Neuroprotective effect of NS-7, a novel Na+ and Ca2+ channel blocker, in a focal ischemic model in the rat

Brain Research 969 (2003) 168–174 www.elsevier.com / locate / brainres

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Neuroprotective effect of NS-7, a novel Na 1 and Ca 21 channel blocker, in a focal ischemic model in the rat Toshiya Katsumata a , *, Hiromi Muramatsu a , Hidenori Nakamura a , Yutaka Nishiyama a , Yasuaki Aoki b , Yasuo Katayama a a

The Second Department of Internal Medicine, Nippon Medical School, 1 -1 -5 Sendagi, Bunkyo-ku, Tokyo, Japan b Research Laboratories, Nippon Shinyaku Co. Ltd., Kyoto, Japan Accepted 8 January 2003

Abstract NS-7 is a novel, voltage-dependent Na 1 and Ca 21 channel blocker. This study evaluated the in vivo neuroprotective effect of NS-7 in a rat transient focal ischemic model when administered during occlusion. Left middle cerebral artery occlusion was induced in adult male Sprague–Dawley rats for 120 min using an intraluminal thread method. The rats received a single intravenous injection of NS-7 or saline (control group) just after the onset of ischemia, and at 30, 60 and 120 min after ischemia. Their brains were removed after 48 h reperfusion, sectioned, and stained with hematoxylin and eosin. Animals were evaluated by neurological examination at 120 min ischemia and 48 h reperfusion. Infarcted cortex and striatum were measured quantitatively and infarction volumes were calculated. Cortical infarction volumes were 128674 (NS-7) and 214664 mm 3 (control) immediately after the ischemia group, 155648 (NS-7) and 225612 mm 3 (control) after the 30 min group, 160654 (NS-7) and 225648 mm 3 (control) after the 60 min group, and 176643 (NS-7) and 223638 mm 3 (control) after the 120 min group. Cortices in NS-7-treated groups were significantly less infarcted than in control groups at all treatment times. There was no significant difference in the striatal infarction volume between the treatment and control groups. Neurological examination showed that hemiparesis and abnormal posture of the NS-7 groups were significantly more improved at 48 h reperfusion than those of the control groups without posture examination in the 120 min group. These observations suggest that NS-7 may be a new potential therapeutic agent for the acute phase of cerebral infarction.  2003 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Long-term potentiation: pharmacology Keywords: Transient cerebral ischemia; NS-7; Na 1 and Ca 21 channel blocker; Neuroprotection

1. Introduction Although much attention has been paid to the mechanism of cerebral ischemia in the last two decades, the precise cellular mechanism underlying ischemic brain damage remains to be elucidated. It is thought that, during and after cerebral ischemia, membrane depolarization caused by ATP depletion activates voltage-dependent Na 1 and Ca 21 channels. The opening of voltage-dependent Na 1 channels may facilitate the accumulation of intracellular *Corresponding author. Tel.: 181-3-3822-2131; fax: 181-3-38224865. E-mail address: [email protected] (T. Katsumata).

Na 1 . Rundown of the Na 1 gradient reverses the direction of glutamate transporters and leads to an elevation of glutamate in the extracellular space [15,19,28]. Glutamate stimulates postsynaptic N-methyl-D-aspartate (NMDA) receptors, which allows Ca 21 to enter the neuron. This plays an important role in the pathogenesis of cerebral ischemia [4,7,23,26,27]. Furthermore, a reversal of the Na 1 / Ca 21 exchanger caused by rundown of the Na 1 gradient and the direct activation of voltage-dependent Ca 21 channels lead to excessive Ca 21 influx [6,34]. Glutamate can also act on metabotropic receptors, leading to the production of diacylglycerol and inositol tri-phosphate, which upon activation of enzymes, leads to the release of Ca 21 from intracellular stores [5,38]. An increase in the intracellular

0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02296-0

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Ca 21 concentration leads to activation of proteases, nucleases, phospholipases, NO synthase and other degenerative enzymes, which can lead to ischemic cell death [17]. NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopntyloxy)pyrimidine hydrochloride] is a novel voltage-dependent Na 1 and Ca 21 channel blocker. This compound blocks the function of voltage-dependent Na 1 channels by acting on the neurotoxin receptor site 2 in the brain [29]. It also inhibits Na 1 and Ca 21 currents through L- and N-type Ca 21 channels in NG108-15 cells [30] and blocks the KCl-induced activation of nitric oxide synthase (NOS) through blockade of L- and P/ Q-type Ca 21 channels in primary cultured neurons [22]. Moreover, a previous in vivo study has reported a reduction of the infarct volume by NS-7 using a permanent middle cerebral artery (MCA) occlusion model in the rat [2]. Since no single animal model represents all aspects of clinical stroke, it has been suggested that neuroprotective agents could be investigated in multiple in vivo cerebral ischemia models and should show lesion reductions in the multiple models [31]. The present study was therefore designed to evaluate the in vivo neuroprotective effect of NS-7 in the acute phase of cerebral ischemia when administered during occlusion. We have examined the effect of NS-7 on the infarction volume and neurological outcome in a transient focal ischemic model in the rat.

2. Materials and methods

2.1. General surgical procedure Experiments were conducted following the Guidelines for Animal Experimentation of the Nippon Medical School. Male Wistar rats weighing 250–350 g were used in the experiments. Animals were fasted for 24 h before investigation. Anesthesia was induced with 2% halothane in a gas mixture of 70% N 2 O and 30% O 2 , and then maintained with 1% halothane using a face mask. The animals were placed on a heating pad to maintain rectal and scalp temperatures (3760.5 8C) during surgery. The caudal artery was cannulated with a PE-50 catheter for continuous monitoring of mean arterial blood pressure and blood collection, and the left femoral vein was cannulated for drug administration. Before and during ischemia, blood was collected through the caudal arterial cannula to monitor blood gases ( pO 2 , pCO 2 ), pH, and blood glucose level.

2.2. Middle cerebral artery ( MCA) occlusion The middle cerebral artery (MCA) was occluded for 120 min using a modified method of Koizumi followed by reperfusion [16]. The rat model, in which the MCA is occluded intraluminally using a silicone rubber cylinder attached to a nylon suture via the carotid artery, has the

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advantage of not requiring a craniotomy, thus eliminating an invasive surgical procedure, and allowing for readily induced reperfusion [13,16]. In human ischemic stroke, recirculation occurs frequently after focal ischemia, particularly in cases of cerebral embolism [25]. We therefore chose this model as appropriate for evaluating the neuroprotective effect of NS-7. We used 120-min transient focal ischemia because this period was sufficient to produce an infarction of maximal size in this model [13]. The left common carotid artery and the bifurcation of the internal and external carotid arteries were exposed, with careful conservation of the vagus nerve. The common carotid and external carotid arteries were ligated with a silk thread. A small incision was made in the common carotid artery near the bifurcation, and a silicone rubber cylinder attached to 4-0 nylon surgical thread (tip ball diameter, 0.2–0.3 mm; nylon surgical thread with a silicon coating of 5 mm) was inserted into the internal carotid artery through the common carotid artery until the MCA was occluded. The wound site was sutured and anesthesia was discontinued. After 120-min ischemia, the surgical thread was removed to allow reperfusion. The animals were allowed free access to food and water during reperfusion.

2.3. Drug administration NS-7 (Nippon Shinyaku, Kyoto, Japan.) was dissolved in physiological saline (Otsuka Pharmaceutical, Japan). Rats were randomly allocated to two groups (NS-7 or saline-treated group). The rats received a single intravenous injection of NS-7 (0.5 mg / kg) or saline (control group) just after the onset of ischemia (NS-7, n59; saline, n59), and at 30 min (NS-7, n59; saline, n59), 60 min (NS-7, n59; saline, n59) and 120 min (NS-7, n59; saline, n59) after the onset of ischemia. The choice of drug dosage (0.5 mg / kg) in this study was governed by a previous study that showed the concentration of NS-7 in the brain after intravenous injection at 0.5 mg / kg reaches a level effective for suppressing calpain activation, which may be implicated in the pathophysiology of ischemiainduced neurodegeneration [32].

2.4. Neurological examination We examined motor performance using a neurological deficit score based on the detection of hemiparesis and abnormal posture following a previous report of 120 min MCA occlusion and 48 h of reperfusion after MCA occlusion [14,36]. For the assessment of abnormal posture, the rats were suspended by their tail, and forelimb flexion and body twisting were evaluated as 0 (normal), 1 (slight twisting), 2 (marked twisting) and 3 (marked twisting and forelimb flexion). For the assessment of hemiparesis, the right hind limb of each rat was extended gently with round-tipped forceps and the flexor response was evaluated as 0 (normal), 1 (slight deficit), 2 (moderate deficit), and 3

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(severe deficit). We evaluated the neurological score by each parameter (hemiparesis, abnormal posture). Neurological examinations were performed in a blinded manner.

2.5. Infarction volume Forty-eight hours after reperfusion, rats were anesthetized with 1% halothane in a gas mixture of 70% N 2 O and 30% O 2 and sacrificed. Brains were quickly removed and frozen by immersion in chilled isopentane (240 8C). Frozen brains were cut into 20 mm coronal sections on a cryostat (Clinicut, Bright Instrument, Cambridge, UK) and stained with hematoxylin and eosin. After staining, each coronal section was then digitized with a microcomputer imaging device (MCID system, Imaging Research, Ontario, Canada). The border between infarcted and noninfarcted tissue was outlined with an image analysis system, and the areas of infarcted cortex and infarcted striatum were measured for the lesioned hemisphere. Infarction volume was calculated by integrating the crosssectional area of ischemic damage at each stereotaxic level and the distances between the levels [24]. The measurement of infarction volumes was performed in a blinded manner. In the present study, the infarction volumes was measured directly without any compensation for cerebral edema.

2.6. Statistical analysis All data are expressed as mean6S.D. Infarction volumes and physiological parameters were first examined by a two-way analysis of variance (ANOVA) followed by Turkey–Kramer’s post-hoc test. Neurological scores were also examined by a two-way analysis of variance (ANOVA) followed by Mann–Whitney U-test. Spearman’s rank test was used to relate neurological scores and infarction volumes. A level of P,0.05 was considered to be significant.

3.2. Infarction volume Cortical infarction volumes were 128674 (NS-7) and 214664 mm 3 (control) in the treatment just after ischemia group; 155648 (NS-7) and 215612 mm 3 (control) in the 30 min group; 160654 (NS-7) and 225648 mm 3 (control) in the 60 min group; and 176643 (NS-7) and 223638 mm 3 (control) in the 120 min group (Fig. 1). The cortical infarction volume of the NS-7-treated group was significantly less than that of the control group at any treatment time. Striatal infarct volumes were 54619 (NS7), and 67611 mm 3 (control) in the treatment just after ischemia group; 74621 (NS-7) and 73627 mm 3 (control) in the 30 min after ischemia group; 76623 (NS-7) and 76612 mm 3 (control) in the 60 min group; and 75611 (NS-7) and 7567 mm 3 (control) in the 120 min group (Fig. 2). There was no difference between the two treatment groups.

3.3. Neurological examinations Table 1 shows the results from the neurological examinations. Posture and hemiparesis at 2 h ischemia were not different between the two groups, but after 48 h reperfusion, both abnormal posture and hemiparesis of the NS-7treated groups were significantly improved in comparison to the control group just after the onset of ischemia, and in the 30 and 60 min ischemia groups. In the 120 min ischemia group, hemiparesis of the NS-7-treated group showed significant improvement at 48 h reperfusion over the control group, and abnormal posture of the NS-7treated group displayed an improving tendency, but did not reach statistical significance when compared to the control group. Correlations were found between the cortical infarction volume and hemiparesis at 48 h (r50.57, P,0.001), and posture at 48 h (r50.62, P,0.001). There were weak correlations between the striatum infarction volume and hemiparsis at 48 h (r50.36, P,0.01), and posture at 48 h (r50.31, P,0.01).

4. Discussion 3. Results

3.1. Physiological parameters Physiological parameters were within normal ranges, i.e. subscalp temperature 36.8–37.1 (before occlusion) and 36.8–37.1 8C (during occlusion), rectal temperature 36.7– 37.0 and 36.8–37.1 8C, mean blood pressure 109–125 and 108–124 mmHg, blood glucose 86–94 and 86–94 mg / dl, pH 7.38–7.41 and 7.37–7.40, pCO 2 39.1–42.5 and 40.2– 43.8 mmHg, and pO 2 119–136 and 114–129 mmHg, respectively. There were no significant differences between the two groups for any of the parameters studied.

The requirement to assess neurological as well as histological outcome in the preclinical evaluation of neuroprotective agents in experimental models of cerebral ischemia is increasingly recognized. In the present study, we evaluated the effect of NS-7 on neurological outcome and infarction volume in a rat transient focal ischemic model when administered during the occlusion. NS-7 is a specific blocker of both voltage-dependent Ca 21 and Na 1 channels [29,30]. Although the cellular mechanism of the neuroprotection produced by NS-7 is not fully understood, NS-7 inhibits tetrodotoxin-sensitive Na 1 currents (IC 50 57.8 mM) and Ca 21 currents through L(IC 50 54.5 mM) and N-type Ca 21 channels (IC 50 57.3

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Fig. 1. Effects of NS-7 on cortical infarction volume after 48 h reperfusion following 2 h of middle cerebral artery occlusion in rats. Each column represents the cortical infarction volume just after ischemia (0 min), and in groups treated at 30, 60 and 120 min. The values are mean6S.D. *P,0.05, **P,0.01 vs. saline (two-way ANOVA, followed by Turkey–Kramer’s post-hoc test).

mM) and K 1 channels (IC 50 5160.5 mM) in NG108-15 cells [30], and blocks KCl-induced activation of nitric oxide synthase (NOS) through blockade of L- and P/ Qtype Ca 21 channels (IC 50 53.1 mM) [22]. In addition, NS-7 inhibits nicotine-induced Na 1 influx via nicotine

receptors in cultured bovine adrenal chromaffin cells (IC 50 515.5 mM) [35], and also suppresses the enhanced activity of calpain, a Ca 21 -activated protease, in cerebral ischemia and suppresses veratridine-evoked glutamate release in the rat cortex [32]. Furthermore, a phar-

Fig. 2. Effects of NS-7 on striatum infarction volume after 48 h reperfusion following 2 h of middle cerebral artery occlusion in rats. Each column represents the striatum infarction volume in the group treated just after ischemia (0 min), and groups treated at 30, 60 and 120 min. The values are mean6S.D. There was no significant difference between the two groups.

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172 Table 1 Neurological outcome 2 h ischemia

48 h reperfusion

Control

NS-7

Control

NS-7

Abnormal posture Just after ischemia 30 min after ischemia 60 min after ischemia 120 min after ischemia

2.360.7 2.860.5 2.760.8 2.760.5

2.060.7 2.660.5 2.660.5 2.660.5

2.160.8 2.760.5 2.560.5 2.660.5

1.460.5* 1.660.7** 1.860.7* 2.460.7

Hemiparesis Just after ischemia 30 min after ischemia 60 min after ischemia 120 min after ischemia

2.160.7 2.560.5 2.660.5 2.760.5

2.360.6 2.360.5 2.460.5 2.360.5

2.160.8 2.560.5 2.360.5 2.960.4

1.260.8* 1.560.8* 1.660.5* 2.160.5**

Values are mean6S.D. *P,0.05, **P,0.01 vs. control group.

macokinetic study showed that a substantial amount of NS-7 can penetrate ischemic brain regions when injected intravenously after MCAO in the rat [12]. Its elimination half-life is about 4 h in normal brain, but increases in ischemic brain. This property may be advantageous for the treatment of ischemic tissue [12]. Taken together, it is reasonable to assume that voltage-dependent Na 1 and Ca 21 channels are effectively blocked by NS-7 in the ischemic region. A single injection of NS-7 not only greatly attenuates cortical infarction volume, but also improves neurological outcome in a rat transient MCA occlusion model. Cortical infarction volume of the NS-7-treated group was reduced 40% by treatment just after ischemia. Interestingly, cortical infarction volume of the NS-7-treated group was reduced by 20% even when administration was delayed by up to 120 min after the onset of ischemia. This observation may support the concept that NS-7 is a potential neuroprotective agent for the acute stage of cerebral infarction, because 120 min was a sufficient period to produce an infarction of maximal size in this model [13]. However, infarction volume was not improved significantly in the striatum. The striatum is generally considered to be the core of the ischemic lesion [9] and previously has proved relatively refractory to neuroprotection. In this model, cerebral infarction develops much earlier in the striatum than in the cortex because of the differences in response to ischemic stress and neuronal vulnerability between cortex and striatum [1,13,21]. Therefore, this difference in the development of infarction may explain, in part, why administration of NS-7 is more effective in reducing cortical infarction volume. Recently, ischemia-induced depolarization has been suggested to play a critical role in the progression of ischemic damage from the ischemic core toward the ischemic border zone in the acute phase of ischemia, and the ischemic lesion volume was significantly correlated with the total depolarization time in cortical peri-focal areas [8,33]. Depolarization activates voltage-dependent

Ca 21 channels and voltage-dependent Na 1 channels with a massive influx of Na 1 and Ca 21 , resulting in a marked increase in the intracellular Ca 21 concentration, which may promote various cytotoxic processes. In addition, repeated depolarization surges are considered to expand the infarcted area [8,33]. During ischemia, Na 1 channel blockers would reduce cellular depolarization, prevent ischemic glutamate release and delay the onset of depolarization [34]. Moreover, Ca 21 channel blockers would prevent the development of neuronal damage processes that could be associated with Ca 21 influx through voltage-dependent Ca 21 channels [17,18]. Under these conditions, a specific blocker of both voltage-dependent Ca 21 and Na 1 channels, such as NS-7, would be expected to inhibit the expansion of infarction damage. In fact, NS-7 suppressed calpain activation, a Ca 21 -activated protease, in cerebral ischemia [32], and also prevented the suppression of the binding activity of PKA (cyclic AMP-dependent protein kinase) to cyclic AMP, which is critical for neuronal survival [33]. These effects may be accompanied by a significant reduction in cortical infarction volume. The utility of a neurological scoring system as a corollary of neuroprotection in rat models of focal ischemia has been highlighted in drug studies. In this study, NS-7 improved the neurological symptoms based on posture and hemiparesis in transient MCA occlusion. While Wahl et al. reported a lack of correlation between neurological deficit and the extent of the infarct [37], others have shown that the neurological deficit significantly correlated with the size of the infarcted area [3,10]. In our study, we observed that neurological score was significantly correlated to cortical infarction volume. It may be postulated that the brain tissue surviving infarction through the protective effect of NS-7 is functional and results in a better neurological outcome. While irreversible neuronal injury due to ischemia is thought to depend largely on an increase in intracellular [Ca 21 ], the mechanisms of Ca 21 entry may differ between white matter and gray matter [11]. In the white matter, pathological Ca 21 influx largely occurs as a result of reversal of the Na 1 / Ca 21 exchanger, which is due to increased intracellular [Na 1 ] and membrane depolarization; although Ca 21 entry via axonal voltage-dependent Ca 21 channels or NMDA receptors cannot be ruled out [11,20]. The prevention of pathological Na 1 influx by Na 1 channel blockade would reduce Ca 21 influx via the Na 1 / Ca 21 exchange mechanism and may be effective in preventing white matter ischemia [11,34]. Therefore, NS-7 may be expected to protect white matter from ischemic injury because it is a specific blocker of voltage-dependent Na 1 channels. One of the advantages of voltage-dependent Na 1 channel blockers is that their neuroprotective effects are compatible with those of NMDA receptor antagonists, without any serious psychomotor or neurotoxic side-effects [33]. Older voltage-dependent Ca 21 channel blockers

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exhibited serious adverse cardiovascular actions such as hypotension and cardiac arrhythmia. NS-7 appears to be free of these side-effects, and therefore it may be safely and efficiently used in ischemic stroke patients [33]. In conclusion, NS-7 protects against ischemic brain damage caused by transient focal ischemia and improves the neurological outcome even if administered up to 120 min after the onset of ischemia. Further study will be necessary to investigate whether treatment with NS-7 after much longer than 2 h after the onset of ischemia still improves neurological outcome and reduces cortical infarction in this model. The present findings suggest that NS-7 is a new potentially useful therapeutic agent for the acute phase of cerebral infarction.

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