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(Stroke. 1997;28:2539-2544.)
© 1997 American Heart Association, Inc.

Articles

The Calmodulin Antagonist Trifluoperazine in Transient Focal Brain Ischemia in Rats

Anti-ischemic Effect and Therapeutic Window

Satoshi Kuroda, MD; Akihito Nakai, MD; Tibor Kristían, PhD; Bo K. Siesjö, MD, PhD

From the Division of Experimental Brain Research, Department of Clinical Neuroscience, Wallenberg Neuroscience Center, University of Lund (Sweden) (S.K., A.N., T.K., B.K.S.); the Department of Neurosurgery, Hokkaido University School of Medicine, Sapporo, Japan (S.K.); the Department of Obstetrics and Gynecology, Nippon Medical School, Tokyo, Japan (A.N.); and the Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic (T.K.).

Correspondence to Satoshi Kuroda, MD, Department of Neurosurgery, Hokkaido University School of Medicine, North 15 West 7, Kita-ku, Sapporo 060, Japan.

	Abstract

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Background and Purpose This study was performed to assess the efficacy and the therapeutic window for the calmodulin antagonist trifluoperazine in experiments involving transient middle cerebral artery (MCA) occlusion.

Methods Male Wistar rats were subjected to transient (2 hours) MCA occlusion by an intraluminal filament technique. Trifluoperazine (5.0 mg · kg^-1) was injected intraperitoneally 5 minutes, 1 hour, or 2 hours after the induction of ischemia. Drug administration was repeated 24 hours after the first injection. Neurological scores and infarct volumes were evaluated at 48 hours of reperfusion. The effect of trifluoperazine on cortical blood flow was studied with continuous laser-Doppler flowmetry.

Results The median value of neurological scores in the control rats (n=7) was 3, while those in the treated groups were 1 (5-minute group; n=7, P<.05) and 2 (1-hour and 2-hour groups; each n=7). The percentage of infarct volume in the control rats was 34.8±4.9% (mean±SD), while those in the treated groups were 11.3±12.3% (5-minute group; P<.01), 24.8±15.1% (1-hour group), and 28.8±8.3% (2-hour group). Trifluoperazine, given at 5 minutes after ischemia, had no influence on blood flow in the neocortical penumbra during and after ischemia.

Conclusions The results demonstrate that trifluoperazine markedly reduces infarct volume after 2 hours of MCA occlusion when given 5 minutes after the induction of ischemia. However, the therapeutic window for trifluoperazine seems narrow since the drug had no significant effect when given after 1 or 2 hours.

Key Words: calcium • calmodulin • cerebral ischemia, focal • middle cerebral artery occlusion • rats

	Introduction

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It is widely held that uncontrolled calcium influx into cells during ischemia is a trigger of neuronal and tissue damage.^1–5 Calcium probably plays an important pathogenetic role by activating Ca²⁺-dependent enzymes such as phospholipases, proteases, and endonucleases and by changing the phosphorylation of proteins. Some of the key enzymes incriminated in this adverse series of events are calmodulin-dependent ones, such as phospholipase A₂ and nitric oxide synthase. This has led to the belief that calcium influx and its binding to calmodulin trigger reactions leading to ischemic cell death. In support of this contention are results showing that calmodulin antagonists, such as trifluoperazine, ameliorate tissue damage due to ischemia/reperfusion, notably in heart tissue.^6–10

Like global or forebrain ischemia, focal ischemia due to MCA occlusion leads to a marked perturbation of cell calcium metabolism. This perturbation involves not only translocation of extracellular Ca²⁺ into intracellular fluids, either permanently (the focus) or intermittently (the penumbra),^11–13 but also an increase in the tissue calcium content secondary to a net influx of Ca²⁺ from blood to cerebral tissues. This occurs during long-lasting ischemia and may continue during reperfusion when extracellular concentration remains reduced.^14,15 Clearly, this influx may grossly aggravate the perturbation of cell calcium metabolism.

One can envisage a series of harmful events, which starts with the influx of calcium into cells, continues with Ca²⁺-dependent activation of metabolic sequences, and ends with ischemic cell death due to the secondary effects of such sequences. Strategies for defining useful therapeutic principles are based on such schemes. For example, drugs have been developed that retard Ca²⁺ influx into cells through voltage-sensitive and agonist-operated calcium channels. However, the therapeutic windows of such drugs are narrow.^5,16–18 Other drugs, like the calmodulin antagonists, act on subsequent steps triggered by Ca²⁺ influx. However, the therapeutic window remains to be defined, and little is known about their effects in brain ischemia.

Recent results suggest that certain therapeutic principles act when instituted after the induction of transient focal ischemia. For example, the spin trap nitrone (PBN) markedly reduces infarct volume, even when given 1 to 3 hours after the start of recirculation, following 2 hours of MCA occlusion.¹⁹ This finding and the metabolic data reported by Folbergrová et al (1995)²⁰ led to the concept of a second window of therapeutic opportunity.^21,22 One could thus envisage that drugs blocking or retarding Ca²⁺ influx act in the first therapeutic window, which is narrow (<1 hour after the induction of ischemia), while PBN acts in a second window, which is wide (3 to 5 hours after the induction of ischemia). Subsequent results showed that the immunosuppressant FK506 acted similarly, ie, it reduced infarct volume when given 1 hour after the start of recirculation, following 2 hours of MCA occlusion.²³ Amelioration of tissue damage by PBN and FK506 is accompanied by improvement of mitochondrial function, as studied in vitro.^24,25

The present study had two objectives. The first one was to assess the therapeutic efficacy of trifluoperazine in transient focal ischemia, the second to define its therapeutic window. Essentially, the question posed was whether trifluoperazine, if forced to be efficacious, acted in the first or second therapeutic window, as defined above.

	Materials and Methods

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Transient MCA Occlusion
Transient MCA occlusion was induced, according to the technique of Koizumi et al²⁶ (1986) as described previously.^24,27,28 Briefly, male Wistar rats (body weight, 300 to 340 g) were fasted overnight but had free access to water. Anesthesia was induced by inhalation of 3% halothane in N₂O/O₂ (70%:30%), and then the animals were intubated. They were ventilated on 1.0% to 1.5% halothane in N₂O/O₂ during operation. The tail artery was cannulated to monitor arterial blood gases, pH, blood glucose, and blood pressure, and 0.1 mL of heparin (300 U · mL^-1) was given just before induction of ischemia. The right common, internal, and external carotid arteries were exposed through a ventral midline neck incision. The external carotid artery was ligated. The proximal common carotid artery was ligated, and the common carotid artery just proximal to its bifurcation was temporarily closed by a microvascular clip. A small incision was made in the common carotid artery, and a filament, which had a round tip and a distal cylinder of silicon rubber (diameter 0.28 mm), was introduced into the internal carotid artery through the common carotid artery. The filament was further advanced approximately 19 mm to occlude the origin of the MCA. Then the animals were extubated and allowed to awaken. Under light reanesthesia, the filament was withdrawn after 2 hours to allow recirculation. Core temperature was maintained at approximately 37.0°C during the operation. After the operation, the animals were cooled by an air-cooling system to avoid the hyperthermia that would otherwise occur.²⁸ Only rats that consistently circled toward the paretic side during 2 hours of MCA occlusion were included in this study. Neurological evaluations were performed at 48 hours of recirculation with the use of the neurological examination grading system described by Bederson et al.²⁹ All procedures followed the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals) and were approved by the Lund University animal ethics committee.

Treatment With Trifluoperazine
Trifluoperazine (Sigma) was dissolved in physiological saline. Four groups of animals (total, n=28) were studied: animals in the control group (n=7) received an intraperitoneal injection of saline 5 minutes after ischemia. In the other groups of animals, 5.0 mg · kg^-1 of trifluoperazine was given intraperitoneally at 5 minutes (group 2; n=7) or 1 hour (group 3; n=7) after induction of ischemia or just after recirculation following 2 hours of ischemia (group 4; n=7). Drug administration was repeated 24 hours after the first injection in the treated groups. The dosage is known to inhibit the activity of brain phospholipase A₂ up to 80% of the control over 48 hours after single intraperitoneal injection.³⁰

Evaluation of Infarct Volume With TTC Staining
The rats were anesthetized by inhalation of 3.0% halothane and killed by decapitation at 48 hours of recirculation. The brain was quickly removed and chilled in ice-cold saline for 10 minutes. Twelve 1-mm coronal slices were cut with a tissue slicer, beginning 1 mm posterior to the anterior pole, and the slices were immersed in a saline solution containing 1.0% TTC (Sigma) at 37°C for 30 minutes and fixed by immersion in 4.0% phosphate-buffered formalin solution.²³ Each brain slice was photographed with black and white film, and the unstained area in each photograph was quantified from the developed film, with the use of a CCD video camera and a video image analyzing system (NIH Image, version 1.55). The infarct area in each slice was calculated by subtracting the normal ipsilateral area from that of the contralateral hemisphere to reduce errors due to cerebral edema and was presented as the percentage of the infarct to the area of the contralateral hemisphere. The total infarct volume was determined by summing the infarct areas of the 12 slices.³¹

`Remote' MCA Occlusion and CBF Measurement
In a separate series of experiments, cortical CBF was continuously monitored by laser-Doppler flowmetry (Periflux PF3, PeriMed) to evaluate the CBF changes after trifluoperazine treatment. CBF monitoring was performed in a total of 9 animals: 5 control animals and 4 treated animals in which trifluoperazine was given 5 minutes after the induction of MCA occlusion.

The animals were kept under artificial ventilation with 1.0% to 1.5% of halothane and continuous infusion of a muscle relaxant (vecuronium bromide) through the experiments. To induce "remote" MCA occlusion of the animals, which were placed in a stereotaxic frame, a special occluding device was constructed, as described by Kohno et al³² (1995) and Röther et al³³ (1996), with some modifications. The occluder filament had a round tip and a distal cylinder of silicon rubber but was 20 cm in length, much longer than the usual occluder filament. Following the same preparation as the usual MCA occlusion, a polyethylene tube (ID 0.58 mm, OD 0.96 mm, length 16 cm) was introduced into the common carotid artery and was advanced just proximal to its bifurcation. The polyethylene tube was fixed to the common carotid artery by a ligature. The occluder filament was advanced to the skull base through the polyethylene tube. Then the animal was placed in a stereotaxic frame. A 2-mm-diameter burr hole was drilled in the skull 5.5 mm lateral to bregma on the ipsilateral side. This area corresponded to the neocortical penumbra.²⁵ The dura mater was exposed but was kept intact to prevent any cortical injury.

Cortical CBF was monitored 10 minutes before MCA occlusion to establish the control values. Remote MCA occlusion was induced by advancing the long filament to occlude the origin of the MCA. A sudden decrease of cortical CBF could be observed when the filament occluded the origin of the MCA. Cortical CBF was continuously monitored through the whole period of ischemia. After 2 hours of ischemia, the filament was withdrawn, and recovery of cortical blood flow was observed for 30 minutes. The changes in cortical CBF were expressed as percentage of the control level.

Statistical Analysis
All data were expressed as mean±SD. One-factor ANOVA followed by Scheffé's F test was used to compare the infarct volumes among the experimental groups. The Kruskal-Wallis test followed by the Mann-Whitney U test was used to compare the neurological scores among the experimental groups. The unpaired t test was performed to compare cortical CBF between control and treated groups at each time point. Differences with a value of P<.05 were considered statistically significant.

	Results

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Physiological Parameters
There were no significant differences in arterial PO₂, PCO₂, pH, glucose, and blood pressure between the experimental groups. No differences in core temperature were observed during the experiments (Table 1).

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters in Rats Subjected to 48-Hour Reperfusion After 2-Hour MCA Occlusion

Neurological Scores
Table 2 shows the effects of trifluoperazine on neurological deficits at 48 hours of reperfusion after 2 hours of MCA occlusion. Control rats showed severe neurological deficits at 48 hours of reperfusion, with median scores of 3. When the rats were treated with trifluoperazine 5 minutes after induction of ischemia, they showed significantly lower scores at 48 hours (median score 1; P<.05). In contrast, there was no significant difference of neurological scores between the control rats and the treated rats when they were treated with trifluoperazine 1 or 2 hours after induction of ischemia (median score 2 in both cases).

View this table: [in this window] [in a new window]   Table 2. Effect of Trifluoperazine on Neurological Scores at 48 Hours of Reperfusion After 2 Hours of MCA Occlusion

Infarct Volumes
Fig 1 demonstrates the effects of trifluoperazine on infarct volume at 48 hours of reperfusion. In the control rats, the infarct was detected in the lateral caudoputamen and the overlying cortex. The percentage of infarct volume varied from 25.6% to 42.5%, with a mean value of 34.8±4.9% of the contralateral hemisphere. Trifluoperazine treatment significantly reduced infarct volume when administered after 5 minutes of ischemia (11.3±12.3%; P<.01). Neocortical infarcts were not observed in 4 of 7 animals (57. 1%); in addition, infarction was not detected in the lateral caudoputamen in 2 of 7 animals (28.5%). However, no significant effects of the drug could be observed when the drug was injected 1 or 2 hours after ischemia. The infarct volumes were 24.8±15.1% and 28.8±8.3%, respectively.

View larger version (42K): [in this window] [in a new window]   Figure 1. Effect of trifluoperazine on infarct volume. Columns indicate the mean values in each group, and open circles represent the values of the individual animals. Trifluoperazine (5.0 mg · kg-1 IP) was injected 5 minutes, 1 hour, or 2 hours after the onset of ischemia. Drug administration was repeated 24 hours after the first injection. **P<.01 against control group (one-factor ANOVA followed by Scheffé's test).

Cortical CBF Measurement
Fig 2 shows relative changes of cortical CBF during ischemia in control and treated animals. Immediately after remote MCA occlusion, cortical CBF decreased to 29.5±7.4% and 35.6±10.3% of the control values, respectively. These decreased CBF values were sustained at 20% to 30% of the control values during the whole periods of ischemia in both groups. Then cortical CBF recovered to 83.1±13.1% and 97.8±18.4% after reperfusion, respectively. No significant changes in cortical CBF were observed after intraperitoneal injection of trifluoperazine.

View larger version (15K): [in this window] [in a new window]   Figure 2. Changes of cortical CBF during 2-hour MCA occlusion and subsequent 30-minute reperfusion. Focal ischemia was induced by a "remote" MCA occlusion (MCAO) technique, and cortical CBF was continuously monitored with laser-Doppler flowmetry. TFP indicates trifluoperazine; rCBF, regional cerebral blood flow.

	Discussion

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Roles of Calcium in Ischemic Brain Damage
The calcium ion (Ca²⁺) is considered an important mediator of ischemic brain damage.^1–5 Ischemia-induced energy failure results in Ca²⁺ influx into the neurons through NMDA receptor–modulated Ca²⁺ channels, which constitute important agonist-operated calcium channels, and through voltage-sensitive calcium channels. Ca²⁺ is also released from intracellular stores such as endoplasmic reticulum through the activation of the inositol 1,4,5-triphosphate receptor. Furthermore, an increase of intracellular Ca²⁺ is accelerated by ischemia-induced deactivation of the efflux systems of Ca²⁺, such as Ca²⁺-activated ATPase and the 3Na⁺/Ca²⁺ exchanger.³⁴ Increased intracellular Ca²⁺ can in part be sequestered by mitochondria,^35,36 but such mitochondrial sequestration of Ca²⁺ leads to uncoupling of respiration and generation of hydroxyl radicals, causing further damage to mitochondria.^14,22

Increased intracellular Ca²⁺ binds to calmodulin, one of the major intracellular binding proteins of Ca.²⁺ It is widely held that the Ca²⁺-calmodulin complex, in turn, triggers tissue-damaging cascades by changing the activities of Ca²⁺-calmodulin–dependent protein kinase II, protein kinase C, phospholipase A₂, proteases (for example, calpain and interleukin-1ß–converting enzyme), nitric oxide synthase, calcineurin, and endonucleases.^1,4,5,34 Indeed, Grotta et al³⁷ (1990) showed that Ca²⁺-calmodulin binding correlated with the severity of delayed neuronal necrosis after reperfusion after forebrain ischemia in rats.

Dense forebrain ischemia is accompanied by a precipitous rise in the intracellular Ca²⁺ concentration, but any rise in the total tissue or mitochondrial calcium content may be delayed by hours or days.^36,38–40 Increases in cellular calcium content and in intracellular Ca²⁺ concentration have also been observed in permanent^41,42 and transient^15,16 focal ischemia. Interestingly, intracellular Ca²⁺ further increases during reperfusion after transient (1 to 2 hours) focal ischemia.^15,16 Therefore, many experimental studies have been aimed at showing that ischemic brain damage due to focal ischemia can be inhibited by blocking Ca²⁺-mediated cascades.³⁴ However, it has recently been recognized that Ca²⁺ channel blockers¹⁶ and NMDA receptor antagonists such as MK-801 have a narrow window of therapeutic opportunity in focal cerebral ischemia.^5,17,18 Therefore, it is clinically of interest to determine whether drugs that can inhibit the Ca²⁺-calmodulin system have a wider therapeutic window than previously examined drugs blocking calcium influx.

The present study showed that trifluoperazine markedly reduced infarct volume when given 5 minutes after the induction of ischemia. Since trifluoperazine had no effects on CBF (Fig 2), its anti-ischemic action must be due to its direct actions on neuronal cells. However, trifluoperazine was not effective when given 1 or 2 hours after ischemia. These results suggest that trifluoperazine does not have as wide a therapeutic window against focal cerebral ischemia as PBN and FK506 and that the Ca²⁺-calmodulin complex and the cascades it elicits are activated at an early time, ie, less than 1 hour after the induction of ischemia.

Protective Effects of Phenothiazines
Phenothiazines such as chlorpromazine and trifluoperazine are known to have protective effects in ischemia/reperfusion in tissues such as heart and liver. Chien et al⁴³ (1977) reported that pretreatment with chlorpromazine reduced cellular and mitochondrial Ca²⁺ overload and ameliorated liver cell injury after ischemia/reperfusion. Pretreatment with either chlorpromazine or trifluoperazine improved functional recovery and reduced enzyme release from the tissue during reperfusion after ischemia in the isolated perfused heart.^6–8 Similar results have been obtained in in vivo experiments, when trifluoperazine was given 30 minutes after the induction of ischemia.^9,10 These drugs could also accelerate the recovery of energy metabolites and myocardial oxygen consumption during reperfusion^7,9,10 and preserve the ultrastructure of mitochondria.⁹ However, the drugs were not effective when treatments were started just before reperfusion.⁶

Both chlorpromazine and trifluoperazine were also reported to reduce neurological deficits in a model of rabbit multiple cerebral embolism, when they were given 5 minutes after embolization.⁴⁴ Yu et al⁴⁵ (1992) showed that pretreatment with a phenothiazine derivative, 2-(10H-phenothiazin-2-yloxy)-N,N-dimethylethanamine hydrochloride, moderately ameliorated brain damage due to forebrain ischemia and transient (2 hours), but not permanent, focal ischemia.

These protective effects of phenothiazines have, at least in part, been assumed to result from their inhibitory actions on lipid peroxidation due to the activated phospholipase A₂ during ischemia/reperfusion. For example, it has been shown that trifluoperazine prevented the release of malondialdehyde from the isolated heart subjected to ischemia/reperfusion.^10,46 It has also been reported that chlorpromazine and trifluoperazine could reduce the degradation of membrane phospholipids during ischemia/reperfusion in the heart, mimicking the effects of the phospholipase A₂ inhibitor quinacrine.^47,48 In vitro studies have supported these results.^49,50 Both chlorpromazine and trifluoperazine can also decrease brain phospholipase A₂ activity in normal rats,³⁰ although ischemia/reperfusion enhances its activity.⁵¹

Furthermore, phenothiazines are known to inhibit Ca²⁺ influx into cells under pathological conditions such as ischemia. That is, pretreatment with chlorpromazine reduced an increase in tissue Ca²⁺ in the ischemic heart.⁴⁷ Chlorpromazine also protected rat hippocampal slices from hypoxic injury by delaying spreading depression and the resulting massive influx of Ca²⁺ into the neurons.⁵²

Hiestand et al⁵³ (1992) also demonstrated that pretreatment with trifluoperazine could reduce the inhibition of Ca²⁺-calmodulin–dependent protein kinase II phosphorylation during reperfusion after forebrain ischemia in gerbils. This effect may be of pathogenetic importance.³⁷ It is less likely that PKC inhibition is involved in its protective effects.⁸

Therapeutic Window in Transient Focal Ischemia
For the treatment of transient focal ischemia, trifluoperazine alone does not qualify as an important therapeutic agent because of its narrow therapeutic window. As remarked above, studies from our laboratory have shown that PBN, a spin trap, has a much wider therapeutic window.¹⁹ This also applies to the immunosuppressant FK506.²³ Both drugs have been shown to prevent a secondary mitochondrial failure during reperfusion, even when given at 1 hour after reperfusion following transient (2 hours) focal ischemia.^24,25 It is most likely that the wider therapeutic windows of these drugs result from their inhibitory actions on free radicals, which are produced over an extended period during reperfusion after ischemia.^54,55 PBN is considered to trap reactive oxygen species.⁵⁶ FK506 can also inhibit calcineurin-mediated activation of nitric oxide synthase, which generates nitric oxide during reperfusion after transient focal ischemia.⁵⁷ Therefore, even if treatment against Ca²⁺-mediated cascades within the first hour after ischemia ("first therapeutic window") are not feasible, there is another opportunity to reduce the infarct volume and to improve the outcome with drugs scavenging free radicals or curbing the secondary inflammatory and immunologic cascades ("second therapeutic window").

	Selected Abbreviations and Acronyms

 

CBF = cerebral blood flow MCA = middle cerebral artery NMDA = N-methyl-D-aspartate PBN = -phenyl-N-tert-butyl nitrone TTC = 2,3,5-triphenyltetrazolium chloride

	Acknowledgments

 
This study was supported by the US Public Health Service through the National Institutes of Health (5 R01NS07838–26), the Swedish Medical Research Council (14X-00263–32), and the Medical Faculty, University of Lund.

Received May 5, 1997; revision received July 30, 1997; accepted September 9, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Choi D. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci. 1988;11:465–469.[Medline] [Order article via Infotrieve]

2. Choi D. Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci. 1995;18:58–60.[Medline] [Order article via Infotrieve]

3. Siesjö BK. Mechanisms of ischemic brain damage. Crit Care Med. 1988;16:954–963.[Medline] [Order article via Infotrieve]

4. Siesjö BK. The role of calcium in cell death. In: Price D, Thoenen H, Aguayo A, eds. Neurodegenerative Disorders: Mechanisms and Prospects for Therapy. New York, NY: John Wiley & Sons Ltd; 1991.

5. Tymianski M, Tator CH. Normal and abnormal calcium homeostasis in neurons: a basis for the pathophysiology of traumatic and ischemic central nervous system injury. Neurosurgery. 1996;38:1176–1195.[Medline] [Order article via Infotrieve]

6. Beresewicz A. Anti-ischemic and membrane stabilizing activity of calmodulin inhibitors. Basic Res Cardiol. 1989;84:631–645.[Medline] [Order article via Infotrieve]

7. Otani H, Engelman RM, Rousou JA, Breyer RH, Clement R, Prasad R, Klar J, Das DK. Improvement of myocardial function by trifluoperazine, a calmodulin antagonist, after acute coronary artery occlusion and coronary revascularization. J Thorac Cardiovasc Surg. 1992;97:267–74.[Abstract]

8. Sargent CA, Sleph PG, Dzwonczyk S, Smith MA, Grover GJ. Effect of calmodulin and protein kinase C inhibitors on globally ischemic rat hearts. J Cardiovasc Pharmacol. 1992;20:251–260.[Medline] [Order article via Infotrieve]

9. Gabauer I, Slezak J, Styk J, Ziegelhoffer A. Protective effect of calmodulin inhibitors on reperfusion injury. Bratisl Lek Listy. 1991;92:184–194.[Medline] [Order article via Infotrieve]

10. Kimura Y, Engelman RM, Rousou J, Flack J, Iyengar J, Das DK. Moderation of myocardial ischemia reperfusion injury by calcium channel and calmodulin receptor inhibition. Heart Vessels. 1992;7:189–195.[Medline] [Order article via Infotrieve]

11. Nedergaard M, Hansen A. Characterization of cortical depolarizations evoked in focal cerebral ischemia. J Cereb Blood Flow Metab. 1993;13:568–574.[Medline] [Order article via Infotrieve]

12. Gill R, Andine O, Hillered L, Persson L, Hagberg H. The effect of MK-801 on cortical spreading depression in the penumbra zone following focal ischemia in the rat. J Cereb Blood Flow Metab. 1992;12:371–379.[Medline] [Order article via Infotrieve]

13. Back T, Kohno K, Hossmann K-A. Cortical negative DC deflections following middle cerebral artery occlusion and KCl-induced spreading depression: effect on blood flow, tissue oxygenation and electroencephalogram. J Cereb Blood Flow Metab. 1994;14:12–19.[Medline] [Order article via Infotrieve]

14. Kristián T, Siesjö BK. Calcium-related damage in ischemia. Life Sci. 1996;59:357–367.[Medline] [Order article via Infotrieve]

15. Kristián T, Gidö G, Kuroda S, Schutz A, Siesjö BK. Calcium metabolism of focal and penumbral tissues in rats subjected to transient middle cerebral artery occlusion. Exp Brain Res. In press.

16. Greenberg JH, Uematsu D, Araki N, Hickey WF, Reivich M. Cytosolic free calcium during focal cerebral ischemia and the effects of nimodipine on calcium and histologic damage. Stroke. 1990;21(suppl IV):IV-72-IV-77.

17. Memezawa H, Zhao Q, Smith M-L, Siesjö BK. Hyperthermia nullifies the ameliorating effect of dizocilpine maleate (MK-801) in focal cerebral ischemia. Brain Res. 1995;670:48–52.[Medline] [Order article via Infotrieve]

18. Margaill I, Parmentier S, Callebert J, Allix M, Boulu R, Plotkine M. Short therapeutic window for MK-801 in transient focal cerebral ischemia in normotensive rats. J Cereb Blood Flow Metab. 1996;16:107–113.[Medline] [Order article via Infotrieve]

19. Zhao Q, Pahlmark K, Smith M-L, Siesjö BK. Delayed treatment with the spin trap -phenyl-N-tert-butyl nitrone (PBN) reduced infarct size following transient middle cerebral artery occlusion in rats. Acta Physiol Scand. 1994;152:349–350.[Medline] [Order article via Infotrieve]

20. Folbergrová J, Zhao Q, Katsura K, Siesjö BK. N-tert-Butyl--phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia. Proc Natl Acad Sci U S A.. 1995;92:5057–5061.[Abstract/Free Full Text]

21. Siesjö BK, Katsura K, Zhao Q, Folbergrova J, Pahlmark K, Siesjö P, Smith M. Mechanisms of secondary brain damages in global and focal ischemia: a speculative synthesis. J Neurotrauma. 1995;12:943–956.[Medline] [Order article via Infotrieve]

22. Siesjö BK, Siesjö P. Mechanisms of secondary brain injury. Eur J Anaesthesiol. 1996;13:247–268.[Medline] [Order article via Infotrieve]

23. Kuroda S, Siesjö BK. Postischemic administration of FK506 reduced infarct volume following transient focal brain ischemia. Neurosci Res Com. 1996;19:83–90.

24. Kuroda S, Katsura K, Hillered L, Bates TE, Siesjö BK. Delayed treatment with -phenyl-N-tert-butyl nitrone (PBN) attenuates secondary mitochondrial dysfunction after transient focal cerebral ischemia in the rat. Neurobiol Dis. 1996;3:149–157.[Medline] [Order article via Infotrieve]

25. Nakai A, Kuroda S, Kristían T, Siesjö BK. The immunosuppressant FK506 ameliorates secondary mitochondrial failure following transient focal cerebral ischemia in the rat. Neurobiol Dis. In press.

26. Koizumi J, Yoshida Y, Nakazawa T, Ooneda G. Experimental studies of ischemic brain edema, I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Jpn J Stroke. 1986;8:1–8.

27. Memezawa H, Minamisawa H, Smith M-L, Siesjö BK. Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res. 1992;89:67–78.[Medline] [Order article via Infotrieve]

28. Zhao Q, Memezawa H, Smith M-L, Siesjö BK. Hyperthermia complicates middle cerebral artery occlusion induced by an intraluminal filament. Brain Res. 1994;649:253–259.[Medline] [Order article via Infotrieve]

29. Bederson J, Pitts L, Tsuji M, Nishimura M, Davis R, Bartkowski H. Rat middle cerebral artery occlusion: evaluation of the model and development of a neurological examination. Stroke. 1986;17:472–476.[Abstract/Free Full Text]

30. Trzeciak HI, Kalacinski W, Malecki A, Kokot D. Effects of neuroleptics on phospholipase A₂ activity in the brain of rats. Eur Arch Psychiatry Clin Neurosci. 1995;245:179–182.[Medline] [Order article via Infotrieve]

31. Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson C, Sharp FR. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab. 1990;10:290–293.[Medline] [Order article via Infotrieve]

32. Kohno K, Back T, Hoehn-Berlage M, Hossmann K-A. A modified rat model of middle cerebral artery thread occlusion under electrophysiological control for magnetic resonance investigations. Magn Reson Imaging. 1995;13:65–71.[Medline] [Order article via Infotrieve]

33. Röther J, de Crespigny A, D'Arceuil H, Moseley M. MR detection of cortical spreading depression immediately after focal ischemia in rats. J Cereb Blood Flow Metab. 1996;16:214–220.[Medline] [Order article via Infotrieve]

34. Siesjö B, Bengtsson F. Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab. 1989;9:127–140.[Medline] [Order article via Infotrieve]

35. Hossmann KA, Ophoff BG, Schmidt-Kastner R, Oschlies U. Mitochondrial calcium sequestration in cortical and hippocampal neurons after prolonged ischemia of the cat brain. Acta Neuropathol (Berl). 1985;68:230–238.[Medline] [Order article via Infotrieve]

36. Zaidan E, Sims N. The calcium content of mitochondria from brain subregions following short-term forebrain ischemia and recirculation in the rat. J Neurochem. 1994;63:1812–1819.[Medline] [Order article via Infotrieve]

37. Grotta JC, Picone CM, Dedman JR, Rhoades HM, Strong RA, Earls RM, Yao LP. Neuronal protection correlates with prevention of calcium-calmodulin binding in rats. Stroke. 1990;21(suppl III):III-28-III-31.

38. Dienel GA. Regional accumulation of calcium in postischemic rat brain. J Neurochem. 1984;43:913–925.[Medline] [Order article via Infotrieve]

39. Deshpande JK, Siesjö BK, Wieloch T. Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia. J Cereb Blood Flow Metab. 1987;7:89–95.[Medline] [Order article via Infotrieve]

40. Dux E, Mies G, Hossmann K-A, Siklos L. Calcium in the mitochondria following brief ischemia of gerbil brain. Neurosci Lett. 1987;78:295–300.[Medline] [Order article via Infotrieve]

41. Rappaport ZH, Young W, Flamm ES. Regional brain calcium changes in the rat middle cerebral artery occlusion model of ischemia. Stroke. 1987;18:760–764.[Abstract/Free Full Text]

42. Shirotani T, Shiama K, Iwata M, Kita H, Chigasaki H. Calcium accumulation following middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. J Cereb Blood Flow Metab. 1994;14:831–836.[Medline] [Order article via Infotrieve]

43. Chien KR, Abrams J, Pfau RG, Farber JL. Prevention by chlorpromazine of ischemic liver cell death. Am J Pathol. 1977;88:539–558.[Medline] [Order article via Infotrieve]

44. Zivin JA, Kochhar A, Saitoh T. Phenothiazines reduce ischemic damage to the central nervous system. Brain Res. 1989;482:189–193.[Medline] [Order article via Infotrieve]

45. Yu MJ, McCowan JR, Smalstig EB, Bennett DR, Roush ME, Clemens JA. Phenothiazine derivative reduced rat brain damage after global or focal ischemia. Stroke. 1992;23:1287–1291.[Abstract/Free Full Text]

46. Beresewicz A, Wasilewska A, Herbaczynskacedro K. Calmodulin antagonist reduced release of malondialdehyde from isolated ischemic/reperfused rat heart. Acta Physiol Pol. 1988;39:442–449.[Medline] [Order article via Infotrieve]

47. Chien KR, Pfau RG, Farber JL. Ischemic myocardial cell injury. Am J Pathol. 1979;97:505–530.[Abstract]

48. Otani H, Prasad MR, Jones RM, Das DK. Mechanism of membrane phospholipid degradation in ischemic-reperfused rat hearts. Am J Physiol. 1989;257:H252–H258.[Abstract/Free Full Text]

49. Smith DS, Rehncrona S, Siesjö BK. Inhibitory effects of different barbiturates on lipid peroxidation in brain tissue in vitro: comparison with the effects of promethazine and chlorpromazine. Anesthesiology. 1980;53:186–194.[Medline] [Order article via Infotrieve]

50. Janero DR, Burghardt B. Prevention of oxidative injury to cardiac phospholipid by membrane-active 'stabilizing agents.' Res Commun Chem Pathol Pharmacol. 1989;63:163–173.[Medline] [Order article via Infotrieve]

51. Rordorf G, Uemura Y, Bonventre J. Characterization of phospholipase A₂ (PLA₂) activity in gerbil brain: enhanced activities of cytosolic, mitochondrial, and microsomal forms after ischemia and reperfusion. J Neurosci. 1991;11:1829–1836.[Abstract]

52. Balestrino M, Somjen GG. Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression-mediated calcium influx. Brain Res. 1986;385:219–226.[Medline] [Order article via Infotrieve]

53. Hiestand DM, Haley BE, Kindy MS. Role of calcium in inactivation of calcium/calmodulin dependent protein kinase II after cerebral ischemia. J Neurol Sci. 1992;113:31–37.[Medline] [Order article via Infotrieve]

54. Matsuo Y, Kihara T, Ikeda M, Ninomiya M, Onodera H, Kogure K. Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation. J Cereb Blood Flow Metab. 1995;15:941–947.[Medline] [Order article via Infotrieve]

55. Dirnagl U, Lindauer U, Them A, Schreiber S, Pfister H-W, Koedel U, Reszka R, Freyer D, Villringer A. Global cerebral ischemia in the rat: online monitoring of oxygen free radical production using chemiluminescence in vivo. J Cereb Blood Flow Metab. 1995;15:929–940.[Medline] [Order article via Infotrieve]

56. Floyd R. Role of oxygen free radicals in carcinogenesis and brain ischemia. FASEB J. 1990;4:2587–2597.[Abstract]

57. Dawson T, Steiner J, Dawson V, Dinerman J, Uhl G, Snyder S. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci U S A. 1993;90:9808–9812.[Abstract/Free Full Text]

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