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Neuroprotective effect of memantine demonstrated in vivo and in vitro
European Journal of Pharmacology, 185 (1990) 19-24
19
Elsevier EJP 51435
Neuroprotective effect of memantine demonstrated in vivo and in vitro M o n a Seif el N a s r , B a r b a r a Peruche, Christine R o g b e r g 1, H a n s - D i e t e r M e n n e l 1 a n d J o s e f Krieglstein Institut J'w"Pharmakologie und Toxikologie, j Abteilung f'tir Neuropathologie, Philipps-Unioersitiit, Ketzerbach 63, D-3550 Marburg; F.R.G.
Received 11 April 1990, accepted 22 May 1990
The purpose of the present study was to test whether the anticonvulsant, memantine (1-amino-3,5-dimethyladamantane), can protect neurons against hypoxic or ischemic damage. To this end, we used a rat model of transient forebrain ischemia and cultured neurons from chick embryo cerebral hemispheres. Ischemia was induced for 10 rain by clamping both carotid arteries and lowering the mean arterial blood pressure to 40 mm Hg; the rats were allowed to recover for 7 days. Cultured neurons were made hypoxic with 1 retool/1 NaCN added to the incubation medium for 30 rain followed by a recovery period of 3 days. The possible effects of memantine were compared with those produced by a typical non-competitive NMDA antagonist, dizocilpine. Similar effects were obtained with both drugs. The drugs reduced the damage caused by transient ischemia to neurons of the hippocampal CA1 subfield. Memantine (10 and 20 mg/kg) had a dose-dependent effect when administered intraperitoneally to the rats 1 h before ischemia. Dizocilpine was active in this model at a dosage of 1 mg/kg. When administered after ischemia, 10 mg/kg memantine significantly protected CA1 neurons against ischemic damage. Furthermore, the drugs protected cultured neurons against hypoxic damage. The lowest effective concentration was 0.1 #tool/1 for dizocilpine and 1 ptmol/l for memantine. Thus, memantine possesses neuroprotective activity but is less potent than dizocilpine.
Adamantane derivatives; Dizocilpine; Cerebral ischemia; Hypoxia; Neurons; (Rat)
1. Introduction Memantine (1-amino-3,5-dimethyladamantane) is used for the therapy of various cerebral disorders such as Parkinson's disease, spasticity and chronic brain syndrome. Experimental studies have shown that the drug interferes with various central transmitter systems (Wesemann et al., 1983).
Correspondence to: M. Seif el Nasr, Institut far Pharmakologie und Toxikologie, Philipps-Universit~it, Ketzerbach 63, D-3550 Marburg, F.R.G.
Memantine acts as a non-competitive antagonist at N-methyl-D-aspartate ( N M D A ) receptors (Bormann, 1989) and inhibits the serotonin-induced rise in cytosolic Ca 2÷ in a neuronal cell line (Reiser and Koch, 1989). These pharmacological and therapeutic properties suggest that memantine could protect neurons against ischemic or hypoxic damage. We therefore examined the neuroprotective activity of this drug using the forebrain ischemia rat model of Smith et al. (1984) a n d cultured neurons from chick embryo cerebral hemispheres (Pettmann et al., 1979; Krieglstein et al., 1988). We compared the possible effects of
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
2o memantine with those of a standard neuroprotective, dizocilpine, an N M D A antagonist.
2. Materials and methods
2.1. Animals Male Wistar rats (Ivanovas, Kisslegg, F.R.G.) weighing 250-300 g were used in all experiments and were maintained under controlled lighting and environmental conditions (12 h d a r k / l i g h t cycle, 23 + I ° C , 55 + 5% rel. humidity). Standard rat chow (Altromin®, Lage, F.R.G.) and tap water were available ad libitum. The rats were fasted overnight before the ischemic procedure.
2.2. Materials Memantine was obtained from Merz (Frankfurt, F.R.G.) and dizocilpine from MSD (Terlings Park, U.K.). Trimethaphan camphor sulphonate (Arfonad ®) and heparin were obtained from Hoffmann-La Roche (Grenzach-Wyhlen, F.R.G.). Celestine blue and acid fuchsin were from Sigma Co (St. Louis, MO, U.S.A.). Paraplast was obtained from Monject Scientific (Kildare, Ireland).
2.3. Physiological variables Arterial PCO2, PO 2, p H (Coming 178, Coming Medical, Giessen, F.R.G.), blood pressure (Statham P23DB, Heto Rey, Puerto Rico; Recorned, Hellige, Freiburg, F.R.G.), and plasma glucose (Beckman Glucose Analyser II, Munich, F.R.G.) were measured with routine methods. The sample size for the determination of blood gases or plasma glucose was 40 gl. Animals with values outside the normal range were not used.
2.4. Surgical procedure and induction of ischemia Forebrain iscliemia was induced according to the method of Smith et al. (1984). Animals were anesthetized with 3.5% halothane, intubated with a plastic tube and connected to a Starling type respirator delivering 0.7% halothane and 70% N20
in 02 . The common carotid arteries and the fight jugular vein were exposed through a neck incision. A silicon catheter was advanced into the inferior caval vein for exsanguination. A tail arterial catheter was inserted for blood pressure recording and blood sampling. Heparin was injected intravenously as anticoagulant. Halothane was then discontinued. Muscle paralysis was maintained with an intravenous injection of 5 m g / k g suxamethonium chloride. The animals were then ventilated with 70% N20 and 30% 02 for 30 min. Body temperature was maintained at 37°C, arterial PO 2 higher than 100 mm Hg, and PCO 2 at 35-40 mm Hg. Ischemia was induced by clamping the carotid arteries and reducing the mean arterial blood pressure to 40 mm Hg; this was achieved by the administration of trimethaphan camphor sulphonate (5 m g / k g ) and blood withdrawal. Ischemia was terminated after 10 min by removal of the carotid clamps and reinfusion of the shed blood: the rats received N a H C O 3 (1 m m o l / k g ) to prevent systemic acidosis. Seven days later the rats were anesthetized with 1% halothane in a 2 : 1 mixture of N 2 0 / O 2. After the brains had been rinsed with about 30 ml physiological saline, they were perfusion-fixed transcardially via the ascending aorta with a fixative containing 4% formaldehyde in a phosphate buffer of p H 7.35. After dehydration in a series of ethanol solutions up to 95%, the brains were embedded in Paraplast and sectioned in 5 g m slices. Slices were stained with a mixture of 1% celestine blue and 1% acid fuchsin (Auer et al., 1984). This staining procedure visualizes necrotic neurons. Intact and necrotic cells in the hlppocampus were counted. The results are given as a percentage of damaged neurons.
2.5. Drug treatment Dizocilpine was applied intraperitoneaUy 1 h before ischemia. In two sets of experiments memantine was injected intraperitoneally 1 h before ischemia in doses of 5, 10, 20 m g / k g . In another set memantine was injected in doses of 10 and 20 m g / k g immediately after ischemia. Drugs were dissolved in saline; the control animals received vehicle.
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2.6. Cell culture technique P r i m a r y n e u r o n a l cell cultures were p r e p a r e d a c c o r d i n g to the m e t h o d d e s c r i b e d b y P e t t m a n n et al. (1979). T h e n e u r o n s were d e r i v e d f r o m m e c h a nically dissociated telencephalons from 7-day-old chick e m b r y o s . T h e n e u r o n a l cell cultures were m a i n t a i n e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m ( D M E M ) s u p p l e m e n t e d with 20% fetal calf serum, 100 u n i t s / m l penicillin a n d 100 # g / m l s t r e p t o m y c i n . T h e m e d i u m was c h a n g e d after 3 days. D r u g s a n d s o d i u m c y a n i d e were dissolved in n u t r i e n t m e d i u m ( D M E M ) a n d sterilized (millip o r e filter) b e f o r e b e i n g a p p l i e d to the culture m e d i u m . H y p o x i a was i n d u c e d after 4 d a y s in culture, b y the a d d i t i o n o f i m m o l / 1 N a C N to the c u l t u r e m e d i u m . A f t e r 30 m i n of h y p o x i a , the c y a n i d e - c o n t a i n i n g m e d i u m was r e p l a c e d with fresh culture m e d i u m . T h e cells were allowed to recover for 3 days. T h e drugs were a d d e d to the c u l t u r e m e d i u m 30 rain b e f o r e the start o f h y p o x i a a n d were p r e s e n t until 1 d a y after h y p o x i a . N o r m o x i c c o n t r o l cultures h a d identical schedules o f m e d i u m exchange.
T o d e t e r m i n e n e u r o n a l viability, cells were c o u n t e d after 7 d a y s in c u l t u r e u n d e r a p h a s e c o n t r a s t m i c r o s c o p e with a c a l i b r a t e d N e u b a u e r h e m o c y t o m e t e r . V i a b i l i t y was assessed using the t r y p a n b l u e exclusion m e t h o d : cells t h a t a p p e a r e d d a r k b l u e were c o n s i d e r e d n o n - v i a b l e . T h e t o t a l n u m b e r of cells includes v i a b l e a n d n o n - v i a b l e cells. F o r A T P analysis, cells were w a s h e d with ice-cold D u l b e c c o ' s b u f f e r e d salt s o l u t i o n (DBSS). A f t e r a d d i t i o n o f 0.3 m o l / 1 p e r c h l o r i c acid, the cell s u s p e n s i o n was h o m o g e n i z e d a n d centrifuged. T h e s u p e r n a t a n t was n e u t r a l i z e d with K O H - i m i d a z o l e s o l u t i o n a n d A T P was m e a s u r e d w i t h the L u c i f e r i n - L u c i f e r a s e assay ( L u m i n o m e t e r L K B Wallac, 1250). P r o t e i n was d e t e r m i n e d a c c o r d i n g to the m e t h o d of L o w r y et al. (1951) a n d b o v i n e s e r u m a l b u m i n was used as a s t a n d a r d .
2. 7. Statistics Differences in h i p p o c a m p a l n e u r o n a l d a m a g e b e t w e e n t r e a t e d a n d c o n t r o l a n i m a l s were a n a l y s e d statistically with the M a n n - W h i t n e y U-test. D a t a o b t a i n e d with the cell cultures a r e p r e s e n t e d as
TABLE 1 Physiological variables of drug-treated and control rats. Physiological parameters were determined 10 n'fin before the induction of ischemia. With the postischemic treatment, the physiological variables were determined 10 min before induction of ischemia (values not shown) and 5 rain after drug application. Physiological parameters of rats treated with other doses of memantine were also measured and there were no significant differences between the drug-treated groups and controls (values not shown). The values are given as means 4- S.D.
PaO2 (mmHg) PaCO2 (mmHg) Art. pH Plasma glucose (rag/100 ml) MA BP (mmHg) Preischemic Postischemic Temperature
Dizocilpine (1 mg/kg 1 h before ischemia)
Memantine (20 mg/kg 1 h before ischemia)
Memantine (20 mg/kg) immediately after ischemia
Control n=6
Treated n=8
Control n=5
Treated n=6
Control n=10
Treated n=10
124.1 4-22.9 33.9 4-3.3 7.41 4-0.05
127.8 4-20.1 35.8 4-2.2 7.38 4-0.04
146.8 4- 37.1 37.6 4-2.0 7.38 4-0.02
151.5 ± 30.0 36.1 4-2.4 7.39 + 0.04
150.9 4- 25.1 36.6 4-2.3 7.38 4-0.03
144.6 4- 21.5 35.4 4- 3.0 7.38 4-0.03
135.7 4-17.5
140.1 4-18.8
119.4 4-17.8
120.7 4-9.0
116.7 4-15.9
136.5 4-13.9
136.4 4-14.4 135.7 4-11.3 37.1 4-0.40
154.4 4-15.9 140.6 4-15.7 37.3 4-0.31
135.0 4-5.0 132.0 4- 8.4 37.2 4-0.13
130.0 + 7.1 125.0 4-9.8 37.1 +0.25
134.0 4-7.4 128.8 4-15.6 36.9 4-0.17
135.0 4-9.7 117.5 4- 20.5 36.8 4-0.26
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means + S.D. Significant differences were determined by using a one-way analysis of variance followed by Duncan's multiple range test.
NEURONAL DAMAGE
3. Results 3.1. Cultured neurons
50
Memantine and dizocilpine protected cultured neurons against hypoxic damage in a dose-dependent manner (table 2). The protein content, the total number of cells per culture and the viability of the neurons increased significantly when the drugs were present 30 rain before to 1 day after the hypoxic period. Dizocilpine was already active at a concentration of 0.1/~mol/1 and memantine at 1.0/~mol/1.
0
I a" k\\~l CA1
CA3
. =
_,.,m..=
.
CA/,
Fig. 1. Protective effect of dizocilpine against neuronal damage in the rat hippocampal CA1 subfield after 10 mill ischemia followed by 7 days of recovery. Values are means 4- S.E.M. of seven (controls, empty columns) and eight (dizocilpine, hatched columns) experiments. Mann-Whitney U-test: * * P < 0.01.
3.2. Forebrain ischemia of the rat
Memantine and dizocilpine did not alter the physiological parameters measured (table 1). Neither drug affected the body temperature of the rats. The drugs prolonged anesthesia by 10-20
min. There was no difference between treated and control rats in the late recovery period. Dizocilpine, 1 mg/kg i.p. (1 h before ischemia), reduced neuronal damage in the CA1 subfield of
TABLE 2 Neuroprotective effects Of memantine and dizocilpine against hypoxic damage of cultured neurons. Neurons were obtained from chick embryo cerebral hemispheres. Hypoxia was induced by adding 1 mmol/1 NaCN to the incubation medium for 30 min after 4 days in culture. Normoxic controls ( o ) received saline only. Vehicle or drugs were added 30 mill before hypoxia and were left in the cultures up to 1 day after hypoxia. The cultures were analysed 3 days after hypoxia. The values are given as means 4- S.D. of five experiments. Duncan's test: " P < 0.01 Drug conc. (#mol/1)
ATP (nmol/mg protein)
Protein (mg/flask)
Cell count (lO s )
Viability (%)
22.43 23.21 23.00 23.10 28.06 27.03
0.185 0.222 0.260 0,295 0,275 0.266
+ 0.010 4- 0.014 a 4- 0.019 a 5=0.012 a 4- 0.011 5- 0.016
4.47 4- 0.11 4.99 4- 0.27 6.01 4- 0.48 a 5.78 4- 0.32 a 7.02 4- 0.91 6.96 + 0.30
60 4- 3 78 4- 6 a 74 5:1 a 82 4- 5 a 84 4- 5 86 4- 3
0.200 4- 0.014 0.199:1:0.010 0.250 + 0.015 a 0.2565-0.010 a 0.273 5:0.013 0.271 4- 0.007
4.14 4- 0.31 4.00 4- 0.50 5.51 4- 0.44 a 5.404-0.31 ~ 5.70 5- 0.31 5.96 4- 0.38
60 4- 4 59 4-1 83 4- 3 a 825:2 a 89 4- 2 88 4- 5
Dizocilpine 0 0.1 1 10 10 o 0o
+ 1.63 4-1.53 4-1.40 4- 0.91 4-1.44 4-1.61
Memantine 0 0.1 1 10 10 o 0°
21.60 4-1.24 21.25 4- 0.51 23.20 4-1.09 23.285:1.40 26.34 4- 0.88 26.72 + 0.91
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the rat hippocampus (fig. 1). Pre-ischemic application of 10 m g / k g i.p. memantine (1 h before ischemia) caused a similar effect; the effect was even more pronounced with 20 m g / k g i.p. At a dose of 5 m g / k g i.p., memantine did not protect pyramidal CA1 neurons against ischemic damage (fig. 2). When administered immediately after ischemia, 10 m g / k g i.p. memantine significantly reduced the neuronal damage whereas 20 m g / k g of the drug did not provide significant protection (fig. 3).
NEURONAL DAMAGE ,\* 100"
50
4. Discussion
We demonstrated that memantine and dizocilpine can protect neurons in vivo and in vitro against damage caused by ischemia and hypoxia, respectively. Dizocilpine is a generally accepted non-competitive N M D A antagonist, and the resuits obtained with this drug are in line with previous findings (for references see Iversen et al., 1989). Thus, dizocilpine prevents neuronal damage in the hippocampal CA1 subfield after global ischemia in gerbils (Gill et al., 1987a; 1988) and NEURONAL DAMAGE % lOO"
--Z-v/..C,/d
50' ~llrA
5
10
I
20 DOSAGE (mg/kg, i.p.}
Fig. 2. Dose-dependent neuroprotective effect of memantine. Ischemia was induced for 10 min and rats were allowed 7 days to recover. Neuronal damage of the CA1 subfield is given. Values are means + S.E.M. of five (controls, empty columns) and 5-10 memantine (hatched columns) experiments. MannWhitney U-test: * * P < 0.01.
lu
20 DOSAGE (rng/kg,i.p.)
Fig. 3. Postischemic administration of memantine protects
neurons against ischemic damage. Forebrain ischemia was induced in rats for 10 min followed by 7 days of recovery. Neuronal damage of the hippocampal CA1 subfield is given. Values are means+S.E.M, of 10 (controls, empty columns) and 10-11 (memantine, hatched columns) experiments. MannWhitney U-test: * P < 0.02. rats (Gill et al., 1987b; Seif el Nasr et al., 1989; Rod and Auer, 1989), and reduces the infarct volume after focal ischemia in rats and cats (Oyzurt et al., 1988; McCulloch et al., 1989). In addition, dizocilpine ameliorates the viability of cultured neurons after hypoxia (Ahlemeyer and Krieglstein, 1989). Memantine binds to the phencyclidine recognition site in the NMDA-operated Ca 2+ channel (Kornhuber et al.,1989) and blocks Ca 2÷ entry through this channel (Bormann, 1989). Memantine also blocks Ca 2÷ entry through serotonin-operated channel (Reiser and Koch, 1989). These pharmacological properties may be the basis for the neuroprotective activity of memantine. Like dizocilpine, memantine reduced the percentage of damaged neurons in the CA1 subfield of the rat hippocampus 7 days after ischemia. This effect of memantine was dose-dependent and was attained after both pre-ischemic and postischemic administration. However, the minimal effective dose was 10 m g / k g for memantine and 1 m g / k g for dizocilpine. Cultured neurons are particularly suitable for demonstrating the neuroprotective effects of N M D A antagonists (Ahlemeyer and Kriegl-
24
stein, 1989; Peruche et al., 1990). We therefore used these cells to test the potential neuroprotective effect of memantine in comparison with that of dizocilpine: both drugs showed the expected effect. However, higher concentrations of memanfine than of dizocilpine were required for a neuroprotective effect. Most ligands of the phencyclidine recognition site seem to cause schizophrenia-like symptoms, so these drugs may have limited value as neuroprotectives for clinical therapy. Psychomimetic effects have not often been observed in patients with organic brain syndrome treated with high doses of memantine (Riederer and Danielczyk, personal communication). In summary, memantine protected neurons in the CA1 subfield of rat hippocampus against ischemic damage and cultured neurons of chick embryo cerebral hemispheres against hypoxic injury. These effects were comparable to those of dizocilpine, although memantine seemed to be less active. Whether memantine could be useful as a neuroprotective for clinical therapy remains to be shown.
Acknowledgements The authors wish to thank Ms. H. Lorenz and Ms. M. Lorenz for their skillful technical assistance.
References A_hlemeyer, B. and J. K.rieglstein, 1989, Testing drug effects against hypoxic damage of cultured neurons during longterm recovery, Life Sei. 45, 835. Auer, R.N., Y. Olsson and B.K. Siesj~, 1984, Hypoglycemic brain damage with the EEG isoelectric time, Diabetes 33, 1090. Bormann, J., 1989, Memantine is a potent blocker of Nmethyl-D-aspartate (NMDA) receptor channel, European J. Pharmacol. 166, 591. Gill, R., A.C. Foster and G.N. Woodruff, 1987a, Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Nenrosci. 7, 3343. Gill, R., A.C. Foster and G.N. Woodruff, 1987b, Systemic administration of MK-801 protects against ischemic neuropathology in the rat, Br. J. Pharmacol. Proc. Suppl. 91, 311 p.
Gill, R., A.C. Foster and G.N. Woodruff, 1988, MK-801 is neuroprotective in gerbils when administered during the postischemic period, Neuroscience 25, 847. Iversen, L.L., G.N. Woodruff, J.A. Kemp, A.C. Foster, R. Gill and E.H.F. Wong, 1989, Nenroprotective properties of the glutamate antagonist dizocilpine maleate (MK-801) in animal models of focal and global ischemia, in: Pharmacology of Cerebral Ischemia 1988, ed. J. Krieglstein (Wissenschaftliche Verlagsgesellschaft, Stuttgart) p. 165. Kornlurber, J., J. Borrnann, W. Retz, M. Hiibers and P. Riederer, 1989, Memantine displaces [3H] MK-801 at therapeutic concentrations in postmortem human frontal cortex, European J. Pbarmacol. 166, 589. Krieglstein, J., H. Brungs and B. Peruche, 1988, Cultured neurons for testing cerebroprotective drug effects in vitro, J. Pharmacol. Methods. 20, 39. Lowry, O.H., N.J. Rosebrough, A.Y. Farr and R. Randel, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. McCulloch, J., C.K. Park, E. Oyzurt, D.G. Nehls, G.M. Teasdale and D.I. Graham, 1989, Effects of MK-801 in experimental models of focal cerebral ischemia in the cat and rat, in: Pharmacology of Cerebral Ischemia 1988, ed. J. Krieglstein (Wissenschaftliche Vedagsgesellschaft, Stuttgart) p. 173. Oyzurt, E., D.I. Graham, G.N. Woodruff and J. McCulloch, 1988, Protective effect of the glutamate antagonist MK-801 in focal cerebral isebemia in the cat, J. Cereb. Blood Flow Metab. 8, 138. Peruche, B., B. Ahlemeyer, H. Brungs, and J. Krieglstein, 1990, An in vitro model for testing drug protection against neuronal damage caused by hypoxia, J. Pharmacol. Meth. 23, 63. Pettmann, B., J.C. Louis and M. Sensenbrermer, 1979, Morphological and biochemical maturation of neurons cultured in the absence of glial cells, Nature 281, 378. Reiser, G. and R. Koch, 1989, Memantine inhibits serotonininduced rise of cytosolic Ca 2+ activity and of cyclic GMP level in a neuronal cell line, European J. Pharmacol. 172, 199. Rod, M.R. and R.N. Auer, 1989, Pre- and post-ischemic administration of dizocilpine (MK-801) reduces cerebral necrosis in the rat, Can. J. Nenrol. Sci. 16, 340. Self el Nasr, M., D. Sauer, C. Rossberg, H.D. Mermel and J. Krieglstein, 1989, Effects of NMDA-antagonists against neuronal damage after forebrain iscbemia in the rat, in: Pharmacology of Cerebral Iscbemia 1988, ed. J. Krieglstein (Wissenschaftliche Verlagsgesellschaft, Stuttgart) p. 211. Smith, M.L.,G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch and B.K. Siesjti, 1984, Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model, Acta Nenrol. Scand. 69, 385. Wesemann, W., K.H. Sontag and J. Maj, 1983, Zur Pharmakodynamik und Pharmakokinetik des Memantin, Ar-znelm. Forseh. 33, 1122.
19
Elsevier EJP 51435
Neuroprotective effect of memantine demonstrated in vivo and in vitro M o n a Seif el N a s r , B a r b a r a Peruche, Christine R o g b e r g 1, H a n s - D i e t e r M e n n e l 1 a n d J o s e f Krieglstein Institut J'w"Pharmakologie und Toxikologie, j Abteilung f'tir Neuropathologie, Philipps-Unioersitiit, Ketzerbach 63, D-3550 Marburg; F.R.G.
Received 11 April 1990, accepted 22 May 1990
The purpose of the present study was to test whether the anticonvulsant, memantine (1-amino-3,5-dimethyladamantane), can protect neurons against hypoxic or ischemic damage. To this end, we used a rat model of transient forebrain ischemia and cultured neurons from chick embryo cerebral hemispheres. Ischemia was induced for 10 rain by clamping both carotid arteries and lowering the mean arterial blood pressure to 40 mm Hg; the rats were allowed to recover for 7 days. Cultured neurons were made hypoxic with 1 retool/1 NaCN added to the incubation medium for 30 rain followed by a recovery period of 3 days. The possible effects of memantine were compared with those produced by a typical non-competitive NMDA antagonist, dizocilpine. Similar effects were obtained with both drugs. The drugs reduced the damage caused by transient ischemia to neurons of the hippocampal CA1 subfield. Memantine (10 and 20 mg/kg) had a dose-dependent effect when administered intraperitoneally to the rats 1 h before ischemia. Dizocilpine was active in this model at a dosage of 1 mg/kg. When administered after ischemia, 10 mg/kg memantine significantly protected CA1 neurons against ischemic damage. Furthermore, the drugs protected cultured neurons against hypoxic damage. The lowest effective concentration was 0.1 #tool/1 for dizocilpine and 1 ptmol/l for memantine. Thus, memantine possesses neuroprotective activity but is less potent than dizocilpine.
Adamantane derivatives; Dizocilpine; Cerebral ischemia; Hypoxia; Neurons; (Rat)
1. Introduction Memantine (1-amino-3,5-dimethyladamantane) is used for the therapy of various cerebral disorders such as Parkinson's disease, spasticity and chronic brain syndrome. Experimental studies have shown that the drug interferes with various central transmitter systems (Wesemann et al., 1983).
Correspondence to: M. Seif el Nasr, Institut far Pharmakologie und Toxikologie, Philipps-Universit~it, Ketzerbach 63, D-3550 Marburg, F.R.G.
Memantine acts as a non-competitive antagonist at N-methyl-D-aspartate ( N M D A ) receptors (Bormann, 1989) and inhibits the serotonin-induced rise in cytosolic Ca 2÷ in a neuronal cell line (Reiser and Koch, 1989). These pharmacological and therapeutic properties suggest that memantine could protect neurons against ischemic or hypoxic damage. We therefore examined the neuroprotective activity of this drug using the forebrain ischemia rat model of Smith et al. (1984) a n d cultured neurons from chick embryo cerebral hemispheres (Pettmann et al., 1979; Krieglstein et al., 1988). We compared the possible effects of
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
2o memantine with those of a standard neuroprotective, dizocilpine, an N M D A antagonist.
2. Materials and methods
2.1. Animals Male Wistar rats (Ivanovas, Kisslegg, F.R.G.) weighing 250-300 g were used in all experiments and were maintained under controlled lighting and environmental conditions (12 h d a r k / l i g h t cycle, 23 + I ° C , 55 + 5% rel. humidity). Standard rat chow (Altromin®, Lage, F.R.G.) and tap water were available ad libitum. The rats were fasted overnight before the ischemic procedure.
2.2. Materials Memantine was obtained from Merz (Frankfurt, F.R.G.) and dizocilpine from MSD (Terlings Park, U.K.). Trimethaphan camphor sulphonate (Arfonad ®) and heparin were obtained from Hoffmann-La Roche (Grenzach-Wyhlen, F.R.G.). Celestine blue and acid fuchsin were from Sigma Co (St. Louis, MO, U.S.A.). Paraplast was obtained from Monject Scientific (Kildare, Ireland).
2.3. Physiological variables Arterial PCO2, PO 2, p H (Coming 178, Coming Medical, Giessen, F.R.G.), blood pressure (Statham P23DB, Heto Rey, Puerto Rico; Recorned, Hellige, Freiburg, F.R.G.), and plasma glucose (Beckman Glucose Analyser II, Munich, F.R.G.) were measured with routine methods. The sample size for the determination of blood gases or plasma glucose was 40 gl. Animals with values outside the normal range were not used.
2.4. Surgical procedure and induction of ischemia Forebrain iscliemia was induced according to the method of Smith et al. (1984). Animals were anesthetized with 3.5% halothane, intubated with a plastic tube and connected to a Starling type respirator delivering 0.7% halothane and 70% N20
in 02 . The common carotid arteries and the fight jugular vein were exposed through a neck incision. A silicon catheter was advanced into the inferior caval vein for exsanguination. A tail arterial catheter was inserted for blood pressure recording and blood sampling. Heparin was injected intravenously as anticoagulant. Halothane was then discontinued. Muscle paralysis was maintained with an intravenous injection of 5 m g / k g suxamethonium chloride. The animals were then ventilated with 70% N20 and 30% 02 for 30 min. Body temperature was maintained at 37°C, arterial PO 2 higher than 100 mm Hg, and PCO 2 at 35-40 mm Hg. Ischemia was induced by clamping the carotid arteries and reducing the mean arterial blood pressure to 40 mm Hg; this was achieved by the administration of trimethaphan camphor sulphonate (5 m g / k g ) and blood withdrawal. Ischemia was terminated after 10 min by removal of the carotid clamps and reinfusion of the shed blood: the rats received N a H C O 3 (1 m m o l / k g ) to prevent systemic acidosis. Seven days later the rats were anesthetized with 1% halothane in a 2 : 1 mixture of N 2 0 / O 2. After the brains had been rinsed with about 30 ml physiological saline, they were perfusion-fixed transcardially via the ascending aorta with a fixative containing 4% formaldehyde in a phosphate buffer of p H 7.35. After dehydration in a series of ethanol solutions up to 95%, the brains were embedded in Paraplast and sectioned in 5 g m slices. Slices were stained with a mixture of 1% celestine blue and 1% acid fuchsin (Auer et al., 1984). This staining procedure visualizes necrotic neurons. Intact and necrotic cells in the hlppocampus were counted. The results are given as a percentage of damaged neurons.
2.5. Drug treatment Dizocilpine was applied intraperitoneaUy 1 h before ischemia. In two sets of experiments memantine was injected intraperitoneally 1 h before ischemia in doses of 5, 10, 20 m g / k g . In another set memantine was injected in doses of 10 and 20 m g / k g immediately after ischemia. Drugs were dissolved in saline; the control animals received vehicle.
21
2.6. Cell culture technique P r i m a r y n e u r o n a l cell cultures were p r e p a r e d a c c o r d i n g to the m e t h o d d e s c r i b e d b y P e t t m a n n et al. (1979). T h e n e u r o n s were d e r i v e d f r o m m e c h a nically dissociated telencephalons from 7-day-old chick e m b r y o s . T h e n e u r o n a l cell cultures were m a i n t a i n e d in D u l b e c c o ' s m o d i f i e d E a g l e ' s m e d i u m ( D M E M ) s u p p l e m e n t e d with 20% fetal calf serum, 100 u n i t s / m l penicillin a n d 100 # g / m l s t r e p t o m y c i n . T h e m e d i u m was c h a n g e d after 3 days. D r u g s a n d s o d i u m c y a n i d e were dissolved in n u t r i e n t m e d i u m ( D M E M ) a n d sterilized (millip o r e filter) b e f o r e b e i n g a p p l i e d to the culture m e d i u m . H y p o x i a was i n d u c e d after 4 d a y s in culture, b y the a d d i t i o n o f i m m o l / 1 N a C N to the c u l t u r e m e d i u m . A f t e r 30 m i n of h y p o x i a , the c y a n i d e - c o n t a i n i n g m e d i u m was r e p l a c e d with fresh culture m e d i u m . T h e cells were allowed to recover for 3 days. T h e drugs were a d d e d to the c u l t u r e m e d i u m 30 rain b e f o r e the start o f h y p o x i a a n d were p r e s e n t until 1 d a y after h y p o x i a . N o r m o x i c c o n t r o l cultures h a d identical schedules o f m e d i u m exchange.
T o d e t e r m i n e n e u r o n a l viability, cells were c o u n t e d after 7 d a y s in c u l t u r e u n d e r a p h a s e c o n t r a s t m i c r o s c o p e with a c a l i b r a t e d N e u b a u e r h e m o c y t o m e t e r . V i a b i l i t y was assessed using the t r y p a n b l u e exclusion m e t h o d : cells t h a t a p p e a r e d d a r k b l u e were c o n s i d e r e d n o n - v i a b l e . T h e t o t a l n u m b e r of cells includes v i a b l e a n d n o n - v i a b l e cells. F o r A T P analysis, cells were w a s h e d with ice-cold D u l b e c c o ' s b u f f e r e d salt s o l u t i o n (DBSS). A f t e r a d d i t i o n o f 0.3 m o l / 1 p e r c h l o r i c acid, the cell s u s p e n s i o n was h o m o g e n i z e d a n d centrifuged. T h e s u p e r n a t a n t was n e u t r a l i z e d with K O H - i m i d a z o l e s o l u t i o n a n d A T P was m e a s u r e d w i t h the L u c i f e r i n - L u c i f e r a s e assay ( L u m i n o m e t e r L K B Wallac, 1250). P r o t e i n was d e t e r m i n e d a c c o r d i n g to the m e t h o d of L o w r y et al. (1951) a n d b o v i n e s e r u m a l b u m i n was used as a s t a n d a r d .
2. 7. Statistics Differences in h i p p o c a m p a l n e u r o n a l d a m a g e b e t w e e n t r e a t e d a n d c o n t r o l a n i m a l s were a n a l y s e d statistically with the M a n n - W h i t n e y U-test. D a t a o b t a i n e d with the cell cultures a r e p r e s e n t e d as
TABLE 1 Physiological variables of drug-treated and control rats. Physiological parameters were determined 10 n'fin before the induction of ischemia. With the postischemic treatment, the physiological variables were determined 10 min before induction of ischemia (values not shown) and 5 rain after drug application. Physiological parameters of rats treated with other doses of memantine were also measured and there were no significant differences between the drug-treated groups and controls (values not shown). The values are given as means 4- S.D.
PaO2 (mmHg) PaCO2 (mmHg) Art. pH Plasma glucose (rag/100 ml) MA BP (mmHg) Preischemic Postischemic Temperature
Dizocilpine (1 mg/kg 1 h before ischemia)
Memantine (20 mg/kg 1 h before ischemia)
Memantine (20 mg/kg) immediately after ischemia
Control n=6
Treated n=8
Control n=5
Treated n=6
Control n=10
Treated n=10
124.1 4-22.9 33.9 4-3.3 7.41 4-0.05
127.8 4-20.1 35.8 4-2.2 7.38 4-0.04
146.8 4- 37.1 37.6 4-2.0 7.38 4-0.02
151.5 ± 30.0 36.1 4-2.4 7.39 + 0.04
150.9 4- 25.1 36.6 4-2.3 7.38 4-0.03
144.6 4- 21.5 35.4 4- 3.0 7.38 4-0.03
135.7 4-17.5
140.1 4-18.8
119.4 4-17.8
120.7 4-9.0
116.7 4-15.9
136.5 4-13.9
136.4 4-14.4 135.7 4-11.3 37.1 4-0.40
154.4 4-15.9 140.6 4-15.7 37.3 4-0.31
135.0 4-5.0 132.0 4- 8.4 37.2 4-0.13
130.0 + 7.1 125.0 4-9.8 37.1 +0.25
134.0 4-7.4 128.8 4-15.6 36.9 4-0.17
135.0 4-9.7 117.5 4- 20.5 36.8 4-0.26
22
means + S.D. Significant differences were determined by using a one-way analysis of variance followed by Duncan's multiple range test.
NEURONAL DAMAGE
3. Results 3.1. Cultured neurons
50
Memantine and dizocilpine protected cultured neurons against hypoxic damage in a dose-dependent manner (table 2). The protein content, the total number of cells per culture and the viability of the neurons increased significantly when the drugs were present 30 rain before to 1 day after the hypoxic period. Dizocilpine was already active at a concentration of 0.1/~mol/1 and memantine at 1.0/~mol/1.
0
I a" k\\~l CA1
CA3
. =
_,.,m..=
.
CA/,
Fig. 1. Protective effect of dizocilpine against neuronal damage in the rat hippocampal CA1 subfield after 10 mill ischemia followed by 7 days of recovery. Values are means 4- S.E.M. of seven (controls, empty columns) and eight (dizocilpine, hatched columns) experiments. Mann-Whitney U-test: * * P < 0.01.
3.2. Forebrain ischemia of the rat
Memantine and dizocilpine did not alter the physiological parameters measured (table 1). Neither drug affected the body temperature of the rats. The drugs prolonged anesthesia by 10-20
min. There was no difference between treated and control rats in the late recovery period. Dizocilpine, 1 mg/kg i.p. (1 h before ischemia), reduced neuronal damage in the CA1 subfield of
TABLE 2 Neuroprotective effects Of memantine and dizocilpine against hypoxic damage of cultured neurons. Neurons were obtained from chick embryo cerebral hemispheres. Hypoxia was induced by adding 1 mmol/1 NaCN to the incubation medium for 30 min after 4 days in culture. Normoxic controls ( o ) received saline only. Vehicle or drugs were added 30 mill before hypoxia and were left in the cultures up to 1 day after hypoxia. The cultures were analysed 3 days after hypoxia. The values are given as means 4- S.D. of five experiments. Duncan's test: " P < 0.01 Drug conc. (#mol/1)
ATP (nmol/mg protein)
Protein (mg/flask)
Cell count (lO s )
Viability (%)
22.43 23.21 23.00 23.10 28.06 27.03
0.185 0.222 0.260 0,295 0,275 0.266
+ 0.010 4- 0.014 a 4- 0.019 a 5=0.012 a 4- 0.011 5- 0.016
4.47 4- 0.11 4.99 4- 0.27 6.01 4- 0.48 a 5.78 4- 0.32 a 7.02 4- 0.91 6.96 + 0.30
60 4- 3 78 4- 6 a 74 5:1 a 82 4- 5 a 84 4- 5 86 4- 3
0.200 4- 0.014 0.199:1:0.010 0.250 + 0.015 a 0.2565-0.010 a 0.273 5:0.013 0.271 4- 0.007
4.14 4- 0.31 4.00 4- 0.50 5.51 4- 0.44 a 5.404-0.31 ~ 5.70 5- 0.31 5.96 4- 0.38
60 4- 4 59 4-1 83 4- 3 a 825:2 a 89 4- 2 88 4- 5
Dizocilpine 0 0.1 1 10 10 o 0o
+ 1.63 4-1.53 4-1.40 4- 0.91 4-1.44 4-1.61
Memantine 0 0.1 1 10 10 o 0°
21.60 4-1.24 21.25 4- 0.51 23.20 4-1.09 23.285:1.40 26.34 4- 0.88 26.72 + 0.91
23
the rat hippocampus (fig. 1). Pre-ischemic application of 10 m g / k g i.p. memantine (1 h before ischemia) caused a similar effect; the effect was even more pronounced with 20 m g / k g i.p. At a dose of 5 m g / k g i.p., memantine did not protect pyramidal CA1 neurons against ischemic damage (fig. 2). When administered immediately after ischemia, 10 m g / k g i.p. memantine significantly reduced the neuronal damage whereas 20 m g / k g of the drug did not provide significant protection (fig. 3).
NEURONAL DAMAGE ,\* 100"
50
4. Discussion
We demonstrated that memantine and dizocilpine can protect neurons in vivo and in vitro against damage caused by ischemia and hypoxia, respectively. Dizocilpine is a generally accepted non-competitive N M D A antagonist, and the resuits obtained with this drug are in line with previous findings (for references see Iversen et al., 1989). Thus, dizocilpine prevents neuronal damage in the hippocampal CA1 subfield after global ischemia in gerbils (Gill et al., 1987a; 1988) and NEURONAL DAMAGE % lOO"
--Z-v/..C,/d
50' ~llrA
5
10
I
20 DOSAGE (mg/kg, i.p.}
Fig. 2. Dose-dependent neuroprotective effect of memantine. Ischemia was induced for 10 min and rats were allowed 7 days to recover. Neuronal damage of the CA1 subfield is given. Values are means + S.E.M. of five (controls, empty columns) and 5-10 memantine (hatched columns) experiments. MannWhitney U-test: * * P < 0.01.
lu
20 DOSAGE (rng/kg,i.p.)
Fig. 3. Postischemic administration of memantine protects
neurons against ischemic damage. Forebrain ischemia was induced in rats for 10 min followed by 7 days of recovery. Neuronal damage of the hippocampal CA1 subfield is given. Values are means+S.E.M, of 10 (controls, empty columns) and 10-11 (memantine, hatched columns) experiments. MannWhitney U-test: * P < 0.02. rats (Gill et al., 1987b; Seif el Nasr et al., 1989; Rod and Auer, 1989), and reduces the infarct volume after focal ischemia in rats and cats (Oyzurt et al., 1988; McCulloch et al., 1989). In addition, dizocilpine ameliorates the viability of cultured neurons after hypoxia (Ahlemeyer and Krieglstein, 1989). Memantine binds to the phencyclidine recognition site in the NMDA-operated Ca 2+ channel (Kornhuber et al.,1989) and blocks Ca 2÷ entry through this channel (Bormann, 1989). Memantine also blocks Ca 2÷ entry through serotonin-operated channel (Reiser and Koch, 1989). These pharmacological properties may be the basis for the neuroprotective activity of memantine. Like dizocilpine, memantine reduced the percentage of damaged neurons in the CA1 subfield of the rat hippocampus 7 days after ischemia. This effect of memantine was dose-dependent and was attained after both pre-ischemic and postischemic administration. However, the minimal effective dose was 10 m g / k g for memantine and 1 m g / k g for dizocilpine. Cultured neurons are particularly suitable for demonstrating the neuroprotective effects of N M D A antagonists (Ahlemeyer and Kriegl-
24
stein, 1989; Peruche et al., 1990). We therefore used these cells to test the potential neuroprotective effect of memantine in comparison with that of dizocilpine: both drugs showed the expected effect. However, higher concentrations of memanfine than of dizocilpine were required for a neuroprotective effect. Most ligands of the phencyclidine recognition site seem to cause schizophrenia-like symptoms, so these drugs may have limited value as neuroprotectives for clinical therapy. Psychomimetic effects have not often been observed in patients with organic brain syndrome treated with high doses of memantine (Riederer and Danielczyk, personal communication). In summary, memantine protected neurons in the CA1 subfield of rat hippocampus against ischemic damage and cultured neurons of chick embryo cerebral hemispheres against hypoxic injury. These effects were comparable to those of dizocilpine, although memantine seemed to be less active. Whether memantine could be useful as a neuroprotective for clinical therapy remains to be shown.
Acknowledgements The authors wish to thank Ms. H. Lorenz and Ms. M. Lorenz for their skillful technical assistance.
References A_hlemeyer, B. and J. K.rieglstein, 1989, Testing drug effects against hypoxic damage of cultured neurons during longterm recovery, Life Sei. 45, 835. Auer, R.N., Y. Olsson and B.K. Siesj~, 1984, Hypoglycemic brain damage with the EEG isoelectric time, Diabetes 33, 1090. Bormann, J., 1989, Memantine is a potent blocker of Nmethyl-D-aspartate (NMDA) receptor channel, European J. Pharmacol. 166, 591. Gill, R., A.C. Foster and G.N. Woodruff, 1987a, Systemic administration of MK-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil, J. Nenrosci. 7, 3343. Gill, R., A.C. Foster and G.N. Woodruff, 1987b, Systemic administration of MK-801 protects against ischemic neuropathology in the rat, Br. J. Pharmacol. Proc. Suppl. 91, 311 p.
Gill, R., A.C. Foster and G.N. Woodruff, 1988, MK-801 is neuroprotective in gerbils when administered during the postischemic period, Neuroscience 25, 847. Iversen, L.L., G.N. Woodruff, J.A. Kemp, A.C. Foster, R. Gill and E.H.F. Wong, 1989, Nenroprotective properties of the glutamate antagonist dizocilpine maleate (MK-801) in animal models of focal and global ischemia, in: Pharmacology of Cerebral Ischemia 1988, ed. J. Krieglstein (Wissenschaftliche Verlagsgesellschaft, Stuttgart) p. 165. Kornlurber, J., J. Borrnann, W. Retz, M. Hiibers and P. Riederer, 1989, Memantine displaces [3H] MK-801 at therapeutic concentrations in postmortem human frontal cortex, European J. Pbarmacol. 166, 589. Krieglstein, J., H. Brungs and B. Peruche, 1988, Cultured neurons for testing cerebroprotective drug effects in vitro, J. Pharmacol. Methods. 20, 39. Lowry, O.H., N.J. Rosebrough, A.Y. Farr and R. Randel, 1951, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193, 265. McCulloch, J., C.K. Park, E. Oyzurt, D.G. Nehls, G.M. Teasdale and D.I. Graham, 1989, Effects of MK-801 in experimental models of focal cerebral ischemia in the cat and rat, in: Pharmacology of Cerebral Ischemia 1988, ed. J. Krieglstein (Wissenschaftliche Vedagsgesellschaft, Stuttgart) p. 173. Oyzurt, E., D.I. Graham, G.N. Woodruff and J. McCulloch, 1988, Protective effect of the glutamate antagonist MK-801 in focal cerebral isebemia in the cat, J. Cereb. Blood Flow Metab. 8, 138. Peruche, B., B. Ahlemeyer, H. Brungs, and J. Krieglstein, 1990, An in vitro model for testing drug protection against neuronal damage caused by hypoxia, J. Pharmacol. Meth. 23, 63. Pettmann, B., J.C. Louis and M. Sensenbrermer, 1979, Morphological and biochemical maturation of neurons cultured in the absence of glial cells, Nature 281, 378. Reiser, G. and R. Koch, 1989, Memantine inhibits serotonininduced rise of cytosolic Ca 2+ activity and of cyclic GMP level in a neuronal cell line, European J. Pharmacol. 172, 199. Rod, M.R. and R.N. Auer, 1989, Pre- and post-ischemic administration of dizocilpine (MK-801) reduces cerebral necrosis in the rat, Can. J. Nenrol. Sci. 16, 340. Self el Nasr, M., D. Sauer, C. Rossberg, H.D. Mermel and J. Krieglstein, 1989, Effects of NMDA-antagonists against neuronal damage after forebrain iscbemia in the rat, in: Pharmacology of Cerebral Iscbemia 1988, ed. J. Krieglstein (Wissenschaftliche Verlagsgesellschaft, Stuttgart) p. 211. Smith, M.L.,G. Bendek, N. Dahlgren, I. Rosen, T. Wieloch and B.K. Siesjti, 1984, Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model, Acta Nenrol. Scand. 69, 385. Wesemann, W., K.H. Sontag and J. Maj, 1983, Zur Pharmakodynamik und Pharmakokinetik des Memantin, Ar-znelm. Forseh. 33, 1122.