The Calmodulin Antagonist Trifluoperazine in Transient Focal Brain Ischemia in Rats

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 Under light reanesthesia, the filament was withdrawn after 2 hours to allow recirculation.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.

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⇓).

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).

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.

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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.

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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.

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-1β–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”).