Effects of nilvadipine on neuronal function in the ischemic cat brain

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255

Effects of Nilvadipine on Neuronal Function in the Ischemic Cat Brain Shoji Takakura, Masanobu

M.Sc., Yasuhisa Furuichi, M.Sc., Hisashi Satoh, Ph.D., Jo Mori, Ph.D., and

Kohsaka, Ph.D.

Department of Pharmacology, Product Development Laboratories, Fujisawa Pharmaceutical Co., Osaka, Japan

Takakura S, Furuichi Y, Satoh H, Mori J, Kohsaka M. Effects of nilvadipine on neuronal function in the ischemic cat brain. Surg Neurol 1992;37:255-60. The effect of nilvadipine, a dihydropyridine-type calcium entry blocker, on neuronal function during and following ischemia was investigated with a model of focal cerebral ischemia in cats and was compared with that of nicardipine. Drugs were given intravenously 30 minutes before occlusion of the left middle cerebral artery (MCA). Occlusion of the left MCA for 60 minutes was followed by reperfusion for 90 minutes. The amplitude of the somatosensory evoked potentials (SEPs), and the residual relative regional cortical blood flow in the left ectosylvian gyrus and the left posterior sigmoid gyrus, were higher or had a tendency toward higher values in the nilvadipine-treated (32 gig/kg) group than in other groups. After reperfusion, the amplitude of SEPs rapidly recovered in the nilvadipine-treated groups. When administered before MCA occlusion, nilvadipine improved neuronal function measured by SEPs both during and following the ischemic period. Thus, nilvadipine is effective against neuronal dysfunction in focal cerebral ischemia. WORDS: Calcium entry blocker; Nilvadipine; Cerebral blood flow; Middle cerebral artery; Somatosensory evoked potentials KEY

Calcium entry blockers may have two beneficial effects in cerebral ischemia: (1) relaxation of vascular smooth muscle by calcium entry blockade and subsequent increase of cerebral blood flow [19,25]; and (2) preservation o f cellular function in cerebral tissues by preventing the accumulation of intracellular calcium [ 1,10], which may serve as a trigger of irreversible cellular injury [7,20,23].

Address reprint requests to: Shoji Takakura, M.Sc., Department of Pharmacology,Product Development Laboratories,FujisawaPharmaceutical Co., Ltd., 2-1-6, Kashima, Osaka 532, Japan. Received August 9, 1991; accepted September 4, 1991.

© 1992 by ElsevierSciencePublishingCo., Inc.

Middle cerebral artery (MCA) occlusion is a widely used animal model in experimental research on neuronal metabolism and dysfunction and cerebral circulation during ischemia. With somatosensory evoked potentials (SEPs) used as a measure of neuronal function, it was found that pretreatment of animals with a calcium entry blocker reduced the decrease in SEPs during ischemia and facilitated the return of neuronal function after reperfusion in comparison with the course o f untreated animals [9,11]. Nilvadipine, a dihydropyridine-type calcium entry blocker, has a possible role in cerebral vascular disease because it has selective and long-lasting effects on cerebral arteries in vivo [19]. It has also been reported that nilvadipine exhibits a widespread distribution into various tissues, including brain [27]. Shiino et al [22] reported that nilvadipine reduced the infarct size produced by MCA occlusion in spontaneously hypertensive rats. Moreover, a protective effect of nilvadipine on ischemic degradation of cytoskeletal protein was reported by Kuwaki et al [16]. The present study was conducted to examine whether nilvadipine ameliorates the disturbance of cerebral circulation and impairments o f neuronal function induced by MCA occlusion in cats. The effects of nilvadipine were compared with those ofnicardipine hydrochloride (nicardipine), a selective cerebral vasodilator [25].

Materials and Methods Male and female adult cats weighing 2.2-4.1 kg were used. After induction of anesthesia with an intramuscular injection of ketamine hydrochloride (30 mg/kg), the animals were immobilized with an intravenous injection of pancuronium bromide (0.2-0.3 mg/kg initially with additions thereafter when needed) and were mechanically ventilated with room air by an animal ventilator (model 662, Harvard, South Hatick, MA). Systemic arterial blood pressure and heart rate were continuously monitored by a catheter in the left femoral artery that was connected to a pressure transducer (TP-200T, Ni0090-3019/92/$5.00

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hon Kohden, Tokyo, Japan). Body temperature was kept at around 37.5°C with a heating blanket with rectal thermometer (CFP8185, Bioscience, Kent, UK). Blood gases were intermittently measured with a blood gas analyzer (model 288, Ciba-Corning, Medfield, MA), and hematocrit values were also intermittently measured. Ventilation was controlled so that the Paco 2 was maintained at 28-34 mm Hg.

Occlusion of the Middle Cerebral Artery After positioning the head fixation in a stereotactic instrument (SN-3, Narishige, Tokyo,Japan), the left MCA was temporarily occluded via a transorbital approach [18]. The left eyeball and orbital contents were excised. With an operating microscope (OME, Olympus, Tokyo, Japan) and a dental drill a small craniectomy was made to enlarge the optic foramen. The dura mater was opened and the proximal segment of the MCA was exposed. The arachnoid membrane covering the MCA was carefully dissected, and the MCA was occluded close to its origin from the internal carotid artery by means of a Yasargil microclip.

Measurement of Relative Regional Cortical Blood Flow Relative regional cortical blood flow (CBF) was measured by laser-Doppler flowmetry [4,5] with an Advance laser-Doppler flowmeter (ALF2100, Advance, Tokyo, Japan). Standard probes (needle type, Advance, Tokyo, Japan) were placed over the left ectosylvian gyrus and the left posterior sigmoid gyrus (dura intact) according to the atlas of Snider and Niemer [24]. The blood flow thus obtained served as baseline and was designated 100%.

Recording of Somatosensory Evoked Potentials Somatosensory evoked potentials were recorded from the left sensory cortex after stimulation of the contralateral median nerve. The silver ball recording electrode was placed on the dura mater over the posterior sigmoid gyrus that corresponds to the primary somatosensory cortex for the forelimb in the cat [15]. The reference electrode was placed at the chin. Square wave stimuli (0.1 ms in duration) were applied (SEN-3201, Nihon Kohden, Tokyo, Japan) at 4 Hz with supramaximal voltage. Somatosensory evoked responses were amplified with a bandpass of 16-3,000 Hz and averaged for 64 sweeps in a 100-ms time window (EEG-2414 and ATAC-450, Nihon Kohden, Tokyo, Japan). Averaged tracings were copied with an x-y plotter (7225A, Hew-

Takakura et al

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Figure 1. Time course of drug-induced changes in mean arterial blood pressure (MABP). (0) Control. (R) Nilvadipine 10 Izg/kg. (1) Nilvadipine 32/zg/kg. (A) Nicardipine 32 ~g/kg. (A) Nicardipine 100/zg/kg. * p < 0.05 and **p < 0.01 comparedwith control.

lett Packard, USA) and only the peak-to-peak amplitude of the positive/negative sequence of the primary evoked response [ 15 ] was recorded directly from the hard copy. Drug Administration Nilvadipine was prepared in our laboratories and nicardipine hydrochloride was purchased from Sigma. Both calcium entry blockers were dissolved in polyethyleneglyco1400 (PEG 400) to the concentrations needed. Cats were given an intravenous bolus injection of 0.1 mL/kg of the drug solution or vehicle alone. Administration of the drug and the vehicle was made 30 minutes prior to an occlusion of the MCA. Statistical Analysis Statistical differences were assessed with Dunnett-type multiple comparisons for nonparametric data, which were performed for differences among the groups at each time. A p value < 0.05 was regarded as statistically significant. All values were given as the mean - standard error of the mean.

Results Physiological Parameters Figure 1 shows the changes of mean arterial blood pressure during the experiment. Transient hypotension was observed after intravenous injection of nilvadipine and nicardipine. Hypotension induced by both drugs was dose-dependent. Five minutes after the drug injection, mean arterial blood pressure in animals treated with 10 ~g/kg and 32 /zg/kg doses of nilvadipine was reduced to 15.7% and 28.0% of baseline, respectively; and in animals treated with 32/zg/kg and 100 ~g/kg doses of

Effects of Nilvadipine in Ischemia

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Table 1. PhysiologicalParametersfor Cats Subjectedto MCA Occlusion Before occlusion Experimental group Control

Parameter

Paco 2 (mm Hg) Pao 2 (mm Hg) pH Hct (%)

Nilvadipine (10 ~g/kg)

PaCO 2 (ram Hg) PaO 2 (mm Hg) pH Hct (%)

Nilvadipine (32/xg/kg)

PaCo 2 (mm Hg) PaO2 (ram Hg) pH Hct

(%) Nicardipine (32 ~g/kg)

PaCO 2 (mm Hg) Pao2 (mm Hg) pH Hct (%)

Nicardipine (100/~g/ kg)

PaCo 2 (mm Hg) Pao 2 (mm Hg) pH Hct (%)

- 15 min

- 30 min

31.6 1.0 103.1 -+ 1.9 7.345 -0.010 38.1 -+ 1.1 -+

31.1 0.4 101.1 ± 0.8 7.363 ± 0.019 38.5 4 2.5 ±

32.7 0.6 98.5 -+ 1.3 7.322 -0.012 42,6 -+

±

1.1

31.0 0.7 100.5 -+ 1.9 7.363 -+ 0.014 40.7 + 1.9

-+

30.5 0.7 102.2 -+ 1.2 7.378 -+ 0.013 37.9 2.1 -+

33.1 0.9 101.8 1.4 +7.334 0.011 ± 35.9 1.2 ± -+

32.5 0.5 98.3 1.1 ± 7.341 0.017 -+ 37.2 2.7 ±

34.9 0.4 99.0 1.4 -+ 7.289* -+ 0.007 41.0 _+ 1.2 -+

31.4 0.4 97.2 2.0 ± 7.345 0.016 -+ 39.8 2.1 -+ -+

33.2 0.5 101.7 + 1.1 7.325 0.008 -+ 36.1 2.2 --+

During occlusion (30 min)

29.5 1.0 105.7 ± 2.4 7.354 ± 0.009 37.4 ± 1.1 ±

30.5 0.6 100.1 ± 1.8 7.352 ± 0.016 37.9 ± 2.6

±

31.4 0.6 100.0 ± 1.4 7.328 ± 0.002 42.1 ± 1.4 ±

± ± ± ±

30.1 0.6 97.8 2.0 7.371 0.016 40.4 2.0

31.3 0.4 100.2 ± 1.3 7.350 ± 0.010 36.7 ± 2.2 ±

Reperfusion 150 rain

90 min

3O.3 0.8 101.5 -+ 2.3 7.324 ± 0.008 35.0 -+ 1.8 ±

30.3 0.6 98.6 -+ 2.3 7.349 -+ 0.018 37.4 +2.6

-+

31.4 0.9 100.2 -+ 2.6 7.331 ± 0.010 42.3* ± 1.0 -+

29.6 0.8 99.6 -+ 2.3 7.379* -+ 0.016 40.2 + 2.0

-+

31,8 0.6 100.3 -+ 1.3 7.357 + 0.012 35.5 -+ 1.9 -+

29.1 0.7 103.3 + 1.6 7.342 -+ 0.009 35.5 -+ 1.4

+-

31.4 0.9 97,7 -+ 1.9 7.347 + 0.020 37.1 -+ 2.4

-+

32.0 1.5 98.7 -+ 1.4 7.330 + 0.012 42.4 ± 1.3 -+

29.7 0.7 100.2 -+ 2.3 7.386 -+ 0.014 39.9 -+ 2.2

+

29.4 0.8 103.1 -+ 1.7 7.376 -+ 0.016 34.0 -+ 2.2 ±

Data are mean -+ SEM. *p < 0.05 compared with control.

nicardipine to 29.0% and 38.8% of baseline, respectively. With a dose of 32 /~g/kg, both drugs induced almost the same hypotension, but the duration of hypotension induced by nilvadipine was much longer than that by nicardipine. The heart rate in all groups measured in this study showed no significant change throughout the experiment. Table 1 summarizes the physiological parameters, except for mean arterial blood pressure and heart rate. The parameters remained in the normal range throughout the experiment.

Relative Regional Cortical Blood Flow Figure 2 and Figure 3 show the changes of relative rCBF in the left ectosylvian gyrus and the left posterior sigmoid gyrus, respectively. In both sites, rCBF in drugtreated groups increased by approximately 20% of the baseline level, and the increases lasted to the time of the left MCA occlusion. In the control group, which received vehicle alone, an occlusion of the left MCA caused a rapid and severe reduction of the rCBF both in the left ectosylvian gyrus and in the left posterior sig-

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rCBF

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Takakura et al

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Figure 2. Relative cerebral bloodflow (rCBF) in the left ectosy&iangyrus. During ischemia flow was reduced to approximately 20% of the pretreated level in the control group. (0) Control (El) Nilvadipine 10 tzg/kg. (I) Nilvadipine 32/*g/kg. (A) Nicardipine 32/*g/kg. (it) Nicardipine 100 gtg/kg. * p < 0.05 and ** p < 0.01 compared with control.

moid gyrus. In the left ectosylvian gyrus, the degree of rCBF reduction after the left MCA occlusion was much milder in the group treated with 32 ~g/kg doses of nilvadipine than that in the control group. However, the difference in the degree of residual rCBF between the two groups was not significant. Residual rCBF in the left posterior sigmoid gyrus during left MCA occlusion was much higher in two groups (nilvadipine 10 ~*g/kg and nicardipine 100 tzg/kg) than that of the control group, and the differences were significant at 45 minutes and 55 minutes after the occlusion. In the nilvadipine 32 /*g/kg group residual rCBF during left MCA occlusion showed a strong tendency to increase, but the change did not reach the level of statistical significance. After release of the occlusion, transient hyperperfusion occurred in the left ectosylvian gyrus, while in the

Figure 3. Relative cerebral bloodflow (rCBF; in the left postsigmoid gyrus. During ischemia flow was reduced to approximately 20% of the pretreated level in the control group. After reperfusion, there was an immediate hyperemia in the control group. (0) Control. ([]) Nilvadipine 10/*g/kg. ( i ) Nilvadipine 32/*g/kg. (A) Nicardipine 32/*g/kg. (it) Nicardipine 100 tzg/kg. * P < 0.05 and ** p < 0.01 compared with control

0

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45

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65

75

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Time (min)

Figure 4. SEPs during experiment. After MCA occlusion, SEPs immediately decreased to less than 10% of pretreated levels in the control group. After reperfusion SEPs started to recover in all groups. * p < 0.05 compared u ith control.

left posterior sigmoid gyrus hyperperfusion occurred with long duration. In the drug-treated groups, obvious hyperperfusion in the left posterior sigmoid gyrus was not observed. Somatosensory Evoked Potentials

Administration of drugs had little effect on SEPs prior to occlusion. Occlusion of the left MCA caused a severe decrease in the amplitude of the SEPs in the control group. In animals treated with nilvadipine and nicardipine the reduction of SEPs induced by the left MCA occlusion was delayed in a dose-dependent manner. In the animals treated with 3 2 / , g / k g doses of nilvadipine the retention of SEPs was significantly higher than that in control animals. After reperfusion, the amplitude of SEPs recovered rapidly to the preocclusion levels in animals treated with nilvadipine. In the control group the recovery of the amplitude was delayed, reaching about 80% of the preocclusion level at 90 minutes after release of the occlusion. Nicardipine-treated groups did not show the rapid recovery after reperfusion that was shown in the nilvadipine-treated groups (Figure 4).

rCBF 220 -

Discussion

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The origins of SEPs, which apparently represent one of the most reliable parameters for assessing neuronal function, have been well characterized in patients. In animal experiments change in SEPs during ischemia induced by MCA occlusion not only is a sensitive indicator of neuronal function [17,21] but it also correlates with the metabolic alterations in ischemic brain [ 13]. Cortical blood flow also influences the SEPs [2,3], and the changes of SEPs during ischemia are widely used in the assessment of drugs, including Ca 2+ blockers [6,9,11].

Effects of Nilvadipine in lschemia

In this report we have compared the effects of nilvadipine and nicardipine on neuronal dysfunction induced by MCA occlusion. After intravenous injection of both drugs, in spite of a decrease in mean arterial blood pressure, rCBF increased approximately 20% in the two cortical areas where rCBF were measured. In the left ectosylvian gyrus (MCA territory), the significant increases in rCBF were not dose-related. This phenomenon may be due to the autoregulation of cerebral circulation. The increase in rCBF was not accompanied by Paco 2 changes and may have been due to the vasodilating effect of the drugs. After temporary occlusion of the left MCA, rCBF was markedly reduced in both gyri. Two calcium entry blockers, nilvadipine and nicardipine, reduced the degree of decrease in the rCBF in the ipsilateral hemisphere. Similar observations have also been reported for other calcium entry blockers [11,12] and might be due to an improvement in the collateral blood flow. After reperfusion of the area previously rendered ischemic, transient hyperperfusion was observed in the left ectosylvian gyrus, and a long-lasting hyperperfusion was observed in the left posterior sigmoid gyrus in the control group. Hyperperfusion after transient ischemia is thought to aggravate cerebral edema [26]. Both calcium entry blockers inhibited the long-lasting hyperperfusion in the left posterior sigmoid gyrus, suggesting that calcium entry blockers may reduce postischemic edema. Occlusion of the left MCA reduced the amplitude of the SEPs immediately in the control group, whereas the reduction of SEPs was delayed in cats pretreated with nilvadipine and nicardipine. In the animals pretreated with 32 Izg/kg doses of nilvadipine the amplitude of the SEPs remained significantly higher than that in control animals. The effect of both calcium entry blockers correlated with the rCBF in the left ectosylvian gyrus. The effects of drugs on SEPs may be related by their effects on blood flow during ischemia. Graf et al [8] demonstrated that the functional impairment found in the cortical ischemic area is caused by injury of subcortical structures that leads to a cortical deafferentation rather than by the degree of rCBF in the primary somatosensory cortex. Neuronal function was influenced by damage in the subcortical region that was induced by MCA occlusion, regardless of the degree of the ischemia in the cortical neurons. Pretreatment with 32 tzg/kg doses of nilvadipine may protect neurons from lesions induced by MCA occlusion by preserving the rCBF in afferent pathways to the cortex, as was observed in the left ectosylvian gyrus. After reperfusion, SEPs recovered gradually in the control and nicardipine-treated groups. In contrast, in groups pretreated with nilvadipine SEPs recovered more rapidly. The effect of nilvadipine on the amplitude of

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the SEPs during the experiment may correlate with the degree of neuronal injury that was represented by SEP amplitude during a period of left MCA occlusion rather than the rCBF after reperfusion. It has been reported that the degree of electrophysiological changes in the electroencephalogram and that of the SEPs were closely correlated with biochemical alterations, such as the tissue acidosis, adenosine triphosphate depletion, decrease of tissue potassium content, and suppression of protein synthesis [ 13]. The effect of nilvadipine on alteration of SEPs during and after ischemia may reflect a protective effect not only on neuronal function but also on neuronal metabolism. During cerebral ischemia, when the plasma membrane is depolarized because of energy depletion, a large amount of Ca 2÷ enters through the voltage-sensitive Ca 2÷ channels. Recently, Uematsu et al [28] demonstrated that nimodipine, a dihydropyridine-type calcium entry blocker, attenuated both increase in cytosolic free calcium and histological damage in the cortex following focal cerebral ischemia and reperfusion in cats. Nimodipine was also shown to have favorable effects on the recovery of electroencephalogram amplitude following reperfusion as well as on the extent of focal histological damage. The protective effect of nilvadipine against focal cerebral ischemia, which was demonstrated in this study, might be associated with an inhibitory effect on ischemia-induced increases in cytosolic calcium. Nilvadipine was more potent than nicardipine in this study. The effects of nilvadipine on cerebral arteries are more selective and long-lasting than those of nicardipine [19]. Nilvadipine has also been reported to be distributed well into the brain, whereas the distribution of nicardipine into the brain may be less effective because of its water solubility [14]. Brain concentrations of nilvadipine are indeed higher than those of nicardipine after administration of equivalent doses, and the halflife of nilvadipine in the brain has been found to be longer that that of nicardipine (T. Fujiwara et al, unpublished data). These differences may explain differences in the effects between the two drugs. The effects of the drugs tested in this study of neuronal dysfunction against ischemia may therefore reflect not only their cerebral vasodilatory effects but also their ability to inhibit calcium influx into neurons. In the present study nilvadipine showed a beneficial effect on the SEPs during and after ischemia in this experimental model of MCA occlusion in cats. Nilvadipine was more potent than nicardipine in this study, suggesting that nilvadipine could be more effective against neuronal dysfunction in cerebral ischemia. We thank ProfessorJ. Handa, M.D., Department of Neurosurgery, ShigaUniversityof MedicalScience,for helpingus learnthe technique

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of middle cerebral artery occlusion in cats. We also thank Dr. Steven Butcher for reading the manuscript.

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