No animal in this group had any residual neurological deficits on
postoperative day 7. In addition, this was the only group with a
statistically significant improved
electrophysiological recovery. The
hyperglycemic and hypotensive effect of MgCl2
present in this study had already been observed by several
investigators.32 40 Magnesium-induced hyperglycemia was
increased by hypothermia, probably by further suppression of insulin
release from pancreatic islet cells and increased
glycogenolysis.41 No evidence of hyperglycemia has been
found in human studies with magnesium,13 but hyperglycemia
and arterial hypotension might have reduced the efficacy of
this treatment strategy in our study.
Combination of Hypothermia and Pharmacotherapy
Most studies that evaluated the effects of hypothermia combined
with pharmacological neuroprotection were performed with NMDA
antagonists in models of global ischemia. MK-801
combined with moderate hypothermia (30°C) resulted in an overall
enhanced neuroprotection in rats after forebrain
ischemia.42 43 The combination of
postischemic hypothermia (30°C) and dextromethorphan, a
noncompetitive NMDA antagonist and calcium channel blocker,
was synergistically protective in a rat model of bilateral carotid
artery occlusion plus hypotension.44 The neuroprotective
activity of memantine, another noncompetitive NMDA
antagonist, was increased by hypothermia in a rat model of
forebrain ischemia.45 The efficacy of CGS-19755, a
competitive NMDA antagonist, was significantly enhanced
when combined with mild, intraischemic hypothermia (34°C) in
gerbils surviving up to 1 month after forebrain
ischemia.46 47 In contrast, protection with
postischemic hypothermia alone was not evident after 1
month.48 Similarly, Coimbra et al49 found
that treatment with postischemic hypothermia alone delayed
neuronal damage after global ischemia in rats, but combined
treatment with hypothermia and dipyrone, an anti-inflammatory drug,
diminished neuronal damage by >50% at both 7 days and 2 months of
recovery.
There is little published information about the effects of combined hypothermia and pharmacological protection in focal ischemia. In a rat permanent focal ischemia model, it appears that hypothermia and MK-801 offer similar cerebroprotective effects when administered separately but do not yield additive effects when used in combination.50 In transient focal ischemia, mannitol provided no further significant protection to hypothermia in rats,51 and triple therapy with hypertension, mannitol, and hypothermia in rabbits resulted in a smaller infarct volume than each single therapy alone, but the difference was not significant.52
In summary, it is not possible from our study to point to a single mechanism that underlies the marked neuroprotection provided by combination pharmacotherapy and hypothermia. Because tirilazad and magnesium increased the neuroprotective efficacy of hypothermia despite its multiple protective mechanisms, it is possible that the main mechanisms responsible for hypothermia-induced neuroprotection are not antioxidative or antiexcitotoxic. For a review of alternative mechanisms, see Colbourne et al16 and Dietrich et al.18 The remarkable benefit is likely due to a multitude of actions, and this may explain why the effectiveness of each single therapy is limited. Interestingly, low levels of serum ionized magnesium have been found in patients early after stroke, which resulted in a rapid elevation of cytosolic free calcium and spasm in cerebral vascular muscle cells.53 In addition to magnesium's role in the regulation of neurotransmitter-linked ionotropic channels, magnesium is essential for many critical cellular processes, including glycolysis, oxidative phosphorylation, ATPase function, membrane integrity, and protein synthesis.54 Therefore, any decline in free magnesium levels may adversely affect these processes.55 Likewise, superoxide dismutase activity in serum is reduced in stroke patients.56 Thus, when the endogenous defense mechanisms are overwhelmed, replacement could be beneficial in the acute treatment of cerebral ischemia.
Albeit a beneficial effect of tirilazad monotherapy in stroke patients could not be demonstrated,57 it reduced mortality rates and increased recovery in patients with aneurysmal SAH.12 A large, multicenter clinical trial testing the efficacy of magnesium (IMAGES: Intravenous Magnesium Efficacy in Stroke Trial) is underway and should report within 4 years.13 Achieving subnormal brain temperature is difficult in the awake stroke patient, but it is an option for comatose patients with severe ischemic stroke or patients undergoing cerebrovascular procedures.4 17 19
Conclusions
To our knowledge, this is the first report on combination
pharmacotherapy plus mild hypothermia in cerebral ischemia.
Even the most potent therapeutic approach, mild hypothermia, can be
improved by additional pharmacotherapy. In contrast to many
experimental agents, this treatment strategy offers the advantage that
its components are clinically available. This multifactorial strategy
might be an interesting candidate for clinical evaluation in
cerebrovascular surgery. Further experiments under the setting of
permanent focal ischemia and with a posttreatment regimen are
planed to define its role for stroke patients.
|
Acknowledgments |
|---|
This work was supported by the Deutsche Forschungsgemeinschaft (Schm1067/2-1) and Friedrich Baur Stiftung (to R.N. Plesnila, MD).
Received January 20, 1999; revision received April 29, 1999; accepted June 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Kristian T, Siesjö BK. Calcium in ischemic cell death. Stroke. 1998;29:705–718.
2. Bogousslavsky J, Dekeyser J, Diener HC, Fieschi C, Hacke W, Kaste M, Orgogozo JM, Pulsinelli W, Wahlgren NG. Neuroprotection as initial therapy in acute stroke: third report of an Ad Hoc Consensus Group Meeting. Cerebrovasc Dis. 1998;8:59–72.[Medline] [Order article via Infotrieve]
3.
Fisher M, Bogousslavsky J. Further evolution toward
effective therapy for acute ischemic stroke. JAMA. 1998;279:1298–1303.
4. Koroshetz WJ, Moskowitz MA. Emerging treatments for stroke in humans. Trends Pharmacol Sci. 1996;17:227–233.[Medline] [Order article via Infotrieve]
5. Zivin JA. Current status of new drug development for ischemic stroke. In: Hsu CY, ed. Ischemic Stroke: From Basic Mechanisms to New Drug Development. Monographs in Clinical Neuroscience Volume 16. Basel, Switzerland: Karger; 1998:128–138.
6. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen HJ. Monotherapy with dextromethorphan or tirilazad, but not a combination of both, improves outcome after transient focal cerebral ischemia in rats. Exp Brain Res. 1998;122:121–127.[Medline] [Order article via Infotrieve]
7. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A, Reulen HJ. Neuroprotective effects of combination therapy with tirilazad and magnesium in rats subjected to reversible focal cerebral ischemia. Neurosurgery. 1999;44:163–172.[Medline] [Order article via Infotrieve]
8. Hall ED, McCall JM, Means ED. Therapeutic potential of the lazaroids (21-aminosteroids) in acute central nervous system trauma, ischemia and subarachnoid hemorrhage. Adv Pharmacol. 1994;28:221–268.
9. Altura BT, Altura BM. Interactions of Mg and K on cerebral vessels: aspects in view of stroke: review of present status and new findings. Magnesium. 1984;3:195–211.[Medline] [Order article via Infotrieve]
10.
Villa RF, Gorini A. Pharmacology of lazaroids and brain
energy metabolism: a review. Pharmacol Rev. 1997;49:99–136.
11. Muir KW. New experimental and clinical data on the efficacy of pharmacological magnesium infusions in cerebral infarcts. Magnes Res. 1998;11:43–56.[Medline] [Order article via Infotrieve]
12. Kassell NF, Haley EC, Apperson-Hansen C, Stat M, Alves WM, Dorsch NW, Fabinyi G, Matheson J, Reilly P, Siu K, Stokes B, Stuart G, Koos W, Calliauw L, Selosse P, Astrup J, Gjerris F, Mendelow AD, Castel JP, Christiaens JL, Cophignon J, Keravel Y, Lagarrigue J, Mourier K, Philippon J, Brandt L, Vonessen C, Persson L, Brock M, Fahlbusch P, Gilsbach J, Hassler W, Perneczky A, Samii M, Schmiedek P, Mee E, Arista A, Cantore G, Carteri A, Collice M, Dapian R, Marini G, Menonna P, Baena RRY, Matteo PS, Testa PC, Villani R, Antunes JL. Randomized, double-blind, vehicle-controlled trial of tirilazad mesylate in patients with aneurysmal subarachnoid hemorrhage: a cooperative study in Europe, Australia, and New Zealand. J Neurosurg. 1996;84:221–228.[Medline] [Order article via Infotrieve]
13.
Muir KW, Lees KR. Dose optimization of
intravenous magnesium sulfate after acute stroke.
Stroke. 1998;29:918–923.
14.
Muir KW, Lees KR. Clinical experience with excitatory
amino acid antagonist drugs. Stroke. 1995;26:503–513.
15. Barone FC, Feuerstein GZ, White RF. Brain cooling during transient focal ischemia provides complete neuroprotection. Neurosci Biobehav Rev. 1997;21:31–44.[Medline] [Order article via Infotrieve]
16. Colbourne F, Sutherland G, Corbett D. Postischemic hypothermia: a critical appraisal with implications for clinical treatment. Mol Neurobiol. 1997;14:171–201.[Medline] [Order article via Infotrieve]
17.
Schwab S, Schwarz S, Spranger M, Keller E, Bertram M,
Hacke W. Moderate hypothermia in the treatment of patients with severe
middle cerebral artery infarction. Stroke. 1998;29:2461–2466.
18. Dietrich WD, Busto R, Globus MY, Ginsberg MD. Brain damage and temperature: cellular and molecular mechanisms. Adv Neurol. 1996;71:177–194.[Medline] [Order article via Infotrieve]
19. Hindman BJ, Todd MM, Gelb AW, Loftus CM, Craen RA, Schubert A, Mahla ME, Torner JC. Mild hypothermia as a protective therapy during intracranial aneurysm surgery: a randomized prospective pilot trial. Neurosurgery. 1999;44:23–33.[Medline] [Order article via Infotrieve]
20.
Schmid-Elsaesser R, Zausinger S, Hungerhuber E,
Baethmann A, Reulen HJ. A critical reevaluation of the intraluminal
thread model of focal cerebral ischemia: evidence of
inadvertent premature reperfusion and subarachnoid
hemorrhage by laser Doppler flowmetry.
Stroke. 1998;29:2162–2170.
21.
Lin TN, He YY, Wu G, Khan M, Hsu CY. Effect of brain
edema on infarct volume in a focal cerebral ischemia model in
rats. Stroke. 1993;24:117–121.
22. Karlsson BR, Loberg EM, Grogaard B, Steen PA. The antioxidant tirilazad does not affect cerebral blood flow or histopathologic changes following severe ischemia in rats. Acta Neurol Scand. 1994;90:256–262.[Medline] [Order article via Infotrieve]
23.
Xue D, Slivka A, Buchan AM. Tirilazad reduces cortical
infarction after transient but not permanent focal cerebral
ischemia in rats. Stroke. 1992;23:894–899.
24. Chi OZ, Pollak P, Weiss HR. Effects of magnesium sulfate and nifedipine on regional cerebral blood flow during middle cerebral artery ligation in the rat. Arch Int Pharmacodyn Ther. 1990;304:196–205.[Medline] [Order article via Infotrieve]
25.
Nishio A, Gebrewold A, Altura BT, Altura BM.
Comparative effects of magnesium salts on reactivity of arterioles and
venules to constrictor agents: an in situ study on microcirculation.
J Pharmacol Exp Ther. 1988;246:859–865.
26. Rosomoff HL, Holaday DA. Cerebral blood flow and cerebral oxygen consumption during hypothermia. Am J Physiol. 1954;179:85–88.
27. Hägerdal M, Harp J, Nilsson L, Siesjö BK. The effect of induced hypothermia upon oxygen consumption in the rat brain. J Neurochem. 1975;24:311–316.[Medline] [Order article via Infotrieve]
28.
Lo EH, Steinberg GK. Effects of hypothermia on evoked
potentials, magnetic resonance imaging, and blood flow in focal
ischemia in rabbits. Stroke. 1992;23:889–893.
29.
Steen PA, Soule EH, Michenfelder JD. Detrimental effect
of prolonged hypothermia in cats and monkeys with and without regional
cerebral ischemia. Stroke. 1979;10:522–529.
30. Astrup J, Skovsted P, Gjerris F, Sorensen HR. Increase in extracellular potassium in the brain during circulatory arrest: effects of hypothermia, lidocaine, and thiopental. Anesthesiology. 1981;55:256–262.[Medline] [Order article via Infotrieve]
31. Platner WS, Hosko MJ Jr. Mobility of serum magnesium in hypothermia. Am J Physiol. 1953;174:273–276.
32. Davis M, Perry RH, Mendelow AD. The effect of non-competitive N-methyl-D-aspartate receptor antagonism on cerebral oedema and cerebral infarct size in the aging ischaemic brain. Acta Neurochir Suppl Wien. 1997;70:30–33.[Medline] [Order article via Infotrieve]
33.
Baldwin WA, Kirsch JR, Hurn PD, Toung WS, Traystman
RJ. Hypothermic cerebral reperfusion and recovery from
ischemia. Am J Physiol. 1991;261:H774–H781.
34.
Oku K, Sterz F, Safar P, Johnson D, Obrist W, Leonov Y,
Kuboyama K, Tisherman SA, Stezoski SW. Mild hypothermia after cardiac
arrest in dogs does not affect postarrest multifocal cerebral
hypoperfusion. Stroke. 1993;24:1590–1597.
35.
Busto R, Globus MY, Dietrich WD, Martinez E, Valdes I,
Ginsberg MD. Effect of mild hypothermia on ischemia-induced
release of neurotransmitters and free fatty acids in rat brain.
Stroke. 1989;20:904–910.
36. Morikawa E, Ginsberg MD, Dietrich WD, Duncan RC, Kraydieh S, Globus MY, Busto R. The significance of brain temperature in focal cerebral ischemia: histopathological consequences of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1992;12:380–389.[Medline] [Order article via Infotrieve]
37.
Kuluz JW, Prado R, Chang J, Ginsberg MD, Schleien CL,
Busto R. Selective brain cooling increases cortical cerebral blood flow
in rats. Am J Physiol. 1993;265:H824–H827.
38. Morimoto T, Globus MY, Busto R, Martinez E, Ginsberg MD. Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab. 1996;16:92–99.[Medline] [Order article via Infotrieve]
39. Fukuyama N, Takizawa S, Ishida H, Hoshiai K, Shinohara Y, Nakazawa H. Peroxynitrite formation in focal cerebral ischemia-reperfusion in rats occurs predominantly in the peri-infarct region. J Cereb Blood Flow Metab. 1998;18:123–129.[Medline] [Order article via Infotrieve]
40. Izumi Y, Roussel S, Pinard E, Seylaz J. Reduction of infarct volume by magnesium after middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab. 1991;11:1025–1030.[Medline] [Order article via Infotrieve]
41. Steffen JM. Glucose, glycogen, and insulin responses in the hypothermic rat. Cryobiology. 1988;25:94–101.[Medline] [Order article via Infotrieve]
42. Dietrich WD, Lin BW, Globus MYT, Green EJ, Ginsberg MD, Busto R. Effect of delayed MK-801 (Dizocilpine) treatment with or without immediate postischemic hypothermia on chronic neuronal survival after global forebrain ischemia in rats. J Cereb Blood Flow Metab. 1995;15:960–968.[Medline] [Order article via Infotrieve]
43. Green EJ, Pazos AJ, Dietrich WD, McCabe PM, Schneiderman N, Lin B, Busto R, Globus MYT, Ginsberg MD. Combined postischemic hypothermia and delayed MK-801 treatment attenuates neurobehavioral deficits associated with transient global ischemia in rats. Brain Res. 1995;702:145–152.[Medline] [Order article via Infotrieve]
44. Ginsberg MD, Globus MY, Busto R, Dietrich WD. The potential of combination pharmacotherapy in cerebral ischemia. In: Krieglstein J, Oberpichler H, eds. Pharmacology of Cerebral Ischemia 1990. Stuttgart, Germany: Wissenschaftliche Verlagsgesellschaft mbH; 1990:499–510.
45. Krieglstein J, Seif el Nasr M, Lippert K. Neuroprotection by memantine as increased by hypothermia and nimodipine. Eur J Pharm Sci. 1997;5:71–77.
46. Shuaib A, Ijaz S, Mazagri R, Senthilsevlvan A. CGS-19755 is neuroprotective during repetitive ischemia: this effect is significantly enhanced when combined with hypothermia. Neuroscience. 1993;56:915–920.[Medline] [Order article via Infotrieve]
47. McCrea S, Shuaib A, Wishart T, Ijaz S. Mortality in gerbils with repetitive ischemia: CGS-19755/hypothermia therapy. Neurosci Lett. 1994;170:133–135.[Medline] [Order article via Infotrieve]
48. Shuaib A, Waqar T, Wishart T, Kanthan R. Post-ischemic therapy with CGS-19755 (alone or in combination with hypothermia) in gerbils. Neurosci Lett. 1995;191:87–90.[Medline] [Order article via Infotrieve]
49.
Coimbra C, Drake M, Boris Moller F, Wieloch T.
Long-lasting neuroprotective effect of postischemic
hypothermia and treatment with an anti-inflammatory/antipyretic drug:
evidence for chronic encephalopathic processes following
ischemia. Stroke. 1996;27:1578–1585.
50. Frazzini VI, Winfree CJ, Choudhri HF, Prestigiacomo CJ, Solomon RA. Mild hypothermia and MK-801 have similar but not additive degrees of cerebroprotection in the rat permanent focal ischemia model. Neurosurgery. 1994;34:1040–1045.[Medline] [Order article via Infotrieve]
51. Karibe H, Zarow GJ, Weinstein PR. Use of mild intraischemic hypothermia versus mannitol to reduce infarct size after temporary middle cerebral artery occlusion in rats. J Neurosurg. 1995;83:93–98.[Medline] [Order article via Infotrieve]
52. Ogilvy CS, Chu D, Kaplan S. Mild hypothermia, hypertension, and mannitol are protective against infarction during experimental intracranial temporary vessel occlusion. Neurosurgery. 1996;38:1202–1209.[Medline] [Order article via Infotrieve]
53. Altura BT, Memon ZI, Zhang A, Cheng TP, Silverman R, Cracco RQ, Altura BM. Low levels of serum ionized magnesium are found in patients early after stroke which result in rapid elevation in cytosolic free calcium and spasm in cerebral vascular muscle cells. Neurosci Lett. 1997;230:37–40.[Medline] [Order article via Infotrieve]
54. Grubbs RD, Maguire ME. Magnesium as a regulatory cation: criteria and evaluation. Magnesium. 1987;6:113–127.[Medline] [Order article via Infotrieve]
55. Heath DL, Vink R. Neuroprotective effects of MgSO4 and MgCl2 in closed head injury: a comparative phosphorus NMR study. J Neurotrauma. 1998;15:183–189.[Medline] [Order article via Infotrieve]
56.
Spranger M, Krempien S, Schwab S, Donneberg S, Hacke W.
Superoxide dismutase activity in serum of patients with acute cerebral
ischemic injury: correlation with clinical course and infarct
size. Stroke. 1997;28:2425–2428.
57.
The RANTTAS investigators. A randomized trial of
tirilazad mesylate in patients with acute stoke (RANTTAS).
Stroke. 1996;27:1453–1458.
Editorial Comment
A Promising Treatment Strategy for Reversible, Focal Cerebral Ischemia
Department of Neurosciences, University of California, San Diego, La Jolla, California
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
It is well established that hypothermia can reduce stroke-induced neurological damage in animal models. Whether hypothermia produces a permanent reduction in the ultimate injury is not yet clear. However, even if hypothermia only delays the onset of permanent damage, it may well increase the "window of opportunity" for some other form of therapy.
Tirilazad is a drug that was tested extensively in animal stroke models1 and in clinical trials.2 3 It has free radical–scavenging properties that were thought to be its mechanism of action. Although the preclinical studies were encouraging, the trials for acute stroke therapy were disappointing, and the drug was shown not to be a useful form of therapy for ischemic stroke. It is possible that for patients, the drug has beneficial effects that are too small to be detected in the types of clinical trials that were conducted.
Magnesium has also been shown to be effective in reducing neurological damage in several animal models.4 This is presumably related to its effects in reducing glutamate-related neurotoxicity. The safety of MgCl2 is well established, since it has been used for many years as a treatment for eclampsia. It is now being tested in a clinical trial for acute stroke.5
The article by Schmid-Elsaesser and colleagues demonstrates that in a standard rat model of middle cerebral artery occlusion, the combination of drugs and hypothermia reduced damage better than did the individual agents. The authors suggest that this strategy may be useful as a method of preventing cerebral injury during cerebrovascular surgery. They also propose that this treatment regimen may be useful for acute stroke management.
A problem for use of this technique for acute stroke therapy is that the time required to induce hypothermia in patients is likely to be considerably longer than for rats. Humans have a much smaller surface-to-volume ratio than do rats, and therefore, surface cooling is much slower. To substantially increase the rate of hypothermia induction in humans, it will almost certainly be necessary to use some sort of invasive procedure, such as a heat exchanger to cool the circulation. Furthermore, people find even small decrements of core body temperature to be quite uncomfortable, and thus, they will have to be sedated in some fashion.
It has become a common expectation that combination therapy is likely to be better than monotherapy for treatment of strokes, but it is far from certain that many combinations will be effective. For example, hypothermia may reduce the efficacy of some neuroprotective drugs if the beneficial effects of therapy are substantially diminished by a decreased metabolic rate. Therefore, careful testing of combinations will be essential.
Received January 20, 1999; revision received April 29, 1999; accepted June 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
|---|
1. Hall ED, Braughler JM, McCall JM. Role of oxygen radicals in stroke: effects of the 21-aminosteroids (lazaroids): a novel class of antioxidants. Prog Clin Biol Res. 1990;361:351–362.[Medline] [Order article via Infotrieve]
2. Haley EC, Kassell NF, Alves WM, Weir BK, Hansen CA. Phase II trial of tirilazad in aneurysmal subarachnoid hemorrhage: a report of the Cooperative Aneurysm Study. J Neurosurg. 1995;82:786–790.[Medline] [Order article via Infotrieve]
3.
Johnston KC, Li JY, Lyden PD, Hanson SK, Feasby TE,
Adams RJ, Fraught RE, Haley EC Jr. Medical and neurological
complications of ischemic stroke: experience from the RANTTAS
trial: RANTTAS investigators. Stroke. 1998;29:447–453.
4. Muir KW. New experimental data on the efficacy of pharmacological magnesium infusions in cerebral infarcts. Magnes Res. 1998;11:43–56.
5. Muir KW, Lees KR. Dose optimization of intravenous magnesium sulfate after acute stroke. Stroke. 1998;29:918–923.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||