Turkish Neurosurgery 12: 22 - 25, 2002 Ars/al1fa~: Lifariziue alld Nil1lOdil'iuf ill Focal Cere/'r,,1 /scl,elllia

Effects of Lifarizine and Nimodipine on Forebrain LactateLevels in a Rat Model of Focal Cerebral Ischemia

LaktatEtkileri

Modelinde On Beyinve Nimodipin'in

Fokal Serebral iskeminin RatSeviyeleri Uzerine Lifarizin

ALi ARSLANT A$, HAKAN BOZOGLU

Department of Neurosurgery, Osmangazi University (AA, HB), Eski~ehir, Turkey

Received: 10.01.2002 ¢::> Accepted: 05.03.2002

Abstract: Objective: The effects of lifarizine (RS-87476)and nimodipine on lactate concentrations in ischemicforebrain were evaluated in a rat model of bilateral carotid

artery ligation.Methods: Eighteen rats were randomly assigned to threegroups (control, lifarizine treatment, and nimodipinetreatment). The animals were anesthetized, the arterieswere ligated, and assigned treatments were administered15 min. later. After 72 hours of assigned treatment andreperfusioll, the rats were decapitated and forebrain lactatelevels were measured.

Results: The lactate levels in the lifarizine group weresignificantly lower than control levels (p<0.05). The levelsin the nimodipine group were lower than control levels,but the difference was not significant (p>0.05).Conclusion: These results suggest that lifarizine is morelleuroprotective than llimodipille in the setting of focalcerebral ischemia in the rat.

Ozet: AI/!af: Lifarizin ve nimodipill'in on-beyin iskemisiuzerine etkileri, ratlarda iki tarafh karotis arterlerinbaglanmasl ile olu~turulan deneysel modelde ara~tmldl.Yon tel/!: Rasgele sec;ilmi~18rat Hekontrol, lifarizin teda\live nimodipin tedavi gruplan olu~turuldu. Anestezi ediliparterler balamp 15 dakika sonra tedavilerine ba~landl.Yetmi~iki saatlik tedavi ve reperfUzyon sonrasmda ratlardekapite edildi \Ie on-beyin laktat olc;umleri yaplldl.SOli/If: Lifarizin tedavisi grubunda laktat miktan kontrolgrubundan onemli derecede du~uk bulunmu~tur.(p<0.05). Nimodipin grubunda se\liyeler kontrol grubunaoranla du~uk bulunmu~ ancak bu fark anlaml!bulunmaml~tJr (p>0.05).YOrlll/!: Bu bulgular lifarizin'in nimodipine oranla dahaetkili noroprotektif bir ajan oldugunu du~i.indi.irmektedir.

Key W 0 r d s: focal cerebral ischemia, lifarizille,neuroprotective effect, nimodipine

Anahtar kelimeler: fokal serebral iskemi, lifarizin,noroprotektif etki, nimodipin

INTRODUCTION

Stroke is defined as a sudden development of afocal neurologic deficit due to occlusion of a cerebralvessel, or spontaneous intracranial artery rupturewith consequent hemorrhage in the brain

parenchyma (4). Recently, a considerable amount ofresearch has focused on pharmacologicalinterventions that might help improve clinicaloutcome following stroke. Although varioustreatment modalities are used, there is still noclinically effective treatment for acute stroke (9).

22

Tllrkish Neurosllrgery 11: 11 - 15, 2002

Accumulation of sodium and calcium in the cellis a critical step in the process that leads to neuronaldamage. Most pharmacological studies that haveinvestigated treatment of neurological damage dueto trauma or ischemia have focused on preventinginfluxes of calcium using calcium antagonists, suchas calcium-channel blockers and N-methyl-D­aspartate (NMDA) receptor antagonists (5,14). Inaddition to calcium antagonists, some researchershave used sodium-channel blockers to treat cerebralischemia. Reports have documented that lifarizine,a centrally active neuronal sodium-channel blocker,has neuroprotective efficacy in several in vivo modelsof focal and global cerebral ischemia, including four­vessel occlusion in the rat 0), and middle cerebralartery occlusion in the cat (6) and mouse (3).

The aim of this study was to compare the effectsof this sodium/ calcium-channel modulatorOifarizine, RS-87476) and a widely used calcium­channel antagonist (nimodipine) on forebrain lactatelevels in the setting of focal ischemia in the rat.

MATERIAL AND METHODS

Eighteen adult Sprague-Dawley rats weighing330-450g (mean 385.5 g) were used. Each animal wasanesthetized with an intraperitoneal (IP) injection ofthiopental sodium (0.1 mg/kg). The femoral arterywas cannulated to record mean arterial blood

pressure (MABP), and 0.2 ml of heparinized saline(l00 mU/ml) was administered. Blood pressure wasmeasured with a pressure transducer (PI 000 B,Narcobiosystem) and recorded with a four-channelphysiograph (MKIII, Narcobiosystem). Bodytemperature was monitored with a rectal probe, andwas kept between 36.5 °C and 37°C with a heatingpad.

Surgical ProcedureA vertical ventral midline incision was made

in the skin of the anterior part of the neck. The leftand right common carotid arteries were exposed inthe para tracheal region, and were carefully dissectedfree of the accompanying vagosympathetic nervetrunks. A loose ligature was placed around each ofthe arteries. The rats were randomly assigned to oneof three groups of six. All 18 animals underwent thecarotid ligation procedure described above. Thecontrol group (Group 1) underwent ligation butreceived no drug treatment. The nimodipinetreatment group (Group 2) received 0.5-mg/kgnimodipine IP twice daily for 3 days. The lifarizinetreatment group (Group 3) received 0.5-mg/kg

Arslnlltn~: Lifnrizille nlld Nilllodipille ill Focnl Cere/mIl ischemitl

lifarizine IP three times daily for 3 days. After 72 hrsof treatment and reperfusion, each rat was re­anesthetized with thiopental sodium (0.1 mg/kg) IPand then decapitated. The heads were immediatelyplaced in liquid nitrogen. Once they were frozen,each brain was removed and forebrain specimenswere preserved in liquid nitrogen to halt enzymaticreactions prior to lactate level analysis. The forebrainspecimens were homogenized, and the tissueconcentration of lactate was measured byspectrophotometric methods (7,8).

Drug and SolutionsLifarizine, an analogue of diphenylpiperazine

in a 100 mg/ ml formulation (RS-87476, which is 1­[DiphenylmethylJ-[ 4-methyH2-4-[methyphenyl] 4­methyl imidazol-5-yl)); (Syntex®, USA) \·vasprepared as a solution with 5% ethanol and sterilenormal saline (0.9%NaCI). Nimodipidine (Nimotop­Bayer®, Germany) was used in 100 mg/ml form.Thiopental sodium USP (PENTOTHAL SODIUM 500mg, Abbott) was used to anesthetize the rats, andwas prepared by diluting with normal saline to aconcentration of 0.1 mg/ml.

Statistical AnalysisThe lactate and MABP data were statistically

evaluated using the student's t-test and one-wayANOV A. All group data were expressed asmean±SD. A p value <0.05 was consideredstatistically significant.

RESULTS

In all three groups, MABP decreasedimmediately after the carotid arteries were ligated(l11±4.52 mmHg before ligation and 81.67±S.16mmHg after ligation; p>0.05). Table 1 shows theforebrain tissue lactate concentrations for each group.The lactate levels in both the lifarizine and

nimodipine treatment groups were lower than thecontrol level. The level in the lifarizine treatment

group was significantly lower than that in the

Table 1: The forebrain lactate concentrations for thethree study groups (}lmol/ g). Values arelisted as mean±SD. F=5.157, P <0.05

GroupIlmollactate/wet wt

Control

43.03±1.68

Nimodipine

41.65±1.91Lifarizine

35.20±1.44

23

Tllrkish Nellrosllrgery 12: 22 - 25, 2002

controls (p<0.05), whereas the lactate level in thenimodipine treatment group was not (p>0.05).

DISCUSSION

Many pathophysiological processes in the brainare associated with a rise in tissue lactate content,

and one of these is hypoxia. If cellular oxygen fallsbelow the critical level needed for mithochondrial

oxidative phosphorylation, the level of the reducedform of nicotinamide adenine dinucleotide-NADH

rises. This leads to altered glucose degradation, withpyruvate being reduced to form lactate, instead ofdecarboxylated to form acetyl-CoA. Thus, lactatelevels are considered good indicators of inadequateoxygen supply to tissue (0). Increased lactateproduction is usually considered to occur withactivation of anaerobic glucose metabolism due toinadequate oxygen supply, and the eventual resultofJactate build-up is tissue acidosis (10). In our study,the forebrains of the rats that were treated with

lifarizine after ischemic injury contained significantlylower lactate levels than the tissues from the control

and nimodipine-treated animals.

The tissue's ability to recover after cerebralischemia may depend on the level of lactic acidosisthat develops during an ischemic episode. Even verymild or short-term ischem.ia causes significantdamage to pyramidal neurons in the CA 1 and CA4regions of the hippocampus, and other neuronal andglial cells.

Experimental studies on ischemia have shownthat certain kinds of drugs may greatly reduce thetissue injury caused by cerebral infarction. L-type andpre-synaptic (N-type) voltage-regulator calcium­channel antagonists, and anticonvulsants such asbarbiturates and phenytoin reduce brain damagewhen they are administered at the start of or just afteran ischemic event. Specifically, the L-type calcium­channel blocker nimodipine prevents contraction ofsmooth muscle in artery walls; superoxide dismutase,a free radical scavenger, increases the half-life of nitricoxide; and MK-801, an NMDA receptor/channelantagonist, increases cerebral blood flow in ischemiczones (11).

As mentioned above, accumulation of sodium

and calcium in the cell is an important step in thepathological process that leads to cell death aftercerebral ischemia (5,12,13,14). Lifarizine helpsminimize the recurrent depolarization induced byischemia because it blocks neuronal sodium and

24

Ars!nllfn~: Lifnri::illc nllli Nil1lodipillc ill Foca! Cere/1m! l.<c!IClllill

calcium channels, thus preventing influxes of sodiumand calcium ions into the cell (3). In vitro studies haveshown that lifarizine causes allosteric activation of

toxin zone 2 in sodium channels. This agent has alsobeen shown to effectively block the sodium currentin N1E-115 neuroblastoma cells (9).

Experiments on lifarizine treatment of ischemiacaused by occlusion of the middle cerebral artery incats revealed that this drug reduced the infarct areaand cerebral edema, and stabilized energy reservesin the brain tissue (6). Other research on globalischemia caused by four-artery occlusion in ratsshowed that this agent prevented late neurona I deathafter 10 min of carotid ligation (1). In a study thatinvolved magnetic resonance imaging, Brown et a1.demonstrated that lifarizine treatment for cerebralischemia reduced the amount of edema that

developed, and also protected against striataldamage despite insufficient collateral blood supplyto this brain area. The authors' conclusion was that

lifarizine is effective at preventing neuronal deathin the CAI region after bilateral carotid occlusion inthe brains of rats and other rodents (2). Blockage ofsodium channels in neuronal tissues is a effective

mechanism for reducing neuron damage aftercerebral ischemia (3).

This is the first study that has investigated howlifarizine effects lactate levels in the ischemic ratbrain. Our results indicate that lifarizine treatment

and nimodipine treatment are both associated withreduced lactate production after forebrain ischemiain this animal model; however, nimodipine appearsto be less effective than lifarizine. Lifarizine maysomehow block the production of metabolites afterinitial ischemic injury. Our results suggest thatlifarizine is a more effective neuroprotective agentthan nimodipine in the setting of rat forebrainischemia induced by bilateral carotid artery ligation.

Correspondence: Ali Arslanta~Hasan Polatkan Bulvan.No: 102/C K:4, 0:8Esh;ehir, TurkeyTel: 0 533 611 3535Fax: 0222239 3774

E-mail: aali@ogu.edu.tr

REFERENCES

1. Alps BJ, Calder C, Wilson AD, Pascal JC: Generalprotection with a novel calcium blocker in rats. XXth IntCongr Med (lSIM Satellite), Stockholm, Sweden. 1990

Turkish Neurosurgery 12: 22 - 25, 2002

2. Brown CM, Calder C, Alps BJ,Spedding M: The effectof lifarizine (RS-87476), a novel sodium and calciumchannel modulator, on ischaemic dopamine depletionin the corpus striatum of the gerbil. Br J Pharmacol109: 175-177, 1993

3. Brown CM, Calder C, Linton C, Small C,.Kenny BA,Spedding M, Patmore L. Neuroprotective propertiesof lifarizine compared with those of other agents in amouse model of focal cerebral ischemia. BrJ Pharmacol115:1425-1432, 1995

4. Garcia JH: Experimental ischemic stroke: A review.Stroke 15: 5-14, 1984

5. Gelmers HJ. Calcium channel blockers: effects oncerebral blood flow and potential uses for acute stroke.Am J Cardiol55: 144B-148B, 1985

6. Kucharczyk J, Mintorovitch J, Moseley ME, Asgari HS,Sevick RJ, Derugin N, Norman D: Ischaemic braindamage: reduction by sodium-calcium ion channelmodulator RS-87476. Radiology 179: 221-227,1991

7. Livesley B, Atkinson L: Accurate quantitativeestimation of lactate in whole blood. Clin Chern 20:154­158, 1974

8. Marbach EM, Wells MH: Rapid enzymatic

Arslnlltn§: Lijnrizille nlld Nimodipille ill Focal Cerebral lscilell/in

measurement of blood lactate and pyruvate. Clin Chern13:314-325, 1967

9. McGivern JG, Patmore L, Sheridan RD. Actions of thenovel neuroprotective agent lifarizine (RS-87476) onvoltage-dependent sodium currents in theneuroblastoma cell line, NIE-115. Bri J Pharmacol 114:1738-1744,1995

10. Paschen W, Djuricic B, Mies G, Schmidt-Kastner R,Linn F: Lactate and pH in the brain: Association anddissociation in different pathophysiological states.Journal of Neurochemistry 48: 154-158, 1987

11. Pulsinelli W: Pathophysiology of acute ischaemicstroke. Lancet 339: 533-6, 1992

12. Siesjo BK. Cell damage in the brain: a speculativesynthesis. J Cereb Blood Flow Metab 1: 155-185, 1981

13. Simon RP, Griffiths T, Evans MC, Swan JH, MeldrumBS.Calcium overload in selectively vulnerable neuronsof the hippocampus during and after ischaemia: anelectron microscopy study in the rat. J Cereb BloodFlow Metab 4: 350-360, 1984

14. Spedding M, Kilpatrick AT, Alps BJ: Activators andinactivators of calcium channels: effect in the central

nervous system. Fundam Clin Pharmacol3: 3-29, 1989

J Assoc Physicians India 2002 Feb;50:250-B

Neuroprotective agents in acute ischemic stroke.

Sareen D.

Ten classes of neuroprotective agents have reached phaseIII efficacy trials. They included calcium channel antagonists,NMDA receptor antagonists, lubeluzole, CDP-choline, thefree radical scavenger tirilazad and ebselen, enlimomab,GABA agonist c1omethiazole, the sodium channel antagonistfosphenytoin, magnesium, glycine site antagonist GV150526and piracetam.

25