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(Stroke. 2003;34:2246.)
© 2003 American Heart Association, Inc.

Original Contributions

Combination Drug Therapy and Mild Hypothermia After Transient Focal Cerebral Ischemia in Rats

Stefan Zausinger, MD; Karsten Schöller, MD; Nikolaus Plesnila, MD Robert Schmid-Elsaesser, MD, PhD

From the Department of Neurosurgery (S.Z., K.S., R.S.-E.) and Institute for Surgical Research (N.P.), Ludwig-Maximilians-Universität, Klinikum Grosshadern, Munich, Germany.

Correspondence to Dr Stefan Zausinger, Department of Neurosurgery, Ludwig-Maximilians-University, Klinikum Grosshadern, Marchioninistr 15, 81377 Munich, Germany. E-mail Stefan.Zausinger{at}nc.med.uni-muenchen.de

	Abstract

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Background and Purpose— We have recently demonstrated that pretreatment with magnesium (calcium and glutamate antagonist) and tirilazad (antioxidant) in combination with intraischemic mild hypothermia (33°C) (MTH) offers superior neuroprotective efficacy in a rat model of focal transient cerebral ischemia. In the present study, we investigated the time window of this treatment strategy with a posttreatment regimen to define its role for stroke patients.

Methods— We subjected 48 Sprague-Dawley rats to 90 minutes of middle cerebral artery occlusion by an intraluminal filament. Bilateral regional cerebral blood flow was continuously recorded by laser Doppler flowmetry. Combination therapy with MTH was started at 0, 1, 3, and 5 hours after induction of ischemia. Drugs were given in 1-hour intervals, and hypothermia was maintained for 2 hours. Neurological deficits were assessed daily. Infarct size was planimetrically determined on postoperative day 7.

Results— Combination therapy with MTH significantly reduced infarct volume compared with normothermic controls by -74%, -49%, and -45% when applied at 0, 1, and 3 hours after induction of ischemia. Furthermore, these treatment groups showed less neurological deficits on postischemic days 1 and 2 (P<0.05). Onset of treatment 5 hours after middle cerebral artery occlusion failed to significantly reduce infarct formation and neurological deficits.

Conclusions— The therapeutic window of the new combination therapy is at least 3 hours after onset of ischemia, comparable to that of moderate hypothermia (30°C), a grade of hypothermia associated with higher risks of severe side effects.

Key Words: cerebral ischemia, focal • drug therapy, combination • hypothermia • neuroprotection • therapeutics • rats

	Introduction

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Acritical factor in developing a therapeutic strategy against focal ischemic cerebral damage is the therapeutic time window available. This is important not only in the clinical situation of stroke treatment but also for neurosurgical patients. Thrombosis, embolism, vasospasm, or dissection of a brain-supplying artery with subsequent stenosis or occlusion can occur as a result of intraoperative manipulation, initially unrecognized by the surgeon. Because the onset of ischemia can occur during the operation, hours can pass until the operation is finished and the clinical signs are recognizable or, in the case of persistent unconscious patients, until signs appear on MR or CT scans.

Animal research and clinical studies in patients^1,2 suffering from focal cerebral ischemia suggest that moderate hypothermia may improve outcome by attenuating the deleterious metabolic processes in neuronal injury. It has been speculated that if the therapeutic window of mild hypothermia can be increased by additional pharmacotherapy, respective hypothermia could extend the therapeutic window of other treatment modalities.^3–5

We have shown that combined administration of tirilazad (antioxidant) and magnesium (calcium and glutamate antagonist) provides an overall enhanced neuroprotective effect in rats subjected to transient focal cerebral ischemia.⁶ Furthermore, the neuroprotective potential of intraischemic mild hypothermia was significantly increased if combined with pretreatment with magnesium and tirilazad.⁷ This combined approach—magnesium, tirilazad, and hypothermia (MTH)—offered superior neuroprotective efficacy compared with the customary treatment options used in neurosurgery during temporary occlusion of a cerebral artery.⁸

The objective of this study was to determine the neuroprotective efficacy of this newly developed treatment strategy in a posttreatment regimen to define its therapeutic window with regard to a possible use for stroke patients.

	Materials and Methods

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We studied 48 male Sprague-Dawley rats (270- to 300-g body weight). Animals were purchased from Charles River Laboratory and cared for before and at all stages during the experiment in compliance with applicable institutional guidelines and regulations of the government of Bavaria.

Animal Preparation and Monitoring
After induction of anesthesia with 4% halothane, the animals were intubated and ventilated with 0.8% halothane in a mixture of 70% N₂O and 30% O₂ under maintenance of normal blood gases as previously described.⁹ Temporalis muscle and rectal temperature (probe inserted 6 cm into the rectum¹⁰) were monitored and regulated throughout the experiment by use of a heating lamp and pad. In the hypothermic groups, whole-body hypothermia was induced with the use of ice packs until 33°C rectal and temporalis muscle temperatures were reached and maintained. Before induction of ischemia, a 20-minute interval was allowed for physiological stabilization. Rewarming (1°C every 10 minutes) was started 30 minutes after reperfusion. Femoral vein and tail artery were cannulated for blood sampling, monitoring of arterial blood pressure, and administration of fluids and drugs. Blood gases, serum glucose, hemoglobin, and hematocrit were measured before, during, and after ischemia. Continuous laser Doppler flowmetry (LDF; MBF3D, Moor Instruments Ltd, England) was used to monitor local cerebral blood flow (LCBF) in the area of 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 animal was placed supine, and the head was firmly immobilized in a stereotaxic frame. A micromanipulator was used to position a rectangularly bent LDF probe over each brain hemisphere. Cortical CBF was continuously measured (2-Hz sampling rate) from before the onset of ischemia until 1 hour after reperfusion.

MCA Occlusion
All animals were subjected to 90 minutes of MCA occlusion by introduction of a silicone-coated 4-0 nylon monofilament inserted via the external carotid artery as previously described.¹¹ Briefly, the filament was advanced until LDF showed a sharp decrease in the ipsilateral LCBF to 20% of baseline, indicating adequate occlusion of the MCA. Five animals were excluded and replaced because LDF of the contralateral hemisphere also showed a sharp drop on filament insertion, indicating vessel perforation with subsequent subarachnoid hemorrhage. Reperfusion was established by withdrawal of the filament back into the external carotid artery after 90 minutes.

Drug Administration and Treatment Arms
Animals were randomly assigned to 1 of 5 groups: (1) normothermic vehicle (3.0 mL/kg of 0.02 mol/L citric acid), treated controls (n=12) or (2) MgCl₂ (Sigma-Aldrich Chemie) (2x1 mmol · L^-1 · kg^-1)+tirilazad (Freedox, Upjohn; 2x3 mg/kg)+hypothermia (33°C) (MTH) beginning at ischemia onset (n=10) or after a delay of (3) 1 hour (n=10), (4) 3 hours (n=10), or (5) 5 hours (n=6). Drugs were given in 1-hour intervals, and hypothermia was maintained for 2 hours. Animals in the control group received 4.5 to 7.5 hours of halothane-anesthesia proportionally to the duration of anesthesia of the various treatment groups. A study flow diagram is presented in Figure 1.

View larger version (24K): [in this window] [in a new window]   Figure 1. Study flow diagram of the acute phase depicting the course of experimental ischemia and timing of hypothermia, drug administration, and monitoring.

Neurological Evaluation
Functional neurological deficits and infarct volumes were assessed by a person blinded to treatment. Postoperatively, the neurological function of all animals was evaluated daily with a 6-point scale¹²: 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.

Quantification of Ischemic Damage
Seven days after ischemia, the brains were subjected to perfusion fixation, cut into 24 coronal sections in 400-µm intervals, and stained with hematoxylin and eosin. The infarct areas were assessed planimetrically (OPTIMAS 5.1, BioScan Inc). Total infarct volume (I_T) expressed (in mm³) was calculated as the sum of the infarct areas of all slices (I_n) multiplied by the distance (400 µm) between successive slices [I_T=0.4(I₁+I₂+...I₂₄) mm³]. The infarct volume of cortex and basal ganglia was determined by measuring the area of infarct in sections obtained 2, 3.6, 5.2, 6.8, and 8.4 mm from the frontal pole. Cortex and basal ganglia were determined according to a stereotactic atlas of rat brain.¹³

Statistical Analysis
Statistical analysis was performed with SigmaStat 2.0 Statistical Software (Jandel Scientific). The physiological data of each time point and the infarct volumes were analyzed by 1-way analysis of variance (ANOVA), the LDF data by 2-way ANOVA for repeated measures, and the neurological function scores by Kruskal-Wallis ANOVA on ranks for each of the 7 postoperative days. If multiple comparisons were indicated, Dunnett’s test or the Student-Newman-Keuls test for neurological function scores was applied. Differences were considered significant at P<0.05. Results are presented as mean±SD.

	Results

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Mortality
One animal in the control group with >5 hours of halothane-anesthesia died within the first 2 days after ischemia. Two animals in group 5 (start of therapy 5 hours after induction of ischemia) died within 1 day after ischemia. In light of the increased mortality and missing improvement in neurological recovery, group 5 was terminated after 6 animals because of restrictions formulated in the permission to conduct animal experiments.

Physiological Variables
The experimental groups did not differ with respect to preischemic, intraischemic, or postischemic blood pressure, arterial blood gases, hemoglobin, or hematocrit. As previously observed,⁸ blood glucose levels significantly increased after drug administration in hypothermic rats receiving MgCl₂, whereas blood glucose decreased in the vehicle-treated normothermic animals. Blood pressure dropped by 10 to 15 mm Hg during infusion of MgCl₂ without significantly influencing the mean values (the Table).

View this table: [in this window] [in a new window]   Physiological Variables

Laser Doppler Flowmetry
Within the normothermic control group, MCA occlusion resulted in an immediate reduction in LCBF to 20% of baseline in the territory supplied by the ipsilateral artery, whereas contralateral blood flow remained unchanged throughout the experiment. A short period of postischemic hyperemia evolved on reperfusion, followed by a decrease in ipsilateral LCBF to 70% baseline. The delayed hypoperfusion persisted until the end of the postischemic observation period. In all treated groups, the contralateral LCBF significantly decreased to 80% during cooling and recovered to baseline with rewarming. In group 2 with the start of treatment at MCA occlusion, ipsilateral flow recovered to 85% at reperfusion, whereas with the beginning of rewarming 30 minutes later, ipsilateral flow further improved gradually to 105%, which was significantly higher compared with normothermic controls. In the groups with a later start of hypothermia, ipsilateral flow fell significantly compared with controls by 20% during cooling and recovered with rewarming to its level (80% baseline) before cooling, again reaching an LCBF comparable to that of controls.

Functional Outcome
Animals treated with MTH with treatment delayed until 3 hours after induction of MCA occlusion showed a significantly improved neurological recovery on postischemic days 1 and 2 (P<0.05). These groups recovered better than the control group over the complete observation period of 7 days but missed significance at later time points. Initiation of treatment 5 hours after induction of MCA occlusion resulted in only minimal improvement without significance compared with controls (Figure 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Neurological recovery during 7 days after ischemia. Nonparametric data are presented as line-and-scatter plot for clarity. *P<0.05 vs vehicle-treated controls at corresponding phase of the study for each day (Kruskal-Wallis ANOVA on ranks followed by the Student-Newman-Keuls test).

Infarct Volume
There was no difference in brain size between 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.¹⁴ The lesions involved mainly the frontoparietal cortex, thalamic region, lateral caudoputamen, and internal capsule.

Total infarct volume was 84.5±37.3 mm³ in vehicle-treated controls, 21.9±17.7 mm³ (-74%) in animals treated with hypothermia and drug administration at onset of ischemia, 43.1±30.3 mm³ (-49%) in animals treated after a delay of therapy of 1 hour, 46.7±42.4 mm³ (-45%) with therapy delayed 3 hours, and 62.2±31.1 mm³ (-26%) with therapy delayed 5 hours. In groups with up to 3 hours of delay, the total infarct volumes were significantly (P=0.004) smaller than in the control group (Figure 3).

View larger version (18K): [in this window] [in a new window]   Figure 3. Total infarct volume at 7 days after 90 minutes of MCA occlusion of untreated normothermic control animals vs animals treated with MTH at various time points. Infarct volume (mm3) is expressed as mean±SD. *P<0.05 vs vehicle-treated controls (1-way ANOVA followed by Dunnett’s test).

In the separate analysis of cortical brain tissue and basal ganglia, we found that administration of MTH significantly (P=0.005) attenuated cortical infarction if applied up to 3 hours after onset of ischemia. Cortical infarct volume was 40.1±26.2 mm³ in vehicle-treated controls, 5.4±11.3 mm³ (-87%) in animals treated at onset of ischemia, 12.6±18.1 mm³ (-69%) in those with a delay of 1 hour, 14.5±26.4 mm³ (-64%) those with a delay of 3 hours, and 38.3±21.2 mm³ (-5%) in the 4 surviving animals with a delay of 5 hours.

Compared with vehicle-treated controls (40.7±17.3 mm³), infarction in the basal ganglia in rats treated at onset of ischemia was significantly (P=0.025) limited to 15.5±11.7 mm³ (-59%). Longer delays of treatment initiation of 1 to 5 hours did not result in a significant attenuation of infarction within the basal ganglia (27.6±10.1 to 30.5±16.2 mm³ (-25% to -32%) (Figure 4).

View larger version (24K): [in this window] [in a new window]   Figure 4. Treatment effects on infarct volume in the cortex (Co) and basal ganglia (BG) 7 days after MCA occlusion. *P<0.05 vs normothermic vehicle-treated controls (1-way ANOVA followed by Dunnett’s test).

	Discussion

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We found that our combined approach, mild hypothermia of 33°C maintained for 2 hours combined with MgCl₂ and tirilazad (MTH), significantly reduced morphological damage and improved neurological recovery even if applied 180 minutes after the onset of ischemia. The therapeutic window of MTH is comparable to that of moderate hypothermia (30°C) alone,¹⁵ a grade of hypothermia associated with higher risks of severe side effects.¹⁶ The pathophysiological and therapeutical implications of these treatment modalities alone and in combination have been discussed in detail.^6–8 All components are clinically licensed, making this combination therapy a promising candidate for clinical evaluation.

Most experimental studies on hypothermia in focal cerebral ischemia were performed in rats with 120-minute MCA occlusion and mild hypothermia of 32°C to 33°C for 1 to 5 hours.^4,15,17–22 Although application of intraischemic hypothermia—the minimum effective duration seems to be 1 hour²⁰—evolved to be neuroprotective in all of these studies, the capacity of mild hypothermia to provide a protective effect when administered after the onset of ischemia seems to be limited. Data in animal models suggest that irreversible ischemic injury begins as early as 5 minutes and that the damage is complete within 6 hours.^23–25 Investigating the therapeutic window of mild hypothermia, Kawai et al¹⁹ found reduced ischemic brain damage when 4 hours of postischemic mild hypothermia was applied 90 minutes after onset of ischemia. Huh et al¹⁷ and Maier et al⁴ even saw significant neuroprotection when mild hypothermia for 3 and 2 hours, respectively, began 120 minutes after the onset of ischemia. In the latter study, a further delay in the start of hypothermia up to 180 minutes failed to improve behavioral deficits or histopathological changes. On the other hand, Kollmar et al²² and Zhang et al¹⁵ found significant neuroprotective effects of hypothermia, even if applied 3 hours after onset of 120 minutes of MCA occlusion in rats. However, these groups used 5 hours of mild hypothermia (33°C) and applied deeper hypothermia (30°C) for 3 hours, respectively. An important point is that cellular damage seems to be blocked and not merely delayed by mild hypothermia.^4,26

The fact that significant neuroprotection was achieved even with delayed application of MTH cannot be attributed to prolonged halothane-anesthesia. Postischemic application of halothane does not lead to enhanced neuroprotection,²⁷ nor did we observe differences in the physiological variables between treatment groups. Furthermore, consequent control of adequate MCA occlusion by continuous LDF was accomplished within all groups over the complete duration of the experiment, ruling out the severe effects of filament dislocation with intermittent reperfusion or vessel perforation.¹¹

Therefore, it is conceivable that pharmacotherapy-related mechanisms of MTH are responsible for a therapeutic window over several hours. It was shown that the therapeutic window of magnesium in rats subjected to focal cerebral ischemia by embolization of the MCA with an autologous thrombus is 6 hours after onset of ischemia.²⁸ Small clinical trials in acute stroke have reported reduced odds of death or dependence with administration of magnesium; definitive data from ongoing larger trials are awaited.^29,30 Although the RANTTAS trial of tirilazad, administered beginning at a median of 4.3 hours after stroke, was halted in 1996 when an interim analysis indicated that tirilazad was unlikely to provide benefit,³¹ experimental data suggest a therapeutic window of at least 3 hours after the onset of transient focal ischemia.³² It has been speculated that a reason for the failure of tirilazad in clinical stroke trials—while exhibiting protective effects after subarachnoid hemorrhage³³—was the fact that more than three quarters of patients received tirilazad >3 hours after stroke.³⁴ Furthermore, tirilazad seems to exert its beneficial properties predominantly under conditions of transient but not permanent focal cerebral ischemia.³⁵ This is consistent with findings that free radical mechanisms may be active mainly in the aggravation of injury in the ischemic penumbra under conditions of reperfusion.^36,37

In the clinical situation, reperfusion within 3 hours after stroke becomes more and more likely; clot lysis with tissue plasminogen activator is increasingly used for patients with embolic occlusion of a cerebral artery.³⁸ Christou and coworkers³⁹ administered a tissue plasminogen activator bolus at 132±54 minutes from symptom onset. Recanalization on transcranial Doppler sonography was found at the mean time of 251±171 minutes after stroke onset. Furthermore, there are more reports of methods to induce comparably rapid cooling down of the patients. Schwab et al⁴⁰ used cooling blankets, alcohol, and ice bags in 50 patients with cerebral infarction. Time required for cooling to <33°C varied from 3.5 to 11 hours. Kammersgaard et al⁴¹ examined feasibility and safety of inducing modest hypothermia in awake patients with acute stroke through surface cooling. Patients were given hypothermic treatment for 6 hours by the "forced air" method, a surface cooling method that uses a cooling blanket with a flow of cool air (10°C). Hypothermia (35.5°C) was present until 4 hours after therapy. Georgiadis et al⁴² evaluated the feasibility of inducing and maintaining moderate hypothermia with the use of endovascular rather than surface cooling. Hypothermia was induced by circulating temperature-adjusted normal saline in a closed-loop system entailing 3 balloons located near the tip of a central line, which dwelled in the inferior vena cava. The mean initial temperature of the patients was 37±1°C. The pace of cooling was 1.4±0.6°C/h, and target temperature was reached after 3±1 hours. Singultus was the only device-related complication encountered. Pulmonary infection, arterial hypotension, bradycardia, arrhythmia, and thrombocytopenia were the most common side effects of hypothermia.

In conclusion, our data suggest that patients suffering from focal cerebral ischemia who come to treatment hours after the initial insult might profit from delayed administration of MTH. In contrast to many other experimentally studied agents, MTH has the advantage that all of its components are clinically available with tolerable side effects. Considering that, apart from thrombolytic therapy, 1 agent alone has never been shown to significantly reduce cerebral infarction in humans, this combination therapy may be a promising candidate for clinical evaluation in stroke patients.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (Schm1067/2–1) and Friedrich Baur Stiftung.

Received September 20, 2002; revision received March 25, 2003; accepted April 22, 2003.

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