Journal of Cerebral Blood Flow and Metabolism
10:654--659 © 1990 Raven Press, Ltd., New York
Protective Effect of Nimodipine Against Ischemic Neuronal Damage in Rat Hippocampus Without Changing Postischemic Cerebral Blood Flow
Jorg Nuglisch, Chourouk Karkoutly, *Hans Dieter Mennel, *Christine RoBberg, and Josef Krieglstein Institut fur Pharmakologie und Toxikologie and *Abteilung Neuropathologie, Philipps-Universitiit, Marburg, F.R.G.
Summary: The purpose of the present study was to in vestigate the neuroprotective action of nimodipine. Fur thermore, the influence of nimodipine on postischemic local CBF (LCBF) was examined. Forebrain ischemia of the rat was performed for 10 min by bilateral carotid clamping, administration of trimethaphan, and blood withdrawal to obtain an MABP of 40 mm Hg. LCBF was measured after 10 min of postischemic recirculation by injecting [14C]iodoantipyrine in saline solution. Nimo dipine (0. 1, 0.3, and 1.0 mg/kg) was suspended in miglyol oil and applied orally 60 min prior to ischemia. Histolog ical evaluation was performed 7 days after ischemia. Hip-
pocampal neuronal damage was determined as the per centage of necrotic neurons. After preischemic applica tion of nimodipine, neuronal damage was significantly reduced in the hippocampal CAl subfield. Postischemic LCBF was not affected by treatment with nimodipine. These findings show that nimodipine is able to protect neurons against ischemic damage. The neuroprotective effect of nimodipine was not mediated by a postischemic cerebral vasodilation, but by a direct action on the neu rons. Key Words: Forebrain ischemia-Local cerebral blood flow-Neuronal damage-Nimodipine.
It is widely accepted that calcium plays an impor tant role in the deleterious progress of neuronal damage. Furthermore, it is known that ischemia re sults in an inhibition of ATP-dependent ionic pumps and causes a substantial increase in cytoplasmic Ca2+ concentration (Farber, 1981; Siesj6, 1981; Siesj6 and Bengtsson, 1989). This elevated intra cellular Ca2+ level is supposed to contribute to sev eral processes leading to neuronal death (Siesj6, 1981; Borgers et aI., 1983; Choi, 1988). Calcium an tagonists are known to block voltage-sensitive cal cium channels, reduce cerebrovascular resistance under normal and pathological conditions, and im prove the resistance of the brain to the effects of an ischemic stroke (Kazda et aI., 1982; Nakayama et aI., 1989). The neuroprotective effect of calcium an tagonists has already been demonstrated in earlier work; e.g., flunarizine or emopamil significantly re-
duced neuronal damage in the hippocampus (Desh pande and Wieloch, 1985, 1986; Beck et aI., 1988; Bielenberg et aI., 1989a) or reduced infarct size af ter occlusion of the middle cerebral artery of rats (Nakayama et aI., 1989). Furthermore, evidence has been presented that the reduction in neuronal damage is caused by a direct effect of the calcium antagonists on cerebral parenchyma and is not me diated by a vascular action (Bielenberg et aI., 1987, 1989a; Beck et aI., 1988). Nimodipine is a calcium antagonist of the 1,4dihydropyridine type with potent selective effect on cerebral vessels at doses insufficient to affect sys temic arterial blood pressure (Kazda and Towart, 1982; Kazda et aI., 1982; Scriabine et aI., 1985). The presence of nimodipine-specific binding sites on se lectively vulnerable neurons in the hippocampus and the cortex (Quirion et aI., 1985; Spedding and Middlemiss, 1985) and the behavioral effects after nimodipine treatment observed by Hoffmeister et aI. (1982) suggest that nimodipine, in addition to its cerebral vasodilating effect, may modify neuronal function via a direct action. In the isolated perfused rat brain, Bielenberg et aI. (1989b) have found a
Received September 18, 1989; revised February 19, 1990; ac cepted February 21, 1990. Address correspondence and reprint requests to Dr. J. Nuglisch at Institut fiir Pharmakologie und Toxikologie, Phil . ipps-Universitat, Ketzerbach 63, 3550 Marburg, F.R. G. Abbreviation used: LCBF, local CBF.
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EFFECT OF NIMODIPINE reduction in postischemic lactate levels indepen dent of the mean postischemic perfusion rate. On the other hand, nimodipine has also been shown to prevent postischemic hypoperfusion following tran sient cerebral ischemia in dogs (Steen et aI., 1983, 1984; Newberg et aI., 1986), in cats (Kazda et aI., 1982), and in rats (Smith et aI., 1983; Young et aI., 1987). The purpose of the present study was to demon strate that nimodipine is able to reduce postisch emic neuronal damage in the hippocampus. Fur thermore, the possible correlation of a neuroprotec tive action of nimodipine with an influence on postischemic regional CBF was investigated.
MATERIALS AND METHODS Animals Male Wistar rats (250-300 g; Ivanovas, Kissleg, F.R.G) were used in all experiments. They were maintained un der controlled lighting and environmental conditions (12h dark-light cycle, 23 ± lOC, 55 ± 5% relative humidity). The rats were kept on standard diet (Altromin, Lage, F.R.G.) and tap water ad libitum. Preceding the experi ments the rats were fasted overnight. Rats were divided into two groups. In group 1 histological evaluation was performed on 37 rats. The measurement of CBF was car ried out in 30 rats of the second group.
Surgical procedure and induction of ischemia The experiments were performed according to Smith et al. (1984). Rats were anesthetized with 3.5% halothane, intubated, and connected to a Starling-type respirator de livering 0.7% halothane and 30% O2 in nitrous monoxide. The jugular vein (silicone catheter) and the tail artery (polyethylene catheter) were catheterized for withdrawal of blood and for monitoring blood pressure or collecting arterial blood samples. Anticoagulation was achieved by intravenous heparin (200 IV/kg) application. Blood gases, blood pH (Coming 178; Corning Medical, GieBen, F.R.G.), blood pressure (Statham P23DB, Heto Rey, Pu erto Rico; HSE-Electromanometer, Hugstetten, F.R.G.), and plasma glucose (Beckman Glucose Analyzer II, Mu nich, F.R.G.) were measured 5 min prior to ischemia and 10 min after ischemia. Halothane (but not nitrous monoxide) was discontin ued and the rats allowed to recover for 30 min. During this period muscle paralysis was maintained with 5 mg/kg suxamethonium chloride (Asta Pharma AG, Frankfurt, F.R.G.), repeated every 15 min. Ischemia was induced by injection of 5 mg/kg trimethaphan camphor sulfonate (Hoff mann-La Roche, Grenzach-Wyhlen, F.R.G.), occlusion of both common carotid arteries, and exsanguination to a blood pressure of 40 mm Hg. After 10 min of ischemia, blood pressure was restored by removing the carotid clamps and reinfusing the shed blood. Rats then received 1 mmol/kg sodium bicarbonate to prevent systemic aci dosis. Body temperature was held at -37°C with a heat ing lamp. The femoral artery and the femoral vein were additionally cannulated in rats used for the determination
655
of local CBF (LCBF) to allow blood sampling and injec tion of the isotope solution. Ten minutes after ischemia CBF was measured in arti ficially ventilated rats receiving the nitrous monoxide/ oxygen mixture (70:30) until decapitation.
LCBF LCBF was determined by injecting [14C]iodoantipyrine in saline solution (Sakurada et aI., 1978). The femoral vessels were cannulated and 150 fLCi/kg e4C]iodo antipyrine (spec. act. 60 mCi/mmol; NEN, Dreieich, F.R.G.) was infused over 1 min using a ramp function. During infusion 10 timed arterial blood samples were col lected and analyzed for [14C]iodoantipyrine content by liquid scintillation counting. The rats were decapitated at the end of infusion; the brains were dissected within 4560 s, frozen in isopentane, and processed for quantitative autoradiography according to standard procedures by cryosectioning (mod. 2700; Reichert-Jung, NuBloch, F.R.G.). Then 20-fLm brain slices were exposed to Osray M3 film (Agfa-Gaevert, Leverkusen, F.R.G.) for 10 days. Regional gray values of the autoradiograms were mea sured with an image analyzer (IBAS; Kontron, Eching, F.R.G.) and transformed to CBF values.
Histological assessment of ischemic cell damage For histological evaluation animals were anesthetized with halothane on day 7 after ischemia and perfused trans cardially via the ascending aorta with a phosphate buffer of pH 7.35 containing 4% formaldehyde after rinsing the brain with -30 ml physiological saline solution. Brains were removed, embedded in Paraplast, and sectioned in 5-fLm slices. Then the slices were stained with a mixture of 1% celestine blue and 1% acid fuchsin (Auer et aI., 1984). Hippocampal intact and necrotic cells were counted in a section 6 mm rostral to the tentorial incision. Cell loss was determined as the percentage of acid stainable cells.
Drug administration Nimodipine was suspended in miglyol oil and was given orally 1 h prior to ischemia in dosages of 0.1, 0.3, and 1.0 mg/kg. Solutions containing nimodipine were carefully protected from light. Controls received miglyol oil only.
Statistics Values are presented as means ± SD. In all experi ments mUltiple comparisons were made by the Kruskal Wallis H test combined with the Duncan test.
RESULTS Physiological variables
Nimodipine dosages of 0.1 and 0.3 mg/kg did not influence the physiological variables (Table 1). Ad ministration of 1.0 mg/kg nimodipine lowered post ischemic arterial blood pressure measured 10 min after ischemia significantly (Table 1). Other physi ological parameters were not affected by treatment with 1.0 mglkg nimodipine (Table 1). J Cereb Blood Flow Metab, Vol.
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J. NUGLISCH ET AL.
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TABLE 1. Physiological variables after treatment with nimodipine Nimodipine (mg/kg) Control (n 12) =
Arterial blood pressure (mm Hg) Before ischemia After ischemia Plasma glucose (mg/IOO ml) P02 (mm Hg) Pco2 (mm Hg) pH Rectal temperature eC)
127 130
±
127 123 37 7.38
±
37.0
±
17 10
0.1 (n
124 130
±
11 12 2 0.04
134 124 37 7. 33
±
0.0
37.0
± ±
=
± ±
0.3 8)
(n
12 10
123 131
=
± ±
1.0 8)
(n
=
9)
117 ± 14 115 ± lla
15 14
±
8 23 2 0.06
119 ± 13 130 ± 35 37 ± 2 7.35 ± 0.04
125 ± 17 130 ± 32 38 ± 4 7.32 ± 0.06
±
0.0
36.9
36.9
± ± ±
±
0.2
±
0.1
Physiological variables were determined 5 min prior to ischemia. In addition, postischemic arterial blood pressure was measured 10 min after ischemia. Values are presented as means ± SD from n experiments. a Different from control: p < 0.05 (Kruskal-Wallis H test).
Histological evaluation of neuronal necrosis
Oral application of 0.1 or 0.3 mg/kg nimodipine significantly inhibited postischemic neuronal dam age in the hippocampal CAl region (Fig. O. Neuro nal necrosis in the CA3 and CA4 subfields of the hippocampus was only marginal and was not atten uated by nimodipine (Fig. l). The oral dosage of 1.0 mg/kg nimodipine did not reduce the neuronal dam age significantly, but the mean value of the percent age of neuronal necrosis in the hippocampal CAl region at this dosage was diminished in comparison with the ischemic control group (Fig. I). LCBF
LCBF was determined 10 min after ischemia with preischemic application of the oral dosages of 0.1, 0.3, or 1.0 mg/kg nimodipine (Table 2). None of NEURONAL DA ... AGE % 70
D CONTROL
60 50
•
•
�
NIMODIPINE
DISCUSSION
Nimodipine is a calcium antagonist with a prefer ential vasodilatory effect on cerebral vessels and with less effect on peripheral arteries (Kazda and Towart, 1982; Towart et aI., 1982; Scriabine et aI., 1985). Contradictory results were found concerning the effect of nimodipine on CBF in vivo. In anes thetized rabbits intracarotid application of nimo dipine produced an increase in CBF ipsilateral to the side of infusion (Haws and Heistad, 1984). A significant increase in cortical arteriolar diameter and in regional CBF was found in anesthetized cats after intravenous infusion of nimodipine (Schmidli et aI., 1985). Dose-dependent effects of nimodipine on CBF were found by Mohamed et a1. (1984) in rats and by McCalden et a1. (1984) in baboons. Low doses significantly increased blood flow, whereas higher doses resulted in a decreased systemic arte-
� (O.lmgikg.p.QI
IIIII!I!IIII NIMOOIPINE
IIIIII!IIII (O.3mgikg.p.QI
30
these oral dosages changed postischemic blood flow in the hippocampus or in other measured areas of the brain.
� NIMODIPINE
� (1.0mgikg,p.o.l
20 10 o FIG. 1. Nimodipine protects neurons against ischemia. His
tological demonstration of neuronal damage in the subfields of the hippocampus after 10 min of forebrain ischemia and 7 days of recovery. Nimodipine was given orally 1 h prior to ischemia in dosages of 0. 1, 0.3, and 1.0 mg/kg. Values are means ± SO of 12 control and 8-9 nimodipine-treated rats. 'p < 0.05, Kruskal-Wallis H test.
J Cereb Blood Flow Metab. Vol. 10. No.5, 1990
TABLE 2. Local CBF 10 min after ischemia and oral preischemic treatment with nimodipine 0"1 i :--.. v"--Z vUe ��. . . ,/-. . , Nimodipine
N·/
.�
c/'
, 1..5-
Brain structure Occipital cortex Parietal cortex Frontal cortex Cingulate cortex Retrosplenial cortex Hippocampus CAl Hippocampus CA3 Hippocampus CA. Dentate gyrus
wo-
Control (n 8)
(mg/kg)
0. 1
=
(n
224 ± 88 317 ± 66 290 ± 58 335 ± 96 192 ± 113 205 ± 88 173 ± 62 188 ± 6 1 194 ± 67
2 19 285 272 312 204 184 169 179 189
=
± ± ± ± ± ± ± ± ±
7)
34 47 48 67 60 28 30 45 49
(n
0.3
244 362 314 353 150 193 159 174 182
=
± ± ± ± ± ± ± ± ±
8)
31 74 63 61 76 38 41 54 50
(n
234 345 304 356 219 2 19 185 211 223
1.0 -7) =
± ± ± ± ± ± ± ± ±
43 64 50 76 143 69 66 83 94
Values are means ± SD from n experiments expressed as mlllOO g/min. After 10 min of forebrain ischemia, brains were recirculated for 10 min. Nimodipine was applied I h prior to ischemia.
EFFECT OF NIMODIPINE rial blood pressure and a return to CBF control lev els. In conscious rats Kanda and Flaim (1986) did not find an alteration in CBF or in cerebral vascular resistance after infusion of nimodipine. In addition, no effect on CBF was found by Forsman et ai. (1986) in unanesthetized dogs when nimodipine was applied intravenously. In contrast to these results are data obtained by Mohamed et ai. (1985a). These authors showed a significantly increased level of CBF in 24 brain regions in conscious rats after in fusion of nimodipine. In accordance with these re sults are experiments from Haws et al. (1983), who demonstrated an increase in CBF in anesthetized and unanesthetized cats. Contrary results were obtained concerning the ef ficacy of nimodipine treatment associated with isch emia. Preischemic treatment with nimodipine im proved CBF in middle cerebral artery-occluded rats (Mohamed et aI., 1985b) and tissue pH in rab bits (Tally et aI., 1989). An increase in CBF in the middle cerebral artery occlusion model of the rat was not found when nimodipine was given after in duction of ischemia (Graham et aI., 1985; Gotoh et al., 1986). Adverse results were obtained in neuro pathological quantification of the ischemic damage, too. Protective effects in the volume of infarcted area after middle cerebral artery occlusion were seen after preischemic treatment with nimodipine (Graham et aI. , 1985; Mohamed et aI. , 1985b), whereas application of nimodipine after occlusion of the middle cerebral artery showed no ameliora tive effect (Graham et al., 1985; Gotoh et aI., 1986). In a model of complete cerebral ischemia of the dog, Steen et ai. (1983, 1984) demonstrated that pre or postischemic treatment with nimodipine in creased postischemic CBF, but only preischemic application improved neurologic outcome in a his topathological scoring system. On the other hand, Steen et ai. (1985) showed that nimodipine im proved neurological function and histopathological score when given after complete cerebral ischemia in pigtailed monkeys. Parenteral treatment with ni modipine after ischemia failed to prevent hippocam pal neuronal damage in the four- and two-vessel occlusion model and in the model of forebrain isch emia in the gerbil (van Reempts et aI., 1986; Vilbuls resth et aI., 1987; Alps et aI., 1988). Nimodipine prevents postischemic cerebrovascu lar spasm and improves postischemic impaired reperfusion in various experimental models includ ing the same model as used in the present study (Kazda et aI., 1982; Smith et aI., 1983; Steen et aI., 1983; Newberg et aI., 1986; Young et aI., 1987). In these studies the authors assumed that an amelio ration in neurologic recovery is probably mediated
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by the prevention of delayed postischemic hypo perfusion. On the other hand, the behavioral effects in ni modipine-treated rats obtained by Hoffmeister et ai. (1982) suggest that nimodipine has other pharmaco logical properties besides its vasoactive action. Berger and Hakim (1988) demonstrated that nimo dipine normalizes local cerebral pH without chang ing LCBF in middle cerebral artery-occluded rats when applied after ischemia. Protective effects of nimodipine on postischemic brain energy metabo lism were found by Bielenberg et ai. (1987, 1989b). These authors showed that nimodipine reduced postischemic lactate levels independently of the corresponding flow values, and it was suggested, therefore, that the drug improves postischemic mi tochondrial function by a direct action on cerebral parenchyma. Our study demonstrates that if dose and applica tion are appropriately chosen, nimodipine can re duce neuronal damage in the hippocampus. Postischemic MABP was not influenced by the lower oral nimodipine doses, but the higher dose of 1. 0 mg/kg caused a significant decrease in the post ischemic MABP, measured 10 min after ischemia. The dosage of 1.0 mg/kg nimodipine seems to be the critical value to affect MABP. These data support the earlier finding that nimodipine is not a selective cerebral vasodilator but in higher doses affects sys temic arterial blood pressure as well (Mohamed et aI., 1984). Histological examination of neuronal damage 7 days after ischemia showed a significant reduction of neuronal necrosis in the hippocampal CAl sub field after oral pretreatment with 0.1 or 0.3 mg/kg nimodipine. A clear dose-response relationship concerning the reduction of hippocampal neuronal damage was not demonstrable. It is assumed that pharmacoki netic reasons could be responsible for that. Nimo dipine does not accumulate in brain tissue after a single oral dose (Suwelack et aI., 1985) so that tis sue levels of the drug may vary considerably. Therefore, a very clear-cut dose-response relation ship could not be expected for the neuroprotective effect of nimodipine. The peak plasma level of radioactivity is attained 40-60 min after administration of a single oral dose l of [ 4C]nimodipine in rats (Maruhn et al., 1985). Ra dioactivity half-life in the plasma is 4.6-9.2 h, but unchanged nimodipine represents only a part (22% in rats) of the total radioactivity (Maruhn et aI., 1985). Thus, it has to be assumed that an oral dose of nimodipine given 1 h prior to ischemia affects LCBF in the period of postischemic recirculation. J Cereb Blood Flow Metab, Vol.
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Therefore, measurements of LCBF were performed 10 min after ischemia during the period of relative hyperemia (Kagstrom et al., 1983) with the oral doses of 0.1, 0.3, and 1.0 mg/kg nimodipine. None of these dosages were able to influence postisch emic LCBF 10 min after ischemia in any areas mea sured, including the subfields of the hippocampus. These findings indicate that nimodipine is able to reduce ischemia-induced neuronal necrosis. The cerebroprotective effect of the drug was not medi ated by a postischemic cerebral vasodilation, but by a direct action on the neurons. Acknowledgment: The authors wish to express their gratitude to Miss L. Schombert, Mrs. H. Lorenz, and Mrs. R. Seidel for skillful technical assistance. These ex periments were supported by the Deutsche Forschungs gemeinschaft.
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10:654--659 © 1990 Raven Press, Ltd., New York
Protective Effect of Nimodipine Against Ischemic Neuronal Damage in Rat Hippocampus Without Changing Postischemic Cerebral Blood Flow
Jorg Nuglisch, Chourouk Karkoutly, *Hans Dieter Mennel, *Christine RoBberg, and Josef Krieglstein Institut fur Pharmakologie und Toxikologie and *Abteilung Neuropathologie, Philipps-Universitiit, Marburg, F.R.G.
Summary: The purpose of the present study was to in vestigate the neuroprotective action of nimodipine. Fur thermore, the influence of nimodipine on postischemic local CBF (LCBF) was examined. Forebrain ischemia of the rat was performed for 10 min by bilateral carotid clamping, administration of trimethaphan, and blood withdrawal to obtain an MABP of 40 mm Hg. LCBF was measured after 10 min of postischemic recirculation by injecting [14C]iodoantipyrine in saline solution. Nimo dipine (0. 1, 0.3, and 1.0 mg/kg) was suspended in miglyol oil and applied orally 60 min prior to ischemia. Histolog ical evaluation was performed 7 days after ischemia. Hip-
pocampal neuronal damage was determined as the per centage of necrotic neurons. After preischemic applica tion of nimodipine, neuronal damage was significantly reduced in the hippocampal CAl subfield. Postischemic LCBF was not affected by treatment with nimodipine. These findings show that nimodipine is able to protect neurons against ischemic damage. The neuroprotective effect of nimodipine was not mediated by a postischemic cerebral vasodilation, but by a direct action on the neu rons. Key Words: Forebrain ischemia-Local cerebral blood flow-Neuronal damage-Nimodipine.
It is widely accepted that calcium plays an impor tant role in the deleterious progress of neuronal damage. Furthermore, it is known that ischemia re sults in an inhibition of ATP-dependent ionic pumps and causes a substantial increase in cytoplasmic Ca2+ concentration (Farber, 1981; Siesj6, 1981; Siesj6 and Bengtsson, 1989). This elevated intra cellular Ca2+ level is supposed to contribute to sev eral processes leading to neuronal death (Siesj6, 1981; Borgers et aI., 1983; Choi, 1988). Calcium an tagonists are known to block voltage-sensitive cal cium channels, reduce cerebrovascular resistance under normal and pathological conditions, and im prove the resistance of the brain to the effects of an ischemic stroke (Kazda et aI., 1982; Nakayama et aI., 1989). The neuroprotective effect of calcium an tagonists has already been demonstrated in earlier work; e.g., flunarizine or emopamil significantly re-
duced neuronal damage in the hippocampus (Desh pande and Wieloch, 1985, 1986; Beck et aI., 1988; Bielenberg et aI., 1989a) or reduced infarct size af ter occlusion of the middle cerebral artery of rats (Nakayama et aI., 1989). Furthermore, evidence has been presented that the reduction in neuronal damage is caused by a direct effect of the calcium antagonists on cerebral parenchyma and is not me diated by a vascular action (Bielenberg et aI., 1987, 1989a; Beck et aI., 1988). Nimodipine is a calcium antagonist of the 1,4dihydropyridine type with potent selective effect on cerebral vessels at doses insufficient to affect sys temic arterial blood pressure (Kazda and Towart, 1982; Kazda et aI., 1982; Scriabine et aI., 1985). The presence of nimodipine-specific binding sites on se lectively vulnerable neurons in the hippocampus and the cortex (Quirion et aI., 1985; Spedding and Middlemiss, 1985) and the behavioral effects after nimodipine treatment observed by Hoffmeister et aI. (1982) suggest that nimodipine, in addition to its cerebral vasodilating effect, may modify neuronal function via a direct action. In the isolated perfused rat brain, Bielenberg et aI. (1989b) have found a
Received September 18, 1989; revised February 19, 1990; ac cepted February 21, 1990. Address correspondence and reprint requests to Dr. J. Nuglisch at Institut fiir Pharmakologie und Toxikologie, Phil . ipps-Universitat, Ketzerbach 63, 3550 Marburg, F.R. G. Abbreviation used: LCBF, local CBF.
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EFFECT OF NIMODIPINE reduction in postischemic lactate levels indepen dent of the mean postischemic perfusion rate. On the other hand, nimodipine has also been shown to prevent postischemic hypoperfusion following tran sient cerebral ischemia in dogs (Steen et aI., 1983, 1984; Newberg et aI., 1986), in cats (Kazda et aI., 1982), and in rats (Smith et aI., 1983; Young et aI., 1987). The purpose of the present study was to demon strate that nimodipine is able to reduce postisch emic neuronal damage in the hippocampus. Fur thermore, the possible correlation of a neuroprotec tive action of nimodipine with an influence on postischemic regional CBF was investigated.
MATERIALS AND METHODS Animals Male Wistar rats (250-300 g; Ivanovas, Kissleg, F.R.G) were used in all experiments. They were maintained un der controlled lighting and environmental conditions (12h dark-light cycle, 23 ± lOC, 55 ± 5% relative humidity). The rats were kept on standard diet (Altromin, Lage, F.R.G.) and tap water ad libitum. Preceding the experi ments the rats were fasted overnight. Rats were divided into two groups. In group 1 histological evaluation was performed on 37 rats. The measurement of CBF was car ried out in 30 rats of the second group.
Surgical procedure and induction of ischemia The experiments were performed according to Smith et al. (1984). Rats were anesthetized with 3.5% halothane, intubated, and connected to a Starling-type respirator de livering 0.7% halothane and 30% O2 in nitrous monoxide. The jugular vein (silicone catheter) and the tail artery (polyethylene catheter) were catheterized for withdrawal of blood and for monitoring blood pressure or collecting arterial blood samples. Anticoagulation was achieved by intravenous heparin (200 IV/kg) application. Blood gases, blood pH (Coming 178; Corning Medical, GieBen, F.R.G.), blood pressure (Statham P23DB, Heto Rey, Pu erto Rico; HSE-Electromanometer, Hugstetten, F.R.G.), and plasma glucose (Beckman Glucose Analyzer II, Mu nich, F.R.G.) were measured 5 min prior to ischemia and 10 min after ischemia. Halothane (but not nitrous monoxide) was discontin ued and the rats allowed to recover for 30 min. During this period muscle paralysis was maintained with 5 mg/kg suxamethonium chloride (Asta Pharma AG, Frankfurt, F.R.G.), repeated every 15 min. Ischemia was induced by injection of 5 mg/kg trimethaphan camphor sulfonate (Hoff mann-La Roche, Grenzach-Wyhlen, F.R.G.), occlusion of both common carotid arteries, and exsanguination to a blood pressure of 40 mm Hg. After 10 min of ischemia, blood pressure was restored by removing the carotid clamps and reinfusing the shed blood. Rats then received 1 mmol/kg sodium bicarbonate to prevent systemic aci dosis. Body temperature was held at -37°C with a heat ing lamp. The femoral artery and the femoral vein were additionally cannulated in rats used for the determination
655
of local CBF (LCBF) to allow blood sampling and injec tion of the isotope solution. Ten minutes after ischemia CBF was measured in arti ficially ventilated rats receiving the nitrous monoxide/ oxygen mixture (70:30) until decapitation.
LCBF LCBF was determined by injecting [14C]iodoantipyrine in saline solution (Sakurada et aI., 1978). The femoral vessels were cannulated and 150 fLCi/kg e4C]iodo antipyrine (spec. act. 60 mCi/mmol; NEN, Dreieich, F.R.G.) was infused over 1 min using a ramp function. During infusion 10 timed arterial blood samples were col lected and analyzed for [14C]iodoantipyrine content by liquid scintillation counting. The rats were decapitated at the end of infusion; the brains were dissected within 4560 s, frozen in isopentane, and processed for quantitative autoradiography according to standard procedures by cryosectioning (mod. 2700; Reichert-Jung, NuBloch, F.R.G.). Then 20-fLm brain slices were exposed to Osray M3 film (Agfa-Gaevert, Leverkusen, F.R.G.) for 10 days. Regional gray values of the autoradiograms were mea sured with an image analyzer (IBAS; Kontron, Eching, F.R.G.) and transformed to CBF values.
Histological assessment of ischemic cell damage For histological evaluation animals were anesthetized with halothane on day 7 after ischemia and perfused trans cardially via the ascending aorta with a phosphate buffer of pH 7.35 containing 4% formaldehyde after rinsing the brain with -30 ml physiological saline solution. Brains were removed, embedded in Paraplast, and sectioned in 5-fLm slices. Then the slices were stained with a mixture of 1% celestine blue and 1% acid fuchsin (Auer et aI., 1984). Hippocampal intact and necrotic cells were counted in a section 6 mm rostral to the tentorial incision. Cell loss was determined as the percentage of acid stainable cells.
Drug administration Nimodipine was suspended in miglyol oil and was given orally 1 h prior to ischemia in dosages of 0.1, 0.3, and 1.0 mg/kg. Solutions containing nimodipine were carefully protected from light. Controls received miglyol oil only.
Statistics Values are presented as means ± SD. In all experi ments mUltiple comparisons were made by the Kruskal Wallis H test combined with the Duncan test.
RESULTS Physiological variables
Nimodipine dosages of 0.1 and 0.3 mg/kg did not influence the physiological variables (Table 1). Ad ministration of 1.0 mg/kg nimodipine lowered post ischemic arterial blood pressure measured 10 min after ischemia significantly (Table 1). Other physi ological parameters were not affected by treatment with 1.0 mglkg nimodipine (Table 1). J Cereb Blood Flow Metab, Vol.
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TABLE 1. Physiological variables after treatment with nimodipine Nimodipine (mg/kg) Control (n 12) =
Arterial blood pressure (mm Hg) Before ischemia After ischemia Plasma glucose (mg/IOO ml) P02 (mm Hg) Pco2 (mm Hg) pH Rectal temperature eC)
127 130
±
127 123 37 7.38
±
37.0
±
17 10
0.1 (n
124 130
±
11 12 2 0.04
134 124 37 7. 33
±
0.0
37.0
± ±
=
± ±
0.3 8)
(n
12 10
123 131
=
± ±
1.0 8)
(n
=
9)
117 ± 14 115 ± lla
15 14
±
8 23 2 0.06
119 ± 13 130 ± 35 37 ± 2 7.35 ± 0.04
125 ± 17 130 ± 32 38 ± 4 7.32 ± 0.06
±
0.0
36.9
36.9
± ± ±
±
0.2
±
0.1
Physiological variables were determined 5 min prior to ischemia. In addition, postischemic arterial blood pressure was measured 10 min after ischemia. Values are presented as means ± SD from n experiments. a Different from control: p < 0.05 (Kruskal-Wallis H test).
Histological evaluation of neuronal necrosis
Oral application of 0.1 or 0.3 mg/kg nimodipine significantly inhibited postischemic neuronal dam age in the hippocampal CAl region (Fig. O. Neuro nal necrosis in the CA3 and CA4 subfields of the hippocampus was only marginal and was not atten uated by nimodipine (Fig. l). The oral dosage of 1.0 mg/kg nimodipine did not reduce the neuronal dam age significantly, but the mean value of the percent age of neuronal necrosis in the hippocampal CAl region at this dosage was diminished in comparison with the ischemic control group (Fig. I). LCBF
LCBF was determined 10 min after ischemia with preischemic application of the oral dosages of 0.1, 0.3, or 1.0 mg/kg nimodipine (Table 2). None of NEURONAL DA ... AGE % 70
D CONTROL
60 50
•
•
�
NIMODIPINE
DISCUSSION
Nimodipine is a calcium antagonist with a prefer ential vasodilatory effect on cerebral vessels and with less effect on peripheral arteries (Kazda and Towart, 1982; Towart et aI., 1982; Scriabine et aI., 1985). Contradictory results were found concerning the effect of nimodipine on CBF in vivo. In anes thetized rabbits intracarotid application of nimo dipine produced an increase in CBF ipsilateral to the side of infusion (Haws and Heistad, 1984). A significant increase in cortical arteriolar diameter and in regional CBF was found in anesthetized cats after intravenous infusion of nimodipine (Schmidli et aI., 1985). Dose-dependent effects of nimodipine on CBF were found by Mohamed et a1. (1984) in rats and by McCalden et a1. (1984) in baboons. Low doses significantly increased blood flow, whereas higher doses resulted in a decreased systemic arte-
� (O.lmgikg.p.QI
IIIII!I!IIII NIMOOIPINE
IIIIII!IIII (O.3mgikg.p.QI
30
these oral dosages changed postischemic blood flow in the hippocampus or in other measured areas of the brain.
� NIMODIPINE
� (1.0mgikg,p.o.l
20 10 o FIG. 1. Nimodipine protects neurons against ischemia. His
tological demonstration of neuronal damage in the subfields of the hippocampus after 10 min of forebrain ischemia and 7 days of recovery. Nimodipine was given orally 1 h prior to ischemia in dosages of 0. 1, 0.3, and 1.0 mg/kg. Values are means ± SO of 12 control and 8-9 nimodipine-treated rats. 'p < 0.05, Kruskal-Wallis H test.
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TABLE 2. Local CBF 10 min after ischemia and oral preischemic treatment with nimodipine 0"1 i :--.. v"--Z vUe ��. . . ,/-. . , Nimodipine
N·/
.�
c/'
, 1..5-
Brain structure Occipital cortex Parietal cortex Frontal cortex Cingulate cortex Retrosplenial cortex Hippocampus CAl Hippocampus CA3 Hippocampus CA. Dentate gyrus
wo-
Control (n 8)
(mg/kg)
0. 1
=
(n
224 ± 88 317 ± 66 290 ± 58 335 ± 96 192 ± 113 205 ± 88 173 ± 62 188 ± 6 1 194 ± 67
2 19 285 272 312 204 184 169 179 189
=
± ± ± ± ± ± ± ± ±
7)
34 47 48 67 60 28 30 45 49
(n
0.3
244 362 314 353 150 193 159 174 182
=
± ± ± ± ± ± ± ± ±
8)
31 74 63 61 76 38 41 54 50
(n
234 345 304 356 219 2 19 185 211 223
1.0 -7) =
± ± ± ± ± ± ± ± ±
43 64 50 76 143 69 66 83 94
Values are means ± SD from n experiments expressed as mlllOO g/min. After 10 min of forebrain ischemia, brains were recirculated for 10 min. Nimodipine was applied I h prior to ischemia.
EFFECT OF NIMODIPINE rial blood pressure and a return to CBF control lev els. In conscious rats Kanda and Flaim (1986) did not find an alteration in CBF or in cerebral vascular resistance after infusion of nimodipine. In addition, no effect on CBF was found by Forsman et ai. (1986) in unanesthetized dogs when nimodipine was applied intravenously. In contrast to these results are data obtained by Mohamed et ai. (1985a). These authors showed a significantly increased level of CBF in 24 brain regions in conscious rats after in fusion of nimodipine. In accordance with these re sults are experiments from Haws et al. (1983), who demonstrated an increase in CBF in anesthetized and unanesthetized cats. Contrary results were obtained concerning the ef ficacy of nimodipine treatment associated with isch emia. Preischemic treatment with nimodipine im proved CBF in middle cerebral artery-occluded rats (Mohamed et aI., 1985b) and tissue pH in rab bits (Tally et aI., 1989). An increase in CBF in the middle cerebral artery occlusion model of the rat was not found when nimodipine was given after in duction of ischemia (Graham et aI., 1985; Gotoh et al., 1986). Adverse results were obtained in neuro pathological quantification of the ischemic damage, too. Protective effects in the volume of infarcted area after middle cerebral artery occlusion were seen after preischemic treatment with nimodipine (Graham et aI. , 1985; Mohamed et aI. , 1985b), whereas application of nimodipine after occlusion of the middle cerebral artery showed no ameliora tive effect (Graham et al., 1985; Gotoh et aI., 1986). In a model of complete cerebral ischemia of the dog, Steen et ai. (1983, 1984) demonstrated that pre or postischemic treatment with nimodipine in creased postischemic CBF, but only preischemic application improved neurologic outcome in a his topathological scoring system. On the other hand, Steen et ai. (1985) showed that nimodipine im proved neurological function and histopathological score when given after complete cerebral ischemia in pigtailed monkeys. Parenteral treatment with ni modipine after ischemia failed to prevent hippocam pal neuronal damage in the four- and two-vessel occlusion model and in the model of forebrain isch emia in the gerbil (van Reempts et aI., 1986; Vilbuls resth et aI., 1987; Alps et aI., 1988). Nimodipine prevents postischemic cerebrovascu lar spasm and improves postischemic impaired reperfusion in various experimental models includ ing the same model as used in the present study (Kazda et aI., 1982; Smith et aI., 1983; Steen et aI., 1983; Newberg et aI., 1986; Young et aI., 1987). In these studies the authors assumed that an amelio ration in neurologic recovery is probably mediated
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by the prevention of delayed postischemic hypo perfusion. On the other hand, the behavioral effects in ni modipine-treated rats obtained by Hoffmeister et ai. (1982) suggest that nimodipine has other pharmaco logical properties besides its vasoactive action. Berger and Hakim (1988) demonstrated that nimo dipine normalizes local cerebral pH without chang ing LCBF in middle cerebral artery-occluded rats when applied after ischemia. Protective effects of nimodipine on postischemic brain energy metabo lism were found by Bielenberg et ai. (1987, 1989b). These authors showed that nimodipine reduced postischemic lactate levels independently of the corresponding flow values, and it was suggested, therefore, that the drug improves postischemic mi tochondrial function by a direct action on cerebral parenchyma. Our study demonstrates that if dose and applica tion are appropriately chosen, nimodipine can re duce neuronal damage in the hippocampus. Postischemic MABP was not influenced by the lower oral nimodipine doses, but the higher dose of 1. 0 mg/kg caused a significant decrease in the post ischemic MABP, measured 10 min after ischemia. The dosage of 1.0 mg/kg nimodipine seems to be the critical value to affect MABP. These data support the earlier finding that nimodipine is not a selective cerebral vasodilator but in higher doses affects sys temic arterial blood pressure as well (Mohamed et aI., 1984). Histological examination of neuronal damage 7 days after ischemia showed a significant reduction of neuronal necrosis in the hippocampal CAl sub field after oral pretreatment with 0.1 or 0.3 mg/kg nimodipine. A clear dose-response relationship concerning the reduction of hippocampal neuronal damage was not demonstrable. It is assumed that pharmacoki netic reasons could be responsible for that. Nimo dipine does not accumulate in brain tissue after a single oral dose (Suwelack et aI., 1985) so that tis sue levels of the drug may vary considerably. Therefore, a very clear-cut dose-response relation ship could not be expected for the neuroprotective effect of nimodipine. The peak plasma level of radioactivity is attained 40-60 min after administration of a single oral dose l of [ 4C]nimodipine in rats (Maruhn et al., 1985). Ra dioactivity half-life in the plasma is 4.6-9.2 h, but unchanged nimodipine represents only a part (22% in rats) of the total radioactivity (Maruhn et aI., 1985). Thus, it has to be assumed that an oral dose of nimodipine given 1 h prior to ischemia affects LCBF in the period of postischemic recirculation. J Cereb Blood Flow Metab, Vol.
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Therefore, measurements of LCBF were performed 10 min after ischemia during the period of relative hyperemia (Kagstrom et al., 1983) with the oral doses of 0.1, 0.3, and 1.0 mg/kg nimodipine. None of these dosages were able to influence postisch emic LCBF 10 min after ischemia in any areas mea sured, including the subfields of the hippocampus. These findings indicate that nimodipine is able to reduce ischemia-induced neuronal necrosis. The cerebroprotective effect of the drug was not medi ated by a postischemic cerebral vasodilation, but by a direct action on the neurons. Acknowledgment: The authors wish to express their gratitude to Miss L. Schombert, Mrs. H. Lorenz, and Mrs. R. Seidel for skillful technical assistance. These ex periments were supported by the Deutsche Forschungs gemeinschaft.
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